Detailed Description
Provided herein are LNPs comprising phospholipids containing sterol moieties. LNP can be loaded with mRNA, such as in mRNA vaccine technology. Sterol-modified phospholipids stabilize the bilayer, but do not exchange freely between membranes like cholesterol does. The mRNA-loaded LNP exhibits the ability to increase protein expression in target cells compared to mRNA-loaded LNP with more conventional phospholipids. Increasing protein expression helps to increase the effectiveness and efficiency of mRNA-based therapies and therapies.
I. Definition of the definition
As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" means that the material is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition for administration to a patient without causing any significant undesirable biological effect or interacting in a deleterious manner with any of the other components of the composition in which it is contained. The pharmaceutically acceptable carrier or excipient preferably meets the required criteria for toxicology and manufacturing testing and/or is incorporated in the inactive ingredient guidance (INACTIVE INGREDIENT Guide) made by the U.S. food and drug administration (U.S. food and Drug Administration).
As used herein, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable substrate, composition, or vehicle used in the course of drug delivery, which carrier may have one or more ingredients including, but not limited to, one or more excipients, one or more binders, one or more diluents, one or more solvents, one or more fillers, and/or one or more stabilizers.
As used herein, the term "lipid" refers to a group of compounds including, but not limited to, fats, sterols, waxes, fat-soluble vitamins, monoglycerides, diglycerides, sphingolipids, and phospholipids. In the context of the present disclosure, phospholipids, ionizable lipids, polymer-bound lipids, and lipid stabilizers are considered lipids.
As used herein, the term "ionizable lipid" refers to a lipid having a non-zero net charge at physiological pH. The term includes cationic lipids, including lipids having a partial positive charge at physiological pH. The term also includes mixtures of ionizable lipids, which may contain two or more ionizable lipids. In each instance of an embodiment, the term "ionizable lipid" is used, as is the "cationic lipid" as if all embodiments were specifically and individually listed with both ionizable lipids and cationic lipids.
As used herein, the term "polymer-bound lipid" refers to a lipid comprising a polymer moiety. The term includes pegylated lipids, including pegylated phosphatidylethanolamine, pegylated phosphatidic acid, pegylated ceramide, pegylated dialkylamine, pegylated diacylglycerol, and pegylated dialkylglycerol. The term also includes mixtures of polymer-bound lipids, which may contain two or more polymer-bound lipids. In each instance of an embodiment, the term "polymer-bound lipid" is considered as also with "pegylated lipid" as if all embodiments were specifically and individually listed with both polymer-bound lipids and pegylated lipids.
As used herein, the term "lipid stabilizing agent" refers to a component of a lipid nanoparticle that is believed to help stabilize the LNP structure. Without being bound by theory, it is believed that the lipid stabilizer component of the LNP helps support the liquid ordered phase of the lipid membrane in the LNP. See, e.g., albertsen, H.C. et al, month 9 of ,"The role of lipid components in lipid nanoparticles for vaccines and gene therapy."Adv Drug Deliv Rev.2022, section 3.3.1 of 188:114416. Compounds that can act as lipid stabilizers include sterols, corticosteroids, vitamins, and other compounds that contain a steroid core.
As used herein, the term "alkyl" refers to a chain of carbon atoms in which all bonds between carbon atoms in the alkyl group are single bonds. The term includes both straight and branched chains (e.g., the term includes n-propyl and isopropyl).
As used herein, the term "Cx-Cy alkyl" refers to an alkyl group having at least x carbon atoms and no more than y carbon atoms in the alkyl chain. For example, the term "C1-C3 alkyl" includes, but is not limited to, methyl, ethyl, n-propyl, and isopropyl.
As used herein, the term "alkylene" refers to an alkyl chain attached to other chemical groups at least two positions. "Cx-Cy alkylene" refers to an alkylene group having at least x carbon atoms and no more than y carbon atoms in the alkylene chain. For example, the term "C1-C3 alkylene" includes, but is not limited to, methylene, ethylene, n-propylene, and isopropylene.
As used herein, the term "alkenyl" refers to a chain of carbon atoms having at least one double bond between two carbon atoms in the chain. The term includes both straight and branched chains (e.g., the term includes 1-propenyl and isopropenyl).
As used herein, the term "Cx-Cy alkenyl" refers to alkenyl groups having at least x carbon atoms and no more than y carbon atoms in the alkenyl chain. For example, the term "C2-C4 alkenyl" includes, but is not limited to, vinyl and 1-propenyl.
As used herein, the term "alkenylene" refers to an alkenyl chain attached to other chemical groups at least two positions. "Cx-Cy alkenylene" refers to alkenylene having at least x carbon atoms and no more than y carbon atoms.
As used herein, the term "alkynyl" refers to a chain of carbon atoms having at least one triple bond between two carbon atoms in the chain. The term includes both straight and branched chains (e.g., the term includes 1-propynyl and isopropanynyl).
As used herein, the term "Cx-Cy alkynyl" refers to an alkynyl group having at least x carbon atoms and no more than y carbon atoms in the alkynyl chain.
As used herein, the term "cycloalkyl" refers to a cyclic group of carbon atoms, wherein all bonds between carbon atoms are single bonds. The term "Cx-Cy cycloalkyl" refers to cycloalkyl groups having at least x carbon atoms and no more than y carbon atoms. For example, the term "C6-C10 cycloalkyl" includes, but is not limited to, cyclohexyl and cyclooctyl. The term "cycloalkylene" has the same meaning as cycloalkyl except that the cycloalkylene is attached to at least two other chemical groups.
As used herein, the term "cycloalkenyl" refers to a cyclic group of carbon atoms wherein at least one bond between two carbon atoms in the cycloalkenyl is a double bond. The term "Cx-Cy cycloalkenyl" refers to cycloalkenyl groups having at least x carbon atoms and no more than y carbon atoms.
The term "Cx-Cy aryl" refers to an aryl group having at least x carbon atoms and no more than y carbon atoms. For example, the term "C6-C10 aryl" includes, but is not limited to, phenyl and naphthyl. The term "arylene" has the same meaning as aryl except that the arylene is attached to at least two other chemical groups.
As used herein, the term "heterocycloalkyl" refers to a cyclic group of atoms, wherein all bonds between atoms in the ring are single bonds. The term "Cx-Cy heterocycloalkyl" refers to heterocycloalkyl having at least x atoms and no more than y atoms. For example, the term "C5-C6 heterocycloalkyl" includes, but is not limited to, pyrrolidinyl and 1, 4-dioxanyl. The term "heterocycloalkylene" has the same meaning as heterocycloalkyl except that the heterocycloalkylene is attached to at least two other chemical groups.
The term "x to y membered heteroaryl" refers to a cyclic atomic group having at least x atoms and no more than y atoms. For example, 5-or 6-membered heteroaryl groups include, but are not limited to, pyridinyl and furanyl.
As used herein, the term "carbocycle" refers to a cycloalkyl or aryl group. Likewise, the term "heterocycle" refers to a heterocycloalkyl or heteroaryl group.
Possible atoms constituting the ring in the heterocycloalkyl and heteroaryl groups and derivatives thereof include, but are not limited to, carbon, nitrogen, oxygen, and sulfur.
As used herein, the term "optionally substituted" means that the indicated group may be substituted or unsubstituted. The term substituted refers to the modification of another chemical moiety of the indicated group by substitution of one H atom. For example, ethanol is an example of ethane substituted with OH. In some embodiments, the optionally substituted group is optionally substituted with chloro, fluoro, bromo, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C6-C10 aryl, or 5 or 6 membered heteroaryl.
The terms "individual," "subject," and "patient" are used interchangeably herein to describe a mammal, including a human. In some embodiments, the individual is in need of treatment, e.g., the individual may have been diagnosed with or suspected of having cancer.
It is to be understood that the embodiments of the invention described herein include "consisting of" and/or "consisting essentially of" embodiments.
References herein to "about" a value or parameter include (and describe) deviations from the value or parameter itself. For example, a description referring to "about X" includes a description of "X". In some embodiments, the term "about" a value or parameter refers to a range of 20% of the value or parameter in either direction.
As used herein, references to "not being" a value or parameter generally mean and describe "in addition to" a value or parameter.
As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
As used herein and in the appended claims, the mole percentages of lipid and lipid stabilizing agent in the lipid nanoparticle are calculated based on the total moles of components in the lipid nanoparticle.
II Lipid Nanoparticles (LNP)
Lipid Nanoparticles (LNPs) herein comprise a phospholipid containing a sterol moiety and optionally one or more of an ionizable lipid, a polymer-bound lipid, and a lipid stabilizer. In some embodiments, the LNP comprises a phospholipid comprising a sterol moiety, an ionizable lipid, and a polymer-bound lipid. In some embodiments, the LNP comprises a phospholipid comprising a sterol moiety, a polymer-bound lipid, and a lipid stabilizing agent. In some embodiments, the LNP comprises a phospholipid comprising a sterol moiety, an ionizable lipid, and a lipid stabilizing agent. In some embodiments, the LNP comprises a phospholipid comprising a sterol moiety, an ionizable lipid, a polymer-bound lipid, and a lipid stabilizing agent.
In some embodiments, the phospholipid comprises 1 to 30mol% of the total lipids in the LNP. In some embodiments, the phospholipid comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30mol% of the total lipid in the LNP. In some embodiments, the phospholipid comprises 2 to 25mol% of the total lipids in the LNP. In some embodiments, the phospholipid comprises 3 to 20mol% of the total lipids in the LNP. In some embodiments, the phospholipid comprises 5 to 10, 15, 20, 25, or 30 mole% of the total lipids in the LNP. In some embodiments, the phospholipid comprises 10 to 15, 20, 25, or 30mol% of the total lipids in the LNP. In some embodiments, the phospholipid comprises 15 to 20, 25, or 30mol% of the total lipids in the LNP. In some embodiments, the phospholipid comprises 20 to 25 or 30mol% of the total lipids in the LNP. In some embodiments, the phospholipid comprises 25 to 30mol% of the total lipids in the LNP.
In some embodiments, the ionizable lipid comprises 40 to 80mol% of the total lipids in the LNP. In some embodiments, the ionizable lipid comprises 40 to 50, 60, 70, or 80mol% of the total lipids in the LNP. In some embodiments, the ionizable lipid comprises 45 to 50, 60, 70, 75, or 80mol% of the total lipids in the LNP. In some embodiments, the ionizable lipid comprises 50 to 60, 70, or 80mol% of the total lipids in the LNP. In some embodiments, the ionizable lipid comprises 60 to 65, 70, or 80mol% of the total lipids in the LNP. In some embodiments, the ionizable lipid comprises 70 to 80mol% of the total lipids in the LNP. In some embodiments, the ionizable lipid comprises about 40, 50, 60, 70, or 80mol% of the total lipids in the LNP. In some embodiments, the ionizable lipid comprises about 50, 60, or 70mol% of the total lipids in the LNP.
In some embodiments, the LNP has a molar ratio of ionizable lipid to phospholipid of 20:1 to 2:1. In some embodiments, the molar ratio is 20:1 to 15:1, 10:1, 5:1, or 2:1. In some embodiments, the molar ratio is 18:1 to 2.5:1. In some embodiments, the molar ratio is 16:1 to 4:1. In some embodiments, the molar ratio is 15:1 to 10:1, 5:1, or 2:1. In some embodiments, the molar ratio is 10:1 to 5:1 or 2:1. In some embodiments, the ratio is 5:1 to 2:1. In some embodiments, the ratio is 15:1 to 5:1.
In some embodiments, the polymer-bound lipids have a molar ratio of 0.5 to 5mol% of the total lipids in the LNP. In some embodiments, the polymer-bound lipids have a molar ratio of 1 to 2mol% of the total lipids in the LNP. In some embodiments, the polymer-bound lipid has a molar ratio of 1.5mol% of the total lipid in the LNP.
In some embodiments, the molar ratio of polymer-bound lipid to phospholipid is from 1:2 to 1:20. In some embodiments, the molar ratio of polymer-bound lipid to phospholipid is from 1:3 to 1:18. In some embodiments, the molar ratio of polymer-bound lipid to phospholipid is from 1:5 to 1:10.
In some embodiments, the lipid stabilizing agent comprises 5 to 50mol% of the total lipids in the LNP. In some embodiments, the lipid stabilizing agent comprises 5 to 10, 20, 30, 40, or 50mol% of the total lipids in the LNP. In some embodiments, the lipid stabilizing agent comprises 8 to 20, 30, 40, or 50mol% of the total lipids in the LNP. In some embodiments, the lipid stabilizing agent comprises 10 to 20, 30, 40, or 50mol% of the total lipids in the LNP. In some embodiments, the lipid stabilizing agent comprises 20 to 30, 40, or 50 mole% of the total lipids in the LNP. In some embodiments, the lipid stabilizing agent comprises 30 to 40 or 50mol% of the total lipids in the LNP. In some embodiments, the lipid stabilizing agent comprises 40 to 50mol% of the total lipids in the LNP. In some embodiments, the lipid stabilizing agent comprises about 5, 10, 20, 30, 40, or 50mol% of the total lipids in the LNP.
It will be appreciated that in any embodiment describing the lipid composition of LNP described herein, any of the mole percentages or mole ratios described above for the sterol-containing phospholipid, the ionizable lipid, the polymer-bound lipid, and the lipid stabilizing agent can be combined with one another such that each combination is contemplated as if each combination were specifically and individually disclosed.
In some embodiments, the phospholipid comprises 1 to 30mol% of the total lipids in the LNP, the ionizable lipid comprises 40 to 80mol%, the polymer-bound lipid comprises 1 to 2mol%, and the lipid stabilizing agent comprises 5 to 50mol%. In some embodiments, the phospholipid comprises 5 to 25mol% of the total lipids in the LNP, the ionizable lipid comprises 45 to 75mol%, the polymer-bound lipid comprises 1 to 2mol%, and the lipid stabilizing agent comprises 20 to 40%. In some embodiments, the phospholipid comprises 5 to 15mol% of the total lipids in the LNP, the ionizable lipid comprises 40 to 60mol%, the polymer-bound lipid comprises 1 to 2mol%, and the lipid stabilizing agent comprises 20 to 40mol%. In some embodiments, the phospholipid comprises about 10mol% of the total lipids in the LNP, the ionizable lipid comprises about 50mol%, the polymer-bound lipid comprises about 38.5mol%, and the lipid stabilizing agent comprises about 1.5mol%.
In some embodiments, the LNP has a size of 20 to 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, or 300nm. In some embodiments, the size is 40 to 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, or 300nm. In some embodiments, the size is 50 to 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, or 300nm. In some embodiments, the size is 60 to 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, or 300nm. In some embodiments, the size is 70 to 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, or 300nm. In some embodiments, the size is 80 to 90, 100, 110, 120, 130, 140, 150, 200, 250, or 300nm. In some embodiments, the size is 90 to 100, 110, 120, 130, 140, 150, 200, 250, or 300nm. In some embodiments, the size is 100 to 110, 120, 130, 140, 150, 200, 250, or 300nm. In some embodiments, the size is 110 to 120, 130, 140, 150, 200, 250, or 300nm. In some embodiments, the size is 120 to 130, 140, 150, 200, 250, or 300nm. In some embodiments, the size is 130 to 140, 150, 200, 250, or 300nm. In some embodiments, the size is 140 to 150, 200, 250, or 300nm. In some embodiments, the size is 150 to 200, 250, or 300nm. In some embodiments, the size is 200 to 300nm. In some embodiments, the size is 60 to 150nm. In some embodiments, the size is 65 to 90nm. In some embodiments, the size is 70 to 80nm. In some embodiments, the size is about 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, or 115nm. In some embodiments, the size is about 85 or about 90nm. In some embodiments, the size is about 85nm. In some embodiments, the size is about 90nm.
A. Phospholipid containing sterol moiety
LNP herein comprises a phospholipid containing a sterol moiety. A phospholipid containing a sterol moiety is any phospholipid that incorporates the sterol moiety into the lipid structure. In some embodiments, sterol moieties are combined to replace one or more alkyl chains on the phospholipid. In some embodiments, the sterol moiety is incorporated into one alkyl chain of the phospholipid. In some embodiments, the sterol moiety is attached through an O atom of the sterol (e.g., converting the sterol moiety to an-O-atom of an ester attached to the rest of the phospholipid). In some embodiments, the sterol moiety is cholesterol. In some embodiments, the sterol moiety is a cholesterol moiety linked through an O atom of the sterol (e.g., by converting the sterol O atom of the cholesterol to an-O-atom of an ester linked to the rest of the phospholipid).
In some embodiments, the phospholipid has a structure selected from the group consisting of:
And
B. Ionizable lipids
In some embodiments, the LNP comprises an ionizable lipid. In some embodiments, the ionizable lipid is a cationic lipid. In some embodiments, the cationic lipid is a cationic lipid described in International patent publication No. WO 2021/204175, which is incorporated herein by reference in its entirety.
In some embodiments, the cationic lipid is a compound of formula (01-I):
Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:
Each of G1 and G2 is independently a bond, C2-C12 alkylene or C2-C12 alkenylene, wherein one or more of the alkylene or alkenylene groups-CH2 -is optionally replaced by-O-;
l1 is –OC(＝O)R1、-C(＝O)OR1、-OC(＝O)OR1、-C(＝O)R1、-OR1、-S(O)xR1、-S-SR1、-C(＝O)SR1、-SC(＝O)R1、-NRaC(＝O)R1、-C(＝O)NRbRc、-NRaC(＝O)NRbRc、-OC(＝O)NRbRc、-NRaC(＝O)OR1、-SC(＝S)R1、-C(＝S)SR1、-C(＝S)R1、-CH(OH)R1、-P(＝O)(ORb)(ORc)、-(C6-C10 arylene) -R1, - (6 to 10 membered heteroarylene) -R1 or R1;
L2 is –OC(＝O)R2、-C(＝O)OR2、-OC(＝O)OR2、-C(＝O)R2、-OR2、-S(O)xR2、-S-SR2、-C(＝O)SR2、-SC(＝O)R2、-NRdC(＝O)R2、-C(＝O)NReRf、-NRdC(＝O)NReRf、-OC(＝O)NReRf、-NRdC(＝O)OR2、-SC(＝S)R2、-C(＝S)SR2、-C(＝S)R2、-CH(OH)R2、-P(＝O)(ORe)(ORf)、-(C6-C10 arylene) -R2, - (6 to 10 membered heteroarylene) -R2 or R2;
R1 and R2 are each independently C6-C32 alkyl or C6-C32 alkenyl;
Ra、Rb、Rd and Re are each independently H, C1-C24 alkyl or C2-C24 alkenyl;
rc and Rf are each independently C1-C32 alkyl or C2-C32 alkenyl;
G3 and C2-C24 alkylene, C2-C24 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenyl;
R3 is-N (R4)R5;
R4 is C3-C8 cycloalkyl, C3-C8 cycloalkenyl, 4 to 8 membered heterocyclyl or C6-C10 aryl, or R4、G3 or a portion of G3 together with the nitrogen to which it is attached form a cyclic moiety;
R5 is C1-C12 alkyl or C3-C8 cycloalkyl, or R4、R5 together with the nitrogen to which it is attached form a cyclic moiety;
x is 0, 1 or 2, and
Wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.
In some embodiments, the cationic lipid is a compound of formula (01-II):
Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:
Is a single bond or a double bond;
Each of G1 and G2 is independently a bond, C2-C12 alkylene or C2-C12 alkenylene, wherein one or more of the alkylene or alkenylene groups-CH2 -is optionally replaced by-O-;
L1 is –OC(＝O)R1、-C(＝O)OR1、-OC(＝O)OR1、-C(＝O)R1、-OR1、-S(O)xR1、-S-SR1、-C(＝O)SR1、-SC(＝O)R1、-NRaC(＝O)R1、-C(＝O)NRbRc、-NRaC(＝O)NRbRc、-OC(＝O)NRbRc、-NRaC(＝O)OR1、-SC(＝S)R1、-C(＝S)SR1、-C(＝S)R1、-CH(OH)R1、-P(＝O)(ORb)(ORc)、-(C6-C10 arylene) -R1, - (6 to 10 membered heteroarylene) -R1 or R1;
L2 is –OC(＝O)R2、-C(＝O)OR2、-OC(＝O)OR2、-C(＝O)R2、-OR2、-S(O)xR2、-S-SR2、-C(＝O)SR2、-SC(＝O)R2、-NRdC(＝O)R2、-C(＝O)NReRf、-NRdC(＝O)NReRf、-OC(＝O)NReRf、-NRdC(＝O)OR2、-SC(＝S)R2、-C(＝S)SR2、-C(＝S)R2、-CH(OH)R2、-P(＝O)(ORe)(ORf)、-(C6-C10 arylene) -R2, - (6 to 10 membered heteroarylene) -R2 or R2;
R1 and R2 are each independently C6-C32 alkyl or C6-C32 alkenyl;
Ra、Rb、Rd and Re are each independently H, C1-C24 alkyl or C2-C24 alkenyl;
rc and Rf are each independently C1-C32 alkyl or C2-C32 alkenyl;
G4 is a bond, C1-C23 alkylene, C2-C23 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenyl;
R3 is-N (R4)R5;
R4 is C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, 4-to 8-membered heterocyclyl or C6-C10 aryl, or R4、G3 or a portion of G3 together with the nitrogen to which it is attached form a cyclic moiety;
R5 is C1-C12 alkyl or C3-C8 cycloalkyl, or R4 and R5 together with the nitrogen to which they are attached form a cyclic moiety;
x is 0, 1 or 2, and
Wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.
In some embodiments, the cationic lipid is a compound of formula (01-I-B), (01-I-B'), (01-I-B "), (01-I-C), (01-I-D), or (01-I-E):
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.
In some embodiments, G1 and G2 are each independently C3-C7 alkylene. In some embodiments, G1 and G2 are each independently C5 alkylene. In some embodiments, G3 is C2-C4 alkylene. In some embodiments, G3 is C2 alkylene. In some embodiments, G3 is C4 alkylene.
In some embodiments, R3 has one of the following structures:
in some embodiments, R1、R2、Rc and Rf are each independently branched C6-C32 alkyl or branched C6-C32 alkenyl. In some embodiments, R1、R2、Rc and Rf are each independently branched C6-C24 alkyl or branched C6-C24 alkenyl. In some embodiments, R1、R2、Rc and Rf are each independently-R7-CH(R8)(R9), wherein R7 is C0-C5 alkylene and R8 and R9 are independently C2-C10 alkyl. In some embodiments, R1、R2、Rc and Rf are each independently-R7-CH(R8)(R9), wherein R7 is C0-C1 alkylene and R8 and R9 are independently C4-C8 alkyl.
In some embodiments, the cationic lipid is a compound of table 1, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
Table 1.
In some embodiments, the cationic lipid is a cationic lipid described in international patent application No. PCT/CN2022/072694, which is incorporated herein by reference in its entirety. In some embodiments, the cationic lipid is a compound of formula (02-I):
Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:
Each of G1 and G2 is independently C2-C12 alkylene or C2-C12 alkenylene, wherein one or more of G1 and G2 -CH2 -is optionally replaced by-O-, -C (=o) O-or-OC (=o) -;
Each L1 is independently –OC(＝O)R1、-C(＝O)OR1、-OC(＝O)OR1、-C(＝O)R1、-OR1、-S(O)xR1、-S-SR1、-C(＝O)SR1、-SC(＝O)R1、-NRaC(＝O)R1、-C(＝O)NRbRc、-NRaC(＝O)NRbRc、-OC(＝O)NRbRc、-NRaC(＝O)OR1、-SC(＝S)R1、-C(＝S)SR1、-C(＝S)R1、-CH(OH)R1、-P(＝O)(ORb)(ORc)、-NRaP(＝O)(ORb)(ORc);
Each L2 is independently –OC(＝O)R2、-C(＝O)OR2、-OC(＝O)OR2、-C(＝O)R2、-OR2、-S(O)xR2、-S-SR2、-C(＝O)SR2、-SC(＝O)R2、-NRdC(＝O)R2、-C(＝O)NReRf、-NRdC(＝O)NReRf、-OC(＝O)NReRf、-NRdC(＝O)OR2、-SC(＝S)R2、-C(＝S)SR2、-C(＝S)R2、-CH(OH)R2、-P(＝O)(ORe)(ORf)、-NRdP(＝O)(ORe)(ORf);
R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;
Ra、Rb、Rd and Re are each independently H, C1-C24 alkyl or C2-C24 alkenyl;
rc and Rf are each independently C1-C24 alkyl or C2-C24 alkenyl;
G3 is C2-C12 alkylene or C2-C12 alkenylene, wherein part or all of the alkylene or alkenylene is optionally replaced by C3-C8 cycloalkylene or C3-C8 cycloalkenyl;
R3 is-N (R4)R5、-OR6 or-SR6;
R4 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4 to 8 membered heterocycloalkyl;
R5 is H, C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4-to 8-membered heterocycloalkyl;
R6 is hydrogen, C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C10 aryl;
x is 0, 1 or 2, and
Wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted.
In some embodiments, the cationic lipid is a compound of formula (02-II):
Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:
Each of G1 and G2 is independently C2-C12 alkylene or C2-C12 alkenylene, wherein one or more of G1 and G2 -CH2 -is optionally replaced by-O-, -C (=o) O-or-OC (=o) -;
Each L1 is independently –OC(＝O)R1、-C(＝O)OR1、-OC(＝O)OR1、-C(＝O)R1、-OR1、-S(O)xR1、-S-SR1、-C(＝O)SR1、-SC(＝O)R1、-NRaC(＝O)R1、-C(＝O)NRbRc、-NRaC(＝O)NRbRc、-OC(＝O)NRbRc、-NRaC(＝O)OR1、-SC(＝S)R1、-C(＝S)SR1、-C(＝S)R1、-CH(OH)R1、-P(＝O)(ORb)(ORc)、-NRaP(＝O)(ORb)(ORc);
Each L2 is independently –OC(＝O)R2、-C(＝O)OR2、-OC(＝O)OR2、-C(＝O)R2、-OR2、-S(O)xR2、-S-SR2、-C(＝O)SR2、-SC(＝O)R2、-NRdC(＝O)R2、-C(＝O)NReRf、-NRdC(＝O)NReRf、-OC(＝O)NReRf、-NRdC(＝O)OR2、-SC(＝S)R2、-C(＝S)SR2、-C(＝S)R2、-CH(OH)R2、-P(＝O)(ORe)(ORf)、-NRdP(＝O)(ORe)(ORf);
R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;
Ra、Rb、Rd and Re are each independently H, C1-C24 alkyl or C2-C24 alkenyl;
rc and Rf are each independently C1-C24 alkyl or C2-C24 alkenyl;
G3 is C2-C12 alkylene or C2-C12 alkenylene, wherein part or all of the alkylene or alkenylene is optionally replaced by C3-C8 cycloalkylene or C3-C8 cycloalkenyl;
R3 is-N (R4)R5、-OR6 or-SR6;
R4 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4 to 8 membered heterocycloalkyl;
R5 is H, C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4-to 8-membered heterocycloalkyl;
R6 is hydrogen, C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C10 aryl;
x is 0, 1 or 2, and
Wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted.
In some embodiments, the compound is A compound of formulA (02-V-A), (02-V-B), (02-V-C), (02-V-D), (02-V-E), (02-V-F):
Wherein z is an integer of 2 to 12,
X0 is an integer from 1 to 11;
y0 is an integer from 1 to 11;
x1 is an integer from 0 to 9;
y1 is an integer from 0 to 9;
x2 is an integer from 2 to 9;
x3 is an integer from 1 to 5;
x4 is an integer from 0 to 3;
y2 is an integer from 2 to 9;
y3 is an integer from 1 to 5, and
Y4 is an integer from 0 to 3;
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.
In some embodiments, z is an integer from 2 to 6. In some embodiments, z is 2, 4, or 5. In some embodiments, x0 and y0 are independently 2 to 6. In some embodiments, x0 and y0 are independently 4 or 5. In some embodiments, x1 and y1 are independently 2 to 6. In some embodiments, x1 and y1 are independently 4 or 5. In some embodiments, x2 and y2 are independently integers from 2 to 8. In some embodiments, x2 and y2 are independently 3,5, or 7. In some embodiments, x3 and y3 are both 1. In some embodiments, x4 and y4 are independently 0 or 1.
In some embodiments, each L1 is independently-OR1、-OC(＝O)R1 OR-C (=o) OR1, and each L2 is independently-OR2、-OC(＝O)R2 OR-C (=o) OR2. In some embodiments, R1 and R2 are independently straight chain C6-C10 alkyl or-R7-CH(R8)(R9), wherein R7 is C0-C5 alkylene, and R8 and R9 are independently C2-C10 alkyl or C2-C10 alkenyl.
In some embodiments, the compound is a compound of formula (02-VI-A), (02-VI-B), (02-VI-C), (02-VI-D), (02-VI-E), or (02-VI-F):
Wherein z is an integer from 2 to 12;
y is an integer from 2 to 12;
x0 is an integer from 1 to 11;
x1 is an integer from 0 to 9;
x2 is an integer from 2 to 5;
x3 is an integer from 1 to 5, and
X4 is an integer from 0 to 3;
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.
In some embodiments, z is an integer from 2 to 6. In some embodiments, z is 2, 4, or 5. In some embodiments, x0 is 4 or 5. In some embodiments, x1 is 4 or 5. In some embodiments, x2 is an integer from 2 to 5. In some embodiments, x2 is 3 or 5. In some embodiments, x3 is 0 or 1. In some embodiments, y is an integer from 2 to 6. In some embodiments, y is 5.
In some embodiments, each L1 is independently-OR1、-OC(＝O)R1 OR-C (=o) OR1, and L2 is-OC (=o) R2 OR-C (=o) OR2、-NRdC(＝O)R2 OR-C (=o) NReRf. In some embodiments, R1 is straight chain C6-C10 alkyl or-R7-CH(R8)(R9), wherein R7 is C0-C5 alkylene, and R8 and R9 are independently C2-C10 alkyl or C2-C10 alkenyl. In some embodiments, each of R2 and Rf is independently a linear C6-C18 alkyl group, C6-C18 alkenyl or-R7-CH(R8)(R9), wherein R7 is C0-C5 alkylene, and R8 and R9 are independently C2-C10 alkyl or C2-C10 alkenyl. In some embodiments, Rd and Re are each independently H.
In some embodiments, the compound is a compound of table 2, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
Table 2.
In some embodiments, the cationic lipids described herein are those described in international patent publication No. WO 2022/152109, which is incorporated herein by reference in its entirety.
In some embodiments, the cationic lipid is a compound of formula (03-I):
Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:
Each of G1 and G2 is independently a bond, C2-C12 alkylene, or C2-C12 alkenylene, wherein one or more of G1 and G2 -CH2 -is optionally replaced by-O-;
each L1 is independently –OC(＝O)R1、-C(＝O)OR1、-OC(＝O)OR1、-C(＝O)R1、-OR1、-S(O)xR1、-S-SR1、-C(＝O)SR1、-SC(＝O)R1、-NRaC(＝O)R1、-C(＝O)NRbRc、-NRaC(＝O)NRbRc、-OC(＝O)NRbRc、-NRaC(＝O)OR1、-SC(＝S)R1、-C(＝S)SR1、-C(＝S)R1、-CH(OH)R1、-P(＝O)(ORb)(ORc)、-NRaP(＝O)(ORb)(ORc)、-(C6-C10 arylene) -R1, - (6-to 10-membered heteroarylene) -R1, - (4-to 8-membered heterocyclylene) -R1 or R1;
Each L2 is independently –OC(＝O)R2、-C(＝O)OR2、-OC(＝O)OR2、-C(＝O)R2、-OR2、-S(O)xR2、-S-SR2、-C(＝O)SR2、-SC(＝O)R2、-NRdC(＝O)R2、-C(＝O)NReRf、-NRdC(＝O)NReRf、-OC(＝O)NReRf、-NRdC(＝O)OR2、-SC(＝S)R2、-C(＝S)SR2、-C(＝S)R2、-CH(OH)R2、-P(＝O)(ORe)(ORf)、-NRdP(＝O)(ORe)(ORf)、-(C6-C10 arylene) -R2, - (6-to 10-membered heteroarylene) -R2, - (4-to 8-membered heterocyclylene) -R2 or R2;
r1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;
Ra、Rb、Rd and Re are each independently H, C1-C24 alkyl or C2-C24 alkenyl;
rc and Rf are each independently C1-C24 alkyl or C2-C24 alkenyl;
G3 is C2-C12 alkylene or C2-C12 alkenylene, wherein part or all of the alkylene or alkenylene is optionally replaced by C3-C8 cycloalkylene, C3-C8 cycloalkenylene, C3-C8 cycloalkynylene, 4-to 8-membered heterocyclylene, C6-C10 arylene or 5-to 10-membered heteroarylene;
R3 is hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C3-C8 cycloalkynyl, 4 to 8 membered heterocyclyl, C6-C10 aryl, or 5 to 10 membered heteroaryl, or R3 and a portion of G1 or G1 together with the nitrogen to which they are attached form a cyclic moiety, or R3 and a portion of G3 or G3 together with the nitrogen to which they are attached form a cyclic moiety;
R4 is C1-C12 alkyl or C3-C8 cycloalkyl;
x is 0, 1 or 2;
n is 1 or 2;
m is 1 or 2, and
Wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkylene, alkenylene, cycloalkylene, cycloalkenyl, cycloalkynylene, heterocyclylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.
In some embodiments, the cationic lipid is a compound of formula (03-II-A):
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.
In some embodiments, the cationic lipid is a compound of formula (03-II-B):
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.
In some embodiments, the cationic lipid is a compound of formula (03-II-C):
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.
In some embodiments, the cationic lipid is a compound of formula (03-II-D):
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.
In some embodiments, G1 and G2 are each independently C2-C12 alkylene. In some embodiments, G1 and G2 are each independently C5 alkylene. In some embodiments, G3 is C2-C6 alkylene.
In some embodiments, R3 is C1-C12 alkyl, C2-C12 alkenyl, or C3-C8 cycloalkyl. In some embodiments, R3 is C3-C8 cycloalkyl. In some embodiments, R3 is unsubstituted. In some embodiments, R4 is substituted C1-C12 alkyl. In some embodiments, R4 is-CH2CH2 OH.
In some embodiments, L1 is-OC (=o) R1、-C(＝O)OR1、-NRaC(＝O)R1 or-C (=o) NRbRc, and L2 is-OC (=o) R2、-C(＝O)OR2、-NRdC(＝O)R2 or-C (=o) NReRf. In some embodiments, each of R1、R2、Rc and Rf is independently a linear C6-C18 alkyl group, Linear C6-C18 alkenyl or-R7-CH(R8)(R9), wherein R7 is C0-C5 alkylene and R8 and R9 are independently C2-C10 alkyl or C2-C10 alkenyl. In some embodiments, each of R1、R2、Rc and Rf is independently a linear C7-C15 alkyl group, Linear C7-C15 alkenyl or-R7-CH(R8)(R9), wherein R7 is C0-C1 alkylene and R8 and R9 are independently C4-C8 alkyl or C6-C10 alkenyl. In some embodiments, Ra、Rb、Rd and Re are each independently H.
In some embodiments, the cationic lipid is a compound of table 3, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
TABLE 3 Table 3
In some embodiments, the cationic lipid is a cationic lipid described in international patent application No. PCT/CN2022/094227, which is incorporated herein by reference in its entirety.
In some embodiments, the cationic lipid is a compound of formula (04-I):
Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:
Each of G1 and G2 is independently a bond, C2-C12 alkylene, or C2-C12 alkenylene;
L1 is –OC(＝O)R1、-C(＝O)OR1、-OC(＝O)OR1、-C(＝O)R1、-OR1、-S(O)xR1、-S-SR1、-C(＝O)SR1、-SC(＝O)R1、-NRaC(＝O)R1、-C(＝O)NRbRc、-NRaC(＝O)NRbRc、-OC(＝O)NRbRc、-NRaC(＝O)OR1、-SC(＝S)R1、-C(＝S)SR1、-C(＝S)R1、-CH(OH)R1、-P(＝O)(ORb)(ORc)、-(C6-C10 arylene) -R1, - (6 to 10 membered heteroarylene) -R1 or R1;
L2 is –OC(＝O)R2、-C(＝O)OR2、-OC(＝O)OR2、-C(＝O)R2、-OR2、-S(O)xR2、-S-SR2、-C(＝O)SR2、-SC(＝O)R2、-NRdC(＝O)R2、-C(＝O)NReRf、-NRdC(＝O)NReRf、-OC(＝O)NReRf、-NRdC(＝O)OR2、-SC(＝S)R2、-C(＝S)SR2、-C(＝S)R2、-CH(OH)R2、-P(＝O)(ORe)(ORf)、-(C6-C10 arylene) -R2, - (6 to 10 membered heteroarylene) -R2 or R2;
R1 and R2 are each independently C5-C32 alkyl or C5-C32 alkenyl;
Ra、Rb、Rd and Re are each independently H, C1-C24 alkyl or C2-C24 alkenyl;
rc and Rf are each independently C1-C32 alkyl or C2-C32 alkenyl;
R0 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4 to 8 membered heterocycloalkyl;
G3 is C2-C12 alkylene or C2-C12 alkenylene;
R4 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4 to 8 membered heterocycloalkyl;
R5 is C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4-to 8-membered heterocycloalkyl;
x is 0, 1 or 2;
s is 0 or 1, and
Wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, arylene, and heteroarylene is independently optionally substituted.
In some embodiments, the cationic lipid is a compound of formula (04-III):
Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:
R1 and R2 are each independently C5-C32 alkyl or C5-C32 alkenyl;
R0 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4 to 8 membered heterocycloalkyl;
G3 is C2-C12 alkylene or C2-C12 alkenylene;
G4 is C2-C12 alkylene or C2-C12 alkenylene;
R3 is-N (R4)R5 OR-OR6;
R4 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4 to 8 membered heterocycloalkyl;
R5 is C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4 to 8 membered heterocycloalkyl, or R4 and R5 together with the nitrogen to which they are attached form a cyclic moiety;
R6 is hydrogen, C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C10 aryl, and
Wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, and cyclic moiety is independently optionally substituted.
In some embodiments, the cationic lipid is a compound of formula (04-IV):
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.
In some embodiments, G3 is C2-C4 alkylene. In some embodiments, G4 is C2-C4 alkylene.
In some embodiments, R0 is C1-C6 alkyl. In some embodiments, R3 is —oh. In some embodiments, R3 is-N (R4)R5. In some embodiments, R4 is C3-C8 cycloalkyl, in some embodiments, R4 is unsubstituted, in some embodiments, R5 is-CH2CH2 OH.
In some embodiments, L1 is-OC (=o) R1、-C(＝O)OR1、-C(＝O)R1、-C(＝O)NRbRc or R1, and L2 is-OC (=o) R2、-C(＝O)OR2、-C(＝O)R2、-C(＝O)NReRf or R2. In some embodiments, R1 and R2 are each independently branched C6-C24 alkyl or branched C6-C24 alkenyl. in some embodiments, R1 and R2 are each independently-R7-CH(R8)(R9), wherein R7 is C1-C5 alkylene and R8 and R9 are independently C2-C10 alkyl or C2-C10 alkenyl. In some embodiments, R1 is straight chain C6-C24 alkyl and R2 is branched chain C6-C24 alkyl. In some embodiments, R1 is straight chain C6-C24 alkyl and R2 is-R7-CH(R8)(R9), wherein R7 is C1-C5 alkylene and R8 and R9 are independently C2-C10 alkyl.
In some embodiments, the cationic lipid is a compound of table 4, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
Table 4.
In some embodiments, the cationic lipids contained in the particles or compositions provided herein are those described in U.S. patent No. US10442756B2, US9868691B2, or US9868692B2, which are incorporated herein by reference in their entirety.
In some embodiments, the cationic lipid is a compound of formula (05-I):
or a salt or isomer thereof, wherein
L is selected from 1,2, 3, 4 and 5;
m is selected from 5, 6, 7, 8 and 9;
m1 is a bond or M';
R4 is unsubstituted C1-C3 alkyl or -(CH2)nOH、-NHC(S)N(R)2、-NHC(O)N(R)2、-N(R)C(O)R、-N(R)S(O)2R、-N(R)R8、-NHC(＝NR9)N(R)2、-NHC(＝CHR9)N(R)2、-OC(O)N(R)2、-N(R)C(O)OR、-N(OR)C(O)R、-N(OR)S(O)2R、-N(OR)C(O)OR、-N(OR)C(O)N(R)2、-N(OR)C(S)N(R)2、-N(OR)C(＝NR9)N(R)2、-N(OR)C(＝CHR9)N(R)2 or heteroaryl, and each n is selected from 1,2, 3, 4 or 5;
m and M ' are independently selected from the group consisting of-C (O) O-, -OC (O) -, -C (O) N (R ') -, -P (O) (OR ') O-, -S-S-, aryl, and heteroaryl; and
R2 and R3 are both C1-C14 alkyl or C2-C14 alkenyl,
R8 is selected from the group consisting of C3-C6 carbocycle and heterocycle;
R9 is selected from the group consisting of H, CN, NO2、C1-C6 alkyl, -OR, -S (O)2R、-S(O)2N(R)2、C2-C6 alkenyl, C3-C6 carbocycle, and heterocycle;
Each R is independently selected from the group consisting of C1-C3 alkyl, C2-C3 alkenyl, and H, and
R' is a straight chain alkyl group.
In some embodiments, the cationic lipid is SM102 or lipid 5:
In some embodiments, the cationic lipid is a cationic lipid described in U.S. patent No. US10166298B2, the entire teachings of which are incorporated herein by reference.
In some embodiments, the cationic lipid is a compound of formula (06-I):
Or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:
One of L1 or L2 is -O(C═O)-、-(C═O)O-、-C(═O)-、-O-、-S(O)x-、-S-S-、-C(═O)S-、SC(═O)-、-NRaC(═O)-、-C(═O)NRa-、NRaC(═O)NRa-、-OC(═O)NRa- or-NRa C (═ O) O-, and the other of L1 or L2 is -O(C═O)-、-(C═O)O-、-C(═O)-、-O-、-S(O)x-、-S-S-、-C(═O)S-、SC(═O)-、-NRaC(═O)-、-C(═O)NRa-、NRaC(═O)NRa-、-OC(═O)NRa- or-NRa C (═ O) O-or a direct bond;
Each of G1 and G2 is independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene;
G3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenyl;
ra is H or C1-C12 alkyl;
r1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;
r3 is H, OR5、CN、-C(＝O)OR4、-OC(＝O)R4 or-NR5C(＝O)R4;
R4 is C1-C12 alkyl;
R5 is H or C1-C6 alkyl, and
X is 0, 1 or 2.
In some embodiments, the cationic lipid is a compound of table 5, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
Table 5.
In some embodiments, the cationic lipids of the present disclosure are the same as those disclosed in international application publication No. WO 2010/144740, the entire teachings of which are incorporated herein by reference. For example, the cationic lipid is a compound represented by the formula (07-I), also referred to as compound 07-I:
Preferably, the ionizable lipids used in the LNP according to the invention are selected from:
a compound of formula (01-I-O):
Wherein y and z are each independently integers from 4 to 6,
S is an integer of 2 to 4,
T is an integer from 1 to 3, and
R1 and R2 are each independently C12-C22 alkyl;
R4 is C3-C8 cycloalkyl;
R6 is hydrogen or hydroxy,
A compound of formula 05-I:
Wherein the method comprises the steps of
L is selected from 1,2, 3, 4 and 5;
m is selected from 5, 6, 7, 8 and 9;
M1 is-C (O) O-;
R4 is- (CH2)n OH) and n is selected from 1, 2, 3, 4 or 5;
m is-OC (O) -, and
R2 and R3 are both C6-10 alkyl,
A compound of formula (06-I):
Wherein the method comprises the steps of
L1 and L2 are-O (C ═ O) -;
Each of G1 and G2 is independently unsubstituted C4-C8 alkylene;
G3 is C3-C8 alkylene;
R1 and R2 are each independently C12-C22 alkyl;
R3 is H or OH,
Compounds of formula (02-V-B)
Wherein the method comprises the steps of
Each L1 is independently-OC (=o) R1;
each L2 is independently-OC (=o) R2;
R1 and R2 are each independently C6-C24 alkyl;
R3 is-OR6;
r6 is hydrogen;
z is an integer from 2 to 12;
x1 is an integer from 0 to 9;
y1 is an integer from 0 to 9;
compounds of formula (02-VI-F)
Wherein the method comprises the steps of
Each L1 is independently-OC (=o) R1;
each L2 is independently-OC (=o) R2;
r1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;
R3 is-OR6;
r6 is hydrogen;
z is an integer from 2 to 12;
y is an integer from 2 to 12;
x1 is an integer from 2 to 5;
Compounds of formula (02-V-F)
Wherein the method comprises the steps of
Each L1 is independently-OC (=o) R1;
each L2 is independently-OC (=o) R2;
R1 and R2 are each independently C6-C24 alkyl;
R3 is-OR6;
r6 is hydrogen;
z is an integer from 2 to 12;
x2 is an integer from 2 to 9;
x4 is an integer from 0 to 3;
y2 is an integer from 2 to 9;
y4 is an integer from 0 to 3,
Compounds of formula (03-I)
Wherein the method comprises the steps of
Each of G1 and G2 is independently C3-C8 alkylene;
Each L1 is independently-OC (=o) R1 OR-C (=o) OR1;
Each L2 is independently-C (=o) OR2 OR-OC (=o) R2;
R1 is independently C6-C24 alkyl;
R2 is independently C6-C24 alkyl;
G3 is C2-C12 alkylene;
r3 is C3-C8 cycloalkyl;
r4 is C1-C4 hydroxyalkyl;
n is 1 or 2;
m is 1 or 2, and
Compound 07-I:
more preferably, the ionizable lipids used in the LNP according to the invention are selected from the following compounds:
And
C. Polymer-bound lipids
In some embodiments, the LNP comprises a polymer-bound lipid. Polymers that may be incorporated into the polymer-bound lipids include polyamines, polyethers, polyamides, polyesters, polyurethanes, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethylenimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. For example, the polymer may include poly (caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly (lactic acid) (PLA), poly (L-lactic acid) (PLLA), poly (glycolic acid) (PGA), poly (lactic-co-glycolic acid) (PLGA), poly (L-lactic-co-glycolic acid) (PLLGA), poly (D, L-lactide) (PDLA), poly (L-lactide) (PLLA), poly (D, L-lactide-co-caprolactone-co-glycolide), poly (D, L-lactide-co-PEO-co-D, L-lactide), poly (D, L-lactide-co-PPO-co-D, L-lactide), polyalkylcyanoacrylate, polyurethane, poly L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly (L-glutamic acid), poly (hydroxy acid), polyanhydrides, polyorthoesters, poly (ester amides), polyamides, poly (ether esters), polycarbonates, poly (ethylene and poly (ethylene) glycols, such as polyethylene glycol), poly (ethylene glycol), such as PEO (PEO) oxide, poly (PEO) and poly (ethylene oxide) Polyalkylene terephthalates such as poly (ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters such as poly (vinyl acetate), polyvinyl halides such as poly (vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropyl celluloses, carboxymethyl celluloses, acrylic polymers such as poly (methyl (meth) acrylate) (PMMA), poly (ethyl (meth) acrylate), and poly (butyl (meth) acrylate), poly (isobutyl (meth) acrylate), poly (hexyl (meth) acrylate), poly (isodecyl (meth) acrylate), poly (lauryl (meth) acrylate), poly (phenyl (meth) acrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), poly (octadecyl acrylate) and copolymers and mixtures thereof), polydioxanone and copolymers thereof, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamer (poloxamer), poloxamine (polyoxamine), poly (o) esters, poly (butyric acid), poly (valeric acid), poly (lactide-co-caprolactone), trimethylene carbonate or polyvinylpyrrolidone.
In some embodiments, the polymer-bound lipid is a pegylated lipid (PEG lipid). Without being bound by theory, it is expected that the polymer-bound lipid component in the LNP may improve colloidal stability of the nanoparticle and/or reduce protein absorption. Exemplary pegylated lipids that can be used in conjunction with the present disclosure include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG-conjugated lipid may be PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, or PEG-modified dialkylglycerol. In some embodiments, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG 2000, or Chol-PEG2000.
In some embodiments, the pegylated lipid is a pegylated diacylglycerol (PEG-DAG) (such as 1- (monomethoxy-polyethylene glycol) -2, 3-dimyristoylglycerol (PEG-DMG)), a pegylated phosphatidylethanolamine (PEG-PE), a PEG succinic diacylglycerol (PEG-S-DAG) (such as 4-O- (2 ',3' -di (tetradecyloxy) propyl-1-O- (ω -methoxy (polyethoxy) ethyl) succinate (PEG-S-DMG)), a pegylated ceramide (PEG-cer) or a PEG dialkoxypropyl carbamate (such as ω -methoxy (polyethoxy) ethyl-N- (2, 3-di (tetradecyl) propyl) carbamate or 2, 3-di (tetradecyl) propyl-N- (ω -methoxy (polyethoxy) ethyl) carbamate).
In some embodiments, the pegylated lipid is present at a concentration in the range of 1.0 to 2.5 mole percent. In some embodiments, the polymer-bound lipid is present at a concentration of about 1.7 mole percent. In some embodiments, the polymer-bound lipid is present at a concentration of about 1.5 mole percent.
In some embodiments, the molar ratio of the ionizable lipid to the polymer-bound lipid is in the range of about 20:1 to about 100:1. In some embodiments, the molar ratio of the ionizable lipid to the polymer-bound lipid is in the range of about 25:1 to about 80:1. In some embodiments, the molar ratio of the ionizable lipid to the polymer-bound lipid is in the range of about 30:1 to about 60:1. In some embodiments, the molar ratio of the ionizable lipid to the polymer-bound lipid is in the range of about 30:1 to about 50:1.
In some embodiments, the pegylated lipid has the following structural formula:
Or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:
R12 and R13 are each independently a straight-chain or branched alkyl or alkenyl chain having from 10 to 30 carbon atoms, where the alkyl chain is optionally interrupted by one or more ester bonds, and
W has an average value in the range of 30 to 60.
In some embodiments, R12 and R13 are each independently a straight saturated alkyl chain containing from 12 to 16 carbon atoms. In other embodiments, the average w is in the range of 42 to 55, e.g., the average w is 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55. In some embodiments, the average w is about 49.
In some embodiments, the pegylated lipid has the following structural formula:
or a pharmaceutically salt thereof, wherein the average w is about 49.
D. Lipid stabilizing agent
In some embodiments, the LNP comprises a lipid stabilizing agent. In some embodiments, the lipid stabilizing agent comprises a sterol. In some embodiments, the lipid stabilizing agent comprises a corticosteroid. In some embodiments, the lipid stabilizing agent comprises two or more components. In some embodiments, the lipid stabilizing agent comprises a corticosteroid and a sterol. In some embodiments, the sterol is selected from the group consisting of cholesterol, fecal sterols, sitosterols, ergosterols, campesterols, stigmasterols, and brassicasterol. In some embodiments, the corticosteroid is selected from the group consisting of prednisolone (prednisolone), dexamethasone (dexamethasone), prednisone (prednisone), and hydrocortisone (hydrocortisone). In some embodiments, the lipid stabilizing agent comprises lycorine, lycopersicin, ursolic acid, or alpha-tocopherol. In some embodiments, the lipid stabilizing agent comprises one or more compounds selected from the group consisting of cholesterol, fecal sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, prednisolone, dexamethasone, prednisone, hydrocortisone, lycorine, lycopersicin, ursolic acid, and alpha-tocopherol. In some embodiments, the lipid stabilizing agent is cholesterol.
E. LNP comprising mRNA
In some embodiments, the LNPs disclosed herein further comprise a therapeutic payload. The payload may be any substance or compound having a therapeutic or prophylactic effect. In some embodiments, the therapeutic payload is a small molecule, cytotoxin, radioactive ion, chemotherapeutic compound, vaccine, or a compound that elicits an immune response.
In some embodiments, an LNP disclosed herein comprises a nucleic acid. In some embodiments, the nucleic acid is DNA. In some embodiments, the DNA is catalytic DNA, plasmid DNA, aptamer, or complementary DNA (cDNA). In some embodiments, the nucleic acid is RNA. In some embodiments, the RNA is messenger RNA (mRNA), antisense oligonucleotide, microrna (miRNA), miRNA inhibitor (e.g., an Da can be amanita (antagomir) or ampere amanita (antimir)), messenger RNA interfering complementary RNA (microrna), multivalent RNA, dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), antisense RNA, transfer RNA (tRNA), asymmetric interfering RNA (aiRNA), ribozyme, aptamer, or vector. In some embodiments, the RNA is an mRNA hybrid. In some embodiments, the nucleic acid is mRNA. In some embodiments, the mRNA encodes a protein. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the LNP comprises an RNAi agent or an RNAi-inducing agent. Preferably, the weight ratio of ionizable lipid to therapeutic payload is from 5:1 to 20:1.
F. compositions comprising Lipid Nanoparticles (LNPs)
The present disclosure includes compositions comprising the LNPs described herein. In some embodiments, the composition comprises a plurality of LNPs. In some embodiments, the plurality of LNPs has a polydispersity index (PDI) of 0.001 to 0.2. In some embodiments, the plurality of LNPs has a polydispersity index (PDI) of 0.001 to 0.1. In some embodiments, the LNP has a PDI of 0.005 to 0.05.
In some embodiments, some of the LNPs of the LNP composition comprise mRNA. In some embodiments, the mRNA encapsulation efficiency of the LNP composition (EE% -i.e., the percentage of individual LNPs in the composition that encapsulate the mRNA) is 70% to 100%. In some embodiments, the EE% of the LNP composition is 80 to 95%. In some embodiments, the EE% of the LNP composition is 85 to 95%. In some embodiments, the EE% of the LNP composition is 90 to 95%. In some embodiments, EE% is 80% or greater. In some embodiments, EE% is 85% or greater. In some embodiments, EE% is 90% or greater. In some embodiments, EE% is 95% or greater. In some embodiments, EE% is 80% -100%. In some embodiments, EE% is 85% -100%. In some embodiments, EE% is 90% -100%. In some embodiments, EE% is 95% -100%.
In some embodiments, the LNP composition is capable of increasing protein expression compared to a similar LNP composition that does not comprise a steroid-containing phospholipid.
It will be appreciated that in any embodiment describing the composition of LNP described herein, any of the mole percentages or mole ratios described above for the sterol-containing phospholipid, the ionizable lipid, the polymer-bound lipid, and the lipid stabilizing agent can be combined with one another such that each combination is contemplated as if each combination were specifically and individually disclosed.
G. methods of making LNP and compositions thereof
The LNP herein may be manufactured according to methods well known in the art.
In some embodiments, the method comprises dissolving lipid components (e.g., phospholipids, ionizable lipids, polymer-bound lipids, and lipid stabilizers) in a solvent. In some embodiments, the method comprises the steps of:
(a) Dissolving a lipid component (e.g., a phospholipid, an ionizable lipid, a polymer-bound lipid, and optionally a lipid stabilizing agent) in a solvent to produce a lipid mixture;
(b) Diluting mRNA in a solvent to produce a mRNA mixture, and
(C) The lipid mixture was mixed with the mRNA mixture to obtain an mRNA LNP mixture.
In some embodiments, the solvent in step (a) is a polar solvent. In some embodiments, the solvent in step (a) is an alcohol solvent. In some embodiments, the solvent in step (a) is methanol, ethanol, n-propanol, or isopropanol. In some embodiments, the solvent in step (a) is ethanol.
In some embodiments, the solvent in step (b) is an aqueous solvent. In some embodiments, the solvent in step (b) is an aqueous buffer. In some embodiments, the solvent in step (b) is a citrate buffer. In some embodiments, the citrate buffer has a citrate concentration of 5 to 100 mM. In some embodiments, the citrate buffer has a citrate concentration of 10 to 50 mM. In some embodiments, the aqueous solvent of step (b) has a pH of 2 to 6. In some embodiments, the aqueous solvent of step (b) has a pH of 3 to 5.
In some embodiments, the mixing of step (c) is performed at a lipid to mRNA weight ratio of 10:1 to 30:1. In some embodiments, the mixing of step (c) is performed at a lipid to mRNA volume ratio of 1:1 to 1:5. In some embodiments, the volume ratio is 1:2 to 1:4. In some embodiments, the volume ratio is about 1:3. In some embodiments, the mixing of step (c) is performed using a microfluidic device. In some embodiments, the microfluidic device has a flow rate of 9 to 30 mL/min.
In some embodiments, the mRNA mixture has an mRNA concentration of 1 to 3, 5, 7, 10, 12, 15, 20, 30, 40, or 50 mM. In some embodiments, the concentration is 3 to 5, 7, 10, 12, 15, 20, or 30mM. In some embodiments, the concentration is 5 to 7, 10, 12, 15, 20, or 30mM. In some embodiments, the concentration is 7 to 10, 12, 15, 20, or 30mM. In some embodiments, the concentration is 10 to 12, 15, 20, or 30mM. In some embodiments, the concentration is 12 to 15, 20, or 30mM. In some embodiments, the concentration is 15 to 20 or 30mM. In some embodiments, the concentration is 20 to 30mM. In some embodiments, the concentration is about 5, 7, 10, 12, 15, or 20mM. In some embodiments, the concentration is about 5, 7, 10, 12, or 15mM. In some embodiments, the concentration is about 10mM. In some embodiments, the concentration is about 12mM. In some embodiments, the concentration is about 15mM. In some embodiments, the concentration is about 20mM.
In some embodiments, the method further comprises step (d):
(d) The lipid nanoparticles were filtered through a sterile filter.
In some embodiments, the sterile filter is a 0.2 μm sterile filter.
Methods relating to Lipid Nanoparticles (LNP)
The present disclosure includes methods of utilizing the LNPs described herein that may be useful.
In some embodiments, a method is used to express a protein in a cell, wherein the method comprises introducing into the cell an LNP or a composition thereof as described above. In some embodiments, the cell is a mammalian cell. In some embodiments, the LNP or a composition thereof is administered systemically to the mammal. In some embodiments, the mammal is a human.
In some embodiments, the LNP composition is capable of increasing protein expression compared to a similar LNP composition that does not comprise a steroid-containing phospholipid.
V. kit comprising phospholipids containing sterol moieties
The present disclosure includes a kit comprising a phospholipid containing a sterol moiety and a package of the phospholipid. In some embodiments, the kit further comprises an ionizable lipid. In some embodiments, the kit further comprises a polymer-bound lipid. In some embodiments, the kit further comprises a lipid stabilizing agent. In some embodiments, the kit further comprises an ionizable lipid, a polymer-bound lipid, and a lipid stabilizing agent. In some embodiments, the ionizable lipid is a cationic lipid. In some embodiments, the polymer-bound lipid is a pegylated lipid. In some embodiments, the lipid stabilizing agent is cholesterol. In some embodiments, the kit further comprises a cationic lipid, a pegylated lipid, and cholesterol.
It is to be understood that any embodiment of a compound provided herein as set forth above, and any particular substituent and/or variable of a compound provided herein as set forth above, may independently be combined with other embodiments of a compound and/or substituents and/or variables to form embodiments not specifically set forth above. Furthermore, where a list of substituents and/or variables is listed for any particular group or variable, it is to be understood that each individual substituent and/or variable may be deleted from a particular embodiment and/or technical scheme and that the remaining list of substituents and/or variables is to be considered within the scope of embodiments provided herein.
It is to be understood that in this specification, combinations of substituents and/or variables of the formulae shown are permissible only if such contributions result in stable compounds.
Exemplary embodiments
The following exemplary embodiments are provided herein:
Embodiment 1. A Lipid Nanoparticle (LNP) comprising:
Phospholipids containing sterol moieties;
an ionizable lipid, and
Polymer-bound lipids.
Embodiment 2. The LNP of embodiment 1 wherein the phospholipid has a structure selected from the group consisting of:
And
Embodiment 3. The LNP of embodiment 1 or 2 wherein the phospholipid has the following structure:
embodiment 4. The LNP of embodiment 1 or 2 wherein the phospholipid has the following structure:
Embodiment 5. The LNP of any one of embodiments 1 to 4, wherein the LNP has a molar ratio of the ionizable lipid to the phospholipid of 20:1 to 2:1.
Embodiment 6. The LNP of embodiment 5, wherein the molar ratio of the ionizable lipid to the phospholipid is 15:1 to 5:1.
Embodiment 7. The LNP of any one of embodiments 1 to 4, wherein the ionizable lipid comprises 40 to 80mol% of the total amount of lipids in the LNP.
Embodiment 8. The LNP of embodiment 7 wherein the ionizable lipid comprises 50 to 70mol% of the total amount of the lipids in the LNP.
Embodiment 9. The LNP of any of embodiments 1 to 8, wherein the ionizable lipid is a cationic lipid.
Embodiment 10. The LNP of any of embodiments 1 to 8, wherein the ionizable lipid is a compound according to any of the formulae selected from 01-I, 01-II, 02-I, 02-II, 03-I, 03-II-a, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV, 05-I, 06-I, and the subformulae thereof, or wherein the ionizable lipid is a cationic lipid selected from the compounds listed in any of tables 1 to 5.
Embodiment 11. The LNP of any one of embodiments 1 to 10 wherein the polymer bound lipid comprises 1 to 2% of the total amount of lipids in the LNP.
Embodiment 12. The LNP of embodiment 11 wherein the polymer bound lipid comprises 1.5% of the total amount of the lipid in the LNP.
Embodiment 13. The LNP of any one of embodiments 1 to 12, wherein the LNP has a molar ratio of the polymer-bound lipid to the phospholipid of 1:5 to 1:10.
Embodiment 14. The LNP of any of embodiments 1 to 13 wherein the polymer-bound lipid is a pegylated lipid.
Embodiment 15. The LNP of any of embodiments 1 to 13, wherein the polymer-bound lipid is a pegylated lipid having the structure:
or a pharmaceutically acceptable salt thereof, wherein
R12 and R13 are each independently a straight-chain or branched alkyl or alkenyl chain having from 10 to 30 carbon atoms, where the alkyl chain is optionally interrupted by one or more ester bonds, and
W is an integer in the range of 30 to 60.
Embodiment 16. The LNP of any of embodiments 1 to 15, wherein the polymer-bound lipid is a pegylated lipid having the structure:
or a pharmaceutically acceptable salt thereof, wherein
W is an integer in the range of 30 to 60.
Embodiment 17. The LNP of embodiment 15 or 16 wherein w is an integer in the range of 45 to 55.
Embodiment 18. The LNP of embodiment 15 or 16, wherein w is about 49.
Embodiment 19. The LNP of any of embodiments 1 to 14, wherein the polymer-bound lipid is DMG-PEG or DMPE-PEG.
Embodiment 20. The LNP of any one of embodiments 1 to 19 further comprising a lipid stabilizing agent.
Embodiment 21. The LNP of embodiment 20, wherein the LNP has a molar ratio of the lipid stabilizing agent to the phospholipid of 10:1 to 1:4.
Embodiment 22. The LNP of embodiment 21, wherein the molar ratio of the lipid stabilizing agent to the phospholipid is from 5:1 to 1:1.
Embodiment 23. The LNP of embodiment 21, wherein the molar ratio of the lipid stabilizing agent to the phospholipid is from 4:1 to 3:1.
Embodiment 24. The LNP of any one of embodiments 20 to 23, wherein the lipid stabilizing agent comprises 5 to 50mol% of the total amount of lipids in the LNP.
Embodiment 25. The LNP of embodiment 24, wherein the lipid stabilizing agent comprises 8 to 40mol% of the total amount of lipids in the LNP.
Embodiment 26. The LNP of embodiment 24, wherein the lipid stabilizing agent comprises 10 to 30mol% of the total amount of lipids in the LNP.
Embodiment 27. The LNP of any one of embodiments 1 to 26, wherein the phospholipid comprises 1 to 30mol% of the total amount of lipids in the LNP.
Embodiment 28. The LNP of embodiment 27, wherein the phospholipid comprises 2 to 25mol% of the total amount of lipids in the LNP.
Embodiment 29. The LNP of embodiment 27, wherein the phospholipid comprises 3 to 20mol% of the total amount of lipids in the LNP.
Embodiment 30. The LNP of embodiment 27, wherein the phospholipid comprises 5 to 15mol% of the total amount of lipids in the LNP.
Embodiment 31. The LNP of embodiment 27, wherein the phospholipid comprises about 10mol% of the total amount of lipids in the LNP.
Embodiment 32. The LNP of any one of embodiments 1 to 31, wherein the LNP has a size of 50nm to 150nm as determined using dynamic light scattering.
Embodiment 33. The LNP of embodiment 32, wherein the size is 60nm to 140nm.
Embodiment 34. The LNP of embodiment 32, wherein the size is 80nm to 100nm.
Embodiment 35. The LNP of embodiment 32, wherein the size is 85nm to 95nm.
Embodiment 36. The LNP of any one of embodiments 1 to 35, wherein the LNP encapsulates mRNA.
Embodiment 37. A composition comprising Lipid Nanoparticles (LNPs), wherein each LNP is an LNP as described in any one of embodiments 1 to 36.
Embodiment 38 the composition of embodiment 37, wherein at least 80% of the LNP encapsulates mRNA.
Embodiment 39 the composition of embodiment 37, wherein at least 85% of the LNP encapsulates mRNA.
Embodiment 40. A method for expressing a protein in a cell, the method comprising introducing into the cell an LNP as described in embodiment 36 or a composition as described in embodiment 38 or 39.
Embodiment 41. The method of embodiment 40, wherein the cell is a mammalian cell.
Embodiment 42. A method for delivering a protein to a subject, the method comprising administering to the individual an LNP as described in embodiment 36 or a composition as described in embodiment 38 or 39, wherein the mRNA encodes the protein.
Embodiment 43 the method of embodiment 42 wherein said LNP or said composition is administered systemically.
Embodiment 44. The method of embodiment 42, wherein the subject is a mammal.
Embodiment 45. The method of embodiment 42, wherein the subject is a human.
Embodiment 46. A Lipid Nanoparticle (LNP) comprising a phospholipid, wherein the phospholipid has a structure selected from the group consisting of:
And
Embodiment 47. The LNP of embodiment 46, wherein the phospholipid has the following structure:
embodiment 48. The LNP of embodiment 46, wherein the phospholipid has the structure:
Embodiment 49 the LNP of any of embodiments 46-48, further comprising an ionizable lipid.
Embodiment 50. The LNP of embodiment 49, wherein the LNP has a molar ratio of the ionizable lipid to the phospholipid of 20:1 to 2:1.
Embodiment 51. The LNP of embodiment 50 wherein the molar ratio of ionizable lipid to phospholipid is from 15:1 to 5:1.
Embodiment 52. The LNP of embodiment 49, wherein the ionizable lipid comprises 40 to 80mol% of the total amount of lipids in the LNP.
Embodiment 53. The LNP of embodiment 52, wherein the ionizable lipid comprises 50 to 70mol% of the total amount of said lipids in said LNP.
Embodiment 54 the LNP of any of embodiments 49-53, wherein the ionizable lipid is a compound according to a formula selected from the group consisting of 01-I, 01-II, 02-I, 02-II, 03-I, 03-II-A, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV, 05-I, 06-I, and any of the sub-formulae thereof, or wherein the ionizable lipid is a cationic lipid selected from the group consisting of the compounds listed in any of tables 1-5.
Embodiment 55. The LNP of any of embodiments 49-54, wherein the ionizable lipid is a cationic lipid.
Embodiment 56 the LNP of any one of embodiments 46-55 further comprising a polymer-bound lipid.
Embodiment 57. The LNP of embodiment 56 wherein the polymer bound lipid comprises 1 to 2% of the total amount of lipids in the LNP.
Embodiment 58. The LNP of embodiment 57, wherein the polymer-bound lipid comprises 1.5% of the total amount of the lipid in the LNP.
Embodiment 59. The LNP of any one of embodiments 46 to 58, wherein the LNP has a molar ratio of the polymer-bound lipid to the phospholipid of 1:5 to 1:10.
Embodiment 60. The LNP of any of embodiments 56-59 wherein the polymer-bound lipid is a pegylated lipid.
Embodiment 61 the LNP of any of embodiments 56-60 wherein the polymer-bound lipid is a pegylated lipid having the structure:
or a pharmaceutically acceptable salt thereof, wherein
R12 and R13 are each independently a straight-chain or branched alkyl or alkenyl chain having from 10 to 30 carbon atoms, where the alkyl chain is optionally interrupted by one or more ester bonds, and
W is an integer in the range of 30 to 60.
Embodiment 62. The LNP of any of embodiments 56-61 wherein the polymer-bound lipid is a pegylated lipid having the structure:
or a pharmaceutically acceptable salt thereof, wherein
W is an integer in the range of 30 to 60.
Embodiment 63 the LNP of embodiment 61 or 62, wherein w is an integer in the range of 45 to 55.
Embodiment 64 the LNP of embodiment 61 or 62, wherein w is about 49.
Embodiment 65 the LNP of any of embodiments 56-60, wherein the polymer-bound lipid is DMG-PEG or DMPE-PEG.
Embodiment 66. The LNP of any of embodiments 46-65, further comprising a lipid stabilizing agent.
Embodiment 67. The LNP of embodiment 66, wherein the LNP has a molar ratio of the lipid stabilizing agent to the phospholipid of 10:1 to 1:4.
Embodiment 68. The LNP of embodiment 67, wherein the molar ratio of the lipid stabilizing agent to the phospholipid is from 5:1 to 1:1.
Embodiment 69. The LNP of embodiment 67 wherein the molar ratio of the lipid stabilizing agent to the phospholipid is from 4:1 to 3:1.
Embodiment 70. The LNP of any of embodiments 66-69 wherein the lipid stabilizing agent comprises 5 to 50mol% of the total amount of lipids in the LNP.
Embodiment 71. The LNP of embodiment 70, wherein the lipid stabilizing agent comprises 8 to 40mol% of the total amount of lipids in the LNP.
Embodiment 72. The LNP of embodiment 70, wherein the lipid stabilizing agent comprises 10 to 30 mole% of the total amount of lipids in the LNP.
Embodiment 73. The LNP of any one of embodiments 46 to 72, wherein the phospholipid comprises 1 to 30mol% of the total amount of lipids in the LNP.
Embodiment 74. The LNP of embodiment 73, wherein the phospholipid comprises 2 to 25mol% of the total amount of lipids in the LNP.
Embodiment 75. The LNP of embodiment 73, wherein the phospholipid comprises 3 to 20mol% of the total amount of lipids in the LNP.
Embodiment 76. The LNP of embodiment 73, wherein the phospholipid comprises 5 to 15mol% of the total amount of lipids in the LNP.
Embodiment 77. The LNP of embodiment 73, wherein the phospholipid comprises about 10mol% of the total amount of lipids in the LNP.
Embodiment 78. The LNP of any one of embodiments 46 to 77, wherein the LNP has a size of 50nm to 150nm as determined using dynamic light scattering.
Embodiment 79. The LNP of embodiment 78, wherein the size is 60nm to 140nm.
Embodiment 80. The LNP of embodiment 78, wherein the size is 80nm to 100nm.
Embodiment 81. The LNP of embodiment 78, wherein the size is 85nm to 95nm.
Embodiment 82. The LNP of any one of embodiments 46 to 81, wherein the LNP encapsulates mRNA.
Embodiment 83. A composition comprising Lipid Nanoparticles (LNPs), wherein each LNP is an LNP of any one of embodiments 46 to 82.
Embodiment 84 the composition of embodiment 83, wherein at least 80% of the LNP encapsulates mRNA.
Embodiment 85 the composition of embodiment 83, wherein at least 85% of said LNP encapsulates mRNA.
Embodiment 86. A method for expressing a protein in a cell, the method comprising introducing into the cell an LNP as described in embodiment 82 or a composition as described in embodiment 84 or 85.
Embodiment 87. The method of embodiment 86, wherein the cell is a mammalian cell.
Embodiment 88 a method for delivering a protein to a subject, comprising administering to the individual an LNP as described in embodiment 82 or a composition as described in embodiment 84 or 85, wherein the mRNA encodes the protein.
Embodiment 89 the method of embodiment 88 wherein said LNP or said composition is administered systemically.
Embodiment 90 the method of embodiment 88 wherein the subject is a mammal.
Embodiment 91. The method of embodiment 88, wherein the subject is a human.
Examples
The application will be more fully understood with reference to the following examples. However, they should not be construed as limiting the scope of the application. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
General preparative High Performance Liquid Chromatography (HPLC) purification is carried out on a INERTSIL PRE-C8 OBD column on a Waters 2767 equipped with a Diode Array Detector (DAD), typically using water containing 0.1% trifluoroacetic acid (TFA) as solvent A and acetonitrile as solvent B.
LCMS analysis is performed on a typical liquid chromatography-mass spectrometry (LCMS) system. Chromatography was performed on SunFire C18, typically using water containing 0.1% formic acid as solvent a and acetonitrile containing 0.1% formic acid as solvent B.
Abbreviations OChemsPC refer to the following compounds:
Abbreviations PChemsPC refer to the following compounds:
abbreviations DChemsPC refer to the following compounds:
"DSPC" refers to distearoyl phosphatidylcholine. "Compound 01-1" refers to Compound 01-1 in Table 1."Chol" is an abbreviation for cholesterol. "DMG-PEG" refers to 1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000. ALC-0315 refers to compound 06-1, and MC3 refers to compound 07-I.
EXAMPLE 1 preparation of Compounds 02-1 and 02-3
Compound 02-1 was prepared according to the following scheme.
Compounds of formula (I) 02-1：1H NMR(400MHz,CDCl3)δ:0.86-0.90(m,12H),1.27-1.63(m,53H),1.97-2.01(m,2H),2.28-2.64(m,14H),3.52-3.58(m,2H),4.00-4.10(m,8H).LCMS:Rt:1.080min;MS m/z(ESI):826.0[M+H]+.
Compound 02-3 was prepared in a similar manner to compound 02-1 using the corresponding starting materials.
EXAMPLE 2 preparation of Compound 02-2
Compound 02-2 was prepared according to the following scheme.
Compounds of formula (I) 02-2：1H NMR(400MHz,CDCl3)δ:0.86-0.90(m,12H),1.28-1.67(m,54H),1.88-2.01(m,7H),2.28-2.56(m,18H),3.16-3.20(m,1H),3.52-3.54(m,2H),4.00-4.10(m,8H).LCMS:Rt:1.060min;MS m/z(ESI):923.0[M+H]+.
EXAMPLE 3 preparation of Compounds 02-4
Compound 02-4 was prepared according to the following scheme.
Compounds of formula (I) 02-4：1H NMR(400MHz,CDCl3)δ:0.86-0.90(m,9H),1.26-1.32(m,34H),1.41-1.49(m,4H),1.61-1.66(m,15H),2.00-2.03(m,1H),2.21-2.38(m,8H),2.43-2.47(m,4H),2.56-2.60(m,2H),3.50-3.54(m,2H),4.03-4.14(m,8H).LCMS:Rt:1.030min;MS m/z(ESI):798.0[M+H]+.
EXAMPLE 4 preparation of Compounds 02-9 and 02-14
Compounds 02-9 were prepared according to the following scheme.
Compounds of formula (I) 02-9：1H NMR(400MHz,CDCl3)δ:0.86-0.90(m,12H),1.28-1.30(m,33H),1.58-2.01(m,18H),2.30-2.54(m,18H),3.10-3.19(m,1H),3.52-3.68(m,8H),4.09-4.20(m,8H).LCMS:Rt:1.677min;MS m/z(ESI):927.7[M+H]+.
Compounds 02 to 14 were prepared in a similar manner to compounds 02 to 9 using the corresponding starting materials.
EXAMPLE 5 preparation of Compounds 02-10 and 02-11
Compounds 02-10 were prepared according to the following scheme.
Compounds of formula (I) 02-10：1H NMR(400MHz,CDCl3)δ:0.86-0.90(m,12H),1.26-1.41(m,48H),1.51-1.72(m,11H),1.94-2.03(m,1H),2.29-2.32(m,6H),2.41-2.91(m,5H),3.51-3.76(m,2H),3.96-4.10(m,6H).LCMS:Rt:1.327min;MS m/z(ESI):782.6[M+H]+.
Compounds 02 to 11 were prepared in a similar manner to compounds 02 to 10 using the corresponding starting materials.
EXAMPLE 6 preparation of Compounds 02-12
Compounds 02-12 were prepared according to the following scheme.
Compounds of formula (I) 02-12：1H NMR(400MHz,CDCl3)δ:0.86-0.89(m,18H),1.25-1.35(m,53H),1.41-1.48(m,8H),1.56-1.61(m,20H),1.95-2.01(m,2H),2.28-2.35(m,6H),2.43-2.46(m,4H),2.56-2.58(m,2H),3.51-3.54(m,2H),4.00-4.10(m,8H).LCMS:Rt:0.080min;MS m/z(ESI):1050.8[M+H]+.
EXAMPLE 7 preparation of Compounds 02-20
Compounds 02-20 were prepared according to the following procedure.
Compounds of formula (I) 02-20：1H NMR(400MHz,CDCl3)δ：0.86-0.90(m,9H),1.25-1.36(m,48H),1.41-1.48(m,5H),1.60-1.62(m,8H),1.97-2.00(m,1H),2.27-2.32(m,6H),2.43-2.46(m,4H),2.56-2.59(m,2H),3.52-3.54(m,2H),4.01-4.10(m,6H).LCMS：Rt:0.093min;MS m/z(ESI):782.6[M+H]+.
EXAMPLE 8 preparation of Compound 04-1
Compound 04-1 was prepared according to the following scheme.
LCMS-Rt of Compound 1-1 of example 8: 0.750min, MS m/z (ESI): 206.2[ M+H ]+.
LCMS-Rt of Compound 1-2 of example 8: 0.87min, MS m/z (ESI): 448.3[ M+H ]+.
LCMS-Rt of Compounds 1-3 of example 8: 1.360min, MS m/z (ESI): 616.5[ M+H ]+.
Compounds of formula (I) 04-1：1H NMR(400MHz,CDCl3)δ：0.79-0.83(m,6H),1.14-1.26(m,38H),1.47-1.61(m,6H),1.86-1.96(m,4H),2.51-2.58(m,4H),3.17(s,1H),3.32-3.44(m,5H),3.51-3.66(m,3H).LCMS:Rt:0.94min;MS m/z(ESI):526.5[M+H]+.
EXAMPLE 9 preparation of Compound 04-2
Compound 04-2 was prepared according to the following scheme.
LCMS-Rt of Compound 2-1 of example 9 1.340min, MS m/z (ESI): 630.5[ M+H ]+.
Compounds of formula (I) 04-2：1H NMR(400MHz,CDCl3)δ:0.86-0.90(m,6H),1.25-1.33(m,35H),1.50-1.69(m,7H),1.87-1.99(m,1H),2.00-2.08(m,2H),2.33(t,J＝7.6Hz,2H),2.56-2.81(m,4H),3.17-3.27(m,1H),3.38-3.48(m,3H),3.50-3.65(m,3H),5.08-5.14(m,1H).LCMS:Rt:1.180min;MS m/z(ESI):540.4[M+H]+.
EXAMPLE 10 preparation of Compound 04-7
Compound 04-7 was prepared according to the following scheme.
LCMS-LCMS of Compound 7-1 of example 10 Rt:0.780min, MS m/z (ESI): 427.4[ M+H ]+.
Compounds of formula (I) 04-7：1H NMR(400MHz,CDCl3)δ:0.86-0.90(m,9H),1.26-1.35(m,45H),1.41-1.67(m,7H),2.28-2.32(m,3H),2.36-2.70(m,11H),2.79-2.83(m,2H),3.35-3.46(m,4H),3.77-3.85(m,1H),3.96-3.97(m,2H).LCMS:Rt:1.220min;MS m/z(ESI):669.6[M+H]+.
EXAMPLE 11 preparation of Compound 04-8
Compound 04-8 was prepared according to the following scheme.
LCMS-LCMS of Compound 8-1 of example 11 Rt:0.730min, MS m/z (ESI) 371.3[ M+H ]+.
Compounds of formula (I) 04-8：1H NMR(400MHz,CDCl3)δ:0.86-0.90(m,9H),1.25-1.27(m,47H),1.40-1.49(m,4H),1.56-1.73(m,8H),2.30(t,J＝7.6Hz,3H),2.40-2.82(m,10H),3.32-3.38(m,1H),3.43-3.46(m,3H),3.70-3.80(m,1H),3.92-3.97(m,2H).LCMS:Rt:1.090min;MS m/z(ESI):709.6[M+H]+.
EXAMPLE 12 preparation of Compounds 04-65, 04-66 and 04-67
Compounds 04-65 were prepared according to the following procedure.
Compounds of formula (I) 04-65：1H NMR(400MHz,CCl3D):δ:0.79-0.83(m,12H),1.23-1.27(m,62H),1.29-1.37(m,2H),1.51-1.61(m,2H),1.76-1.93(m,7H),2.13-2.16(m,4H),2.17-2.25(m,3H),2.41-2.51(m,7H),3.05-3.06(m,1H),3.52-3.54(m.2H),3.92-4.03(m,4H).LCMS:Rt:0.588min;MS m/z(ESI):863.6[M+H]+.
Compounds 04-66 and 04-67 were prepared in a similar manner to compound 04-65 using the corresponding starting materials.
EXAMPLE 13 preparation of Compounds 04-68
Compound 04-68 was prepared according to the following procedure.
Compound 68-2 of example 131H NMR-1H NMR(400MHz,CDCl3)δ:0.86-0.90(m,12H),1.26-1.46(m,53H),1.56-1.62(m,2H),1.83(s,2H),1.96-2.02(m,1H),2.23-2.24(m,4H),3.64(s,2H),4.02-4.11(m,4H).
Compounds of formula (I) 04-68：1H NMR(400MHz,CDCl3)δ:0.83-0.92(m,12H),1.17-1.37(m,56H),1.38-1.45(m,2H),1.64-1.67(m,2H),1.70-1.86(m,6H),1.92-2.04(m,2H),2.19-2.26(m,4H),2.40-2.49(m,3H),2.57-2.65(m,2H),3.41-3.51(m,2H),3.97-4.12(m,4H).LCMS:Rt:0.080min;MS m/z(ESI):778.5[M+H]+.
EXAMPLE 14 preparation of Compounds 04-69, 04-79 and 04-80
Compounds 04-69 were prepared according to the following procedure.
LCMS-Rt of compound 69-1 of example 14: 1.290min, MS m/z (ESI): 750.7[ M+H ]+.
Compounds of formula (I) 04-69：1H NMR(400MHz,CDCl3)δ:0.83-0.92(m,12H),0.98-1.06(m,3H),1.17-1.47(m,52H),1.54-1.72(m,5H),1.78-2.06(m,8H),2.20-2.27(m,4H),2.37-2.46(m,4H),2.49-2.66(m,5H),3.01-3.12(m,1H),3.52-3.59(m,2H),3.98-4.11(m,4H).LCMS:Rt:0.093min;MS m/z(ESI):821.6[M+H]+.
Compounds 04-79 and 04-80 were prepared in a similar manner to compound 04-69 using the corresponding starting materials.
Example 15 initial screening for LNP containing steroid-containing Phospholipids
Briefly, a specified amount of lipid component was dissolved in ethanol at a specified molar ratio (see table 6). mRNA was diluted in 10 to 50mM citrate buffer (ph=3-5). LNP was prepared by mixing an ethanol lipid solution with an aqueous mRNA solution at a volume ratio of 1:3 using a microfluidic device at a total lipid to mRNA weight ratio of about 10:1 to 30:1, with a total flow rate in the range of 9-30 mL/min. Ethanol was thus removed and replaced with Dulbecco' sphosphate-buffered saline (DPBS) using dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 μm sterile filter.
Lipid nanoparticle size was determined by dynamic light scattering using Malvern Zetasizer Nano ZS (Malvern UK) using a 173 ° back scattering detection mode. The encapsulation efficiency of lipid nanoparticles was determined using the Quant-it Ribogreen RNA quantitative assay kit (Thermo FISHER SCIENTIFIC, UK) according to the manufacturer's instructions.
To measure the size and polydispersity index (PDI), the lipid nanoparticle formulation was diluted 20-fold in PBS and transferred 1mL in a measurement cuvette. LNP encapsulation efficiency (EE%) was determined using the Quant-it RiboGreen RNA analysis kit and LNP formulations were diluted to 0.5 μg/mL in Tris-EDTA and 0.1% Triton, respectively. To determine the free and total RNA fluorescence intensities, ribogreen reagents were diluted 200-fold with Tris-EDTA buffer and mixed in the same volume as the diluted LNP formulation. Fluorescence intensities were measured at room temperature in a Molecular Devices Spectramax iD spectrometer using excitation and emission wavelengths of 488nm and 525 nm. EE% was calculated based on the ratio of encapsulated RNA to total RNA fluorescence intensity.
TABLE 6 expression levels of LNP comprising steroid-containing phospholipids
Lipid nanoparticles encapsulating human erythropoietin (hEPO) mRNA were prepared as described above and administered systemically to 6-8 week old female ICR mice (Shanghai Sipuler-Bikai laboratory animal Co., ltd. (Xipuer-Bikai, shangghai)) by tail vein injection at a dose of 0.5 mg/kg. Mice were euthanized by CO2 excess 6 hours after administration, and blood samples were taken for hEPO measurement. Specifically, serum was separated from total blood by centrifugation at 5000g for 10 minutes at 4 ℃, flash frozen and stored at-80 ℃ for analysis. Serum hEPO levels were measured using a commercial kit (DEP 00, R & D systems) by ELISA analysis according to the manufacturer's instructions. The hEPO expression levels (μg/ml) measured from the test groups are plotted in fig. 1 and summarized in table 6.
As shown, DSPC was replaced with PChemsPC and the molar ratio was adjusted to significantly increase protein expression in EPO-LNP by a factor of 2-3.
EXAMPLE 16 expression level of LNP-loaded comprising steroid-containing Phospholipids
This study examined the in vivo expression levels of LNP formulations with DSPC or PChemsPC containing 45% -75% of compound 01-1. Lipid nanoparticles containing human erythropoietin (hEPO) mRNA were prepared as described in example 15. The results are shown in fig. 2 and table 7.
Of the DSPC LNPs tested, LNPs containing 45-60mol% of compound 01-1 produced the highest protein expression levels. For PCHEMSPC LNP, 60-75mol% of compound 01-1 produced the highest protein expression level.
TABLE 7 other expression levels of LNP containing steroid-containing phospholipids
EXAMPLE 17 screening of PChemsPC percent for optimal protein expression
Further screening was performed to examine the in vivo expression levels of LNP formulations containing 5-25mol% PChemSPC with varying amounts of Compound 01-1. Lipid nanoparticles containing human erythropoietin (hEPO) mRNA were prepared as described in example 15. The results are shown in fig. 3 and table 8.
Of PCHEMSPC LNP tested, 5-10mol% PChemSPC had the highest protein expression level. The optimum amount of PChemsPC varies with the amount of compound 01-1. When the mol% of PChemsPC exceeds 15%, the protein expression seems to be reduced.
Table 8. Other expression levels of LNP comprising steroid containing phospholipids.
EXAMPLE 18 screening of LNP compositions with steroid-containing phospholipids for optimal protein expression
This study examined the in vivo EPO expression levels of PCHEMSPC LNP with different compositions. Lipid nanoparticles containing human erythropoietin (hEPO) mRNA were prepared as described in example 15. In this study, the molar ratios of 50% -75% of compound 01-1, 18.5% -38.5% cholesterol, 5-15% PChemSPC were tested to achieve acceptable levels of protein expression. The corresponding LNP characterizations are listed in table 9.
Table 9. Other expression levels of LNP comprising steroid containing phospholipids with different molar ratios.
As can be seen from table 9, high protein expression can be produced in LNP formulations comprising 60-75mol% cationic lipid, 5-10mol% pchemspc, and 18.5-28.5mol% cholesterol, and their physicochemical properties (such as size, polydispersity index, and encapsulation efficiency) are acceptable.
Example 19 tissue-specific expression of nucleic acid molecules delivered in lnp formulations.
To study tissue biodistribution of LNP in mice, LNP formulations listed in table 10 containing mRNA encoding luciferase were prepared as described in example 15.
Table 10 lnp composition and physical characterization
Each formulation was administered systemically to 6-8 week old female ICR mice (Shanghai Sipuler-BiKai laboratory animal Co., ltd.) by tail vein injection at a dose of 0.25 mg/kg. After 6 hours, xenoLight D-fluorescein (potassium salt), a substrate for luciferase that catalyzes the production of luminescence, was subcutaneously administered to mice. Mice were then euthanized 15min later by CO2 excess. Mouse tissues were harvested and placed in a luminescence imaging scanner to measure the expression levels of luciferase in each tissue. Luminescence levels measured from harvested liver tissue are plotted in fig. 4, showing mean and Standard Deviation (SD) of at least five replicate animals per group.
As shown in fig. 4, LNP composed of steroid-containing phospholipids (e.g., PChemsPC, OChemsPC, DChemsPC) produced higher liver signals than DSPC controls. Compound 01-1 plus PChemsPC in a molar ratio of 50% -75% produced a higher hepatic luminescence signal.
The percent luminous intensity in the different tissues was calculated and plotted in fig. 5.
As shown in fig. 5, DSPC LNP produced 96% liver distribution and about 3% spleen distribution after injection, while steroid-containing phospholipid LNP showed 98-99% liver distribution and 0.4% spleen distribution. It can be seen that steroid-modified phospholipid LNPs show better hepatic tropism.
Example 20 characterization of sterol-modified phospholipid LNPs with different ionizable lipids
LNP formulations containing PChemsPC were prepared using different ionizable lipids. The LNP formulation consisted of 65% mole ratio of ionizable lipids, 10% mole ratio PChemsPC lipids, 23.5% mole ratio cholesterol-based lipids, and 1.5% mole ratio pegylated lipids. Lipid nanoparticles containing human erythropoietin (hEPO) mRNA were prepared as described in example 15.
Compound 01-1 plus the DSPC control was used in this study as the comparative group here. The corresponding LNP characterization for the different ionizable lipids is listed in table 11.
Table 11 LNP characterization for different ionizable lipids.
As shown in table 11, most of the ionizable lipids showed better protein expression levels than the compound 01-1 control, indicating that PChemsPC was widely compatible with most of the ionizable lipids. PChemsPC in combination with compound 02-3 can produce up to 2.8 fold increase in protein expression levels.
In addition, the effect of using PChemsPC for DSPC instead of protein expression levels in vivo was also studied for the commercial lipids ALC-0315 and MC 3. In this study, the LNP formulation consisted of 50% -65% mole of ionizable lipids, 10% mole of phospholipids, 23.5% -38.5% mole of cholesterol-based lipids, and 1.5% mole of pegylated lipids. The LNP characterization is listed in table 12.
Table 12. Physical characterization of alc-0315 and MC3 LNP.
| Lipid component | Molar ratio of | Size (nm) | PDI | EE(%) |
| ALC-0315/DSPC/Chol/DMG-PEG | 50/10/38.5/1.5 | 67.53 | 0.123 | 95.40 |
| ALC-0315/PChemsPC/Chol/DMG-PEG | 65/10/23.5/1.5 | 86.62 | 0.091 | 87.46 |
| MC3/DSPC/Chol/DMG-PEG | 50/10/38.5/1.5 | 72.76 | 0.102 | 98.80 |
| MC3/PChemsPC/Chol/DMG-PEG | 65/10/23.5/1.5 | 92.83 | 0.011 | 99.20 |
Fold changes in vivo hEPO expression levels are shown in figure 6.
As shown in fig. 6, replacement of DSPC with PChemsPC significantly improved protein expression levels in vivo by a factor of 1.6-1.8.
EXAMPLE 21 characterization of serum cytokines in vivo after injection of the steroid-containing phospholipid LNP
As an allogeneic substance, LNP injection would result in a significant increase in pro-inflammatory cytokines such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (INF-gamma), interferon-alpha (IFN-alpha), which may cause innate immune responses and produce undesirable side effects. This study assessed serum cytokine levels following systemic administration of LNP, comparing serum cytokines enhanced by DSPC LNP and PCHEMSPC LNP.
Lipid nanoparticles containing human erythropoietin (hEPO) mRNA were prepared as described in example 15 and administered systemically to 6-8 week old female ICR mice (shanghai-bikei laboratory animal limited) by tail vein injection at a dose of 0.5 mg/kg. Mice were euthanized by CO2 overdose 6 hours after administration and blood samples were taken for cytokine measurement. Specifically, serum was separated from total blood by centrifugation at 5000g for 10 minutes at 4 ℃, flash frozen and stored at-80 ℃ for analysis.
DSPC LNP consists of 50% mole ratio of ionizable lipids, 10% mole ratio of DSPC, 38.5% mole ratio of cholesterol, and 1.5% mole ratio of pegylated lipids. PCHEMSPC LNP consists of 65% by mole of ionizable lipids, 10% by mole of PChemsPC, 23.5% and 1.5% by mole of cholesterol and pegylated lipids, respectively. Several ionizable lipids (e.g., compound 01-1, lipid 5, SM-102, ALC-0315, compound 03-135) were tested in this study.
The results are shown in fig. 7 to 11. As shown in fig. 7-11, PCHEMSPC LNP can significantly reduce IL-6 levels compared to the corresponding DSPC control. In particular for ALC-0315LNP, a reduction in IL-6 levels of about 9-fold was observed, indicating that PCHEMSPC LNP has better safety after administration.
Example 22 characterization of serum cytokines in vivo after administration of steroid-containing phospholipid LNP with self-amplified mRNA (saRNA)
In this study, lipid nanoparticles containing human erythropoietin (hEPO) self-amplified mRNA were prepared as described in example 15. After tail vein injection, mice were euthanized by CO2 overdose 6 hours after administration, and blood samples were taken for cytokine measurement. Cytokine levels were measured and plotted in fig. 12 and 13.
As shown in fig. 12 and 13, PCHEMSPC LNP produced significantly lower IL-6, IFN- α, TNF- α levels.
EXAMPLE 23 characterization of sterol-modified phospholipid LNP with CD3-CD19 mRNA
In this study, DSPC LNP consisted of 50% compound 01-1, 10% DSPC, 38.5% cholesterol, and 1.5% pegylated lipid. PCHEMSPC LNP consists of compound 01-1 in a molar ratio of 65%, PChemsPC in a molar ratio of 10%, cholesterol and pegylated lipid in molar ratios of 23.5% and 1.5%, respectively.
Lipid nanoparticles encapsulating CD19-CD3 mRNA were prepared as described in example 15 and administered systemically to 6-8 week old female Balb/c mice (Shanghai Sipuler-BiKai laboratory animal Co., ltd.) by tail vein injection at a dose of 0.3 mg/kg. Mice were euthanized by CO2 overdose 6 hours after administration and blood samples were taken for antibody measurement. Specifically, serum was separated from total blood by centrifugation at 5000g for 10 minutes at 4 ℃, flash frozen and stored at-80 ℃ for analysis. Serum antibody levels are shown in fig. 14.
As shown in fig. 14, PCHEMSPC LNP can significantly improve CD3-CD19 antibody expression levels after administration.