Biodegradable lipids for delivery of active agents

Abstract

The problem to be solved by the present invention is to provide cationic lipids for oligonucleotide delivery which can provide a high drug: lipid ratio, which protect the nucleic acid from degradation and clearance in serum, which are suitable for systemic delivery, which can result in intracellular delivery of the nucleic acid, and which are well tolerated, provide an adequate therapeutic index, and treatment of patients with an effective dose of the nucleic acid is not associated with significant toxicity and / or risk to the patient.SOLUTION: The present invention relates to cationic lipids having one or more biodegradable groups located in the lipid portion (e.g., hydrophobic chain) of the cationic lipid. These cationic lipids can be incorporated into lipid particles for the delivery of active agents such as nucleic acids. The present invention also relates to a lipid particle comprising a neutral lipid, a lipid capable of reducing aggregation, a cationic lipid of the invention and optionally a sterol. The lipid particles may further comprise a therapeutic agent, such as a nucleic acid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 739,548, filed October 1, 2018, the entire contents of which are hereby incorporated by reference.

[0002] The present invention relates to biodegradable lipids and their use for the delivery of active agents, such as nucleic acids. [Background technology]

[0003] Therapeutic nucleic acids include, for example, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides, ribozymes, plasmids, immunostimulatory nucleic acids, antisense, antagomir, antimir, microRNA mimics, supermir, U1 adaptors, and aptamers. In the case of siRNA or miRNA, these nucleic acids can downregulate the intracellular levels of specific proteins through a process called RNA interference (RNAi). Because siRNA and miRNA constructs can be synthesized using any nucleotide sequence directed against a target protein, the therapeutic applications of RNAi are very broad. To date, siRNA constructs have demonstrated the ability to specifically downregulate target proteins in both in vitro and in vivo models. Furthermore, siRNA constructs are currently being evaluated in clinical trials.

[0004] However, two problems currently faced by siRNA or miRNA constructs are, first, their susceptibility to nuclease digestion in plasma, and, second, when administered systemically as free siRNA or miRNA, their limited ability to access intracellular compartments where they can bind to the protein RISC. To facilitate cellular uptake of oligonucleotides, lipid nanoparticles formed from cationic lipids along with cholesterol and PEG lipids and other lipid components of oligonucleotides (such as siRNA and miRNA) have been used.

[0005] There remains a need for improved cationic lipids and lipid nanoparticles for oligonucleotide delivery. Preferably, these lipid nanoparticles provide a high drug:lipid ratio, protect the nucleic acid from degradation and clearance in serum, are suitable for systemic delivery, and provide intracellular delivery of the nucleic acid. Furthermore, these lipid-nucleic acid particles should be well tolerated, provide an appropriate therapeutic index, and allow treatment of patients with effective doses of nucleic acid without significant toxicity and / or risk to the patient. Summary of the Invention [Means for solving the problem]

[0006] The present invention relates to improved lipid particles and improved cationic lipids.

[0007] One embodiment is a lipid particle comprising a biodegradable cationic lipid, a neutral lipid, a sterol, and an aggregation-reducing lipid (e.g., a PEG-modified lipid), wherein the molar ratio of the biodegradable cationic lipid to the sterol ranges from about 1.6:1 to about 2.0:1, and / or the molar ratio of the biodegradable cationic lipid to the neutral lipid ranges from about 5.5:1 to about 5.9:1. The inventors surprisingly found that lipid particles having a certain higher content of biodegradable cationic lipid relative to the amount of sterol and / or neutral lipid exhibit enhanced efficacy for delivery of an active agent (e.g., siRNA). In one embodiment, the biodegradable cationic lipid comprises a lipid moiety having one or more biodegradable groups, such as an ester group (-C(O)O- or -OC(O)-). In one preferred embodiment, the aggregation-reducing lipid is 1,2-dimyristoyl-sn-glycerol-methoxypolyethylene glycol (PEG-DMG), such as a PEG-DMG with an average polyethylene glycol molecular weight of 2000.

[0008] In a further embodiment, the molar ratio of biodegradable cationic lipid to sterol is about 1.7 to about 1.9:1, such as about 1.9:1. In another embodiment, the molar ratio of biodegradable cationic lipid to neutral lipid is in the range of about 5.5:1 to about 5.8:1, such as about 5.8:1.

[0009] In one embodiment, the lipid particles comprise about 55 to about 60 mol%, e.g., about 58 mol%, of a biodegradable cationic lipid (relative to 100 mol% of the lipid components in the lipid particles). The lipid particles may comprise about 28 to about 33 mol%, e.g., about 28 to about 32 mol%, of a sterol (relative to 100 mol% of the lipid components in the lipid particles). In one embodiment, the lipid particles comprise about 3 to about 12 mol%, e.g., about 5 to about 12 mol%, about 8 to about 12 mol%, or about 9 to about 11 mol%, of a neutral lipid (relative to 100 mol% of the lipid components in the lipid particles). In another embodiment, the lipid particles comprise about 10 mol% of a neutral lipid (relative to 100 mol% of the lipid components in the lipid particles). In yet another embodiment, the lipid particle comprises about 0.5 to about 10 mol %, for example, about 0.5 to about 5 mol %, or about 1 to about 3 mol %, of an aggregation-reducing lipid (e.g., a PEG-modified lipid) (based on 100 mol % of the lipid components in the lipid particle).

[0010] Another embodiment is a lipid particle comprising a biodegradable cationic lipid, a neutral lipid, a sterol, and an aggregation-reducing lipid (e.g., a PEG-modified lipid), wherein the lipid particle comprises about 55 to about 60 mol% of the biodegradable cationic lipid and about 33 to about 28 mol% of the sterol (relative to 100 mol% of the lipid components in the lipid particle). In one embodiment, the lipid particle comprises about 58 mol% of the biodegradable cationic lipid (relative to 100 mol% of the lipid components in the lipid particle). In another embodiment, the lipid particle comprises about 3 to about 12 mol% of the neutral lipid and about 0.5 to about 10 mol% of the aggregation-reducing lipid (relative to 100 mol% of the lipid components in the lipid particle). In yet another embodiment, the lipid particle comprises about 10 mol% of the neutral lipid (relative to 100 mol% of the lipid components in the lipid particle). In yet another embodiment, the lipid particle comprises about 2 mol% of the aggregation-reducing lipid (relative to 100 mol% of the lipid components in the lipid particle). In one embodiment, the lipid particles comprise (relative to 100 mol % of the lipid components in the lipid particles) about 55 to about 60 mol % cationic lipid, about 3 to about 12 mol % neutral lipid, about 28 to about 33 mol % sterol, and about 0.5 to about 10 mol % aggregation-reducing lipid. In another embodiment, the lipid particles comprise (relative to 100 mol % of the lipid components in the lipid particles) about 58% cationic lipid, about 10% neutral lipid, about 30% sterol, and about 2% aggregation-reducing lipid. In yet another embodiment, the lipid particles comprise (relative to 100 mol % of the lipid components in the lipid particles) about 55% cationic lipid, about 10% neutral lipid, about 33% sterol, and about 2% aggregation-reducing lipid. In a preferred embodiment, the aggregation-reducing lipid is 1,2-dimyristoyl-sn-glycerol-methoxypolyethylene glycol (PEG-DMG), e.g., PEG-DMG having an average polyethylene glycol molecular weight of 2000.

[0011] One embodiment is a compound of formula (A): [ka] or a salt thereof (e.g., a pharmaceutically acceptable salt thereof) having the formula: R' is absent, hydrogen, or alkyl (e.g., C1-C4 alkyl); Regarding R1 and R2, (i) R1 and R2 are each independently an optionally substituted alkyl, alkenyl, alkynyl, cycloalkylalkyl, heterocycle, or R10; (ii) R1 and R2 together with the nitrogen atom to which they are attached form an optionally substituted heterocyclic ring; or (iii) one of R1 and R2 is an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, or heterocycle, and the other forms a 4-10 membered heterocyclic ring or heteroaryl (e.g., a 6-membered ring) having (a) an adjacent nitrogen atom and (b) an (R)a group adjacent to the nitrogen atom; each occurrence of R is independently -(CR3R4)-; Each occurrence of R3 and R4 is independently H, halogen, OH, alkyl, alkoxy, -NH2, R10, alkylamino, or dialkylamino (in one preferred embodiment, each occurrence of R3 and R4 is independently H or C1-C4 alkyl); each occurrence of R10 is independently selected from PEG and polymers based on poly(oxazoline), poly(ethylene oxide), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide], and poly(amino acid), (i) the PEG or polymer is linear or branched, (ii) the PEG or polymer is polymerized with n subunits, (iii) n is a number average degree of polymerization from 10 to 200 units, and (iv) the compound of formula has at most two R10 groups (preferably at most one R10 group); A dashed line to Q is absent or a bond; if there is no break to Q, then Q is absent or is -O-, -NH-, -N(R5)-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R4)-, -N(R5)C(O)-, -SS-, -OC(O)O-, -ON=C(R5)-, -C(R5)=NO-, -OC(O)N(R5)-, -N(R5)C(O)N(R5)-, -N(R5)C(O)O-, -C(O)S-, -C(S)O- or -C(R5)=NOC(O)-; or When the dashed line to Q is a bond, (i) b is 0, and (ii) Q and its adjacent tertiary carbon (C*) form a substituted or unsubstituted, monocyclic or bicyclic heterocyclic group having 5 to 10 ring atoms (e.g., heteroatoms in heterocyclic groups are selected from O and S, preferably O); each occurrence of R5 is independently H or alkyl (e.g., C1-C4 alkyl); X is alkylene or alkenylene (e.g., C4-C20 alkylene or C4-C20 alkenylene); M1 is a biodegradable group (for example, -OC(O)-, -C(O)O-, -SC(O)-, -C(O)S-, -OC(S)-, -C(S)O-, -SS-, -C(R5)=N-, -N=C(R5)-, -C(R5)=NO-, -ON=C(R5)-, -C(O)(NR 5)-, -N(R5)C(O)-, -C(S)(NR5)-, -N(R5)C(O)-, -N(R5)C(O)N(R5)-, -OC(O)O-, -OSi(R5)2O-, -C(O)(CR3R4)C(O)O-, -OC(O)(CR3R4)C(O)- or [ka] (wherein R11 is C2-C8 alkyl or alkenyl); a is 1, 2, 3, 4, 5 or 6; b is 0, 1, 2 or 3; Z1 is a C6 to C14 branched alkyl group; Z2 is a C4-C20 alkenyl, where the alkenyl group can be optionally substituted with one or two fluorine atoms alpha to the double bond between the double bond and the terminus of Z2 (e.g., [ka] )

[0012] The R'R1R2N-(R)aQ-(R)b- group can be any of the head groups and salts thereof described herein, including those shown in Table 1 below. In a preferred embodiment, R'R1R2N-(R)aQ-(R)b- is (CH3)2N-(CH2)2-, (CH3)2N-(CH2)3-C(O)O-, (CH3)2N-(CH2)2-NH-C(O)O-, (CH3)2N-(CH2)2-OC(O)-NH-, or (CH3)2N-(CH2)3-C(CH3)=NO-. In a preferred embodiment, R'R1R2N-(R)aQ-(R)b- is (CH3)2N-(CH2)2-.

[0013] In one embodiment, R1 and R2 are both alkyl (e.g., methyl, ethyl, or a combination thereof). In one embodiment, R1 and R2 are both methyl. In another embodiment, one of R1 and R2 is methyl and the other of R1 and R2 is ethyl.

[0014] In a further embodiment, a is 2. In another embodiment, b is 0. In another embodiment, Q is absent. In yet another embodiment, a is 2, b is 0, and Q is absent. In yet another embodiment, a is 4, b is 0, and Q is -O-.

[0015] In another embodiment, X is -(CH2)n-, where n is 4 to 20, e.g., 4 to 18, 4 to 16, or 4 to 12. In one embodiment, n is 4, 5, 6, 7, 8, 9, or 10. In one embodiment, X is -(CH2)7-9-. In an exemplary embodiment, X is -(CH2)7-. In an exemplary embodiment, X is -(CH2)8-. In an exemplary embodiment, X is -(CH2)9-.

[0016] In further embodiments, M is -OC(O)- or -C(O)O-. For example, in one embodiment, M is -C(O)O-. In another embodiment, M is -OC(O)-.

[0017] In another embodiment, Z1 is a C6-C10 branched alkyl group, such as -CH(CH2CH3)(CH2CH2CH2CH3), -CH2CH(iPr)(CH2CH2iPr), or -CH2CH(n-Bu)2.

[0018] In another embodiment, Z2 is a C19 alkenyl containing one or two double bonds. For example, Z2 is -(CH2)9CH=CHCH2CH=CH(CH2)4CH3.

[0019] Yet another embodiment is [ka] [ka] [ka] and salts thereof (e.g., pharmaceutically acceptable salts thereof).

[0020] A further embodiment is a compound of formula (AI): [ka] or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein: s, t, u, v, and q are each independently 0, 1, 2, 3, 4, 5, 6, or 7; W is a head group (eg, a protonatable amine group having a pKa of about 4 to about 11, eg, about 4 to about 7, about 5 to about 7, or about 5.5 to about 6.8).

[0021] Suitable head groups include any of those described herein (see, eg, Table 1A).

[0022] In certain embodiments, the head group is (CH3)2N-(CH2)2-, (CH3)2N-(CH2)3-C(O)O-, (CH3)2N-(CH2)2-NH-C(O)O-, (CH3)2N-(CH2)2-OC(O)-NH-, or (CH3)2N-(CH2)3-C(CH3)=NO-.

[0023] In one embodiment, the head group is (CH3)2N-(CH2)2-.

[0024] In one embodiment, variable s is 3 to 5, for example 4.

[0025] In one embodiment, variable t is 4 to 6, for example 5.

[0026] In one embodiment, the variable q is 2 to 4, for example 3.

[0027] In one embodiment, the variable u is 0 to 2, for example 1.

[0028] In one embodiment, the variable v is 0 to 2, for example 1.

[0029] Yet another embodiment is a compound of formula (A-II): [ka] or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein: b is 0, 1, 2 or 3; s is 0, 1, 2, 3, 4 or 5; R200 is C12-C22 alkyl, C12-C22 alkenyl, or C12-C22 alkynyl; R100 is C5 to C15 alkyl, C5 to C15 alkenyl, or C5 to C15 alkynyl.

[0030] In one embodiment, b is 1.

[0031] In one embodiment, s is 2, 3 or 4, for example 3.

[0032] In one embodiment, R100 is C8-C12 alkyl.

[0033] In one embodiment, R200 is C12-C14 alkyl, C12-C14 alkenyl, or C12-C14 alkynyl.

[0034] In another embodiment, R200 is C18-C20 alkyl, C18-C20 alkenyl, or C18-C20 alkynyl.

[0035] These cationic lipids described herein can be incorporated into lipid particles. One embodiment is a lipid particle comprising cationic lipids such as those described above (including those of formula (A), (AI) and (A-II)). Each of the cationic lipids described herein comprises one or more biodegradable groups. The biodegradable groups are located in the lipid portion (e.g., hydrophobic chain) of the cationic lipid. These cationic lipids can be incorporated into lipid particles for delivering active substances such as nucleic acids (e.g., siRNA). The incorporation of biodegradable groups into the lipid results in faster metabolism and elimination of the lipid from the body after delivery of the active substance to the target area. As a result, these lipids are less toxic than similar lipids without biodegradable groups.

[0036] The lipid particles described herein may further comprise an active substance. Non-limiting examples of the active substance include nucleic acids, such as plasmids, immunostimulatory oligonucleotides, siRNA, antisense oligonucleotides, microRNA, antagomir, aptamers, and ribozymes. In a preferred embodiment, the nucleic acid is siRNA.

[0037] The lipid particles described herein can be incorporated into pharmaceutical compositions. One embodiment is a pharmaceutical composition comprising the lipid particles described herein and a pharmaceutically acceptable carrier. The lipid particles preferably comprise an active substance, such as a nucleic acid. In a preferred embodiment, the active substance is siRNA.

[0038] In another embodiment, the active agent is a nucleic acid and is an siRNA.

[0039] In yet another embodiment, there is provided a method of treating a disease or disorder characterized by overexpression of a polypeptide in a subject, comprising providing to the subject a pharmaceutical composition described herein. In one embodiment, the active agent is a nucleic acid selected from the group consisting of an siRNA, a microRNA, and an antisense oligonucleotide, wherein the siRNA, microRNA, or antisense oligonucleotide comprises a polynucleotide that specifically binds to a polynucleotide encoding the polypeptide, or a complement thereof.

[0040] Another embodiment is a method of treating a disease or disorder characterized by underexpression of a polypeptide in a subject, comprising providing to the subject a pharmaceutical composition described herein, hi a preferred embodiment, the active agent is a plasmid encoding the polypeptide or a functional variant or fragment thereof.

[0041] Another embodiment is a method of inducing an immune response in a subject, comprising providing to the subject a pharmaceutical composition described herein, hi a preferred embodiment, the active agent is an immunostimulatory oligonucleotide. [Brief explanation of the drawings]

[0042] [Figure 1] FIG. 1 is a bar graph showing relative Factor VII protein levels following administration of the lipid formulations described in Example 4. [Figure 2] FIG. 2 is a bar graph showing relative Factor VII protein levels following administration of the lipid formulations described in Example 4. [Figure 3] FIG. 3 is a bar graph showing relative Factor VII protein levels following administration of the lipid formulations described in Example 4. [Figure 4] FIG. 4 is a bar graph showing relative Factor VII protein levels following administration of the lipid formulations described in Example 4. [Figure 5] FIG. 5 is a graph showing relative Factor XII plasma levels after administration of the lipid formulations described in Example 5. [Figure 6] Figure 6A is a graph showing relative plasma levels of factor XII after administration of lipid formulations AF-094 (0.03, 0.1, and 0.3 mg / kg) and AF-011 (0.3 mg / kg) described in Example 6. Figure 6B is a graph showing relative plasma levels of factor XII after administration of lipid formulations AF-079 (0.3 mg / kg) and AF-011 (0.3 mg / kg) described in Example 6. [Figure 7] FIG. 7 is a graph showing relative factor XII plasma levels after administration of the lipid formulations AF-073 (0.03, 0.1, and 0.3 mg / kg) and AF-011 (0.3 mg / kg) described in Example 6. [Figure 8] FIG. 8 is a graph showing relative Factor XII plasma levels following administration of the lipid formulations AF-093 (0.03, 0.1, and 0.3 mg / kg) and AF-011 (0.3 mg / kg) described in Example 6. [Figure 9]FIG. 9 is a graph showing relative Factor XII plasma levels following administration of the lipid formulations AF-083 (0.1 and 0.3 mg / kg) and AF-011 (0.3 mg / kg) described in Example 6. DETAILED DESCRIPTION OF THE INVENTION

[0043] cationic lipids The cationic lipid may be any biodegradable cationic lipid known in the art, such as those described in International Publication Nos. 2011 / 153493, 2013 / 086322, 2013 / 086354 and 2013 / 086373, U.S. Patent Nos. 9,012,498, 9,061,063 and 9,463,247, and U.S. Patent Application Publication No. 2014 / 0308304, which are hereby incorporated by reference in their entirety. These biodegradable cationic lipids contain one or more biodegradable groups. As a result, the cationic lipids are metabolized and removed from the body more quickly after delivery of the active substance to the target area. As a result, the toxicity of these cationic lipids is substantially lower than that of similar cationic lipids without biodegradable groups. In one embodiment, one or more biodegradable groups are located in the central or distal section of the lipid portion (eg, the hydrophobic chain) of the cationic lipid.

[0044] In one embodiment, the biodegradable cationic lipid has the formula (A): [ka] or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein: R' is absent, hydrogen, or alkyl (e.g., C1-C4 alkyl); Regarding R1 and R2, (i) R1 and R2 are each independently an optionally substituted alkyl, alkenyl, alkynyl, cycloalkylalkyl, heterocycle, or R10; (ii) R1 and R2 together with the nitrogen atom to which they are attached form an optionally substituted heterocyclic ring; or (iii) one of R1 and R2 is an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, or heterocycle, and the other, together with (a) the adjacent nitrogen atom and (b) the (R)a group adjacent to the nitrogen atom, forms a 4- to 10-membered heterocyclic ring or heteroaryl (e.g., a 6-membered ring); each occurrence of R is independently -(CR3R4)-; Each occurrence of R3 and R4 is independently H, halogen, OH, alkyl, alkoxy, -NH2, R10, alkylamino, or dialkylamino (in one preferred embodiment, each occurrence of R3 and R4 is independently H or C1-C4 alkyl); each occurrence of R10 is independently selected from PEG and polymers based on poly(oxazoline), poly(ethylene oxide), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide], and poly(amino acid), wherein (i) the PEG or polymer is linear or branched, (ii) the PEG or polymer is polymerized with n subunits, (iii) n is a number average degree of polymerization between 10 and 200 units, and (iv) the compound of formula has at most two R10 groups (preferably at most one R10 group); A dashed line to Q is absent or a bond; If there is no break to Q, then Q is either absent or -O-, -NH-, -N(R5)-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R4)-, -N(R5)C(O)-, -SS-, -OC(O)O-, -ON=C(R5)-, -C(R5)=NO-, -OC(O)N(R5)-, -N(R5)C(O)N(R5)-, -N(R5)C(O)O-, -C(O)S-, -C(S)O- or -C(R5)=NOC(O)-; or When the dashed line to Q is a bond, (i) b is 0, and (ii) Q and its adjacent tertiary carbon (C*) form a substituted or unsubstituted monocyclic or bicyclic heterocyclic group having 5 to 10 ring atoms (e.g., the heteroatoms in the heterocyclic group are selected from O and S, preferably O); each occurrence of R5 is independently H or alkyl (e.g., C1-C4 alkyl); X is alkylene or alkenylene (e.g., C4-C20 alkylene or C4-C20 alkenylene); M1 is a biodegradable group (for example, -OC(O)-, -C(O)O-, -SC(O)-, -C(O)S-, -OC(S)-, -C(S)O-, -SS-, -C(R5)=N-, -N=C(R5)-, -C(R5)=NO-, -ON=C(R5)-, -C(O)(NR 5)-, -N(R5)C(O)-, -C(S)(NR5)-, -N(R5)C(O)-, -N(R5)C(O)N(R5)-, -OC(O)O-, -OSi(R5)2O-, -C(O)(CR3R4)C(O)O-, -OC(O)(CR3R4)C(O)- or [ka] (wherein R11 is C2-C8 alkyl or alkenyl); a is 1, 2, 3, 4, 5 or 6; b is 0, 1, 2 or 3; Z1 is a C6 to C14 branched alkyl group; Z2 is a C4-C20 alkenyl, which can be optionally substituted with one or two fluorine atoms alpha to the double bond between the double bond and the terminus of Z2 (e.g., [ka] )

[0045] The R'R1R2N-(R)aQ-(R)b- group can be any of the head groups and salts thereof described herein, including those shown in Table 1 below. In a preferred embodiment, R'R1R2N-(R)aQ-(R)b- is (CH3)2N-(CH2)2-, (CH3)2N-(CH2)3-C(O)O-, (CH3)2N-(CH2)2-NH-C(O)O-, (CH3)2N-(CH2)2-OC(O)-NH-, or (CH3)2N-(CH2)3-C(CH3)=NO-. In a preferred embodiment, R'R1R2N-(R)aQ-(R)b- is (CH3)2N-(CH2)2-.

[0046] In one embodiment, R1 and R2 are both alkyl (e.g., methyl, ethyl, or a combination thereof). In one embodiment, R1 and R2 are both methyl. In another embodiment, one of R1 and R2 is methyl and the other of R1 and R2 is ethyl.

[0047] In a further embodiment, a is 2. In another embodiment, b is 0. In another embodiment, Q is absent. In yet another embodiment, a is 2, b is 0, and Q is absent. In yet another embodiment, a is 4, b is 0, and Q is -O-.

[0048] In another embodiment, X is -(CH2)n-, where n is 4 to 20, e.g., 4 to 18, 4 to 16, or 4 to 12. In one embodiment, n is 4, 5, 6, 7, 8, 9, or 10. In one embodiment, X is -(CH2)7-9-. In an exemplary embodiment, X is -(CH2)7-. In an exemplary embodiment, X is -(CH2)8-. In an exemplary embodiment, X is -(CH2)9-.

[0049] In further embodiments, M is -OC(O)- or -C(O)O-. For example, in one embodiment, M is -C(O)O-. In another embodiment, M is -OC(O)-.

[0050] In another embodiment, Z1 is a C6-C10 branched alkyl group, for example, -CH(CH2CH3)(CH2CH2CH2CH3), -CH2CH(iPr)(CH2CH2iPr), or -CH2CH(n-Bu)2.

[0051] In another embodiment, Z2 is a C19 alkenyl having one or two double bonds. For example, Z2 can be -(CH2)9CH=CHCH2CH=CH(CH2)4CH3-.

[0052] Yet another embodiment is [ka] [ka] [ka] and salts thereof (e.g., pharmaceutically acceptable salts thereof).

[0053] In certain embodiments, the biodegradable group present in the cationic lipid is an ester (e.g., —C(O)O— or —OC(O)—), a disulfide (—SS—), an oxime (e.g., —C(H)═NO— or —ON═C(H)—), —C(O)—O—, —OC(O)—, —C(R5)═N—, —N═C(R5)—, —C(R5)═NO—, —ON═C(R5)—, — is selected from -OC(O)O-, -C(O)N(R), -N(R)C(O)-, -C(S)(NR)-, (NR)C(S)-, -N(R)C(O)N(R)-, -C(O)S-, -SC(O)-, -C(S)O-, -OC(S)-, -OSi(R)O-, -C(O)(CRR)C(O)O- or -OC(O)(CRR)C(O)-.

[0054] In one embodiment, the aliphatic group in one or both of the hydrophobic tails of the cationic lipid contains at least one carbon-carbon double bond.

[0055] Suitable cholesterol moieties for cationic lipids (including compounds of formula (A), (AI) and (A-II)) have the formula: [ka] It has.

[0056] Further embodiments include cationic lipids having a head group, one or more hydrophobic tails, and a linker between the head group and one or more tails. The head group can be an amine, e.g., an amine with a desired pKa. The pKa can be influenced by the structure of the lipid, particularly the nature of the head group; for example, the presence, absence, and position of functional groups, such as anionic functional groups, hydrogen bond donor functional groups, hydrogen bond acceptor functional groups, hydrophobic groups (e.g., aliphatic groups), hydrophilic groups (e.g., hydroxyl or methoxy), or aryl groups. The head group amine can be a cationic amine; primary, secondary, or tertiary amine; the head group can contain one amine group (monoamine), two amine groups (diamine), three amine groups (triamine), or more amine groups, such as in oligoamines or polyamines. The head group can also contain a functional group with a lower basic strength than an amine, such as an imidazole, pyridine, or guanidinium group. The head group can be zwitterionic. Other head groups are also suitable.

[0057] One or more hydrophobic tails may comprise two hydrophobic chains, which may be the same or different. The tails may be aliphatic, e.g., they may be either saturated or unsaturated but lacking aromatic rings and composed of carbon and hydrogen. The tails may be fatty acid tails. Some such groups include octanyl, nonanyl, decyl, lauryl, myristyl, palmityl, stearyl, α-linoleyl, stearidonyl, linoleyl, γ-linolenyl, aracadonyl, and oleyl. Other hydrophobic tails are also suitable.

[0058] Linkers can include, for example, glyceride linkers, acyclic glyceride analog linkers, or cyclic linkers (including spiro linkers, bicyclic linkers, and polycyclic linkers). Linkers can include functional groups such as ethers, esters, phosphates, phosphonates, phosphorothioates, sulfonates, disulfides, acetals, ketals, imines, hydrazones, or oximes. Other linkers and functional groups are also suitable.

[0059] In one embodiment, the cationic lipid is a racemic mixture.In another embodiment, the cationic lipid is enriched in one diastereomer, for example, the cationic lipid has at least 95%, at least 90%, at least 80% or at least 70% diastereomeric excess.In yet another embodiment, the cationic lipid is enriched in one enantiomer, for example, the lipid has at least 95%, at least 90%, at least 80% or at least 70% enantiomeric excess.In yet another embodiment, the cationic lipid is chirally pure, for example, a single optical isomer.In yet another embodiment, the cationic lipid is enriched in one optical isomer.

[0060] Where double bonds are present (e.g., carbon-carbon double bonds or carbon-nitrogen double bonds), there can be isomerism in configuration about the double bond (i.e., cis / trans or E / Z isomerism). Where a double bond configuration is illustrated in a chemical structure, it is understood that the corresponding isomers can also exist. The amount of isomers present can vary depending on the relative stability of the isomers and the energy required to convert between isomers. Thus, some double bonds, for practical purposes, exist in only a single configuration, while others (e.g., having similar relative stability and lower energy of conversion) can exist as an inseparable equilibrium mixture of configurations.

[0061] In some cases, the double bond unsaturation can be replaced by cyclic unsaturation. The cyclic unsaturation can be alicyclic unsaturation, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl groups. In some cases, the cyclic group can be a polycyclic group, such as a bicyclic group or a tricyclic group. The bicyclic group can be bridged, fused, or have a spiro structure.

[0062] In some cases, the double bond moiety may be replaced by a cyclopropyl moiety, e.g. [ka] teeth, [ka] For example, the moiety shown below has two carbon-carbon double bonds, each of which can be independently substituted with a cyclic moiety, such as a cyclopropyl moiety. Thus: [ka] The substituents for [ka] may be mentioned.

[0063] For further examples, [ka] The substituents for [ka] Examples include:

[0064] For further examples, [ka] The substituents for [ka] Examples include:

[0065] For further examples, [ka] The substituents for [ka] Examples include:

[0066] The cationic lipid comprises one or more biodegradable groups. The biodegradable group comprises one or more bonds that can undergo bond-breaking reactions in a biological environment, for example, in an organism, organ, tissue, cell, or organelle. Functional groups containing biodegradable bonds include, for example, esters, dithiols, and oximes. Biodegradation can be a factor that affects the clearance of a compound from the body when administered to a subject. Biodegradation can be measured in a cell-based assay, where a formulation containing a cationic lipid is exposed to cells and samples are collected at various time points. Lipid fractions can be extracted from the cells, separated, and analyzed by LC-MS. From the LC-MS data, the rate of biodegradation (e.g., as a t value) can be measured.

[0067] For example, the compound [ka] contains an ester bond in each aliphatic chain, which can undergo hydrolysis in the biological environment, for example, when exposed to lipases or esterases. The structure of the compound will, of course, affect the rate at which the compound undergoes biodegradation. Thus, related compounds, e.g. [ka] etc. are expected to exhibit different rates of biodegradation. A greater effect on the rate is expected from changes in the structure of the compound at the hydrolysis site. One modification that can affect the rate of hydrolysis, and thereby the rate of biodegradation and clearance from the subject's body, is to have a primary alcohol as the leaving group for the hydrolysis reaction, rather than a secondary alcohol.

[0068] In one embodiment, the cationic lipids of any of the embodiments described herein have an in vivo half-life (t) (e.g., in the liver, spleen, or plasma) of less than about 3 hours, e.g., less than about 2.5 hours, less than about 2 hours, less than about 1.5 hours, less than about 1 hour, less than about 0.5 hours, or less than about 0.25 hours. The cationic lipids preferably have a half-life sufficient to remain intact or to form stable lipid nanoparticles that effectively deliver the desired active pharmaceutical ingredient (e.g., nucleic acid) to its target, but then rapidly degrade to minimize any adverse effects on the subject. For example, in mice, the cationic lipids preferably have a t in the spleen of about 1 to about 7 hours.

[0069] In another embodiment, a cationic lipid of any of the embodiments described herein that contains a biodegradable group has an in vivo half-life (t) (e.g., in the liver, spleen, or plasma) that is less than about 10% (e.g., less than about 7.5%, less than about 5%, less than about 2.5%) of the same cationic lipid without the biodegradable group.

[0070] Some cationic lipids can be conveniently represented as a hydrophobic group coupled to a head group via a central moiety (such as a carbon atom). [ka] can be thought of as a combination of a head group, a central portion and two hydrophobic groups as follows: [ka]

[0071] Suitable cationic lipids include compounds composed of any combination of the head groups and hydrophobic groups listed below (in combination with a central moiety, such as a central carbon atom):

[0072] Some suitable heads include those shown in Table 1A:

[0073] [Table 1]

[0074] [Table 2]

[0075] Suitable head groups include, but are not limited to, those that are combinations of head groups and central carbon atoms from Table 1A. Other suitable head groups include those in Table 1B below:

[0076] [Table 3]

[0077] Some suitable hydrophobic tail groups include those shown in Table 2:

[0078] [Table 4]

[0079] [Table 5]

[0080] [Table 6]

[0081] Cationic lipids include those with alternative fatty acid groups and other dialkylamino groups, including those with different alkyl substituents (e.g., N-ethyl-N-methylamino-, N-propyl-N-ethylamino-, etc.). For embodiments in which R1 and R2 are both long-chain alkyl, alkenyl, alkynyl, or cycloalkylalkyl groups, these groups may be the same or different. Generally, lipids (e.g., cationic lipids) with less saturated acyl chains are more easily sized to less than about 0.3 microns, particularly for filter stabilization purposes. Cationic lipids containing unsaturated fatty acids with carbon chain lengths ranging from C10 to C20 are typical. Other scaffolds can also be used to separate the amino group (e.g., the amino group of the cationic lipid) and the fatty acid or fatty alkyl portion of the cationic lipid. Suitable scaffolds are known to those skilled in the art.

[0082] In certain embodiments, cationic lipid has at least one protonizable or deprotonizable group, and lipid is positively charged at a pH below physiological pH (for example, pH 7.4), and neutral at a second pH, preferably above physiological pH.Such lipid is also called cationic lipid.Of course, it is understood that the addition or removal of protons as a function of pH is an equilibrium process, and the reference to charged or neutral lipid refers to the nature of the main species, and does not require that all lipids exist in charged or neutral form.Lipid can have multiple protonizable or deprotonizable groups or can be zwitterionic.

[0083] In certain embodiments, the protonatable lipid (i.e., cationic lipid) has a pKa of the protonatable group in the range of about 4 to about 11. Typically, the pKa of the lipid, when incorporated into lipid particles, is about 4 to about 7, e.g., about 5 to 7, e.g., about 5.5 to 6.8. Such lipids are cationic at lower pH formulation stages, while the particles are primarily (if not completely) surface-neutral at physiological pH around pH 7.4. One advantage of a pKa in the range of about 4 to 7 is that at least some nucleic acids associated with the outer surface of the particles lose their electrostatic interactions at physiological pH and can be removed by simple dialysis; thus, the susceptibility of the particles to clearance is significantly reduced. Measurement of the pKa of lipids within lipid particles can be performed, for example, by using the fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) using the method described in Cullis et al., (1986) Chem Phys Lipid 40, 127-144, which is incorporated by reference in its entirety.

[0084] In certain embodiments, the lipid is a charged lipid. As used herein, the term "charged lipid" is meant to include lipids having one or two fatty acyl or fatty alkyl chains and a quaternary amino head group. The quaternary amine carries a permanent positive charge. The head group may optionally contain an ionizable group, such as a primary, secondary, or tertiary amine, that can be protonated at physiological pH. The presence of a quaternary amine may change the pKa of the ionizable group compared to the pKa of the group in a structurally similar compound that lacks a quaternary amine (e.g., the quaternary amine is replaced by a tertiary amine). In some embodiments, the charged lipid is referred to as an "amino lipid." See, for example, U.S. Provisional Patent Application No. 61 / 267,419, filed December 7, 2009, which is incorporated by reference in its entirety.

[0085] In one embodiment, the cationic lipid is a compound of formula (I) having a branched alkyl at the alpha position adjacent to the biodegradable group (between the biodegradable group and the tertiary carbon): [ka] or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein: R' is absent, hydrogen, or alkyl (e.g., C1-C4 alkyl); Regarding R1 and R2, (i) R1 and R2 are each independently an optionally substituted alkyl, alkenyl, alkynyl, cycloalkylalkyl, heterocycle, or R10; (ii) R1 and R2 together with the nitrogen atom to which they are attached form an optionally substituted heterocyclic ring; or (iii) one of R1 and R2 is an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, or heterocycle, and the other, together with (a) the adjacent nitrogen atom and (b) the (R)a group adjacent to the nitrogen atom, forms a 4- to 10-membered heterocyclic ring or heteroaryl (e.g., a 6-membered ring); each occurrence of R is independently -(CR3R4)-; each occurrence of R3 and R4 is independently H, halogen, OH, alkyl, alkoxy, -NH2, R10, alkylamino, or dialkylamino (in one preferred embodiment, each occurrence of R3 and R4 is independently H or C1-C4 alkyl); each occurrence of R10 is independently selected from PEG and polymers based on poly(oxazoline), poly(ethylene oxide), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide], and poly(amino acid), (i) the PEG or polymer is linear or branched, (ii) the PEG or polymer is polymerized with n subunits, (iii) n is a number average degree of polymerization from 10 to 200 units, and (iv) the compound of formula has at most two R10 groups (preferably at most one R10 group); A dashed line to Q is absent or a bond; if there is no break to Q, then Q is absent or is -O-, -NH-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R4)-, -N(R5)C(O)-, -SS-, -OC(O)O-, -ON=C(R5)-, -C(R5)=NO-, -OC(O)N(R5)-, -N(R5)C(O)N(R5)-, -N(R5)C(O)O-, -C(O)S-, -C(S)O- or -C(R5)=NOC(O)-; or When the dashed line to Q is a bond, (i) b is 0, and (ii) Q and its adjacent tertiary carbon (C*) form a substituted or unsubstituted, monocyclic or bicyclic heterocyclic group having 5 to 10 ring atoms (e.g., the heteroatoms in the heterocyclic group are selected from O and S, preferably O); each occurrence of R5 is independently H or alkyl (e.g., C1-C4 alkyl); X and Y are each independently alkylene or alkenylene (e.g., C4-C20 alkylene or C4-C20 alkenylene); M1 and M2 each independently represent a biodegradable group (e.g., -OC(O)-, -C(O)O-, -SC(O)-, -C(O)S-, -OC(S)-, -C(S)O-, -SS-, -C(R5)=N-, -N=C(R5)-, -C(R5)=NO-, -ON=C(R5)-, -C(O)(NR5)-, -N(R5)C(O)-, -C(S)(NR5)-, -N(R5)C(O)-, -N(R5)C(O)N(R5)-, -OC(O)O-, -OSi(R5)2O-, -C(O)(CR3R4)C(O)O-, -OC(O)(CR3R4)C(O)- or [ka] (wherein R11 is C2-C8 alkyl or alkenyl); each occurrence of Rz is independently C1-C8 alkyl (e.g., methyl, ethyl, isopropyl, n-butyl, n-pentyl, or n-hexyl); a is 1, 2, 3, 4, 5 or 6; b is 0, 1, 2 or 3; Z1 and Z2 are each independently a C8-C14 alkyl or a C8-C14 alkenyl, and the alkenyl group can optionally be substituted with one or two fluorine atoms alpha to the double bond between the double bond and the terminus of Z1 or Z2 (e.g., [ka] )

[0086] The R'R1R2N-(R)aQ-(R)b- group can be any of the head groups and salts thereof described herein, including those shown below in Table 1. In a preferred embodiment, R'R1R2N-(R)aQ-(R)b- is (CH3)2N-(CH2)3-C(O)O-, (CH3)2N-(CH2)2-NH-C(O)O-, (CH3)2N-(CH2)2-OC(O)-NH-, or (CH3)2N-(CH2)3-C(CH3)=NO-.

[0087] In one embodiment, R1 and R2 are both alkyl (eg, methyl).

[0088] In a further embodiment, a is 3. In another embodiment, b is 0.

[0089] In a further embodiment, a is 3, b is 0, and R is -CH2-. In yet a further embodiment, a is 3, b is 0, R is -CH2-, and Q is -C(O)O-. In another embodiment, R1 and R2 are methyl, a is 3, b is 0, R is -CH2-, and Q is -C(O)O-.

[0090] In another embodiment, X and Y are each independently -(CH2)n-, where n is 4 to 20, e.g., 4 to 18, 4 to 16, or 4 to 12. In one embodiment, n is 4, 5, 6, 7, 8, 9, or 10. In a representative embodiment, X and Y are -(CH2)6-. In another embodiment, X and Y are -(CH2)7-. In yet another embodiment, X and Y are -(CH2)9-. In yet another embodiment, X and Y are -(CH2)8-.

[0091] In further embodiments, M1 and M2 are each independently -OC(O)- or -C(O)O-. For example, in one embodiment, M1 and M2 are each -C(O)O-.

[0092] In one embodiment, the cationic lipid comprises a compound of formula (V) having an alkoxy or thioalkoxy (i.e., -S-alkyl) group substitution on at least one tail: [ka] or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein: R', R1, R2, R, R3, R4, R10, Q, R5, M1, M2, a and b are defined as in formula (I); X and Y are each independently alkylene (e.g., C6-C8 alkylene) or alkenylene, where the alkylene or alkenylene group is optionally substituted with 1 or 2 fluorine atoms alpha to the M1 or M2 group (e.g., [ka] ); Z1 and Z2 are each independently a C8-C14 alkyl or a C8-C14 alkenyl, wherein (i) at least one of Z1 and Z2, the C8-C14 alkyl or C8-C14 alkenyl, is substituted with one or more alkoxy (e.g., a C1-C4 alkoxy, such as -OCH3) or thioalkoxy (e.g., a C1-C4 thioalkoxy, such as -SCH3) groups, and (ii) the alkenyl group can be optionally substituted with one or two fluorine atoms alpha to the double bond between the double bond of Z1 or Z2 and the terminal end (e.g., [ka] )

[0093] In one embodiment, the alkoxy substitution at Z1 and / or Z2 is in a position beta from the M1 and / or M2 group.

[0094] In another embodiment, X and Y are each independently -(CH2)n- (wherein n is 4 to 20, e.g., 4 to 18, 4 to 16, or 4 to 12). In one embodiment, n is 4, 5, 6, 7, 8, 9, or 10. In a representative embodiment, X and Y are -(CH2)6-. In another embodiment, X and Y are -(CH2)7-. In yet another embodiment, X and Y are -(CH2)9-. In yet another embodiment, X and Y are -(CH2)8-.

[0095] The R'R1R2N-(R)aQ-(R)b- group can be any of the head groups and salts thereof described herein, including those shown below in Table 1. In a preferred embodiment, R'R1R2N-(R)aQ-(R)b- is (CH3)2N-(CH2)3-C(O)O-, (CH3)2N-(CH2)2-NH-C(O)O-, (CH3)2N-(CH2)2-OC(O)-NH-, or (CH3)2N-(CH2)3-C(CH3)=NO-.

[0096] In one embodiment, the cationic lipid has one or more fluoro substituents on at least one tail, at a position that is either alpha to a double bond or alpha to a biodegradable group, a compound of formula (VIA): [ka] or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein: R1, R2, R, a and b are as defined for formula (I); Q is absent or is -O-, -NH-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R)-, -N(R)C(O)-, -SS-, -OC(O)O-, -ON=C(R)-, -C(R)=NO-, -OC(O)N(R)-, -N(R)C(O)N(R)-, -N(R)C(O)O-, -C(O)S-, -C(S)O- or -C(R)=NOC(O)-; R' is absent, hydrogen, or alkyl (e.g., C1-C4 alkyl); each of R9 and R10 is independently a) a C12-C24 alkyl (e.g., C12-C20 alkyl), C12-C24 alkenyl (e.g., C12-C20 alkenyl), or C12-C24 alkoxy (e.g., C12-C20 alkoxy) having one or more biodegradable groups, and (b) optionally substituted with one or more fluorine atoms (i) at a position alpha to the biodegradable group and between the biodegradable group and the tertiary carbon atom marked with an asterisk (*), or (ii) at a position alpha to the carbon-carbon double bond and between the double bond and the end of the R9 or R10 group; each biodegradable group independently interrupts or is substituted at the end of the C12-C24 alkyl, alkenyl, or alkoxy group; (i) at least one of R9 and R10 contains a fluoro group; (ii) the compound is a moiety of: [ka] (In the formula, [ka] is an optional bond); (iii) The termini of R9 and R10 are separated from the tertiary carbon atom marked with an asterisk (*) by a chain of 8 or more atoms (e.g., 12 or 14 or more atoms).

[0097] In a preferred embodiment, the termini of R9 and R10 are separated from the tertiary carbon atom marked with an asterisk (*) by a chain of 18 to 22 carbon atoms (eg, 18 to 20 carbon atoms).

[0098] In another embodiment, the terminus of R9 and / or R10 has the formula -C(O)O-CF3.

[0099] In another embodiment, the cationic lipid has one or more fluoro substituents on at least one tail, either alpha to the double bond or alpha to the biodegradable group, a compound of formula (VIB): [ka] or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein: R', R1, R2, R, R3, R4, R10, Q, R5, M1, M2, a and b are defined as in formula (I); X and Y are each independently alkylene (e.g., C6-C8 alkylene) or alkenylene, where the alkylene or alkenylene group is optionally substituted with 1 or 2 fluorine atoms alpha to the M1 or M2 group (e.g., [ka] ); Z1 and Z2 are each independently a C8-C14 alkyl or a C8-C14 alkenyl, the C8-C14 alkenyl optionally being substituted with one or more fluorine atoms at the position alpha to the double bond (e.g., [ka] ) wherein at least one of X, Y, Z1 and Z2 contains a fluorine atom.

[0100] In one embodiment, at least one of Z1 and Z2 is substituted with two fluoro groups either alpha to the double bond or alpha to the biodegradable group, hi one embodiment, at least one of Z1 and Z2 has a terminal -CF3 group alpha to the biodegradable group (i.e., at least one of Z1 and Z2 terminates in a -C(O)OCF3 group).

[0101] For example, at least one of Z1 and Z2 may include one or more of the following moieties: [ka] In one embodiment, X and Y are each independently -(CH2)n-, where n is 4 to 20, e.g., 4 to 18, 4 to 16, or 4 to 12. In one embodiment, n is 4, 5, 6, 7, 8, 9, or 10. In one exemplary embodiment, X and Y are -(CH2)7-. In another exemplary embodiment, X and Y are -(CH2)9-. In yet another embodiment, X and Y are -(CH2)8-.

[0102] The R'R1R2N-(R)aQ-(R)b- group can be any of the head groups and salts thereof described herein, including those shown below in Table 1. In a preferred embodiment, R'R1R2N-(R)aQ-(R)b- is (CH3)2N-(CH2)3-C(O)O-, (CH3)2N-(CH2)2-NH-C(O)O-, (CH3)2N-(CH2)2-OC(O)-NH-, or (CH3)2N-(CH2)3-C(CH3)=NO-.

[0103] In one embodiment, the cationic lipid has an acetal group as the biodegradable group in at least one tail, a compound of formula (VII): [ka] or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein: R', R1, R2, R, R3, R4, R10, Q, R5, a and b are defined as in formula (I); X and Y are each independently alkylene (e.g., C6-C8 alkylene) or alkenylene, and the alkylene or alkenylene group is optionally substituted with one or two fluorine atoms (e.g., alpha to the M1 or M2 group). [ka] ); M1 and M2 each independently represent a biodegradable group (e.g., -OC(O)-, -C(O)O-, -SC(O)-, -C(O)S-, -OC(S)-, -C(S)O-, -SS-, -C(R5)=N-, -N=C(R5)-, -C(R5)=NO-, -ON=C(R5)-, -C(O)(NR5)-, -N(R5)C(O)-, -C(S)(NR5)-, -N(R5)C(O)-, -N(R5)C(O)N(R5)-, -OC(O)O-, -OSi(R5)2O-, -C(O)(CR3R4)C(O)O-, -OC(O)(CR3R4)C(O)- or [ka] (wherein R11 is C4-C10 alkyl or C4-C10 alkenyl); However, at least one of M1 and M2 is [ka] and; Z1 and Z2 are each independently a C4-C14 alkyl or a C4-C14 alkenyl, which alkenyl group can be optionally substituted with one or two fluorine atoms alpha to the double bond between the double bond of Z1 or Z2 and the terminal end (e.g., [ka] )

[0104] In one embodiment, each of M1 and M2 is [ka] is.

[0105] In another embodiment, X and Y are each independently -(CH2)n-, where n is 4 to 20, e.g., 4 to 18, 4 to 16, or 4 to 12. In one embodiment, n is 4, 5, 6, 7, 8, 9, or 10. In a representative embodiment, X and Y are -(CH2)6-. In another embodiment, X and Y are -(CH2)7-. In yet another embodiment, X and Y are -(CH2)9-. In yet another embodiment, X and Y are -(CH2)8-.

[0106] The R'R1R2N-(R)aQ-(R)b- group can be any of the head groups and salts thereof described herein, including those shown below in Table 1. In a preferred embodiment, R'R1R2N-(R)aQ-(R)b- is (CH3)2N-(CH2)3-C(O)O-, (CH3)2N-(CH2)2-NH-C(O)O-, (CH3)2N-(CH2)2-OC(O)-NH-, or (CH3)2N-(CH2)3-C(CH3)=NO-.

[0107] In another embodiment, the cationic lipid or salt thereof is (i) the central carbon atom, (ii) a nitrogen-containing head group directly attached to the central carbon atom; and (iii) two hydrophobic tails directly attached to the central carbon atom wherein each hydrophobic tail has the formula -Re-M-Rf, where Re is a C4-C14 alkyl or alkenyl, M is a biodegradable group, and Rf is a branched alkyl or alkenyl (e.g., a C10-C20 alkyl or a C10-C20 alkenyl), such that (i) the chain length of -Re-M-Rf is at most 20 atoms (i.e., the overall length of the tail from the first carbon atom after the central carbon atom to the end of the tail is at most 20), and (ii) the group -Re-M-Rf has at least 20 carbon atoms (e.g., at least 21 atoms). Optionally, the alkyl or alkenyl group in Re can be substituted with one or two fluorine atoms in the alpha position relative to the M1 or M2 group (e.g., [ka] Optionally, the alkenyl group in Rf can also be substituted with one or two fluorine atoms alpha to the double bond between the double bond and the terminus of Rf (e.g., [ka] ).

[0108] In one embodiment, the cationic lipids (such as those of Formulas I-VII) have asymmetric hydrophobic groups (i.e., the two hydrophobic groups have different chemical formulas). For example, the cationic lipids have the formula: [ka] or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein: G is a branched or unbranched C3-C15 alkyl, alkenyl, or alkynyl (e.g., n-C8 alkyl, n-C9 alkyl, or n-C10 alkyl); R12 is a branched or unbranched alkylene or alkenylene (e.g., C6 to C20 alkylene or C6 to C20 alkenylene, such as C12 to C20 alkylene or C12 to C20 alkenylene); M1 is a biodegradable group (for example, -OC(O)-, -C(O)O-, -SC(O)-, -C(O)S-, -OC(S)-, -C(S)O-, -SS-, -C(R5)=N-, -N=C(R5)-, -C(R5)=NO-, -ON=C(R5)-, -C(O)(NR 5)-, -N(R5)C(O)-, -C(S)(NR5)-, -N(R5)C(O)-, -N(R5)C(O)N(R5)-, -OC(O)O-, -OSi(R5)2O-, -C(O)(CR3R4)C(O)O-, -OC(O)(CR3R4)C(O)- or [ka] (wherein R11 is C2-C8 alkyl or alkenyl); R3 and R4 are defined as in formula (I); each occurrence of R5 is independently H or alkyl (e.g., C1-C4 alkyl); R13 is a branched or unbranched C3-C15 alkyl, alkenyl, or alkynyl; [ka] may have a protonatable group having a pKa of about 4 to about 13, more preferably about 5 to about 8 (for example, about 5 to about 7, or about 5 to about 6.5, or about 5.5 to about 6.5, or about 6 to about 6.5).

[0109] In one embodiment, the main group comprises a central moiety (e.g., a central carbon atom) to which both (i) the head group and (ii) the hydrophobic tail are directly attached. Exemplary central moieties include, but are not limited to, a central carbon atom, a central nitrogen atom, a central carbocyclic group, a central aryl group, a central hetrocyclic group (e.g., a central tetrahydrofuranyl group or a central pyrrolidinyl group), and a central heteroaryl group.

[0110] Representative [ka] As for [ka] (wherein n is 0 to 6), but is not limited thereto.

[0111] Representative asymmetric cationic lipids include: [ka] (wherein w is 0, 1, 2, or 3; x and y are each independently 1, 2, 3, 4, 5, 6, or 7).

[0112] In certain embodiments, each R is independently -(CR3R4)-, where R3 and R4 are each independently H or alkyl (e.g., C1-C4 alkyl). For example, in one embodiment, each R is independently -(CHR4)-, where each R4 is independently H or alkyl (e.g., C1-C4 alkyl). In another embodiment, each R is independently -CH2-, -C(CH3)2-, or -CH(iPr)-, where iPr is isopropyl. In another embodiment, each R is -CH2-.

[0113] In another embodiment, R5, at each occurrence, is hydrogen or methyl. For example, R5, at each occurrence, can be hydrogen.

[0114] In one embodiment, Q is absent, -C(O)O-, -OC(O)-, -C(O)N(R)-, -N(R)C(O)-, -SS-, -OC(O)O-, -C(R)=NO-, -OC(O)N(R)-, -N(R)C(O)N(R)-, -N(R)C(O)O-, -C(O)S-, -C(S)O-, or -C(R)=NOC(O)-. In one embodiment, Q is -C(O)O-.

[0115] In one embodiment, there is no dash to Q, b is 0, and R'R1R2N-(R)aQ- and the adjacent tertiary carbon (C*) can be selected from the group [ka] (wherein n is 1 to 4 (for example, n is 2)).

[0116] In one embodiment, there is no dash to Q, b is 0, and R'R1R2N-(R)aQ- and the adjacent tertiary carbon atom can be one of the following groups: [ka] wherein n is 1 to 4 (e.g., n is 2), and R1, R2, R, a, and b are as defined for formula (I). In one embodiment, a is 3.

[0117] In one embodiment, there is no dash to Q, b is 0, and R'R1R2N-(R)aQ- and the adjacent tertiary carbon atom can be one of the following groups: [ka] wherein n is 1 to 4 (e.g., n is 2), and R1, R2, R, a, and b are as defined for formula (I). In one embodiment, a is 0. For example, this group can be [ka] It could be.

[0118] In one embodiment, b is 0. In another embodiment, a is 2, 3, or 4, and b is 0. For example, in one embodiment, a is 3 and b is 0. In another embodiment, a is 3, b is 0, and Q is —C(O)O—.

[0119] In certain embodiments, the biodegradable group present in the cationic lipid is an ester (e.g., —C(O)O— or —OC(O)—), a disulfide (—SS—), an oxime (e.g., —C(H)═NO— or —ON═C(H)—), —C(O)—O—, —OC(O)—, —C(R5)═N—, —N═C(R5)—, —C(R5)═NO—, —ON═C(R5)—, is selected from -OC(O)O-, -C(O)N(R), -N(R)C(O)-, -C(S)(NR)-, (NR)C(S)-, -N(R)C(O)N(R)-, -C(O)S-, -SC(O)-, -C(S)O-, -OC(S)-, -OSi(R)2O-, -C(O)(CR3R4)C(O)O- or -OC(O)(CR3R4)C(O)-.

[0120] In preferred embodiments of the biodegradable cationic lipids described above, the logP value of the biodegradable cationic lipid is at least 10.1 (as calculated by software from Molinspiration Cheminformatics of Slovensky Grob, Slovak Republic, available at http: / / www.molinspiration.com / services / logp.html). More preferably, the logP value is at least 10.2 or 10.3.

[0121] In another preferred embodiment of the biodegradable cationic lipid described above, the biodegradable cationic lipid in the lipid nanoparticle has an HPLC retention time (relative to the retention time of cholesterol in the lipid nanoparticle), hereinafter referred to as tlipid-tchol, of at least 1.4. (HPLC parameters are provided in the Examples below. Unless otherwise specified, the lipid nanoparticle formulation used is that described in Example 31.) More preferably, the tlipid-tchol value is at least 1.75, 2.0, or 2.25.

[0122] Another embodiment is a biodegradable cationic lipid having (i) a logP value of at least 10.1 and / or a tlipid-tchol of at least 1.4 and (ii) one or more biodegradable groups (such as ester groups) located in the middle or distal section of the lipid portion (e.g., hydrophobic chain) of the cationic lipid.

[0123] In a preferred embodiment, the cationic lipid comprises a branched alkyl or branched alkenyl group in its biodegradable group. In another preferred embodiment, the logP of the cationic lipid is at least 10.2 or 10.3. In yet another preferred embodiment, the logP of the cationic lipid is at least 1.75, 2.0, or 2.25. The pKa of the cationic lipid is preferably about 4 to about 7 (e.g., 6.0 to 6.5).

[0124] In one embodiment, a cationic lipid having a logP value of at least 10.1 and / or a tripid-tchol of at least 1.4 comprises (a) a head group (preferably a nitrogen-containing head group, such as a head group described herein), (b) at least two hydrophobic tails, each of the formula -(hydrophobic chain)-(biodegradable group)-(hydrophobic chain), and (c) a linker group (e.g., a single central carbon atom) connecting the head group and the hydrophobic tail. The cationic lipid preferably has one, two, three, four or more of the following characteristics: (i) a pKa of about 4 to about 7 (e.g., 6.0 to 6.5); (ii) in at least one hydrophobic tail (and preferably all hydrophobic tails), the biodegradable group is separated from the end of the hydrophobic tail by about 6 to about 12 carbon atoms (e.g., 6 to 8 carbon atoms or 8 to 12 carbon atoms) for at least one hydrophobic tail (and preferably all hydrophobic tails); (iii) for at least one hydrophobic tail (and preferably all hydrophobic tails), the chain length from the linker group to the end of the hydrophobic tail is at most 21 atoms (e.g., at most 20 atoms, or about 17 to about 21 atoms, about 18 to about 20 atoms, or about 16 to about 18 atoms) (atoms in the linker group are not counted when calculating the chain length); (iv) for at least one hydrophobic tail (and preferably all hydrophobic tails), the total number of carbon atoms in the hydrophobic tails is from about 17 to about 26 (e.g., from about 19 to about 26 or from about 21 to about 26); (v) for at least one hydrophobic tail (and preferably all hydrophobic tails), the number of carbon atoms between the linker group and the biodegradable group ranges from about 5 to about 10 (e.g., 6 to 10 or 7 to 9); (vi) for at least one hydrophobic tail (and preferably all hydrophobic tails), the total number of carbon atoms between the linker group and the end of the hydrophobic tail is about 15 to about 20 (e.g., 16 to 20, 16 to 18, or 18 to 20); (vii) for at least one hydrophobic tail (and preferably all hydrophobic tails), the total number of carbon atoms between the biodegradable group and the end of the hydrophobic tail is from about 12 to about 18 (e.g., from 13 to 25); (viii) for at least one hydrophobic tail (and preferably all hydrophobic tails), the terminal hydrophobic chain in the hydrophobic tail is a branched alkyl or alkenyl group, e.g., the branching occurs at the α, β, γ, or δ position on the hydrophobic chain relative to the biodegradable group; (ix) when formulated as lipid nanoparticles (such as in Example 1), the in vivo half-life (t) of the cationic lipid in the liver is less than about 3 hours, e.g., less than about 2.5 hours, less than about 2 hours, less than about 1.5 hours, less than about 1 hour, less than about 0.5 hours, or less than about 0.25 hours; (x) when formulated as lipid nanoparticles (such as in Example 1), cationic lipids are cleared from the liver in mice, resulting in greater than 10-fold reduction in lipid levels relative to Cmax within the first 24 hours after administration; (xi) when formulated as lipid nanoparticles (such as in Example 1), cationic lipids are cleared from the spleen in mice, resulting in a significant reduction in lipid levels by more than 10-fold relative to Cmax within the first 168 hours after administration; (xii) When formulated as lipid nanoparticles (such as in Example 1), the cationic lipids are cleared from plasma with a terminal plasma half-life (t1 / 2β) of 48 hours or less in rodents and non-human primates.

[0125] Suitable cationic lipids include compounds having some or all of the above-mentioned properties in any combination. These properties provide cationic lipids that remain intact until delivery of an active substance, such as a nucleic acid, after which the hydrophobic tail is cleaved in vivo. For example, the compounds may have all of properties (i) to (viii) (in addition to the logP or lipid-tchol value). In another embodiment, the compounds have properties (i), (ii), (iii), and (viii). In yet another embodiment, the compounds have properties (i), (ii), (iii), (v), (vi), and (viii).

[0126] In the case of cationic lipid compounds containing an atom (such as nitrogen atom) carrying a positive charge, the compound also contains a negatively charged counterion.Counterion can be any anion, for example, organic or inorganic anion.Suitable examples of anion include but are not limited to tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate, α-glycerophosphate, halide (for example, chloride), sulfate, nitrate, bicarbonate and carbonate ion.In one embodiment, counterion is halide (for example, Cl).

[0127] Representative central moieties include, but are not limited to, central carbon atoms, central nitrogen atoms, central carbocyclic groups, central aryl groups, central hetrocyclic groups (e.g., central tetrahydrofuranyl groups or central pyrrolidinyl groups), and central heteroaryl groups. Additionally, the central moiety can include, for example, a glyceride linker, an acyclic glyceride analog linker, or a cyclic linker (including spiro linkers, bicyclic linkers, and polycyclic linkers). The central moiety can include functional groups such as ethers, esters, phosphates, phosphonates, phosphorothioates, sulfonates, disulfides, acetals, ketals, imines, hydrazones, or oximes. Other central moieties and functional groups are also suitable.

[0128] In one embodiment, the cationic lipid is a racemic mixture.In another embodiment, the cationic lipid is enriched in one diastereomer, for example, the cationic lipid has at least 95%, at least 90%, at least 80% or at least 70% diastereomeric excess.In yet another embodiment, the cationic lipid is enriched in one enantiomer, for example, the lipid has at least 95%, at least 90%, at least 80% or at least 70% enantiomeric excess.In yet another embodiment, the cationic lipid is chirally pure, for example, a single optical isomer.In yet another embodiment, the cationic lipid is enriched in one optical isomer.

[0129] When double bonds (e.g., carbon-carbon double bonds or carbon-nitrogen double bonds) are present, there can be isomerism in the configuration about the double bond (i.e., cis / trans or E / Z isomerism). When a double bond configuration is exemplified in a chemical structure, it is understood that the corresponding isomers can also exist. The amount of isomers present can vary depending on the relative stability of the isomers and the energy required to convert between the isomers. Thus, some double bonds, for practical purposes, can exist in only a single configuration, while others (e.g., with similar relative stabilities and lower conversion energies) can exist as an inseparable equilibrium mixture of configurations.

[0130] In some cases, the double bond unsaturation is replaced by a cyclic unsaturation. The cyclic unsaturation can be an alicyclic unsaturation, such as a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl group. In some cases, the cyclic group can be a polycyclic group, such as a bicyclic group or a tricyclic group. The bicyclic group can be bridged, fused, or have a spiro structure. In some cases, the double bond moiety can be replaced by a cyclopropyl moiety, such as [ka] teeth, [ka] may be replaced by

[0131] Other suitable tail groups include those of the formula -R12-M1-R13, where R12 is a C4-C14 alkyl or C4-C14 alkenyl, M1 is a biodegradable group as defined above, R13 is a branched alkyl or alkenyl (e.g., a C10-C20 alkyl or a C10-C20 alkenyl), and (i) the chain length of -R12-M1-R13 is at most 21 atoms (i.e., the total length of the tail from the first carbon after the tertiary carbon (marked with an asterisk) to the end of the tail is at most 21), and (ii) the group -R12-M1-R13 has at least 20 carbon atoms (e.g., at least 21 or 22 carbon atoms).

[0132] In a preferred embodiment, the chain length of -R12-M1-R13 is at most 21 (eg, at most 20). For example, the chain length can be from about 17 to about 24 or from about 18 to about 20.

[0133] In one embodiment, the total carbon atom content of each tail (-R12-M1-R13) is from about 17 to about 26. For example, the total carbon atom content can be from about 19 to about 26 or from about 21 to about 26.

[0134] In one embodiment, the tail has the formula: [ka] wherein R13 is an alkyl or alkenyl group having from about 13 to about 17 carbon atoms, and the total carbon length of the tail from the first carbon (top leftmost carbon atom) to the end of the tail is up to 20. Preferably, the tail has from about 22 to about 26 carbon atoms. In one embodiment, the maximum length of R13 from its attachment point to the ester group of the compound is 12 carbon atoms (e.g., the maximum length can be 11 carbon atoms). In a preferred embodiment, the branching in the alkyl or alkenyl group is in the δ-position or after the attachment point of R13 relative to the ester group. Suitable R13 groups include: [ka] These include, but are not limited to:

[0135] For example, cationic lipids include: [ka] or a salt thereof (e.g., a pharmaceutically acceptable salt thereof) (wherein R13 is selected from the groups described above).

[0136] Another example is the formula [ka] wherein R13 is an alkyl or alkenyl group having about 13 to about 15 carbon atoms, and the total carbon length of the tail from the first carbon of the tail (i.e., the left-most carbon atom connected to the tertiary carbon) to the end is a maximum of 20. Preferably, the tail has about 24 to about 26 carbon atoms. In one embodiment, the maximum length of R13 from its attachment point to the ester group of the compound is 10 carbon atoms (e.g., the maximum length can be 9 carbon atoms). In a preferred embodiment, the branching in the alkyl or alkenyl group is at the δ-position or after the attachment point of R13 relative to the ester group. Suitable R13 groups include: [ka] These include, but are not limited to:

[0137] For example, cationic lipids include: [ka] or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein R13 is selected from the groups described above.

[0138] The R13 group can be derived from natural products such as dihydrocitgronellol, lavandulol, phytol, or dihydrophytol. In one embodiment, the R13 group in the tail is a dihydrocitronellol group (either racemic or chirally pure): [ka] is.

[0139] For example, cationic lipids with dihydroitronellol groups are [ka] or a salt thereof (e.g., a pharmaceutically acceptable salt thereof).

[0140] In another embodiment, the R group in the above tail is a lavandulol group or a congener thereof, as shown below: [ka]

[0141] In another embodiment, the R13 group in the above tail is a phytol or dihydrophytol group: [ka] is.

[0142] For example, cationic lipids include: [ka] It could be.

[0143] formula: [ka] Cationic lipids are composed of two parts: a head group, a linker portion, and a hydrophobic chain such as: [ka] It can also be considered as a combination of

[0144] Various head groups, linker moieties, and hydrophobic chains I and II are listed below. Suitable cationic lipids include compounds composed of any combination of the head groups, linker, hydrophobic chain I, and hydrophobic chain II groups listed below.

[0145] [Table 7]

[0146] [Table 8]

[0147] [Table 9]

[0148] [Table 10]

[0149] [Table 11]

[0150] In one embodiment, the cationic lipid is the following compound and salts thereof, including pharmaceutically acceptable salts thereof: This cationic lipid is suitable for forming nucleic acid-lipid particles: [ka]

[0151] Alternatively, the formula [ka] For the compounds above having a head group of (X can be, for example, —C(O)O—), the head group can have one methylene unit between the X group (or other functional group) and the nitrogen atom. For example, the head group can be: [ka] It could be.

[0152] Cationic lipids include those with alternative fatty acid groups and other dialkylamino groups other than those shown, including those with different alkyl substituents (e.g., N-ethyl-N-methylamino- and N-propyl-N-ethylamino-).

[0153] The free form of the cationic lipid described herein and its pharmaceutically acceptable salt and stereoisomer are included in the present invention.The cationic lipid can be the protonated salt of an amine cationic lipid.The term "free form" refers to the amine cationic lipid in non-salt form.The free form can be regenerated by treating the salt with a suitable dilute aqueous base solution, such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium bicarbonate.

[0154] The pharmaceutically acceptable salt of cationic lipid can be synthesized from the cationic lipid that contains basic or acidic moiety by conventional chemical methods.Generally, the salt of basic cationic lipid is prepared by either ion exchange chromatography or by reacting free base with stoichiometric amount or excess amount of desired salt-forming inorganic or organic acid in suitable solvent or various combinations of solvents.Similarly, the salt of acidic compound is formed by reacting with suitable inorganic or organic base.

[0155] Thus, pharmaceutically acceptable salts of cationic lipids include non-toxic salts of cationic lipids such as those formed by reacting the basic cationic lipids with inorganic or organic acids, such as those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid, as well as those prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, and trifluoroacetic acid (TFA).

[0156] When cationic lipid is acidic, suitable " pharmaceutically acceptable salt " refers to the salt prepared from pharmaceutically acceptable non-toxic base, including inorganic base and organic base.The salt derived from inorganic base includes aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganese salt, manganese, potassium, sodium and zinc.In one embodiment, the base is selected from ammonium, calcium, magnesium, potassium and sodium. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, and tromethamine.

[0157] Note also that under physiological conditions, cationic lipids may potentially be internal salts or zwitterions, since deprotonated acidic moieties in the compound, such as carboxyl groups, may be anionic, and this charge may be balanced internally against the cationic charge of protonated or alkylated basic moieties, such as quaternary nitrogen atoms.

[0158] In addition to those specifically mentioned above, one or more additional cationic lipids that have a net positive charge at about physiological pH can also be included in the lipid particles and compositions described herein.Such cationic lipids include N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC"); N-(2,3-dioleyloxy)propyl-N,NN-triethylammonium chloride ("DOTMA"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTAP"); 1,2-dioleyloxy-3-trimethylaminopropane chloride salt ("DOTAP.Cl"); 3β-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol ("DC-Chol"), N-(1-( Examples of suitable cationic lipids include, but are not limited to, (2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate ("DOSPA"), dioctadecylamidoglycylcarboxyspermine ("DOGS"), 1,2-dileoyl-sn-3-phosphoethanolamine ("DOPE"), 1,2-dioleoyl-3-dimethylammonium propane ("DODAP"), N,N-dimethyl-2,3-dioleyloxy)propylamine ("DODMA"), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"). Additionally, many commercially available preparations of cationic lipids may be used, such as, for example, Lipofectin (including DOTMA and DOPE, available from GIBCO / BRL) and Lipofectamine (including DOSPA and DOPE, available from GIBCO / BRL).

[0159] Other lipid components The lipid particles and compositions described herein may also include one or more neutral lipids. The neutral lipid, if present, can be any of a number of lipid species that exist in either uncharged or neutral zwitterionic form at physiological pH. Examples of such lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. In one embodiment, the neutral lipid component is a lipid having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine). In one embodiment, the neutral lipid contains saturated fatty acids with carbon chain lengths ranging from C10 to C20. In another embodiment, the neutral lipid comprises mono- or di-unsaturated fatty acids with carbon chain lengths ranging from C10 to C20. Suitable neutral lipids include, but are not limited to, DSPC, DPPC, POPC, DOPE, DSPC, and SM.

[0160] The lipid particles and compositions described herein may also contain one or more lipids capable of reducing aggregation.Examples of lipids that reduce particle aggregation during formation include polyethylene glycol (PEG)-modified lipids (PEG lipids, such as PEG-DMG and PEG-DMA), monosialoganglioside Gm1 and polyamide oligomers ("PAO") (described in U.S. Patent No. 6,320,017, which is incorporated by reference in its entirety).Suitable PEG lipids include, but are not limited to, PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (such as PEG-CerC14 or PEG-CerC20) (such as those described in U.S. Patent No. 5,820,873, which is incorporated by reference in its entirety), PEG-modified dialkylamine and PEG-modified 1,2-diacyloxypropan-3-amine, PEG-modified diacylglycerol and dialkylglycerol, mPEG(mw2000)-diastearoylphosphatidylethanolamine (PEG-DSPE).

[0161] The lipid particles and compositions may include a sterol, such as cholesterol.

[0162] lipid particles In a further aspect, the present invention relates to lipid particles comprising one or more of the cationic lipids described herein.

[0163] Lipid particles include, but are not limited to, liposomes. As used herein, a liposome is a structure having a lipid-containing membrane enclosing an aqueous interior.

[0164] Another embodiment is a nucleic acid-lipid particle (e.g., SNALP) comprising a cationic lipid, a non-cationic lipid (such as a neutral lipid), optionally a PEG-lipid conjugate (such as a lipid for reducing lipid particle aggregation as discussed herein), optionally a sterol (e.g., cholesterol), and a nucleic acid. As used herein, the term "SNALP" refers to a stable nucleic acid-lipid particle. SNALPs correspond to particles made of lipids, in which a nucleic acid (e.g., an interfering RNA) is encapsulated within the lipid. In certain instances, SNALPs are useful for systemic application because they can exhibit extended circulatory life after intravenous (iv) injection, and they can accumulate at distant sites (e.g., sites physically distant from the administration site), where they can mediate the suppression of target gene expression. Nucleic acids can be complexed with condensing agents and encapsulated within SNALPs as described in International Publication No. WO 00 / 03683, the disclosure of which is incorporated herein by reference in its entirety.

[0165] For example, the lipid particle may comprise a cationic lipid, a fusogenic lipid (e.g., DPPC), a neutral lipid, cholesterol, and a PEG-modified lipid. In one embodiment, the lipid particle comprises the above lipid mixture in a molar ratio (based on 100% total moles of lipids in the lipid particle) of about 20-70% cationic lipid: 0.1-50% fusogenic lipid: 5-45% neutral lipid: 20-55% cholesterol: 0.5-15% PEG-modified lipid.

[0166] In another embodiment of the lipid particle, the cationic lipid is present in a molar percentage between about 20% and about 60% (based on 100% total moles of lipid in the lipid particle); the neutral lipid is present in a molar percentage between about 5% and about 25%; the sterol is present in a molar percentage between about 25% and about 55%; and the PEG lipid is PEG-DMA, PEG-DMG, or a combination thereof, and is present in a molar percentage between about 0.5% and about 15%.

[0167] In certain embodiments, the molar lipid ratio for (mol % cationic lipid / DSPC / Chol / PEG-DMG or PEG-DMA) is approximately 40 / 10 / 40 / 10, 35 / 15 / 40 / 10, or 52 / 13 / 30 / 5. This mixture can be further combined with a fusion-promoting lipid in a molar ratio of 0.1-50%, 0.1-50%, 0.5-50%, 1-50%, 5%-45%, 10%-40%, or 15%-35%. In other words, when a 40 / 10 / 40 / 10 mixture of lipid / DSPC / Chol / PEG-DMG or PEG-DMA is combined with a fusogenic peptide in a 50% molar ratio, the resulting lipid particles can have a total molar ratio of (mol % cationic lipid / DSPC / Chol / PEG-DMG or PEG-DMA / fusogenic peptide) of 20 / 5 / 20 / 5 / 50. In another embodiment, the neutral lipid, DSPC, is replaced with POPC, DPPC, DOPE, or SM in these compositions.

[0168] In one embodiment, the lipid particles comprise a cationic lipid, a neutral lipid, a sterol, and a PEG-modified lipid. In one embodiment, the lipid particles comprise about 25% to about 75% cationic lipid on a molar basis, for example, about 35% to about 65%, about 45% to about 65%, about 60%, about 57.5%, about 57.1%, about 50%, or about 40% on a molar basis. In one embodiment, the lipid particles comprise about 0% to about 15% neutral lipid on a molar basis, for example, about 3% to about 12%, about 5% to about 10%, about 15%, about 10%, about 7.5%, about 7.1%, or about 0% on a molar basis. In one embodiment, the neutral lipid is DPPC. In one embodiment, the neutral lipid is DSPC.

[0169] In one embodiment, the formulation comprises about 5% to about 50% on a molar basis of a sterol, e.g., about 15 to about 45%, about 20 to about 40%, about 48%, about 40%, about 38.5%, about 35%, about 34.4%, about 31.5%, or about 31% on a molar basis. In one embodiment, the sterol is cholesterol.

[0170] The lipid particles described herein may further comprise one or more therapeutic agents. In a preferred embodiment, the lipid particles comprise a nucleic acid (e.g., an oligonucleotide), such as an siRNA or miRNA.

[0171] In one embodiment, the lipid particles contain about 0.1% to about 20% PEG-modified lipid on a molar basis, for example, about 0.5 to about 10%, about 0.5 to about 5%, about 10%, about 5%, about 3.5%, about 1.5%, about 0.5%, or about 0.3% on a molar basis. In one embodiment, the PEG-modified lipid is PEG-DMG. In one embodiment, the PEG-modified lipid is PEG-c-DMA. In one embodiment, the lipid particles contain, on a molar basis, 25 to 75% cationic lipid, 0.5 to 15% neutral lipid, 5 to 50% sterol, and 0.5 to 20% PEG-modified lipid.

[0172] In one embodiment, the lipid particles comprise, on a molar basis, 35-65% cationic lipid, 3-12% neutral lipid, 15-45% sterol, and 0.5-10% PEG-modified lipid. In one embodiment, the lipid particles comprise, on a molar basis, 45-65% cationic lipid, 5-10% neutral lipid, 25-40% sterol, and 0.5-5% PEG-modified lipid. In one embodiment, the PEG-modified lipid comprises PEG molecules with an average molecular weight of 2,000 Da. In one embodiment, the PEG-modified lipid is PEG-distyrylglycerol (PEG-DSG). In a preferred embodiment, the PEG-modified lipid is 1,2-dimyristoyl-sn-glycerol-methoxypolyethylene glycol (PEG-DMG), such as PEG-DMG with an average polyethylene glycol molecular weight of 2,000.

[0173] In one embodiment, the lipid: siRNA ratio (weight [mg]:weight [mg]) is at least about 0.5:1, at least about 1:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 11:1, or at least about 33:1. In one embodiment, the lipid: siRNA ratio is about 1:1 to about 35:1, about 3:1 to about 15:1, about 4:1 to about 15:1, or about 5:1 to about 13:1. In one embodiment, the lipid: siRNA ratio is about 3:1 to about 12:1.

[0174] One embodiment is a lipid particle comprising a biodegradable cationic lipid, a neutral lipid, a sterol, and an aggregation-reducing lipid (e.g., a PEG-modified lipid), wherein the molar ratio of the biodegradable cationic lipid to the sterol ranges from about 1.6:1 to about 2.0:1, and / or the molar ratio of the biodegradable cationic lipid to the neutral lipid ranges from about 5.5:1 to about 5.9:1. The inventors have surprisingly found that lipid particles and / or neutral lipids having certain higher contents of biodegradable cationic lipid relative to the amount of sterol exhibit enhanced efficacy for delivery of an active agent (e.g., siRNA). In one embodiment, the biodegradable cationic lipid comprises a lipid moiety having one or more biodegradable groups, such as an ester group (-C(O)O- or -OC(O)-). In a preferred embodiment, the aggregation-reducing lipid is 1,2-dimyristoyl-sn-glycerol-methoxypolyethylene glycol (PEG-DMG), such as a PEG-DMG having an average polyethylene glycol molecular weight of 2000.

[0175] In a further embodiment, the molar ratio of biodegradable cationic lipid to sterol is from about 1.7 to about 1.9:1, such as about 1.9:1. In another embodiment, the molar ratio of biodegradable cationic lipid to neutral lipid is in the range of from about 5.5:1 to about 5.8:1, such as about 5.8:1.

[0176] In one embodiment, the lipid particles comprise about 55 to about 60 mol%, e.g., about 58 mol%, of a biodegradable cationic lipid (relative to 100 mol% of the lipid components in the lipid particles). The lipid particles may comprise about 28 to about 33 mol%, e.g., about 28 to about 32 mol%, of a sterol (relative to 100 mol% of the lipid components in the lipid particles). In one embodiment, the lipid particles comprise about 3 to about 12 mol%, e.g., about 5 to about 12 mol%, about 8 to about 12 mol%, or about 9 to about 11 mol%, of a neutral lipid (relative to 100 mol% of the lipid components in the lipid particles). In another embodiment, the lipid particles comprise about 10 mol% of a neutral lipid (relative to 100 mol% of the lipid components in the lipid particles). In yet another embodiment, the lipid particle comprises about 0.5 to about 10 mol %, for example, about 0.5 to about 5 mol %, or about 1 to about 3 mol %, of an aggregation-reducing lipid (e.g., a PEG-modified lipid) (based on 100 mol % of the lipid components in the lipid particle).

[0177] Another embodiment is a lipid particle comprising a biodegradable cationic lipid, a neutral lipid, a sterol, and an aggregation-reducing lipid (e.g., a PEG-modified lipid), wherein the lipid particle comprises about 55 to about 60 mol% of the biodegradable cationic lipid and about 33 to about 28 mol% of the sterol (relative to 100 mol% of the lipid components in the lipid particle). In one embodiment, the lipid particle comprises about 58 mol% of the biodegradable cationic lipid (relative to 100 mol% of the lipid components in the lipid particle). In another embodiment, the lipid particle comprises about 3 to about 12 mol% of the neutral lipid and about 0.5 to about 10 mol% of the aggregation-reducing lipid (relative to 100 mol% of the lipid components in the lipid particle). In yet another embodiment, the lipid particle comprises about 10 mol% of the neutral lipid (relative to 100 mol% of the lipid components in the lipid particle). In yet another embodiment, the lipid particle comprises about 2 mol% of the aggregation-reducing lipid (relative to 100 mol% of the lipid components in the lipid particle). In one embodiment, the lipid particles comprise (relative to 100 mol % of the lipid components in the lipid particles) about 55 to about 60 mol % cationic lipid, about 3 to about 12 mol % neutral lipid, about 28 to about 33 mol % sterol, and about 0.5 to about 10 mol % aggregation-reducing lipid. In another embodiment, the lipid particles comprise (relative to 100 mol % of the lipid components in the lipid particles) about 58% cationic lipid, about 10% neutral lipid, about 30% sterol, and about 2% aggregation-reducing lipid. In yet another embodiment, the lipid particles comprise (relative to 100 mol % of the lipid components in the lipid particles) about 55% cationic lipid, about 10% neutral lipid, about 33% sterol, and about 2% aggregation-reducing lipid. In a preferred embodiment, the aggregation-reducing lipid comprises 1,2-dimyristoyl-sn-glycerol-methoxypolyethylene glycol (PEG-DMG), such as PEG-DMG having an average polyethylene glycol molecular weight of 2000.

[0178] In one embodiment, the lipid particles are nanoparticles. In a further embodiment, the lipid particles have an average diameter of about 50 nm to about 300 nm, such as about 50 nm to about 250 nm, such as about 50 nm to about 200 nm.

[0179] In one embodiment, the lipid particles containing the cationic lipids of any of the embodiments described herein have an in vivo half-life (t) (e.g., in the liver, spleen, or plasma) of less than about 3 hours, such as less than about 2.5 hours, less than about 2 hours, less than about 1.5 hours, less than about 1 hour, less than about 0.5 hours, and less than about 0.25 hours.

[0180] In another embodiment, the in vivo half-life (t) (e.g., in the liver, spleen, or plasma) of a lipid particle containing a cationic lipid of any of the embodiments described herein is less than about 10% (e.g., less than about 7.5%, less than about 5%, less than about 2.5%) of that of the same cationic lipid without the biodegradable groups.

[0181] Further components The lipid particles and compositions described herein may further comprise one or more antioxidants. Antioxidants stabilize lipid particles and prevent, reduce, and / or inhibit degradation of cationic lipids and / or active substances present in the lipid particles. Antioxidants can be hydrophilic antioxidants, lipid-soluble antioxidants, metal chelators, primary antioxidants, secondary antioxidants, salts thereof, and mixtures thereof. In certain embodiments, the antioxidant comprises a metal chelator, such as EDTA or a salt thereof, alone or in combination with one, two, three, four, five, six, seven, eight, or more additional antioxidants, such as primary antioxidants, secondary antioxidants, or other metal chelators. In a preferred embodiment, the antioxidant comprises a metal chelator, such as EDTA or a salt thereof, in a mixture with one or more primary antioxidants and / or secondary antioxidants. For example, the antioxidant may include a mixture of EDTA or a salt thereof, a primary antioxidant such as α-tocopherol or a salt thereof, and a secondary antioxidant such as ascorbyl palmitate or a salt thereof. In one embodiment, the antioxidant includes at least about 100 mM citric acid or a salt thereof. Examples of antioxidants include, but are not limited to, hydrophilic antioxidants, lipid-soluble antioxidants, and mixtures thereof. Non-limiting examples of hydrophilic antioxidants include chelating agents (e.g., metal chelating agents) such as ethylenediaminetetraacetic acid (EDTA), citrate, ethylene glycol tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), diethylenetriaminepentaacetic acid (DTPA), 2,3-dimercapto-1-propanesulfonic acid (DMPS), dimercaptosuccinic acid (DMSA), cc-lipoic acid, salicylaldehyde isonicotinoylhydrazone (SIH), hexylthioethylamine hydrochloride (HTA), desferrioxamine, its salts, and mixtures thereof. Additional hydrophilic antioxidants include ascorbic acid, cysteine, glutathione, dihydrolipoic acid, 2-mercaptoethanesulfonic acid, 2-mercaptobenzimidazole sulfonic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, sodium disulfite, its salts and mixtures thereof.Non-limiting examples of fat-soluble antioxidants include vitamin E isomers, such as α-, β-, γ-, and δ-tocopherol and α-, β-, γ-, and δ-tocotrienol; polyphenols, such as 2-tert-butyl-4-methylphenol, 2-tert-butyl-5-methylphenol, and 2-tert-butyl-6-methylphenol; butylated hydroxyanisole (BHA) (e.g., 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole); butylhydroxytoluene (BHT); tert-butylhydroquinone (TBHQ); ascorbyl palmitate; rc-propyl gallate; salts thereof; and mixtures thereof. Suitable antioxidants and formulations containing such antioxidants are described in WO 2011 / 066651, which is hereby incorporated by reference.

[0182] In another embodiment, the lipid particle or composition contains the antioxidant EDTA (or a salt thereof), the antioxidant citric acid (or a salt thereof) or EDTA (or a salt thereof) in combination with one or more (e.g., mixtures thereof) primary and / or secondary antioxidants, such as α-tocopherol (or a salt thereof) and / or ascorbyl palmitate (or a salt thereof).

[0183] In one embodiment, the antioxidant is present in an amount sufficient to prevent, inhibit, or reduce degradation of cationic lipids present in the lipid particles. For example, the antioxidant may be present at a concentration of at least about or about 0.1 mM, 0.5 mM, 1 mM, 10 mM, 100 mM, 500 mM, 1 M, 2 M, or 5 M, or from about 0.1 mM to about 1 M, from about 0.1 mM to about 500 mM, from about 0.1 mM to about 250 mM, or from about 0.1 mM to about 100 mM.

[0184] The lipid particles and compositions described herein can further comprise an apolipoprotein. As used herein, the term "apolipoprotein" or "lipoprotein" refers to apolipoproteins and variants and fragments thereof known to those skilled in the art, and to apolipoprotein agonists, analogs, or fragments thereof as described below.

[0185] In a preferred embodiment, the active agent is a nucleic acid, such as an siRNA. For example, the active agent can be a nucleic acid encoding a product of interest, including, but not limited to, RNA, antisense oligonucleotide, antagomir, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA) (e.g., single-stranded, 17-25 nucleotides in length), transfer RNA (tRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), antigens, fragments thereof, proteins, peptides, vaccines, and small molecules, or mixtures thereof. In a more preferred embodiment, the nucleic acid is an oligonucleotide (e.g., 15-50 nucleotides in length (or 15-30 or 20-30 nucleotides in length)). The siRNA can have a double-stranded region that is, for example, 16-30 nucleotides in length. In another embodiment, the nucleic acid is an immunostimulatory oligonucleotide, a decoy oligonucleotide, a supermir, a miRNA mimic, or a miRNA inhibitor. A supermir refers to a single-stranded, double-stranded, or partially double-stranded oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), or both, or modifications thereof, which has a nucleotide sequence that is substantially identical to an miRNA and antisense to its target. miRNA mimics represent a class of molecules that can be used to mimic the gene-silencing ability of one or more miRNAs. Thus, the term "microRNA mimic" refers to a synthetic non-coding RNA (i.e., a miRNA not obtained by purification from an endogenous miRNA source) that can enter the RNAi pathway and regulate gene expression.

[0186] The nucleic acid present in lipid-nucleic acid particles can be in any form.Nucleic acid can be, for example, single-stranded DNA or RNA, double-stranded DNA or RNA, or DNA-RNA hybrid, including their chemically modified analogs.Non-limiting examples of double-stranded RNA include siRNA.Single-stranded nucleic acids include, for example, antisense oligonucleotides, ribozymes, microRNAs, and triplex-forming oligonucleotides.Lipid particles can also deliver nucleic acids complexed with one or more ligands.

[0187] Pharmaceutical Composition The lipid particles, particularly when associated with a therapeutic agent, can be formulated as a pharmaceutical composition further comprising, for example, a pharmaceutically acceptable diluent, excipient, or carrier, such as physiological brine or phosphate buffer.

[0188] The resulting pharmaceutical product can be sterilized by conventional, well-known sterilization techniques.The aqueous solution can then be packaged for use or filtered under aseptic conditions and lyophilized, and the lyophilized preparation is combined with a sterile aqueous solution before administration.The composition can contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH adjusting and buffering agents and osmolality adjusting agents, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride.In addition, the lipid suspension can contain a lipid protective agent to protect lipids from free radical and lipid peroxidase damage during storage.Lipid-soluble free radical quenchers, such as α-tocopherol and water-soluble iron-specific chelators, such as ferrioxamine, are suitable.

[0189] The concentration of lipid particles or lipid-nucleic acid particles in a pharmaceutical formulation can vary, for example, from less than about 0.01%, to about 0.05-5% by weight or at least about 0.05-5% by weight, to 10-30% by weight.

[0190] Manufacturing method Cationic lipids, lipid particles containing them, and methods of making pharmaceutical compositions containing cationic lipids and / or lipid particles are described, for example, in International Publication Nos. WO 2010 / 054406, WO 2010 / 054401, WO 2010 / 054405, WO 2010 / 054384, WO 2010 / 042877, WO 2010 / 129709, WO 2009 / 086558, and WO 2008 / 042973, and U.S. Patent Application Publication Nos. 2004 / 0142025, 2006 / 0051405, and 2007 / 0042031, each of which is incorporated by reference in its entirety.

[0191] For example, in one embodiment, a solution of one or more lipids (including any of the cationic lipid embodiments described herein) in an organic solution (e.g., ethanol) is prepared. Similarly, a solution of one or more active (therapeutic) agents (e.g., siRNA molecules or a 1:1 molar mixture of two siRNA molecules) in an aqueous buffer (e.g., citrate buffer) is prepared. The two solutions are mixed and diluted to form a colloidal suspension of siRNA-lipid particles. In one embodiment, the average particle size of the siRNA-lipid particles is about 80-90 nm. In a further embodiment, the dispersion may be filtered through a 0.45 / 2 micron filter, concentrated, and diafiltered by tangential flow filtration.

[0192] definition As used herein, the term "cationic lipid" includes lipids having one or two fatty acid or fatty aliphatic chains and an amino acid containing a head group that can be protonated to form a cationic lipid at physiological pH. In some embodiments, the cationic lipid is referred to as an "amino acid-conjugated cationic lipid."

[0193] The subject or patient to whom administration of the compound is an effective treatment regimen for a disease or disorder is preferably a human, but may be any animal, including a laboratory animal in the context of a clinical trial or screening or activity experiment. Thus, as can be readily recognized by those skilled in the art, the methods, compounds, and compositions of the present invention are particularly suitable for administration to any animal, particularly mammals, including but not limited to humans, domestic animals such as feline or canine subjects, livestock such as cattle, horses, goats, sheep, and pigs, wildlife (whether in the wild or in zoos), research animals such as mice, rats, rabbits, goats, sheep, pigs, dogs, and cats, avian species such as chickens, turkeys, and songbirds, i.e., for vertebrate medical use, but not limited to.

[0194] Many of the chemical groups recited in the general formula above are written in a particular order (e.g., -OC(O)-). The chemical groups are intended to be incorporated into the general formula in the order given unless otherwise indicated. For example, a general formula of the form -(R)i-(M1)k-(R)m- (where M1 is -C(O)O- and k is 1) refers to -(R)iC(O)O-(R)m- unless otherwise specified. When chemical groups are written in a particular order, it is understood that the reverse order is also contemplated unless otherwise specified. For example, in the general formula -(R)i-(M1)k-(R)m-, where M1 is defined as -C(O)NH- (i.e., -(R)iC(O)-NH-(R)m-), unless otherwise specified, compounds in which M1 is -NHC(O)- (i.e., -(R)i-NHC(O)-(R)m-) are also contemplated.

[0195] The term "biodegradable cationic lipid" refers to a cationic lipid having one or more biodegradable groups located in the central or distal section of the lipid portion (e.g., hydrophobic chain) of the cationic lipid. The incorporation of biodegradable groups into the cationic lipid results in faster metabolism and elimination of the cationic lipid from the body after delivery of the active pharmaceutical ingredient to the target area.

[0196] As used herein, the term "biodegradable group" refers to a group that includes one or more bonds that can undergo bond-breaking reactions in a biological environment, such as in an organism, organ, tissue, cell, or organelle. For example, a biodegradable group can be metabolized (e.g., by hydrolysis) by the mammalian, e.g., human, body. Some groups that contain biodegradable bonds include, but are not limited to, esters, dithiols, and oximes. Non-limiting examples of biodegradable groups are -OC(O)-, -C(O)O-, -SC(O)-, -C(O)S-, -OC(S)-, -C(S)O-, -SS-, -C(R)=N-, -N=C(R)-, -C(R)=NO-, -ON=C(R)-, -C(O)(NR)-, -N(R)C(O)-, -C(S)(NR)-, -N(R)C(O)-, -N(R)C(O)N(R)-, -OC(O)O-, -OSi(R)O-, -C(O)(CRR)C(O)O-, or -OC(O)(CRR)C(O)-.

[0197] As used herein, an "aliphatic" group is a non-aromatic group in which carbon atoms are linked in a chain and which is either saturated or unsaturated.

[0198] The terms "alkyl" and "alkylene" refer to straight-chain or branched-chain saturated hydrocarbon moieties. In one embodiment, an alkyl group is a straight-chain saturated hydrocarbon. Unless otherwise specified, an "alkyl" or "alkylene" group contains 1 to 24 carbon atoms. Representative saturated straight-chain alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl. Representative saturated branched alkyl groups include isopropyl, sec-butyl, isobutyl, tert-butyl, and isopentyl.

[0199] The term "alkenyl" refers to a straight- or branched-chain hydrocarbon moiety having one or more carbon-carbon double bonds. In one embodiment, an alkenyl group contains 1, 2, or 3 double bonds and is otherwise saturated. Unless otherwise specified, an "alkenyl" group contains 2 to 24 carbon atoms. Alkenyl groups include both cis and trans isomers. Representative straight- and branched-chain alkenyl groups include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, and 2,3-dimethyl-2-butenyl.

[0200] The term "alkynyl" refers to a straight or branched chain hydrocarbon moiety having one or more carbon-carbon triple bonds. Unless otherwise specified, an "alkynyl" group contains 2 to 24 carbon atoms. Representative straight and branched chain alkynyl groups include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, and 3-methyl-1-butynyl.

[0201] Unless otherwise specified, the terms "branched alkyl," "branched alkenyl," and "branched alkynyl" refer to an alkyl, alkenyl, or alkynyl group in which (1) one carbon atom in the group is bonded to at least three other carbon atoms and (2) is not a ring atom of a cyclic group. For example, a spirocyclic group in an alkyl, alkenyl, or alkynyl group is not considered a branch point.

[0202] Unless otherwise specified, the term "acyl" refers to a carbonyl group substituted with hydrogen, alkyl, partially saturated or fully saturated cycloalkyl, partially saturated or fully saturated heterocycle, aryl, or heteroaryl. For example, acyl groups include groups such as (C1-C20)alkanoyl (e.g., formyl, acetyl, propionyl, butyryl, valeryl, caproyl, and t-butylacetyl), (C3-C20)cycloalkylcarbonyl (e.g., cyclopropylcarbonyl, cyclobutylcarbonyl, cyclopentylcarbonyl, and cyclohexylcarbonyl), heterocyclic carbonyl (e.g., pyrrolidinylcarbonyl, pyrrolid-2-one-5-carbonyl, piperidinylcarbonyl, piperazinylcarbonyl, and tetrahydrofuranylcarbonyl), aroyl (e.g., benzoyl), and heteroaroyl (e.g., thiophenyl-2-carbonyl, thiophenyl-3-carbonyl, furanyl-2-carbonyl, furanyl-3-carbonyl, 1H-pyrroyl-2-carbonyl, 1H-pyrroyl-3-carbonyl, and benzo[b]thiophenyl-2-carbonyl).

[0203] The term "aryl" refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system. Unless otherwise specified, an "aryl" group contains 6 to 14 carbon atoms. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, anthracenyl, and pyrenyl.

[0204] The terms "cycloalkyl" and "cycloalkylene" refer to saturated monocyclic or bicyclic hydrocarbon moieties such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Unless otherwise specified, a "cycloalkyl" or "cycloalkylene" group contains from 3 to 10 carbon atoms.

[0205] The term "cycloalkylalkyl" refers to a cycloalkyl group that is bound to an alkyl group, which in turn is bound to the remainder of the molecule.

[0206] The term "heterocycle (or "heterocyclyl")" refers to a non-aromatic 5- to 8-membered monocyclic or 7- to 12-membered bicyclic or 11- to 14-membered tricyclic ring system, either saturated or unsaturated, containing 1 to 3 heteroatoms in the monocyclic, 1 to 6 heteroatoms in the bicyclic, or 1 to 9 heteroatoms in the tricyclic, independently selected from nitrogen, oxygen, and sulfur, where the nitrogen and sulfur heteroatoms can be optionally oxidized and the nitrogen heteroatom can be optionally quaternized. For example, the heterocycle can be a cycloalkoxy group. The heterocycle can be connected to the remainder of the molecule through any heteroatom or carbon atom in the heterocycle. Heterocycles include, but are not limited to, morpholinyl, pyrrolidinoyl, pyrrolidinyl, piperidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, and tetrahydrothiopyranyl.

[0207] The term "heteroaryl" refers to an aromatic 5- to 8-membered monocyclic, 7- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system having 1 to 3 heteroatoms in the monocyclic, 1 to 6 heteroatoms in the bicyclic, or 1 to 9 heteroatoms in the tricyclic, where the heteroatoms are selected from O, N, or S (e.g., carbon atoms and 1 to 3, 1 to 6, or 1 to 9 heteroatoms of N, O, or S in the monocyclic, bicyclic, or tricyclic, respectively). Heteroaryl groups described herein can also contain fused rings that share a common carbon-carbon bond.

[0208] The term "substituted," unless otherwise indicated, refers to the replacement of one or more hydrogen radicals in a given structure, where designated substituent radicals include, but are not limited to, halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, oxo, thioxy, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and aliphatic groups. It is understood that substituents can be further substituted. Representative substituents include amino, alkylamino, dialkylamino, and cyclic amino compounds.

[0209] The term "halogen" or "halo" refers to fluoro, chloro, bromo and iodo.

[0210] The following abbreviations may be used in this application: DSPC: distearoylphosphatidylcholine; DPPC: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; POPC: 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine; DOPE: 1,2-dileoyl-sn-3-phosphoethanolamine; PEG-DMG is generally 1,2-dimyristoyl-sn-glycerol-methoxypolyethylene glycol (e.g., PEG2000); TBDPSCl: tert-butylchlorodiphenylsilane; DMAP: dimethylaminopyridine; HMPA: hexamethylphosphoramide; EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; DIPEA: diisopropylethylamine; DCM: dichloromethane; TEA: triethylamine; TBAF: tetrabutylammonium fluoride.

[0211] Methods for preparing various organic groups and protecting groups are known in the art, and their use and modification are generally within the capabilities of one of ordinary skill in the art (see, for example, Green, T. W. et al., Protective groups in Organic Synthesis (1999); Stanley R. Sandler and Wolf Karo, Organic Functional Group Preparations (1989); Greg T. Hermanson, Bioconjugate Techniques (1996); and Leroy G. Wade, Compendium of Organic Synthesis Methods (1980)). Briefly, a protecting group is any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask that reactivity during certain reactions and then removed to expose the original functional group. In some embodiments, an "alcohol protecting group" is used. An "alcohol protecting group" is any group that reduces or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.

[0212] The compounds may be prepared by at least one of the techniques described herein or by known organic synthesis techniques. [Example]

[0213] Example 1: Synthesis of ether-linked lipids [ka] 10-(4-Bromobutoxy)nonadeca-1,18-diene 3 [ka] To a solution of nonadeca-1,18-dien-10-ol (30 mmol, 8.145 g) in toluene was added NaH (90 mmol, 60%, 3.6 g). The reaction mixture was allowed to stir at 80 °C overnight. After cooling to room temperature, neat 1,4-dibromobutane (300 mmol, 64.78 g) was added to the reaction. The reaction mixture was then heated at 90 °C for 6 h. TLC indicated the reaction was complete. After cooling to room temperature, the reaction mixture was quenched with ice, extracted with EtOAc, and washed with brine. The organic layer was separated and dried over sodium sulfate. The organic layer was filtered and evaporated under reduced pressure. The residue was purified by ISCO (SiO2: 0-10% EtOAc / hexanes) to provide the product (12.46 g) as a colorless oil. 1H NMR (400MHz, chloroform-d)δ 5.81(m,J=16.9,10.2,6.7Hz,2H),5.04-4.88(m,4H),3.65-3.31(m,5H),3.18(p,J=5. 8Hz, 1H), 2.09-1.98 (m, 5H), 2.02-1.90 (m, 3H), 1.75-1.63 (m, 2H), 1.51-1.19 (m, 21H).

[0214] 9-(4-Bromobutoxy)heptadecanedioic acid 4 [ka] To a 1 L RBF was added 10-(4-bromobutoxy)nonadeca-1,18-diene (30 mmol, 12.46 g) in 400 mL (1:1 volume) of anhydrous DCM and acetonitrile, RuCl3 (1.5 mmol, 311 mg), cooled in an ice bath, and a solution of NaIO4 (300 mmol, 64 g) in water was slowly added. The reaction mixture was allowed to stir overnight at room temperature. TLC indicated the reaction was complete. The reaction mixture was diluted with DCM and water. A few drops of 3% sodium sulfite were added for decolorization. The organic layer was separated, dried over sodium sulfate, and evaporated under vacuum. The residue was purified by ISCO (SiO2: 0-10% MeOH / DCM (+0.1% AcOH)) to provide the product (7.9 g) as a colorless oil. MS:M+2=453.1.1H NMR(400MHz,chloroform-d)δ 3.44(m,J=12.3,6.2Hz,4H),3.18(p,J=5.8Hz,1H),3.09(q,J=7.3Hz,3H),2.32(t,J=7.4Hz,5 H),2.07(s,3H),2.00-1.88(m,2H),1.73-1.54(m,7H),1.47-1.39(m,4H),1.47-1.30(m,10H).

[0215] 9-(4-(dimethylamino)butoxy)heptadecanedioic acid 5 [ka] To a 150 mL pressure bottle was added 9-(4-bromobutoxy)heptadecanedioic acid (10 mmol, 4.51 g) in anhydrous THF (80 mL), followed by potassium carbonate (30 mmol, 4.14 g) and a 2 M solution of dimethylamine (50 mmol, 25 mL) in anhydrous THF. The reaction mixture was sealed and allowed to stir at 65 °C overnight. TLC indicated the reaction was complete. The reaction mixture was acidified by adding 1 N HCl until the pH was 4, and the solution was extracted with DCM and water. The organic layer was separated and dried over sodium sulfate. The organic solution was filtered and evaporated under vacuum. The residue was co-evaporated with 3 × 20 mL toluene to provide the product (3.64 g) as a light brown oil.

[0216] 9-(4-(dimethylamino)butoxy)heptadecanedioic acid dichloride 6 [ka] To a solution of 9-(4-(dimethylamino)butoxy)heptadecanedioic acid (8.75 mmol, 3.64 g) in anhydrous DCM (50 mL) was added 5 drops of anhydrous DMF at 0° C., and oxayl chloride (52.5 mmol, 4.58 mL) was added dropwise. The reaction mixture was allowed to stir at room temperature overnight. The reaction was completed by checking MS when a sample was prepared in MeOH, and mass spectrometry indicated the methyl ester mass. The reaction mixture was evaporated under vacuum to provide the product as a red oil, which was used directly in the next step. MS (methyl ester) after treatment with MeOH. MS: M+1=444.3.

[0217] Bis(3-pentyloctyl) 9-(4-(dimethylamino)butoxy)heptadecanedioate (ALNY-651) [ka] To a solution of 9-(4-(dimethylamino)butoxy)heptadecanedioyl dichloride (3.29 mmol, 1.49 g) in DCM (30 mL) was added potassium carbonate (16.45 mmol, 2.27 g), followed by 3-pentyloctan-1-ol (13.16 mmol, 2.64 g). The reaction mixture was allowed to stir at room temperature overnight. TLC showed the reaction was complete. The reaction was quenched with water and extracted with DCM. The organic layer was separated, dried over sodium sulfate, and evaporated under vacuum. The residue was purified by ISCO (SiO2: 30-100% EtOAc / hexane with 3% TEA) to provide a colorless oil (0.538 g). MS:M+1=780.7.1H NMR(500MHz,chloroform-d)δ 4.07(t,J=7.1Hz,6H),3.40(s,2H),2.35-2.23(m,10H),2.21(s,9H),1.78(p,J=7.5Hz,2H),1. 57(tt,J=13.8,6.9Hz,17H),1.46-1.36(m,8H),1.29(t,J=6.9Hz,26H),0.88(t,J=7.1Hz,17H).

[0218] Bis(3-pentyloctyl) 9-(4-(isopropyl(methyl)amino)butoxy)heptadecanedioate (ALNY-659) [ka] A similar procedure was used to synthesize lipid 659. MS: M+1 = 809. 1H NMR (500 MHz, chloroform-d) δ 4.08 (t, J = 7.1 Hz, 6H), 3.40 (t, J = 5.9 Hz, 2H), 3.18 (s, 1H), 2.39-2.24 (m, 10H), 2.19 (s, 4H), 2.18 (m, 1H), 1.65-1.46 (m, 19H), 1.40 (dq, J = 12.6 Hz, 7.5 Hz, 9H), 1.30 (d, J = 7.6 Hz, 25H), 0.98 (t, J = 5.9 Hz, 8H), 0.88 (t, J = 7.0 Hz, 17H).

[0219] Bis(2-isopropyl-5-methylhexyl) 9-(4-(dimethylamino)butoxy)heptadecanedioate (ALNY-652) [ka] MS: M+1 = 697.6. 1H NMR (400 MHz, chloroform-d) δ 4.09-3.88 (m, 4H), 3.40 (s, 2H), 3.18 (t, 1H), 2.33-2.23 (m, 6H), 2.20 (s, 6H), 1.76 (pd, J = 6.9, 4.7 Hz, 4H), 1.66-1.09 (m, 40H), 0.88 (td, J = 6.5, 5.9, 3.0 Hz, 22H). 13C NMR (126 MHz, chloroform-d) δ 174.27, 79.68, 68.87, 65.54, 59.93, 45.69, 43.55, 36.98, 34.70, 34.67, 34.23, 29.93, 29.59, 29.52, 29.39, 28.64, 28.51, 28.37, 26.06, 25.63, 25.23, 25.19, 24.75, 22.92, 22.68, 19.64.

[0220] Bis(2-isopropyl-5-methylhexyl) 9-(4-(isopropyl(methyl)amino)butoxy)heptadecanedioate (ALNY-657) [ka] MS:M+1=725.0.1H NMR(400MHz,chloroform-d)δ 4.07-3.95(m,4H),3.40(s,2H),3.18(s,1H),2.36(s,2H),2.29(t,J=7.5Hz,4H),2.19(s,3H),1.76(qd ,J=6.9,4.7Hz,3H),1.56-1.10(m,42H),0.98(dd,J=6.6,4.6Hz,6H),0.88(td,J=6.5,5.9,3.0Hz,22H). [ka]

[0221] Ethyl 3-pentyloct-2-enoate II [ka] To a solution of triethylphosphonic acetate (100 mmol, 22.42 g) in anhydrous THF (50 mL) at −10° C., 1N NaHMDS (1N in THF, 100 mL) was added dropwise via a dropping funnel. After the addition, the reaction mixture was stirred at −10° C. for 1 hour and then at 0° C. for 1 hour. To this was added 6-undecanone (50 mmol, 8.52 g), warmed to room temperature, and stirred at 45° C. overnight. The reaction mixture was quenched with water, extracted with diethyl ether (2×100 mL), and the combined ether was washed with brine. The organic layer was dried over sodium sulfate and filtered. The organic solution was evaporated. The residue was purified by ISCO (SiO:100% hexanes) to provide the product (11.7 g) as a colorless oil. 1H NMR (400MHz, chloroform-d)δ 5.61(s,1H),4.13(q,J=7.1Hz,2H),2.66-2.49(m,2H),2.12(td,J=7.6,1.2Hz, 2H),1.45(ddt,J=10.1,7.3,4.5Hz,4H),1.38-1.21(m,10H),0.94-0.84(m,7H).

[0222] Ethyl 3-pentyloctanoate III [ka] To a 200 mL RBF was added a solution of ethyl 3-pentyloct-2-enoate (48.6 mmol, 11.7 g) in ethyl acetate (100 mL). The solution was purged with argon three times, Pd / C (5% wt) was added, and the mixture was purged and refilled with argon followed by hydrogen via a hydrogen balloon. The reaction mixture was stirred overnight with a H2 balloon. After filtering through Celite, washing well with ethyl acetate, the combined organic solution was evaporated under reduced pressure to provide the product (11 g) as a colorless oil. 1H NMR (400MHz, chloroform-d)δ 4.12(q,J=7.1Hz,2H),2.21(d,J=6.9Hz,2H),1.84(p,J=5.8Hz,1H),1.27(dddd,J=14.1,11.3,7.1,4.8Hz,19H),0.87(t,J=6.9Hz,6H).

[0223] 3-Pentyloctan-1-ol IV [ka] To a 250 mL RBF was added a solution of ethyl 3-pentyloctanoate (45.4 mmol, 11 g) in anhydrous THF. Cooled in an ice bath and under argon, LAH (2 N THF, 91 mmol, 45.4 mL) was slowly added. Allowed to warm to room temperature and stirred at room temperature for 30 minutes, then at 70 °C overnight. The reaction mixture was cooled to room temperature. A solution of saturated potassium sodium tartrate tetrahydrate was added dropwise at 0 °C until effervescence ceased. The mixture was diluted with diethyl ether and filtered through Celite. The combined ether was dried over sodium sulfate and filtered. The organic solution was evaporated under reduced pressure. Purification of the residue by ISCO (SiO2: 0-10% EtOAc / hexanes) provided 8.26 g of product. 1H NMR (400MHz, chloroform-d)δ 3.61(t,J=7.1Hz,2H),2.08(s,1H),1.49(q,J=6.9Hz,2H),1.38(p,J=4.9,4.0Hz,1H),1.33-1.16(m,16H),0.86(t,J=6.9Hz,6H).

[0224] Example 2: Synthesis of ester-containing lipids [ka] Ethyl (2E,13Z,16Z)-docosa-2,13,16-trienoate [ka] To a 1 L RBF was added 750 mL of DCM and, with stirring, oxayl chloride (239.1 mmol, 20.5 mL). After cooling to -78 °C, DMSO (296.48 mmol, 23.16 g) was added dropwise, followed by the slow addition of a solution of (11Z,14Z)-icosa-11,14-dien-1-ol (119.55 mmol, 35.21 g) in DCM (50 mL) over 5 min. After stirring at -78 °C for 30 min, triethylamine (717.3 mmol, 100 mL) was added. The reaction mixture was allowed to warm to room temperature and stirred for an additional 50 min. Ethoxycarbonylmethyl triphenylphosphonate (135.47 mmol, 51 g) was added to the reaction and allowed to stir at room temperature overnight. TLC indicated the reaction was complete. The reaction mixture was quenched with 1N HCl, extracted with DCM, and washed with brine. The organic layer was separated, dried over sodium sulfate, and evaporated in vacuo. The residue was purified by ISCO (SiO: 0-30% EtOAc / hexanes) to provide the product (26.75 g) as a colorless oil. MS:M+1=363.3.1H NMR(400MHz,chloroform-d)δ 6.96(dt,J=15.7,7.0Hz,1H),5.81(dt,J=15.7,1.6Hz,1H),5.45-5.27(m,5H),4.18(q,J=7.1Hz,2H),2.77(t,J= 6.5Hz,2H),2.19(qd,J=7.2,1.6Hz,2H),2.04(dd,J=7.6,6.1Hz,5H),1.44(q,J=7.2Hz,2H),1.41-1.28(m,22H).

[0225] Ethyl (13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)docosa-13,16-dienoate [ka] To a pre-dried 250 mL RBF, CuBr (0.76 mmol, 109 mg) and LiCl (1.52 mmol, 64.4 mg) in THF (10 mL) were added, stirred at room temperature for 10 minutes, cooled in an ice bath, and ethyl (2E,13Z,16Z)-docosa-2,13,16-trienoate (7.6 mmol, 2.8 g) in DCM (40 mL) was added, followed by TMSCl (8.36 mmol, 908 mg). The reaction mixture was stirred at 0° C. for 15 minutes, after which (8-(1,3-dioxolan-2-yl)octyl)magnesium bromide (11.4 mmol, 3.3 g) in THF (16 mL) was added. The reaction mixture was allowed to stir at 0° C. for 1.5 hours and then stirred at room temperature overnight. TLC indicated the reaction was complete, and the reaction was quenched with saturated NH4Cl, diluted with diethyl ether (200 mL), extracted, separated, and washed with brine. The organic layer was dried over sodium sulfate and evaporated in vacuo. The residue was purified by ISCO (SiO2: 0-20% EtOAc and hexanes) to provide the product (2.16 g) as a colorless oil. 1H NMR (500 MHz, chloroform-d) δ 5.35(dtd,J=18.0,10.6,5.4Hz,4H),4.84(t,J=4.8Hz,2H),4.12(q,J=7.2Hz, 2H),3.90(d,J=59.2Hz,6H),3.56(t,J=6.8Hz,1H),2.77(t,J=6.9Hz,2H),2.21 (d,J=6.9Hz,2H),2.05(q,J=7.2Hz,4H),1.83(t,J=7.3Hz,1H),1.65(dt,J=8.9 ,5.1Hz,3H),1.51(t,J=6.8Hz,1H),1.46-1.26(m,33H),0.89(t,J=6.6Hz,3H).

[0226] (13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)docosa-13,16-dien-1-ol [ka] To a solution of ethyl (13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)docosa-13,16-dienoate (10.87 mmol, 5.97 g) in anhydrous THF (60 mL) at 0 °C, lithium aluminum hydride (1N in THF, 21.74 mmol, 22 mL) was added slowly. The reaction mixture was stirred at 0 °C for 15 minutes and then at room temperature for 4 hours. The mixture was cooled in an ice bath, and saturated potassium sodium tartrate tetrahydrate solution was added dropwise until effervescence ceased. The reaction mixture was diluted with EtOAc and extracted. The organic layer was separated, dried over sodium sulfate, and concentrated to a colorless oil. The residue was coevaporated with toluene to completely remove water. The yield of the product was 3.61 g.

[0227] (13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)docosa-13,16-dien-1-yl methanesulfonate [ka] To a solution of (13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)docosa-13,16-dien-1-ol (7.132 mmol, 3.61 g) in anhydrous DCM (50 mL) at 0 °C, trimethylamine (21.396 mmol, 2.99 mL) was added, followed by MsCl (14.264 mmol, 1.1 mL). The reaction mixture was allowed to stir at room temperature for 5 h. TLC showed the reaction was complete. The reaction was quenched with saturated NaHCO3, diluted with DCM, and extracted. The organic layer was separated and dried over sodium sulfate. The organic solution was filtered and evaporated under reduced pressure to give the product (1.77 g). 1H NMR (500MHz, chloroform-d)δ 5.35(tdt,J=13.8,9.7,5.0Hz,4H),4.84(t,J=4.8Hz,1H),4.24(t,J=7.0Hz,2H),4.12(q,J=7.2Hz,3H),3.96(s,2H),3.90-3.77 (m,2H),3.00(s,3H),2.77(t,J=6.8Hz,2H),2.05(d,J=6.9Hz,9H),1.77-1.59(m,5H),1.52-1.15(m,28H),0.89(t,J=6.8Hz,3H).

[0228] (13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)-N,N-dimethyldocosa-13,16-dien-1-amine [ka] A solution of (13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)docosa-13,16-dien-1-yl methanesulfonate (2.37 mmol, 1.39 g) in THF (20 mL) was added to a 150 mL pressure bottle, followed by dimethylamine (2 M in THF, 14.22 mmol, 7.11 mL). The reaction mixture was sealed and heated at 65 °C overnight. The reaction mixture was cooled to 0 °C, quenched with brine, diluted, and extracted with EtOAc. The organic layer was separated, dried over sodium sulfate, and evaporated under vacuum to a yellow oil. The residue was purified by ISCO (SiO2: 30–100% EtOAc / hexanes with 3% trimethylamine) to provide the product (1.38 g) as a pale yellow oil. 1H NMR (400MHz, chloroform-d)δ 5.35(qd,J=10.9,5.3Hz,4H),4.05-3.76(m,4H),2.77(t,J=6.5Hz,2H),2.20(s,6H), 2.05(q,J=6.9Hz,4H),1.70-1.56(m,2H),1.49-1.21(m,42H),0.89(t,J=6.8Hz,3H).

[0229] (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dienal [ka] To a solution of (13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)-N,N-dimethyldocosa-13,16-dien-1-amine (2.58 mmol, 1.38 g) in acetone (10 mL) was added 1N HCl (7 mL). The reaction mixture was allowed to stir at room temperature overnight. TLC showed the reaction was complete, and the reaction mixture was slowly added with 20% aqueous KCO until the pH reached 10, diluted with EtOAc, and the organic solution was extracted, separated, dried over sodium sulfate, and concentrated to a colorless oil. The residue was dried under high vacuum to give the title product (1.1 g). 1H NMR (500MHz, chloroform-d)δ 9.70(s,1H),5.36(tdd,J=17.9,11.3,7.1Hz,4H),2.77(t,J=6.9Hz,2H),2.42(td,J=7.4,1.9Hz,2H), 2.21(s,8H),2.05(q,J=7.1Hz,4H),1.63(p,J=7.4Hz,2H),1.46-1.14(m,36H),0.89(t,J=6.7Hz,3H).

[0230] (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dien-1-ol [ka] At 0 °C, NaBH (0.653 mmol, 25 mg) was added to a solution of (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dienal (0.327 mmol, 160 mg) in anhydrous MeOH (4 mL). The reaction mixture was stirred at room temperature overnight. TLC indicated the reaction was complete. The reaction mixture was quenched with saturated NH Cl, diluted with EtOAc, and extracted. The organic layer was dried over sodium sulfate and concentrated to a colorless oil. The residue was purified by ISCO (SiO: 30-80% EtOAc / hexanes) to provide 145 mg of product. 1H NMR (500MHz, chloroform-d)δ 5.36(dtd,J=17.9,11.2,7.2Hz,4H),4.12(q,J=7.2Hz,2H),3.64(t,J=6.6Hz,2H),2.77(t,J=6 .9Hz,2H),2.21(s,8H),2.05(d,J=6.5Hz,7H),1.47-1.23(m,34H),0.88(tt,J=9.9,5.2Hz,6H).

[0231] (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dien-1-yl 2-ethylhexanoate (ALNY-654) [ka] To a 100 mL RBF was added a solution of (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dien-1-ol (0.295 mmol, 145 mg) in anhydrous DCM (3 mL), to which K2CO3 (0.885 mmol, 122 mg) was added, cooled in an ice bath, and 2-ethylhexanoyl chloride (0.442 mmol, 71.9 mg) was added. The reaction mixture was allowed to stir overnight at room temperature. The reaction was quenched with water and extracted with DCM. The organic layer was separated and dried over sodium sulfate. The combined organic solution was evaporated under reduced pressure. The residue was purified by ISCO (SiO2: 30-80% EtOAc and hexanes with 3% TEA) to provide the product (78.2 mg) as a colorless oil. MS:M+1=618.7.1H NMR(500MHz,chloroform-d)δ 5.36(tdd,J=17.8,11.4,7.1Hz,4H),4.07(t,J=6.7Hz,2H),2.78(t,J=6.9Hz,2H),2.21 (s,12H),2.05(q,J=7.0Hz,5H),1.44-1.14(m,43H),0.88(ddd,J=7.3,5.5,3.3Hz,11H).

[0232] 9-Bromonanal [ka] To a 200 mL RBF was added a suspension of pyridinium chlorochromate (30 mmol, 6.47 g) and MgSO4 (2.5 g) in DCM (50 mL). Cooled in an ice bath, a solution of 9-bromo-i-nonanol (20 mmol, 4.46 g) in anhydrous DCM (10 mL) was added slowly. Stirred at 0°C for 1 h and then at room temperature for 1 h. Diethyl ether (2 × 50 mL) was added to the reaction mixture, which was stirred vigorously for 5 min and filtered through Celite. The residue was washed with 3 × 50 mL of ether. The combined ether solution was concentrated to give the product (4.06 g) as a brown oil. 1H NMR (400MHz, chloroform-d)δ 9.76(d,J=1.8Hz,1H),3.40(t,J=6.8Hz,3H),2.43(td,J=7.3,1.8Hz,2H), 1.84(p,J=6.9Hz,3H),1.62(p,J=7.2Hz,3H),1.42(dq,J=13.0,6.9Hz,5H).

[0233] 2-(8-bromooctyl)-1,3-dioxolane [ka] To a 250 mL RBF was added a solution of 9-bromononanal (18.09 mmol, 4.0 g) in anhydrous toluene (60 mL), followed by p-TsOH (0.946 mmol, 180 mg), followed by ethylene glycol (53.97 mmol, 3.35 g). The reaction was equipped with a Dean-Stark condenser and refluxed at 130 °C overnight. The reaction mixture was quenched with saturated NaHCO3, extracted with EtOAc, and washed with brine. The organic solution was dried over sodium sulfate and evaporated under reduced pressure to a colorless oil. The residue was purified by ISCO (SiO2: 0-20% EtOAc / hexanes) to provide 3.12 g of product. 1H NMR (400MHz, chloroform-d)δ 4.84(t,J=4.8Hz,1H),4.07-3.76(m,4H),3.40(t,J=6.9Hz,2H),1.84(p,J=7.0Hz,2H),1.70-1.59(m,2H),1.47-1.26(m,10H).

[0234] (8-(1,3-dioxolan-2-yl)octyl)magnesium bromide [ka] To a 100 mL two-neck RBF was added pre-activated magnesium turnings (12.67 mmol, 308 mg) in THF (16 mL), followed by a solution of 2-(8-bromooctyl)-1,3-dioxolane (6.34 mmol, 1.68 g) in THF, followed by 1-2 crystals of iodine. The reaction was stirred at 60 °C for 35 min. The solution was decolorized. It was cooled to room temperature and used as is.

[0235] (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dienoic acid [ka] To a solution of (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dienal (1.45 mmol, 711 mg) in anhydrous DMF (6 mL) was added oxone (1.45 mmol, 892.3 mg). The reaction mixture was allowed to stir at room temperature overnight. TLC showed the reaction was complete, and the reaction was quenched by adding water, diluted and extracted with EtOAc, and washed with brine. The organic layer was separated and dried over sodium sulfate. The combined organic layers were evaporated under reduced pressure. The residue was co-evaporated with toluene to remove all water. The product (733 mg) was obtained. MS: M+1=506.5.

[0236] (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dienoyl chloride [ka] To a solution of (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dienoic acid (1.45 mmol, 733 mg) in anhydrous DCM (8 mL) at 0° C., two drops of anhydrous DMF were added, followed by the dropwise addition of neat oxayl chloride. The reaction mixture was stirred at room temperature overnight. TLC indicated the reaction was complete (a small sample of the reaction mixture was treated with MeOH). The reaction solution was concentrated to a dark red oil.

[0237] 3-Pentyloctyl (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dienoate [ka] To a solution of (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dienoyl chloride (0.944 mmol, 495 mg) in DCM (10 mL) was added K2CO3 (3.30 mmol, 457 mg), followed by a solution of 3-pentyloctan-1-ol (1.89 mmol, 378.1 mg) in DCM (2 mL). The reaction mixture was stirred at room temperature overnight. TLC indicated the reaction was complete. The reaction mixture was quenched with water, diluted with DCM, and extracted. The organic layer was separated and dried over sodium sulfate, and the organic solution was filtered and evaporated under reduced pressure. The residue was purified by ISCO (SiO2: 0-40% EtOAc and hexanes) to provide the product (195.6 mg). MS:M+1=689.7.1H NMR(400MHz,chloroform-d)δ 5.35(qd,J=10.9,5.6Hz,4H),4.10(dt,J=20.3,7.1Hz,4H),2.77(t,J=6.5Hz,2H),2.31-2.24 (m,3H),2.22(s,7H),2.05(d,J=5.5Hz,6H),1.44-1.15(m,55H),0.89(td,J=7.0,2.7Hz,9H).

[0238] 2-Isopropyl-5-methylhexyl (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dienoate [ka] MS:M+1=647.6.1H NMR(400MHz,chloroform-d)δ 5.35(tq,J=10.6,6.3,5.4Hz,4H),4.14-3.91(m,3H),2.77(t,J=6.5Hz,2H),2.29(t,J=7.5Hz,2H),2.22(s ,8H),2.05(q,J=6.9Hz,4H),1.77(pd,J=6.9,4.7Hz,1H),1.47-1.14(m,42H),0.88(td,J=6.2,3.1Hz,17H).

[0239] 3-Pentyloctyl (20Z,23Z)-10-(2-(isopropyl(methyl)amino)ethyl)nonacosa-20,23-dienoate [ka] MS:M+1=717.8.1H NMR(400MHz,chloroform-d)δ 5.36(dq,J=12.4,6.7,5.6Hz,4H),4.20-3.99(m,12H),2.80(dt,J=18.0,6.5Hz,2H),2.30(dt,J=21.5,7.6 Hz,4H),2.19(s,3H),2.04(s,16H),1.34-1.25(m,43H),1.00(d,J=6.5Hz,5H),0.88(h,J=2.9,2.3Hz,9H).

[0240] 2-Isopropyl-5-methylhexyl (20Z,23Z)-10-(2-(isopropyl(methyl)amino)ethyl)nonacosa-20,23-dienoate [ka] MS:M+1=675.7.1H NMR(400MHz,chloroform-d)δ 5.45-5.24(m,4H),4.12(q,J=7.2Hz,9H),4.07-3.94(m,2H),2.80(dt,J=21.8,6.5Hz,3H),2.31(dt,J=20.1,7.7Hz ,4H),2.19(s,3H),2.04(s,16H),1.26(t,J=7.1Hz,21H),1.00(d,J=6.5Hz,6H),0.88(ddt,J=9.3,6.3,2.7Hz,19H). [ka]

[0241] (14Z,17Z)-4-(8-(1,3-dioxolan-2-yl)octyl)tricosa-14,17-dienenitrile [ka] To a solution of (13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)docosa-13,16-dien-1-yl methanesulfonate (5.13 mmol, 3 g) in EtOH (200 proof) was added an aqueous solution of KCN (15.39 mmol, 1.02 g), and the reaction was refluxed at 80 °C overnight. A condenser was connected to a KOH trapper to neutralize the released HCN. The reaction mixture was quenched with saturated NaHCO3, extracted with ether, and washed with brine. The organic layer was dried over sodium sulfate and filtered. The organic solution was evaporated under reduced pressure to a colorless oil. The residue was purified by ISCO (SiO2: 0-20% EtOAc / hexanes) to provide the product (2.08 g). MS:ES-(M-1=514.3).1H NMR(400MHz,chloroform-d)δ 5.34(ddd,J=17.5,11.3,6.6Hz,4H),4.84(t,J=4.8Hz,1H),4.05-3.73(m,4H),2.77(t,J=6.5Hz,2H),2.31(t ,J=7.6Hz,2H),2.14-1.93(m,4H),1.70-1.57(m,4H),1.56(s,2H),1.47-1.18(m,34H),0.89(t,J=6.8Hz,3H).

[0242] (14Z,17Z)-4-(8-(1,3-dioxolan-2-yl)octyl)tricosa-14,17-dien-1-amine [ka] To a solution of (14Z,17Z)-4-(8-(1,3-dioxolan-2-yl)octyl)tricosa-14,17-dienenitrile (4.03 mmol, 2.08 g) in anhydrous THF at 0 °C was added 1N lithium aluminum hydrate in THF (8.06 mmol, 8.06 mL). The reaction mixture was stirred at 0 °C for 25 minutes and then at room temperature for 4 hours. After cooling in an ice bath, saturated potassium sodium tartrate tetrahydrate solution was added dropwise to the reaction mixture until effervescence ceased, resulting in the formation of a ppt. The ppt was filtered through Celite. The solution was extracted with EtOH and brine. The organic layer was separated, dried over sodium sulfate, and filtered. The organic solution was evaporated under reduced pressure to a colorless oil. The residue was dried overnight under high vacuum to give 2 g of product. MS:M+1=520.5.1H NMR(400MHz,chloroform-d)δ 5.35(qd,J=10.8,5.3Hz,4H),4.84(t,J=4.9Hz,1H),4.04-3.80(m,4H),2.77(t,J=6.5Hz,2H),2.66 (t,J=7.1Hz,1H),2.11-1.98(m,4H),1.71-1.59(m,3H),1.47-1.06(m,43H),0.89(t,J=6.8Hz,3H).

[0243] (14Z,17Z)-4-(8-(1,3-dioxolan-2-yl)octyl)-N,N-dimethyltricosa-14,17-dien-1-amine [ka] To a solution of (14Z,17Z)-4-(8-(1,3-dioxolan-2-yl)octyl)tricosa-14,17-dien-1-amine (3.85 mmol, 2 g) in anhydrous MeOH (20 mL) was added formalaldehyde (30% in water, 10 mL), followed by Na(OAC)3BH3 (15.39 mmol, 3.26 g). The reaction mixture was stirred at room temperature overnight. TLC indicated the reaction was complete. The reaction mixture was quenched with 1N NaOH, extracted with EtOAc, and washed with brine. The combined organic layers were dried over sodium sulfate and filtered. The organic solution was evaporated under reduced pressure to a colorless oil. The residue was purified by ISCO (SiO2, 0–80% EtOAc / hexanes) to provide the product (2.3 g). MS:M+1=548.5.1H NMR(400MHz,chloroform-d)δ 5.49-5.23(m,4H),4.96-4.67(m,2H),4.03-3.90(m,2H),3.90-3.76(m,2H),3.39(s,1H),2.77(t,J=6.5Hz,2H),2.25(s,1H),2.1 6(s,1H),2.11(s,1H),2.09-1.99(m,5H),1.78(s,1H),1.69-1.61(m,2H),1.59(s,5H),1.46-1.14(m,37H),0.89(t,J=6.8Hz,3H).

[0244] (20Z,23Z)-10-(3-(dimethylamino)propyl)nonacosa-20,23-dienal [ka] To a solution of (14Z,17Z)-4-(8-(1,3-dioxolan-2-yl)octyl)-N,N-dimethyltricosa-14,17-dien-1-amine (3.65 mmol, 2.3 g) in acetone (25 mL) was added 1N HCl (10 mL). The reaction mixture was stirred at room temperature overnight. The reaction mixture was quenched with 20% aqueous K2CO3 solution, extracted with EtOAc, and washed with brine. The organic layer was dried over sodium sulfate and filtered. The organic solution was evaporated under reduced pressure to a colorless oil. 1.88 g of product was obtained. 1H NMR (400MHz, chloroform-d)δ 9.76(d,J=1.9Hz,1H),5.36(tq,J=11.0,5.6,4.3Hz,4H),4.12(q,J=7.1Hz,1H),2.77(t,J=6.5Hz,2H),2.41(td, J=7.4,1.9Hz,2H),2.22(s,4H),2.10-1.99(m,6H),1.68-1.55(m,2H),1.47-1.10(m,40H),0.89(t,J=6.8Hz,3H).

[0245] (20Z,23Z)-10-(3-(dimethylamino)propyl)nonacosa-20,23-dienoic acid [ka] To a solution of (20Z,23Z)-10-(3-(dimethylamino)propyl)nonacosa-20,23-dienal (3.78 mmol, 1.88 g) in anhydrous DMF (15 mL) was added oxone (3.72 mmol, 2.29 g). The reaction mixture was stirred at room temperature overnight. TLC showed the reaction was complete. The reaction mixture was quenched with brine, diluted with EtOAc, and extracted. The organic layer was separated, dried over sodium sulfate, and filtered. The organic solution was evaporated under reduced pressure to a colorless oil. The residue was co-evaporated with 2 x 20 mL of toluene to completely remove water. 1.99 g of product was obtained. MS: M+1 = 520.5

[0246] (20Z,23Z)-10-(3-(dimethylamino)propyl)nonacosa-20,23-dienoyl chloride [ka] To a solution of (20Z,23Z)-10-(3-(dimethylamino)propyl)nonacosa-20,23-dienoic acid (3.82 mmol, 1.99 g) in anhydrous DCM at 0° C., two drops of anhydrous DMF were added slowly, followed by oxayl chloride (9.57 mmol, 833 μL). The reaction mixture was stirred at room temperature overnight. TLC showed the reaction was complete. The reaction mixture was concentrated to provide the product as a brown-red oil.

[0247] 3-Pentyloctyl (20Z,23Z)-10-(3-(dimethylamino)propyl)nonacosa-20,23-dienoate [ka] To a solution of (20Z,23Z)-10-(3-(dimethylamino)propyl)nonacosa-20,23-dienoyl chloride (2.22 mmol, 1.2 g) in anhydrous DCM (10 mL) was added K2CO3 (7.77 mmol, 1.07 g), followed by a solution of 3-pentyloctan-1-ol (4.46 mmol, 0.893 g) in anhydrous DCM. The reaction mixture was stirred overnight at room temperature. TLC indicated the reaction was complete. The reaction mixture was quenched with brine and diluted with DCM. The organic layer was extracted, separated, and dried over sodium sulfate. The organic solution was filtered and evaporated under reduced pressure. The residue was purified by ISCO (SiO2: 0-40% EtOAc and hexanes) to provide the product (99.5 mg) as a reddish-brown oil. MS:M+1=703.7.1H NMR(400MHz,chloroform-d)δ 5.36(tq,J=10.9,5.5,4.2Hz,4H),4.08(t,J=7.1Hz,2H),2.77(t,J=6.5Hz,2H),2.28(t,J=7.5Hz,2H),2. 21(s,6H),2.05(q,J=6.9Hz,4H),1.57(q,J=6.8Hz,10H),1.44-1.14(m,52H),0.89(td,J=6.9,2.6Hz,8H).

[0248] 2-Isopropyl-5-methylhexyl (20Z,23Z)-10-(3-(dimethylamino)propyl)nonacosa-20,23-dienoate [ka] MS:M+1=661.7.1H NMR(400MHz,chloroform-d)δ 5.35(tq,J=11.1,7.0,5.6Hz,4H),4.15-3.88(m,2H),2.77(t,J=6.5Hz,2H),2.42-2.16(m,9H),2.05(q,J=6.9 Hz,4H),1.77(pd,J=6.9,4.6Hz,1H),1.61(p,J=7.1Hz,2H),1.53-1.03(m,46H),0.88(td,J=6.2,3.1Hz,14H).

[0249] Example 3: Preparation of lipid nanoparticles The cationic lipids described herein are used to formulate liposomes containing AD-1661 duplexes (as shown in the table below) using the in-line mixing method as described in WO 2010 / 088537 (incorporated by reference in its entirety). Unless otherwise indicated, the lipid nanoparticles had the formulation shown in the table below.

[0250] [Table 12]

[0251] The formulations with the formula "58 / 10 / 30 / 2" had the formula shown in the table below.

[0252] [Table 13]

[0253] The formulations with the formula "55 / 10 / 33 / 2" had the formula shown in the table below.

[0254] [Table 14]

[0255] Formulations with a formula of "50 / 10 / 38 / 2" contain 50 mol % cationic lipid, 10 mol % DSPC, 38 mol % cholesterol, and 2 mol % PEG-DMG.

[0256] The siRNA AD-1661 duplex has the sequence shown below.

[0257] [Table 15]

[0258] Lower case letters are 2'OMe modifications, Nf is a 2'F modified nucleobase, dT is deoxythymidine, and s is phosphothioate.

[0259] Lipid nanoparticles were prepared as follows: Cationic lipid, DSPC, cholesterol, and PEG-DMG were solubilized in ethanol in the ratios shown in the table above at a total lipid concentration of 25 mg / mL.

[0260] siRNA stock solutions were prepared by solubilizing siRNA AD-1661 in low pH acetate or citrate buffer (pH=4) at 0.8 mg / mL.

[0261] The stock solution should be completely clear and the lipids should be completely solubilized before combining with the siRNA, therefore, when deemed appropriate, the stock solution was heated to completely solubilize the lipids.

[0262] The individual stock solutions were combined (i.e., in-line mixing) by pumping each solution into a T-junction: Specifically, the ethanol solution (5 mL / min, via 0.01 in. PEEK tubing) and aqueous buffer (15 mL / min, via 0.02 in. PEEK tubing) were mixed through a T-junction (PEEK Tee body, IDEX).

[0263] After the T-junction, a single tube is placed where the combined stream exits. The ethanol is removed and replaced with PBS by dialysis. The lipid formulation is then concentrated to an appropriate working concentration using centrifugation or diafiltration.

[0264] Lipid nanoparticles containing the cationic lipids listed in the table of Example 36 were prepared as described above.

[0265] Example 4: Efficacy of lipid nanoparticles Factor VII (FVII), a prominent protein in the coagulation cascade, is synthesized in the liver (hepatocytes) and secreted into plasma. FVII levels in plasma can be determined by a simple plate-based colorimetric assay. FVII itself represents a convenient model for investigating siRNA-mediated downregulation of hepatocyte-derived proteins.

[0266] The lipid nanoparticle test formulations prepared in Example 3 were evaluated for their FVII knockdown in female C57Bl / 6 mice, 7-9 weeks old, 15-25 g, at 0.01 and 0.03 mg / kg, with three mice per treatment group. All studies included animals receiving either phosphate-buffered saline (PBS, control group) or the benchmark formulation. The formulations were diluted to the appropriate concentration in PBS immediately prior to testing. Mice were weighed and the appropriate dosing volume calculated (10 μL / g body weight). Test and benchmark formulations, as well as PBS (for control animals), were administered intravenously via the posterior tail vein. Animals were anesthetized 24 hours later with an intraperitoneal injection of ketamine / xylazine, and 500-700 μL of blood was collected by cardiac puncture into serum separator tubes (BD Microtainer). Blood was centrifuged at 2,000 x g for 10 minutes at 15°C, and serum was collected and stored at -70°C until analysis. Serum samples were frozen and thawed for 30 minutes at 37°C, diluted in PBS, and dispensed into a 96-well assay plate. Factor VII levels were assessed using a chromogenic assay (Biophen FVII kit, Hyphen BioMed) according to the manufacturer's instructions, and absorbance was measured in a microplate reader equipped with a 405 nm wavelength filter.

[0267] The above procedure was used to determine the efficacy of lipid nanoparticle formulations containing the following cationic lipids: Figure 1 shows relative FVII protein levels on day 3 (at siRNA concentrations of 0.01 and 0.03 mg / kg). Formulation AF-011 contained a cationic lipid known as MC3.

[0268] [Table 16]

[0269] The above procedure was used to determine the efficacy of lipid nanoparticle formulations containing the following cationic lipids. Figure 2 shows the relative FVII protein levels on day 2 (at siRNA concentrations of 0.01 and 0.03 mg / kg). Software available at http: / / www.molinspiration.com / services / logp.html from Molinspiration Cheminformatics of Slovensky Grob, Slovak Republic was used to calculate the logP values ​​for the cationic lipids listed in the table below.

[0270] [Table 17]

[0271] [Table 18]

[0272] Using the above procedure, the efficacy of lipid nanoparticle formulations containing the following cationic lipids was determined. Figure 3 shows the relative FVII protein levels (at siRNA concentrations of 0.01 and 0.03 mg / kg). N / P refers to the ratio of amino groups in the cationic lipid to phosphate groups in the siRNA.

[0273] [Table 19]

[0274] The structure of AD-167990 is shown below.

[0275] [Table 20]

[0276] Lower case letters are 2'OMe modifications, Nf is a 2'F modified nucleobase, dT is deoxythymidine, and s is phosphothioate.

[0277] The above procedure was used to determine the efficacy of lipid nanoparticle formulations containing the following cationic lipids: Figure 4 shows the relative FVII protein levels after 48 hours (at siRNA concentrations of 0.005, 0.01, and 0.03 mg / kg).

[0278] [Table 21]

[0279] Example 5: Non-human primate studies The purpose of this study was to investigate the pharmacodynamics of lipid nanoparticles (LNPs) targeting the F12 protein after a single intravenous infusion in male and / or female cynomolgus monkeys. Five lipid nanoparticle formulations using different lipids (AF-011, AF-070, AF-079, AF-073, and AF-074) carrying F12 siRNA AD-167990 were tested. Each of these formulations was administered to cynomolgus monkeys via intravenous infusion over approximately 60 minutes on Day 1 at a target dose of 0.3 mg / kg (3 animals / group / formulation). Blood samples (approximately 1 mL) were collected from each animal on Days -5, -1, 1 (pre-dose), 2, 3, 5, 8, 15, 22, 29, 36, 43, 50, 57, and 64 for all groups. F12 plasma protein levels were determined using the F12 assay using a human factor XII total antigen assay ELISA kit from Molecular Innovations (HFXIIKT-TOT).

[0280] The formulations listed in the table below were prepared as described in Example 3 using F12 siRNA AD-167990.

[0281] [Table 22]

[0282] The table below provides the protocol used for each formulation. Figure 5 shows relative F12 plasma levels (compared to pre-dose).

[0283] [Table 23]

[0284] Example 6: Non-human primate studies This was a single-dose pharmacodynamics (PD) / pharmacology (PK) study of lipid nanoparticles (LNPs) targeting factor XII (F12) protein after a single intravenous infusion in male and / or female cynomolgus monkeys. Six lipid nanoparticle formulations using different lipids (AF-079, AF-093, AF-0783, AF-073, AF-094, and AF-011) bearing F12 siRNA AD-167990 were tested in this study. Each of these formulations was administered to cynomolgus monkeys on Day 1 by intravenous infusion over approximately 60 minutes at a target dose of 0.03, 0.1, or 0.3 mg / kg (3 animals / group / formulation). F12 plasma protein levels were determined using the F12 assay using a human factor XII total antigen assay ELISA kit from Molecular Innovations (HFXIIKT-TOT), as previously reported.

[0285] The test plan is shown in the table below.

[0286] [Table 24]

[0287] F12 siRNA AD-167990 was used to prepare the formulations listed in the table below as described in Example 3.

[0288] [Table 25]

[0289] Figures 6A, 6B, and 7-9 show the dose response for AF-094, single-dose AF-079, AF-073, AF-093, and AF-083, respectively.

[0290] All references cited herein are incorporated by reference.

Claims

【Request Item 1】 【Chemistry 1】 and salts thereof.

2. The compound is 【Chemistry 2】 or a salt thereof.

3. The compound is 【Transformation 3】 or a salt thereof.

4. The compound is 【Chemistry 4】 or a salt thereof.

5. A lipid particle comprising a neutral lipid, a PEG-lipid, and the compound of claim 1.

6. the neutral lipid is selected from DSPC, DPPC, POPC, DOPE or SM; The lipid particles further comprise a sterol. The lipid particle according to claim 5 .

7. said compound is present in a molar percentage of 20% to 60%; the neutral lipid is present in a molar percentage of 5% to 25%; the sterol is present in a molar percentage of 25% to 55%; the PEG lipid is PEG-DMA, PEG-DMG, or a combination thereof, and is present in a molar percentage of 0.5% to 15%; The lipid particle according to claim 6.

8. The lipid particle of claim 5, wherein the PEG-lipid is PEG-DMG.

9. The lipid particle according to claim 8, wherein (relative to 100% of the lipid constituents in the lipid particle) 50 mol % of the compound, 10% DSPC, 38.5% cholesterol and 1.5% PEG-DMG A lipid particle comprising:

10. The lipid particle according to claim 8, wherein (relative to 100% of the lipid constituents in the lipid particle) 58 mol % of the compound, 10% DSPC, 30% cholesterol and 2% PEG-DMG A lipid particle comprising:

11. The lipid particle of claim 5 , further comprising an active agent.

12. The lipid particle of claim 11 , wherein the active agent is a nucleic acid.

13. The lipid particle of claim 12 , wherein the nucleic acid is single-stranded DNA or single-stranded RNA.

14. 13. The lipid particle of claim 12, wherein the nucleic acid is selected from the group consisting of a plasmid, an immunostimulatory oligonucleotide, an siRNA, an antisense oligonucleotide, a microRNA, an antagomir, an aptamer, and a ribozyme.

15. The lipid particle of claim 14 , wherein the nucleic acid is siRNA.

16. 6. The lipid particle of claim 5 for use in delivering an active agent to a cell.

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