Patent Application
20250235540
The present invention belongs to the field of drug delivery, and overall relates to a PEGylated lipid; specifically, it relates to a non-linear PEGylated lipid which can be used as a component for drug carriers. The present invention also relates to a lipid composition and a lipid pharmaceutical composition, both containing the lipid, and formulation and application thereof.
Liposomes are widely used to deliver nucleic acid drugs, genetic vaccines, anti-tumor drugs, small molecule drugs, polypeptide drugs, or protein drugs. Liposomes formed by lipid compositions have various shapes and structures, among which the lipid nanoparticle (LNP), as an unconventional liposome, is a popular delivery technology at present. In particular, the successful application of LNP-nucleic acid pharmaceutical composition in the vaccines against the novel coronavirus has enhanced the development value of LNP. The LNP used for the delivery of nucleic acid drugs (e.g., mRNA) usually contains cationic lipids, neutral lipids, steroid lipids, and PEGylated lipids; wherein, cationic lipids electrostatically interact with the negatively charged mRNA; neutral lipids are usually phospholipids that prevent lipids from oxidation, or attach ligands to the surface of liposome or LNP, or reduce the aggregation of lipid particles; steroid lipids have stronger membrane fusogenic properties that facilitate cellular uptake and cytosolic entry of mRNA; PEGylated lipids are located at the surface of lipid nanoparticles, wherein the polyethylene glycol (PEG) chains are used for the surface modification of liposomes or LNPs to enhance hydrophilicity, form hydrated films around LNP surfaces, prevent particle aggregation, reduce non-specific cellular uptake during the drug delivery process, achieve the stealth effects of liposomes or LNPs, avoid rapid clearance by the immune system, and therefore improve the efficacy of LNP pharmaceutical formulations.
At present, the PEGylated lipids that have been applied and studied in a wider scope are basically modified with linear polyethylene glycols, with typical examples including DMG-PEG2000 (PEG2k-DMG) and ALC-0159, whereas non-linear PEGylated lipids are rarely used. Polyethylene glycol modification can improve the stability and safety of the modified substances in vivo, but at the same time also reduce the cellular uptake efficiency of drugs. Conventional PEGylated lipids are modified with linear polyethylene glycols which, if replaced by non-linear polyethylene glycols of comparable molecular weight, will cause reduction of the cellular uptake efficiency of the modified substances due to the higher protection ability of its umbrella-like shape (Veronese et al., BioDrugs. 2008, 22, 315-329). Moreover, many studies have revealed the phenomenon that PEGylated lipids can gradually detach from the surface of liposomes or LNPs and, therefore, cause the liposomes or LNPs to partially or completely lose the stealth properties, and such phenomenon becomes more pronounced under the presence of serum proteins and the shear forces in the bloodstream. With the single-chain length being comparable to that of a linear PEGylated lipid, the non-linear PEGylated lipid is easier to detach due to the greater shear forces attributed to the higher molecular weight of the polyethylene glycol moiety in a single lipid molecule (Mastrotto et al., Mol. Pharmaceutics 2020, 17, 472-487).
To solve the aforementioned problems, it is necessary to develop a novel type of non-linear PEGylated lipid.
The present invention provides a novel non-linear PEGylated lipid, particularly a PEGylated lipid containing a non-linear polyethylene glycol moiety and a lipid moiety which are connected by two branching cores. The PEGylated lipid can be applied in the preparation of lipid nanoparticles and pharmaceutical formulations thereof, which can improve the stability of drugs in the systemic circulation, cellular uptake efficiency, biocompatibility, encapsulation efficiency, delivery efficiency, and targeting ability, and meanwhile reduce the toxicity of drugs. Compared with conventional linear PEGylated lipids, the non-linear PEGylated lipids of the present invention exhibit enhanced protection for liposomes or LNPs, and when degradable or stable linking groups are incorporated at specific sites, the PEGylated lipids of the present invention can confer good stability to the liposomes or LNPs during systemic circulation with a higher efficiency of cellular uptake and in particular a higher transfection efficiency of nucleic acid drugs. The non-linear PEGylated lipid of the present invention can also be used in lipid pharmaceutical compositions with targeting properties to improve the targeting efficiency of drugs.
The above-mentioned purpose of the present invention is achieved by the following technical solutions.
An embodiment of the present invention is as follows:
A non-linear PEGylated lipid having the structure represented by the general formula (1) or general formula (2):
The present invention provides another embodiment:
A lipid composition containing the non-linear PEGylated lipid having the structure represented by the general formula (1) or general formula (2).
The present invention provides another embodiment:
A lipid pharmaceutical composition containing the aforementioned lipid composition and drugs.
The present invention provides another embodiment:
A formulation of lipid pharmaceutical composition containing the aforementioned lipid pharmaceutical composition and pharmaceutically acceptable diluents or excipients.
Compared with the Prior Art, the Present Invention has the Following Beneficial Effects:
The present invention provides a non-linear PEGylated lipid, particularly a non-linear PEGylated lipid containing a non-linear polyethylene glycol moiety and a lipid moiety which are connected by two branching cores, as well as a lipid composition containing the non-linear PEGylated lipid, a lipid pharmaceutical composition and formulation thereof. The lipid pharmaceutical composition of the present invention is preferably the LNP pharmaceutical composition, which has a high encapsulation efficiency of drugs, suitable particle sizes, non-toxicity, and good stability in serum. Compared with linear PEGylated lipids, the non-linear PEGylated lipid of the present invention can, even in the cases of lower molar usage and/or shorter PEG chains, realize better protection for the modified LNPs. The present invention also provides an LNP-nucleic acid pharmaceutical composition containing non-linear PEGylated lipids, which exhibits excellent nucleic acid complexation ability and high transfection activity. The present invention also provides a non-linear PEGylated lipid containing targeting groups coupled with its ends, which can significantly enhance the efficiency of targeted delivery of dugs. The present invention also provides a non-linear PEGylated lipid containing specific degradable/stable divalent linking groups, which can enable the lipid pharmaceutical compositions to undergo gradual degradation in the bloodstream and/or complete degradation within cells, therefore enhancing the uptake of drugs and especially the transfection of nucleic acid drugs.
The present invention has provided detailed description for the specific embodiments; however, it should be understood that the description is only provided in an illustrative manner rather than a restrictive manner. Any changes and modifications within the scope of the present invention will be obvious to those skilled in the art.
If a reference cited herein has a description different from the description in the present invention, the present invention shall prevail. This principle applies to all references cited throughout the specification.
In the present invention, unless otherwise described, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. The disclosures of all patents and other publications cited herein are incorporated by reference in their entireties. In the event that any description or interpretation of a term herein conflicts with any document incorporated herein by reference, the following description or interpretation of the term shall prevail. Unless otherwise specified, each term has the following meaning.
In the present invention, unless otherwise specified, the terms “selected from”/“preferably selected from” for any two objects are independent from each other. When multiple levels are available for the selections or preferred selections, those selected or preferably selected for any two objects can be of the same or different levels. For example, the statement “LA and LB are each independently selected from A, B, and C” implies that both LA and LB can be A, or that LA is A while LB is B1 (B1 is a subclass of B). For example, the statement “LA is preferably A (level 1 preferred selection), more preferably selected from A1˜A3 (level 2 preferred selection), and most preferably selected from A11˜A13 (level 3 preferred selection); LB is preferably B (level 1 preferred selection), more preferably selected from B1˜B3 (level 2 preferred selection), and most preferably selected from B11˜B13 (level 3 preferred selection)” implies that the preferred selections can be, that A is selected from A1˜A3 (level 2 preferred selection) while B is selected from B11˜B13 (level 3 preferred selection), or that A and B are both selected from their own level 3 preferred selections.
In the present invention, the terms “each independently”, “each independently selected from”, and “preferably, each independently selected from” mean that different objects can be each independently, or each independently selected from, or preferably, each independently selected from any option within the definitions, which can also be applied to the same object at different locations or occurrences. For example, provided that “a divalent group is selected from the group consisting of —NRcC(═O)—, —C(═O)NRc—, —NRcC(═O)NRc—, —OC(═O)NRc—, —NRcC(═O)O—, —SC(═O)NRc—, —NRcC(═O)S—, —C(Rc)═N—NRc—, and —NRc—N—C(Rc)—; wherein, Re is, at each occurrence, independently H or a C1-12 alkyl group”, such description indicates that the two Rc groups can be the same or different in “—NRc—N═C(Rc)—” (e.g., one Rc is a methyl group while the other Rc is a hydrogen atom or an ethyl group), and that any Rc in “—NRcC(═O)NRc—” can be the same as or different from the Re in “—NRcC(═O)O—”.
In the present invention, when at least two items are listed, the “combination” of the listed items is composed of any two or more of the aforementioned listed items; wherein, the number of an item is not limited, and the number of any item can be one or greater than one; when the number of an item is greater than 1, any two of the items can appear in the same or different specific forms. For example, provided that “a divalent linking group is selected from the group consisting of —CH2—, —O—, —S—, —C(═O)—, —NRc—, and combinations thereof; wherein, Re is, at each occurrence, independently a hydrogen atom or a C1-5 alkyl group”, the divalent linking group can be —CH2—, —O—, —S—, —C(═O)— or —NRc—, or a combination, e.g., —CH2—NH—CH2— (wherein, the number of —CH2— is 2, and the number of —NRc— is 1), (CH2)2—NH—(CH2)4—N(CH3)— (wherein, the number of —CH2— is 6, and the number of —NRc— is 2 with unidentical specific forms), or —(CH2)2—NHC(═O)— (wherein, the number of —CH2— is 2, the number of —NRc— is 1, and the number of —C(═O)— is 1), etc. Particularly, a combination composed of a linking bond and any linking group is still the linking group itself. In the present invention, a “linking group” contains at least one atom by default.
In the present invention, unless otherwise specified, the terms “include”, “contain”, “cover” and similar expressions in the specification and claims herein shall be interpreted as “include(s) but is/are not limited to” or “include(s) without limitation” in an open and inclusive manner.
In the present invention, the term “include(s) but is/are not limited to” followed by a certain range means that the items within the range are selectable but the selections are not limited to the range. Not all structures within the range are applicable, and especially, the items explicitly excluded by the present invention are not for consideration. The basic principle is to use the successful implementation of the present invention as a screening criterion.
In the present invention, the interpretation of numerical intervals includes a numerical interval indicated by a dash (e.g., 1∞6), a numerical interval indicated by a wavy line (e.g., 1˜6), and a numerical interval indicated by “to” (e.g., 1 to 6). Unless otherwise specified, an interval in the form of a range can represent a group consisting of all integers and non-integers within the range including two endpoints. For example, provided that an average number of EO units is selected from 22˜100, the range of selections is not limited to integers but also any non-integers within the interval. For example, the “integer in the range of 1-3” represents a group consisting of 1, 2, and 3. For example, —(CH2)1-4— represents a group consisting of —CH2—, —(CH2)2—, —(CH2)3—, and —(CH2)4—. For example,
represents a group consisting of
The numerical intervals in the present invention, including but not limited to the numerical intervals represented by integers, non-integers, percentages and fractions, all include two endpoints unless otherwise specified.
In the present invention, with respect to the molecular weight of polymers, the terms “about” and “approximately” generally suggest a ±10% numerical range which in some cases can be increased to ±15% but no greater than ±20%, with the preset value as the base value. For example, the deviations of 11 kDa and 12 kDa relative to 10 kDa are 10% and 20%, respectively; therefore, “approximately 10 kDa” includes but is not limited to 11 kDa and 12 kDa. For example, provided that the molecular weight of a PEG component of a general formula is about 5 kDa, the corresponding molecular weight or number average molecular weight is allowed to be 5 kDa±10%, i.e., variable in the range of 4500˜5500 Da.
In the present invention, with respect to percentages, the terms “about” and “approximately” generally suggest a ±(0.5*0.1N) % numerical range when the given value (without “%”) is accurate to the Nth decimal place. For example, approximately 1% means 1±(0.5*0.10) % (i.e., the range of 0.5%-1.5%), approximately 2.2% means 2.2±(0.5*0.11) % (i.e., the range of 2.15%-2.25%), and approximately 2.33% means 2.33±(0.5*0.12) % (i.e., the range of 2.335%-2.325%).
In the present invention, the degree of polymerization of a polyethylene glycol chain is represented by n1 (i is an integer selected from 1, 2, 3, . . . ); unless otherwise specified, “≈” is used to indicate sameness or equality between degrees of polymerization. With respect to monodisperse polymers, “=” can also be used to indicate sameness or equality. ni of the same value in the same structure can interchangeably represent each other; for example, when n2≈n3, n2 can be represented by n3 and n3 can also be represented by n2.
In the present invention, unless otherwise specified, a divalent linking group can be connected to another group using either one of its two connecting ends. For example, when an amide bond is used as a divalent linking group between GroupA and GroupB, both GroupA-C(═O)NH-GroupB and GroupA-NHC(═O)-GroupB are allowable when no specific connecting end is designated.
In the present invention, when it comes to the structural representation of groups, the “” is used to mark a linking bond; for example,
represents the group -G or G-, and
represents the group —CH3 or CH3—.
In the present invention, with respect to a linking bond or group extending out from a cyclic structure, when the connecting end points to the inside of the ring instead of marking any specific ring atom, it means that the linking bond or group can be connected to any appropriate ring atom. Moreover, when the ring is part of a fused ring system, the linking bond or group can be connected to any ring atom of the fused ring system. For example,
represents the group consisting of the structures with any rational chemical connections (including but not limited to
In the present invention, the range of the number of carbon atoms of a group can be represented in the form of a subscript at the subscript position of “C”, and unless otherwise specified, the number of carbon atoms includes no contribution from substituents. For example, C1-12 means “having 1 to 12 carbon atoms”. For example, a C1-10 alkylene group represents any alkylene group having the number of carbon atoms in the range indicated by the subscript, i.e., any one selected from the group consisting of C1, C2, C3, C4, C5, C6, C7, C8, C9, and C10 alkylene groups, including but not limited to a linear C1-10 alkylene group (e.g., —(CH2)6—) and a branched C1-10 alkylene group (e.g., —(CH2)3—CH(CH3)—(CH2)3—). For example, a “substituted C1-12 alkyl group” is selected from the group consisting of C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11 and C12 alkyl groups, each having at least one hydrogen atom replaced by a substituent, without particular restriction on the number of carbon atoms or heteroatoms in the substituent.
In the present invention, when a structure involved has isomers, unless otherwise specified, it can be any of the isomers. For example, with respect to a structure having both cis- and trans-isomers, it can be either a cis structure or a trans structure; with respect to a structure having E/Z isomers, it can be either an (E)-structure or a (Z)-structure; and, when optical activity is involved, a structure can be either levo or dextro.
In the present invention, if a structural representation is inconsistent with the name of the structure, then the structural representation should be prioritized.
In the present invention, the molecular weight of polyethylene glycol and its derivatives refers to the average molecular weight by default. Unless otherwise specified, the “average molecular weight” generally refers to the “number average molecular weight” (Mn). As for the number average molecular weight, it can be used as not only the molecular weight of polydisperse blocks or substances but also the molecular weight of monodisperse blocks or substances. Unless otherwise specified, the unit of measurement for molecular weight is dalton (Da). The molecular weight of polyethylene glycol chain can also be measured by the “degree of polymerization” which is, specifically, the number of repeat units (oxyethylene units). Accordingly, the average value and the number average value of the number of repeat units are preferably represented by “average degree of polymerization” and “number average degree of polymerization”, respectively.
In the present invention, the term “any appropriate” in “any appropriate linking group”, “any appropriate reactive group”, etc., indicates that the structures follow the basic rule of chemical structures and can facilitate the successful implementation of the preparation methods in the present invention. Chemical structures described in this manner can be regarded as having a clear and defined scope.
In the present invention, the terms “stable” and “degradable” regarding a group are a pair of opposing concepts.
In the present invention, the term “degradable” means that a chemical bond in the present invention can be cleaved to obtain at least two independent residues. If a linking group with its structure altered after chemical changes still remains a complete linking group, then it is still within the scope of being “stable”. The conditions for degradation are not particularly limited, which can be physiological conditions in vivo, simulated physiological environments in vitro, or other conditions, preferably physiological conditions in vivo or simulated physiological environments in vitro. The physiological conditions are not particularly limited, which can be intracellular or in the extracellular matrix, in normal physiological tissues or in pathologic tissues (e.g., tumor, inflamed tissue, etc.), and in body parts including but not limited to serum, heart, liver, spleen, lung, kidney, bone, muscle, fat, brain, lymph node, small intestine, gonad, etc. The simulated physiological environment in vitro is not particularly limited, including but not limited to physiological saline, buffer, culture medium, etc. The degradation is not particularly limited with respect to the rate, which can be, for example, rapid degradation via enzymolysis or slow hydrolysis under physiological conditions, etc. The physiological conditions in vivo include physiological conditions during treatment, such as ultraviolet radiation, thermal therapy, etc. The conditions for degradation include but are not limited to light, heat, low temperature, enzyme, oxidation-reduction, acidity, basicity, physiological condition, simulated physiological environment in vitro, etc., preferably light, heat, enzyme, oxidation-reduction, acidity, basicity, etc. The degradation can occur under stimulation by any above-mentioned condition. The light condition includes but is not limited to visible light, ultraviolet light, infrared light, near-infrared light, mid-infrared light, etc. The heat condition refers to a temperature higher than the normal physiological temperature, which is generally above 37° C. and below 45° C., preferably below 42° C. The low temperature condition refers to a temperature below the physiological temperature of human body, preferably below 25° C., and more preferably ≤10° C., with specific examples such as refrigeration temperature, freezer temperature, temperature for liquid nitrogen treatment, 2˜10° C., 4˜8° C., 4° C., 0° C., −20±5° C., etc. The enzyme is not particularly limited, and all enzymes that can be physiologically generated are included, e.g., peptidases, proteases, lyases, etc. The oxidation-reduction is not particularly limited, e.g., oxidation-reduction transformation between a mercapto group and a disulfide bond, and hydrogenation-reduction transformation. The acidic and basic conditions mainly refer to the pH conditions of internal body parts such as normal tissues, pathologic tissues, organs or tissues in treatment, etc.; for example, the stomach has an acidic condition, and tumor sites tend to be acidic as well. The degradation described herein can occur through metabolism in vivo (e.g., physiological effects, enzymes, oxidation-reduction, etc.), or under microenvironment stimulations (e.g., acidity and basicity) in specific in vivo sites, or under clinical therapeutic stimulations (e.g., light, heat, low temperature), etc. It should be noted that some conditions in organic chemistry that are extreme for living organisms, for example, strong acidity, strong basicity, high temperature (e.g., above 100° C.), etc., under which conditions a bond can be cleaved, are not considered to be within the scope of degradation conditions. For example, although an ether bond can be cleaved under strongly acidic conditions such as hydrobromic acid, it is always classified as a stable linking group in the present invention.
In the present invention, the term “stable” indicates that a linking group can keep existing as a complete linking group (i.e., a linking group which is stably and covalently connected to adjacent groups) and therefore it is defined as “stable”, allowing chemical changes without compromising the wholeness of the linking group. The chemical changes are not particularly limited, including but not limited to isomerization, oxidation, reduction, ionization, protonation, deprotonation, substitution, etc. The conditions for stable existence are not particularly limited, including but not limited to light, heat, low temperature, enzyme, oxidation-reduction, neutrality, acidity, basicity, physiological condition, simulated physiological environment in vitro, etc., preferably light, heat, enzyme, oxidation-reduction, acidity, basicity, etc. The stable existence described herein indicates that, without particular stimulation (e.g., pH conditions in particular areas, and light, heat, low temperature in treatment, etc.), the stable connections can be maintained in the metabolic cycle in vivo and the molecular weight is not reduced by bond cleavage (as long as the structural integrity is maintained).
In the present invention, “stable” is not an absolute concept with respect to a specific linking group. For example, an amide bond is much more stable than an ester bond under acidic or basic conditions, and the “stable” linking group of the present invention includes the amide bond. However, the peptide bond, for example, is a kind of amide bond formed via the dehydration condensation of an α-carboxyl group of an amino acid molecule with an α-amino group of another amino acid molecule, which can be cleaved under specific enzymatic conditions and, therefore, is also classified as a “degradable” linking group. Similarly, carbamate group, thiocarbamate group, etc., are linking groups which can be stable or degradable. More commonly, carbamate group, thiocarbamate group, etc., tend to degrade slowly, while non-peptide amide bonds can remain stable in the metabolic cycle in vivo. As another example, a common ester bond can degrade under acidic or basic conditions, while an ester bond contained in a special structure can also degrade under UV exposure. As another example, even though some chemical bonds can degrade under specific enzymatic conditions, they can still be regarded as stable if their circulation in clinical use (e.g., site-directed administration) does not or basically not go through the specific enzymatic conditions.
In the present invention, all compounds of general formula should be regarded as including salts thereof. The term “salt” is selected from the group consisting of acid addition salts formed by the compounds and inorganic and/or organic acids, base addition salts formed by the compounds and inorganic and/or organic bases, and combinations of any two or more thereof. When a compound of general formula contains both a basic moiety (such as, but not limited to, a pyridine or imidazole) and an acidic moiety (such as, but not limited to, a carboxylic acid), a zwitterion (“inner salt”) can be formed and included in the term “salt”. The “salt” can be pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) or otherwise. Salts of compounds of general formula can be formed by reacting the compounds with an amount (e.g., an equivalent) of acid or base in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization. Exemplary acid addition salts include acetates, adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemi sulfates, heptanoates, hexanoates, hydrochlorides, hydrobromides, hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oxalates, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates, sulfonates, tartrates, thiocyanates, toluenesulfonates, undecanoates, etc. Exemplary base addition salts include ammonium salts, alkali metal salts (e.g., sodium, lithium, and potassium salts), alkaline earth metal salts (e.g., calcium and magnesium salts), salts with organic bases (e.g., organic amines), and salts with amino acids (e.g., arginine, lysine). Basic nitrogen-containing groups may be quaternized with agents such as lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g., decyl, lauryl, tetradecyl, and stearyl chlorides, bromides, and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides), etc. The acid addition salts and base addition salts are both preferably pharmaceutically acceptable salts, and should be regarded as equivalent to the free forms of corresponding compounds of general formula for the purpose of this disclosure.
In the present invention, heteroatoms are not particularly limited, including but not limited to O, S, N, P, Si, F, Cl, Br, I, B, etc.
In the present invention, relative to a compound, a group formed after losing part of atoms or groups of the compound is also termed a residue.
In the present invention, groups having a valence greater than or equal to 2 are collectively referred to as “linking groups”. A linking group may have only one atom, such as an ether group (—O—) and a thioether group (—S—).
In the present invention, regarding the valence of a group, the term “multivalent” indicates that the valence is at least 3.
In the present invention, the term “group” for divalent groups can be replaced by the term “bond”, without changing the meanings. For example, a divalent ether group can also be termed an ether bond (—O—), a divalent ester group can also be termed an ester bond (—OC(═O)— or —C(═O)O—), and a divalent carbamate group can also be termed a carbamate bond (—OC(═O)NH— or —NHC(═O)O—). Particularly, the “linking group” can not be termed the “linking bond”.
In the present invention, when the number of atoms in a substituent is 1, the substituent can also be termed a “substituent atom”.
In the present invention, when a compound or group is “substituted”, it means that the compound or group contains one or more substituents.
In the present invention, unless otherwise specified, the terms “amine group” and “amino group” have the same meaning, including monovalent, divalent, trivalent, and quadrivalent neutral or cationic groups, substituted or unsubstituted. For example, the —NH2 in CH3—NH2 can be termed an “amino group”, “amine group”, or “primary amine group”. As another example, the —NH— in CH3—NH—CH3 can be termed a “secondary amine group” or “secondary amino group”, wherein the —NH—CH3 can also be interpreted as a methyl-substituted amine group.
In the present invention, the term “amine group” includes but is not limited to primary amine groups, secondary amine groups, tertiary amine groups, and quaternary ammonium ions. Exemplary amine groups include —NRtRt and —N+RtRtRt, wherein each Rt is independently a hydrogen atom or any hydrocarbon group.
In the present invention, when the valence is undefined, a “hydrocarbon group” could be a monovalent, divalent, trivalent, . . . , or n-valent hydrocarbon group, wherein n is the highest possible valence of the hydrocarbon group.
In the present invention, a “hydrocarbylene group” is a divalent hydrocarbon group.
In the present invention, the secondary amine bond and the hydrazine bond are “—NH—” and “—NH—NH—”, respectively, both capped by hydrocarbon groups at both ends, e.g., —CH2—NH—CH2— and —CH2—NH—NH—CH2—. As a counterexample, —C(═O)—NH— is termed an amide bond, instead of being considered to contain a secondary amine bond.
In the present invention, a “group with functionality” is also termed a “functional group”, which is preferably a reactive group, a protected reactive group, or a precursor of a reactive group, etc. The term “poly” indicates that the number of functional group is at least 3; for example, a polyol is a compound having at least 3 hydroxyl groups, a polythiol is a compound having at least 3 thiol groups, etc. What should be noted is that heterofunctional groups of other types are also allowed. For example, tris(hydroxymethyl)aminomethane is a triol containing also an amino group, and citric acid is a tricarboxylic acid containing also a hydroxyl group.
With respect to the preparation methods in the present invention, unless otherwise specified, reactive groups also include protected forms thereof, and the protected forms may be deprotected in any appropriate step in the actual preparation process to obtain the corresponding reactive forms.
In the present invention, ring atoms are the atoms that together constitute the ring skeleton.
In the present invention, the source of an amino acid is not particularly limited unless otherwise specified, which can be a natural source, a non-natural source, or a mix of both. The structural type of an amino acid in the present invention is not particularly limited unless otherwise specified, which can be L-type, D-type, or a mix of both.
In the present invention, the definitions and illustrations of the skeletons of amino acids, the skeletons of amino acid derivatives, and the skeletons of cyclic monosaccharides in documents including CN104877127A, WO/2016/206540A, CN106967213A, CN108530637A, CN108530617A and all cited documents, are hereby incorporated by reference. Unless otherwise specified, the skeletons are also residues. Wherein, an amino acid residue includes the amino acid with a hydrogen atom removed from its amino group and/or a hydroxyl group removed from its carboxyl group and/or a hydrogen atom removed from its thiol group and/or its amino group being protected and/or its carboxyl group being protected and/or its thiol group being protected. Imprecisely, an amino acid residue can also be termed an amino acid. Specific examples include residues formed by losing a carboxylic hydroxyl group (including every carboxylic hydroxyl group at the C-terminus as well as the carboxylic hydroxyl group in the pendant group of aspartic acid or glutamic acid), a hydrogen atom in a hydroxyl group, a hydrogen atom in a phenolic hydroxyl group (e.g., tyrosine), a hydrogen atom in a thiol group (e.g., cysteine), a hydrogen atom at a nitrogen atom (including every hydrogen atom at the N-terminus as well as the hydrogen atom in an amino group of a pendant group such as the hydrogen atom in the s-amino group of lysine or ornithine and the hydrogen atom in the amino group of the pendant ring of histidine or tryptophan), an amino group in an amide (e.g., asparagine, glutamine), an amino group in a pendant group of a guanidine, or a hydrogen atom in an amino group. Besides having the essential characteristics of an amino acid, a residue of amino acid derivative also has atomic or group moieties with the essential characteristics of a non-amino acid.
In the present invention, the term “bio-related substance” includes but is not limited to the substances described, listed, or referred to in documents including CN104877127A, WO/2016/206540A, CN106967213A, CN108530637A, CN108530617A and all cited documents. In general, bio-related substances include but are not limited to the following substances: drugs, proteins, polypeptides, oligopeptides, protein mimics, fragments and analogs, enzymes, antigens, antibodies and their fragments, receptors, small molecule drugs, nucleosides, nucleotides, oligonucleotides, antisense oligonucleotides, polynucleotides, nucleic acids, aptamers, polysaccharides, proteoglycans, glycoproteins, steroids, steroid compounds, lipid compounds, hormones, vitamins, phospholipids, glycolipids, dyes, fluorescent substances, targeting factors, targeting molecules, cytokines, neurotransmitters, extracellular matrix substances, plant or animal extracts, viruses, vaccines, cells, vesicles, liposomes, micelles, etc. The bio-related substances refer to not only themselves but also their precursors, activated states, derivatives, isomers, mutants, analogs, mimics, polymorphs, pharmaceutically acceptable salts, fusion proteins, chemically modified substances, genetically recombined substances, and also the corresponding agonists, activating agents, activators, inhibitors, antagonists, modulators, receptors, ligands or ligand groups, antibodies and fragments thereof, acting enzymes (e.g., kinases, hydrolases, lyases, oxoreductases, isomerases, transferases, deaminases, deiminases, invertases, synthetases, etc.), enzyme substrates (e.g., coagulation cascade protease substrates, etc.), etc. The derivatives include but are not limited to the derivatives of glycosides, nucleosides, amino acids, and polypeptides. Chemically modified products with newly formed reactive groups, i.e., modified products obtained by changing the types of reactive groups via modification or by introducing extra structures such as functional groups, reactive groups, amino acids or derivatives thereof, polypeptides, etc., are all within the scope of chemically modified substances of bio-related substances. Before or after bio-related substances combine with functionalized compounds, it is allowed that there are target molecules to be combined with, appendages, or delivery vectors to form modified bio-related substances or complex bio-related substances. Wherein, the pharmaceutically acceptable salts can be inorganic salts such as hydrochlorides, sulfates, and phosphates, or organic salts such as oxalates, malates, citrates, etc. Wherein, “drugs” in the present invention include any agents, compounds, compositions, and mixtures with physiological or pharmacological effects in vivo or in vitro, and the effects are usually beneficial. The drugs are not particularly limited with respect to the type, including but not limited to pharmaceuticals, vaccines, antibodies, vitamins, food, food additives, nutritional supplements, nutraceuticals, and other agents providing beneficial effects. The “drugs” are not particularly limited with respect to the range of physiological or pharmacological effects in vivo, which can be effective for the whole body or only at local sites. The “drugs” are not particularly limited with respect to the activity, mainly the active substances capable of interacting with other substances or the inert substances which are non-interactive; however, the inert substances can be transformed into active forms by in vivo effects or certain stimulations. Wherein, “small molecule drugs” are bio-related substances with molecular weight not exceeding 1000 Da, or small molecule mimics or active fragments of any bio-related substance.
In the present invention, the term “oligopeptide” refers to a peptide-type molecule formed by two or more amino acids, wherein the number of amino acid residues constituting the oligopeptide is preferably 2 to 20. An “oligopeptide residue” can be monovalent, divalent, trivalent or higher valent.
In the present invention, unless otherwise specified, the term “monosaccharide group” refers to the residue of monosaccharide, i.e., the skeleton of monosaccharide, including open-chain monosaccharide groups and cyclic monosaccharide groups (e.g., furanose ring and pyranose ring).
Monosaccharide groups in the present invention can be selected from the residues of compounds including but not limited to monosaccharides, sugar alcohols, deoxy sugars, amino sugars, amino sugar derivatives (e.g., amide derivatives), sugar acids, and glycosides, with open-chain or cyclic structures. Examples include the amino residue formed by removing an amino hydrogen atom of an amino sugar, the acyl group formed by removing a carboxylic hydroxyl group of a sugar acid, etc. The monosaccharides include but are not limited to aldoses (polyhydroxy aldehydes) and ketoses (polyhydroxy ketones). Examples also include alkyl ether derivatives, specifically, e.g., methyl ether derivatives, and more specifically, e.g., quebrachitol. Monosaccharide groups in the present invention are not particularly limited with respect to the number of carbon atoms, including but not limited to tetroses, pentoses, hexoses, and heptoses, preferably pentoses and hexoses. Wherein, exemplary tetroses, pentoses, hexoses, heptoses, sugar alcohols, deoxy sugars, amino sugars, amide derivatives of amino sugars, sugar acids, glycosides, etc., include but are not limited to the structures disclosed in CN106967213A.
In the present invention, the “micro-modification” refers to a chemical modification process that can be realized by simple chemical reactions. The simple chemical reactions mainly include protection, deprotection, salt complexation, decomplexation, ionization, protonation, deprotonation, changing leaving groups, etc.
In the present invention, the “micro-modified form” corresponds to the “micro-modification”, referring to a structural form that can be transformed into the target reactive group after simple chemical reactions such as protection, deprotection, salt complexation, decomplexation, ionization, protonation, deprotonation or changing leaving groups, etc. The changing leaving groups, i.e., transformation of leaving groups, includes but is not limited to the transformation of an ester form to an acyl chloride form.
In some specific embodiments of the present invention, the reaction process also involves “protection” and “deprotection” of relevant groups. To avoid the influence of a certain reactive group on the reaction, the reactive group is usually protected. In some specific embodiments of the present invention, when 2 or more reactive groups are present, the target reactive group should be selectively used for the reaction, and therefore the other reactive groups should be protected. The protecting groups not only remain stable during the target reaction process but can also, as needed, be removed by conventional technical means in the art.
In the present invention, the “protection” of a reactive group refers to a strategy for reversibly transforming the reactive group in need of protection to an inert group (i.e., non-reactive group) using particular reagents. Within a protected group, the moiety that distinguishes the protected form from the unprotected form is regarded as the “protecting group”. For example, —OTBS is a protected form of hydroxyl group (—OH), wherein the -TBS group is the hydroxyl protecting group.
In the present invention, the terms “deprotection” and “removal of protection” have the same meaning, both referring to the process of transforming a protected group to its unprotected form.
In the present invention, the term “hydroxyl protecting group” includes all the groups that can be used as common hydroxyl protecting groups, preferably alkanoyl (e.g., acetyl, butyryl), aromatic alkanoyl (e.g., benzoyl), benzyl, triphenylmethyl, trimethylsilyl, t-butyldimethylsilyl, allyl, acetal, or ketal groups. The removal of acetyl groups is generally carried out under basic conditions, most commonly by the ammonolysis with NH3/MeOH or by the methanolysis catalyzed by methanol anions. Benzyl groups can be easily removed via palladium-catalyzed hydrogenolysis in a neutral solution at room temperature, or via reduction reaction with metallic sodium in ethanol or liquid ammonia. Triphenylmethyl groups are generally removed via catalytic hydrogenolysis. Trimethylsilyl groups are generally removed using reagents containing fluoride ions (e.g., tetrabutylammonium fluoride/anhydrous THF, etc.). The t-butyldimethylsilyl ether is relatively stable, which can withstand ester hydrolysis conditions with alcoholic potassium hydroxide as well as mild reduction conditions (e.g., Zn/CH3OH, etc.), but can be removed by fluoride ions (e.g., Bu4N+F−) in a tetrahydrofuran solution or by aqueous acetic acid at room temperature. The protection of diols preferably forms dioxolanes, dioxanes, cyclic carbonates, or cyclic borates.
In the present invention, the term “thiol protecting group” includes all the groups that can be used as common thiol protecting groups. Similar to hydroxyl groups, thiol groups can be protected in the form of thioethers or thioesters. Thiol protecting groups are preferably tert-butyl, benzyl, substituted benzyl, benzhydryl, substituted benzhydryl, trityl, acetyl, benzoyl, tert-butoxycarbonyl, benzyloxycarbonyl, thioacetal, or thioketal groups. The deprotection of thioethers can be realized by acid-catalyzed reduction using Na/NH3, or by reactions with heavy metal ions (e.g., Ag, Hg) followed by treating with hydrogen sulfide. Some groups such as hemi-thioacetals containing S-diphenylmethyl, S-triphenylmethyl thioether, S-2-tetrahydropyranyl, or S-isobutoxymethyl groups can be oxidized to disulfides by (SCN)2, iodine, or thionyl chloride, and further reduced to thiols. The formation of thioester and the corresponding deprotection methods are the same as those of carboxylates.
In the present invention, the term “carboxyl protecting group” refers to a protecting group that can be transformed into a carboxyl group via hydrolysis or via the deprotection of the carboxyl protecting group. A carboxyl protecting group is preferably an alkyl (e.g., methyl, ethyl, and butyl) or aralkyl (e.g., benzyl) group, and more preferably a butyl (tBu), methyl (Me), or ethyl (Et) group. In the present invention, the “protected carboxyl group” is a group formed by protecting a carboxyl group with an appropriate carboxyl protecting group, preferably a methoxycarbonyl group, an ethoxycarbonyl group, a t-butyloxycarbonyl group, or a benzyloxycarbonyl group. The carboxyl protecting groups can be removed through hydrolysis catalyzed by acids or alkalis, or through pyrolysis reactions occasionally. For example, t-butyl groups can be removed under mild acidic conditions, and benzyl groups can be removed by hydrogenolysis. The reagent used for the removal of a carboxyl protecting group is selected from the group consisting of TFA, H2O, LiOH, NaOH, KOH, MeOH, EtOH, and combinations thereof, preferably the combination of TFA and H2O, the combination of LiOH and MeOH, or the combination of LiOH and EtOH. The deprotection of a protected carboxyl group can produce the corresponding free acid, which could be carried out in the presence of an alkali that forms pharmaceutically acceptable salts with the free acid generated during the deprotection.
In the present invention, the term “amino protecting group” is equivalent to “amine protecting group”, including all the groups that can be used as common amino/amine protecting groups, such as aryl C1-6 alkyl groups, C1-6 alkoxy C1-6 alkyl groups, C1-6 alkoxycarbonyl groups, aryloxycarbonyl groups, C1-6 alkylsulfonyl groups, arylsulfonyl groups, methylsilyl groups, etc. An amino protecting group is preferably selected from the group consisting of a t-butoxycarbonyl (Boc) group, a p-methoxybenzyloxycarbonyl (Moz) group, and a 9-fluorenylmethoxycarbonyl (Fmoc) group. The reagent used for the removal of an amino protecting group includes but is not limited to TFA, H2O, LiOH, MeOH, EtOH, and combinations thereof, preferably the combination of TFA and H2O, the combination of LiOH and MeOH, or the combination of LiOH and EtOH. The reagent used for the removal of Boc can be TFA or HCl/EA, preferably TFA. The reagent used for the removal of Fmoc can be the solution of 20% piperidine in N,N-dimethylformamide (DMF).
In the present invention, the term “alkynyl protecting group” includes all the groups that can be used as common alkynyl protecting groups, preferably a trimethylsilyl (TMS), triethylsilyl, tert-butyldimethylsilyl (TBS), or biphenyldimethylsilyl group. The TMS-protected alkynyl groups can be easily deprotected under basic conditions, for example, K2CO3/MeOH or KOH/MeOH. The TBS-protected alkynyl groups can be deprotected in the solution of tetra-n-butylammonium fluoride in tetrahydrofuran (TBAF/THF).
In the present invention, hydroxyl groups that can be protected by hydroxyl protecting groups are not particularly limited, e.g., alcoholic hydroxyl groups and phenolic hydroxyl groups. Amino/amine groups that can be protected by amino protecting groups are not particularly limited, e.g., amino groups of primary amines, secondary amines, hydrazines, and amides. Amine groups in the present invention are not particularly limited, including but not limited to primary amine groups, secondary amine groups, tertiary amine groups, and quaternary ammonium ions.
In the present invention, the deprotection of protected hydroxyl groups is related to the type of hydroxyl protecting group. The type of hydroxyl protecting group is not particularly limited; for example, benzyl, silyl ether, acetal, or tert-butyl groups can be used to protect terminal hydroxyl groups, and the corresponding deprotection methods include but are not limited to the following:
The removal of benzyl groups can be achieved via hydrogenation using hydrogenation reducing agents and hydrogen donors. The water content in this reaction system should be less than 1% so that the reaction can proceed smoothly.
The catalyst for hydrogenation reduction is not particularly limited, preferably palladium or nickel. The catalyst support is not particularly limited, preferably aluminum oxide or carbon, and more preferably carbon. The amount of palladium used is 1 to 100 wt % relative to the compounds containing protected hydroxyl groups, preferably 1 to 20 wt % relative to the compounds containing protected hydroxyl groups.
The reaction solvent is not particularly limited, as long as it can dissolve both the starting materials and the products, but is preferably methanol, ethanol, ethyl acetate, tetrahydrofuran, or acetic acid, more preferably methanol. The hydrogen donor is not particularly limited, but is preferably gaseous hydrogen, cyclohexene, 2-propanol, or ammonium formate, etc. The reaction temperature is preferably in the range of 25 to 40° C.
The reaction time is not particularly limited, which is negatively correlated with the amount of catalyst used and preferably in the range of 1 to 5 hours.
The compounds used for protecting hydroxyl groups in the form of acetals or ketals are preferably ethyl vinyl ether, tetrahydropyran, acetone, 2,2-dimethoxypropane, or benzaldehyde, etc. The deprotection of such acetals or ketals can be realized under an acidic condition wherein the pH of the solution is preferably 0 to 4. The acid used is not particularly limited, but is preferably acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, or nitric acid, and more preferably hydrochloric acid. The reaction solvent is not particularly limited, as long as it can dissolve the reactants and the products, but is preferably water. The reaction temperature is preferably 0 to 30° C.
The protected hydroxyl groups in the form of silyl ethers include trimethylsilyl ether, triethylsilyl ether, tert-butyldimethylsilyl ether, tert-butyldiphenylsilyl ether, etc. The deprotection of such silyl ethers uses compounds containing fluoride ions which are preferably tetrabutylammonium fluoride, tetraethylammonium fluoride, hydrofluoric acid, or potassium fluoride, and more preferably tetrabutylammonium fluoride or potassium fluoride. The molar equivalent of the fluorine-containing compound used is 5 to 20 times that of the protected hydroxyl group, preferably 8 to 15 times. If the molar equivalent of the fluorine-containing compound used is less than 5 times that of the protected hydroxyl group, the deprotonation will be incomplete. If the molar equivalent of the deprotection reagent used exceeds 20 times that of the protected hydroxyl group, the excess reagents or compounds will bring difficulty to purification and will possibly enter the subsequent steps to cause side reactions. The reaction solvent is not particularly limited, as long as it can dissolve the reactants and the products, but is preferably an aprotic solvent and more preferably tetrahydrofuran or dichloromethane. The reaction temperature is preferably 0 to 30° C.; if the temperature is lower than 0° C., the reaction rate will be relatively slow and the protecting group will not be completely removed.
The removal of tert-butyl groups is carried out under an acidic condition wherein the pH of the solution is preferably 0 to 4. The acid used is not particularly limited, but is preferably acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, or nitric acid and more preferably hydrochloric acid. The reaction solvent is not particularly limited, as long as it can dissolve the reactants and the products, but is preferably water. The reaction temperature is preferably 0 to 30° C.
In the present invention, the term “activation of carboxyl group” refers to the activation of carboxyl groups with carboxyl activating agents. The activated carboxyl groups can promote condensation reactions, for example, by inhibiting the generation of racemic impurities, by accelerating the reactions through catalysis, etc. The “carboxyl activating group” is a residue of a carboxyl activating agent. The carboxyl activating agent is selected from the consisting of N-hydroxysuccinimide group (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), N-hydroxy-5-norbornene-2,3-dicarboximide (HONb), N,N′-dicyclohexylcarbodiimide (DCC), and combinations thereof, preferably the combination of NHS/EDCI, NHS/DCC, or HONb/DCC, and more preferably the combination of NHS/EDCI or NHS/DCC.
In the present invention, the condensing agents used in reactions are not particularly limited, preferably selected from the group consisting of N,N′-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl), 0-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), and o-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), most preferably DDC. Generally, the molar equivalent of the condensing agent used is 1 to 20 times that of carboxylic acids, preferably 5 to 10 times. Appropriate catalysts (e.g., 4-dimethylaminopyridine (DMAP)) can be added in this reaction.
In the present invention, the obtained products can be purified via purification methods including but not limited to extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, membrane dialysis, supercritical extraction, etc.
In the present invention, stepwise/multi-step reactions can be completed by a technical personnel in multiple practical operations, or in a single practical operation. For instance, in a reaction system of a practical operation, two reactive groups in one molecule of a lipid compound both undergo coupling reactions with a single-chain polyethylene glycol derivative reagent to obtain a non-linear PEGylated lipid compound with two single-chain PEG arms; considering that the two coupling reactions actually occur successively, the whole process can also be regarded as introducing polyethylene glycol components in a stepwise/multi-step way.
In the present invention, reactions can be solvent-free or using aprotic solvents. The aprotic solvent includes toluene, benzene, xylene, acetonitrile, ethyl acetate, diethyl ether, tert-butyl methyl ether, tetrahydrofuran, chloroform, dichloromethane, dimethylsulfoxide, dimethylformamide, and dimethylacetamide, and is preferably tetrahydrofuran, dichloromethane, dimethylsulfoxide, or dimethylformamide.
In the present invention, the bases used in reactions can be organic bases (e.g., triethylamine, pyridine, 4-dimethylaminopyridine, imidazole, or N,N-diisopropylethylamine, preferably triethylamine or pyridine), or inorganic bases (e.g., K2CO3).
In the present invention, the repeat unit of polyethylene glycol is the oxyethylene unit, i.e., —CH2CH2O— or —OCH2CH2—, also termed the EO unit. The number of repeat units is also termed the number of EO units. The average number of repeat units is also termed the average number of EO units, which is preferably the number-averaged mean value.
In the present invention, for polydisperse cases, the term “identical”, “same” or “equal” (including other forms of equivalent expressions) with respect to the molecular weight/degree of polymerization of a single compound molecule and the number average molecular weight/number average degree of polymerization of a compound component in macroscopic matter, unless otherwise specified, does not indicate a strict equality but a proximity or approximate equality of values, wherein the proximity or approximate equality preferably corresponds to a deviation within ±10% and the preset value is generally used as the base value.
In the present invention, for monodisperse cases, the same or equal numbers of oxyethylene units with respect to a single compound molecule or a general formula are strictly equal in value. For example, provided that the number of EO units of a certain PEG component is 11, the value 12 is not within the preset scope. However, in order to obtain a compound component having the preset number of EO units, particular preparation methods might be used, and therefore the macroscopic matter obtained could also contain components having other numbers of EO units besides the component having the target number of EO units due to the limitations of preparation or purification methods; in this case, if the deviation between the average number of EO units and the preset number of EO units is within ±5% (base value≥10) or within ±0.5 (base value<10), it is considered to have obtained a monodisperse macroscopic matter containing the target component. Moreover, if the content of components having the number or average number of EO units within the allowed deviation reaches specific percentages (preferably ≥90%, more preferably >95%, more preferably >96%, more preferably >98%, and more preferably 99-100%), it is also considered to have obtained the monodisperse macroscopic matter containing the target component; even if the aforementioned content in percentage is not met, the product as well as the component in the form of main product, co-product or by-product, which are insufficient in content, are all within the scope of the present invention as long as they are prepared using the preparation methods of the present invention or any similar methods adopting basically the same ideas, with or without isolation or purification.
In the present invention, when Da, kDa, the number of repeat units, or the number of EO units is used to describe the molecular weight of a compound of general formula for polydisperse components, the value of molecular weight is allowed to be within a certain range around the given value (with endpoints included, and preferably within ±10%) with respect to a single compound molecule. When the number of oxyethylene units is used to describe the preset molecular weight of a compound of general formula for monodisperse components, the number is not a variable value in a range but a discrete point while the average number of EO units of the prepared product could be variable within a certain range (within ±10% or ±1, preferably within ±5% or ±0.5) due to non-uniform molecular weights. For example, provided that the molecular weight of an mPEG (methoxy polyethylene glycol) is 5 kDa, it means that the molecular weight of a single molecule of general formula is in the range of 4500-5500 Da and the average molecular weight of the corresponding components in the product is 5 kDa; i.e., the product with the average molecular weight in the range of 4500-5500 Da is regarded as the target product, and only the components with molecular weight in the range contribute to the content of the target component. As another example, provided that an mPEG is designed to have 22 oxyethylene units, all the compound molecules of general formula should have the number of EO units being strictly 22 while the prepared product could be a mixture of compounds having different numbers of EO units being 20, 21, 22, 23, or 24; meanwhile, if the average number of EO units is within the range of 22±2.2 (preferably in the range of 22=1.1), then the target component is considered obtained and all the components with molecular weight within the range can be regarded as the target component for calculation of purity.
In the present invention, unless otherwise specified, the term “mPEG” refers to a polyethylene glycol chain capped with a methoxy group, having the structure of
wherein ni is the degree of polymerization of the polyethylene glycol chain.
In the present invention, a product with PDI<1.005 can be regarded as monodisperse (PDI=1).
In the present invention, the number of repeat units in a “single-chain component” is at least 4.
In the present invention, the “lipid” includes but is not limited to esters of fatty acids and is generally characterized by poorer solubility in water and being soluble in many nonpolar organics. Although lipids usually have poorer solubility in water, some types of lipids (e.g., lipids modified with polar groups, such as DMG-PEG2000) have limited water solubility and can be dissolved in water under certain conditions. The known types of lipids include biomolecules such as fatty acids, waxes, sterols, fat-soluble vitamins (e.g., vitamins A, D, E, and K), monoglycerides, diglycerides, triglycerides, and phospholipids.
In the present invention, lipids include simple esters, compound esters, and derived lipids. The simple esters are esters formed by fatty acids and alcohols, which can also be classified into three subcategories: fats, oils, and waxes. The compound esters are “lipoid compounds” which are also termed “lipoids”, including phospholipids, sphingolipids, glycolipids, steroids, sterols, and lipoproteins. The derived lipids include simple lipid derivatives and compound lipid derivatives, having the general properties of lipids.
In the present invention, lipids can be synthetic or derived (separated or modified) from natural sources or compounds.
In the present invention, the term “liposome” refers to a closed vesicle formed by the self-assembly of lipids, having one or more lipid bilayer structures.
In the present invention, the term “lipid nanoparticle” or “LNP” refers to a nano-sized (e.g., 1 to 1000 nm) particle that contains one or more types of lipid molecules. In the present invention, the LNP can further contain at least one type of non-lipid payload molecule (e.g., one or more types of nucleic acid molecules). In some embodiments, the LNP contains a non-lipid payload molecule either partially or completely encapsulated inside a lipid shell. Particularly, in some embodiments, the payload is a negatively charged molecule (e.g., mRNA encoding a viral protein), and the lipid components of the LNP include at least one type of cationic lipid and at least one type of PEGylated lipid. It can be expected that the cationic lipids can interact with the negatively charged payload molecules and facilitate the incorporation and/or encapsulation of the payload into the LNP during LNP formation. As provided herein, other lipids that can be part of an LNP include but are not limited to neutral lipids and steroid lipids. In some embodiments, the LNP of the present invention contains one or multiple types of the PEGylated lipids represented by the formula (1) or formula (2) described herein.
In the present invention, the term “cation” refers to a structure that is positively charged either permanently or non-permanently in response to certain conditions (e.g., pH). Therefore, cations include not only permanent cations but also those cationisable. Permanent cations refer to the corresponding compounds, groups, or atoms that are positively charged under conditions of any pH value or hydrogen ion activity of its environment. As a typical example, the presence of a quaternary nitrogen atom results in a positive charge. When a compound carries multiple positive charges, it can also be termed a polycation. A cationisable substance refers to a compound, group, or atom that is positively charged at a lower pH and uncharged at a higher pH of its environment. Moreover, in non-aqueous environments where the pH cannot be determined, a cationisable compound, group, or atom is positively charged at a high concentration of hydrogen ions and uncharged at a low concentration or activity of hydrogen ions. It depends on the individual properties of the cationisable or polycationisable compound, in particular the pKa of the respective cationisable group or atom, at which pH or hydrogen ion concentration the compound is charged or uncharged. In diluted aqueous environments, the fraction of cationisable compounds, groups, or atoms that are positively charged may be estimated using the so-called Henderson-Hasselbalch equation which is well-known to a person skilled in the art. In some embodiments, a cationisable compound or its moiety is positively charged at the physiological pH (e.g., approximately 7.0-7.4). In some preferred embodiments, a cationisable compound or its moiety is neutral at the physiological pH (e.g., approximately 7.0-7.4), but becomes positively charged at a lower pH (e.g., approximately 5.5-6.5). In some embodiments, the preferred range of pKa of the cationisable compound or its moiety is from approximately 5 to approximately 7.
In the present invention, the term “cationic lipid” refers to a lipid that is positively charged at any pH or hydrogen ion activity of its environment, or a cationisable lipid which can be positively charged in response to the pH or hydrogen ion activity of its environment (e.g., the expected environment for its use). Therefore, the term “cationic” includes the scopes of both “permanently cationic” and “cationisable”. In some embodiments, the positive charge of a cationic lipid originates from the existence of a quaternary nitrogen atom. In some embodiments, cationic lipids include zwitterionic lipids that can be positively charged in the expected environment for their use (e.g., at the endosomal pH). In some embodiments, if a liposome or LNP contains cationisable lipids, it is preferred that about 1% to 100% cationisable lipids are cationized at a pH of about 1 to 9, preferably 4 to 9, 5 to 8, or 6 to 8, and more preferably at the endosomal pH (e.g., about 5.5 to 6.5). Cationic lipids include but are not limited to N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTAP), N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy-1-(dimethylamino) propane (DODMA), 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate (DLin-MC3-DMA), (DLin-KC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino) octanoate (SM102), and compositions thereof, as well as the cationic lipids disclosed in CN113402405A and mixtures thereof.
In the present invention, the term “PEGylated lipid” refers to a molecule containing both lipid and polyethylene glycol moieties, which can be classified into linear PEGylated lipid and non-linear PEGylated lipid. PEGylated lipids not only include those described in the present invention, but also include but are not limited to polyethylene glycol-1,2-dimyristoyl-sn-glycerol (PEG-DMG), polyethylene glycol-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE), PEG-cholesterol, polyethylene glycol-diacylglycamide (PEG-DAG), polyethylene glycol-dialkyloxypropyl (PEG-DAA), and specifically, PEG500-dipalmitoylphosphatidylcholine, PEG2000-dipalmitoylphosphatidylcholine, PEG500-distearylphosphatidylethanolamine, PEG2000-distearylphosphatidylethanolamine, PEG500-1,2-oleoylphosphatidylethanolamine, PEG2000-1,2-oleoylphosphatidylethanolamine, PEG2000-2,3-distearoylglycerol (PEG-DMG), etc.
In the present invention, the term “neutral lipid” refers to any of the many types of lipid substances existing in the form of uncharged species or neutral zwitterionic species at a chosen pH, preferably phospholipid. Such lipids include but are not limited to 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (MPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-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-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and compositions thereof. Neutral lipids can be synthetic or naturally sourced.
In the present invention, a “steroid lipid” is selected from the group consisting of cholesterol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol tomatidine, ursolic acid, α-tocopherol, and compositions thereof.
In the present invention, a liposome/lipid nanoparticle can be termed a “PEGylated liposome/lipid nanoparticle” when containing PEGylated lipids, and termed a “cationic liposome/lipid nanoparticle” when containing cationic lipids, and termed either a “PEGylated liposome/lipid nanoparticle” or a “cationic liposome/lipid nanoparticle” when containing both PEGylated lipids and cationic lipids.
In the present invention, the “cationic liposome/lipid nanoparticle” refers to a liposome/lipid nanoparticle containing cationic lipids, which can contain other types of lipids (including but not limited to PEGylated lipids, neutral lipids, steroid lipids, etc.) simultaneously.
In the present invention, the term “N/P ratio” refers to the molar ratio of the nitrogen atoms in cationic lipids to the phosphate groups in nucleic acids.
In the present invention, the term “nucleic acid” refers to DNA, RNA, or a modified form thereof, including purine or pyrimidine bases in DNA (adenine “A”, cytosine “C”, guanine “G”, thymine “T”), and purine or pyrimidine bases in RNA (adenine “A”, cytosine “C”, guanine “G”, uracil “U”).
In the present invention, the term “RNA” refers to a ribonucleic acid that may be naturally or non-naturally existing. For example, an RNA may include modified and/or non-naturally existing components such as one or more nucleobases, nucleosides, nucleotides, or linkers. An RNA may include a cap structure, a chain-terminating nucleoside, a stem loop, a polyadenylation sequence and/or a polyadenylation signal. An RNA can have a nucleotide sequence encoding a polypeptide of interest. For example, an RNA can be a messenger RNA (mRNA). Translation of an mRNA encoding a particular polypeptide, for example, the in vivo translation of an mRNA inside a mammalian cell, can produce the encoded polypeptide. RNAs can be selected from the non-limiting group consisting of small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, single guide RNA (sgRNA), cas9 mRNA, and mixtures thereof.
In the present invention, an antisense oligonucleotide or small interfering RNA (siRNA) can inhibit the expression of the target gene and the target protein in vitro or in vivo.
In the present invention, FLuc mRNA can express the luciferase protein which emits bioluminescence in the presence of a fluorescein substrate, and therefore FLuc is commonly used in the culture of mammalian cells to measure gene expression and cell activity.
In the present invention, the term “inhibiting the expression of a target gene” refers to the ability of nucleic acids to silence, reduce, or inhibit the expression of a target gene. To examine the extent of gene silencing, a test sample (e.g., a sample of cells in culture expressing the target gene) is contacted with nucleic acids that inhibit the expression of the target gene. The expression of the target gene in the test sample or test animal is compared to that in a control sample (e.g., a sample of cells in culture expressing the target gene) which is not contacted with or administered the nucleic acids. The expression of the target gene in the control sample can be assigned a value of 100%. In particular embodiments, inhibition of the expression of the target gene is achieved when the level of target gene expression in the test sample relative to the level of target gene expression in the control sample or the control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%.
In the present invention, methods for determining the level of target gene expression include but are not limited to dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays.
In the present invention, the term “transfection” refers to the introduction of a species (e.g., RNA) into a cell. Transfection may occur, for example, in vitro, ex vivo, or in vivo.
In the present invention, the term “antigen” typically refers to a substance that can be recognized by the immune system, preferably recognized by the adaptive immune system, and can trigger an antigen-specific immune response, for example, forming antibodies and/or antigen-specific T cells as a part of the adaptive immune response. Typically, the antigen may be or may contain a peptide or a protein that can be presented to T cells by MHC. In the sense of the present invention, the antigen may be a translation product of the provided nucleic acid molecule (preferably mRNA as defined herein). In this context, fragments, variants, and derivatives of peptides and proteins containing at least one epitope are also regarded as antigens.
In the present invention, the term “delivery” refers to providing an entity to the target, for example, delivering drugs and/or therapeutic agents and/or prophylactic agents to subjects, the subjects being tissues and/or cells of human and/or other animals.
In the present invention, the term “pharmaceutically acceptable carrier” refers to a diluent, adjuvant, excipient, or vehicle administered together with the therapeutic agent, which is, within the scope of sound medical judgement, suitable for contact with tissues of human and/or other animals without causing any excessive toxicity, irritation, allergic reaction, or other problems or complications corresponding to rational benefit/risk ratios. Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions in the present invention include but are not limited to sterile liquids, such as water and oil, including oils from petroleum, animals, vegetables, or synthetics, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, an exemplary carrier is water. Physiological saline, glucose, and aqueous glycerol solution can also be used as liquid carriers, especially for injection. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, defatted milk powder, glycerol, propylene glycol, water, ethanol, etc. The compositions may also contain a small amount of humectant, emulsifier, or pH buffer as needed. Oral preparations may contain standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Specifically, excipients include but are not limited to anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and water for hydration. More specifically, excipients include but are not limited to butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate, calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, phenyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, and xylitol.
In the present invention, pharmaceutical compositions can act systematically or locally. For this purpose, they can be administered via appropriate routes such as injection (e.g., intravenous, intraarterial, subcutaneous, intraperitoneal, and intramuscular injections, including instillation) or transdermal delivery, or via oral, buccal, transnasal, transmucosal, or topical routes, or in the form of ophthalmic preparation, or by inhalation. Regarding these routes of administration, the pharmaceutical compositions of the present invention can be administered in suitable dosage forms. The dosage forms include but are not limited to tablets, capsules, lozenges, hard sugar agents, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, and syrups.
In the present invention, vaccines are preventive or therapeutic materials that provide at least one antigen or antigenic function. An antigen or antigenic function can stimulate the body's adaptive immune system to provide an adaptive immune response.
In the present invention, treatment refers to patient management and care in order to resist diseases, obstacles, or symptoms, which is intended to delay the development of diseases, obstacles, or symptoms, reduce or alleviate symptoms and complications, and/or cure or eliminate diseases, obstacles, or symptoms. The patients to be treated are preferably mammals, especially humans.
In one embodiment of the present invention:
In the present invention, a compound of the general formula (1) or general formula (2) can exist in an unsolvated or solvated form including the hydrated form. In general, for the purpose of the present disclosure, the solvated forms having pharmaceutically acceptable solvents (e.g., water, ethanol, etc.) are equivalent to the unsolvated forms.
In the present invention, a compound of the general formula (1) or general formula (2) and its salts and solvates can exist in their tautomeric forms including those from keto-enol tautomerism, amide-imidic acid tautomerism, lactam-lactim tautomerism, enamine-imine tautomerism, proton transfer tautomerism and valence tautomerism.
In the present invention, a compound of the general formula (1) or general formula (2) should be considered as including its salts, tautomers, stereoisomers and solvates.
In the present invention, unless otherwise specified, all divalent linking groups are not particularly limited with respect to the stability. Any divalent linking group itself or one that consists of the divalent linking group and its adjacent heteroatom-containing groups are each independently a stable linking group or a degradable linking group.
In the present invention, unless otherwise specified, all divalent linking groups are not particularly limited with respect to the structure which can be a linear structure, a branched structure or a ring-containing structure. The number of non-hydrogen atoms in a divalent linking group is not particularly limited, each independently selected from integers of 0˜20, preferably selected from integers of 1˜10; wherein, the non-hydrogen atom is C, O, S, N, P, Si or B; when the number of non-hydrogen atoms is greater than 1, there are 1 type, 2 types, or more than 2 types of non-hydrogen atoms, and the non-hydrogen atoms are a combination of carbon and carbon atoms, a combination of carbon and hetero atoms, or a combination of hetero and hetero atoms.
In one specific embodiment of the present invention, Lx is, at each occurrence, independently selected from the group consisting of —CH2—, —CH2CH2C(═O)OCH2—, —CH2CH2C(═O)OCH2CH2—, —C(═O)NH—, —C(═O)NH(CH2)4—, —C(═O)—, —C(═O) CH2—, and —C(═O) CH2CH2—, and the left end of Lx is connected to —O—.
In one specific embodiment of the present invention, L01 is selected from the group consisting of a linking bond, —(CH2)t—, —NH(CH2)t—, —NH(CH2)(C(═O)NH(CH2)t—, —O(CH2)t—, —NH(CH2)tC(═O)O(CH2)t—, —OC(═O)(CH2)t—, —OC(═O)O(CH2)t—, —OC(═O)(CH2)tC(═O)—, and —(CH2)tC(═O)NH(CH2)t—, and the left end of L01 is connected to R01; wherein, t is an integer in the range of 1-4; L01 is preferably a linking bond, —NHCH2CH2— or —OCH2CH2—.
In one specific embodiment of the present invention, L1 and L2 are each independently selected from the group consisting of a linking bond, —O—, —C(═O)—, —OC(═O)—, —C(═O)O—, —NHC(═O)—, —C(═O)NH—, —OC(═O)NH—, —NHC(═O)O—, —OC(═O)O—, —C(═O)O(CH2)4OC(═O)—, —C(═O)O(CH2)5OC(═O)—, —C(═O)O(CH2)6OC(═O)—, —C(═O)O(CH2)7OC(═O)—, and —C(═O)O(CH2)8OC(═O)—, and the left end of L1 or L2 is connected to B1 or B2.
In one specific embodiment of the present invention, AA is a residue of an amino acid or derivative thereof, preferably selected from the group consisting of the residues of glycine, alanine, β-alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, ornithine, citrulline, and derivatives thereof; preferably, -(AA)y- is -PheGly-, -GlyGly-, or -GlyGlyGly-; wherein, Gly is the residue of glycine and has the structure of —NHCH2C(═O)—, Phe is the residue of phenylalanine and has the structure of
In one specific embodiment of the present invention, Ld is selected from the group consisting of —OCH2—, —CH2CH2—, —C(═O)NH—, —C(═O)O—, —NHC(═O)—, —OC(═O)—, —C(═O)NHCH2—, —C(═O)OCH2CH2C(═O)NH—, —C(═O)NHCH2CH2C(═O)NH—, —CH2CH2OC(═O)NHCH2—, and —C(═O)(AA)yNH—, and the right end of Ld is connected to X.
In one specific embodiment of the present invention, Lx, L01, L1, L2, Ld, and the group formed by any one thereof together with adjacent heteroatoms, are each independently a degradable or stable group.
In one specific embodiment of the present invention, B1 and B2 are each independently a linking bond or a C1-14 alkylene group; the C1-14 alkylene group has 0-2 hydrogen atoms replaced by 0-2 Rq, and Rq is, at each occurrence, independently —(CH2)tqC[(CH2)tqH]3, (CH2)tqS(CH2)tqH, or —(CH2)tqN[(CH2)tqH]2, wherein tq is, at each occurrence, independently an integer in the range of 0-4; preferably, Rq is —CH3; preferably, B1 and B2 are each independently selected from the group consisting of a linking bond, —CH2—, —(CH2)2—, —(CH2)3—, (CH2)4—, —(CH2)5—, —(CH2)6—, —(CH2)7—, and —(CH2)8—.
In one specific embodiment of the present invention, R1 and R2 are each independently selected from the group consisting of RL, RB, and Rr; preferably, R1 and R2 are each independently RL or RB;
In one specific embodiment of the present invention, n1, n2, and n3 are each independently an integer in the range of 4-100, preferably an integer in the range of 10-60, more preferably an integer in the range of 10-45, and most preferably 10, 11, 12, 20, 21, 22, 23, 24, or 25.
In one specific embodiment of the present invention, the number average molecular weight of
is 0.5 kDa˜20 kDa, preferably 0.5 kDa˜5 kDa, and more preferably 0.5 kDa, 1 kDa, 2 kDa, or 5 kDa.
In one specific embodiment of the present invention, R01 a functional group that can interact with bio-related substances, wherein the interaction is selected from the group consisting of covalent bond formation, hydrogen bond formation, fluorescence effect, and targeting effect; R01 is selected from the group consisting of reactive groups, modified forms of reactive groups, therapeutically targeting functional groups, and fluorescent functional groups; wherein, the modified forms are selected from the group consisting of the precursors of reactive groups, the active forms to which reactive groups are precursors, the active forms in which reactive groups are substituted, and the inactive forms in which reactive groups are protected; wherein, the precursors of reactive groups refer to the structures which can be transformed into the reactive groups through at least one process among oxidation, reduction, hydration, dehydration, electronic rearrangement, structural rearrangement, salt complexation and decomplexation, ionization, protonation, and deprotonation.
In one specific embodiment of the present invention, the aforementioned R01 is selected from the group consisting of the functional groups in the following classes A˜I and modified forms thereof:
In one specific embodiment of the present invention, the aforementioned R01 is selected from the group consisting of the functional groups of the following classes A˜I and modified forms thereof:
In one specific embodiment of the present invention, the aforementioned R01 is preferably selected from the group consisting of a hydroxyl group, a thiol group, a sulfonate group, an amino group, a maleimide group, a succinimide group, a carboxyl group, an acyl chloride group, an aldehyde group, an azido group, a cyano group, an allyl group, a propargyl group, an epoxypropyl group, a folate residue, a biotin residue, and modified forms thereof.
In one specific embodiment of the present invention, the aforementioned R01 is preferably selected from the group consisting of an epoxy group, an alcoholic hydroxyl group, a thiol group, a carboxyl group, an amino group, an azido group, an aldehyde group, an alkenyl group, an alkynyl group, a cyano group, an active ester group, an acetal group, a succinimide group, a maleimide group, an active ester group, an active carbonate group, a p-nitrophenyl group, a carbamate group, an isocyanate group, an isothiocyanate group, an enoate group, a dithiopyridyl group, an α-haloacetyl alkynyl group, a folic acid group, a rhodamine group, a biotin group, a monosaccharide group, a polysaccharide group, and modified forms thereof, or from the group consisting of the following structures:
In one specific embodiment of the present invention, T is a hydrogen atom or a C1-6 alkyl group, preferably a methyl group.
In one specific embodiment of the present invention, a PEGylated lipid can be obtained from a preparation process containing coupling reactions and/or polymerization reactions;
In the present invention, the starting materials used in every preparation methods can be obtained by purchase or synthesis.
In the present invention, with respect to the preparation method of monodisperse PEGylated lipids, the monodisperse starting materials containing polyethylene glycol components can be replaced by polydisperse starting materials containing the same components, therefore obtaining the corresponding polydisperse products; similarly, with respect to the preparation method of polydisperse PEGylated lipids, the polydisperse starting materials containing polyethylene glycol components can be replaced by monodisperse starting materials containing the same components, therefore obtaining the corresponding monodisperse products.
In the present invention, by changing only the number average degree of polymerization and/or PDI of the polyethylene glycol reagent or derivative thereof used as a starting material, without changing the corresponding structural formula, the reaction can be smoothly implemented without requiring any obvious alterations to the reaction conditions, and the alterations do not involve changing the amount of solvent used.
In one specific embodiment of the present invention, polyethylene glycol starting materials including but not limited to linear/non-linear polyethylene glycols and derivatives thereof can be prepared by referring to the methods in CN108530637B, CN110591079A, CN108659227A, CN108530617B, CN1243779C, or CN101029131A.
The intermediates and end-products prepared in the present invention can be purified by a purification method including but not limited to extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, membrane dialysis, supercritical extraction, etc. Characterization methods including but not limited to NMR, electrophoresis, UV-visible spectrophotometer, FTIR, AFM, GPC, HPLC, MALDI-TOF, circular dichroism spectroscopy, etc., can be used in the characterization and determination of the structure and molecular weight of end-products.
In the present invention, the actual preparation process can also include any necessary procedures such as micro-modification, protection/deprotection, intermediate preparation, workup, purification, etc., which are familiar to a person skilled in the art, mentioned or not in the present invention.
The structures of monofunctional lipid small molecule compound LP-1, bifunctional lipid small molecule compound LP-2, and trifunctional lipid small molecule compound LP-3 are as follows:
LP-1 can be synthesized through a coupling reaction between a carbon-branched trifunctional small molecule and a monofunctional aliphatic hydrocarbon derivative:
A specific example is the synthesis of S3-3 in Example 3.1:
In some specific embodiments (e.g., Example 2.1), LP-1 can also be synthesized using phosphorus chloride and hydroxyl group-containing aliphatic hydrocarbon derivative as starting materials, referring to the following method:
LP-1 can further be coupled with a trifunctional small molecule to obtain LP-2:
A specific example is the synthesis of S1-5 in Example 1.1:
LP-1 can further be coupled with a tetrafunctional small molecule to obtain LP-3:
A specific example is as follows:
LP-1, LP-2, and LP-3 can further be coupled with a bifunctional small molecule to obtain a product which still belongs to the scope of LP-1, LP-2, or LP-3; one specific example is the synthesis of S3-7 in Example 3.3:
Introducing a non-linear polyethylene glycol component containing a carbon branching core in a single step. The lipid compound LP-1 undergoes a coupling reaction with a two-arm or three-arm non-linear polyethylene glycol reagent or derivative thereof, therefore obtaining a compound of the general formula (1) or general formula (2). Wherein, the definition of Lm3 is the same as that of the aforementioned Lm1, the definition of FG3 is the same as that of the aforementioned FG1, and the specific forms are based on the smooth implementation of the reactions; FG1 reacts with FG3 to obtain a divalent linking group, which together with Lm1 and Lm3, forms Ld.
A specific example is the synthesis of E3-1 in Example 3.1:
The reactive group FG1 of the lipid compound LP-2 reacts with two RPEG-Lm2-FG2 to
obtain a compound of the general formula (1), wherein RPEG is n1≈n2, the definition of Lm2 is the same as that of the aforementioned Lm1, the definition of FG2 is the same as that of the aforementioned FG1, and the specific forms are based on the smooth implementation of the reactions; FG1 reacts with FG2 to obtain a divalent linking group, which together with Lm1 and Lm2, forms Lx.
A specific example is the synthesis of E1-1 in Example 1.1:
The reactive group FG1 of the lipid compound LP-3 reacts with three RPEG-Lm2-FG2, therefore obtaining a compound of the general formula (2), wherein RPEG is
n1≈n2≈n3, the definition of Lm2 is the same as that of the aforementioned Lm1, the definition of FG2 is the same as that of the aforementioned FG1, and the specific forms are based on the smooth implementation of the reactions; FG1 reacts with FG2 to obtain a divalent linking group, which together with Lm1 and Lm2, forms Lx.
A specific example is as follows:
When the aforementioned FG1 is a hydroxyl group, the lipid compound LP-1, LP-2, or LP-3 can be used as an initiator, which initiates the polymerization of ethylene oxide after deprotonation. The obtained polymerization product is a mixture of alcohol and oxyanion, which is subjected to a capping reaction after complete deprotonation, and the deprotonation reagent is preferably diphenylmethyl potassium (DPMK). A specific example is the synthesis of E1-2 in Example 1.2 via polymerization:
In one specific embodiment of the present invention, the ends of the non-linear PEGylated lipid contain the functional group R01, and R01 is preferably introduced by active polyethylene glycol derivatives in an unprotected or protected manner.
In one specific embodiment of the present invention, starting materials include active polyethylene glycol derivatives. The structure of the polyethylene glycol derivative can be any specific structure of monofunctional non-linear polyethylene glycol documented in CN108530637B, CN110591079A, CN108659227A, CN108530617B, and CN1243779C.
In one specific embodiment of the present invention, the end-functionalization can be further applied to a PEGylated lipid of the general formula (1) or (2), and the obtained structure after the end-functionalization still belongs to the general formula (1) or (2). The method for the end-functionalization is not particularly limited but is related to the type of the final functional group or its protected form, mainly including the functionalization of the terminal hydroxyl group and the transformation from a reactive group to the target functional group or its protected form.
In the present invention, specific preparation methods of the functionalization of the terminal hydroxyl group include but are not limited to those documented in the paragraphs [0960]-[1205] in CN104530417A.
In the present invention, the transformation from a reactive group to the target functional group or its protected form can be realized by any of the following methods:
Method 1: direct modification. The direct modification based on a reactive group produces the target functional group or its protected form. Examples include the transformation of a carboxyl group into an acyl halide group, a hydrazide group, an ester group, a thioester group, or a dithioester group, the transformations of a hydroxyl group, a thiol group, an alkynyl group, an amino group, a carboxyl group, etc., into the corresponding protected forms, and the modifications of a hydroxyl group, an amino group, etc., with anhydrides.
Method 2: coupling reaction between two reactive groups. A heterofunctional reagent is used as a starting material containing 1 type of reactive group as well as the target functional group, and the reactive group reacts with the terminal reactive group of RPEG to introduce the target functional group or its protected form. The reaction of two reactive groups is not particularly limited with respect to the manner or method, and the reaction conditions are relevant to the type of the divalent linking group formed in the reaction. Prior arts such as alkylation, addition of alkenes, addition of alkynes, Schiff base reaction-reduction reaction, condensation, etc., can be used herein. Wherein, the alkylation reaction is preferably based on thiol group or amino group, corresponding to the formation of thioether bond and secondary or tertiary amino group, respectively. Wherein, the condensation reaction includes but is not limited to those producing ester groups, thioester groups, amide groups, imine bonds, hydrazone bonds, carbamate groups, etc. The target functional group or its protected form can also be introduced via click reactions using starting materials including a heterofunctional reagent containing not only a reactive group selected from the group consisting of an azido group, an alkynyl group, an alkenyl group, a tri-thioester group, a thiol group, a diene group, a furyl group, a 1,2,4,5-tetrazinyl group, a cyanate group, etc., but also the target functional group or its protected form. The reaction of two reactive groups is accompanied by the formation of new bonds. Typical representatives of newly formed divalent linking groups include amide bond, urethane bond, ester group, secondary amine bond, thioether bond, triazole group, etc.
Method 3: combination of direct modifications and coupling reactions. The target functional group or its protected form can be obtained via the combination.
In one specific embodiment of the present invention, the structure of any aforementioned non-linear PEGylated lipid conforms to any of the following general formulas:
In one specific embodiment of the present invention, any aforementioned non-linear PEGylated lipid preferably has a structure corresponding to any of the following general formulas:
In one specific embodiment of the present invention, the structure of non-linear PEGylated lipid is represented by the general formula (3):
In one specific embodiment of the present invention, the nonlinear PEGylated lipid is selected from the group consisting of the following structures:
In one embodiment of the present invention, provided herein is a lipid composition containing any aforementioned non-linear PEGylated lipid.
In one specific embodiment of the present invention, the lipid composition contains another one or more types of lipid selected from the group consisting of phospholipid, steroid lipid, and PEGylated lipid, and belongs to any of the following cases:
In one specific embodiment of the present invention, the phospholipid contained in the lipid composition is selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-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-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and compositions thereof; the phospholipid can be synthetic or naturally sourced.
In one specific embodiment of the present invention, the steroid lipid contained in the lipid composition is selected from the group consisting of cholesterol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol tomatidine, ursolic acid, α-tocopherol, and compositions thereof.
In one specific embodiment of the present invention, the cationic lipid contained in the lipid composition is selected from the group consisting of N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTAP), N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy-1-(dimethylamino) propane (DODMA), 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino) octanoate (SM102),
and compositions thereof.
In a more specific embodiment of the present invention, the molar percentages of PEGylated lipid, cationic lipid, phospholipid, and steroid lipid in total lipids of the lipid composition are not particularly limited; wherein,
Lipid compositions of the present invention can be used to deliver bioactive substances to one or more of the following physiological sites of a patient: liver or liver cells (such as hepatocytes), kidney or kidney cells, tumor or tumor cells, CNS (central nervous system, such as brain and/or spinal cord) or CNS cells, PNS (peripheral nervous system) or PNS cells, lung or lung cells, blood vessels or blood vessel cells, skin or skin cells (such as dermal cells and/or follicular cells), eye (such as macula, fovea, cornea, retina) or eye cells, ear or ear cells (such as inner ear, middle ear and/or outer ear cells).
In one embodiment of the present invention,
In one specific embodiment of the present invention, the drug contained in the lipid pharmaceutical composition is a nucleic acid drug selected from the group consisting of DNA, RNA, antisense nucleic acid, plasmid, interfering nucleic acid, aptamer, antagomir, and ribozyme, wherein the RNA is selected from the group consisting of mRNA, saRNA, circRNA, miRNA, and siRNA; the nucleic acid drug is preferably selected from the group consisting of DNA, mRNA, miRNA, and siRNA.
In one specific embodiment of the present invention, the drug contained in the lipid pharmaceutical composition includes but is not limited to doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, streptozotocin, actinomycin D, vincristine, vinblastine, cytosine arabinoside, anthracycline, nitrogen mustard, tioteppa, chlorambucil, rachelmycin, melphalan, carmustine, romustine, busulfan, dibromannitol, mitomycin C, cis-diammineplatinum (II) dichloride, methotrexate, 6-mercaptopurine, 6-thioguanine, cytosine arabinoside, 5-fluorouracil dacarbazine, dibucaine, chlorpromazine, propranolol, demorol, labetalol, clonidine, hydralazine, imipramine, amitriptyline, doxepin, phenytoin, diphenhydramine, chlorpheniramine, promethazine, gentamicin, ciprofloxacin, cefoxitin, miconazole, terconazole, econazole, isoconazole, butoconazole, clotrimazole, itraconazole, nystatin, naftifine, amphotericin B, antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, glaucoma drugs, vitamins, tranquilizers, imaging agents, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, colchicine, daunorubicin, quinizarin, mithramycin, 1-dihydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, puromycin, maytansinoid, and oxaliplatin.
In one specific embodiment of the present invention, the drug contained in the lipid pharmaceutical composition is a nucleic acid drug; the N/P ratio with respect to the cationic lipid and nucleic acid in the composition is preferably (0.1˜100):1, more preferably (0.2˜30):1, and most preferably (0.5˜20):1.
In one specific embodiment of the present invention, the lipid pharmaceutical composition is used as a medicine selected from the group consisting of drugs for treating cancer, anti-infective agents, antibiotic agents, antiviral agents, antifungal agents, and vaccines.
In one specific embodiment of the present invention, the lipid pharmaceutical composition is an LNP-pharmaceutical composition, an LPP-pharmaceutical composition, or a PNP-pharmaceutical composition, preferably an LNP-pharmaceutical composition, more preferably an LNP-nucleic acid pharmaceutical composition, and more preferably an LNP-mRNA composition.
In the present invention, a lipid pharmaceutical composition can form different structures; wherein, the “LNP-pharmaceutical composition” forms the structure of a lipid nanoparticle (LNP), the “LPP-pharmaceutical composition” forms the structure of a lipopolyplex (LPP), and the “PNP-pharmaceutical composition” forms the structure of a polypeptide nanoparticle (PNP); wherein, the “LNP-nucleic acid pharmaceutical composition” forms the structure of a lipid nanoparticle, and the drug contained is a nucleic acid drug; wherein, the “LNP-mRNA composition” is a kind of LNP-nucleic acid pharmaceutical composition, and the nucleic acid drug contained is mRNA.
In one embodiment of the present invention:
In one embodiment of the present invention,
In one specific embodiment of the present invention, the preparation of the formulation of lipid pharmaceutical composition includes the following steps:
In one specific embodiment of the present invention, the preparation of the LNP-nucleic acid pharmaceutical composition includes the following steps:
In one specific embodiment of the present invention, the lipid pharmaceutical composition forms a lipid nanoparticle containing a drug (LNP-pharmaceutical composition), and the particle size of lipid nanoparticle is controlled by ultrasonic, extrusion, or microfluidic devices; the particle size is 1˜1000 nm, preferably 20˜500 nm, more preferably 60˜200 nm, and most preferably 60˜150 nm.
A lipid small molecule containing both a glycerol branching core and one or two reactive groups is synthesized first, which is coupled with a functionalized polyethylene glycol derivative reagent to obtain the non-linear PEGylate lipid of the present invention; or, a lipid small molecule containing two hydroxyl groups can be used as the initiator, which initiates the polymerization of ethylene oxide under basic conditions, and the non-linear PEGylate lipid of the present invention can be obtained after capping.
E1-1 corresponds to the general formula (1); wherein, R1 and R2 are both tridecyl groups, B1 is a methylene group, B2 is a linking bond, L1 and L2 are both —OC(═O)—, Ld is —OCH2—, two Lx are both —CH2—, and two T are both —CH3. The molecular weight of each PEG chain is approximately 1 kDa, corresponding to n1≈n2≈22.
Step a: Under argon atmosphere, glycerol derivative containing a TBS-protected hydroxyl group (S1-1, 2.58 g, 12.5 mmol), myristic acid (S1-2, 7.13 g, 31.3 mmol), and 4-(dimethylamino)pyridine (DMAP, 0.76 g, 6.3 mmol) were successively dissolved in 80 mL dichloromethane, and then the solution of dicyclohexylcarbodiimide (DCC, 7.09 g, 34.4 mmol) in dichloromethane (80 mL) was added dropwise in an ice bath. Upon completion of the addition, the reaction temperature was raised to room temperature, and the reaction was stirred for 24 h. After the reaction was over, the precipitates were removed by filtration. The filtrate was concentrated under reduced pressure, added with 40 mL tetrahydrofuran and further with 40 mL of the 1M solution of tetra-n-butylammonium fluoride (TBAF) in tetrahydrofuran, and the reaction was conducted overnight to remove the TBS protection. After the reaction was over, the reaction solution was concentrated and extracted. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain S1-3 (5.55 g).
Step b: Into the stirred 80 mL anhydrous DMF, compound S1-4 (5.70 g, 12.0 mmol, a glycerol derivative containing two TBS-protected hydroxyl groups and one OTs substituent that replaces another hydroxyl group), compound S1-3 (5.13 g, 10.0 mmol), and potassium carbonate (K2CO3, 2.08 g, 15.0 mmol) were added and reacted for 18 hours at 75° C. After the reaction was over, the mixture was cooled to room temperature, and then extracted with 80 mL dichloromethane. The organic phase was washed with 10% citric acid (40 mL*2) and brine (40 mL*2) successively, and then concentrated under reduced pressure, added with 40 mL tetrahydrofuran and further with the 1M solution of TBAF/THF (40 mL), and the reaction was conducted overnight to remove the TBS protection. After the reaction was over, the reaction solution was concentrated and extracted. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain S1-5 (1.95 g).
Step c: Under anhydrous and oxygen-free conditions, 35 mL tetrahydrofuran was added into a flask, followed by adding diphenylmethyl potassium (DPMK, 1.03 g, 5.0 mmol) and S1-5 (0.59 g, 1.0 mmol), and then, after mixing well, the methoxypolyethylene glycol sulfonate derivative S1-6 (4.80 g, 4.0 mmol, Mn≈1.2 kDa, n1≈22, PDI=1.01) was also added. The reaction was conducted for 12 hours at 30° C. After the reaction was over, the reaction solution was concentrated and extracted. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain the PEGylated lipid E1-1 (1.68 g). The main data of the 1H-NMR spectrum of E1-1 are as follows: 1H NMR (400 MHZ, CDCl3) δ: 5.15-4.96 (m, 1H, >CHOCH2CH<), 4.28-4.12 (m, 2H, >CHCH2OC(═O)—), 3.93-3.40 (m, PEG; 4H, —OCH2CH2OCH2CH<; 3H, >CHOCH2CH<), 3.36 (s, 6H, —OCH3), 2.30 (t, 4H, —OC(═O) CH2—), 1.61-1.13 (m, 40H, —CH2CH2CH2—; 4H, —CH2CH3), 0.85 (t, 6H, —CH2CH3). The molecular weight of E1-1 determined by GPC was approximately 2.6 kDa, PDI=1.02.
Step a: Under anhydrous and oxygen-free conditions, 200 mL tetrahydrofuran, small-molecule initiator S1-5 (1.17 g, 2.5 mmol), and DPMK (0.82 g, 4.0 mmol) were successively added into a flask, and then a calculated amount of ethylene oxide (5.72 mL) was added. The temperature was gradually raised to 60° C., and the reaction was conducted for 48 hours.
Step b: Excess DPMK (8.24 g, 40.0 mmol) was added for complete deprotonation, and then excess capping reagent iodomethane (14.20 g, 100.0 mmol) was added. The reaction was conducted at 30° C. for 12 hours. After the reaction was over, the reaction solution was concentrated and extracted. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated, and purified by column chromatography to obtain the PEGylated lipid E1-1 (4.21 g). The structure of E1-1 was determined by 1H NMR, and the molecular weight determined by GPC was approximately 2.6 kDa, PDI=1.02.
E1-2 corresponds to the general formula (1); wherein, R1 and R2 are both tridecyl groups, B1 is a methylene group, B2 is a linking bond, L1 and L2 are both —OC(═O)—, Ld is —OCH2—, two Lx are both —CH2—, and two T are both —H. The molecular weight of each PEG chain is approximately 1 kDa, corresponding to n1≈n2≈22.
The preparation method is as follows:
Following the preparation method 2 in Example 1.1, the same starting materials and molar equivalents were adopted, and methanol was used as the capping reagent, and therefore the PEGylated lipid E1-2 (4.85 g) was obtained. The main data of the 1H-NMR spectrum of E1-2 are as follows: 1H NMR (400 MHZ, CDCl3) δ: 5.17-4.98 (m, 1H, >CHOCH2CH<), 4.29-4.12 (m, 2H, >CHCH2OC(═O)—), 3.93-3.41 (m, PEG; 4H, —OCH2CH2OCH2CH<; 3H, >CHOCH2CH<), 2.32 (t, 4H, —OC(═O) CH2—), 1.59-1.09 (m, 40H, —CH2CH2CH2—; 4H, —CH2CH3), 0.85 (t, 6H, —CH2CH3) The molecular weight of E1-2 determined by GPC was approximately 2.5 kDa, PDI=1.02.
E1-3 corresponds to the general formula (1); wherein, R1 and R2 are both tridecyl groups, B1 is a methylene group, B2 is a linking bond, L1 and L2 are both —OC(═O)—, Ld is —OCH2—, two Lx are both —CH2CH2C(═O)OCH2—, and two T are both —CH3. The molecular weight of each PEG chain is approximately 1 kDa, corresponding to n1≈n2≈22.
The preparation method is as follows:
Under argon atmosphere, polyethylene glycol propionic acid derivative (S1-8, 4.40 g, 4.0 mmol, Mn≈1.1 kDa, n1≈22, PDI=1.01), S1-5 (0.94 g, 1.6 mmol), and DMAP (0.10 g, 0.8 mmol) were successively dissolved in 30 mL dichloromethane, and then the solution of DCC (0.91 g, 4.4 mmol) in dichloromethane (15 mL) was added dropwise in an ice bath. Upon completion of the addition, the reaction temperature was raised to room temperature, and the reaction was stirred for 24 h. After the reaction was over, the precipitates were removed by filtration. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain the PEGylated lipid E1-3 (1.38 g). The main data of the 1H-NMR spectrum of E1-3 are as follows: 1H NMR (400 MHZ, CDCl3) δ: 5.15-4.96 (m, 1H, >CHOCH2CH<), 4.28-4.12 (m, 2H, >CHCH2OC(═O)—; 4H, —OCH2CH2C(═O)OCH2—), 3.72-3.45 (m, PEG; 4H, —OCH2CH2C(═O)O—; 3H, >CHOCH2CH<), 3.34 (s, 6H, —OCH3), 2.59-2.55 (m, 6H, —OCH2CH2C(═O)O—), 2.30 (t, 4H, —OC(═O) CH2CH2CH2—), 1.62-1.13 (m, 40H, —CH2CH2CH2—; 4H, —CH2CH3), 0.84 (t, 6H, —CH2CH3). The molecular weight of E1-3 determined by GPC was approximately 3.0 kDa, PDI=1.01.
E1-4 corresponds to the general formula (1); wherein, R1 and R2 are both tetradecyl groups, B1 is a methylene group, B2 is a linking bond, L1 and L2 are both —O—, Ld is —OCH2—, two Lx are both —CH2CH2C(═O)OCH2—, and two T are both —CH3. The molecular weight of each PEG chain is approximately 1 kDa, corresponding to n1≈n2≈22.
The preparation method is as follows:
Step a: Under anhydrous and oxygen-free conditions, 100 mL tetrahydrofuran and excess DPMK (8.24 g, 40.0 mmol) were added into a flask, followed by adding the sulfonate derivative of 1-tetradecanol (S1-9, 7.08 g, 19.2 mmol) and S1-1 (1.65 g, 8.0 mmol). The reaction was conducted for 12 hours at 30° C. After the reaction was over, the reaction solution was concentrated and extracted. The obtained crude product was added with 60 mL tetrahydrofuran and further with the 1M solution of TBAF/THF (30 mL), and the reaction was conducted overnight to remove the TBS protection. After the reaction was over, the reaction solution was concentrated and extracted. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the compound S1-10 (3.23 g).
Step b: Under anhydrous and oxygen-free conditions, 80 mL tetrahydrofuran and excess DPMK (6.18 g, 30.0 mmol) were added into a flask, followed by adding the compound S1-10 (2.91 g, 6.0 mmol) and S1-4 (2.85 g, 6.0 mmol). The reaction was conducted for 12 hours at 30° C. After the reaction was over, the reaction solution was concentrated and extracted. The obtained crude product was added with 60 mL tetrahydrofuran and further with the 1M solution of TBAF/THF (30 mL), and the reaction was conducted overnight to remove the TBS protection. After the reaction was over, the reaction solution was concentrated and extracted. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the compound S1-11 (2.72 g).
Step c: Under argon atmosphere, polyethylene glycol propionic acid derivative (S1-8, 6.60 g, 6.0 mmol, Mn≈1.1 kDa, n1≈22, PDI=1.01), S1-11 (1.34 g, 2.4 mmol), and DMAP (0.15 g, 1.2 mmol) were successively dissolved in 50 mL dichloromethane, and then the solution of DCC (1.36 g, 6.6 mmol) in dichloromethane (20 mL) was added dropwise in an ice bath. Upon completion of the addition, the reaction temperature was raised to room temperature, and the reaction was stirred for 24 h. After the reaction was over, the precipitates were removed by filtration. The filtrate was concentrated under reduced pressure, concentrated, and purified by column chromatography to obtain the PEGylated lipid E1-4 (2.03 g). The main data of the 1H-NMR spectrum of E1-4 are as follows: 1H NMR (400 MHz, CDCl3) δ: 4.20-4.08 (m, 4H, —OCH2CH2C(═O)OCH2—), 3.78-3.42 (m, PEG; 4H, —OCH2CH2C(═O)O—; 2H, >CHOCH2CH<; 8H, —OCH2CH(OCH2—) CH2OCH2—), 3.36 (s, 6H, —OCH3), 2.59-2.54 (m, 4H, —OCH2CH2C(═O)O—), 1.52-1.00 (m, 44H, —CH2CH2CH2—; 4H, —CH2CH3), 0.86 (t, 6H, —CH2CH3). The molecular weight of E1-4 determined by GPC was approximately 2.7 kDa, PDI=1.02.
A lipid small molecule containing a phosphate branching core is synthesized first, and then coupled with a non-linear polyethylene glycol reagent, therefore obtaining the non-linear PEGylated lipid of the present invention.
E2-1 corresponds to the general formula (1); wherein, R1 and R2 are both nonyl groups, B1 and B2 are both linking bonds, L1 and L2 are both linking bonds, Ld is —CH2CH2—, and two Lx are both —CH2—. The molecular weight of each PEG chain is approximately 1 kDa, corresponding to n1≈n2≈22.
The preparation method is as follows:
Step a: 1-nonanol (S2-1, 1.37 g, 12.0 mmol) and pyridine (0.64 mL, 8.0 mmol) were dissolved in 15 mL diethyl ether. The solution was placed in an ice bath and added with phosphorus trichloride (PCl3, 0.35 mL, 4.0 mmol) in 1 hour. After the feeding, the reaction solution was slowly raised to room temperature. After 16 hours of reaction with stirring, the reaction solution was filtered. The precipitates were washed with 5 mL*3 diethyl ether. The filtrates were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain the intermediate S2-2 (1.28 g).
Step b: Under nitrogen protection, 15 mL solution of S2-2 (1.00 g, 3.0 mmol) in anhydrous acetonitrile was mixed with 5 mL solution of trichloroisocyanuric acid (TCICA, 0.23 g, 1.0 mmol) in anhydrous acetonitrile, and then reacted for 10 min at room temperature while stirring. White precipitates were formed. The stirring was continued for 2 hours, and then the precipitates were removed by filtration. The filtrate was concentrated and extracted. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain S2-3 (1.01 g).
Step c: Under argon atmosphere, branched polyethylene glycol Y-(mPEG)2-OH (S2-4, 2.10 g, 1.0 mmol, Mn≈2.1 kDa, n1≈n2≈22, PDI=1.02), DMAP (24 mg, 0.2 mmol), and pyridine (0.09 mL, 1.1 mmol) were dissolved in dichloromethane (15 mL). Compound S2-3 (0.74 g, 2.0 mmol) was added to the solution in an ice bath, and then the reaction temperature was raised to room temperature and the reaction was continued for 10 h while stirring. After the reaction was over, the reaction was quenched with water and then extracted. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the PEGylated lipid E2-1 (1.09 g). The main data of the 1H-NMR spectrum of E2-1 are as follows: 1H NMR (400 MHz, CDCl3) δ: 4.03-3.91 (m, 6H, —CH2O(P═O)(OCH2—)2), 3.72-3.45 (m, PEG; 4H, —OCH2CH<), 3.36 (s, 6H, —OCH3), 2.02-1.98 (m, 1H, >CH—), 1.66-1.12 (m, 2H, >CHCH2CH2O—; 24H, —CH2CH2CH2—; 4H, —CH2CH2CH3), 0.86 (t, 6H, —CH2CH3). The molecular weight determined by GPC was approximately 2.4 kDa, PDI=1.02.
E2-2 corresponds to the general formula (1); wherein, R1 and R2 are both
B1 and B2 are both hexylene groups, L1 and L2 are both —OC(═O)—, Ld is —CH2CH2—, and two Lx are both —CH2—. The molecular weight of each PEG chain is approximately 1 kDa, corresponding to n1≈n2≈22.
The preparation method is as follows:
Step a: Under argon atmosphere, 2-hexyldecanoic acid (S2-5, 5.12 g, 20.0 mmol), 1,6-hexanediol (S2-6, 2.83 g, 24.0 mmol), and DMAP (0.49 g, 4.0 mmol) were successively dissolved in 80 mL dichloromethane, and then the solution of DCC (4.53 g, 22.0 mmol) in dichloromethane (60 mL) was added dropwise in an ice bath. Upon completion of the addition, the reaction temperature was raised to room temperature, and the reaction was stirred for 24 h. After the reaction was over, the precipitates were removed by filtration. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain S2-7 (5.36 g).
Step b: S2-7 (4.28 g, 12.0 mmol) and pyridine (0.64 mL, 8.0 mmol) were dissolved in 50 mL diethyl ether. The solution was placed in an ice bath and added with phosphorus trichloride (PCl3, 0.35 mL, 4.0 mmol) in 1 hour. After the feeding, the reaction solution was slowly raised to room temperature. After 16 hours of reaction with stirring, the reaction solution was filtered. The precipitates were washed with 15 mL*3 diethyl ether. The filtrates were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain the intermediate S2-8 (2.82 g).
Step c: Under nitrogen protection, 15 mL solution of S2-8 (2.28 g, 3.0 mmol) in anhydrous acetonitrile was mixed with 5 mL solution of trichloroisocyanuric acid (TCICA, 0.23 g, 1.0 mmol) in anhydrous acetonitrile, and then reacted for 10 min at room temperature while stirring. White precipitates were formed. The stirring was continued for 2 hours. The filtrate was concentrated and extracted. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain S2-9 (2.19 g).
Step d: Under argon atmosphere, branched polyethylene glycol Y-(mPEG)2-OH (S2-4, 2.10 g, 1.0 mmol, Mn≈2.1 kDa, n1≈n2≈22, PDI=1.02), DMAP (24 mg, 0.2 mmol), and pyridine (0.09 mL, 1.1 mmol) were dissolved in dichloromethane (20 mL). Compound S2-9 (1.59 g, 2.0 mmol) was added to the solution in an ice bath, and then the reaction temperature was raised to room temperature and the reaction was continued for 10 h while stirring. After the reaction was over, the reaction was quenched with water and then extracted. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the PEGylated lipid E2-2 (1.32 g). The main data of the 1H-NMR spectrum of E2-2 are as follows: 1H NMR (400 MHZ, CDCl3) δ: 4.09 (t, 4H, —CH2OC(═O)—), 4.05-3.87 (m, 6H, —CH2O(P═O)(OCH2—)2), 3.72-3.42 (m, PEG; 4H, —OCH2CH<), 3.35 (s, 6H, —OCH3), 2.37-2.29 (m, 2H, —OC(═O) CH<), 2.07-1.94 (m, 1H, —OCH2CH<), 1.73-1.16 (m, 2H, >CHCH2CH2O—; 8H, >CHCH2CH2—; 48H, —CH2CH2CH2—; 8H, —CH2CH2CH3), 0.87 (t, 12H, —CH2CH3). The molecular weight determined by GPC was approximately 2.8 kDa, PDI=1.02.
A lipid small molecule containing a glutamate branching core is synthesized first, and then coupled with a non-linear polyethylene glycol reagent containing a lysine branching core, therefore obtaining the non-linear PEGylated lipid of the present invention.
E3-1 corresponds to the general formula (1); wherein, R1 and R2 are both tetradecyl groups, B1 is a linking bond, B2 is an ethylene group, L1 and L2 are both —C(═O)O—, Ld is —C(═O)NH—, and two Lx are —C(═O)NH— and —C(═O)NH(CH2)4—, respectively. The molecular weight of each PEG chain is approximately 1 kDa, corresponding to n1≈n2≈22.
The preparation method is as follows:
Step a: Under argon atmosphere, glutamic acid containing a Boc-protected amino group (S3-1, 0.99 g, 4.0 mmol), 1-tetradecanol (S3-2, 2.05 g, 9.6 mmol), and DMAP (0.20 g, 1.6 mmol) were successively dissolved in 30 mL dichloromethane, and then the solution of DCC (1.81 g, 8.8 mmol) in dichloromethane (30 mL) was added dropwise in an ice bath. Upon completion of the dropwise addition, the reaction temperature was raised to room temperature, and the reaction was stirred for 24 h. After the reaction was over, the precipitates were removed by filtration. The filtrate was concentrated under reduced pressure. The obtained crude product was deprotected with the TFA/DCM mixed solution (1:1 v/v) to remove Boc, washed with purified water, and then extracted with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the intermediate S3-3 (1.83 g) containing a free amino group.
Step b: The non-linear polyethylene glycol carboxylic acid derivative (mPEG)2Lys-COOH (S3-4, 2.20 g, 1.0 mmol, Mn≈2.2 kDa, n1≈n2≈22, PDI=1.02) was dissolved with 15 mL anhydrous dichloromethane, added with N-hydroxysuccinimide (NHS, 0.17 g, 1.5 mmol), and then added with DCC (0.31 g, 1.5 mmol). Into the solution of S3-3 (1.62 g, 3.0 mmol) in 25 mL dichloromethane, DMAP (24 mg, 0.2 mmol) was added. The aforementioned two solutions were mixed and reacted for 24 hours at room temperature while stirring. After the reaction was over, the precipitates were removed by filtration. The filtrate was concentrated and purified by column chromatography to obtain the PEGylated lipid E3-1 (1.17 g). The main data of the 1H-NMR spectrum of E3-1 are as follows: 1H NMR (400 MHZ, CDCl3) δ: 4.33-4.16 (m, 1H, Glu-α-CH<), 4.09-3.85 (m, 4H, —OCH2CH2OC(═O)NH—; 4H, —C(═O)OCH2—), 3.73-3.63 (m, 1H, Lys-α-CH<), 3.62-3.08 (m, PEG; 4H, —OCH2CH2OC(═O)NH—; 6H, —OCH3), 3.02-2.84 (m, 2H, Lys-ε—CH2<), 2.42-2.25 (m, 2H, Glu-γ-CH2—), 2.07-1.91 (m, 1H, Glu-β-CHaHb—), 1.90-1.75 (m, 1H, Glu-β-CHaHb—), 1.72-0.96 (m, 2H, Lys-β-CH2<; 48H, —CH2CH2CH2—; 4H, —CH2CH3), 0.85 (t, 6H, —CH2CH3). The molecular weight determined by GPC was approximately 2.7 kDa, PDI=1.02.
E3-2 corresponds to the general formula (1); wherein, R1 and R2 are both
B1 is a linking bond, B2 is an ethylene group, L1 and L2 are both —C(═O)O(CH2)6OC(═O)—, Ld is —C(═O)NH—, and two Lx are —C(═O)NH— and —C(═O)NH(CH2)4—, respectively. The molecular weight of each PEG chain is approximately 1 kDa, corresponding to n1≈n2≈22.
The preparation method is as follows:
Step a: Under argon atmosphere, S3-1 (0.99 g, 4.0 mmol), S2-7 (3.43 g, 9.6 mmol), and DMAP (0.20 g, 1.6 mmol) were successively dissolved in 50 mL dichloromethane, and then the solution of DCC (1.81 g, 8.8 mmol) in dichloromethane (30 mL) was added dropwise in an ice bath. Upon completion of the dropwise addition, the reaction temperature was raised to room temperature, and the reaction was stirred for 24 h. After the reaction was over, the precipitates were removed by filtration. The filtrate was concentrated under reduced pressure. The obtained crude product was deprotected with the TFA/DCM mixed solution (1:1 v/v) to remove Boc, washed with purified water, and then extracted with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the intermediate S3-5 (2.75 g) containing a free amino group.
Step b: S3-4 (2.20 g, 1.0 mmol, Mn≈2.2 kDa, n1≈n2≈22, PDI=1.02) was dissolved with 15 mL anhydrous dichloromethane, added with NHS (0.17 g, 1.5 mmol), and then added with DCC (0.31 g, 1.5 mmol). Into the solution of S3-5 (2.47 g, 3.0 mmol) in 35 mL dichloromethane, DMAP (24 mg, 0.2 mmol) was added. The aforementioned two solutions were mixed and reacted for 24 hours at room temperature while stirring. After the reaction was over, the precipitates were removed by filtration. The filtrate was concentrated and purified by column chromatography to obtain the PEGylated lipid E3-2 (1.25 g). The main data of the 1H-NMR spectrum of E3-2 are as follows: 1H NMR (400 MHZ, CDCl3) δ: 4.32-4.14 (m, 1H, Glu-α-CH<), 4.08-3.83 (m, 4H, —OCH2CH2OC(═O)NH—; 8H, —C(═O)OCH2—), 3.76-3.65 (m, 1H, Lys-α-CH<), 3.62-3.40 (m, PEG; 4H, —OCH2CH2OC(═O)NH—), 3.26 (s, 6H, —OCH3), 3.05-2.85 (m, 2H, Lys-ε—CH2—), 2.64-2.20 (m, 2H, Glu-β-CH2—; 2H, Glu-γ-CH2—; 2H, —OC(═O) CH<), 1.77-1.14 (m, 2H, Lys-β-CH2<; 8H, —OC(═O)CHCH2—; 52H, —CH2CH2CH2—; 8H, —CH2CH3), 0.85 (t, 12H, —CH2CH3). The molecular weight determined by GPC was approximately 3.0 kDa, PDI=1.02.
E3-3 corresponds to the general formula (1); wherein, R1 and R2 are both tetradecyl groups, B1 is a linking bond, B2 is an ethylene group, L1 and L2 are both —C(═O)O—, Ld is —C(═O)OCH2CH2C(═O)NH—, and two Lx are —C(═O)NH— and —C(═O)NH(CH2)4—, respectively. The molecular weight of each PEG chain is approximately 1 kDa, corresponding to n1≈n2≈22.
The preparation method is as follows:
Step a: 3-Hydroxypropionic acid derivative containing a TBS-protected hydroxyl group (S3-6, 0.82 g, 4.0 mmol) was dissolved with 15 mL anhydrous dichloromethane, added with NHS (0.69 g, 6.0 mmol), and then added with DCC (1.24 g, 6.0 mmol). Into the solution of S3-3 (2.59 g, 4.8 mmol) in 35 mL dichloromethane, DMAP (98 mg, 0.8 mmol) was added. The aforementioned two solutions were mixed and reacted for 24 hours at room temperature while stirring. After the reaction was over, the precipitates were removed by filtration. The filtrate was concentrated under reduced pressure, added with 30 mL tetrahydrofuran and further with 15 mL of the 1M solution of TBAF/THE, and the reaction was conducted overnight to remove the TBS protection. After the reaction was over, the reaction solution was concentrated and extracted. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain S3-7 (2.24 g).
Step b: Under argon atmosphere, S3-7 (01.84 g, 3.0 mmol), S3-4 (2.20 g, 1.0 mmol, Mn≈2.2 kDa, n1≈n2≈22, PDI=1.02), and DMAP (24 mg, 0.2 mmol) were successively dissolved in 30 mL dichloromethane, and then the solution of DCC (0.23 g, 1.1 mmol) in dichloromethane (5 mL) was added dropwise in an ice bath. Upon completion of the dropwise addition, the reaction temperature was raised to room temperature, and the reaction was stirred for 24 h. After the reaction was over, the precipitates were removed by filtration. The filtrate was concentrated and purified by column chromatography to obtain the PEGylated lipid E3-3 (1.08 g). The main data of the 1H-NMR spectrum of E3-3 are as follows: 1H NMR (400 MHZ, CDCl3) δ: 4.35-4.20 (m, 1H, Glu-α-CH<; 2H, —C(═O)OCH2CH2C(═O)NH—), 4.10-3.83 (m, 4H, —OCH2CH2OC(═O)NH—; 4H, —C(═O)OCH2CH2CH2—), 3.77-3.65 (m, 1H, Lys-α-CH<), 3.62-3.41 (m, PEG; 4H, —OCH2CH2OC(═O)NH—), 3.37 (s, 6H, —OCH3), 3.13-2.96 (m, 2H, Lys-ε—CH2—), 2.66-2.10 (m, 2H, Glu-β-CH2—; 2H, Glu-γ-CH2—; 2H, —C(═O)OCH2CH2C(═O)NH—), 1.71-0.99 (m, 2H, Lys-β-CH2—; 48H, —CH2CH2CH2—; 4H, —CH2CH3), 0.83 (t, 6H, —CH2CH3). The molecular weight determined by GPC was approximately 2.8 kDa, PDI=1.02.
E3-4 corresponds to the general formula (1); wherein, R1 and R2 are both tetradecyl groups, B1 is a linking bond, B2 is an ethylene group, L and L2 are both —C(═O)O—, Ld is —C(═O)PheGlyNH— wherein Phe is the residue of phenylalanine and Gly is the residue of glycine, and two Lx are —C(═O)NH— and —C(═O)NH(CH2)4—, respectively. The molecular weight of each PEG chain is approximately 1 kDa, corresponding to n1≈n2≈22.
The preparation method is as follows:
Step a: Phenylalanine-glycine dipeptide derivative containing a Boc-protected amino group (S3-8, 1.29 g, 4.0 mmol) was dissolved with 20 mL anhydrous dichloromethane, added with NHS (0.69 g, 6.0 mmol), and then added with DCC (1.24 g, 6.0 mmol). Into the solution of S3-3 (2.59 g, 4.8 mmol) in 40 mL dichloromethane, DMAP (98 mg, 0.8 mmol) was added. The aforementioned two solutions were mixed and reacted for 24 hours at room temperature while stirring. After the reaction was over, the precipitates were removed by filtration. The filtrate was concentrated under reduced pressure. The obtained crude product was deprotected with the TFA/DCM mixed solution (1:1 v/v) to remove Boc, washed with purified water, and then extracted with dichloromethane. The extract was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain S3-9 (2.59 g).
Step b: S3-4 (2.20 g, 1.0 mmol, Mn≈2.2 kDa, n1≈n2≈22, PDI=1.02) was dissolved with 15 mL anhydrous dichloromethane, added with NHS (0.17 g, 1.5 mmol), and then added with DCC (0.23 g, 1.1 mmol). Into the solution of S3-9 (2.23 g, 3.0 mmol) in 35 mL dichloromethane, DMAP (24 mg, 0.2 mmol) was added. The aforementioned two solutions were mixed and reacted for 24 hours at room temperature while stirring. After the reaction was over, the precipitates were removed by filtration. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain the PEGylated lipid E3-4 (1.05 g). The main data of the 1H-NMR spectrum of E3-4 are as follows: 1H NMR (400 MHz, CDCl3) δ: 7.35-7.26 (m, 1H, Ph), 7.25-7.19 (m, 4H, Ph), 4.54-4.42 (m, 1H, Phe-α-CH<), 4.34-4.23 (m, 1H, Glu-α-CH<), 4.10-3.91 (m, 4H, —OCH2CH2OC(═O)NH—; 4H, —C(═O)OCH2—), 3.87-3.79 (m, 1H, Lys-α-CH<), 3.74-3.70 (m, 1H, Gly-CHaHb—), 3.66-3.62 (m, 1H, Gly-CHaHb—), 3.61-3.44 (m, PEG), 3.44-3.40 (4H, —OCH2CH2OC(═O)NH—), 3.23 (s, 6H, —OCH3), 3.13-2.70 (m, 2H, Lys-ε—CH2—; 2H, Phe-CH2—), 2.44-2.24 (m, 2H, Glu-γ-CH2—), 2.11-1.78 (m, 2H, Glu-β-CH2—), 1.74-1.01 (m, 2H, Lys-β-CH2—; 48H, —CH2CH2CH2—; 4H, —CH2CH3), 0.85 (t, 6H, —CH2CH3). The molecular weight determined by GPC was approximately 2.9 kDa, PDI=1.02.
E3-5 corresponds to the general formula (1); wherein, R1 and R2 are both tetradecyl groups, B1 is a linking bond, B2 is an ethylene group, L1 and L2 are both —C(═O)O—, Ld is —C(═O)GlyGlyGlyNH— wherein Gly is the residue of glycine, and two Lx are —C(═O)NH— and —C(═O)NH(CH2)4—, respectively. The molecular weight of each PEG chain is approximately 1 kDa, corresponding to n1≈n2≈22.
The preparation method is as follows:
Step a: Glycine-glycine-glycine tripeptide derivative containing a Boc-protected amino group (S3-10, 1.16 g, 4.0 mmol) was dissolved with 20 mL anhydrous dichloromethane, added with NHS (0.69 g, 6.0 mmol), and then added with DCC (1.24 g, 6.0 mmol). Into the solution of S3-3 (2.59 g, 4.8 mmol) in 40 mL dichloromethane, DMAP (98 mg, 0.8 mmol) was added. The aforementioned two solutions were mixed and reacted for 24 hours at room temperature while stirring. After the reaction was over, the precipitates were removed by filtration. The filtrate was concentrated under reduced pressure. The obtained crude product was deprotected with the TFA/DCM mixed solution (1:1 v/v) to remove Boc, washed with purified water, and then extracted with dichloromethane. The extract was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain S3-11 (2.44 g).
Step b: S3-4 (2.20 g, 1.0 mmol, Mn≈2.2 kDa, n1≈n2≈22, PDI=1.02) was dissolved with 15 mL anhydrous dichloromethane, added with NHS (0.17 g, 1.5 mmol), and then added with DCC (0.23 g, 1.1 mmol). Into the solution of S3-11 (2.13 g, 3.0 mmol) in 35 mL dichloromethane, DMAP (24 mg, 0.2 mmol) was added. The aforementioned two solutions were mixed and reacted for 24 hours at room temperature while stirring. After the reaction was over, the precipitates were removed by filtration. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain the PEGylated lipid E3-5 (1.22 g). The main data of the 1H-NMR spectrum of E3-5 are as follows: 1H NMR (400 MHz, CDCl3) δ: 4.34-4.21 (m, 1H, Glu-α-CH<), 4.12-3.89 (m, 4H, —OCH2CH2OC(═O)NH—; 4H, —C(═O)OCH2—), 3.86-3.74 (m, 1H, Lys-α-CH<), 3.73-3.39 (m, PEG; 4H, —OCH2CH2OC(═O)NH—; 6H, Gly-CH2—), 3.35 (s, 6H, —OCH3), 3.06-2.90 (m, 2H, Lys-ε—CH2—), 2.47-2.23 (m, 2H, Glu-β-CH2—; 2H, Glu-γ-CH2—), 1.77-1.01 (m, 2H, Lys-β-CH2—; 48H, —CH2CH2CH2—; 4H, —CH2CH3), 0.85 (t, 6H, —CH2CH3). The molecular weight determined by GPC was approximately 2.9 kDa, PDI=1.02.
A lipid small molecule containing an amino group is synthesized first, which reacts with a non-linear polyethylene glycol reagent or derivative thereof, therefore obtaining a non-linear PEGylated lipid containing a carbamate bond or amide bond in the linking group (Ld) between polyethylene glycol and lipid moieties.
E4-1 corresponds to the general formula (1); wherein, R1 and R2 are both tetradecyl groups, B1 is a methylene group, B2 is a linking bond, L1 and L2 are both —O—, Ld is —CH2CH2OC(═O)NHCH2—, two Lx are both —CH2—, and two T are both —CH3. The molecular weight of each PEG chain is approximately 1 kDa, corresponding to n1≈n2≈22.
The preparation method is as follows:
Step a: Under anhydrous and oxygen-free conditions, 60 mL tetrahydrofuran and excess DPMK (4.12 g, 20.0 mmol) were added into a flask, followed by adding S1-9 (3.54 g, 9.6 mmol) and 3-amino-1,2-propanediol containing a Boc-protected amino group (S4-1, 0.76 g, 4.0 mmol). The reaction was conducted for 12 hours at 30° C. After the reaction was over, the reaction solution was washed and concentrated. The obtained crude product was deprotected with the TFA/DCM mixed solution (1:1 v/v) to remove Boc, washed with purified water, and then extracted with dichloromethane. The extract was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain S4-2 (1.55 g).
Step b: The branched polyethylene glycol Y-(mPEG)2-OH (S2-4, 2.10 g, 1.0 mmol, Mn≈2.1 kDa, n1≈n2≈22, PDI=1.02) was dissolved in 15 mL anhydrous acetonitrile, added with triethylamine (TEA, 0.17 mL, 1.2 mmol) and N,N′-disuccinimidyl carbonate (DSC, 0.31 g, 1.2 mmol), and then reacted at room temperature while stirring overnight. Then, the reaction solution was concentrated under reduced pressure. The residue was dissolved with 15 mL dichloromethane and then washed with saturated sodium bicarbonate solution (5 mL*3). The organic phase was dried over anhydrous sodium sulfate, filtered, and then washed with dichloromethane (5 mL*3). The organic phases were combined, and added with S4-2 (1.45 g, 3.0 mmol) and TEA (0.17 mL, 1.2 mmol). The reaction was conducted at room temperature for 2 hours, and then the reaction solution was concentrated and extracted. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to obtain the PEGylated lipid E4-1 (1.38 g). The main data of the 1H-NMR spectrum of E4-1 are as follows: 1H NMR (400 MHZ, CDCl3) δ: 4.12 (t, 2H, >CHCH2CH2OC(═O)NH—), 3.72-3.38 (m, PEG; 4H, —OCH2CH<; 3H, —OC(═O)NHCH2CHCH2—; 4H, —OCH2CH2CH2—), 3.36 (s, 6H, —OCH3), 3.01-2.97 (m, 2H, —OC(═O)NHCH2—), 2.01-1.98 (m, 1H, —OCH2CH<), 1.69-1.66 (m, 2H, >CHCH2CH2—), 1.52-1.11 (m, 44H, —CH2CH2CH2—; 4H, —CH2CH3), 0.88 (t, 6H, —CH2CH3). The molecular weight of E4-1 determined by GPC was approximately 2.6 kDa, PDI=1.02.
E4-2 corresponds to the general formula (1); wherein, R1 and R2 are both tridecyl groups, B1 is a methylene group, B2 is a linking bond, L1 and L2 are both —OC(═O)—, Ld is —CH2CH2OC(═O)NHCH2—, two Lx are both —CH2—, and two T are both —CH3. The molecular weight of each PEG chain is approximately 1 kDa, corresponding to n1≈n2≈22.
The preparation method is as follows:
Step a: Under argon atmosphere, S4-1 (0.96 g, 5.0 mmol), S1-2 (2.85 g, 12.5 mmol), and DMAP (0.31 g, 2.5 mmol) were successively dissolved in 50 mL dichloromethane, and then the solution of DCC (2.83 g, 13.8 mmol) in dichloromethane (50 mL) was added dropwise in an ice bath. Upon completion of the dropwise addition, the reaction temperature was raised to room temperature, and the reaction was stirred for 24 h. After the reaction was over, the precipitates were removed by filtration. The filtrate was concentrated, and the obtained crude product was deprotected with the TFA/DCM mixed solution (1:1 v/v) to remove Boc, washed with purified water, and then extracted with dichloromethane. The extract was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain S4-3 (2.07 g).
Step b: The branched polyethylene glycol Y-(mPEG)2-OH (S2-4, 2.10 g, 1.0 mmol, Mn≈2.1 kDa, n1≈n2≈22, PDI=1.02) was dissolved in 15 mL anhydrous acetonitrile, added with triethylamine (TEA, 0.17 mL, 1.2 mmol) and DSC (0.31 g, 1.2 mmol), and then reacted at room temperature while stirring overnight. After the reaction was over, the reaction solution was concentrated under reduced pressure. The residue was dissolved with 15 mL dichloromethane and then washed with saturated sodium bicarbonate solution (5 mL*3). The organic phase was dried over anhydrous sodium sulfate, filtered, and then washed with dichloromethane (5 mL*3). The organic phases were combined, and added with S4-3 (1.54 g, 3.0 mmol) and TEA (0.17 mL, 1.2 mmol). The reaction was conducted at room temperature for 2 hours, and then the reaction solution was concentrated and extracted. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to obtain the PEGylated lipid E4-2 (1.12 g). The main data of the 1H-NMR spectrum of E4-2 are as follows: 1H NMR (400 MHz, CDCl3) δ: 5.09-5.01 (m, 1H, —OC(═O)NHCH2CH<), 4.27-4.06 (m, 2H, >CHCH2CH2OC(═O)NH—; 2H, >CHCH2OC(═O)—), 3.73-3.42 (m, PEG; 4H, —OCH2CH<), 3.37-3.20 (m, 6H, —OCH3; 2H, —OC(═O)NHCH2CH<), 2.30 (t, 4H, —OC(═O) CH2—), 2.01-1.97 (m, 1H, —OCH2CH<), 1.67-1.65 (m, 2H, >CHCH2CH2—), 1.60-1.13 (m, 40H, —CH2CH2CH2—; 4H, —CH2CH3), 0.85 (t, 6H, —CH2CH3). The molecular weight of E4-2 determined by GPC was approximately 2.6 kDa, PDI=1.02.
E4-3 corresponds to the general formula (1); wherein, R1 and R2 are both tetradecyl groups, B1 is a methylene group, B2 is a linking bond, L1 and L2 are both —O—, Ld is —C(═O)NHCH2—, two Lx are —C(═O)NH— and —C(═O)NH(CH2)4—, respectively, and two T are both —CH3. The molecular weight of each PEG chain is approximately 1 kDa, corresponding to n1≈n2≈22.
The preparation method is as follows:
The non-linear polyethylene glycol carboxylic acid derivative (mPEG)2Lys-COOH (S3-4, 2.20 g, 1.0 mmol, Mn≈2.2 kDa, n1≈n2≈22, PDI=1.02) was dissolved with 15 mL anhydrous dichloromethane, added with NHS (0.17 g, 1.5 mmol), and then added with DCC (0.31 g, 1.5 mmol). Into the solution of S4-2 (1.45 g, 3.0 mmol) in 25 mL dichloromethane, DMAP (24 mg, 0.2 mmol) was added. The aforementioned two solutions were mixed and reacted for 24 hours at room temperature while stirring. After the reaction was over, the precipitates were removed by filtration. The filtrate was concentrated and purified by column chromatography to obtain the PEGylated lipid E4-3 (1.02 g). The main data of the 1H-NMR spectrum of E4-3 are as follows: 1H NMR (400 MHZ, CDCl3) δ: 4.26-4.07 (m, 4H, —OCH2CH2OC(═O)NH—), 3.96-3.84 (m, 1H, Lys-α-CH<), 3.80-3.28 (m, PEG; 4H, —OCH2CH2OC(═O)NH—; 6H, —OCH3; 3H, —C(═O)NHCH2CH<; 4H, >CHCH2OCH2—; 2H, >CHOCH2—), 3.17-2.95 (m, 2H, Lys-ε—CH2—), 1.75-1.10 (m, 2H, Lys-β-CH2—; 48H, —CH2CH2CH2—; 4H, —CH2CH3), 0.86 (t, 6H, —CH2CH3). The molecular weight determined by GPC was approximately 2.7 kDa, PDI=1.02.
E4-4 corresponds to the general formula (1); wherein, R1 and R2 are both tridecyl groups, B1 is a methylene group, B2 is a linking bond, L1 and L2 are both —OC(═O)—, Ld is —C(═O)NHCH2—, two Lx are —C(═O)NH— and —C(═O)NH(CH2)4—, respectively, and two T are both —CH3. The molecular weight of each PEG chain is approximately 1 kDa, corresponding to n1≈n2≈22.
The preparation method is as follows:
The non-linear polyethylene glycol carboxylic acid derivative (mPEG)2Lys-COOH (S3-4, 2.20 g, 1.0 mmol, Mn≈2.2 kDa, n1≈n2≈22, PDI=1.02) was dissolved with 15 mL anhydrous dichloromethane, added with NHS (0.17 g, 1.5 mmol), and then added with DCC (0.31 g, 1.5 mmol). Into the solution of S4-3 (1.54 g, 3.0 mmol) in 25 mL dichloromethane, DMAP (24 mg, 0.2 mmol) was added. The aforementioned two solutions were mixed and reacted for 24 hours at room temperature while stirring. After the reaction was over, the precipitates were removed by filtration. The filtrate was concentrated and purified by column chromatography to obtain the PEGylated lipid E4-3 (0.92 g). The main data of the 1H-NMR spectrum of E4-3 are as follows: 1H NMR (400 MHZ, CDCl3) δ: 4.27-3.98 (m, 4H, —OCH2CH2OC(═O)NH—), 3.97-3.64 (m, 1H, Lys-α-CH<), 3.62-3.41 (m, PEG; 4H, —OCH2CH2OC(═O)NH—), 3.36 (s, 6H, —OCH3), 3.18-2.93 (m, 2H, Lys-ε—CH2—), 2.32 (t, 4H, —OC(═O) CH2—), 1.73-1.15 (m, 2H, Lys-β-CH2—; 44H, —CH2CH2CH2—; 4H, —CH2CH3), 0.87 (t, 6H, —CH2CH3). The molecular weight determined by GPC was approximately 2.7 kDa, PDI=1.02.
The PEGylated lipid of the present invention containing reactive groups as the ends of the polyethylene glycol moiety can be coupled with bioactive small molecules (including but not limited to folic acid), therefore obtaining a non-linear PEGylated lipid with targeting properties.
E5-1 corresponds to the general formula (1); wherein, R1 and R2 are both tridecyl groups, B1 is a methylene group, B2 is a linking bond, L1 and L2 are both —OC(═O)—, Ld is —OCH2—, two Lx are both —CH2—, and two T are both R01-L01-, wherein R01 is the residue of folic acid and L01 is-NHCH2CH2—. The molecular weight of each PEG chain is approximately 1 kDa, corresponding to n1≈n2≈22.
The preparation method is as follows:
Step a: Under anhydrous and oxygen-free conditions, 50 mL tetrahydrofuran was added into a flask, followed by adding DPMK (2.06 g, 10.0 mmol) and S1-5 (1.17 g, 2.0 mmol), and then, after mixing well, the methoxypolyethylene glycol sulfonate derivative S5-1 (6.50 g, 5.0 mmol, Mn≈1.3 kDa, n1≈22, PDI=1.01) containing a Boc-protected amino group was also added. The reaction was conducted for 12 hours at 30° C. After the reaction was over, the reaction solution was washed and concentrated. The obtained crude product was deprotected with the TFA/DCM mixed solution (1:1 v/v) to remove Boc, washed with purified water, and then extracted with dichloromethane. The extract was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain S5-2 (3.80 g).
Step b: Folic acid (S5-3, 1.32 g, 3.0 mmol) was dissolved with 80 mL anhydrous dichloromethane, added with NHS (0.41 g, 3.6 mmol), and then added with DCC (0.74 g, 3.6 mmol). Into the solution of S5-2 (2.34 g, 0.9 mmol, Mn˜2.6 kDa, n1≈n2≈22, PDI=1.01) in 20 mL dichloromethane, DMAP (73 mg, 0.6 mmol) was added. The aforementioned two solutions were mixed and reacted for 24 hours at room temperature while stirring. After the reaction was over, the precipitates were removed by filtration. The filtrate was concentrated and purified by column chromatography to obtain the PEGylated lipid E5-1 (1.39 g). The main data of the 1H-NMR spectrum of E5-1 are as follows: 1H NMR (400 MHZ, CDCl3) δ: 8.71 (s, 2H, pterinC7), 7.65-7.63 (m, 4H, Ph), 6.68-6.66 (m, 4H, Ph-H), 5.13-4.93 (m, 1H, >CHOCH2CH<), 4.51-4.42 (m, 4H, —CH2NHPh-), 4.32-4.11 (m, 2H, —CH(COOH)—; 2H, >CHCH2OC(═O)—), 3.92-3.40 (m, PEG; 4H, —OCH2CH2OCH2CH<; 3H, >CHOCH2CH<; 8H, —C(═O)NH(CH2)2O—), 2.36-2.26 (m, 4H, —OC(═O) CH2—; 4H, >CHCH2CH2C(═O)NH—), 2.08-1.90 (m, 4H, >CHCH2CH2C(═O)NH—), 1.62-0.99 (m, 40H, —CH2CH2CH2—; 4H, —CH2CH3), 0.85 (t, 6H, —CH2CH3). The molecular weight determined by GPC was approximately 3.5 kDa, PDI=1.01.
In this embodiment, LNP-mRNA pharmaceutical compositions containing Fluc-mRNA (LNP/Fluc-mRNA) were prepared; wherein, the phospholipids contained were all DSPC, the steroid lipids contained were all cholesterol, the cationic lipids contained were all CL-1, but the PEGylated lipids contained were different; the CL-1 was obtained by referring to the preparation method disclosed by CN113402405A, and its structure is as follows:
The preparation method of LNP/Fluc-mRNA is as follows:
Step (1): A certain amount of stock solutions of CL-1, DSPC, cholesterol and PEGylated lipid were pipetted to be dissolved in ethanol, wherein the molar ratio of CL-1, DSPC, cholesterol and PEGylated lipid was 50:10:38:1.5, and the ethanol phase solution was obtained (specific lipid formulation of each group is illustrated in Table 1 of Example 7; the PEGylated lipid of the control group L-0 is PEG2k-DMG (abbreviated as DMG); the PEGylated lipids of the experimental groups are the non-linear PEGylated lipids of the present invention).
Step (2): The Fluc-mRNA was added into a 10˜50 mM citrate buffer (pH=4) to obtain the aqueous phase solution.
Step (3): The ethanol phase solution and the aqueous phase solution were mixed (1:3 v/v) to prepare the LNP/Fluc-mRNA. The DPBS ultrafiltration was performed multiple times to wash and remove ethanol and free molecules. Finally, the solution was passed through a 0.2 μm sterile filter for further use.
Determination of nucleic acid complexation ability: The gel electrophoresis experiment was carried out to examine the nucleic acid complexation ability of LNP/Fluc-mRNA. 0.8 g agarose was weighed and dissolved in 40 mL solution of TAE. The solution was heated in a microwave until the granules of agarose were completely dissolved, and then cooled. 5 μL nucleic acid dye (GelGreen) was added in the cooled agarose gel, and then the gel was added into the gel slot and dried with natural air. The mixed solution of LNP/Fluc-mRNA and 2 μL loading buffer was added into the agarose gel well, and the electrophoresis voltage was set to 90 V. The electrophoresis was carried out at room temperature for 10 min. The result showed that free Fluc-mRNA hardly existed in both the experimental groups (L1-1˜L3-6) and the control group (L-0), indicating that the LNP containing the non-linear PEGylated lipid of the present invention has a good ability to complex with nucleic acids.
Determination of encapsulation efficiency: The LNP/Fluc-mRNA was ultracentrifuged (4° C., 60000 rpm, 1 h) with an ultracentrifuge. The concentration of unencapsulated Fluc-mRNA in the supernatant was determined by a nucleic acid quantifier. The encapsulation efficiency of LNP for Fluc-mRNA was calculated. The results are summarized in Table 1, showing that the LNPs of the present invention have higher encapsulation efficiency for nucleic acid drugs; wherein, the encapsulation efficiency of the experimental groups containing PEGylated lipids with a molecular weight of approximately 1 kDa per arm are all above 93%, and the encapsulation efficiency of the experimental group containing the PEGylated lipid with a molecular weight of approximately 0.5 kDa per arm (L3-6) is also above 80%. Particularly, the molar ratio of PEGylated lipid in total lipids of L3-1-half is only half of that of the control group L-0, but the corresponding encapsulation efficiency is even slightly higher than that of L-0, indicating that the non-linear PEGylated lipid of the present invention can be used in an appropriately reduced amount without affecting the encapsulation efficiency of LNP for Fluc-mRNA.
Determination of particle size: According to the literature (Hassett et al., J. Controlled Release 2021, 335, 237-246), an LNP formulation loaded with nucleic acid drugs can exhibit better pharmaceutical efficacy when its particle size is in the range of 60˜150 nm. In this embodiment, the particle size of LNP/Fluc-mRNA was determined by dynamic light scattering (DLS). The measured sizes of LNP/Fluc-mRNA were relatively uniform, and the PDI values were all smaller than 0.3. The results showed that the particle sizes of LNP/Fluc-mRNA prepared with the PEGylated lipid nanoparticles of the present invention were ranging from approximately 64 nm to approximately 103 nm, which were all within the range of the particle size that can achieve better pharmaceutical efficacy.
Unmodified cationic lipids are easily adsorbed to negatively charged serum proteins in the systemic circulation, forming large-sized aggregates which can be cleared by the mononuclear phagocytic system, therefore causing a lower genetic transfection efficiency of liposome or LNP. Compared with conventional linear PEGylated lipids, the non-linear PEGylated lipid of the present invention can confer an enhanced “stealth effect” to LNP.
The aforementioned LNP/Fluc-mRNA (1 control group L-0 and 19 experimental groups L1-1˜L3-6) were respectively added to the culture media containing 10% fetal bovine serum (FBS), stirred at 37° C., and sampled at a regular interval to determine the change of particle size of LNP/Fluc-mRNA to analyze the stability of nucleic acid pharmaceutical formulation in serum. The results showed that, in 7 days, the experimental group L3-6 with a molecular weight of approximately 0.5 kDa per arm of polyethylene glycol component had a relatively large change in particle size (14%); L3-3 with the Ld containing an ester bond had a change of 8% in particle size; the rest experimental groups with the Ld containing an ether bond, amide bond, carbamate bond or peptide bond, as well as the control group, had a change smaller than 5% in particle size; particularly, L3-1 almost had no change in particle size (approximately 0%); L3-1-half had a change of approximately 4% in particle size, same with L-0, while the amount of PEGylated lipid used in L3-1-half was only half of that of L-0. These results indicate that the LNP-nucleic acid pharmaceutical compositions prepared with the non-linear PEGylated lipids of the present invention have good stability in serum; when the molecular weight of PEGylated lipid is close to one another (e.g., PEG2k-DMG and the non-linear two-arm PEGylated lipids of the present invention with a molecular weight of approximately 1 kDa per arm), the non-linear structures can achieve better stability of lipid pharmaceutical composition in serum.
The cells used were Hela cells, and the method used was to measure cell viability by CCK-8 kit.
The commercial transfection reagent Lipofectamine 2000 (L2K) was used to prepare the L2K/Fluc-mRNA complex according to the method in Example 6.
HeLa cells were inoculated onto a 96-well plate at 6,000 cells/well, 100 μL per well, and then divided into the control group (blank control group), the L2K/Fluc-mRNA group (positive control group) and the LNP/Fluc-mRNA groups (experimental groups L1-1˜L5-1), and then incubated at 37° C., 5% CO2. After 24 hours of cell incubation, into the blank control group was added 10 μL PBS solution, into the positive control group and the experimental groups were added 3.3 μg/mL L2K/Fluc-mRNA (10 μL) and 3.3 μg/mL LNP/Fluc-mRNA (10 μL), respectively, and the incubation was continued at 37° C., 5% CO2. After another 24 hours of incubation, the 96-well plate was retrieved while protected from light. The culture medium was aspirated, and then a diluted solution of CCK-8 was added 120 μL per well. Then, the incubation was continued for 1-4 hours at 37° C., 5% CO2.
The 96-well plate was retrieved, and a microplate reader was used to measure the absorbance of each well at a wavelength of 450 nm.
The results of three repeated assays were averaged, and showed that the cell viability of the positive control group L2K/Fluc-mRNA was 92%, those of the experimental groups were all above 90%; in particular, the cell viability of L3-4 and L3-5 with the PEGylated lipid containing peptide bonds were 100% and 98%, respectively.
These results indicate that, compared with the L2K/Fluc-mRNA which contains the commercial transfection reagent Lipofectamine 2000, the LNP/Fluc-mRNA of the present invention also has no obvious cytotoxicity toward HeLa cells.
When the PEGylated lipid used in an LNP contains no degradable linking groups, the LNP has high stability under physiological conditions, but can also simultaneously reduce the efficiency of cellular uptake to a certain extent, therefore affecting the efficiency of drug delivery. In the present invention, some non-linear PEGylated lipids are designed to contain degradable groups (e.g., ester bonds that undergo slow degradation in the bloodstream, peptide bonds that can be degraded within cells, etc.) in the polyethylene glycol components, Ld linking groups, and/or lipid components; such PEGylated lipids can achieve higher efficiency of drug delivery, especially the transfection efficiency of nucleic acid drugs, while maintaining the stability of LNPs.
In order to examine the transfection efficiency of mRNA at cell level for each group of LNP/Fluc-mRNA composition prepared in Example 6, assays were performed on the basis of luciferase bioluminescence. The formulation of LNP/Fluc-mRNA composition was dissolved in the culture medium to the required dose, and HeLa cells, as a cell model, were inoculated at a density of 6,000 cells/well. The cell suspension was inoculated at 100 μL/well onto a 96-well black frame plate with clear wells. After the inoculation, the cells were incubated for 24 h in a cell culture incubator. Then, a dose of 0.2 μg mRNA per well was given, while the blank control group was added with free Fluc-mRNA of the corresponding dose. After 24 hours of transfection, the old culture medium was removed and replaced with a new medium containing the D-fluorescein sodium substrate (1.5 mg/mL). After 5 minutes of incubation, bioluminescence was detected using a microplate reader. Stronger fluorescence meant more Fluc-mRNA were transported into the cytoplasm and translated into the corresponding fluorescent protein. The results are shown in Table 2, wherein, the relative value of fluorescence intensity is the ratio of the fluorescence intensity of each group to that of the blank control group. The results indicate that the LNP/Fluc-mRNA compositions prepared in the present invention all have excellent transfection efficiency in vitro, i.e., the LNPs of the control group and the experimental groups are all effective carriers for delivery, and most of the experimental groups have higher transfection efficiency than the control group. According to the relative value of fluorescence intensity, the LNP/Fluc-mRNA containing degradable groups in non-linear PEGylated lipid thereof has a better performance in transfection, compared with L-0 which contains DMG as the PEGylated lipid; particularly, L3-4 (containing carbamate bonds in the PEG component, peptide bonds in Ld, and ester bonds in the lipid component), L3-5 (containing carbamate bonds in the PEG component, peptide bonds in Ld, and ester bonds in the lipid component), and L1-3 (containing ester bonds in both PEG and lipid components) exhibited the best transfection efficiency.
The above results show that the LNP of the present invention containing non-linear PEGylated lipids can effectively deliver the Fluc-mRNA into cells and then realize a successful transfection. In particular, the rational collocation and design based on the degradable/stable groups within the polyethylene glycol component, Ld, and lipid component can enable the lipid-nucleic acid pharmaceutical composition to exhibit better efficacy.
This embodiment examines the targeting ability of the LNP containing E5-1 which is modified by folic acid at the ends of polyethylene glycol, and compares it with E1-1, a structure most similar in structure except for the ends of PEG.
I. Preparation of the formulation of PEGylated lipid nanoparticle/oxaliplatin composition:
Step a: DSPC (4 g) and cholesterol (667 mg) were dissolved in 60 mL dichloromethane, added with 20 mL aqueous solution of 80 mg oxaliplatin, stirred for 30 min, and ultrasonicated for 20 min. The organic solvent was removed by rotary evaporation at 40° C. After the gel was collapsed, components including E1-1 (0.52 g, 0.2 mmol) and 50 mL aqueous solution of poloxamer F68 (400 mg) were added respectively. The evaporation was continued for 30 min, followed by high-pressure homogenization at 400 bar for 5 min. The volume was adjusted to 100 mL with water, and then the tangential flow ultrafiltration was used to isolate oxaliplatin. A tangential-flow membrane pack with a molecular weight cut-off of 10 kDa was used for the ultrafiltration. The ultrafiltration was performed repeatedly for 3 times, using water as the displacement liquid. The ultrafiltration centrifuge tube was used to measure the encapsulation efficiency, and the high performance liquid chromatography was used to measure the final concentration of drug. The encapsulation efficiency was 95.33%. The particle size was 124 nm. The concentration of drug in the LNP suspension was adjusted to 500 μg/mL with water, therefore obtaining the formulation of PEGylated lipid nanoparticle/oxaliplatin composition, LP-1.
Step b: Using the same preparation method as that of the abovementioned experimental group, E5-1 (0.70 g, 0.2 mmol) was used to replace E1-1 in the preparation to obtain the formulation of folic acid-coupled PEGylated lipid nanoparticle/oxaliplatin composition, LP-2.
II. The HCT-116 human colon cancer cell line was recovered and cultured, and the HCT-116 human colon cancer tumor-bearing nude mouse model was established. The nude mice were inoculated and grew naturally, and the tumor volume was measured with a vernier caliper. After the tumor grew to 50-75 mm3, the mice were randomly divided into 4 groups on the basis of the tumor volume:
Those described above are only embodiments of the present invention, not limiting the patent scope of the present invention. Any transformation of equivalent structure or equivalent route based on the specification content of the present invention, directly or indirectly applied in other related arts, should be included in the protected patent scope of the present invention in the same way.
For those skilled in the art, without departing from the purpose and the scope of the present invention, and without unnecessary experimentation, the present invention can be implemented in a wider range with equivalent parameters, concentrations, and conditions. While the present invention has given particular examples, it should be understood that the present invention can be further modified. In conclusion, in accordance with the principles of the present invention, the present application is intended to cover any alterations, uses, or improvements of the present invention, including changes departing from the scope disclosed in this application but made using conventional techniques known in the art.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210380598.6 | Apr 2022 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2023/087698 | 4/11/2023 | WO |
