Disclosure of Invention
The present disclosure provides ionizable cationic lipids capable of multi-tertiary amine multi-tail chains, compositions comprising the same, and uses thereof. The ionizable cationic lipid provided by the disclosure can be used for delivering therapeutic agents and/or preventive agents such as nucleic acid molecules, small-molecule compounds, polypeptides or proteins, and the like, has the advantages of simple preparation method, strong targeting, low organ off-target accumulation, capability of carrying a drug active ingredient to transfect cells with higher transfection efficiency, lower cytotoxicity and capability of improving delivery efficiency and safety.
The technical scheme provided by the disclosure may include, for example:
[1] a (ionizable cationic lipid) compound or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein said compound has a structure according to formula (I):
,
wherein:
r1 is a substituted or unsubstituted N-containing heterocycle, or a disubstituted amino;
R2 is a substituted or unsubstituted C5-20 linear or branched alkene, or a substituted or unsubstituted C5-20 linear or branched alkane;
R3 is a substituted or unsubstituted C5-20 linear or branched alkene, or a substituted or unsubstituted C5-20 linear or branched alkane, or H, or default;
R4 is a substituted or unsubstituted C5-20 linear or branched alkane;
L1 is unsubstituted C2-10 linear alkylene, or-C (O) (CH2)n -, n is an integer from 2 to 10;
L2 and L3 are each independently unsubstituted C2-10 linear alkylene, or default;
M1 is-ch=ch-, -C (O) O-or-OC (O) -;
M2 is-ch=ch-, -C (O) O-, or default;
m3 is-OC (O) -;
M4 is-OC (O) -, or default;
When L2 and M2 are absent at the same time, L3 and/or M4 are also absent.
The technical scheme [1] provided by the disclosure comprises a compound shown in the formula (I) and any N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof.
In some preferred embodiments, stereoisomers of the compounds of formula (I) do not include compounds having the structure shown in formula (II):
The inventor discovers that LNPs prepared by the compound of the formula (II) which does not have chirality has the advantages of good targeting property, low organ off-target accumulation, low probability of organ toxicity, low cytotoxicity and the like, but the expression level of the carried nucleic acid in a target position (such as muscle tissue at an injection site) is low.
In the compounds of formula (I) provided herein, "default" means that the corresponding group is absent from that position. For example, if M4 is default, then L3 is directly connected to R4.
The present disclosure provides compounds of formula (I) wherein the "substituted group" (e.g., substituted N-containing heterocycle, substituted C5-20 linear or branched alkene, substituted C5-20 linear or branched alkane, etc.) may be employed to refer to a group wherein the H in a group is substituted with another element or group (commonly referred to as a "substituent"). When a substituted group is used at a certain position, the substituent contained in the group may be selected from any one or a combination of at least two of halogen, hydroxy, amino, mercapto and the like. The present disclosure is not particularly limited with respect to the number and positions of substituents in the substituted group. For example, if R2 is a substituted C10 straight chain alkyl group, R2 in the compound of formula (I) may be a group obtained by substituting at least one H on any one or more C with the above substituent.
In the compounds of formula (I) provided herein, R1 may be a substituted N-containing heterocycle, an unsubstituted N-containing heterocycle, or a di-substituted amino group.
"N-containing heterocyclic ring" refers to a cyclic structural group that forms a backbone from C and non-C atoms (also referred to as "heteroatoms," which may be N, S, O, P, etc.), and wherein at least one heteroatom is N. For example, the N-containing heterocycle may contain 1,2 or 3N. Preferably, the N-containing heterocycle does not contain heteroatoms other than N. The total number of C and heteroatoms in the backbone structure of the preferred N-containing heterocycle may be 3-8, for example 3, 4, 5, 6, 7 or 8.
"Disubstituted amino" means that both H's in the amino (-NH2) group are substituted with substituents. For example, a disubstituted amino group may be formed by substitution of both H's in the amino group with any one selected from the group consisting of halogen, C1-5 straight or branched alkyl, C1-5 straight or branched alkene, and the like. In the disubstituted amino group, the two substituents may be the same or different.
In the compounds of formula (I) provided in the present disclosure, the number of carbon atoms of R2 may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or may be a range formed by any two of the above values, or any integer intermediate value in the range.
In the compounds of formula (I) provided in the present disclosure, when R3 is not H, but is not absent, the number of carbon atoms of R3 may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or may be a range formed by any two of the foregoing values, or any integer intermediate value in the range.
In the compounds of formula (I) provided in the present disclosure, the number of carbon atoms of R4 may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or may be a range formed by any two of the above values, or any integer intermediate value in the range.
In the compounds of formula (I) provided by the present disclosure, R2、R3 and R4 may be the same or different.
In the compounds of formula (I) provided in the present disclosure, if L1 is unsubstituted C2-10 straight-chain alkylene, the number of carbon atoms may be 2,3, 4,5, 6, 7, 8, 9, 10, or may be a range formed by any two values, or any integer intermediate value in the range.
In the compounds of formula (I) provided by the present disclosure, if L1 is-C (O) (CH2)n -, n may be 2, 3, 4, 5, 6, 7, 8, 9, 10, or may be a range formed by any two values, or any integer intermediate value in the range.
In the compounds of formula (I) provided in the present disclosure, if L2 and/or L3 are not default, the number of carbon atoms may be 2, 3, 4, 5, 6, 7, 8, 9, 10, or may be a range formed by any two values, or any integer intermediate value in the range.
In the compounds of formula (I) provided by the present disclosure, L1、L2 and L3 may be the same or different.
In the compounds of formula (I) provided by the present disclosure, M1、M2、M3 and M4 may be the same or different.
[2] The compound according to [1], or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R2 is unsubstituted C5-15 linear alkane, or unsubstituted C10-20 branched alkyl, or unsubstituted C5-10 linear alkenyl;
And/or R3 is unsubstituted C5-20 linear alkane, or unsubstituted C15-20 branched alkyl, or unsubstituted C5-10 linear alkenyl, or H;
And/or R4 is unsubstituted C5-15 linear alkane, or unsubstituted C10-20 branched alkyl.
[3] The compound according to [1] or [2], or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R1 is、Or (b);
And/or R2 is、Or unsubstituted C8-11 linear alkanes;
And/or R3 is、、Unsubstituted C8-11 linear alkane, or H;
and/or R4 isOr unsubstituted C8-10 linear alkanes;
And/or L1 is-C (O) (CH2)5-、-(CH2)3 -, or- (CH2)8 -;
And/or L2 is- (CH2)5-、-(CH2)8 -or by default;
And/or L3 is- (CH2)5 -or by default).
[4] The compound according to any one of [1] to [3], or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound has a structure of any one of the following (YK-1601 to YK-1618):
、
、
、
、
、
、
、
、
、
、
、
、
、
、
、
、
And
。
In some particularly preferred embodiments, the compound has the structure of any one of YK-1603, YK-1604, YK-1605, YK-1606, YK-1610, YK-1612, YK-1613, YK-1615, and YK-1618.
[5] A carrier comprising a cationic lipid provided by a compound of any one of [1] to [4], or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof.
[6] The vector according to [5], wherein the vector further comprises a neutral lipid;
And/or, the carrier further comprises a structural lipid;
And/or the carrier further comprises a polymer conjugated lipid;
and/or the carrier further comprises one or more other ionizable lipid compounds.
In the carrier provided by the present disclosure, "other ionizable lipid compound" refers to any kind of ionizable lipid compound other than the compound of any one of [1] to [4] provided by the present disclosure, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof provided cationic lipid. Any ionizable lipid compound available in the art for use in lipid delivery systems may be suitable for use in the carriers of the present disclosure, and the present disclosure is not particularly limited as to the source of the other ionizable lipid compounds, e.g., commercially available, and also prepared by itself according to the prior art.
In some preferred embodiments, the other ionizable lipid compound may be an ionizable cationic lipid compound and/or an ionizable anionic lipid compound.
[7] The carrier according to [5] or [6], wherein the molar percentage of the cationic lipid in the carrier is 25% to 75%;
and/or, in the carrier, the neutral lipid accounts for 5-25% of the mole percentage of the carrier;
and/or, in the carrier, the structural lipid accounts for 15-65% of the mole of the carrier;
and/or, the polymer conjugated lipid accounts for 0.5-10% of the carrier in mole percent.
In the present disclosure, the mole percent of a certain lipid in the carrier refers to the percentage of the mole of the lipid in the carrier based on the mole of all the lipids in the carrier. For example, when all lipids in the carrier are 100mol, in which the content of the cationic lipid is 30mol, the mole percentage of the cationic lipid in the carrier is 30%.
For example, the molar percentage of the cationic lipid in the carrier may be 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or may be a range formed by any two of the above values, or any intermediate value in the range.
For example, the carrier may have a mole percentage of neutral lipids of 5%, 8%, 10%, 12%, 15%, 18%, 20%, 22%, 25%, or may have a range of any two of the above values, or any intermediate value in the range.
For example, the molar percentage of structural lipid in the carrier may be 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or may be a range consisting of any two of the above values, or any intermediate value in the range.
For example, the molar percentage of the polymer conjugated lipid in the carrier may be 0.5%、0.6%、0.7%、0.8%、0.9%、1%、1.1%、1.2%、1.3%、1.4%、1.5%、1.6%、1.7%、1.8%、1.9%、2%、2.5%、3%、3.5%、4%、5%、6%、7%、8%、9%、10%, or may be a range of any two of the values mentioned above, or any intermediate value in the range.
In some preferred embodiments, the molar percentage of the polymer conjugated lipid in the carrier may be 0.5-2.5%.
In a particularly preferred embodiment, the molar percentage of polymer conjugated lipid in the carrier may be 1.5%.
[8] The carrier according to any one of [5] to [7], wherein a molar ratio of the cationic lipid to the neutral lipid in the carrier is 1:1 to 15:1;
And/or, the molar ratio of cationic lipid to structural lipid in the carrier is 0.5:1-3:1;
and/or, the molar ratio of the cationic lipid to the polymer conjugated lipid in the carrier is 4:1-35:1.
For example, the molar ratio of cationic lipid to neutral lipid in the carrier may be 1:1、2:1、2.5:1、3:1、3.2:1、3.4:1、3.6:1、3.8:1、4:1、4.2:1、4.4:1、4.6:1、4.8:1、5:1、5.5:1、6:1、7:1、8:1、9:1、10:1、11:1、12:1、13:1、14:1、15:1, or may be a range of any two ratios described above, or any intermediate ratio in the range. Preferably the molar ratio of cationic lipid to neutral lipid is 4-5:1.
For example, the molar ratio of cationic lipid to structural lipid in the carrier may be 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.2:1, 1.4:1, 1.6:1, 1.8:1, 2:1, 2.5:1, 3:1, or may be a range of any two ratios described above, or any intermediate ratio in the range.
For example, the molar ratio of cationic lipid to polymer conjugated lipid in the carrier may be 4:1、4.1:1、4.2:1、4.3:1、4.4:1、4.5:1、4.6:1、4.7:1、4.8:1、4.9:1、5:1、5.5:1、6:1、7:1、8:1、9:1、10:1、15:1、20:1、25:1、30:1、31:1、32:1、32.1:1、32.2:1、32.3:1、32.4:1、32.5:1、32.6:1、32.7:1、32.8:1、32.9:1、33:1、33.5:1、34:1、34.5:1、35:1, or may be a range of any two ratios described above, or any intermediate ratio in the range.
[9] The carrier according to any one of [5] to [8], wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the polymer conjugated lipid in the carrier is (25-75): 5-25): 15-65): 0.5-10.
[10] The carrier according to any one of [5] to [9], wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the polymer conjugated lipid in the carrier is (35-49): 7.5-15): 35-55): 1-5.
In a particularly preferred embodiment, the molar ratio of cationic lipid, neutral lipid, structural lipid and polymer conjugated lipid in the carrier is 49:10:39.5:1.5.
[11] The carrier according to any one of [ 5-10 ], wherein the neutral lipid is selected from any one or a combination of at least two of the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof;
and/or the structural lipid is selected from any one or a combination of at least two of cholesterol, non-sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycorine, ursolic acid, alpha-tocopherols, and corticosteroids;
And/or the polymer conjugated lipid is selected from any one or a combination of at least two of the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.
[12] The carrier according to any one of [5] to [11], wherein the neutral lipid is any one or a combination of at least two selected from the group consisting of 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1, 2-dimyristoyl-sn-glycero-phosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-dioleoyl-sn-glycero-3-phosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1-palmitoyl-sn-glycero-3-phosphorylcholine, 1-hexadecyl-sn-3-glycero-3-phosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1, 2-dioleoyl-glycero-3-phosphorylcholine, 1-dioleoyl-sn-glycero-3-phosphorylcholine, 2-dioleoyl-glycero-3-phosphorylcholine, 1, 2-dioleoyl-glycero-3-phosphorylcholine 1, 2-di-phytoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-di-stearoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-di-oleoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-di-linolenoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-di-arachidonoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-di-docosahexaenoic acid-sn-glycerol-3-phosphoethanolamine, 1, 2-di-oleoyl-sn-glycerol-3-phospho-rac- (1-glycerol) sodium salt, di-palmitoyl phosphatidylglycerol, palmitoyl-base oil phosphatidylethanolamine, di-stearoyl-phosphatidylethanolamine, di-palmitoyl phosphatidylethanolamine, di-myristoyl-phosphoethanolamine, 1-stearoyl-2-oleoyl-stearoyl-ethanolamine, 1-stearoyl-2-oleoyl-phosphatidylethanolamine, sphingomyelin, phosphatidylethanolamine, phosphatidyl-phosphatidylethanolamine, phosphatidyl choline, phosphatidylserine, phosphatidylcholine, lysophosphatidylcholine, phosphatidylcholine and lysophosphatidylcholine;
And/or, the structural lipid is cholesterol;
And/or the polymer conjugated lipid is selected from any one or a combination of at least two of distearyl phosphatidylethanolamine polyethylene glycol 2000, dimyristoyl glycerol-3-methoxy polyethylene glycol 2000 and methoxy polyethylene glycol ditetradecylacetamide.
[13] The carrier according to any one of [5] to [12], wherein the neutral lipid is selected from 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine and/or 1, 2-distearoyl-sn-glycero-3-phosphorylcholine.
[14] A composition comprising a carrier and an active ingredient, wherein the carrier comprises the carrier of any one of [5] to [13 ].
[15] The composition according to [14], wherein the composition is a nanoparticle preparation having an average particle diameter of 10nm to 300nm, and a polydispersity index (PDI) of 0.5 or less.
For example, the nanoparticle formulation may have an average particle diameter of 10nm、15nm、20nm、25nm、30nm、35nm、40nm、45nm、50nm、60nm、70nm、80nm、81nm、82nm、83nm、84nm、85nm、86nm、87nm、88nm、89nm、90nm、91nm、92nm、93nm、94nm、95nm、96nm、97nm、98nm、99nm、100nm、110nm、120nm、130nm、140nm、150nm、160nm、170nm、180nm、190nm、200nm、250nm、300nm, or may be in a range consisting of any two of the above values, or any intermediate value in the range.
For example, the nanoparticle formulation may have a PDI of 0.0001、0.001、0.01、0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.25、0.3、0.35、0.4、0.45、0.5, or may be in the range of any two values described above, or any intermediate value in the range.
In the present disclosure, parameters such as average particle size and polydispersity index of the nanoparticle formulation have general meanings in the art, for example, average particle size refers to the average of particle sizes of all nanoparticles in a certain amount of the nanoparticle formulation, and polydispersity index (PDI) refers to the ratio of standard deviation of particle sizes and average particle sizes of all nanoparticles in a certain amount of the nanoparticle formulation, for characterizing the uniformity of size distribution of nanoparticles, and smaller PDI indicates narrower size distribution of nanoparticles.
In the present disclosure, parameters such as average particle size and polydispersity of the nanoparticle formulation may be detected by conventional methods in the art. For example, a laser particle analyzer may be used to detect the nanoparticle formulation (diluted to a concentration of about 0.02 mg/mL of active component (e.g., mRNA)) and the test results for the corresponding parameters may be read directly by the instrument or its associated software. The detection conditions may be those commonly used in the art, for example, the detection temperature is room temperature (e.g., 25 ℃) and the scattering angle is 90 °.
[16] The composition according to [14] or [15], wherein the nanoparticle preparation has an average particle diameter of 40nm to 240nm, and the polydispersity of the nanoparticle preparation is not more than 0.4.
[17] The composition of any one of [14] to [16], wherein the active component comprises a therapeutic agent and/or a prophylactic agent.
[18] The composition of any one of [14] to [17], wherein the therapeutic and/or prophylactic agent is selected from any one or a combination of at least two of the group consisting of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.
[19] The composition of any one of [14] to [18], wherein the therapeutic and/or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.
[20] The composition of any one of [14] to [19], wherein the therapeutic and/or prophylactic agent is a nucleic acid.
[21] The composition of any one of [14] to [20], wherein the therapeutic and/or prophylactic agent is RNA.
[22] The composition of any one of [14] to [21], wherein the RNA is any one or a combination of at least two selected from the group consisting of small interfering RNA, asymmetric interfering RNA, microRNA, dicer-substrate RNA, small hairpin RNA, messenger RNA.
[23] The composition of any one of [14] to [22], wherein the RNA is messenger RNA.
[24] The composition according to any one of [14] to [23], wherein the mass ratio of carrier to active ingredient in the composition is 10:1 to 30:1.
For example, the mass ratio of carrier to active ingredient in the composition may be 10:1、10.5:1、11:1、11.5:1、12:1、12.5:1、13:1、13.5:1、14:1、14.5:1、15:1、15.5:1、16:1、16.5:1、17:1、17.5:1、18:1、18.5:1、19:1、19.5:1、20:1、21:1、22:1、23:1、24:1、25:1、26:1、27:1、28:1、29:1、30:1, or may be in the range of any two ratios described above, or any intermediate ratio in the range.
[25] The composition according to any one of [14] to [24], wherein the mass ratio of carrier to active ingredient in the composition is 12.5:1 to 20:1.
[26] The composition according to any one of [14] to [25], wherein the mass ratio of carrier to active ingredient in the composition is 13:1 to 17:1.
[27] The composition of any one of [14] to [26], wherein the composition further comprises a pharmaceutically acceptable excipient and/or diluent.
[28] Use of a compound of any one of [1] to [4] or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a vector of any one of [5] to [13], or a composition of any one of [14] to [27] for increasing the transfection efficiency of cells.
Transfection efficiency refers to the ratio of the number of cells transfected with a substance (e.g., an active ingredient) desired to be introduced into the cells to the total number of cells during cell transfection. I.e. using a compound of the present disclosure or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a carrier, or a composition, enabling the active ingredient (e.g. as mentioned in the foregoing technical solutions) to enter into more cells.
[29] The use according to [28], wherein the use is a use for improving cell transfection efficiency in vitro.
For example, the use may comprise increasing the proportion of transfected cells in an in vitro cell transfection assay in all experimental cells.
[30] Use of the compound of any one of [1] to [4], or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or the vector of any one of [5] to [13], or the composition of any one of [14] to [27], for increasing the targeting of a drug to a target, wherein the target is selected from any one of the group consisting of a target organ, a target tissue and a target cell, or a combination of at least two.
In this disclosure, increasing the targeting of a drug to a target refers to increasing the amount of drug that enters the target in the administered drug, i.e., increasing the ratio of the amount of drug that enters the target to the amount of drug that enters the non-target location, or decreasing the ratio of the amount of drug that enters the non-target location to the amount of drug that enters the target. Preferably, the medicament comprises at least the active ingredient of the aforementioned composition.
[31] The use according to [30], wherein the target tissue is selected from muscle;
and/or, the target cell is selected from a muscle cell.
The inventors have found that when a lipid composition is used to encapsulate a pharmaceutically active ingredient to form a pharmaceutical composition, the incorporation of a compound of the present disclosure into the lipid composition is effective to enhance targeting of the pharmaceutical composition to muscle tissue, such that the pharmaceutical composition remains more at the site of administration (e.g., injection site), while the amount of the pharmaceutical composition that is off-target into internal organs (e.g., heart, liver, spleen, lung, kidney, etc., particularly liver and spleen) is greatly reduced, thereby effectively avoiding side effects or organ toxicity of the drug.
[32] Use of the compound of any one of [1] to [4], or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or the vector of any one of [5] to [13], or the composition of any one of [14] to [27] to increase the ratio of the expression amount of a nucleic acid in a target to the expression amount in a non-target, wherein the target is selected from any one of the group consisting of a target organ, a target tissue and a target cell, or a combination of at least two.
In the present disclosure, increasing the ratio of the amount of expression of a nucleic acid in a target to the amount of expression of a non-target refers to the ratio of the amount of nucleic acid entering the target and expressed at the target to the amount of nucleic acid exiting the target to the non-target and expressed. Non-target (where) i.e. nucleic acid is administered to a subject, anywhere else (e.g. any other tissue, organ, etc.) than the target location. Preferably, the non-target comprises any one or a combination of at least two of the group consisting of organs such as heart, liver, spleen, lung and kidney. More preferably including the liver and/or spleen.
[33] The use according to [32], wherein the target tissue is selected from muscle;
and/or, the target cell is selected from a muscle cell.
[34] Use of a compound of any one of [1] to [4] or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a carrier of any one of [5] to [13], or a composition of any one of [14] to [27] for reducing organ toxicity of a medicament.
In this disclosure, reducing organ toxicity of a drug refers to reducing the adverse effect on non-target organs due to drug off-target entry into non-target organ accumulation. Preferably, the medicament comprises at least the active ingredient of the aforementioned composition.
Preferably, the organ toxicity of the drug includes cardiotoxicity, hepatotoxicity, splenic toxicity, pulmonary toxicity, renal toxicity, etc., i.e., side effects caused by off-target entry of the drug into the organ.
Preferably, the organ toxicity of the drug is selected from liver toxicity and/or spleen toxicity, more preferably liver toxicity.
[35] Use of a compound of any one of [1] to [4] or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a carrier of any one of [5] to [13], or a composition of any one of [14] to [27] in the manufacture of a medicament.
[36] The use according to [35], wherein the active component of the medicament is selected from any one or a combination of at least two of the group consisting of nucleic acids, small molecule compounds, polypeptides or proteins.
[37] The use according to [35] or [36], wherein accumulation of the active ingredient of the medicament in an organ causes organ toxicity.
[38] The use of any one of [35] to [37], wherein the organ is selected from any one or a combination of at least two of the group consisting of heart, liver, spleen, lung and kidney.
[39] Use of a compound of any one of [1] to [4] or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a carrier of any one of [5] to [13], or a composition of any one of [14] to [27] in the manufacture of a medicament for treating a disease or disorder in a subject in need thereof.
[40] The use according to [39], wherein the disease or disorder is characterized by malfunction or abnormality of a protein or polypeptide.
That is, the disease or disorder may be caused by a malfunction or abnormality of a protein or polypeptide.
[41] The use according to [39] or [40], wherein the disease or disorder is selected from any one or a combination of at least two of the group consisting of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases and metabolic diseases.
[42] The use of any one of [39] to [41], wherein the subject is a mammal.
For example, the subject may be a human, a companion animal (e.g., cat, dog, etc.), a farm animal (e.g., cow, horse, pig, etc.), a laboratory animal (e.g., rat, mouse, rabbit, monkey, etc.), or the like.
Preferably, the subject is a human.
[43] The use according to any one of [39] to [42], wherein the medicament is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation, preferably intramuscularly and/or subcutaneously. The medicament may be administered, for example, by intramuscular/subcutaneous injection.
[44] The use according to any one of [39] to [43], wherein the medicament is administered in an amount such that a dose of about 0.001mg/kg body weight to about 10mg/kg body weight of the active ingredient (e.g. therapeutic and/or prophylactic agent) is administered to the subject.
For example, the amount of drug administered is such that the dosage of the active ingredient administered to the subject is about 0.001mg/kg body weight, about 0.005mg/kg body weight, about 0.008mg/kg body weight, about 0.01mg/kg body weight, about 0.05mg/kg body weight, about 0.08mg/kg body weight, about 0.1mg/kg body weight, about 0.5mg/kg body weight, about 1mg/kg body weight, about 1.5mg/kg body weight, about 2mg/kg body weight, about 2.5mg/kg body weight, about 3mg/kg body weight, about 3.5mg/kg body weight, about 4mg/kg body weight, about 4.5mg/kg body weight, about 5mg/kg body weight, about 6mg/kg body weight, about 6.5mg/kg body weight, about 7mg/kg body weight, about 7.5mg/kg body weight, about 8mg/kg body weight, about 8.5mg/kg body weight, about 9mg/kg body weight, about 9.5mg/kg body weight, about 10mg/kg body weight, or any intermediate value therebetween.
Further, the present disclosure also provides the following technical solutions:
[45] A method of increasing the efficiency of cell transfection, the method comprising:
i. Cell transfection after encapsulating the substance to be transfected with a carrier comprising the compound of any one of [1] to [4] or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or
Encapsulating the substance to be transfected with the vector of any one of [5] to [13], followed by cell transfection, or
Cell transfection with the composition of any one of [14] to [27 ].
In the methods provided by the present disclosure, the material to be transfected, i.e., the material to be transfected into the cells, may be any active ingredient useful in the art for cell transfection. For example, nucleic acids, proteins, peptides, small molecule compounds, and the like can be used. The choice of the substance to be transfected is also referred to the active ingredients of the aforementioned compositions and will not be described in detail here.
[46] The method according to [45], wherein the method is a method for improving the transfection efficiency of cells in vitro.
[47] A method of cell transfection, the method comprising:
i. Encapsulating a substance to be transfected with a carrier comprising the compound of any one of [1] to [4] or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or
Encapsulating a substance to be transfected with the vector of any of [5] to [13], or
Providing a composition as described in any one of [14] to [27 ].
[48] The method according to [47], wherein the method further comprises a step of performing cell transfection after the step of encapsulating the substance to be transfected or providing the composition. In the methods of the present disclosure, cell transfection may be performed in any manner known in the art using liposome transduction.
[49] The method according to [47] or [48], wherein the method is a method of cell transfection in vitro.
[50] A method of increasing targeting of a drug to a target selected from any one or a combination of at least two of the group consisting of a target organ, a target tissue, and a target cell;
The method comprises the following steps:
i. encapsulating an active ingredient of a drug with a carrier comprising the compound of any one of [1] to [4] or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or
Encapsulating a pharmaceutical active ingredient with the carrier of any one of [5] to [13], or
Providing a composition of any one of [14] to [27], the active component of the composition comprising the active component of the medicament.
[51] The method of [50], wherein the target tissue is selected from the group consisting of muscle;
and/or, the target cell is selected from a muscle cell.
[52] A method of increasing the expression level of a drug at a target selected from any one or a combination of at least two of the group consisting of a target organ, a target tissue, and a target cell;
The method comprises the following steps:
i. encapsulating an active ingredient of a drug with a carrier comprising the compound of any one of [1] to [4] or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or
Encapsulating a pharmaceutical active ingredient with the carrier of any one of [5] to [13], or
Providing a composition of any one of [14] to [27], the active component of the composition comprising the active component of the medicament.
[53] The method of [51], wherein the target tissue is selected from the group consisting of muscle;
and/or, the target cell is selected from a muscle cell.
[54] A method of reducing organ toxicity/side effects of a drug, the method comprising:
i. encapsulating an active ingredient of a drug with a carrier comprising the compound of any one of [1] to [4] or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or
Encapsulating a pharmaceutical active ingredient with the carrier of any one of [5] to [13], or
Providing a composition of any one of [14] to [27], the active component of the composition comprising the active component of the medicament.
Preferably, the medicament comprises at least the active ingredient of the aforementioned composition.
Preferably, the organ toxicity of the drug includes cardiotoxicity, hepatotoxicity, splenic toxicity, pulmonary toxicity, renal toxicity, etc., i.e., side effects caused by off-target entry of the drug into the organ.
Preferably, the organ toxicity of the drug is selected from liver toxicity and/or spleen toxicity, more preferably liver toxicity.
[55] A method of treating a disease or disorder in a subject in need thereof, the method comprising:
(1) Providing a medicament comprising:
i. encapsulating an active ingredient of a drug with a carrier comprising the compound of any one of [1] to [4] or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or
Encapsulating a pharmaceutical active ingredient with the carrier of any one of [5] to [13], or
Providing a composition of any one of [14] to [27], the active component of the composition comprising the active component of the medicament;
(2) The medicament is administered to the subject in a therapeutically effective amount.
[56] The method of [55], wherein the disease or disorder is characterized by a malfunction or abnormality of a protein or polypeptide.
That is, the disease or disorder may be caused by a malfunction or abnormality of a protein or polypeptide.
[57] The method of [55] or [56], wherein the disease or disorder is selected from any one or a combination of at least two of the group consisting of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.
[58] The method of any one of [55] to [57], wherein the subject is a mammal.
For example, the subject may be a human, a companion animal (e.g., cat, dog, etc.), a farm animal (e.g., cow, horse, pig, etc.), a laboratory animal (e.g., rat, mouse, rabbit, monkey, etc.), or the like.
Preferably, the subject is a human.
[59] The method of any one of [55] to [58], wherein the drug is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation, preferably intramuscularly and/or subcutaneously. The medicament may be administered, for example, by intramuscular injection.
[60] The method of any one of [55] to [59], wherein the drug is administered in an amount such that a dose of about 0.001mg/kg body weight to about 10mg/kg body weight of the active ingredient (e.g., therapeutic and/or prophylactic agent) is administered to the subject.
For example, the amount of drug administered is such that the dosage of the active ingredient administered to the subject is about 0.001mg/kg body weight, about 0.005mg/kg body weight, about 0.008mg/kg body weight, about 0.01mg/kg body weight, about 0.05mg/kg body weight, about 0.08mg/kg body weight, about 0.1mg/kg body weight, about 0.5mg/kg body weight, about 1mg/kg body weight, about 1.5mg/kg body weight, about 2mg/kg body weight, about 2.5mg/kg body weight, about 3mg/kg body weight, about 3.5mg/kg body weight, about 4mg/kg body weight, about 4.5mg/kg body weight, about 5mg/kg body weight, about 6mg/kg body weight, about 6.5mg/kg body weight, about 7mg/kg body weight, about 7.5mg/kg body weight, about 8mg/kg body weight, about 8.5mg/kg body weight, about 9mg/kg body weight, about 9.5mg/kg body weight, about 10mg/kg body weight, or any intermediate value therebetween.
It should be understood that the above uses and methods provided by the present disclosure may include both therapeutic and diagnostic uses or methods, as well as non-therapeutic and non-diagnostic uses or methods. For example, therapeutic aspects may include packaging and delivering a (pharmaceutical) active ingredient into a target organ/tissue/cell with a compound provided by the present disclosure, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or with a lipid composition (i.e., carrier) provided by the present disclosure, or using a composition of the present disclosure to deliver an active ingredient contained therein into a target organ/tissue/cell, thereby achieving the effect of treating a disease, ameliorating a disorder, modulating physiological activity in vivo, or the like; diagnostic uses may include packaging an active ingredient for disease diagnosis in a compound provided by the present disclosure, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer or a lipid composition thereof, such that the active ingredient is delivered into a target organ/tissue/cell for disease diagnosis purposes, and non-therapeutic/non-diagnostic aspects may include using a compound provided by the present disclosure, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or encapsulating an active ingredient with a lipid composition provided by the present disclosure, such that it is delivered into a target organ/tissue/cell for non-therapeutic, non-diagnostic tasks such as performing disease mechanism studies, drug mechanism studies, developing new drugs, drug screening, etc.
The beneficial effects of the present disclosure include at least:
the cationic lipid compounds and lipid composition carriers comprising the same can be used for packaging pharmaceutically active ingredients such as nucleic acids (e.g. mRNA and the like), and the encapsulation efficiency reaches a higher level (e.g. more than 90%).
The mRNA-LNP composition prepared by the cationic lipid has the advantages of being capable of remarkably improving the protein expression quantity in vivo (such as the protein expression quantity in a mouse), meanwhile being remarkable in muscle targeting, being small in organ off-target accumulation, particularly remarkably reducing off-target accumulation in liver compared with the prior art, so that drug hepatotoxicity is avoided or slowed down, and being capable of remarkably improving the protein expression quantity in vivo (in vivo) and in vitro (in intro).
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present disclosure more apparent, the technical solutions of the embodiments of the present disclosure will be described below with reference to the accompanying drawings. It will be apparent that the described embodiments are some, but not all, of the embodiments of the present disclosure. Based on the described embodiments of the present disclosure, one of ordinary skill in the art may implement other specific forms without departing from the basic attributes and gist of the present disclosure. It is to be understood that any and all embodiments of the present disclosure may be combined with any other embodiment or features of multiple other embodiments to yield yet further embodiments without conflict. The present disclosure includes additional embodiments resulting from such combinations.
All publications and patents mentioned in this disclosure are incorporated herein by reference in their entirety. If a use or term used in any of the publications and patents incorporated by reference conflicts with the use or term used in the present disclosure, the use or term of the present disclosure controls.
The section headings used herein are for purposes of organizing articles only and should not be construed as limiting the subject matter.
Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning in the art to which the claimed subject matter belongs. In case there are multiple definitions for a term, the definitions herein control.
Except in the operating examples, or where otherwise indicated, all numbers expressing quantities of quantitative properties such as dosages set forth in the specification and claims are to be understood as being modified in all instances by the term "about". It should also be understood that any numerical range recited in this disclosure is intended to include all sub-ranges within that range and any combination of the individual endpoints of that range or sub-range. When a range of values is disclosed herein, the range is considered to be continuous and includes both the minimum and maximum values for the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
The use of the terms "comprising," "including," or "containing," and the like, in this disclosure, are intended to cover an element listed after that term and its equivalents, but do not exclude the presence of other elements. The terms "comprising," "including," or "comprising," as used herein, may be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of, or" consisting of.
The term "pharmaceutically acceptable" in this disclosure means that the compound or composition is chemically and/or toxicologically compatible with the other components of the formulation and/or with the human or mammal with which the disease or disorder is to be prevented or treated.
The term "subject" or "patient" includes humans and mammals in this disclosure.
The term "treatment" as used herein refers to the administration of one or more pharmaceutical substances to a patient or subject suffering from or having symptoms of a disease, to cure, alleviate, ameliorate or otherwise affect the disease or symptoms of the disease. In the context of the present disclosure, the term "treatment" may also include prophylaxis, unless specifically stated to the contrary.
The term "solvate" refers in the present disclosure to a complex of a compound of formula (I) or a pharmaceutically acceptable salt thereof, in association with a solvent (e.g., ethanol or water). It will be appreciated that any solvate of the compound of formula (I) used in the treatment of a disease or condition, although potentially providing different properties (including pharmacokinetic properties), will result in the compound of formula (I) once absorbed into a subject, such that the use of the compound of formula (I) encompasses the use of any solvate of the compound of formula (I), respectively.
The term "hydrate" refers to the case where the solvent in the above term "solvate" is water.
It is further understood that the compound of formula (I) or a pharmaceutically acceptable salt thereof may be isolated in the form of a solvate, and thus any such solvate is included within the scope of the present disclosure. For example, a compound of formula (I) or a pharmaceutically acceptable salt thereof may exist in unsolvated forms or solvated forms in which are associated with pharmaceutically acceptable solvents (such as water, ethanol, and the like).
The term "pharmaceutically acceptable salt" refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound of the present disclosure. See, for example, S.M. Bere et al, "Pharmaceutical Salts", J.Pharm. Sci.1977, 66, 1-19. Among them, inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid, etc., organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4-hydroxybenzoyl) -benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectic acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptonic acid, glyceric acid, aspartic acid, sulfosalicylic acid, etc. For example, HCl (or hydrochloric acid), HBr (or hydrobromic acid solution), methanesulfonic acid, sulfuric acid, tartaric acid, or fumaric acid may be used to form pharmaceutically acceptable salts with the compounds of formula (I).
The nitrogen-containing compounds of formula (I) of the present disclosure may be converted to N-oxides by treatment with an oxidizing agent (e.g., m-chloroperoxybenzoic acid, hydrogen peroxide, ozone). Thus, the presently claimed compounds include not only nitrogen-containing compounds of the formula, but also N-oxide derivatives thereof, where valence and structure permit.
Certain compounds of the present disclosure may exist in the form of one or more stereoisomers. Stereoisomers include geometric isomers diastereoisomers and enantiomers. Thus, the presently claimed compounds also include racemic mixtures, single stereoisomers, and optically active mixtures. It will be appreciated by those skilled in the art that one stereoisomer may have better efficacy and/or lower side effects than the other stereoisomers. The single stereoisomers and the mixture with optical activity can be obtained by chiral source synthesis methods, chiral catalysis methods, chiral resolution methods and the like. The racemate can be chiral resolved by chromatographic resolution or chemical resolution. For example, separation can be performed by adding chiral acid resolving agents such as chiral tartaric acid, chiral malic acid, and the like to form salts with the compounds of the present disclosure, utilizing differences in the physicochemical properties of the products, such as solubility.
The present disclosure also includes all suitable isotopic variations of the compounds of the present disclosure. Isotopic variations are defined as compounds in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found or predominantly present in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, and oxygen, such as2 H (deuterium),3 H (tritium),11C、13C、14C、15N、17 O, and18 O, respectively.
The term "alkyl" is meant in this disclosure to include both branched and straight chain saturated aliphatic monovalent hydrocarbon groups having the specified number of carbon atoms. The term "alkylene" is meant in this disclosure to include both branched and straight chain saturated aliphatic divalent hydrocarbon groups having the specified number of carbon atoms. Cn-m is meant to include groups having a number of carbon atoms from n to m. For example, C2-5 alkylene includes C2 alkylene, C3 alkylene, C4 alkylene, C5 alkylene.
The alkyl (or alkylene) group may be unsubstituted, or the alkyl (or alkylene) group may be substituted, wherein at least one hydrogen is replaced with another chemical group.
A "therapeutically effective amount" is an amount of a therapeutic agent that, when administered to a patient, ameliorates a disease or condition. A "prophylactically effective amount" is an amount of a prophylactic agent that, when administered to a subject, prevents a disease or condition. The amount of therapeutic agent constituting the "therapeutically effective amount" or the amount of prophylactic agent of the "prophylactically effective amount" varies with the therapeutic agent/prophylactic agent, the disease state and severity thereof, the age, weight, etc. of the patient/subject to be treated/prevented. One of ordinary skill in the art can routinely determine therapeutically effective and prophylactically effective amounts based on their knowledge and disclosure.
In the present disclosure, when the names of the compounds are not identical to the structural formula, the structural formula is taken into consideration.
It is to be understood that the term "compounds of the present disclosure" as used in the present disclosure may include, depending on the context, compounds of formula (I), N-oxides thereof, solvates thereof, pharmaceutically acceptable salts thereof, stereoisomers thereof, and mixtures thereof.
The term "cationic lipid" as used herein refers to a lipid that is positively charged at a selected pH value or range.
Cationic lipids readily bind to negatively charged nucleic acids, i.e., interact with negatively charged phosphate groups in nucleic acids by electrostatic forces, forming Lipid Nanoparticles (LNPs).
The inventors found that when screening a large number of compounds, it was very difficult to screen suitable cationic lipid compounds that met the structural differences from the cationic lipids of the prior art, and that have high transfection efficiency and low cytotoxicity, and high and sustained expression in mice (especially at the target site). The inventors have found in a number of studies that certain compounds, such as YK-1603, YK-1604, YK-1605, YK-1606, YK-1608, YK-1610, YK-1612, YK-1613, YK-1614, YK-1615, and YK-1618, etc., are capable of delivering biologically active molecules such as nucleic acids with significantly improved intracellular transfection efficiency, lower levels of cytotoxicity, significantly improved expression levels in animals (especially at target sites), and muscle targeting, as compared to cationic lipids in the prior art.
The present disclosure is based at least on the following findings:
The cationic lipid compounds of the present disclosure can be used to deliver nucleic acid molecules, small molecule compounds, polypeptides, or proteins. Compared with the known cationic lipid compounds, the cationic lipid compounds of the present disclosure exhibit higher transfection efficiency and less cytotoxicity, and significantly increase the expression level in animal muscle, improving delivery efficiency.
The present disclosure also provides a carrier comprising a cationic lipid provided by a compound of formula (I) of the present disclosure, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof.
The present disclosure further provides a composition comprising the above-described carrier.
In one embodiment, the composition is a nanoparticle formulation having an average size of 10nm to 300nm, preferably 40nm to 240m, and a polydispersity of 0.5 or less, preferably 0.4 or less.
The present disclosure further provides uses and related methods of the above cationic lipid compounds, carriers, or compositions.
Cationic lipids
In one embodiment of the compounds/carriers/compositions of the present disclosure, the cationic lipid comprises one or more selected from the group consisting of compounds of formula (I) of the present disclosure or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof. In one embodiment, the cationic lipid is a compound of formula (I) selected from the group consisting of those described above. For example, the cationic lipid is a compound of formula (I). In a preferred embodiment, the cationic lipid is any one of compounds YK-1601 to YK-1618. In a more preferred embodiment, the cationic lipid is any one of the compounds YK-1603, YK-1604, YK-1605, YK-1606, YK-1608, YK-1610, YK-1612, YK-1613, YK-1614, YK-1615, and YK-1618.
In another embodiment of the carrier/composition of the present disclosure, the cationic lipid comprises (a) one or more selected from the compounds of formula (I) above or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof, and optionally (b) one or more other ionizable lipid compounds different from (a). (b) The cationic lipid compound may be a commercially available cationic lipid, or a cationic lipid compound reported in the literature. For example, (b) the cationic lipid compound may be SM-102 in CN201880017979.X, MC3 in CN201080026228.8, E10-1 in CN 202380014467.9.
In one embodiment, the cationic lipid comprises 25% to 75%, such as 30%, 40%, 50%, 55%, 60%, 65%, 70% of the carrier by mole.
The carrier may be used for delivery of active ingredients such as therapeutic and/or prophylactic agents. The active ingredient may be enclosed within the carrier or combined with the carrier in any form.
For example, examples of the therapeutic agent, the prophylactic agent may be one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein. Such nucleic acids include, but are not limited to, single-stranded DNA, double-stranded DNA, and RNA. Suitable RNAs include, but are not limited to, small interfering RNAs (sirnas), asymmetric interfering RNAs (airnas), micrornas (mirnas), dicer-substrate RNAs (dsRNA), small hairpin RNAs (shrnas), messenger RNAs (mrnas), mixtures thereof, and the like.
Neutral lipids
The carrier may comprise neutral lipids. Neutral lipids in the present disclosure refer to lipids that exist in an uncharged form or in a neutral ionic form at a selected pH or range. The neutral lipids may modulate nanoparticle mobility into lipid bilayers (lipid bilayer) and increase efficiency by promoting lipid phase changes, while also potentially affecting target organ specificity.
In one embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 1:1-15:1, such as about 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, and 1:1. Also for example, in a preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 4.9:1.
For example, the neutral lipids may include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramides, sterols, and derivatives thereof.
The carrier component of the cationic lipid-containing composition may comprise one or more neutral lipid-phospholipids, such as one or more (poly) unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids may include a phospholipid moiety and one or more fatty acid moieties.
Neutral lipids may be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid may be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Non-natural species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, the phospholipid may be functionalized with or crosslinked with one or more alkynes (e.g., alkenyl groups with one or more double bonds replaced with triple bonds). Under appropriate reaction conditions, alkynyl groups may undergo copper-catalyzed cycloaddition reactions upon exposure to azide. These reactions can be used to functionalize the lipid bilayer of the composition to facilitate membrane permeation or cell recognition, or to couple the composition with a useful component such as a targeting or imaging moiety (e.g., dye).
Neutral lipids useful in these compositions may be selected from the non-limiting group consisting of: 1, 2-Dioleoyl-sn-glycero-3-phosphorylcholine (DLPC), 1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dioleoyl-sn-glycero-phosphorylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-octadecenyl-sn-glycero-3-phosphorylcholine (18:0 DietherPC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-3-phosphorylcholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphorylcholine (C), 1, 2-dioleoyl-glycero-3-phosphorylcholine, 1-dioleoyl-sn-3-phosphorylcholine (CPC), 1, 2-dioleoyl-glycero-3-phosphorylcholine, 1-dioleoyl-2-glycero-3-phosphorylcholine (POPC) 1, 2-dioleoyl-sn-glycero-3-phosphate ethanolamine (DOPE), 1, 2-di-phytanoyl-sn-glycero-3-phosphate ethanolamine (ME 16.0 PE), 1, 2-di-stearoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-oleoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-linolenoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-arachidonoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-docosahexaenoic acyl-sn-glycero-3-phosphate ethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphate sodium salt (DOPG), 1, 2-di-oleoyl-rac-3-phosphate sodium salt (DOPG) dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl-based phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof.
In some embodiments, the neutral lipid comprises DSPC. In certain embodiments, the neutral lipid comprises DOPE. In some embodiments, the neutral lipid comprises both DSPC and DOPE.
Structured lipids
The carrier of the composition comprising the cationic lipid may also comprise one or more structural lipids. Structured lipids refer in the present disclosure to lipids that enhance the stability of the nanoparticle by filling the interstices between the lipids.
In one embodiment, the molar ratio of the cationic lipid to the structural lipid is about 1:1-5:1, e.g., about 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1.
The structural lipid may be selected from the group consisting of, but not limited to, cholesterol, non-sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycorine, ursolic acid, alpha-tocopherols, corticosteroids, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipids include cholesterol and corticosteroids (such as prednisolone (prednisolone), dexamethasone, prednisone (prednisone), and hydrocortisone (hydrocortisone), or combinations thereof.
Polymer conjugated lipids
The carrier of the composition comprising the cationic lipid may also comprise one or more polymer conjugated lipids. The polymer conjugated lipid mainly refers to polyethylene glycol (PEG) modified lipid. Hydrophilic PEG stabilizes LNP, regulates nanoparticle size by limiting lipid fusion, and increases nanoparticle half-life by reducing non-specific interactions with macrophages.
In one embodiment, the polymer conjugated lipid is selected from one or more of PEG modified phosphatidylethanolamine, PEG modified phosphatidic acid, PEG modified ceramide, PEG modified dialkylamine, PEG modified diacylglycerol, and PEG modified dialkylglycerol. The PEG modified PEG molecular weight is typically 350-5000Da.
For example, the polymer conjugated lipid is selected from one or more of distearoyl phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG 2000), dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000), and methoxypolyethylene glycol ditetradecylamide (ALC-0159).
In one embodiment of the composition/carrier of the present disclosure, the polymer conjugated lipid is DMG-PEG2000.
In one embodiment of the composition/carrier of the present disclosure, the carrier comprises neutral lipids, structural lipids, and polymer conjugated lipids, the molar ratio of the cationic lipids, the neutral lipids, the structural lipids, and the polymer conjugated lipids is (25-75): (5-25): (15-65): (0.5-10), such as (30-49): (7.5-15): (35-55): (1-5), more preferably (40-49): (8-12): (39-45): (1-3). The sum of the molar ratios of the cationic lipid, the neutral lipid, the structural lipid, and the polymer conjugated lipid is 100
In one embodiment of the composition/carrier of the present disclosure, the carrier comprises a neutral lipid, a structural lipid, and a polymer conjugated lipid, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer conjugated lipid being 40:10:48.5:1.5 or 49:10:39.5:1.5.
Therapeutic and/or prophylactic agent
The composition may include one or more therapeutic and/or prophylactic agents (as active components). In one embodiment, the mass ratio of carrier to the therapeutic and/or prophylactic agent is 10:1-30:1, e.g., 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1.
In one embodiment, the mass ratio of carrier to the therapeutic and/or prophylactic agent is 12.5:1 to 20:1, preferably 13 to 17:1, more preferably 15:1.
The therapeutic and/or prophylactic agents include, but are not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.
For example, the therapeutic and/or prophylactic agent is a vaccine or compound capable of eliciting an immune response.
The vectors of the present disclosure may deliver therapeutic and/or prophylactic agents to mammalian cells or organs, and thus the present disclosure also provides methods of treating a disease or disorder in a mammal in need thereof, comprising administering to the mammal a composition comprising a therapeutic and/or prophylactic agent and/or contacting mammalian cells with the composition. Accordingly, the present disclosure provides for the use of a compound of the present disclosure or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a composition of the present disclosure in the manufacture of a medicament for treating a disease or disorder in a subject in need thereof.
The present disclosure also provides the use of a compound of the present disclosure or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a composition of the present disclosure in the preparation of a nucleic acid drug, vaccine, chemical drug, polypeptide drug or protein drug.
Therapeutic and/or prophylactic agents include bioactive substances and may alternatively be referred to as "active agents," "active components," "active ingredients," and the like. The therapeutic and/or prophylactic agent can be a substance that, upon delivery to a cell or organ, causes a desired change in the cell or organ or other body tissue or system. Such species may be used to treat one or more diseases, disorders or conditions. In some embodiments, the therapeutic and/or prophylactic agent is a small molecule drug that can be used to treat a particular disease, disorder, or condition. Examples of drugs that may be used in the compositions include, but are not limited to, antineoplastic agents (e.g., vincristine, doxorubicin (doxorubicin), mitoxantrone (mitoxantrone), camptothecin, cisplatin (cispratin), bleomycin (bleomycin), cyclophosphamide (cyclophosphamide), methotrexate, and streptozotocin), antineoplastic agents (e.g., actinomycin D (actinomycin D)), Vincristine, vinblastine (vinblastine), cytosine arabinoside (cytosine arabinoside), anthracyclines (anthracyclines), alkylating agents, platinoids, antimetabolites and nucleoside analogues such as methotrexate and purine and pyrimidine analogues), anti-infective agents, local anesthetics (e.g. dibucaine (dibucaine) and chlorpromazine (chlorpromazine)), beta-adrenergic blockers (e.g. propranolol (propranolol), molol (timolol) and labetalol (labetalol)), Antihypertensives (e.g., clonidine and hydralazine (hydralazine)), antidepressants (e.g., imipramine (imipramine), amitriptyline (AMITRIPTYLINE) and doxepin (doxepin)), antispasmodics (e.g., phenytoin (phenytoin)), antihistamines (e.g., diphenhydramine (DIPHENHYDRAMINE), chlorpheniramine (chlorpheniramine) and promethazine (promethazine)), antibiotics/antibacterial agents (e.g., gentamicin (gentamycin), antibiotics/antibacterial agents (e.g., gentamicin), Ciprofloxacin (ciprofloxacin) and cefoxitin (cefoxitin)), antifungal agents (e.g., miconazole (miconazole), terconazole (terconazole), econazole (econazole), econazole (isoconazole), butoconazole (butaconazole), clotrimazole (clotrimazole), itraconazole (itraconazole), nystatin (nystatin), netifen (naftifine) and amphotericin B (amphotericin B)), and pharmaceutical compositions, Antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, anti-glaucoma agents, vitamins, sedatives and imaging agents.
In some embodiments, the therapeutic and/or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that elicits an immune response, and/or another therapeutic and/or prophylactic agent. Cytotoxins or cytotoxic agents include any agent that is detrimental to cells. Examples include, but are not limited to, taxol (taxol), cytochalasin B (cytochalasin B), gramicidin D (gramicidin D), ethidium bromide (ethidium bromide), canamycin (emetine), mitomycin (mitomycin), etoposide (etoposide), teniposide (teniposide), vincristine, vinblastine, colchicine (colchicine), doxorubicin, daunorubicin (daunorubicin), dihydroxyanthracene dione (dihydroxy anthracin dione), and, Mitoxantrone, mithramycin (mithramycin), actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine (procaine), tetracaine (tetracaine), lidocaine (lidocaine), propranolol, puromycin, maytansinoids (maytansinoids) such as maytansinol (maytansinol), azithromycin (rachelmycin) (CC-1065), and analogs or homologs thereof. Radioions include, but are not limited to, iodine (e.g., iodine 125 or iodine 131), strontium 89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, samarium 153, and praseodymium. Examples of vaccines include compounds and formulations capable of providing immunity against one or more conditions associated with infectious diseases such as influenza, measles, human Papilloma Virus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis and tuberculosis, for example may include mRNA encoding a pathogenic antigen (pathogenic antigen) and/or an epitope thereof, and may also include compounds and formulations that direct an immune response against cancer cells, for example may include mRNA encoding a tumor cell-derived antigen, epitope and/or neoepitope. Compounds that elicit an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other species. In some embodiments, a vaccine and/or compound capable of eliciting an immune response is administered intramuscularly through a composition comprising a compound according to formula (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg) or (III) (e.g., compound 3, 18, 20, 25, 26, 29, 30, 60, 108-112 or 122). Other therapeutic and/or prophylactic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine), alkylating agents (e.g., nitrogen mustard (mechlorethamine), thiotepa (thiotepa), chlorambucil (chlorambucil), azithromycin (CC-1065), melphalan (melphalan), carmustine (carmustine, BSNU), robustin (lomustine, CCNU), and combinations thereof, cyclophosphamide, busulfan (busulfan), dibromomannitol, streptozotocin, mitomycin C and cisplatin (II) (DDP), cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin (daunomycin)) and doxorubicin), antibiotics (e.g., dactinomycin (dactinomycin) (formerly dactinomycin), bleomycin, mithramycin (mithramycin) and aflatoxin (anthramycin, AMC)), and antimitotics (e.g., vincristine, vinblastine, taxol and maytansinoids).
In other embodiments, the therapeutic and/or prophylactic agent is a protein. Therapeutic proteins useful in the nanoparticles in the present disclosure include, but are not limited to, gentamicin, amikacin (amikacin), insulin, erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), factor VIR, luteinizing Hormone Releasing Hormone (LHRH) analogs, interferons, heparin, hepatitis b surface antigens, typhoid vaccines, and cholera vaccines.
In some embodiments, the therapeutic and/or prophylactic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term "polynucleotide" is intended to include in its broadest sense any compound and/or substance that is or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), including messenger mRNA (mRNA), hybrids thereof, RNAi-inducing factors, RNAi factors, siRNA, shRNA, miRNA, antisense RNA, ribozyme, catalytic DNA, RNA that induces triple helix formation, aptamers, and the like. In some embodiments, the therapeutic and/or prophylactic agent is RNA. RNAs useful in the compositions and methods described in this disclosure may be selected from the group consisting of, but not limited to shortmer, antagomir, antisense RNA, ribozymes, small interfering RNAs (siRNAs), asymmetric interfering RNAs (aiRNAs), microRNAs (miRNAs), dicer-substrate RNAs (dsRNAs), small hairpin RNAs (shRNAs), transfer RNAs (tRNA), messenger RNAs (mRNAs), and mixtures thereof. In certain embodiments, the RNA is mRNA.
In certain embodiments, the therapeutic and/or prophylactic agent is mRNA. The mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA may be of any size and may have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA may have a therapeutic/prophylactic effect when expressed in a cell.
In other embodiments, the therapeutic and/or prophylactic agent is an siRNA. siRNA is capable of selectively reducing expression of a gene of interest or down-regulating expression of the gene. For example, the siRNA can be selected such that a gene associated with a particular disease, disorder, or condition is silenced after administration of a composition comprising the siRNA to a subject in need thereof. The siRNA may comprise a sequence complementary to an mRNA sequence encoding a gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.
In certain embodiments, the therapeutic and/or prophylactic agent is sgRNA and/or cas9 mRNA. sgRNA and/or cas9 mRNA may be used as a gene editing tool. For example, the sgRNA-cas9 complex can affect mRNA translation of cellular genes.
In some embodiments, the therapeutic and/or prophylactic agent is an shRNA or a vector or plasmid encoding the same. shRNA may be produced inside the target cell after delivery of the appropriate construct into the nucleus. Constructs and mechanisms related to shRNA are well known in the relevant arts.
Diseases or conditions
The compounds/carriers/compositions of the present disclosure may deliver therapeutic or prophylactic agents to a subject or patient. The therapeutic and/or prophylactic agents include, but are not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein. Thus, the compounds/vectors/compositions of the present disclosure may be used to prepare nucleic acid drugs, genetic vaccines, small molecule drugs, polypeptides or protein drugs. Because of the wide variety of therapeutic and/or prophylactic agents described above, the compositions of the present disclosure are useful in the treatment and/or prevention of a variety of diseases or conditions.
In one embodiment, the disease or disorder is characterized by dysfunctional or abnormal protein or polypeptide activity.
For example, the disease or condition is selected from the group consisting of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.
In one embodiment, the infectious disease is selected from the group consisting of diseases caused by coronavirus, influenza virus, or HIV virus, pediatric pneumonia, rift valley fever, yellow fever, rabies, and various herpes.
Other components
The composition may include one or more components other than those described in the preceding section. For example, the composition may include one or more hydrophobic small molecules, such as vitamins (e.g., vitamin a or vitamin E) or sterols.
The composition may also include one or more permeability enhancing molecules, carbohydrates, polymers, surface modifying agents, or other components. The permeability enhancing molecule may be, for example, a molecule described in U.S. patent application publication No. 2005/0222064. Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen, and derivatives and analogs thereof).
Surface modifying agents may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyl dioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamers), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain (bromelain), papain, dyers woad (clerodendrum), bromhexine (bromhexine), carbocisteine (carbocisteine), eplerenone (eprazinone), mesna (mesna), ambroxol (ambroxol), sibiriol (sobrerol), polymitols (domiodol), letrostat (letosteine), setronine (stepronin), tiopronin (tiopronin), gelsolin (gelsolin), thymosin (thymosin) β4, streptococcal dnase α (dornase alfa), netilmicin (neltenexine), and polysteine (erdosteine), and dnase (e.g., rhDNA enzymes). The surface modifying agent may be disposed within and/or on the nanoparticle of the composition (e.g., by coating, adsorption, covalent attachment, or other means).
The composition may further comprise one or more functionalized lipids. For example, the lipid may be functionalized with an alkynyl group that may undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions. In particular, lipid bilayers can be functionalized in this manner with one or more groups effective to facilitate membrane permeation, cell recognition, or imaging. The surface of the composition may also be conjugated to one or more useful antibodies. Functional groups and conjugates useful for targeted cell delivery, imaging, and membrane permeation are well known in the art.
In addition to these components, the composition may include any substance useful in pharmaceutical compositions. For example, the composition may include one or more pharmaceutically acceptable excipients or auxiliary ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersing aids, suspending aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonic agents, thickening or emulsifying agents, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, and the like. Excipients such as starch, lactose or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, e.g., remington' S THE SCIENCE AND PRACTICE of Pharmacy, 21 st edition, a.r. gennaro; lippincott, williams & Wilkins, baltimore, MD, 2006).
Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, dibasic calcium phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and/or combinations thereof.
In some embodiments, compositions comprising one or more lipids described herein may also comprise one or more adjuvants, such as Glucopyranosyl Lipid Adjuvants (GLA), cpG oligodeoxyribonucleotides (e.g., class a or class B), poly (I: C), aluminum hydroxide, and Pam3CSK4.
The compositions of the present disclosure may be formulated in solid, semi-solid, liquid or gaseous form, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, aerosols. The compositions of the present disclosure may be prepared by methods well known in the pharmaceutical arts. For example, sterile injectable solutions can be prepared by incorporating the therapeutic or prophylactic agent in the required amount with various of the other ingredients described above in the appropriate solvent such as sterile distilled water and then filter-sterilizing. Surfactants may also be added to promote the formation of a uniform solution or suspension.
For example, the compositions of the present disclosure may be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In some embodiments, the composition is administered intramuscularly and/or subcutaneously.
The compositions of the present disclosure are administered in therapeutically effective amounts, which may vary not only with the particular agent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and may ultimately be at the discretion of the attendant physician or clinician. For example, a dose of about 0.001mg/kg to about 10mg/kg of the therapeutic and/or prophylactic agent may be administered to a subject (preferably a mammal, such as a human).
Examples
The present disclosure is further described below in connection with the following examples, but the present disclosure is not limited to the following examples. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions which are not noted are conventional conditions in the industry. In the specific examples of the present disclosure, all raw materials used are commercially available. All temperatures are given in degrees celsius unless otherwise indicated. Technical features of the various embodiments of the present disclosure may be combined with each other as long as they do not collide with each other.
In the examples below, the abbreviations represent the following meanings:
NaBH4 sodium borohydride, boc2 O di-tert-butyl dicarbonate, DMAP 4-dimethylaminopyridine, EDCI 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, HATU 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate, DIEA N, N-diisopropylethylamine, DCM dichloromethane, DMF N, N-dimethylformamide, THF tetrahydrofuran, meOH, methanol.
EXAMPLE 1 Synthesis of cationic lipid Compound
1.1 Synthesis of intermediate INT-1
Step 1 Synthesis of INT-1-PM1
(S) -1-amino-3-chloro-2-propanol hydrochloride (10.00 g,68.49 mmol) was dissolved in methylene chloride (150 mL), triethylamine (27.72 g,273.95 mmol) was added, di-tert-butyl dicarbonate (59.75 g,273.95 mmol) was slowly added dropwise, the temperature was raised to 40℃for 24h, and TLC was monitored until the reaction was completed. The heating was stopped, the reaction was quenched by adding saturated aqueous sodium bicarbonate solution, the liquid was separated, the aqueous phase was extracted 2 times with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was dried by spin-drying under reduced pressure, the obtained residue was purified by silica gel column chromatography (0-18% ethyl acetate/n-hexane), and the product was collected and concentrated to give INT-1-PM1 (9.10 g,43.40mmol, 63.4%). C8H16ClNO3,MS(ES):m/z(M+H+) 210.1.
Step 2 Synthesis of INT-1-PM2
INT-1-PM1 (6.00 g,28.62 mmol) was dissolved in acetonitrile (60 mL), followed by the addition of dimethylamine hydrochloride (2.32 g,28.62 mmol), potassium carbonate (11.86 g,85.85 mmol) and potassium iodide (0.95 g,5.72 mmol), followed by a reaction at 70℃for 24h, and TLC monitoring until the starting material was complete. Stopping heating, filtering, concentrating the filtrate under reduced pressure, purifying the residue by silica gel column chromatography (0-12% methanol/dichloromethane), collecting the product, concentrating to obtain INT-1-PM2（5.20g,23.82mmol,83.2%）.C10H22N2O3,MS(ES):m/z(M+H+)219.2.
Step 3 Synthesis of INT-1
INT-1-PM2 (3.00 g,13.74 mmol) and a solution of hydrochloric acid in 1, 4-dioxane (30 mL) were added and reacted at room temperature for 2h, and LCMS was monitored until the starting material was reacted. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in methanol, and then concentrated under reduced pressure, and repeated 2 times to give INT-1 (1.60 g,13.54mmol, 98.5%). C5H14N2O,MS(ES):m/z(M+H+) 119.1.
1.2 Synthesis of intermediate INT-2
Step 1 Synthesis of INT-2-PM1
Using INT-1-PM1 (4.50 g,21.46 mmol) as raw material, and according to the synthesis method of INT-1-PM2 INT-2-PM1（3.00g,10.97mmol,51.1%）.C13H27N3O3,MS(ES):m/z(M+H+)274.2.
Step 2 Synthesis of INT-2
INT-2 (1.90 g,10.97mmol, 99.9%) was obtained by the synthesis of INT-1 starting from INT-2-PM1 (3.00 g,10.97 mmol). C8H19N3O,MS(ES):m/z(M+H+) 174.1.
1.3 Synthesis of intermediate INT-3
Step 1 Synthesis of INT-3-PM1
Using INT-1-PM1 (4.50 g,21.46 mmol) as raw material, and according to the synthesis method of INT-1-PM2 INT-3-PM1（2.50g,10.15mmol,47.3%）.C12H26N2O3,MS(ES):m/z(M+H+)247.2.
Step 2 Synthesis of INT-3
INT-3 (1.20 g,8.21mmol, 91.9%) was obtained by the synthesis of INT-1 starting from INT-3-PM1 (2.20 g,8.93 mmol). C7H18N2O,MS(ES):m/z(M+H+) 147.2.
1.4 Synthesis of intermediate INT-4
2-Hexyldecanol (24.24 g,99.97 mmol) was dissolved in cyclohexane (250 mL), 6-bromohexanoic acid (23.40 g,119.97 mmol) and p-toluenesulfonic acid hydrate (0.28 g,1.49 mmol) were added, a water separator was installed, and the reaction was carried out at 110℃for 7h, and TLC monitoring was carried out until the starting material reaction was completed. The heating was stopped, and the reaction was quenched by adding saturated aqueous sodium bicarbonate solution, separating the solution, extracting the aqueous phase with n-hexane for 1 time, combining the organic phases, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure to give INT-4 (41.42 g,98.74mmol, 98.7%). C22H43BrO2,MS(ES):m/z(M+H+) 419.2.
1.5 Synthesis of intermediate INT-5
INT-5 (47.31 g,153.97mmol, 97.5%) was obtained as a starting material from n-decanol (25.00 g,157.95 mmol) according to the method for the synthesis of INT-4. C14H27BrO2,MS(ES):m/z(M+H+) 307.1.
1.6 Synthesis of intermediate INT-6
Heptadecan-9-ol (5.00 g,19.50 mmol) was dissolved in dichloromethane (60 mL), 6-bromohexanoic acid (4.18 g,21.45 mmol), EDCI (5.61 g,29.24 mmol) and DMAP (0.48 g,3.90 mmol) were added, reacted at room temperature for 16h, and TLC monitoring the reaction until the starting material was complete. The reaction was quenched by adding saturated aqueous sodium bicarbonate, separating the liquid, extracting the aqueous phase with dichloromethane 1 time, combining the organic phases, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure, purifying the resulting residue by column chromatography on silica gel (0-10% ethyl acetate/n-hexane), and concentrating the collected product to give INT-6 (8.00 g,18.45mmol, 94.6%). C23H45BrO2,MS(ES):m/z(M+H+) 433.2.
1.7 Synthesis of intermediate INT-7
Step 1 Synthesis of INT-7-PM1
Using tert-butyl 6-hydroxycaproate (2.00 g,10.62 mmol) as a starting material, INT-7-PM1 (4.40 g,10.31mmol, 97.1%) was obtained according to the INT-6 synthesis method. C26H50O4,MS(ES):m/z(M+H+) 427.3.
Step 2 Synthesis of INT-7
INT-7-PM1 (2.00 g,4.69 mmol) was dissolved in dichloromethane (20 mL) and trifluoroacetic acid (4 mL) was added and reacted at room temperature for 5h, and TLC monitoring was used until the starting material was complete. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in methylene chloride and concentrated under reduced pressure to give INT-7 (1.70 g,4.59mmol, 97.9%). C22H42O4,MS(ES):m/z(M+H+) 371.3.
1.8 Synthesis of intermediate INT-8
Step 1 Synthesis of INT-8-PM1
Using tert-butyl 6-hydroxycaproate (400 mg,2.12 mmol) as a starting material, INT-8-PM1 (750 mg,2.19mmol, 103.0%) was obtained according to the synthesis method of INT-6. C20H38O4,MS(ES):m/z(M+H+) 343.3.
Step 2 Synthesis of INT-8
INT-8 (248 mg,0.85mmol, 97.6%) was obtained by the synthesis of INT-7 starting from INT-8-PM1 (300 mg,0.88 mmol). C16H30O4,MS(ES):m/z(M+H+) 287.2.
1.9 Synthesis of intermediate INT-9
Ind-9 (7.10 g,22.10mmol, 76.4%) was obtained by the synthesis of INT-6 starting from undecanol (5.00 g,29.02 mmol). C15H29BrO2,MS(ES):m/z(M+H+) 321.2.
1.10 Synthesis of YK-1601
Step 1 Synthesis of YK-1601-PM1
INT-1 (300 mg,2.54 mmol) was dissolved in acetonitrile (5 mL), INT-4 (1.06 g,2.54 mmol), potassium carbonate (1.05 g,7.61 mmol) and potassium iodide (42 mg,0.25 mmol) were added sequentially, the temperature was raised to 70℃for 7h, and LCMS was monitored until the starting material was reacted. Stopping heating, filtering, concentrating the filtrate under reduced pressure, purifying the residue by silica gel column chromatography (0-10% methanol/dichloromethane), collecting the product, concentrating to obtain YK-1601-PM1（260mg,0.57mmol,22.4%）.C27H56N2O3,MS(ES):m/z(M+H+)457.5.
Step 2 Synthesis of YK-1601-PM2
YK-1601-PM1 (260 mg,0.57 mmol) was dissolved in acetonitrile (3 mL), then INT-5 (175 mg,0.57 mmol), potassium carbonate (236 mg,1.71 mmol) and potassium iodide (9 mg,0.06 mmol) were added sequentially, the temperature was raised to 70℃for 8h, and LCMS was monitored until the starting material was reacted. Stopping heating, filtering, concentrating the filtrate under reduced pressure, purifying the residue by silica gel column chromatography (0-10% methanol/dichloromethane), collecting the product, concentrating to obtain YK-1601-PM2（90mg,0.13mmol,23.1%）.C41H82N2O5,MS(ES):m/z(M+H+)683.6.
Step 3 Synthesis of YK-1601
YK-1601-PM2 (90 mg,0.13 mmol) was dissolved in dichloromethane (2 mL), then INT-7 (49 mg,0.13 mmol), EDCI (38 mg,0.20 mmol) and DMAP (2 mg,0.01 mmol) were added, reacted for 15h at room temperature and TLC monitored until the starting material was complete. Adding saturated sodium bicarbonate water solution into the reaction system, quenching, separating, extracting water phase with dichloromethane for 1 time, mixing organic phases, drying with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure, purifying the residue by silica gel column chromatography (0-8% methanol/dichloromethane), collecting the product, concentrating to obtain the final product YK-1601（90mg,0.08mmol,65.9%）.C63H122N2O8,MS(ES):m/z(M+H+)1036.1.1H NMR (400 MHz, CDCl3) δ 5.09 (dd,J= 11.9, 6.0 Hz, 1H), 4.08 (q,J= 6.6 Hz, 4H), 3.99 (d,J= 5.7 Hz, 2H), 2.61 – 2.40 (m, 12H), 1.79 – 1.56 (m, 14H), 1.50 – 1.26 (m, 74H), 0.91 (t,J= 6.8 Hz, 15H).
1.11 Synthesis of YK-1602
Step 1 Synthesis of YK-1602-PM1
INT-1 (240 mg,2.03 mmol) was used as the starting material, and was synthesized according to the method of YK-1601-PM1 YK-1602-PM1（270mg,0.57mmol,28.2%）.C28H58N2O3,MS(ES):m/z(M+H+)471.5.
Step 2 Synthesis of YK-1602-PM2
The YK-1602-PM1 (270 mg,0.57 mmol) is used as raw material, and is obtained according to the synthesis method of YK-1601-PM2 YK-1602-PM2（90mg,0.13mmol,22.1%）.C43H86N2O5,MS(ES):m/z(M+H+)711.6.
Step 3 Synthesis of YK-1602
Using YK-1602-PM2 (90 mg,0.13 mmol) as raw material, the preparation method of YK-1601 is followed YK-1602（25mg,0.02mmol,18.6%）.C65H126N2O8,MS(ES):m/z(M+H+)1064.1.1H NMR (400 MHz, CDCl3) δ 4.22 (t,J= 5.1 Hz, 1H), 4.06 (d,J= 7.7 Hz, 4H), 3.52 (d,J= 5.4 Hz, 1H), 2.44 (s, 2H), 2.38 – 2.21 (m, 10H), 2.00 (s, 2H), 1.68 – 1.58 (m, 10H), 1.48 (d,J= 12.5 Hz, 8H), 1.45 – 1.37 (m, 7H), 1.28 (d,J= 15.7 Hz, 58H), 1.17 (d,J= 10.0 Hz, 2H), 0.92 – 0.84 (t, 15H).
1.12 Synthesis of YK-1603
Step 1 Synthesis of YK-1603-PM1
Using INT-1 (141 mg,1.19 mmol) as a starting material, YK-1603-PM1 (82 mg,0.13mmol, 11.1%) was obtained according to the method for synthesizing YK-1601-PM 1. C41H82N2O, MS(ES):m/z(M+H+) 619.6.
Step 2 Synthesis of YK-1603
Using YK-1603-PM1 (82 mg,0.13 mmol) as raw material, the preparation method of YK-1601 is followed YK-1603（74mg,0.07mmol,56.8%）.C63H122N2O4,MS(ES):m/z(M+H+)972.1.1H NMR (400 MHz, CDCl3) δ 5.36 – 5.32 (m, 4H), 4.08 – 4.28 (dd,J= 12.9, 6.4 Hz, 3H), 2.48 – 2.41 (m, 3H), 2.33 – 2.22 (m, 9H), 2.09 – 1.93 (m, 7H), 1.72 – 1.52 (m, 8H), 1.46 – 1.37 (m, 8H), 1.34 – 1.25 (m, 63H), 0.89 – 0.85 (t, 12H).
1.13 Synthesis of YK-1604
Step 1 Synthesis of YK-1604-PM1
Using INT-1 (141 mg,1.19 mmol) as a starting material, YK-1604-PM1 (102 mg,0.16mmol, 13.9%) was obtained according to the method for synthesizing YK-1601-PM 1. C41H78N2O,MS(ES):m/z(M+H+) 615.6.
Step 2 Synthesis of YK-1604
Using YK-1604-PM1 (102 mg,0.16 mmol) as raw material, the preparation method of YK-1601 is followed YK-1604（55mg,0.06mmol,34.3%）.C63H118N2O4,MS(ES):m/z(M+H+)968.1.1H NMR (400 MHz, CDCl3) δ 4.11 – 4.03 (m, 4H), 2.40 – 2.30 (m, 6H), 2.30 – 2.20 (m, 3H), 1.75 – 1.51 (m, 20H), 1.43 (ddd,J= 15.8, 10.9, 6.1 Hz, 11H), 1.30 (dd,J= 14.7, 7.4 Hz, 56H), 1.25 (s, 6H), 0.91 – 0.84 (t, 12H).
1.14 Synthesis of YK-1605
Step 1 Synthesis of YK-1605-PM1
INT-2 (317 mg,1.83 mmol) was used as the starting material, and was synthesized according to the method of YK-1601-PM1 YK-1605-PM1（294mg,0.57mmol,31.4%）.C30H61N3O3,MS(ES):m/z(M+H+)512.5.
Step 2 Synthesis of YK-1605PM2
The YK-1605-PM1 (254 mg,0.57 mmol) is used as a raw material, and is obtained according to the synthesis method of YK-1601-PM2 YK-1605-PM2（160mg,0.22mmol,37.7%）.C44H87N3O5, MS(ES):m/z(M+H+)738.6.
Step 3 Synthesis of YK-1605
Using YK-1605-PM2 (160 mg,0.22 mmol) as raw material, the preparation method of YK-1601 is followed YK-1605（51mg,0.05mmol,21.6%）.C66H127N3O8,MS(ES):m/z(M+H+)1091.1.1H NMR (400 MHz, CDCl3) δ 5.05 – 5.02 (m, 1H), 4.09 – 4.03 (m, 4H), 3.96 (d,J= 5.7 Hz, 2H), 2.48 (tdd,J= 18.1, 12.2, 6.0 Hz, 7H), 2.34 – 2.14 (m, 12H), 1.70 (ddd,J= 15.0, 11.4, 5.2 Hz, 9H), 1.67 – 1.57 (m, 5H), 1.46 – 1.37 (m, 9H), 1.33 – 1.25 (m, 62H), 0.90 – 0.86 (t, 15H).
1.15 Synthesis of YK-1606
Step 1 Synthesis of YK-1606-PM1
INT-2 (380 mg,2.20 mmol) was used as the starting material, and was synthesized according to the method of YK-1601-PM1 YK-1606-PM1（360mg,0.68mmol,31.2%）.C31H63N3O3,MS(ES):m/z(M+H+)526.5.
Step 2 Synthesis of YK-1606-PM2
The YK-1606-PM1 (360 mg,0.68 mmol) is used as a raw material, and is obtained according to the synthesis method of YK-1601-PM2 YK-1606-PM2（210mg,0.27mmol,40.0%）.C46H91N3O5,MS(ES):m/z(M+H+)766.7.
Step 3 Synthesis of YK-1606
Using YK-1606-PM2 (90 mg,0.12 mmol) as raw material, the preparation method of YK-1601 is followed YK-1606（103mg,0.09mmol,78.4%）.C68H131N3O8,MS(ES):m/z(M+H+)1119.1.1H NMR (400 MHz, CDCl3) δ 5.19 – 4.99 (m, 1H), 4.89 (p,J= 6.3 Hz, 1H), 4.09 (q,J= 6.8 Hz, 4H), 2.67 (s, 2H), 2.59 – 2.44 (m, 10H), 2.37 – 2.27 (m, 8H), 1.80 – 1.62 (m, 11H), 1.57 – 1.39 (m, 11H), 1.35 (dd,J= 7.8, 3.6 Hz, 12H), 1.30 (s, 48H), 0.92 (t,J= 6.7 Hz, 15H).
1.16 Synthesis of YK-1607
Using YK-1606-PM2 (90 mg,0.12 mmol) as raw material, the preparation method of YK-1601 is followed YK-1607（105mg,0.10mmol,89.0%）.C62H121N3O6,MS(ES):m/z(M+H+)1005.1.1H NMR (400 MHz, CDCl3) δ 5.05 (dd,J= 11.9, 6.1 Hz, 1H), 4.86 (dd,J= 12.5, 6.3 Hz, 1H), 4.06 (d,J= 6.8 Hz, 2H), 2.60 (s, 2H), 2.50 (d,J= 7.1 Hz, 4H), 2.41 (d,J= 6.1 Hz, 5H), 2.34 – 2.21 (m, 6H), 1.66 – 1.54 (m, 7H), 1.53 – 1.46 (m, 5H), 1.45 – 1.37 (m, 5H), 1.34 – 1.23 (m, 66H), 0.92 – 0.84 (t, 15H).
1.17 Synthesis of YK-1608
Step 1 Synthesis of YK-1608-PM1
Using INT-2 (126 mg,0.73 mmol) as the starting material, YK-1608-PM1 (140 mg,0.21mmol, 28.6%) was obtained according to the method for synthesizing YK-1601-PM 1. C44H87N3O,MS(ES):m/z(M+H+) 674.7.
Step 2 Synthesis of YK-1608
Using YK-1608-PM1 (70 mg,0.10 mmol) as raw material, the preparation method of YK-1601 is followed YK-1608（53mg,0.05mmol,49.7%）.C66H127N3O4,MS(ES):m/z(M+H+)1027.1.1H NMR (400 MHz, CDCl3) δ 5.38 (m, 4H), 5.07 (d,J= 5.8 Hz, 1H), 4.05 (t, 2H), 2.57 – 2.36 (m, 12H), 2.03 (d,J= 5.8 Hz, 7H), 1.72 – 1.52 (m, 7H), 1.46 – 1.35 (m, 10H), 1.34 – 1.18 (m, 69H), 0.92 – 0.84 (t, 12H).
1.18 Synthesis of YK-1609
Using YK-1608-PM1 (70 mg,0.10 mmol) as raw material, the preparation method of YK-1601 is followed YK-1609（33mg,0.04mmol,34.8%）.C60H117N3O2,MS(ES):m/z(M+H+)913.1.1H NMR (400 MHz, CDCl3) δ 5.36 (dt,J= 9.9, 4.3 Hz, 3H), 5.15 – 5.04 (m, 1H), 2.56 – 2.36 (m, 11H), 2.29 (d,J= 6.5 Hz, 3H), 2.06 – 2.00 (m, 5H), 2.00 – 1.92 (m, 2H), 1.69 – 1.52 (m, 4H), 1.44 (d,J= 8.4 Hz, 2H), 1.38 (dd,J= 15.8, 8.0 Hz, 8H), 1.34 – 1.25 (m, 60H), 1.25 – 1.23 (m, 3H), 0.92 – 0.81 (s, 12H).
1.19 Synthesis of YK-1610
Step 1 Synthesis of YK-1610-PM1
Using INT-2 (126 mg,0.73 mmol) as a starting material, YK-1610-PM1 (150 mg,0.22mmol, 30.8%) was obtained according to the method for synthesizing YK-1601-PM 1. C44H83N3O,MS(ES):m/z(M+H+) 670.7.
Step 2 Synthesis of YK-1610
Using YK-1610-PM1 (70 mg,0.10 mmol) as raw material, the preparation method of YK-1601 is followed YK-1610（48mg,0.05mmol,44.9%）.C66H123N3O4,MS(ES):m/z(M+H+)1023.1.1H NMR (400 MHz, CDCl3) δ 5.44 – 5.27 (m, 8H), 4.10 – 4.02 (t, 2H), 2.81 – 2.73 (t, 4H), 2.53 (dd,J= 13.1, 4.5 Hz, 5H), 2.50 – 2.38 (m, 8H), 2.35 – 2.25 (m, 6H), 2.10 – 2.00 (m, 8H), 1.71 – 1.60 (m, 5H), 1.60 – 1.51 (m, 2H), 1.47 – 1.33 (m, 15H), 1.33 – 1.23 (m, 47H), 0.93 – 0.84 (m, 12H).
1.20 Synthesis of YK-1611
Using YK-1610-PM1 (70 mg,0.10 mmol) as raw material, the preparation method of YK-1601 is followed YK-1611（50mg,0.05mmol,52.7%）.C60H113N3O2,MS(ES):m/z(M+H+)909.1.1H NMR (400 MHz, CDCl3) δ 5.45 – 5.32 (m, 8H), 2.84 – 2.73 (m, 7H), 2.63 – 2.53 (m, 6H), 2.29 (ddd,J= 25.1, 14.3, 5.7 Hz, 3H), 2.08 (q,J= 7.0 Hz, 8H), 1.61 (dd,J= 15.0, 7.9 Hz, 4H), 1.48 (d,J= 7.5 Hz, 3H), 1.43 – 1.27 (m, 59H), 0.92 (dd,J= 11.5, 6.0 Hz, 12H).
1.21 Synthesis of YK-1612
Step 1 Synthesis of YK-1612-PM1
INT-1-PM2 (600 mg,2.19 mmol) was used as the starting material, and was synthesized according to the method of YK-1601 YK-1612-PM1（840mg,1.64mmol,74.8%）.C29H57N3O4,MS(ES):m/z(M+H+)512.4.
Step 2 Synthesis of YK-1612-PM2
Using YK-1612-PM1 (120 mg,0.23 mmol) as raw material, and according to the synthesis method of INT-1 YK-1612-PM2（95mg,0.23mmol,98.4%）.C24H49N3O2,MS(ES):m/z(M+H+)412.4.
Step 3 Synthesis of YK-1612
Using YK-1612-PM2 (95 mg,0.23 mmol) as raw material, the preparation method of YK-1601 was followed YK-1612（55mg,0.07mmol,31.2%）.C46H89N3O5,MS(ES):m/z(M+H+)764.7.1H NMR (400 MHz, CDCl3) δ 6.12 (d,J= 5.8 Hz, 1H), 5.04 (t,J= 5.8 Hz, 1H), 4.06 (t,J= 6.7 Hz, 2H), 3.58 – 3.39 (m, 2H), 2.71 – 2.55 (m, 5H), 2.55 – 2.43 (m, 4H), 2.31 (p,J= 5.4 Hz, 2H), 2.16 (t, 2H), 1.65 (ddd,J= 53.1, 30.1, 23.9 Hz, 8H), 1.49 – 1.34 (m, 7H), 1.32 – 1.17 (m, 43H), 0.92 – 0.82 (t, 12H).
1.22 Synthesis of YK-1613
Step 1 Synthesis of YK-1613-PM1
INT-3 (500 mg,3.42 mmol) was used as the starting material, and was synthesized according to the method of YK-1601-PM1 YK-1613-PM1（420mg,0.87mmol,25.3%）.C29H60N2O3,MS(ES):m/z(M+H+)485.5.
Step 2 Synthesis of YK-1613-PM2
Using YK-1613-PM1 (420 mg,0.87 mmol) as raw material, the method for synthesizing YK-1601-PM2 is performed YK-1613-PM2（420mg,0.59mmol,68.2%）.C43H86N2O5,MS(ES):m/z(M+H+)711.6.
Step 3 Synthesis of YK-1613
Using YK-1613-PM2 (80 mg,0.11 mmol) as raw material, the preparation method of YK-1601 was followed YK-1613（50mg,0.05mmol,41.8%）.C65H126N2O8,MS(ES):m/z(M+H+)1064.1.1H NMR (400 MHz, CDCl3) δ 4.10 – 4.01 (m, 4H), 3.96 (d,J= 5.8 Hz, 2H), 2.60 – 2.38 (m, 11H), 2.34 – 2.25 (m, 7H), 1.71 – 1.53 (m, 12H), 1.47 – 1.35 (m, 7H), 1.33 – 1.23 (m, 59H), 1.03 – 0.95 (t, 6H), 0.92 – 0.81 (t, 15H).
1.23 Synthesis of YK-1614
Step 1 Synthesis of YK-1614-PM1
INT-3 (100 mg,0.68 mmol) was used as the starting material, and was synthesized according to the method of YK-1601-PM1 YK-1614-PM1（120mg,0.15mmol,21.3%）.C51H102N2O5,MS(ES):m/z(M+H+)823.8.
Step 2 Synthesis of YK-1614
Using YK-1614-PM1 (120 mg,0.15 mmol) as raw material, according to the method of YK-1601 YK-1614（100mg,0.10mmol,70.2%）.C61H120N2O6,MS(ES):m/z(M+H+)978.1.1H NMR (400 MHz, CDCl3) δ 4.08 – 4.00 (m, 3H), 3.96 (d,J= 5.7 Hz, 3H), 3.63 – 3.28 (m, 3H), 2.55 (s, 10H), 2.35 – 2.25 (m, 7H), 1.71 – 1.55 (m, 12H), 1.29 (dd,J= 14.5, 7.3 Hz, 60H), 1.06 – 0.98 (m, 6H), 0.92 – 0.84 (t, 15H).
1.24 Synthesis of YK-1615
Step 1 Synthesis of YK-1615-PM1
INT-3 (300 mg,2.05 mmol) was used as the starting material, and was synthesized according to the method of YK-1601-PM1 YK-1615-PM1（200mg,0.40mmol,19.5%）.C30H62N2O3,MS(ES):m/z(M+H+)499.5.
Step 2 Synthesis of YK-1615-PM2
The YK-1615-PM1 (200 mg,0.40 mmol) is used as a raw material, and is obtained according to the synthesis method of YK-1601-PM2 YK-1615-PM2（156mg,0.21mmol,52.6%）.C45H90N2O5,MS(ES):m/z(M+H+)739.7.
Step 3 Synthesis of YK-1615
Using YK-1615-PM2 (80 mg,0.11 mmol) as raw material, the preparation method of YK-1601 was followed YK-1615（100mg,0.09mmol,84.6%）.C67H130N2O8,MS(ES):m/z(M+H+)1092.1.1H NMR (400 MHz, CDCl3) δ 5.01 (d,J= 6.0 Hz, 1H), 4.88 (d,J= 6.2 Hz, 1H), 4.10 – 4.01 (m, 4H), 2.60 – 2.38 (m, 11H), 2.36 – 2.23 (m, 7H), 1.78 – 1.67 (m, 3H), 1.67 – 1.61 (m, 7H), 1.60 (d,J= 4.0 Hz, 2H), 1.50 (dd,J= 12.4, 6.3 Hz, 4H), 1.47 – 1.35 (m, 8H), 1.33 – 1.28 (m, 60H), 1.03 – 0.95 (t, 6H), 0.92 – 0.84 (t, 15H).
1.25 Synthesis of YK-1616
Step 1 Synthesis of YK-1616-PM1
Using INT-3 (118 mg,0.81 mmol) as a starting material, YK-1616-PM1 (238 mg,0.37mmol, 45.6%) was obtained according to the method for synthesizing YK-1601-PM 1. C43H86N2O,MS(ES):m/z(M+H+) 647.7.
Step 2 Synthesis of YK-1616
Using YK-1616-PM1 (80 mg,0.12 mmol) as raw material, the preparation method of YK-1601 is followed YK-1616（45mg,0.05mmol,36.4%）.C65H126N2O4,MS(ES):m/z(M+H+)1000.1.1H NMR (400 MHz, CDCl3) δ 5.41 – 5.31 (m, 3H), 4.10 – 4.02 (m, 3H), 2.55 – 2.47 (m, 5H), 2.40 (d,J= 7.3 Hz, 3H), 2.35 – 2.24 (m, 3H), 2.05 (d,J= 4.9 Hz, 4H), 2.01 (dd,J= 12.5, 6.4 Hz, 6H), 1.70 – 1.60 (m, 6H), 1.60 – 1.51 (m, 2H), 1.47 – 1.35 (m, 10H), 1.32 – 1.23 (m, 60H), 1.03 – 0.95 (m, 6H), 0.92 – 0.84 (t, 12H).
1.26 Synthesis of YK-1617
Step 1 Synthesis of YK-1617-PM1
Using INT-3 (120 mg,0.81 mmol) as the starting material, YK-1617-PM1 (200 mg,0.31mmol, 37.9%) was obtained according to the method for synthesizing YK-1601-PM 1. C43H82N2O,MS(ES):m/z(M+H+) 643.7.
Step 2 Synthesis of YK-1617
Using YK-1617-PM1 (80 mg,0.12 mmol) as raw material, the preparation method of YK-1601 is followed YK-1617（52mg,0.05mmol,42.0%）.C65H122N2O4,MS(ES):m/z(M+H+)996.1.1H NMR (400 MHz, CDCl3) δ 5.41 – 5.27 (m, 8H), 4.17 – 3.85 (m, 2H), 2.81 – 2.73 (m, 4H), 2.47 (d,J= 49.9 Hz, 9H), 2.35 – 2.21 (m, 2H), 2.05 (dd,J= 13.8, 6.9 Hz, 8H), 1.71 – 1.51 (m, 8H), 1.49 – 1.36 (m, 10H), 1.33 (dd,J= 14.4, 7.6 Hz, 14H), 1.28 (dd,J= 9.9, 7.1 Hz, 39H), 1.02 (d,J= 7.3 Hz, 6H), 0.88 (dd,J= 13.0, 6.5 Hz, 12H).
1.27 Synthesis of YK-1618
Using YK-1613-PM2 (74 mg,0.10 mmol) as raw material, the preparation method of YK-1601 is followed YK-1618（100mg,0.10mmol,98.1%）.C59H114N2O8,MS(ES):m/z(M+H+)980.1.1H NMR (400 MHz, CDCl3) δ 4.09 – 4.01 (t, 4H), 3.96 (d,J= 5.8 Hz, 2H), 2.60 – 2.47 (m, 7H), 2.43 (dd,J= 17.2, 8.2 Hz, 4H), 2.34 – 2.26 (m, 8H), 1.73 – 1.55 (m, 14H), 1.45 – 1.37 (m, 4H), 1.29 (m, 53H), 1.03 – 0.95 (t, 6H), 0.92 – 0.84 (t, 12H).
1.28 Synthesis of lipid-028
According to In vivo genome editing of human hematopoietic STEM CELLS for treatment of blood
Lipid by MRNA DELIVERY, saijuan Xu et al, bioRxiv preprint, doi: https:// doi.org/10.1101/2024.10.28.620445 Lipid-028 (Lipid-028) was synthesized to give Lipid-028 (Lipid-028) of 76 mg.
1.29 Synthesis of E10-1
According to the E10-1 synthesis method in CN202380014467.9, E10-1 of 43 mg is synthesized.
1.30 Synthesis of E24-1
According to the E24-1 synthesis method in CN202380014467.9, E24-1 of 55 mg is synthesized.
1.31 Synthesis of E7-1
According to the method of E7-1 synthesis in PCT/CN2023/098155, E7-1 of 36 mg is synthesized.
1.32 Synthesis of Compound 92-12
According to the synthesis method of compound 92-12 in PCT/CN2023/098155, compound 92-12 of 23 mg is synthesized.
1.33 Synthesis of Compound 5
Compound 5 of 107 mg was synthesized according to the method for synthesizing compound 5 in CN 202410487810.8.
1.34 Synthesis of Compound 9
Compound 9 of 45 mg was synthesized according to the method for synthesizing compound 9 in CN 202410487810.8.
1.35 Synthesis of control 1
The procedure for the synthesis of YK-1606 in example 1 was followed, except that the starting material for INT-1 was preparedReplaced byControl 1 of 110 mg was synthesized.
Example 2 mRNA-LNP formulation optimization
2.1 Vector (liposome) and mRNA ratio optimization
Step 1, according to the mole ratio of the cationic lipid to DSPC to cholesterol to DMG-PEG2000 of 49:10:39.5:1.5, the cationic lipid YK-1602 or YK-1605 synthesized in example 1 was dissolved in ethanol with DSPC (Ai Weita (Shanghai) medical science and technology Co., ltd.), cholesterol (Ai Weita (Shanghai) medical science and technology Co., ltd.) and DMG-PEG2000, respectively, to obtain a solution A. Solution a was added rapidly to citrate buffer (ph=4.5±0.5) by ethanol injection and vortexed for 30s to obtain an ethanol lipid solution.
Step 2. EGFP-mRNA (Shanghai laboratory Start. Reagent Co., ltd.) was diluted in citrate buffer (pH=4.5.+ -. 0.5) to give an aqueous eGFP-mRNA solution.
And 3, mixing the ethanol lipid solution prepared in the step 1 with the mRNA aqueous solution prepared in the step 2 at a flow rate of 10mL/min by using a microfluidic device according to the mass ratio of the carrier to the mRNA of 5:1, 10:1, 15:1, 20:1, 30:1 and 35:1 to prepare a corresponding liposome solution. After dilution of the liposome solution to 10 volumes using PBS, ultrafiltration was performed using a 300 KDa ultrafiltration tube to remove ethanol. The mixture was then subjected to PBS to a proper volume and filtered through a 0.2 μm sterile filter to obtain mRNA-LNP preparations encapsulating eGFP-mRNA with a molar ratio of cationic lipid (YK-1602 or YK-1605)/DSPC/cholesterol/DMG-PEG 2000 of 49:10:39.5:1.5, respectively.
The results of cell transfection experiments show that when the mass ratio of the vector to the mRNA is in the range of 10:1-30:1, the corresponding mRNA-LNP composition has a good transfection effect, wherein the transfection effect is preferably 15:1, and when the mass ratio is 5:1 and 35:1, the proper transfection effect cannot be obtained.
2.2 Cationic lipid and neutral lipid ratio optimization
MRNA-LNP compositions encapsulating eGFP-mRNA were prepared in analogy to the procedure in 2.1, with the molar ratios of cationic lipids (YK-1602 or YK-1605) to neutral lipid DSPC adjusted to 1:1, 3:1, 3.5:1, 4:1, 4.9:1, 10:1, 15:1 and 20:1, respectively.
Cell transfection experiments show that when the molar ratio of cationic lipid to neutral lipid is in the range of 1:1-15:1, the corresponding mRNA-LNP composition is capable of transfecting cells, wherein the transfection efficiency is 4.9:1 at the highest.
2.3 Optimization of carrier proportion of polymer conjugated lipid
MRNA-LNP compositions encapsulating eGFP-mRNA were prepared in analogy to the procedure in 2.1, wherein the cationic lipids were YK-1602, YK-1603, YK-1604 or YK-1607 and the polymer conjugated lipids DMG-PEG2000 were present in the carrier in mole percentages of 0.5%, 1.5%, 2.5%, 3.5%, 5%, 10% and 15%, respectively.
Cell transfection experiment results show that when the mole percentage of the polymer conjugated lipid in the carrier is in the range of 0.5% -10%, the corresponding mRNA-LNP composition can be used for transfecting cells, and the transfection efficiency is highest at 1.5% and lowest at 10%.
2.4 Optimization of the ratio of the components in the carrier
MRNA-LNP preparations encapsulating eGFP-mRNA were prepared in analogy to the procedure in 2.1, with cationic lipid (YK-1602 or YK-1605), neutral lipid DSPC, structured lipid cholesterol and polymer conjugated lipid DMG-PEG2000 molar ratios of 75:5:15:5, 65:8:25:2, 49:10:39.5:1.5, 45:10:43.5:1.5, 45:25:20:10, 40:10:48.5:1.5, 35:10:53.5:1.5 and 25:5:65:5, respectively, in step 1.
As can be seen from cell transfection experiments, the cationic lipid, neutral lipid, structural lipid and polymer conjugated lipid have good transfection effect in the range of the ratio of (35-49): (7.5-15): (35-55): (1-5) in the molar ratio of (75:5:15:5), 65:8:25:2, 49:10:39.5:1.5, 45:10:43.5:1.5, 45:25:20:10, 40:10:48.5:1.5, 35:10:53.5:1.5 and 25:5:65:5, and the transfection effect is the best when the molar ratio is 49:10:39.5:1.5.
Example 3 cell transfection of mRNA-LNP preparation encapsulating eGFP-mRNA
And 1, cell resuscitating and passaging, namely resuscitating HEK293T cells, and culturing and passaging the HEK293T cells to the required cell number in a culture dish.
Step 2, plating, namely digesting and counting cells in a culture dish, plating 1 ten thousand cells per hole in a 96-well plate, and culturing overnight until the cells adhere to the wall.
Step 3 cell transfection the transfection efficiency was examined by fluorescence intensity after adding 1.5. Mu.g of the mRNA-LNP preparation prepared in example 2 and encapsulating eGFP-mRNA, wherein the cationic lipid was YK-1602 or YK-1605, respectively, to the cell culture broth of a 96-well plate and further culturing for 24 hours.
Based on transfection efficiency results, an mRNA-LNP preparation with a carrier to mRNA mass ratio of 15:1, a cationic lipid to neutral lipid molar ratio of 4.9:1, a polymer conjugated lipid molar ratio of 1.5% to liposomes, and a cationic lipid, neutral lipid, structural lipid and polymer conjugated lipid molar ratio of 49:10:39.5:1.5 was selected for the examples below.
EXAMPLE 4 preparation of mRNA-LNP preparation
TABLE 1 cationic lipid Compounds
4.1 The corresponding ethanolic lipid solutions of the cationic lipids in table 1 were prepared according to the method in step 1 of example 2.1.
4.2 EGFP-mRNA (Shanghai Start-Up reagent Co., ltd.) or Fluc-mRNA (Shanghai Start-Up reagent Co., ltd.) was diluted in citrate buffer (pH=4.5.+ -. 0.5) to give a corresponding aqueous mRNA solution.
4.3 The ethanol lipid solution obtained in 4.1 was mixed with the eGFP-mRNA aqueous solution or Fluc mRNA aqueous solution obtained in 4.2 at a volume ratio of 1:3 using a microfluidic device at a flow rate of 10 mL/min, and the corresponding liposome solution was prepared at a mass ratio of carrier (liposome) to mRNA of about 15:1. After dilution of the liposome solution to 10 volumes using PBS, ultrafiltration was performed using a 300 KDa ultrafiltration tube to remove ethanol. The mixture was then sized to a suitable volume with PBS and filtered through a 0.2 μm sterile filter to give a cationic lipid, DSPC, cholesterol, DMG-PEG2000, at a molar ratio of 49:10:39.5:1.5, respectively, as an mRNA-LNP preparation (in the form of a dispersion, wherein the mRNA content is about 0.1 mg/mL) encapsulating eGFP-mRNA or Fluc-mRNA.
Example 5 determination of mRNA-LNP particle size, polydispersity index (PDI) and encapsulation efficiency
Particle size and Polydispersity (PDI) were determined using dynamic light scattering using a malvern laser particle sizer.
25. Mu.L of the mRNA-LNP preparation prepared in example 4 was diluted to 125. Mu.L with RNase-free deionized water, and the mixture was added to a sample well, and the measurement was repeated 3 times for each sample, and the average value was taken as a measurement result. The assay conditions were 90℃scattering angle, 25℃and the LNP encapsulation efficiency was determined using Quant itRibogreenRNA quantitative assay kit (ThermoFisherScientific, UK) according to the manufacturer's instructions. The test results are shown in Table 2:
TABLE 2 mRNA particle size, polydispersity (PDI), encapsulation efficiency of LNP
As can be seen from Table 2, the nanolipid particles prepared in example 4, having a particle size between 80 and 110 nm, can be used to deliver mRNA. The polydispersity numbers were all less than 0.15, indicating good particle size uniformity. And has higher encapsulation efficiency, and the encapsulation efficiency is more than 90 percent.
Example 6 in vitro delivery Performance and toxicity of LNP
Methods of cell resuscitating and passaging and plating refer to step 1 and step 2 of example 3.
After supplementing a 96-well plate obtained in the step 2 and cultured with HEK293T cells with a proper volume of HEK293T cell culture solution and adding an mRNA-LNP preparation (prepared in example 4) containing 0.3. Mu.g of Fluc-mRNA to the 96-well plate and continuing to culture for 24h, adding a corresponding reagent according to the specification of Gaussia Luciferase Assay Kit, detecting the relative fluorescence intensity of each well by an IVIS fluorescence detection system, and finally adding 10. Mu.L of CCK-8 solution to each well of the above-mentioned 24-hour-cultured well plate, incubating the culture plate in an incubator for 1 hour, and measuring the absorbance at 450 nm by an enzyme-labeled instrument, thereby detecting the cell survival rate. The results of relative fluorescence intensity and cell viability are shown in Table 3 and FIGS. 1-2.
TABLE 3 fluorescent detection results of Fluc-mRNA
The relative fluorescence intensities (corresponding to translation efficiencies of mRNA) of the above mRNA-LNP compositions were significantly different, and the relative fluorescence intensities of the mRNA-LNP compositions prepared from YK-1603, YK-1604, YK-1605, YK-1606, YK-1608, YK-1610, YK-1612 and YK-1614 were significantly higher than those of the mRNA-LNP compositions prepared from SM-102, MC3, lipid-028, E10-1, E24-1, E7-1, compound 92-12, compound 5, compound 9 and control 1, specifically:
1. the cell transfection efficiency of the mRNA-LNP composition prepared by YK-1603, YK-1604, YK-1605, YK-1606, YK-1608, YK-1610, YK-1612 and YK-1614 is obviously improved compared with that of the representative cationic lipid in the prior art. For example, YK-1606 can be transfected with 1.37 times as high as SM-102 and 2.85 times as high as MC 3.
2. The transfection efficiency of the mRNA-LNP composition cells prepared by YK-1603 and YK-1604 is remarkably improved compared with E10-1, E7-1, compound 5 and compound 9 cationic lipid which also have dimethylamino head structures. For example, YK-1603 has cell transfection efficiencies of up to 1.88, 2.47, 1.56 and 1.77 times that of E10-1, E7-1, compound 5 and Compound 9, respectively.
3. The cell transfection efficiency of the mRNA-LNP composition prepared by YK-1605, YK-1606, YK-1608, YK-1610 and YK-1612 is remarkably improved compared with E24-1 and compound 92-12 cationic lipid which also have 4-methylpiperazine head structure. For example, YK-1606 has cell transfection efficiencies of 2.49 and 3.19 times that of E24-1 and 92-12, respectively.
4. The cell transfection efficiency of the mRNA-LNP composition prepared by YK-1614 was significantly improved compared to the lipid-028 cationic lipid which also had a diethylamino head structure. For example, YK-1614 can be transfected up to 1.80 times more efficiently than lipid-028.
5. Compared with other positions with the same structure, the cell transfection efficiency of the mRNA-LNP composition prepared by YK-1606 is obviously improved by only using the cationic lipid of the control 1 with different chiralities. For example, YK-1606 can achieve 2.58 times the cell transfection efficiency of control 1.
Example 7 in vivo delivery Performance of LNP
The Fluc-mRNA-LNP composition prepared in example 4 was intramuscular injected into female BALB/c albino mice of age 4-6 weeks and weight 17-19 g (amount of Fluc-mRNA/mouse is about 5 μg), and a fluorogenic substrate was injected into the mice by intraperitoneal injection 6 hours after administration, the mice were free to move for 5 minutes, and then the total radiation intensity (corresponding to the fluorescent protein expression intensity, i.e., protein expression amount) of the protein expressed in the mice by mRNA carried by the mRNA-LNP composition was detected by IVIS Spectrum small animal in vivo imager. After the sampling is completed, the mice are sacrificed and dissected, and the internal organs of the mice, such as heart, liver, spleen, lung and kidney, are precisely isolated. The total radiation intensity (corresponding to the fluorescent protein expression intensity, i.e., protein expression level) of the Fluc-mRNA expressed in the mouse injection site and in each organ was examined by IVIS Spectrum small animal in vivo imaging. From the test results, the relative fluorescence intensities of heart, liver, spleen, lung, kidney with respect to the injection site were calculated (formula reference formula 1). The results are shown in Table 4 and FIG. 3.
Relative fluorescence intensity with respect to injection site = total radiation intensity of protein expressed in organ/total radiation intensity of protein expressed at injection site—formula 1
TABLE 4 Experimental data on imaging of mice living and organs
The fluorescence imaging of mice living and heart, liver, spleen, lung and kidney after intramuscular injection of the Fluc-mRNA encapsulated mRNA-LNP composition prepared based on YK-1605 and YK-1606 to 6 h in mice is shown schematically in fig. 4, where it can be seen that there is very strong fluorescence at the mouse injection site, whereas no apparent fluorescence is found in organs such as heart, liver, spleen, lung and kidney.
MRNA-LNP compositions prepared from the cationic lipid compounds of the present disclosure can target mRNA to muscle with high efficiency, with significantly enhanced delivery and targeting compared to SM-102, MC3, lipid-028, E10-1, E24-1, E7-1, compound 92-12, compound 5, compound 9, and control 1. Specifically:
1. Except at the injection site, the mRNA-LNP composition prepared from the compound of the present disclosure was not significantly expressed in heart, liver, spleen, lung and kidney, whereas the mRNA-LNP composition prepared from SM-102, MC3, lipid-028, E10-1, E24-1, E7-1, compound 92-12, compound 5, compound 9 was significantly expressed in organs, especially liver and spleen, for example, the mRNA-LNP composition prepared from YK-1606, and the total radiation intensity at the liver site was 3%, 5%, 11%, 12%, 13%, 20%, 50%, 40% of the total radiation intensity at the liver site of mice injected with the mRNA-LNP compositions prepared from SM-102, MC3, lipid-028, E10-1, E24-1, E7-1 and compound 92-12, compound 5 and compound 9, respectively. After injection of the mRNA-LNP compositions prepared from the compounds of the present disclosure, the ratio of the total radiation intensity of each organ of the mice to the total radiation intensity of the injection site was lower than 0.1, and it can be seen that these mRNA-LNP compositions have extremely high targeting to muscles, and the amount of off-target into the heart, liver, spleen, lung, kidney is extremely small, indicating that the possibility of causing organ toxicity is greatly reduced.
Although the ratio of the total radiation intensity of each organ of the mRNA-LNP compositions prepared from compound 5 and compound 9 to the total radiation intensity at the injection site is also at a lower level than other prior art lipid prepared mRNA-LNP compositions, the total radiation intensity at the injection site is lower. For example, the total radiation intensity of the mRNA-LNP composition prepared with YK-1612 at the injection site of mice was 1.5 times and 2.7 times that of the mRNA-LNP composition prepared with Compound 5 and Compound 9, respectively. Moreover, the ratio of total radiation intensity for each organ to total radiation intensity at the injection site was still higher (i.e., greater likelihood of organ toxicity occurring) for the mRNA-LNP compositions prepared with compound 5 and compound 9 as compared to the compounds of the present disclosure. For example, the relative fluorescence intensities of the mRNA-LNP compositions prepared with Compound 5 and Compound 9 increased by 88% and 106%, respectively, the relative fluorescence intensities of the liver increased by 120% and 403%, respectively, the relative fluorescence intensities of the spleen increased by 18% and 168%, respectively, the relative fluorescence intensities of the lung increased by 375% and 1075%, respectively, and the relative fluorescence intensities of the kidney increased by 225% and 500%, respectively, as compared to the mRNA-LNP compositions prepared with YK-1612 (note: relative fluorescence intensity of the mRNA-LNP composition prepared with Compound 5 or Compound 9 = (relative fluorescence intensity of the mRNA-LNP composition prepared with YK-1612)/(relative fluorescence intensity of the mRNA-LNP composition prepared with YK-1612. Times.100%).
2. In addition to the extremely low ratio of total radiation intensity at each organ to total radiation intensity at the injection site, the preparation of mRNA-LNP compositions from the compounds of the present disclosure has the advantage of achieving higher levels of expression at the injection site (muscle).
For example, the total radiation intensity at the injection site of the mRNA-LNP compositions prepared by YK-1603, YK-1604, YK-1605, YK-1606, YK-1608, YK-1610, YK-1612, YK-1613, YK-1614, YK-1615 and YK-1618 reached 2.63×108p/s、3.77×108p/s、4.66×108p/s、4.89×108p/s、1.99×108p/s、2.83×108p/s、2.3×108p/s、2.79×108p/s、1.23×108p/s、3.05×108p/s、3.56×108p/s,, respectively, and even exceeded several times the expression level of SM-102, which was most expressed at the injection site in the control compound. Whereas the control compound SM-102 had a significant accumulation of liver and spleen (ratio of total radiation intensity to injection site of 0.958, 0.458, respectively), was significantly improved compared to the mRNA-LNP composition prepared from the compound of the present disclosure.
As another example, the total radiation intensity of the muscle site of the mRNA-LNP composition prepared by YK-1606 is 2.3-fold, 4.1-fold, 5.9-fold, 6.4-fold, 7.3-fold, 6.9-fold, 8.4-fold, 3.2-fold and 5.8-fold, respectively, of the mRNA-LNP composition prepared by SM-102, MC3, lipid-028, E10-1, E24-1, E7-1, compound 92-12, compound 5 and compound 9.
3. In addition, the test results also show that the total radiation intensity at the muscle site is significantly enhanced by the mRNA-LNP composition prepared from YK-1606 of the present disclosure compared to the mRNA-LNP composition prepared from control 1, which is identical in structure at other locations, but completely devoid of chirality. The total radiation intensity of the muscle portion of the mRNA-LNP composition prepared by the control 1 was only 1.03X108 p/s, while the total radiation intensity of the muscle portion of the mRNA-LNP composition prepared by the YK-1606 was 4.7 times that of the control 1.
Furthermore, the mRNA-LNP composition prepared by YK-1606 was expressed in lower amounts in each organ than in control 1. For example, the relative fluorescence intensities of the mRNA-LNP composition prepared with control 1 increased by 217%, 142%, 138%, 150%, 217% in heart, liver, spleen, lung, kidney, respectively, as compared to the mRNA-LNP composition prepared with YK-1606 (note: relative fluorescence intensity increase= (relative fluorescence intensity of the mRNA-LNP composition prepared with control 1-relative fluorescence intensity of the mRNA-LNP composition prepared with YK-1606)/(relative fluorescence intensity of the mRNA-LNP composition prepared with YK-1606. Times.100%).