HK1245271B - Amine cationic lipids and uses thereof - Google Patents

Description

Amine cationic lipids and uses thereof

This application is a divisional application of international application number PCT/US2012/060875, with an international application date of October 18, 2012, entering the Chinese national phase on June 11, 2014, application number 201280061076.4, and invention name “Amine cationic lipids and their uses”.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/548,598, filed October 18, 2011, the disclosure of which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates in part to amine cationic lipid compounds and their formulations, and their use for delivering therapeutic agents, such as nucleic acid molecules, to cells.

Background Art

Nucleic acid molecules cannot easily pass through the cell membrane because of their size and hydrophilicity. Therefore, delivery has become one of the major challenges of nucleic acid therapy, such as antisense payloads and RNAi technology. In order to trigger RNase H activity or RNAi activity after systemic administration, preparations containing nucleic acid molecules must not only (1) prevent enzymatic and non-enzymatic degradation of the load and (2) provide appropriate biodistribution of the preparation, but also (3) allow cellular uptake or internalization of the preparation and (4) promote the delivery of the nucleic acid payload to the cytoplasm of the cell. Many preparations that are excellent in terms of the above-mentioned criteria 1 and 2 are defective in terms of criteria 3 and 4, so many nucleic acid preparations show excellent biodistribution, but fail to knock out the target gene due to the lack of systemic delivery and local delivery.

Therefore, there is a need for new compounds for the delivery of therapeutic agents, such as RNAi agents. Specifically, compounds that can function as cationic lipids can be used in lipid particle formulations to deliver nucleic acid payloads to cells.

Summary of the Invention

We have developed novel amine-based lipid compounds, including amino-amine and aminoamide cationic lipids, and formulations thereof, for the delivery of one or more therapeutic agents. Specifically, compounds of the invention (e.g., compounds of Formula (I) or II(a)-II(k)) can be used to deliver polycationic payloads or antisense payloads (e.g., nucleic acid molecules or RNAi agents) to cells and silence target genes.

In one aspect, the invention features a compound having the formula:

or a pharmaceutically acceptable salt thereof, wherein

each R ¹ and R ² is independently optionally substituted C _11-24 alkyl, optionally substituted C _11-24 alkenyl, optionally substituted C _11-24 alkynyl, optionally substituted C _11-24 heteroalkyl, optionally substituted C _11-24 heteroalkenyl, or optionally substituted C _11-24 heteroalkynyl, wherein R ¹ and R ² are not substituted with oxygen on adjacent carbons of >CHNR ³ R ⁴ ;

^R3 is H or optionally substituted _C1-6 alkyl; and

R ⁴ is unsubstituted C _1-6 alkyl substituted by -NR ^4a R ^4b , substituted C _1-6 alkyl further substituted by -NR ^4a R ^4b , or optionally substituted C _3-7 heterocyclyl, wherein each R ^4a and R ^4b is independently H, C(═NH)NH ₂ or optionally substituted C _1-6 alkyl, or wherein R ^4a and R ^4b are combined to form an optionally substituted C _3-7 heterocyclyl; and wherein R ³ and R ⁴ may be combined to form an optionally substituted C _3-7 heterocyclyl,

wherein R ³ and R ⁴ do not join together to form an optionally substituted imidazolyl or an optionally substituted benzimidazolyl or an optionally substituted succinimidyl; one and only one primary amine may be present on R ³ or R ⁴ , or no primary amine is present on R ³ or R ⁴ ; and wherein neither R ³ nor R ⁴ is an optionally substituted amide; and

wherein when R ¹ or R ² is a saturated C ₁₁ alkyl group or a saturated C ₁₅ alkyl group, R ³ is not H; wherein when R ¹ or R ² is a saturated C ₁₆ alkyl group or a saturated C ₁₇ alkyl group, R ¹ and R ² are not substituted with hydroxyl groups; wherein when R ¹ or R ² is a saturated C ₁₇ alkyl group, R ³ or R ⁴ are not substituted with hydroxyl groups; and wherein when R ¹ or R ² is a saturated C ₁₈ alkyl group, R ⁴ is not substituted with optionally substituted imidazolyl groups.

In some embodiments, R ³ is C _1-6 alkyl substituted with -NR ^3a R ^3b and wherein each R ^3a and R ^3b is independently H or optionally substituted C _1-6 alkyl. In certain embodiments, each R ^3a and R ^3b is independently H or C _1-6 alkyl.

In some embodiments, ^R is unsubstituted C _1-6 alkyl substituted with -NR ^4a R ^4b . In specific embodiments, ^R is substituted C _1-6 alkyl (e.g., substituted C _1-3 alkyl, substituted C _1-2 alkyl, substituted C 1 alkyl, substituted C ₂ alkyl, or substituted C ₃ alkyl) or C _1-6 aminoalkyl further substituted with -NR ^4a R ^4b . In some embodiments _, ^R is C _1-6 alkyl substituted with oxo and further substituted with -NR ^4a R ^4b . In some embodiments, R ^4a and R ^4b are combined to form an optionally substituted C _3-7 heterocyclyl (e.g., optionally substituted pyrrolidinyl, optionally substituted imidazolidinyl, optionally substituted pyrazolidinyl, optionally substituted piperidinyl, optionally substituted piperazinyl, optionally substituted azepanyl, optionally substituted pyrrolyl, optionally substituted imidazolyl, or optionally substituted pyrazolyl). In some embodiments, each R ^4a and R ^4b is independently optionally substituted C _1-6 alkyl. In some embodiments, R ⁴ is unsubstituted C _1-6 alkyl substituted with an optionally substituted C _3-7 heterocyclyl (e.g., any one described herein). In some embodiments, R ⁴ is substituted C 1-6 alkyl (e.g., substituted with oxygen) or C _{1-6 aminoalkyl further substituted with an optionally substituted C 3-7} heterocyclyl (e.g., optionally substituted pyrrolidinyl, optionally substituted imidazolidinyl, optionally substituted pyrazolidinyl, optionally substituted piperidinyl, optionally substituted piperazinyl, optionally substituted azepanyl, optionally substituted pyrrolyl, optionally substituted imidazolyl, optionally substituted pyrazolyl, optionally substituted _pyridinyl , optionally substituted pyrazinyl, optionally substituted _pyrimidinyl , or optionally substituted pyridazinyl).

In some embodiments, ^R and ^R are taken together to form an optionally substituted _C3-7 heterocyclyl (e.g., optionally substituted pyrrolidinyl, optionally substituted imidazolidinyl, optionally substituted pyrazolidinyl, optionally substituted piperidinyl, optionally substituted piperazinyl, optionally substituted azepanyl, optionally substituted pyrrolyl, optionally substituted imidazolyl, optionally substituted pyrazolyl, optionally substituted pyridinyl, optionally substituted pyrazinyl, optionally substituted pyrimidinyl, or optionally substituted pyridazinyl).

In some embodiments, the compound has the formula:

or a pharmaceutically acceptable salt thereof, wherein each R ¹ and R ² are independently optionally substituted C _11-24 alkyl, optionally substituted C _11-24 alkenyl, optionally substituted C _11-24 alkynyl, optionally substituted C _11-24 heteroalkyl, optionally substituted C _11-24 heteroalkenyl, or optionally substituted C _11-24 heteroalkynyl; each n1 and n2 are independently an integer from 0 to 2 (e.g., n1 and n2 are both 1, or n1 is 1 and n2 is 2); and R ⁵ is selected from the group consisting of H, optionally substituted C _1-6 alkyl, and optionally substituted heterocyclyl (e.g., unsubstituted C _1-6 alkyl or C 1-6 alkyl substituted with optionally substituted pyrrolyl, optionally substituted imidazolyl, optionally substituted pyrazolyl, optionally substituted pyridinyl, optionally substituted pyrazinyl, optionally substituted _pyrimidinyl , or optionally substituted pyridazinyl). In some embodiments, the compound is selected from the group consisting of L-2, L-5, L-6, L-22, L-23, L-24, L-25, L-26, L-28, L-29, L-45 and L-48, or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound has the formula:

or a pharmaceutically acceptable salt thereof, wherein each R ¹ and R ² is optionally substituted C _11-24 alkyl, optionally substituted C _11-24 alkenyl, optionally substituted C _11-24 alkynyl, optionally substituted C _11-24 heteroalkyl, optionally substituted C _{11-24 heteroalkenyl, optionally substituted C 11-24 heteroalkynyl} ; each n1 and n2 is independently an integer from 0 to 2 (e.g., n1 and n2 are both 1, or n1 is 1 and n2 is 2); and R ⁵ is selected from the group consisting of H, optionally substituted C _1-6 alkyl, and optionally substituted heterocyclyl (e.g., unsubstituted C _1-6 alkyl or C 1-6 alkyl substituted with optionally substituted pyrrolyl, optionally substituted imidazolyl, optionally substituted pyrazolyl, optionally substituted pyridinyl, optionally substituted pyrazinyl, optionally substituted _pyrimidinyl , or optionally substituted pyridazinyl). In some embodiments, the compound is selected from the group consisting of L-27 and L-47, or pharmaceutically acceptable salts thereof.

In some embodiments of any one of the formulae described herein (e.g., formulae (I), (IIa), and (IIb)), ^R is C _1-6 alkyl substituted with NR ^5a R ^5b , wherein each R ^5a and R ^5b is independently H, optionally substituted C _1-6 alkyl (e.g., optionally substituted C _1-6 alkyl), and wherein ^{R 5a} and R ^5b can be combined to form an optionally substituted C _3-7 heterocyclyl. In some embodiments, ^R is optionally substituted heterocyclyl (e.g., optionally substituted pyrrolidinyl, optionally substituted imidazolidinyl, optionally substituted pyrazolidinyl, optionally substituted piperidinyl, optionally substituted piperazinyl, optionally substituted azepanyl, optionally substituted pyrrolyl, optionally substituted imidazolyl, optionally substituted pyrazolyl, optionally substituted pyridinyl, optionally substituted pyrazinyl, optionally substituted pyrimidinyl, or optionally substituted pyridazinyl).

In some embodiments, the compound has the formula:

or a pharmaceutically acceptable salt thereof, wherein each R ¹ and R ² is an optionally substituted C _11-24 alkyl, an optionally substituted C _11-24 alkenyl, an optionally substituted C _11-24 alkynyl, an optionally substituted C _11-24 heteroalkyl, an optionally substituted C _11-24 heteroalkenyl, an optionally substituted C _11-24 heteroalkynyl; and each n1 and n2 are independently an integer from 0 to 2 (e.g., n1 and n2 are both 1, or n1 is 1 and n2 is 2). In some embodiments, the compound is L-46 or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound has the formula:

or a pharmaceutically acceptable salt thereof, wherein each R ¹ and R ² is independently optionally substituted C _11-24 alkyl, optionally substituted C _11-24 alkenyl, optionally substituted C _11-24 alkynyl, optionally substituted C _11-24 heteroalkyl, optionally substituted C _11-24 heteroalkenyl, or optionally substituted C _11-24 heteroalkynyl; R ³ is H or optionally substituted C 1-6 alkyl; L ¹ is optionally substituted C _1-6 alkylene; and each R ⁵ and R ⁶ is independently H or optionally substituted C _1-6 alkyl, or wherein R ⁵ and R ⁶ combine to form an optionally substituted C _{3-7 heterocyclyl} .

In some embodiments, the compound has the formula:

or a pharmaceutically acceptable salt thereof, wherein each R ¹ and R ² is independently optionally substituted C _11-24 alkyl, optionally substituted C _11-24 alkenyl, optionally substituted C _11-24 alkynyl, optionally substituted C _11-24 heteroalkyl, optionally substituted C _11-24 heteroalkenyl, or optionally substituted C _11-24 heteroalkynyl; R ³ is H or optionally substituted C 1-6 alkyl; L ¹ is optionally substituted C _1-6 alkylene; and each R ⁵ and R ⁶ is independently H or optionally substituted C _1-6 alkyl, or wherein R ⁵ and R ⁶ combine to form an optionally substituted C _{3-7 heterocyclyl} .

In some embodiments of Formula (IId) or (IIe), R ⁵ and R ⁶ combine to form optionally substituted pyrrolidinyl, optionally substituted imidazolidinyl, optionally substituted pyrazolidinyl, optionally substituted piperidinyl, optionally substituted piperazinyl, or optionally substituted azepanyl.

In some embodiments, the compound is selected from the group consisting of L-1, L-3, L-4, L-7, L-9, L-10, L-11, L-12, L-15, L-16, L-17, L-18, L-19, L-30, L-31, L-32, L-33, L-34, L-42, L-43 and L-49, or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound has the formula:

or a pharmaceutically acceptable salt thereof, wherein each R ¹ and R ² are independently optionally substituted C _11-24 alkyl, optionally substituted C _11-24 alkenyl, optionally substituted C _11-24 alkynyl, optionally substituted C _11-24 heteroalkyl, optionally substituted C _11-24 heteroalkenyl, or optionally substituted C _11-24 heteroalkynyl; R ³ is H or optionally substituted C _1-6 alkyl; L ¹ is optionally substituted C _1-6 alkylene; each n3 and n4 are independently an integer from 0 to 2; and R ⁵ is H or optionally substituted C _1-6 alkyl.

In some embodiments, the compound is selected from the group consisting of L-14, L-21, and L-36, or a pharmaceutically acceptable salt thereof.

In some embodiments of any one of the formulae described herein (e.g., formula (IId)-(IIj), e.g., formula (IId)-(IIg)), ^R is C _1-6 alkyl substituted with -NR ^3a R ^3b , and wherein each R ^3a and R ^3b are independently H or optionally substituted C _1-6 alkyl. In some embodiments, ^R is unsubstituted C _1-6 alkyl.

In some embodiments of any one of the formulae described herein (e.g., formula (IId)-(IIj), e.g., formula (IId)-(IIg)), L ¹ is C _1-6 alkylene substituted with methyl, ethyl, propyl, or -NR ^{La RLb} , wherein each R ^La and ^RLb is independently H or optionally substituted C _1-6 alkyl.

In some embodiments, the compound has the formula:

or a pharmaceutically acceptable salt thereof, wherein each R ¹ and R ² is independently optionally substituted C _11-24 alkyl, optionally substituted C _11-24 alkenyl, optionally substituted C _11-24 alkynyl, optionally substituted C _11-24 heteroalkyl, optionally substituted C _11-24 heteroalkenyl, or optionally substituted C _11-24 heteroalkynyl; R ³ is H or optionally substituted C _1-6 alkyl; L ¹ is optionally substituted C _1-6 alkylene; and R ⁵ is H or optionally substituted C _1-6 alkyl.

In some embodiments, L ¹ is attached to the imidazolyl group at the 4-position.

In some embodiments, the compound is selected from the group consisting of L-8, L-13, L-20, L-35, and L-44, or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound has the formula:

or a pharmaceutically acceptable salt thereof, wherein each R ¹ and R ² is independently optionally substituted C _11-24 alkyl, optionally substituted C _11-24 alkenyl, optionally substituted C _11-24 alkynyl, optionally substituted C _11-24 heteroalkyl, optionally substituted C _11-24 heteroalkenyl, or optionally substituted C _11-24 heteroalkynyl; R ³ is H or optionally substituted C 1-6 alkyl; L ¹ is optionally substituted C _1-6 alkylene; _and each R ⁵ and R ⁶ is independently H or optionally substituted C _1-6 alkyl.

In some embodiments, the compound has the formula:

or a pharmaceutically acceptable salt thereof, wherein each R ¹ and R ² is independently optionally substituted C _11-24 alkyl, optionally substituted C _11-24 alkenyl, optionally substituted C _11-24 alkynyl, optionally substituted C _11-24 heteroalkyl, optionally substituted C _11-24 heteroalkenyl, or optionally substituted C _11-24 heteroalkynyl; R ³ is H or optionally substituted C _1-6 alkyl; and each R ⁵ and R ⁶ is independently H or optionally substituted C _1-6 alkyl.

In some embodiments of any one of the formulae described herein (e.g., formula (IId)-(IIk), e.g., formula (IIi)-(IIk)), each R ⁵ and R ⁶ is independently C _1-6 alkyl substituted with -NR ^5a R ^5b , and wherein each R ^5a and R ^5b is independently H or optionally substituted C _1-6 alkyl.

In some embodiments, the compound is selected from the group consisting of L-37, L-38, L-39, L-40, and L-41, or a pharmaceutically acceptable salt thereof.

In some embodiments of any one of the formulae described herein (eg, formulae (IId)-(IIk)), L ¹ is optionally substituted C _1-6 alkylene.

In some embodiments of any one of the formulae described herein (e.g., formula (I) or (IIa)-(IIk)), R ³ is optionally substituted C _1-6 alkyl. In some embodiments, each R ¹ and R ² is independently an unsubstituted C _11-24 alkenyl or unsubstituted C _11-24 heteroalkenyl, including straight and branched forms (e.g., each R ¹ and R ² is independently an unsubstituted C _{11-24 alkenyl or unsubstituted C 11-24} heteroalkenyl containing one or more double bonds). In some embodiments, one of R ¹ or R ² is not a saturated C _11-24 alkyl. In some embodiments, neither R ¹ nor R ² is a saturated C _11-24 alkyl. In some embodiments, each R ¹ and R 2 is an unsubstituted C 11-24 alkenyl or unsubstituted C _11-24 heteroalkenyl. ² are independently selected from the group consisting of: linenyl (C18:3), linenyloxy (C18:3), linolenoyl (C18:3), linoleyl (C18:2), linoleyloxy (C18:2), linoleoyl (C18:2), oleyl (C18:1), oleyloxy (18:1), oleyloxymethylene (18:1), oleoyl (C18:1), oleoylmethylene (C18:1), stearyl (C18:0), stearyloxy ( C18:0), stearoyl (C18:0), palmityl (C16:0), palmityloxy (C16:0), palmitoyl (C16:0), palmitoylmethylene (C16:0), myristyl (C14:0), myristyloxy (C14:0), myristoyl (C14:0), lauryl (C12:0), lauryloxy (C12:0) and lauroyl (C12:0), such as linoleyl (C18:2) or oleyl (C18:1). In some embodiments, ^R1 and ^R2 are the same or different.

In some embodiments of any one of the formulae described herein (e.g., formulae (I) or (IIa)-(IIk)), R ³ or R ⁴ , but not both R ³ and R ⁴ , are simultaneously substituted with a primary amine. In some embodiments, R ³ and R ⁴ are not simultaneously substituted with a primary amine.

In some embodiments of any one of the formulae described herein (e.g., Formulae (I) or (IIa)-(IIk)), ^R3 and ^R4, together with the N to which they are attached, comprise a head group selected from one of H- ¹ to H-52 of Tables 2 and 3. In some embodiments, each of R1 and ^R2 is independently selected from the group consisting of linenyl (C18:3), linenyloxy (C18:3), linolenoyl (C18:3), linoleenyl (C18:2), linoleyloxy (C18:2), linoleoyl (C18:2), oleyl (C18:1), oleyloxymethylene (18:1), oleoyl (C18:1), oleoylmethylene (C18:1), stearyl (C18:3), linenyloxy (C18:3), linolenoyl (C18:3), linolenoyl (C18:2), linoleyloxy (C18:2), linoleoyl (C18:2), oleyl (C18:1), oleyloxymethylene (18:1), oleoyl (C18:1), oleoylmethylene (C18:1), stearyl (C18:3), linenyloxy (C18:3), linenyloxy (C18:3), linolenoyl (C18:3), linolenoyl (C18:2), linoleyloxy (C18:2), linoleoyl (C18:2), oleyl (C18:1), oleyloxymethylene (C18:1), oleoyl (C18:1), oleoylmethylene (C18:1), linalool (C18:1), linalool (C18:1), linalool (C18:1), linalool (C18:1), linalool (C :0), stearyloxy (C18:0), stearoyl (C18:0), palmityl (C16:0), palmityloxy (C16:0), palmitoyl (C16:0), palmitoylmethylene (C16:0), myristyl (C14:0), myristyloxy (C14:0), myristoyl (C14:0), lauryl (C12:0), lauryloxy (C12:0) and lauroyl (C12:0), for example, each of R ¹ and R ² is independently linoleyl (C18:2) or oleyl (C18:1).

In another aspect, compounds of the invention include R ¹ R ² -CH-A, wherein R ¹ and R ² are tail groups (e.g., any of those described herein, e.g., in Table 4), and A is a head group (e.g., any of those described herein, e.g., in Tables 2 and 3). In some embodiments, the head group is one of H-1 to H-52, e.g., H-2, H-5, H-6, H-19, H-26, or H-43 (e.g., H-5 or H-43).

In another aspect, the compound of the invention is any compound provided in Table 1 or a pharmaceutically acceptable salt thereof.

In one aspect, the invention features a formulation that includes any compound described herein (eg, one or more compounds provided in Table 1), or a pharmaceutically acceptable salt thereof.

In some embodiments, the formulation includes two or more of the compounds, for example, 2, 3, 4, 5, 6, 7 or more of the compounds.

In some embodiments, the formulation includes from about 10% to about 80% of the compound, for example, from about 10% to about 15%, from about 10% to about 20%, from about 10% to about 25%, from about 10% to about 30%, from about 10% to about 35%, from about 15% to about 20%, from about 15% to about 25%, from about 15% to about 30%, from about 15% to about 35%, from about 15% to about 40%, from about 20% to about 25%, from about 20% to about 30%, from about 20% to about 35%, from about 20% to about 40%, from about 25% to about 30%, from about 25% to about 35%, from about 25% to about 40%, from about 30% to about 35%, from about 30% to about 40%, or from about 35% to about 40% of one or more compounds of this invention.

In some embodiments, the formulation further comprises a cationic lipid (e.g., DODMA, DOTMA, DPePC, DODAP, or DOTAP), a neutral lipid (e.g., DSPC, POPC, DOPE, or SM), and optionally a sterol derivative (e.g., cholesterol; cholestanone; cholestenone; coprostanol; 3β-[-(N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol); biguanide-trisaminoethylamine-cholesterol (BGTC); (2S,3S)-2-(((3S,10R,13R,17R (2S,3S)-((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)carbonylamino)ethyl 2,3,4,4-tetrahydroxybutyrate (DPC-1); (2S,3S)-((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14 bis((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) 2,3,4,4-trihydroxyglutarate (DPC-3); or bis(((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) 2,3,4-trihydroxyglutarate (DPC-3); In some embodiments, the formulation further comprises a PEG-lipid conjugate (e.g., PEG-DMG, PEG-DMPE, PEG-DPPE, PEG-DPG, PEG-DOPE, or PEG-DOG).

In some embodiments, the formulation includes about 10 mol% to about 40 mol% of one or more compounds of the invention (e.g., one or more compounds described herein, e.g., in Table 1), about 10 mol% to about 40 mol% of one or more cationic lipids or one or more compounds of the invention (e.g., one or more compounds described herein, e.g., in Table 1), about 1 mol% to about 20 mol% of one or more PEG-lipid conjugates, about 5 mol% to about 20 mol% of one or more neutral lipids, and about 20 mol% to about 40 mol% of one or more sterol derivatives.

In certain embodiments, the formulation includes about 10 mol% to about 80 mol% (e.g., about 40 mol% to about 55 mol%, such as about 48 mol%) of one or more cationic lipids (e.g., a compound of the invention and/or other cationic lipids as described herein), about 1 mol% to about 20 mol% of one or more PEG-lipid conjugates, about 5 mol% to about 20 mol% of one or more neutral lipids, and about 20 mol% to about 40 mol% of one or more sterol derivatives. In some embodiments, the formulation includes about 10 mol% to about 30 mol% (e.g., about 22 mol%) of one or more compounds of the invention (e.g., L-6, L-30, and/or any described herein), about 15 mol% to about 35 mol% (e.g., about 26 mol%) of one or more cationic lipids (e.g., DODMA, or any described herein), about 3 mol% to about 9 mol% (e.g., about 6 mol%) of one or more PEG-lipid conjugates (e.g., PEG-DSPE, PEG-DMPE, and/or any described herein), about 10 mol% to about 20 mol% (e.g., about 14 mol%) of one or more neutral lipids (e.g., DSPC, or any described herein), and about 20 mol% to about 40 mol% (e.g., about 29 mol% to about 33 mol%, for example, about 33 mol%) of one or more sterol derivatives (e.g., cholesterol, a derivative thereof, or any described herein).

In some embodiments, one or more compounds of the present invention are present in an amount of about 10 mol% to about 40 mol%, for example, about 10 mol% to about 15 mol%, about 10 mol% to about 20 mol%, about 10 mol% to about 25 mol%, about 10 mol% to about 30 mol%, about 10 mol% to about 35 mol%, about 15 mol% to about 20 mol%, about 15 mol% to about 25 mol%, about 15 mol% to about 30 mol%, about 15 mol% to about 35 mol%, about 15 mol% to about 40 mol%, about 20 mol% to about 25 mol%, about 20 mol% to about 30 mol%, about 2 % to about 35 mol%, about 20 mol% to about 40 mol%, about 25 mol% to about 30 mol%, about 25 mol% to about 35 mol%, about 25 mol% to about 40 mol%, about 30 mol% to about 35 mol%, about 30 mol% to about 40 mol%, or about 35 mol% to about 40 mol% (e.g., about 21.0 mol%, 21.2 mol%, 21.4 mol%, 21.6 mol%, 21.8 mol%, 22 mol%, 25 mol%, 26 mol%, 26 mol%, 30 mol%, 35 mol% or 40 mol%) of one or more compounds of the present invention. In some embodiments, one or more compounds of the present invention are present in an amount of about 10 mol% to about 80 mol%, for example, about 10 mol% to about 15 mol%, about 10 mol% to about 20 mol%, about 10 mol% to about 25 mol%, about 10 mol% to about 30 mol%, about 10 mol% to about 35 mol%, about 10 mol% to about 40 mol%, about 10 mol% to about 45 mol%, about 10 mol% to about 50 mol%, about 10 mol% to about 55 mol%, about 10 mol% to about 60 mol%, about 10 mol% to about 65 mol%, about 10 mol% to about 70 mol%, about 10 mol% to about 75 mol%, about 15 mol% to about 20 mol%, about 15 mol% to about 25 mol%, about 15 mol% to about 30 mol%, about 15 mol% to about 35 mol%, about 15 mol% to about 40 mol%, about 15 mol% to about 45 mol%, about 15 mol% to about 50 mol%, about 15 mol% to about 55 mol%, about 15 mol% to about 60 mol%, about 15 mol% to about 65 mol% %, about 15 mol% to about 70 mol%, about 15 mol% to about 75 mol%, about 15 mol% to about 80 mol%, about 20 mol% to about 25 mol%, about 20 mol% to about 30 mol%, about 20 mol% to about 35 mol%, about 20 mol% to about 40 mol%, about 20 mol% to about 45 mol%, about 20 mol% to about 50 mol%, about 20 mol% to about 55 mol%, about 20 mol% to about 60 mol%, about 20 mol% to about 65 mol%, about 20 mol% to about 70 mol%. mol%, about 20 mol% to about 75 mol%, about 20 mol% to about 80 mol%, about 25 mol% to about 30 mol%, about 25 mol% to about 35 mol%, about 25 mol% to about 40 mol%, about 25 mol% to about 45 mol%, about 25 mol% to about 50 mol%, about 25 mol% to about 55 mol%, about 25 mol% to about 60 mol%, about 25 mol% to about 65 mol%, about 25 mol% to about 70 mol%, about 25 mol% to about 75 mol%, about 25 mol% to about 8 0 mol%, about 30 mol% to about 35 mol%, about 30 mol% to about 40 mol%, about 30 mol% to about 45 mol%, about 30 mol% to about 50 mol%, about 30 mol% to about 55 mol%, about 30 mol% to about 60 mol%, about 30 mol% to about 65 mol%, about 30 mol% to about 70 mol%, about 30 mol% to about 75 mol%, about 30 mol% to about 80 mol%, about 35 mol% to about 40 mol%, about 35 mol% to about 45 mol%, about 35 mol% to about to about 50 mol%, about 35 mol% to about 55 mol%, about 35 mol% to about 60 mol%, about 35 mol% to about 65 mol%, about 35 mol% to about 70 mol%, about 35 mol% to about 75 mol%, or about 35 mol% to about 80 mol%, about 40 mol% to about 45 mol%, about 40 mol% to about 50 mol%, about 40 mol% to about 55 mol%, about 40 mol% to about 60 mol%, about 40 mol% to about 65 mol%, about 40 mol% to about 70 mol%, about 40 ... mol% to about 75mol%, about 40mol% to about 80mol%, about 45mol% to about 50mol%, about 45mol% to about 55mol%, about 45mol% to about 60mol%, about 45mol% to about 65mol%, about 45mol% to about 70mol%, about 45mol% to about 75mol%, or about 45mol% to about 80mol%, about 50mol% to about 55mol%, about 50mol% to about 60mol%, about 50mol% to about 65mol%, about 50mol% to about 70mol%, about % to about 75 mol%, or about 50 mol% to about 80 mol% (e.g., about 21.0 mol%, 21.2 mol%, 21.4 mol%, 21.6 mol%, 21.8 mol%, 22 mol%, 25 mol%, 26 mol%, 26 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 48 mol%, 49 mol%, 50 mol%, 55 mol%, 60 mol%, 65 mol%, 70 mol% or 75 mol%) of one or more compounds of the invention.

In some embodiments, the one or more cationic lipids are present in an amount of about 10 mol% to about 40 mol%, for example, about 10 mol% to about 15 mol%, about 10 mol% to about 20 mol%, about 10 mol% to about 25 mol%, about 10 mol% to about 30 mol%, about 10 mol% to about 35 mol%, about 15 mol% to about 20 mol%, about 15 mol% to about 25 mol%, about 15 mol% to about 30 mol%, about 15 mol% to about 35 mol%, about 15 mol% to about 40 mol%, about 20 mol% to about 25 mol%, about 20 mol% to about 30 mol%, about 20 mol% to about 35 mol%, about 20 mol% to about 40 mol%, about 2 % to about 30 mol%, about 25 mol% to about 35 mol%, about 25 mol% to about 40 mol%, about 30 mol% to about 35 mol%, about 30 mol% to about 40 mol%, or about 35 mol% to about 40 mol% (e.g., about 25.1 mol%, 25.2 mol%, 25.3 mol%, 25.4 mol%, 25.5 mol%, 25.6 mol%, 25.7 mol%, 25.8 mol%, 25.9 mol%, 26.0 mol%, 26.2 mol%, 26.4 mol%, 26.6 mol%, 26.8 mol%, or 27 mol%) of one or more cationic lipids (e.g., DODMA or any one described herein, e.g., in Table 1).

In some embodiments, one or more PEG-lipid conjugates are present in an amount from about 1 mol% to about 20 mol%, for example, from about 1 mol% to about 5 mol%, from about 1 mol% to about 10 mol%, from about 1 mol% to about 15 mol%, from about 2 mol% to about 5 mol%, from about 2 mol% to about 10 mol%, from about 2 mol% to about 15 mol%, from about 2 mol% to about 20 mol%, from about 5 mol% to about 10 mol%, from about 5 mol% to about 15 mol%, from about 5 mol% to about 20 mol%, from about 10 mol% to about 15 mol%, from about 10 mol% to about 20 mol%, from about 15 mol%. To about 20 mol% (e.g., about 2.5 mol%, 2.6 mol%, 2.7 mol%, 2.8 mol%, 2.9 mol%, 3 mol%, 3.5 mol%, 4 mol%, 4.3 mol%, 4.5 mol%, 4.7 mol%, 5 mol%, 5.3 mol%, 5.5 mol%, 5.7 mol%, 6 mol%, 6.5 mol%, 6.7 mol%, 7 mol%, 7.5 mol%, 8 mol%, 8.5 mol% or 9 mol%) of one or more PEG-lipid conjugates (e.g., PEG-DSPE, PEG-DMPE and/or any described herein).

In some embodiments, the one or more neutral lipids are present in an amount from about 5 mol% to about 20 mol%, e.g., from about 5 mol% to about 10 mol%, from about 5 mol% to about 15 mol%, from about 5 mol% to about 20 mol%, from about 7 mol% to about 10 mol%, from about 7 mol% to about 15 mol%, from about 7 mol% to about 20 mol%, from about 10 mol% to about 15 mol%, from about 10 mol% to about 20 mol%, from about 15 mol% to about 20 mol% (e.g., about 13.0 mol%, 13.2 mol%, 13.4 mol%, 13.6 mol%, 13.8 mol%, 14 mol%, 14.1 mol%, 14.3 mol%, 14.5 mol%, 14.7 mol%, or 14.9 mol%) of one or more neutral lipids (e.g., DSPC or any described herein).

In some embodiments, the one or more sterol derivatives are present in an amount from about 20 mol% to about 40 mol%, for example, from about 20 mol% to about 25 mol%, from about 20 mol% to about 30 mol%, from about 20 mol% to about 35 mol%, from about 25 mol% to about 30 mol%, from about 25 mol% to about 35 mol%, from about 25 mol% to about 40 mol%, from about 30 mol% to about 35 mol%, from about 30 mol% to about 40 mol%, or from about 35 mol% to about 40 mol% (e.g., about 28.4 mol%, 28.6 mol%, 28.8 mol%, 29.0 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 33.2 mol%, 33.4 mol%, 33.6 mol%, 33.8 mol%, 34 mol%, 34.4 mol%, 34.7 mol%, or 34.9 mol%) of one or more sterol derivatives (e.g., cholesterol or any described herein).

In some embodiments, the formulation comprises one or more lipid particles comprising one or more RNA binders and one or more transfection lipids, wherein the one or more RNA binders comprise about 10 mol% to about 40 mol% of one or more cationic lipids or one or more compounds of the invention and about 0.5 mol% to about 10 mol% of one or more PEG-lipids; and wherein the one or more transfection lipids comprise about 10 mol% to about 40 mol% of one or more compounds of the invention, about 5 mol% to about 20 mol% of one or more neutral lipids, about 0.5 mol% to about 10 mol% of one or more PEG-lipid conjugates, and about 20 mol% to about 40 mol% of one or more sterol derivatives. Other formulations and percentages are as described herein.

In some embodiments, the formulation further comprises a polycationic payload or an antisense payload. In some embodiments, the polycationic payload is an RNAi agent (e.g., dsRNA, siRNA, miRNA, shRNA, ptgsRNA or DsiRNA, such as DsiRNA). In some embodiments, in some embodiments, the RNAi agent has a length of 10 to 40 nucleotides, such as 10 to 15 nucleotides, 10 to 20 nucleotides, 10 to 25 nucleotides, 10 to 30 nucleotides, 10 to 35 nucleotides, 15 to 20 nucleotides, 15 to 25 nucleotides, 15 to 30 nucleotides, 15 to 35 nucleotides, 15 to 40 nucleotides, 16 to 20 nucleotides, 16 to 25 nucleotides, 16 to 30 nucleotides, 16 to 35 nucleotides, 16 to 40 nucleotides, 20 to 25 nucleotides, 18 to 20 nucleotides, 18 to 25 nucleotides, 18 to 2 or a length of 35 to 40 nucleotides, for example a length of 25 to 35 nucleotides, for example a length of 16 to 30 nucleotides, for example a length of 19 to 19 nucleotides. In some embodiments, the antisense payload has a length of 8 to 50 nucleotides (e.g., 8 to 10 nucleotides, 8 to 15 nucleotides, 8 to 15 nucleotides, 8 to 20 nucleotides, 8 to 25 nucleotides, 8 to 30 nucleotides, 8 to 35 nucleotides, 8 to 40 nucleotides, or 8 to 45 nucleotides in length), e.g., a length of 14 to 35 nucleotides (e.g., 14 to 15 nucleotides, 14 to 20 nucleotides, 14 to 25 nucleotides, 14 to 30 nucleotides in length), e.g., a length of 17 to 24 nucleotides, e.g., a length of 17 to 20 nucleotides).

In some embodiments, the formulation comprises a ratio of about 1:10 (w/w) to about 1:100 (w/w) of the polycationic payload to the total lipid present in the formulation, for example, a ratio of about 1:10 (w/w) to about 1:15 (w/w), a ratio of about 1:10 (w/w) to about 1:20 (w/w), a ratio of about 1:10 (w/w) to about 1:40 (w/w), a ratio of about 1:10 (w/w) to about 1:50 (w/w), a ratio of about 1:10 (w/w) to about 1:60 (w/w), a ratio of about 1:10 (w/w) to about 1:70 (w/w), a ratio of about 1:10 (w/w) to about 1:80 (w/w), a ratio of about 1:10 (w/w) to about 1:90 (w/w). w) ratio, about 1:10 (w/w) to about 1:95 (w/w) ratio, about 1:20 (w/w) to about 1:40 (w/w) ratio, about 1:20 (w/w) to about 1:50 (w/w) ratio, about 1:20 (w/w) to about 1:60 (w/w) ratio, about 1:20 (w/w) to about 1:70 (w/w) ratio, about 1:20 (w/w) to about 1:80 (w/w) ratio, about 1:20 (w/w) to about 1:90 (w/w) ratio, about 1:20 (w/w) to about 1:95 (w/w) ratio, about 1:20 (w/w) to about 1:100 (w/w) ratio, about 1:40 (w/w) to about 1:50 (w/w) ratio, about 1:40 (w/w) to about 1:60 (w/w) ratio, about 1:40 (w/w) to about 1:70 (w/w) ratio, about 1:40 (w/w) to about 1:80 (w/w) ratio, about 1:40 (w/w) to about 1:90 (w/w) ratio, about 1:40 (w/w) to about 1:95 (w/w) ratio, about 1:40 (w/w) to about 1:100 (w/w) ratio, about 1:50 (w/w) to about 1:60 (w/w) ratio, about 1:50 (w/w) to about 1:70 (w/w) ratio, about 1:50 (w/w) to about 1:80 (w/w) ratio, about 1:50 (w/w) to about 1:90 (w/w) ratio, about 1:50 (w/w) to about 1:100 (w/w) ratio The invention also provides a polycationic payload to total lipid present in the formulation in a ratio of about 1:95 (w/w), about 1:50 (w/w) to about 1:100 (w/w) ratio, about 1:60 (w/w) to about 1:70 (w/w) ratio, about 1:60 (w/w) to about 1:80 (w/w) ratio, about 1:60 (w/w) to about 1:90 (w/w) ratio, about 1:60 (w/w) to about 1:95 (w/w) ratio, about 1:60 (w/w) to about 1:100 (w/w) ratio, about 1:80 (w/w) to about 1:90 (w/w) ratio, about 1:80 (w/w) to about 1:95 (w/w) ratio, or about 1:80 (w/w) to about 1:100 (w/w) ratio.

In some embodiments, the formulation comprises a liposome (eg, lipid nanoparticle), a lipoplex, or a micelle.

In one aspect, the invention features a pharmaceutical composition comprising any compound described herein (e.g., one or more compounds provided in Table 1) or a pharmaceutically acceptable salt thereof, or any formulation described herein; and a pharmaceutically acceptable excipient.

In another aspect, the invention features a method of treating or prophylactically treating a disease in a subject, the method comprising administering to the subject any compound described herein (e.g., one or more compounds provided in Table 1) or a pharmaceutically acceptable salt thereof, any formulation described herein, or any composition described herein, in an amount sufficient to treat the disease (e.g., liver cancer (e.g., hepatocellular carcinoma, hepatoblastoma, cholangiocarcinoma, angiosarcoma, or malignant hemangioma), lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), prostate cancer, or neuroblastoma). The invention further features methods for the therapeutic or prophylactic treatment of neoplastic diseases and related complications, including but not limited to cancer (e.g., lung, breast, pancreas, colon, hepatocellular, kidney, female genital tract, prostate, squamous cell carcinoma in situ), lymphoma (e.g., histiocytic lymphoma, non-Hodgkin lymphoma), MEN2 syndrome, multiple neurofibromatosis (including Schwann cell tumor), myelodysplastic syndrome, leukemia, tumor angiogenesis, thyroid cancer, liver, bone, skin, brain, central nervous system, pancreas, lung (e.g., small cell lung cancer, non-small cell lung cancer), breast cancer, gland, colon, bladder, prostate, gastrointestinal tract, endometrium, fallopian tube, testicle and ovary, gastrointestinal stromal tumors (GIST), prostate tumors, mast cell tumors (including canine mast cell tumors), acute myeloid myelofibrosis, leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, melanoma, mastocytosis, glioma, glioblastoma, astrocytoma, neuroblastoma, sarcoma (e.g., sarcoma of neuroectodermal origin or leiomyosarcoma), metastasis of tumors to other tissues, and chemotherapy-induced hypoxia.

In another aspect, the invention features a method of modulating expression of a target nucleic acid in a subject, the method comprising administering an agent sufficient to decrease expression of the target gene (e.g., any one described herein, e.g., one or more target genes selected from the group consisting of: ABL1, AR, β-catenin (CTNNB1), BCL1, BCL2, BCL6, CBFA2, CBL, CSF1R, ERBA1, ERBA2, ERBB1, ERBB2, ERBB3, ERBB4, ETS1, ETS2, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MET, MDM2, MLL1, MLL2, MLL3, MYB, MYC, M YCL1, MYCN, NRAS, PIM1, PML, RET, SRC, TAL1, TAL2, TCL3, TCL5, YES, BRCA1, BRCA2, MADH4, MCC, NF1, NF2, RB1, TP53, WT1, ApoB100, CSN5, CDK6, ITGB1, TGFβ1, cyclin D1, hepcidin, PCSK9, TTR, PLK1, and KIF1-binding protein) in an amount that increases or decreases the expression of a target gene in a subject.

In another embodiment, the invention features administering to a subject a dose of a polycationic payload or antisense payload of the invention one or more times per day (e.g., 1, 2, 3, or 4 times per day), one or more times per week (e.g., 2, 3, 4, 5, 6, or 7 times per week), or one or more times per month (e.g., 2, 3, 4, 5, 6, 7, or 10 times per week). A subject can receive a dose of a polycationic payload or antisense payload ranging from about 0.0001 to about 10 mg/kg, e.g., about 0.0001 to about 1 mg/kg, about 0.0001 to about 5 mg/kg, about 0.001 to about 1 mg/kg, about 0.001 to about 5 mg/kg, about 0.001 to about 10 mg/kg, about 0.01 to about 1 mg/kg, about 0.01 to about 5 mg/kg, about 0.001 to about 10 mg/kg, about 0.01 to about 1 mg/kg, about 0.01 to about 5 mg/kg, about 0.01 to about 10 mg/kg, about 1 to about 5 mg/kg, or about 1 to about 10 mg/kg, at any dosing schedule, e.g., one or more times per day (e.g., 1, 2, 3, 4, 5, 6, or 7 times per week), or one or more times per month (e.g., 2, 3, 4, 5, 6, 7, or 10 times per week).

In some embodiments, the invention features administering a formulation of the invention to a subject one or more times per day (e.g., 1, 2, 3, or 4 times per day), one or more times per week (e.g., 2, 3, 4, 5, 6, or 7 times per week), or one or more times per month (e.g., 2, 3, 4, 5, 6, 7, or 10 times per week). The subject can receive about 0.001 to about 200 mg/kg, for example, about 0.001 to about 1 mg/kg, about 0.001 to about 10 mg/kg, about 0.001 to about 20 mg/kg, about 0.001 to about 50 mg/kg, about 0.001 to about 100 mg/kg, about 0.01 to about 1 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 50 mg/kg, about 0.01 to about 100 mg/kg, about 0.01 to about 1 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 50 mg/kg, about 0.01 to about 100 mg/kg, [0014] The present invention relates to a formulation dosage ranging from about 1% to about 2% of the total dose, or about 1% to about 2% of the total dose, of a formulation of claim 1, wherein the dosage range is about 1% to about 2% of the total dose, or about 1% to about 2% of the total dose, of a formulation of claim 1, wherein the dosage range is about 1% to about 2% of the total dose, or about 1% to about 2% of the total dose, of a formulation of claim 1, wherein the dosage range is about 1% to about 2% of the total dose, or about 1% to about 2% of the total dose, of a formulation of claim 1, wherein the dosage range is about 1% to about 2% of the total dose, or about 1% to about 2% of the total dose, of a formulation of claim 1, wherein the dosage range is about 1% to about 2% of the total dose, or about 1% to about 2% of the total dose, of a formulation of claim 1, wherein the dosage range is about 1% to about 2% of the total dose, of a formulation of claim 1,

On the other hand, the present invention is characterized in that polycationic payload or antisense payload are delivered to the method for specific tissue type.The example of the specific tissue type to which the payload can be delivered includes but is not limited to liver, pancreas, lung, prostate, kidney, bone marrow, spleen, thymus, lymph node, brain, spinal cord, heart, skeletal muscle, skin, oral mucosa, esophagus, stomach, ileum, small intestine, colon, bladder, cervix uteri, ovary, testis, mammary gland, adrenal gland, adipose tissue (white and/or brown), blood (for example, hematopoietic cell, for example human hematopoietic progenitor cell, human hematopoietic stem cell, CD34+ cell, CD4+ cell), lymphocyte and other blood cell lineages.

In any of the above aspects, the compound of the invention includes two unsaturated lipid tail groups (e.g., each R ¹ and R ² is independently optionally substituted C _11-24 alkenyl, optionally substituted C _11-24 alkynyl, optionally substituted C _11-24 heteroalkenyl, or optionally substituted C _11-24 heteroalkynyl).

In any of the above aspects, the compounds of the invention include lipid tail groups, wherein these groups do ^not include an oxygen adjacent to ^-CHR3R4 (e.g., each ^R1 and ^R2 is independently optionally substituted _C11-24 alkyl, optionally substituted _C11-24 alkenyl, or optionally substituted _C11-24 alkynyl).

In any of the above aspects, the compounds of the invention include lipid tail groups, wherein these groups do not include one or more biodegradable groups (eg, one or more ester groups).

In any of the above aspects, the compound of the invention includes two lipid tail groups having more than 11, 12, 13, 14, 15, 16, or 18 carbons (e.g., each R ¹ and R ² is independently optionally substituted C _17-24 alkenyl, optionally substituted C _15-24 alkynyl, optionally substituted C _15-24 heteroalkenyl, or optionally substituted C _15-24 heteroalkynyl; each R ¹ and R ² is independently optionally substituted C _16-24 alkenyl, optionally substituted C 16-24 alkynyl, optionally substituted C _16-24 heteroalkenyl, or optionally substituted C _16-24 heteroalkynyl; each R ¹ and R ² is independently optionally substituted C _{17-24 alkenyl, optionally substituted C 17-24} alkynyl, optionally substituted C _17-24 heteroalkenyl, or optionally substituted C _17-24 heteroalkynyl; or each R ¹ and R 2 is independently optionally substituted C 17-24 alkenyl, optionally substituted C _17-24 alkynyl, optionally substituted C _{17-24 heteroalkenyl, or optionally substituted C 17-24} heteroalkynyl; ² is independently optionally substituted C _18-24 alkenyl, optionally substituted C _18-24 alkynyl, optionally substituted C _18-24 heteroalkenyl, or optionally substituted C _18-24 heteroalkynyl).

In any of the above aspects, the compounds of the invention do not contain urea groups (e.g., neither R ³ nor R ⁴ is an optionally substituted amide). In some embodiments, the compounds do not contain carbamoyl groups. In some embodiments, the compounds do not contain more than one primary amine group (e.g., there are not two primary amine groups or no primary amine groups in one or more of R ¹ -R ⁶ , such as in R ³ or R ⁴ ). In specific embodiments, the compounds include only one primary amine or no primary amine (e.g., there is only one primary amine or no primary amine in one or more of R ¹ -R ⁶ , such as in R ³ or R ⁴ ).

In any of the above aspects, the compound of the invention does not contain hydroxyl groups (e.g., neither R ¹ or R ² is substituted with 1, 2, or 3 hydroxyl groups; or neither R ³ or R ⁴ is substituted with 1, 2, or 3 hydroxyl groups). In some embodiments, when R ¹ or R ² is a saturated C _11-24 alkyl group (e.g., a saturated C ₁₅ alkyl group, a saturated C ₁₆ alkyl group, a saturated C ₁₇ alkyl group, or a saturated C ₁₈ alkyl group), R ¹ and/or R ² is not substituted with 1, 2, or 3 hydroxyl groups. In some embodiments, when R ¹ or R ² is a saturated C _11-24 alkyl group (e.g., a saturated C ₁₅ alkyl group, a saturated C ₁₆ alkyl group, a saturated C ₁₇ alkyl group, or a saturated C ₁₈ alkyl group), R ³ and/or R ⁴ is not substituted with 1, 2, or 3 hydroxyl groups.

In any of the above aspects, the compounds of the invention include no more than two amide groups (e.g., no more than two or one amide groups in the head group of the compound). In other embodiments, the compounds include 0, 1, or 2 amide groups in one or more of R ¹ -R ⁶ (e.g., 0, 1, or 2 amide groups in R ³ or R ⁴ ). In yet other embodiments, the compounds may include one and only one amide group (e.g., one and only one amide group may be included in R ³ or R ⁴ ). In other embodiments, the compounds include one and only one amide group or no amide group (e.g., one and only one amide group or no amide group in R ³ or R ⁴ ).

In any of the above aspects, the compounds of the present invention exclude N-(4-N’,N’-dimethylamino)butanoyl-(6Z,9Z,28Z,31Z)-triacontane-6,9,28,31-tetraen-19-amine or N-(3-N’,N’-dimethylamino)propanoyl-(6Z,9Z,28Z,31Z)-triacontane-6,9,28,31-tetraen-19-amine or a salt thereof. In some embodiments, the compounds of the present invention exclude N-methyl-N-(4-N’,N’-dimethylamino)butanoyl-(6Z,9Z,28Z,31Z)-pentatriacontane-6,9,28,31-tetraen-19-amine or N-methyl-N-(3-N’,N’-dimethylamino)propanoyl-(6Z,9Z,28Z,31Z)-pentatriacontane-6,9,28,31-tetraen-19-amine or a salt thereof.

In any of the above aspects, the compounds of the present invention exclude N-(4-N',N'-dimethylamino)butyryl-(6Z,9Z,28Z)-pentatriacontane-6,9,28-trien-19-amine, N-methyl-N-(4-N',N'-dimethylamino)butyryl-(6Z,9Z,28Z)-pentatriacontane-6,9,28-trien-19-amine, N-(4-N',N'-dimethylamino)butyryl-(6Z,9Z,28Z,31Z,34Z)-pentatriacontane-6,9,28,31,34-pentacontane-19-amine, N-methyl-N-(4-N',N'-dimethylamino)butyryl-(6Z,9Z,28Z,31Z,34Z)-pentatriacontane-6,9,28,31,34-pentacontane-19-amine, -pentaen-19-amine, N-(3-N', N'-dimethylamino) propionyl-(6Z,9Z,28Z)-pentatriaconta-6,9,28-trien-19-amine, N-methyl-N-(3-N', N'-dimethylamino) propionyl-(6Z,9Z,28Z)-pentatriaconta-6,9,28-trien-19-amine, N-(3-N', N'-dimethylamino) propionyl-(6Z,9Z,28Z)-pentatriaconta-6,9,28-trien-19-amine, N-methyl-N-(3-N', N'-dimethylamino)propionyl-(6Z,9Z,28Z,31Z,34Z)-pentatriacontane-6,9,28,31,34-pentaen-19-amine, N-methyl-N-(3-N',N'-dimethylamino)propionyl-(6Z,9Z,28Z,31Z,34Z)-pentatriacontane-6,9,28,31,34-pentaen-19-amine or a salt thereof.

In any of the above aspects, the compounds of the present invention exclude di((Z)-non-2-en-1-yl) 9-((3-(dimethylamino)propionyl)amino)heptadecanedioate, di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)amino)heptadecanedioate, di((Z)-non-2-en-1-yl) 9-((5-(dimethylamino)pivaloyl)amino)heptadecanedioate, or salts thereof.

In any of the above aspects, the compounds of the invention have a pKa value less than 6.2 and greater than 6.5 (e.g., a pKa value of 4.0 to 6.2, e.g., 4.0 to 5.2, 4.0 to 5.6, or 4.0 to 5.8; or 6.5 to 8.5, e.g., 6.5 to 7.0, 6.5 to 7.5, or 6.5 to 8.0). In certain embodiments, the pKa value is from about 5.0 to about 6.0 (e.g., 5.0 to 5.5, 5.0 to 5.6, 5.0 to 5.7, 5.0 to 5.8, 5.0 to 5.9, 5.0 to 6.0, 5.2 to 5.5, 5.2 to 5.6, 5.2 to 5.7, 5.2 to 5.8, 5.2 to 5.9, 5.2 to 6.0, 5.4 to 5.5, 5.4 to 5.6, 5.4 to 5.7, 5.4 to 5.8, 5.4 to 5.9, 5.4 to 6.0, 5.6 to 5.7, 5.6 to 5.8, 5.6 to 5.9, or 5.6 to 6.0). The pKa value can be determined by any useful method, for example, by measuring the fluorescence of 2-(p-toluidinyl)-6-naphthalenesulfonic acid (TNS), zeta potential measurements, etc. In certain embodiments, the pKa value is the ratio of the concentration of charged cationic lipid to the concentration of uncharged lipid (eg, as determined by in situ TNS fluorescence titration, where the pKa is determined as the pH at half-maximal fluorescence intensity).

definition

As used herein, the term "about" means the mean ± 10% of the recited value.

Unless otherwise indicated, "alkenyl" refers to a monovalent straight or branched chain group of 2 to 24 carbon atoms containing one or more carbon-carbon double bonds. Examples of alkenyl groups include ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, oleyl, linoleyl, linenyl, and the like. The term " _Cxy alkenyl" represents an alkenyl group having from x to y carbon atoms. Exemplary values for x are 2, 3, 4, 5, and 11; exemplary values for y are 3, 4, 5, 6, and 24; and exemplary values for x to y are 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 10 to 24, 11 to 24, 12 to 24, 14 to 24, 16 to 24, 18 to 24, 10 to 22, 11 to 22, 12 to 22, 14 to 22, 16 to 22, 18 to 22, 10 to 20, 11 to 20, 12 to 20, 14 to 20, 16 to 20, or 18 to 20. In some embodiments, the alkenyl group can be further substituted with 1, 2, 3, or 4 substituents as defined herein for an alkyl group.

Unless otherwise indicated, "alkyl" means a monovalent straight or branched chain saturated radical of 1 to 24 carbon atoms. Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, neopentyl, lauryl, myristyl, palmityl, stearyl, and the like, and may be optionally substituted with 1, 2, 3, or, in the case of an alkyl group of two or more carbon atoms, 4 substituents independently selected from the group consisting of: (1) alkoxy; (2) amino as defined herein; (3) halogen, such as F, Cl, Br, or I; (4) (heterocyclyl)oxy; (5) heterocyclyl; (6) alkyl; (7) alkenyl; (9) alkynyl; (10) cycloalkyl; (11) hydroxy; (12) nitro; or (13) oxygen (e.g., aldehyde or acyl). In some embodiments, each of these groups may be further substituted as described herein. The term " _Cxyalkyl " represents an alkyl group having from x to y carbon atoms. Exemplary values for x are 1, 2, 3, 4, 5, and 11; exemplary values for y are 2, 3, 4, 5, 6, and 24; and exemplary values for x to y are 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 10 to 24, 11 to 24, 12 to 24, 14 to 24, 16 to 24, 18 to 24, 10 to 22, 11 to 22, 12 to 22, 14 to 22, 16 to 22, 18 to 22, 10 to 20, 11 to 20, 12 to 20, 14 to 20, 16 to 20, or 18 to 20.

As used herein, the term "alkylene" and the prefix "alk-" represent a polyvalent (e.g., divalent) hydrocarbon radical derived from a straight or branched chain hydrocarbon by removing two hydrogen atoms. Examples of alkylene groups are methylene, ethylene, isopropylene, and the like. The term "C _xy alkylene" represents an alkylene group having x to y carbon atoms. Exemplary values for x are 1, 2, 3, 4, and 5, and exemplary values for y are 2, 3, 4, 5, and 6. In some embodiments, an alkylene group may be further substituted with 1, 2, 3, or 4 substituents as defined herein for an alkyl group.

Unless otherwise indicated, "alkynyl" refers to a monovalent straight or branched chain radical of 2 to 24 carbon atoms containing one or more carbon-carbon triple bonds. Examples of alkynyl groups are acetylene, 1-propynyl, and the like. The term " _Cxyalkynyl " represents an alkynyl radical having x to y carbon atoms. Exemplary values for x are 2, 3, 4, 5, and 11; exemplary values for y are 3, 4, 5, 6, and 24; and exemplary values for x to y are 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 10 to 24, 11 to 24, 12 to 24, 14 to 24, 16 to 24, 18 to 24, 10 to 22, 11 to 22, 12 to 22, 14 to 22, 16 to 22, 18 to 22, 10 to 20, 11 to 20, 12 to 20, 14 to 20, 16 to 20, or 18 to 20. In some embodiments, the alkynyl group can be further substituted with 1, 2, 3, or 4 substituents as defined herein for an alkyl group.

"Amide" means an amine group, as defined herein, appended to the parent molecular moiety through a carbonyl group.

As used herein, "amino" refers to -N( ^RN1 ) ₂ , wherein each ^RN1 is independently H, OH, _NO2 , N( ^RN2 ) ₂ , _SO2ORN2 , ^SO2RN2 , _SORN2 , ^an N-protecting group, an alkyl group, ^an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, an alkaryl group, a cycloalkyl group, an alkacycloalkyl group, a heterocyclyl group (e.g., a heteroaryl group), an alkheterocyclyl group (e.g., an alkheteroaryl group), or two ^{RN1 groups} are combined to form a heterocyclyl group or an N-protecting group, and wherein each ^RN2 is independently H, an alkyl group, or an aryl group. In a preferred embodiment, _the amino group is _-NH2 or ^-NHRN1 , wherein ^RN1 is independently OH, _NO2 , NH2, _NRN22 , ^SO2ORN2 , _SO2RN2 , ^SORN2 , ^an alkyl ^group , or an aryl group, and each ^RN2 can be H, an alkyl _group , or an aryl group. "Primary amine" means a group having the structure _-NH2 .

As used herein, the term "aminoalkyl" represents an alkyl group, as defined herein, substituted with an amino group, as defined herein. Alkyl and amino groups may each be further substituted with 1, 2, 3 or 4 substituents as described herein for each group.

As used herein, the term "carbamyl" refers to a carbamate group having the structure -NR ^N1 C(=O)OR or -OC(=O)N(R ^N1 ) ₂ , wherein each R ^N1 has the meaning as provided herein for "amino", and R is alkyl, cycloalkyl, alkoxycycloalkyl, aryl, alkaryl, heterocyclyl (e.g., heteroaryl), or alkheterocyclyl (e.g., alkheteroaryl), as defined herein.

As used herein, the term "carbonyl" represents a C(O) group, which may also be represented as C=O.

"Cycloalkyl" refers to a monovalent saturated or partially unsaturated 3- to 10-membered monocyclic or polycyclic (e.g., bicyclic or tricyclic) hydrocarbon ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

As used herein, the term "halogen" refers to a halogen selected from bromine, chlorine, iodine or fluorine.

"Heteroalkenyl" means an alkenyl group as defined herein, wherein one or more of the constituent carbon atoms have been individually replaced by O, N, or S. Exemplary heteroalkenyl groups include alkenyl groups as described herein that are substituted with an oxy group and/or attached to the parent molecular group through an oxygen atom. In some embodiments, the heteroalkenyl group can be further substituted with 1, 2, 3, or 4 substituents as described herein for alkyl groups.

"Heteroalkyl" means an alkyl group, as defined herein, in which one or more of the constituent carbon atoms have been replaced by O, N, or S. Exemplary heteroalkyl groups include alkyl groups as described herein that are substituted with oxy groups and/or attached to the parent molecular group through an oxygen atom. In some embodiments, the heteroalkyl group can be further substituted with 1, 2, 3, or 4 substituents as described herein for the alkyl group.

As used herein, the term "heteroalkylene" refers to an alkylene group as defined herein, wherein one or two of the constituent carbon atoms have each been replaced by O, N, or S. In some embodiments, the heteroalkylene group may be further substituted with one, two, three, or four substituents as described herein for alkylene groups. The term " _Cxy heteroalkylene" represents a heteroalkylene group having from x to y carbon atoms. Exemplary values for x are 1, 2, 3, 4, 5, and 11; exemplary values for y are 2, 3, 4, 5, 6, and 24; and exemplary values for x to y are 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 10 to 24, 11 to 24, 12 to 24, 14 to 24, 16 to 24, 18 to 24, 10 to 22, 11 to 22, 12 to 22, 14 to 22, 16 to 22, 18 to 22, 10 to 20, 11 to 20, 12 to 20, 14 to 20, 16 to 20, or 18 to 20.

"Heteroalkynyl" means an alkynyl group as defined herein, wherein one or more of the constituent carbon atoms have been replaced by O, N, or S. Exemplary heteroalkynyl groups include alkynyl groups as described herein that are substituted with an oxy group and/or attached to the parent molecular group through an oxygen atom. In some embodiments, the heteroalkynyl group can be further substituted with 1, 2, 3, or 4 substituents as described herein for alkyl groups.

As used herein, the term "heteroaryl" refers to a subset of heterocyclyl groups as defined herein that are aromatic: that is, they contain 4n+2pi electrons within a monocyclic or polycyclic ring system. In some embodiments, the heteroaryl group is substituted with 1, 2, 3, or 4 substituents as described herein for heterocyclyl.

Unless otherwise indicated, the term "heterocyclyl" as used herein refers to a 3-, 4-, 5-, 6-, 7- or 8-membered ring containing 1, 2, 3 or 4 heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur. A heterocyclyl group can be saturated or unsaturated and contain 0 to 3 unsaturated bonds. For example, a 5-membered ring has 0 to 2 double bonds, and a 6- and 7-membered ring has 0 to 3 double bonds. Certain heterocyclyl groups include 2 to 9 carbon atoms, for example 3 to 7 carbon atoms. Other such groups can include up to 12 carbon atoms. The term "heterocyclyl" also refers to a heterocyclic compound having a bridged polycyclic structure in which one or more carbon and/or heteroatom bridges two non-adjacent members of a monocyclic ring, for example a quinuclidine group. Examples of heterocyclic groups include aziridinyl, azetidinyl, pyrrolinyl, pyrrolyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridinyl, pyrimidinyl, piperidinyl, azepanyl, pyrazinyl, piperazinyl, diazepanyl, morpholinyl, tetrahydrofuranyl, dihydrofuranyl, and the like.

As used herein, the term "(heterocyclyl)oxy" represents a heterocyclyl, as defined herein, attached to the parent molecular group through an oxygen atom. In some embodiments, the heterocyclyl group may be substituted with 1, 2, 3, or 4 substituents, as defined herein.

As used herein, the term "(heterocyclyl)acyl" refers to a heterocyclyl, as defined herein, attached to the parent molecular group through a carbonyl group. In some embodiments, the heterocyclyl group may be substituted with 1, 2, 3, or 4 substituents, as defined herein.

As used herein, the term "hydroxy" refers to an -OH group.

"Linker" means an optionally substituted multivalent (eg, divalent) group containing one or more atoms. Examples of linkers include optionally substituted alkylene and heteroalkylene groups as defined herein.

As used herein, the term "N-protecting group" refers to those groups intended to protect an amino group from undesirable reactions during synthesis. Commonly used N-protecting groups are disclosed in Greene, "Protective Groups in Organic Synthesis," 3rd edition (John Wiley & Sons, New York, 1999), which is incorporated herein by reference. N-protecting groups include acyl, aryl acyl, or carbamoyl groups, such as formyl, acetyl, propionyl, pivaloyl, tert-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthaloyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliaries, such as protected or unprotected D, L or D, L-amino acids such as alanine, leucine, phenylalanine, etc.; sulfonyl-containing groups such as benzylsulfonyl, p-toluenesulfonyl, etc.; carbamate-forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, diphenylmethyloxycarbonyl, tert-butyloxycarbonyl, diisopropylmethoxycarbonyl, Isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and the like, alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl and the like, and silyl groups such as trimethylsilyl and the like. Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl, tert-butylacetyl, alanyl, benzylsulfonyl, benzyl, tert-butyloxycarbonyl (Boc) and benzyloxycarbonyl (Cbz).

As used herein, the term "oxo" refers to =0.

The term "urea" refers to a group having the structure NR ^N1 C(=O)NR ^N1 , where each R ^N1 has the meaning as defined for "amino" provided herein.

A "sufficient amount" of an agent refers to an amount of the agent sufficient to achieve a beneficial or desired result, such as a clinical result, and therefore, a sufficient amount depends on the context in which it is used. For example, in the context of administering an agent that reduces the expression level of a target gene, a sufficient amount of the agent is an amount sufficient to achieve a decrease in the expression level of the target gene compared to the response obtained when the agent is not administered.

"Anionic lipid" means any lipid molecule having a net negative charge at physiological pH.

As used herein, the term "antisense compound" or "antisense payload" specifically encompasses single-stranded antisense oligonucleotides (DNA, DNA-like, RNA, RNA-like) or certain double-stranded or self-hybridizing constructs comprising an antisense targeting oligonucleotide, an antisense PNA, a ribozyme, and an external guide sequence (a sequence that recruits RNase P, as described, for example, in Guerrier-Takada et al., Proc. Natl. Acad. Sci. USA 94:8468, 1997). Antisense compounds can exert their effects in many ways. One way is antisense-mediated targeting of endogenous nucleases, such as RNase H in eukaryotes or RNase P in prokaryotes (Chiang et al., J. Biol. Chem. 1266:18162, 1991; Forster et al., Science, 249:783, 1990).

"Cationic lipid" means any lipid molecule having a net positive charge at physiological pH. Exemplary cationic lipids include any of those described herein, e.g., in Table 1.

"Dicer-substrate RNA" or "DsiRNA" refers to a class of 25-35 (eg, 25-27, such as 27) nucleotide double-stranded molecules capable of gene silencing. Due to its longer length compared to other RNAi agents, DsiRNA is likely a substrate for Dicer.

By "double-stranded molecule" is meant a double-stranded RNA:RNA or RNA:DNA molecule that can be used to silence a gene product by RNA interference.

"Expression" means detecting a gene or polypeptide by methods known in the art. For example, DNA expression is often detected by Southern blotting or polymerase chain reaction (PCR), and RNA expression is often detected by Northern blotting, RT-PCR, gene chip technology or RNase protection assay. Methods for measuring protein expression levels generally include, but are not limited to, Western blotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, surface plasmon resonance, chemiluminescence, fluorescence polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, microcytometry, microscopy, fluorescence activated cell sorting (FACS) and flow cytometry, as well as assays based on protein properties, including but not limited to enzyme activity or interactions with other protein partners.

"Hybridization" means that sufficiently complementary polynucleotides, as defined herein, or portions thereof, pair together to form a double-stranded molecule under various stringency conditions. (See, e.g., Wahl et al., Methods Enzymol. 152:399 (1987); Kimmel, Methods Enzymol. 152:507 (1987)). For example, high stringency salt concentrations are typically less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of an organic solvent, such as formamide, while high stringency hybridization can be achieved in the presence of at least about 35% formamide or at least about 50% formamide. High stringency temperature conditions will typically include temperatures of at least about 30° C., 37° C., or 42° C. Various other parameters, such as hybridization time, concentration of detergents such as sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. In one embodiment, hybridization is carried out at 30 ℃ in 750mM NaCl, 75mM trisodium citrate and 1% SDS. In an optional embodiment, hybridization is carried out at pH 6.4, at 50 ℃ or 70 ℃, in 400mM NaCl, 40mMPIPES and 1mM EDTA, and hybridization continues after 12-16 hour, washes. Extra preferred hybridization conditions are included in 70 ℃ in 1xSSC or at 50 ℃ in 1xSSC, 50% formamide, wash in 0.3xSSC at 70 ℃ subsequently, or hybridize in 4xSSC or at 50 ℃ in 4xSSC, 50% formamide at 70 ℃, wash in 1xSSC at 67 ℃ subsequently. It is particularly obvious for those skilled in the art to change usefully for these conditions. One such exemplary variation includes evaluating hybridization under conditions designed to mimic physiological intracellular conditions, wherein cations and anions are coordinated in the following ratios: sodium:potassium:calcium:magnesium 10:160:2:26 for cations; and chloride:bicarbonate:phosphate:sulfate:gluconate 3:10:100:20:65 for anions.

"Lipid carrier" means a liposome, lipoplex, micelle, lipid nanoparticle, core-based particle, particle comprising an RNA binder-RNA aggregate associated with a transfection lipid, or vesicle-based particle comprising one or more compounds of the invention.

"MicroRNA" (miRNA) refers to single-stranded RNA molecules that can be used to silence gene products through RNA interference.

"Regulate " refers to gene expression, or the level of RNA molecules encoding one or more proteins or protein subunits or equivalent RNA molecules or the activity of one or more proteins or protein subunits is raised or lowered so that expression, level or activity are greater or less than that observed in the absence of a modulator. For example, the term regulate can include inhibition or gene silencing, and the gene expression level or RNA molecule level or its equivalent is reduced by at least 10% (e.g., 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%) compared to a control.

"Neutral lipid" refers to any lipid molecule that exists in an uncharged or neutral zwitterionic form at physiological pH.

"Polycationic payload" refers to a chemical moiety comprising multiple negatively charged atoms that can be added to a formulation. Examples of polycationic payloads include nucleic acids, RNAi agents, siRNA, dsRNA, miRNA, shRNA, DsiRNA, and antisense payloads.

"RNA binder" means any agent or combination of agents capable of binding to or hybridizing a nucleic acid, such as a nucleic acid payload of a therapeutic formulation. RNA binders include any lipid described herein (e.g., one or more cationic lipids, combinations of one or more cationic lipids, such as those described herein or in Table 1, and combinations of one or more cationic lipids and any other lipid, such as a neutral lipid or a PEG-lipid conjugate). RNA binders can form any useful structure within the formulation, such as internal aggregates.

"RNAi agent" refers to any agent or compound that exerts a gene silencing effect by hybridizing to a target nucleic acid. RNAi agents include any nucleic acid molecule capable of mediating sequence-specific RNAi (e.g., under stringent conditions), e.g., short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotides, short interfering nucleic acids, short interfering modified oligonucleotides, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and Dicer-substrate RNA (DsiRNA).

"Short hairpin RNA" or "shRNA" refers to an RNA sequence that forms a tight hairpin loop and is capable of gene silencing.

"Sense region" refers to a nucleotide sequence of a nucleic acid of the present invention that is sufficiently complementary to the antisense region of another nucleic acid. In addition, the sense region of a nucleic acid of the present invention may include a nucleotide sequence that has homology to the nucleotide sequence of a target gene. "Antisense region" refers to a nucleotide sequence of a nucleic acid of the present invention that is sufficiently complementary to the nucleotide sequence of a target gene.

"Silencing" or "gene silencing" means that the expression of a gene or the level of an RNA molecule encoding one or more proteins is reduced in the presence of an RNAi agent to below that observed under control conditions (e.g., in the absence of an RNAi agent or in the presence of an inactivating or attenuating molecule, such as an RNAi molecule with a scrambled sequence or mismatch). Gene silencing can reduce gene product expression by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% (i.e., complete inhibition).

"Small inhibitory RNA," "short interfering RNA," or "siRNA" refers to a class of 10-40 (e.g., 15-25, such as 21) nucleotide double-stranded molecules capable of gene silencing. Most notably, siRNAs are typically involved in the RNA interference (RNAi) pathway, whereby siRNAs interfere with the expression of specific gene products.

"Substantial identity" or "substantially identical" refers to polypeptide or polynucleotide sequences that have the same polypeptide or polynucleotide sequence, respectively, as a reference sequence, or that have a specified percentage of amino acid residues or nucleotides, respectively, that are identical at corresponding positions within the reference sequence when the two sequences are optimally aligned. For example, an amino acid sequence that is "substantially identical" to a reference sequence has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the reference amino acid sequence. For polypeptides, the length of the comparison sequence is generally at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids, more preferably at least 25, 50, 75, 90, 100, 150, 200, 250, 300, or 350 contiguous amino acids, and most preferably the full-length amino acid sequence. For nucleic acids, the length of the comparison sequence is generally at least 5 continuous nucleotides, preferably at least 10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 or 25 continuous nucleotides, and most preferably full-length nucleotide sequences. Sequence analysis software can be used to measure sequence identity based on default settings (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). This software can be used to mate similar sequences by assigning homology degrees to various replacements, disappearances and other modifications.

By "sufficiently complementary" is meant a polynucleotide sequence that has an exact complementary polynucleotide sequence to a target nucleic acid, or has a specified percentage of nucleotides that are exactly complementary to corresponding positions within the target nucleic acid when the two sequences are optimally aligned. For example, a polynucleotide sequence that is "substantially complementary" to a target nucleic acid sequence has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to the target nucleic acid sequence. For RNAi agents having a length of 10 to 40 nucleotides, sufficiently complementary sequences include those having 1, 2, 3, 4, or 5 non-complementary nucleotides. In fact, in certain embodiments including, for example, DsiRNA agents, active double-stranded RNAi agents can have as few as 15 to 19 consecutive nucleotides of a guide strand that is sufficiently complementary to a target nucleic acid, without requiring the remainder of the guide strand to have any degree of complementarity to the target nucleic acid (although in certain embodiments, the remainder of the guide strand can be partially or completely complementary to the targeted nucleic acid (e.g., mRNA).

"Target nucleic acid" refers to any nucleic acid sequence whose expression or activity is to be regulated. The target nucleic acid can be DNA or RNA. In certain embodiments, the target nucleic acid is a target mRNA.

"Transfection lipid" means any lipid or combination of lipids capable of delivering a nucleic acid, such as a nucleic acid payload (optionally, the nucleic acid payload is associated with an RNA binder, such as one or more cationic lipids). Transfection lipids include any lipid described herein (e.g., one or more cationic lipids, combinations of one or more cationic lipids, such as those described herein or in Table 1, and combinations of one or more cationic lipids and any other lipid or agent, such as a neutral lipid, an anionic lipid, a PEG-lipid conjugate, or a sterol derivative). The transfection lipid or combination comprising such a transfection lipid can form any useful structure within the formulation, such as an external aggregate surface.

"Pharmaceutical composition" means a composition containing a compound described herein, formulated with a pharmaceutically acceptable excipient and approved by a governmental regulatory agency for manufacture or sale as part of a therapeutic regimen for treating a disease in a mammal. The pharmaceutical composition can be formulated for oral administration, for example, in unit dosage form (e.g., tablets, capsules, film-coated tablets (caplets), gelcaps (gelcaps), or syrup); for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution free of embolic particles and in a solvent system suitable for intravenous use); or in any other formulation described herein.

"Pharmaceutically acceptable excipient" means any ingredient other than a compound described herein that has non-toxic and non-inflammatory properties in a patient (e.g., a vehicle capable of suspending or dissolving an active compound). Excipients may include, for example, anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colorants), softeners, emulsifiers, fillers (diluents), film formers or coatings, flavorings, fragrances, glidants (flow enhancers), lubricants, preservatives, inks, absorbents, suspending or dispersing agents, extenders, and water of hydration. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, cross-linked carboxymethylcellulose, cross-linked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl bonitoate, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl bonitoate, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

"Pharmaceutically acceptable salts" means those salts which are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, etc., within the scope of sound medical judgment and commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharm. Sci. 66(1): 1, 1977 and Pharmaceutical Salts: Properties, Selection, and Use, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008. Salts can be prepared in situ during the final isolation and purification of the compounds of the invention or separately by reacting the free base with a suitable organic acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, and the like.

"Subject" refers to a human or non-human animal (eg, a mammal).

As used herein and as known in the art, "treatment" is a method for obtaining a beneficial or desired result, such as a clinical result. Beneficial or desired results can include, but are not limited to, alleviating or alleviating one or more symptoms or conditions; reducing the extent of a disease, disorder, or condition; stabilizing the state of a disease, disorder, or condition (i.e., not worsening); preventing the spread of a disease, disorder, or condition; delaying or slowing the progression of a disease, disorder, or condition; alleviating or alleviating a disease, disorder, or condition; and eliminating (partially or completely), whether detectable or undetectable. "Alleviating" a disease, disorder, or condition means that the extent and/or unwanted clinical manifestations of the disease, disorder, or condition are reduced and/or the time course is slowed or prolonged compared to the extent or time course in the absence of treatment. "Treating cancer," "preventing cancer," or "inhibiting cancer" means causing a decrease in tumor size or the number of cancer cells, slowing or inhibiting an increase in tumor size or cancer cell proliferation, increasing the disease-free survival time between the disappearance of a tumor or other cancer and its recurrence, preventing or reducing the likelihood of the initial or subsequent appearance of a tumor or other cancer, or reducing adverse symptoms associated with a tumor or other cancer. In one embodiment desired, as measured using any standard assay, the percentage of tumors or cancer cells that survive treatment is at least 20, 40, 60, 80, or 100% lower than the initial number of tumors or cancer cells. Desirably, the reduction in the number of tumors or cancer cells induced by administering the compounds of the invention is at least 2, 5, 10, 20, or 50 times the reduction in the number of non-tumor or non-cancerous cells. Desirably, as measured using standard methods, the methods of the invention result in a 20, 40, 60, 80, or 100% reduction in tumor size or number of cancer cells. Desirably, at least 20, 40, 60, 80, 90, or 95% of the treated subjects have complete elimination in which all evidence of the tumor or cancer disappears. Desirably, the tumor or cancer does not reappear, or reappears after no less than 5, 10, 15, or 20 years. "Prophylactic treatment" of a subject's disease or condition (e.g., cancer) means reducing the occurrence (i.e., incidence) of the disease or condition before symptoms of the disease appear or reducing its severity. Prophylactic treatment can completely prevent or reduce the occurrence of a disease or its symptoms, and/or can be therapeutic in terms of partially or completely curing a disease and/or attributing adverse effects of a disease. Prophylactic treatment can include reducing or preventing the occurrence of a disease or condition (e.g., preventing cancer) in an individual who may be susceptible to the disease but has not yet been diagnosed with the disease.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows exemplary embodiments of amino-amides (labeled "amides") and amino-amines (labeled "amines"). For these compounds, ^R and ^R can be any tail group described herein, such as optionally substituted C _11-24 alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, or heteroalkynyl.

2A-2B show exemplary compounds L-1 to L-14.

FIG3 shows exemplary compounds L-15 to L-21 having a tertiary amine.

FIG4 shows exemplary analogs of L-2, including compounds L-5, L-6, L-22, and L-23.

FIG5 shows exemplary analogs of L-6, including compounds L-24 to L-29.

FIG6 shows exemplary amide analogs of L-9, including compounds L-30 to L-36.

Figure 7 shows compounds L-37 to L-41 having a piperazinyl group, wherein each R ¹ and R ² is independently any tail group described herein, such as optionally substituted C _11-24 alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, or heteroalkynyl.

FIG8 shows other exemplary amidoamine cationic lipid structures L-42, L-43, and L-44.

FIG9 shows other exemplary cationic lipid structures of L-6, L-45, L-46 and L-47 of the present invention.

Figure 10 is a graph showing in vitro knockdown of lipid particles containing compound L-1 or L-2 compared to untreated controls.

11 is a graph showing in vivo knockdown of HPRT1 mRNA in mouse liver using a single dose of lipid particles containing L-1, L-2, L-5, L-6, L-7, L-8, L-22, or L-30 and 5 mg/kg of DsiRNA, followed by tissue collection 48 hours later.

12 is a graph showing in vivo knockdown of HPRT1 mRNA in mouse liver using a single dose of lipid particles containing L-2, L-5, L-6, or L-30 and 1 mg/kg or 5 mg/kg of DsiRNA, followed by tissue collection 48 hours later.

13 is a graph showing in vivo knockdown of HPRT1 mRNA in mouse liver and orthotopic Hep3B tumors using two doses of lipid particles formulated with L-6 or L-30 and 5 mg/kg of DsiRNA, followed by tissue collection 48 hours later.

14 is a graph showing in vivo knockdown of HPRT1 mRNA in mouse liver and orthotopic HepG2 tumors using two doses of lipid particles formulated with L-6 or L-30 and 5 mg/kg of DsiRNA, followed by tissue collection 48 hours later.

FIG15 is a graph showing the effect of lipid particle formulations L-6 and L-30 with active DsiRNA ("L-6 Active" and "L-30 Active") on serum α-fetoprotein (AFP) levels in an in vivo Hep3B model. Controls are provided for formulations without DsiRNA ("L-6 Control" and "L-30 Control") and buffer ("PBS").

Figure 16 is a graph showing the effect of lipid particle formulations L-6 and L-30 with active DsiRNA ("L-6 Active" and "L-30 Active") on tumor weight in an in vivo Hep3B model. Controls are provided for formulations without DsiRNA ("L-6 Control" and "L-30 Control") and buffer ("PBS").

FIG17 shows exemplary compounds L-48 and L-49 having a dioleyl tail group.

FIG18 is a graph showing in vivo knockdown of HPRT1 mRNA in mouse liver and orthotopic Hep3B tumors (0.5 g ± 0.1) using a single IV dose of lipid particles containing L-30 and 1, 3, or 10 mg/kg of DsiRNA, followed by tissue collection 48 hours later. N=7/group liver and tumor (mean with SEM). Two formulations were tested: L-30 [1] and L-30 [2] as described herein.

FIG19 is a graph showing in vivo knockdown of HPRT1 mRNA in multiple orthotopic liver cancer models using a single dose of lipid particles containing the L-30[1] formulation and DsiRNA. Hep3B and HepG2 models were implanted as cell suspensions; and HuH1, HuH7, and MHCC97H were implanted as trocar fragments. N=5-7/group, 5 mg/kg IV, Q2D x 1. Target knockdown in all groups was significant relative to PBS (p<0.05).

FIG20 is a graph showing in vivo knockdown of various mRNAs in an orthotopic Hep3B HCC tumor model using a single dose of lipid particles containing L-30[1] formulation and various independent DsiRNAs. N=6-7/group, 5 mg/kg IV, TIW x 2; all given sorafenib at 10 mg/kg orally, QD x 14. **=p<0.01, *=p<0.05.

FIG21 is a graph showing in vivo knockdown of HPRT1 mRNA in Hep3B HCC tumor tissue using a single dose of lipid particles containing 5 mg/kg of DsiRNA in seven different formulations of L-30 ([A] to [G]), followed by tissue collection 72 hours later. Bar graph values with error represent mean + SEM (N = 7/group).

Figure 22 is a graph showing in vivo knockdown of HPRT1 mRNA in H1975 NSCLC SC xenograft lung tumor tissue using lipid particles containing 10 mg/kg of DsiRNA in two formulations, L-6[2] and L-30[2], on days 1 and 3, followed by tissue collection on day 5. Bar graph values with error bars represent mean + SEM (N = 7/group).

Figure 23 is a graph showing in vivo knockdown of HPRT1 mRNA in 22Rv1 prostate cancer SC xenografts using lipid particles containing 10 mg/kg of DsiRNA, two formulations, L-6[2] and L-30[2], on days 1 and 3, followed by tissue collection on day 5. Bar graph values with error represent mean + SEM (N = 7/group).

FIG24 is a graph showing in vivo knockdown of HPRT1 mRNA in 22Rv1 prostate cancer cells implanted in the liver using lipid particles containing 10 mg/kg of DsiRNA at three formulations, L-6[1], L-30[A], and L-30[E], on days 1 and 3, followed by tissue collection on day 5. Bar graph values with error bars represent mean + SEM (N = 7/group).

DETAILED DESCRIPTION

We have now developed amino-amine and amino-amide cationic lipids that can be formulated into lipid particles. The preparation of the present invention can be used to deliver polycationic payloads (e.g., nucleic acid molecules or RNAi agents) to cells (e.g., in vitro or in vivo in a subject). The delivery of polycationic payloads can achieve sequence-specific gene silencing in cells.

Amino-amine and amino-amide lipids

The compounds of the present invention include any compound of formula (I). In certain embodiments, the compound is selected from Table 1.

Table 1.

The compounds of the invention (e.g., as provided in Table 1) can be prepared by methods similar to those established in the art, for example, by the reaction sequences shown in Schemes 1 to 4. Exemplary lipids produced by these reaction sequences or adjustments thereof are provided in Figures 1 to 9 and Figure 17.

Solution 1

Secondary amines of formula C1 can be prepared by treating a ketone A1 wherein ^R1 and ^R2 are lipid tail groups as described herein with a primary amine B1 wherein ^R4 is as described herein under reductive amination conditions. The reductive amination conditions comprise mixing the ketone A1 and the primary amine B1 with a reducing agent, such as sodium cyanoborohydride or sodium triacetoxyborohydride, in an appropriate solvent. In certain embodiments, the amino-amine lipid of C1 is further oxidized to form a corresponding amino-amide lipid having an oxy group on the carbon adjacent to the nitrogen in ^R3 . In other embodiments, the amino-amine lipid of C1 is further alkylated on the nitrogen or any carbon of ^R4 . Exemplary compounds that can be produced using this protocol are provided in Figure 1.

Option 2

Tertiary amines of formula E2 can be prepared by treating a ketone A2 wherein R ¹ and R ² are lipid tail groups as described herein with a secondary amine D2 wherein R ³ and R ⁴ are as described herein under reductive amination conditions. The reductive amination conditions comprise mixing the ketone A2 and the primary amine D2 with a reducing agent, such as sodium cyanoborohydride or sodium triacetoxyborohydride, in a suitable solvent. In some embodiments of D2, R ³ and R ⁴ combine to form a heterocycle containing one or more heteroatoms, and the resulting tertiary amine E2 comprises such R ³ and R ⁴ groups. In certain embodiments, the amino-amine lipid of E2 is further oxidized to form a corresponding amino-amide lipid having an oxy group on the carbon adjacent to the nitrogen in R ³ or R ^4. In other embodiments, the amino-amine lipid of E2 is further alkylated on any carbon of R ³ and/or R ⁴ .

Option 3

The amine of formula F3 can be prepared by mixing ketone A3, ammonia, dihydrogen and catalyst in a suitable solvent, optionally under high pressure. The amino-amide lipid of formula H3 can be prepared by mixing amine F3 with activated carboxylic acid G3 in a suitable solvent, wherein LG is a leaving group and R ⁴ is as described herein. Exemplary LG includes halogen (e.g., chlorine, bromine or iodine), toluenesulfonate and trifluoromethanesulfonate. The amino-amine lipid of I3 can be prepared by mixing amide H3 with a reducing agent (e.g., lithium aluminum hydride, borane-tetrahydrofuran or borane-dimethyl sulfide). In specific embodiments, the amino-amide lipid of H3 further undergoes alkylation on any carbon of nitrogen or R ^{4 '} . In other embodiments, the amino-amine lipid of I3 further undergoes alkylation on any carbon of nitrogen or R ⁴ .

Option 4

An amino-amide lipid of formula K4 can be prepared by mixing ketone A4 and amine J4 in a suitable solvent, wherein LG is a leaving group and R ¹ , R ² and R ⁴ are as described herein. Exemplary LGs include halogens (e.g., chlorine, bromine or iodine), toluenesulfonates and trifluoromethanesulfonates. An amino-amine lipid of L4 can be prepared by mixing amide K4 with a reducing agent (e.g., lithium aluminum hydride, borane-tetrahydrofuran or borane-dimethyl sulfide). In other embodiments, the amino-amide lipid of K4 is further alkylated on any carbon of nitrogen or R ^{4 '} . In other embodiments, the amino-amine lipid of L4 is further alkylated on any carbon of nitrogen or R ^{4 '} . Exemplary compounds prepared by this method are provided in Figure 7.

In any of the above schemes, ^R4 can be optionally substituted heterocyclyl, optionally substituted ^{-L1- NR5R65 ,} optionally substituted -C(O) ^{-L1- NR5R6} , or optionally substituted ^-L1 - ^heterocyclyl as described herein.

In any of the above schemes, the compound can be further alkylated to introduce an optionally substituted C _1-6 alkyl group on N (ie, R ³ is an optionally substituted C _1-6 alkyl group) to form a tertiary amine. Exemplary compounds with tertiary amines are provided in FIG3 .

Any of the lipids described herein, eg, in Figures 1-9 and Figure 17, can be prepared by applying the synthetic schemes provided above or in Examples 1-5 and by making adjustments, if necessary, known to those skilled in the art.

lipid head group

The compounds of the present invention generally include a lipid head group, a head segment (headpiece) and one or more lipid tail groups. The head segment, for example, >CH-, connects the head group to the tail group. In certain embodiments, the head group includes two or more nitrogen atoms. Any of the head groups described herein, for example, in Table 2 or 3, can optionally be substituted with one or more substituents (e.g., one or more substituents described herein for alkyl).

A non-limiting list of head groups having an amine group is provided in Table 2. Any of the head groups described herein, such as head groups H-1 to H-39 in Table 2, can be combined with any of the tail groups described herein, such as those in Table 4, via a head>CH- segment to form inventive compounds.

Table 2: Examples of lipid head groups

A non-limiting list of head groups having an amide group is provided in Table 3. Any of the head groups described herein, such as head groups H-40 to H-52 in Table 3, can be combined with any of the tail groups described herein, such as in Table 4, via a head>CH- segment to form inventive compounds.

Table 3: Examples of amide-containing lipid head groups

lipid tail

As described herein, the compounds of the present invention generally include one or more tail groups, which may optionally include one or more heteroatoms. For each compound, the tail groups may be the same or different. Any of the tail groups described herein, such as those in Table 4, may optionally be substituted with one or more substituents (e.g., one or more substituents described herein for alkyl).

Exemplary tail groups include saturated and unsaturated groups having carbon or one or more heteroatoms (e.g., O), such as linenyl (C18:3), linenyloxy (C18:3), linolenoyl (C18:3), linoleyl (C18:2), linoleyloxy (C18:2), and linoleoyl (C18:2); and any heteroatom tail group described herein that is attached to the head segment via a methylene group, such as a tail group selected from the group consisting of linenyloxymethylene (C18:3), linolenoylmethylene (C18:3), and linoleyloxymethylene (C18:2) or linoleoylmethylene (C18:2). Table 4 provides a further non-limiting list of lipid tail groups.

Table 4: Examples of lipid tail groups

preparation

The compounds of this invention can be combined with one or more lipid molecules (e.g., cations, anions or neutral lipids) to produce formulations. The formulations can also include one or more components (e.g., sterol derivatives, PEG-lipid conjugates, polyamide-lipid conjugates, gangliosides, antioxidants, surfactants, amphipathic agents or salts) and/or one or more polycationic payloads (e.g., one or more nucleic acids or RNAi agents). Methods for formulating lipids to incorporate nucleic acid payloads have been described, see, for example, Judge et al., J. Clin. Invest. 119(3):661, 2009; Noble et al., Cancer Chemother. Pharmacol. 64(4):741, 2009; Abrams et al., Mol. Ther. 18(1):171, 2009; Yagi et al., Cancer Res. 69(16):6531, 2009; Ko et al., J. Control. Release 133(2):132, 2009; Mangala et al., Methods Mol. Biol. 555:29, 2009, which are incorporated herein by reference.

Formulations with more than one lipid molecule

The formulations of the invention can include any useful combination of lipid molecules (e.g., a compound of the invention, a cationic lipid (optionally including one or more cationic lipids, e.g., one or more cationic lipids of the invention as described herein and/or optionally including one or more cationic lipids known in the art), a neutral lipid, an anionic lipid, and a PEG-lipid conjugate), including polypeptide-lipid conjugates and other components that assist in the formation or stability of the lipid carrier as described herein. One skilled in the art will know how to optimize combinations that are beneficial for encapsulation of a particular agent, stability of the lipid formulation, scale-up reaction conditions, or any other relevant factors. The formulations of the invention can include other components that assist in formation or stability.

The percentage of each component in the formulation can be balanced to produce a lipid carrier capable of encapsulating the RNAi agent and transfecting the agent into cells. Exemplary formulations include about 10 mol % to about 40 mol % of one or more compounds of the invention, about 10 mol % to about 40 mol % of one or more cationic lipids, about 1 mol % to about 20 mol % of one or more PEG-lipid conjugates, about 5 mol % to about 20 mol % of one or more neutral lipids, and about 20 mol % to about 40 mol % of one or more sterol derivatives. In certain embodiments, the formulation includes about 20 mol% to about 25 mol% (e.g., about 21.0 mol%, 21.2 mol%, 21.4 mol%, 21.6 mol%, 21.8 mol% or 22 mol%) of one or more compounds of the invention, about 25 mol% to about 30 mol% (e.g., about 25.1 mol%, 25.2 mol%, 25.3 mol%, 25.4 mol%, 25.5 mol%, 25.6 mol%, 25.7 mol%, 25.8 mol%, 25.9 mol%, 26.0 mol%, 26.2 mol%, 26.4 mol%, 26.6 mol%, 26.8 mol% or 27 mol%) of one or more cationic lipids (e.g., DODMA), about 10 mol% to about 15 mol% (e.g., %, about 13.0 mol%, 13.2 mol%, 13.4 mol%, 13.6 mol%, 13.8 mol%, 14 mol%, 14.1 mol%, 14.3 mol%, 14.5 mol%, 14.7 mol% or 14.9 mol%) of one or more neutral lipids (e.g., DSPC), about 2.5 mol% to about 10 mol% (e.g., about 2.5 mol%, 2.6 mol%, 2.7 mol%, 2.8 mol%, 2.9 mol%, 3 mol%, 3.5 mol%, 4 mol%, 4.3 mol%, 4.5 mol%, 4.7 mol%, 5 mol%, 5.3 mol%, 5.5 mol%, 5.7 mol%, 6 mol%, 6.5 mol%, 6.7 mol%, 7 mol%, 7.5 mol%, 8 m % PEG2000-DSPE and/or PEG2000-DMPE and/or 3 mol%, 3.5 mol%, 3.7 mol%, 3.9 mol%, 4 mol%, 4.1 mol%, 4.3 mol%, 4.5 mol%, 4.7 mol%, 4.9 mol%, 5 mol%, 5.1 mol%, 5.3 mol%, 5.5 mol%, 5.7 mol%, 5.9 mol%, 6 mol%, 6.3 mol%, 6.5 mol%, 6.7 mol% or 7 mol% of one or more PEG-lipid conjugates (e.g., about 2.8 mol%, 2.9 mol%, 3.0 mol%, 3.5 mol%, 3.7 mol%, 3.9 mol%, 4 mol%, 4.1 mol%, 4.3 mol%, 4.5 mol%, 4.7 mol%, 4.9 mol%, 5 % PEG2000-DMG) and about 25 mol% to about 35 mol% (e.g., about 28.4 mol%, 28.6 mol%, 28.8 mol%, 29.0 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 33.2 mol%, 33.4 mol%, 33.6 mol%, 33.8 mol%, 34 mol%, 34.4 mol%, 34.7 mol% or 34.9 mol%) of a sterol derivative (e.g., cholesterol).

Preparation can comprise one or more cationic lipids of any useful amount.In some embodiments, in preparation, the content of cationic lipid is that about 10mol% is to about 40mol% (for example, about 10mol% to 15mol%, about 15mol% to 20mol%, about 20mol% to 25mol%, about 25mol% to 30mol%, about 30mol% to 35mol% and about 35mol% to 40mol%).In specific embodiments, the cationic lipid (for example, the L-1 of 10.8mol% and the L-2 of 10.8mol%) of mixing has been used.

In some embodiments, the formulation includes lipid particles having one or more RNA-binding agents and one or more transfection lipids, wherein the one or more RNA-binding agents include about 10 mol% to about 40 mol% of one or more cationic lipids (e.g., DODMA) and about 0.5 mol% to about 10 mol% of one or more PEG-lipid conjugates (e.g., PEG-DSPE, e.g., PEG2000-DSPE, and/or PEG-DMPE, e.g., PEG2000-DMPE); and wherein the one or more transfection lipids include about 10 mol% to about 40 mol% of one or more compounds of the invention (e.g., L-6, L-30, or any of Table 1), about 5 mol% to about 20 mol% of one or more neutral lipids (e.g., DSPC), about 0.5 mol% to about 10 mol% of one or more PEG-lipid conjugates (e.g., PEG-DSPE, e.g., PEG2000-DSPE and/or PEG-DMPE, e.g., PEG2000-DMPE), and about 20 mol% to about 40 mol% of one or more sterol derivatives (e.g., cholesterol).

The RNA binding agent of lipid particles can include any useful lipid and conjugate combination. In specific embodiments, the content of cationic lipid (e.g., DODMA) is from about 10 mol% to about 40 mol% (e.g., from about 20 mol% to 40 mol%, 20 mol% to 35 mol%, 20 mol% to 30 mol%, 15 mol% to 40 mol%, 15 mol% to 35 mol%, 15 mol% to 25 mol% or 15 mol% to 20 mol%). In some embodiments, PEG-lipid conjugates (e.g., PEG-DSPE, e.g., PEG2000-DSPE, and/or PEG-DMPE, e.g., PEG2000-DMPE) are from about 0.5 mol% to about 10 mol% (e.g., from about 0.5 mol% to 1 mol%, 0.5 mol% to 5 mol%, 0.5 mol% to 10 mol%, 1 mol% to 5 mol% or 1 mol% to 10 mol%).

In some embodiments, the transfection lipid of lipid granule can comprise the combination of any useful lipid and conjugate.In specific embodiments, the content of one or more compounds of this invention (for example, any one in L-6, L-30 or table 1) is that about 10mol% is to about 40mol% (for example, about 10mol% to 20mol%, 10mol% to 30mol%, 10mol% to 35mol%, 15mol% to 20mol%, 15mol% to 25mol%, 15mol% to 30mol%, 15mol% to 35mol%, 15mol% to 40mol%, 20mol% to 25mol%, 20mol% to 30mol%, 20mol% to 35mol%, 20mol% to 40mol%, 25mol% to 30mol%, 25mol% to 35mol% or 25mol% to 40mol%). In some embodiments, the content of one or more neutral lipids (e.g., DSPC) is from about 5 mol% to about 20 mol% (e.g., from about 5 mol% to 10 mol%, from 5 mol% to 15 mol%, from 7 mol% to 10 mol%, from 7 mol% to 15 mol%, from 7 mol% to 20 mol%, from 10 mol% to 15 mol%, or from 10 mol% to 20 mol%). In some embodiments, the content of one or more PEG-lipid conjugates (e.g., PEG-DSPE, e.g., PEG2000-DSPE, and/or PEG-DMPE, e.g., PEG2000-DMPE) is from about 0.5 mol% to about 10 mol% (e.g., from about 0.5 mol% to 1 mol%, from 0.5 mol% to 5 mol%, from 0.5 mol% to 10 mol%, from 1 mol% to 5 mol%, or from 1 mol% to 10 mol%). In some embodiments, the content of one or more sterol derivatives (e.g., cholesterol) is from about 20 mol% to about 40 mol% (e.g., from about 20 mol% to 25 mol%, from 20 mol% to 30 mol%, from 20 mol% to 35 mol%, from 20 mol% to 40 mol%, from 25 mol% to 30 mol%, from 25 mol% to 35 mol%, or from 25 mol% to 40 mol%).

In other embodiments, the compounds of the invention are used in the formulation of RNA binders (e.g., about 25.9 mol% L-6, L-30, L-48, or L-49). In certain embodiments, the compounds of the invention used in the formulation of RNA binders are different from the compounds of the invention used in the formulation of transfection lipids (e.g., 25.9 mol% L-48 as the RNA binder and 21.6 mol% L-30 as the transfection lipid). In some embodiments of the formulation, one or more RNA binders form an internal aggregate and one or more transfection lipids form an external aggregate surface. In certain embodiments, the external aggregate surface is not a membrane, lipid bilayer, and/or multilayer.

Preparation can also include one or more PEG-lipid conjugates of any useful amount.In some embodiments, the content of PEG-lipid conjugates is about 1mol% to about 20mol% (for example, about 1mol% to about 2mol%, about 2mol% to about 4mol%, about 2mol% to about 7mol%, about 4mol% to about 8mol%, about 8mol% to about 12mol%, about 12mol% to about 16mol% or about 16mol% to about 20mol%) in preparation.In other embodiments, the content of PEG-lipid conjugates is about 7mol%, 6mol%, 3.0mol% or 2.5mol%.And, PEG-lipid content can be by suitably adjusting DSPC or cholesterol or both content and change, about 1mol% to about 20mol%. The PEG-lipid can be varied by using C14:0 (e.g., PEG-DSPE or PEG-DMPE, etc., as in Table 4), C16 (PEG-DPPE, PEG-DPG, etc.), C18:0 (PEG-DSPE, PEG-DSG, etc.), or C18:1 (PEG-DOPE, PEG-DOG, etc.). Furthermore, PEG moieties of varying molecular weights can be used (PEG2000, PEG3400, PEG5000, etc.). In particular embodiments, as used herein, mixed PEG-conjugates are used. In particular embodiments, PEG2000-DSPE is used. In particular embodiments, PEG2000-DMPE is used.

Preparations containing RNAi agents

The formulations of the invention can be formulated with amino-amine cationic lipids and/or amino-amide lipids and RNAi agents by any of the methods described herein. For example, see: Judge et al., J. Clin. Invest. 119(3): 661, 2009; Noble et al., Cancer Chemother. Pharmacol. 64(4): 741, 2009; Abrams et al., Mol. Ther. 18(1): 171, 2009; Yagi et al., Cancer Res. 69(16): 6531, 2009; Ko et al., J. Control. Release 133(2): 132, 2009; Mangala et al., Methods Mol. Biol. 555: 29, 2009, which are incorporated herein by reference.

The formulation can include any useful ratio of RNAi agent and lipid molecules and/or one or more components. Exemplary ratios include about 1:10 to about 1:100 (w/w) (e.g., about 1:10 to about 1:50, such as about 1:20) RNAi agent:total lipid ratio (w/w) ratio, wherein the total lipid ratio is the weight of the combination of one or more lipid molecules (e.g., cations, anions, or neutral lipids) and one or more components (e.g., sterol derivatives, PEG-lipid conjugates, polyamide-lipid conjugates, gangliosides, antioxidants, surfactants, amphiphiles, or salts).

The formulation can include the RNAi agent at a dosage of any RNAi agent described herein ranging from about 1 mg/kg to about 10 mg/kg. Exemplary dosages include 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, and 10 mg/kg of RNAi agent in the formulation.

Method for preparing the preparation

Any useful method can be used to prepare the preparation of the present invention.In an exemplary program, the component of preparation (for example, one or more RNA binding agents, transfection lipids or any lipid described herein) is dissolved in a solvent (for example, an aqueous solvent, a non-aqueous solvent or its solvent mixture). The lipid suspension obtained can optionally be filtered, mixed (for example, batch mixing, online mixing and/or vortex), evaporated (for example, using nitrogen or argon stream), resuspended (for example, in an aqueous solvent, a non-aqueous solvent or its solvent mixture), freeze-thawed, extruded and/or ultrasonic. Moreover, lipid suspension can optionally be processed to produce final suspension by adding any desired component (for example, one or more RNAi agents, RNA binding agents, transfection lipids and/or any lipid described herein). One or more desired components can be provided in the same or different solvents in the form of suspension. For example, the lipid suspension can be provided in a first solvent or solvent system (e.g., one or more aqueous or non-aqueous solvents, such as water, water-HCl, water-ethanol, a buffer (e.g., phosphate-buffered saline (PBS), Hank's balanced salt solution (HBSS), Dulbecco's phosphate-buffered saline (DPBS), Earle's balanced salt solution (EBSS), carbonate, lactate, ascorbate, and citrate, such as 5 mM, 10 mM, 50 mM, 75 mM, 100 mM, or 150 mM)), a physiological osmotic pressure solution (290 mOsm/kg, such as 0.9% saline, 5% dextrose, and 10% sucrose), saline, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, glycerol, ethylene glycol, propylene glycol, polyethylene glycol, chloroform, dichloromethane, hexane, cyclohexane, acetone, ethyl ether, diethyl ether, dioxane, isopropyl ether, tetrahydrofuran, or a combination thereof), And the RNAi agent can be provided in a second solvent or solvent system, for example, one or more aqueous or non-aqueous solvents, such as water, water-HCl, water-ethanol, a buffer (e.g., phosphate-buffered saline (PBS), Hank's balanced salt solution (HBSS), Dulbecco's phosphate-buffered saline (DPBS), Earle's balanced salt solution (EBSS), carbonate, lactate, ascorbate, and citrate, e.g., 5 mM, 10 mM, 50 mM, 75 mM, 100 mM, or 150 mM)), a physiological osmotic pressure solution (290 mOsm/kg, e.g., 0.9% saline, 5% dextrose, and 10% sucrose), saline, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, glycerol, ethylene glycol, propylene glycol, polyethylene glycol, chloroform, dichloromethane, hexane, cyclohexane, acetone, ethyl ether, diethyl ether, dioxane, isopropyl ether, tetrahydrofuran, or a combination thereof). Exemplary concentrations of aqueous solvents and/or buffers include about 4% to about 8% ethanol (e.g., about 4% to 5%, 5% to 6%, 6% to 7%, or 7% to 8%), about 10 mM to about 100 mM citrate (e.g., about 10 mM to 30 mM, 30 mM to 50 mM, 50 mM to 70 mM, 70 mM to 90 mM, or 90 mM to 100 mM). Any solvent or solvent system can include one or more stabilizers, such as antioxidants, salts (e.g., sodium chloride), citric acid, ascorbic acid, glycine, cysteine, ethylenediaminetetraacetic acid (EDTA), mannitol, lactose, trehalose, maltose, glycerol, and/or glucose. In other examples, one or more RNA binders are introduced into a lipid suspension using a first solvent or solvent system, and then one or more transfection lipids are added in a second solvent or solvent system, wherein the first and second solvents or solvent systems are the same or different (e.g., the first solvent or solvent system is any one described herein; and the second solvent or solvent system is any one described herein). In certain embodiments, the second solvent or solvent system comprises one or more aqueous or non-aqueous solvents selected from the group consisting of saline, a buffer (e.g., citrate or PBS), water, and ethanol. The final suspension can optionally be separated (e.g., by ultracentrifugation), mixed (e.g., batch mixing, online mixing, and/or vortexing), resuspended, adjusted (e.g., with one or more solvents or buffer systems), sonicated, freeze-thawed, extruded, and/or purified.

Cationic lipids

One or more cationic lipids may be included in the formulation. In addition to the compounds of the present invention, other cationic lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), N,N-distearyl-N,N-dimethylammonium (DDAB), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP, including chiral forms R-DOTAP and S-DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium (DOSPA), dioctadecylamidoglycylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DOD AP), N,N-dimethyl-(2,3-dioleyloxy)propylamine (DODMA), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium (DMRIE), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLenDMA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1, 2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dipalmitoyl-sn-glycero-O-ethyl-3-phosphocholine (DPePC), distearyldimethylammonium chloride (DSDMA), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (12:0EPC, for example, or its chloride salt), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (DPePC), distearyldimethylammonium chloride (DSDMA), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DPePC, for example, or its chloride salt), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (DPePC), :0EPC, for example, or its chloride salt), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (18:0EPC, for example, or its chloride salt), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1EPC, for example, or its chloride salt), dipalmitoylphosphatidylethanolamidospermine (DPPES), dipalmitoylphosphatidylethanolamido L-lysine (DPPEL), 1-[2-dioleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), (1-methyl-4-(cis-9-dioleyl)methyl-pyridinium chloride)) (SAINT) and C12-200, as described in Love et al., Proc Natl Acad Sci U S A, 107(5): 1864-1869 (2010), which is incorporated herein by reference.

Cationic lipids include those in different chiral forms (e.g., the R or S form of any cationic lipid described herein) or in any salt form (e.g., the chloride, bromide, trifluoroacetate, or methanesulfonate salt of any cationic lipid described herein).

In addition, many commercial preparations of cationic lipids can be included in the formulation. Such commercial preparations include, but are not limited to: Lipofectamine ^™ (a combination of DOSPA and DOPE) and FLEXTRA® (a combination of DOTMA and DOPE) from Invitrogen Corp.; and FLEXTRA® (a composition including DOGS) and Transfast ^™ from Promega Corp.

Anionic lipids

One or more anionic lipids may be included in the formulation. Such anionic lipids include, but are not limited to, phosphatidylglycerol (PG), cardiolipin (CL), diacylphosphatidylserine (PS), diacylphosphatidic acid (PA), phosphatidylinositol (PI), N-acylphosphatidylethanolamine (NAPE), N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, and palmitoyloleoylphosphatidylglycerol (POPG), as well as different chiral forms (e.g., R or S forms), salt forms (e.g., chloride, bromide, trifluoroacetate, or methanesulfonate), and mixtures thereof.

neutral lipids

One or more neutral lipids may be included in the formulation. Such neutral lipids include, but are not limited to, ceramides, sphingomyelins (SMs), diacylglycerols (DAGs), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, including chiral forms R-DSPC and S-DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-glycero-sn-3-phosphocholine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE ... The lipid granules of the present invention can be choline granules of the present invention.

In some embodiments, the neutral lipid component present in the formulation includes one or more phospholipids. In other embodiments, the neutral lipid component includes a mixture of one or more phospholipids and cholesterol. In some embodiments, pharmacokinetic and/or pharmacodynamic properties, such as lipid particle size and stability in the bloodstream, are considered to guide the selection of neutral lipids for the formulation.

Sterol derivatives

One or more sterol derivatives may be included in the formulation. Without wishing to be bound by theory, sterol derivatives may be used to stabilize the formulation and/or increase transfection. Exemplary sterol derivatives include cholesterol, cholesterol derivatives (e.g., cholestanone, cholestenone, or coprostanol); 3β-[-(N-(N', N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-cholesterol, e.g., its hydrochloride); biguanide-triaminoethylamine-cholesterol (BGTC); (2S, 3S)-2-(((3S, 10R, 13R, 17R)-10,13-dimethyl-17-((R)-6-methylheptyl-2- ((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)carbonylamino)ethyl 2,3,4,4-tetrahydroxybutyrate (DPC-1); (2S,3S)-((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)carbonylamino)ethyl 2,3,4,4-tetrahydroxybutyrate (DPC-1); 1,2,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) 2,3,4,4-tetrahydroxybutyrate (DPC-2); bis((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-2,3,4,4-tetrahydroxybutyrate and 6-(((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yloxy)phosphoryloxy)-2,3,4,5-tetrahydroxyhexanoate (DPC-4).

PEG-lipid conjugates

One or more PEG-lipid conjugates can be included in the formulation. Without wishing to be bound by theory, PEG-lipid conjugates can reduce aggregation of lipid carriers. PEG-lipid conjugates are described in U.S. Patent No. 5,885,613 and U.S. Patent Publication No. 2003/0077829, which are incorporated herein by reference.

PEG-lipid conjugates that can be included in the formulation include, but are not limited to, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(carbonyl-methoxy-polyethylene glycol) (PEG-DMPE) (e.g., 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(carbonyl-methoxy-polyethylene glycol-2000) (PEG-2000-DMPE)), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- (carbonyl-methoxy-polyethylene glycol) (PEG-DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(carbonyl-methoxy-polyethylene glycol) (PEG-DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carbonyl-methoxy-polyethylene glycol) (PEG-DOPE), 1,2-dimyristoyl-sn-glycero-3-(methoxy-polyethylene glycol) (PEG-DMG) (e.g., 1,2- Dimyristoyl-sn-glycerol-3-(methoxy-polyethylene glycol) (PEG-2000-DMG)), 1,2-dipalmitoyl-sn-glycerol-3-(methoxy-polyethylene glycol) (PEG-DPG), 1,2-distearoyl-sn-glycerol-3-(methoxy-polyethylene glycol) (PEG-DSG), 1,2-dioleoyl-sn-glycerol-3-(methoxy-polyethylene glycol) (PEG-DOG), 3-N-[(ω-methoxypoly(ethylene glycol)] )2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA), R-3-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine (PEG-2000-C-DOMG), and PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20, which are described in U.S. Pat. No. 5,820,873, which is incorporated herein by reference). Other PEG-lipid conjugates include PEG conjugated to any lipid described herein, such as phosphatidylethanolamine or ceramide (see, U.S. Pat. Nos. 5,820,873; 5,534,499; and 5,885,613, which are incorporated herein by reference), as well as salt forms (e.g., sodium, ammonium, or trimethylammonium salts) of any PEG-lipid conjugate described herein.

PEG-lipid conjugates can include one or more different modifications, such as substitution with any lipid molecule described herein or PEG moieties of different molecular weights (e.g., 300 to 5,000 Daltons). Exemplary substitutions include the use of one or more C14:0 (as in Table 4), C16 (PEG-DPPE, PEG-DPG, etc.), C18:0 (PEG-DSPE, PEG-DSG, etc.), or C18:1 (PEG-DOPE, PEG-DOG, etc.) in combination with polyethylene glycol moieties (e.g., PEG2000, PEG3400, PEG5000, etc.) to form PEG-lipid conjugates (e.g., mPEG2000-DMG). Examples of PEG moieties with different molecular weights include PEG350, PEG550, PEG750, PEG1000, PEG2000, PEG3000, PEG3400, PEG4000, and PEG5000.

Other components

The formulation may include any other components to help stabilize the lipid carrier, reduce aggregation of the lipid carrier, and/or deliver a therapeutic agent (e.g., an RNAi agent). Exemplary components include polyamide-lipid conjugates (ATTA-lipids) based on ω-amino (oligoethylene glycol) alkanoic acid monomers, such as those described in U.S. Patent Nos. 6,320,017 and 6,586,559, which are incorporated herein by reference; gangliosides (e.g., asialoganglioside GM1 or GM2; disialoganglioside GD1a, GD1a-NAcGal, GD1-b, GD2, or GD3; erythroside, monosialoganglioside GM1, GM2, or GM3, tetrasialoganglioside GQ1b, and trisialoganglioside GT1a or GT1b); antioxidants (e.g., α-tocopherol or β-hydroxytoluidine); one or more surfactants (e.g., sorbitan monopalmitate or sorbitan monopalmitate); monopalmitate, oily sucrose esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, oxyethylene fatty acid esters, polyoxyethylene alkyl ethers, polyoxyethylene sterol ethers, polyoxyethylene-polypropoxyalkyl ethers, block polymers and cetyl ethers as well as polyoxyethylene castor oil or hydrogenated castor oil derivatives and polyglycerol fatty acid esters, for example or (for example EL having as main components glycerol-polyethylene glycol ricinoleate and polyethylene glycol fatty acid esters); one or more amphiphilic agents (for example vegetable oils, for example soybean oil, safflower oil, olive oil, sesame oil, borage oil, castor oil and cottonseed oil; mineral oil and marine oils, hydrogenated and/or fractionated triglycerides from such sources; medium chain triglycerides (MCT-oil, for example, ) and various synthetic or semi-synthetic mono-, di- or triglycerides, for example WO 92/05571, and an acetylated monoglyceride or an alkyl ester of a fatty acid, such as isopropyl myristate, ethyl oleate (see EP 0 353 267), or a fatty acid alcohol, such as oleyl alcohol, cetyl alcohol); and one or more salts, such as any salt described herein. Typically, the concentration of the lipid component selected to reduce aggregation is about 1 mol% to 15 mol%.

lipid carriers

The formulations of the invention can include one or more compounds of the invention (e.g., compounds of Formula (I) or selected from Table 1) and any lipid-based composition capable of transporting a therapeutic agent (e.g., an RNAi agent). Exemplary lipid-based compositions include one or more lipid molecules (e.g., a compound of the invention, a cationic lipid, an anionic lipid, or a neutral lipid) and/or one or more components (e.g., a sterol derivative and/or a PEG-lipid conjugate).

Any biocompatible lipid or lipid combination capable of forming a lipid carrier (e.g., liposomes, liposome complexes, and micelles) can be used to form a lipid carrier. Encapsulating the therapeutic agent in a lipid carrier can protect the therapeutic agent from damage or degradation, or promote its entry into cells. The lipid carrier interacts and fuses with the cell membrane due to charge interaction (e.g., cationic lipid carriers and anionic cell membranes), thereby releasing the therapeutic agent into the cytoplasm. Liposomes are double-layer capsules comprising one or more compounds of the present invention, lipid molecules, and/or components. Lipid nanoparticles are liposomes ranging in size from about 1 nm to about 1,000 nm. Liposome complexes are liposomes formed with cationic lipid molecules to give the liposome an overall positive charge. Micelles are capsules with single-layer lipid molecules.

liposomes

In certain embodiments, the lipid carrier is a liposome. Generally, the lipid used can form a double layer and is cationic. The category of lipid molecules that are suitable includes phospholipids (e.g., phosphatidylcholine), fatty acids, glycolipids, ceramides, glycerides and cholesterol or any combination thereof. Alternatively or additionally, the lipid carrier can include a neutral lipid (e.g., dioleoylphosphatidylethanolamine (DOPE)). Other lipids that can form lipid carriers are known in the art and are described herein.

As used herein, " lipid molecule " is the molecule with hydrophobic head part and hydrophilic tail part, and can form liposome, comprises any cation, neutral or anionic lipid described in the compounds of this invention or this paper.Lipid molecule can optionally be modified to comprise hydrophilic polymer group.The example of this type of lipid molecule comprises 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-[methoxyl group (polyethylene glycol)-2000] (PEG2000-DSPE), for example its ammonium salt) and 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-[carboxyl (polyethylene glycol)-2000] (PEG2000-DSPE carboxyl).

Examples of lipid molecules include natural lipids such as cardiolipin (CL), phosphatidic acid (PA), phosphatidylcholine (PC), lysolecithin (LPC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI) and phosphatidylserine (PS); lipid mixtures such as lecithin (lechitin); sphingolipids such as sphingosine, ceramide, sphingomyelin, cerebrosides, sulfheptazoline, gangliosides and phytosphingosine; cationic lipids such as 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), dimethyldioctadecyl ammonium bromide (DDAB), 3-β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-[1-(2,3,-dioleyloxy)propyl]-N,N-dimethylaminoethylammonium bromide (DMRIE), methyl-N-hydroxyethylammonium bromide (DORIE) and 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA); phosphatidylcholines, such as 1,2-dilauroyl-sn-glycero-3-ethyl choline phosphate, 1,2-dilauroyl-sn-glycero-3-choline phosphate (DLPC), 1,2-dimyristoyl-sn-glycero-3-choline phosphate (DMPC), 1,2-dipalmitoyl-sn-glycero-3-choline phosphate (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); phosphoethanolamines such as 1,2-dibutyryl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine Phosphatidic acids, such as dihexadecyl phosphate (DCP), 1,2-dimyristoyl-sn-glyceroyl-3-phosphoethanolamine (DPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl); phosphatidic acids, such as dihexadecyl phosphate (DCP), 1,2-dimyristoyl-sn-glyceroyl-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl); phosphatidylglycerols, such as dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylglycerol (DOPG), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) and 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol); phosphatidylserines, such as 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol); myristoyl-sn-glycero-3-phospho-L-serine, 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine, and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine; cardiolipins, such as 1',3'-bis[1,2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol; and PEG-lipid conjugates, such as 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750] 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000].

The compounds of the present invention can be combined with any useful lipid composition, including commercially available lipid compositions. Examples of such compositions include Lipofectamine ^™ (a combination of DOSPA and DOPE) and (a combination of DOTMA and DOPE) from Invitrogen Corp.; (compositions including DOGS) and Transfast ^™ from Promega Corp.; NeuroPORTER ^™ and Escort ^™ from Sigma-Aldrich Co.; 6 from Roche; and known lipid compositions from Strategene include Trojan Horse liposome technology, as described in Boado, Pharm. Res. 24: 1772-1787 (2007).

Liposomes may also include other components that aid in liposome formation or stability. Examples of components include cholesterol, antioxidants (eg, α-tocopherol or β-hydroxytoluidine), surfactants, and salts.

Liposomes can be any useful composition comprising lipid molecules, including one or more compounds of the invention and other lipid components that assist in liposome formation or stability. One skilled in the art will know how to optimize combinations that contribute to specific agent encapsulation, liposome stability, scale-up reaction conditions, or any other relevant factors. Exemplary combinations are described in Boado, Pharm. Res. 24: 1772-1787 (2007).

Preparation liposomes is usually carried out by a general two-step process. In the first step, lipids and lipid components are mixed in a volatile organic solvent or solvent mixture to ensure a uniform lipid mixture. Examples of solvents include chloroform, methanol, cyclohexane and n-butanol. The solvent is then removed to form a dry lipid mixture of film, powder or sheet form. Solvents can also be removed by using any known analytical technique, for example, by using nitrogen, rotary evaporation, spray drying, lyophilization and vacuum drying.

In the second step, the dry lipid mixture is hydrated with an aqueous solution to form liposomes. The agent can be added to the aqueous solution, which results in the formation of liposomes with the encapsulated agent. Alternatively, liposomes are first formed with a first aqueous solution and then exposed to another aqueous solution containing the agent. Encapsulation of the agent can be promoted by any known technique, such as by repeated freeze-thaw cycles, ultrasound or mixing. Other examples of this method are described in Boado, Pharm. Res. 24: 1772-1787 (2007). Alternatively, the agent is coupled to a hydrophobic portion (e.g., cholesterol) to produce a lipophilic derivative, and the lipophilic derivative is used together with other lipid molecules to form liposomes.

During the second step, the dry lipid mixture may or may not contain polypeptide-lipid conjugates. The process may optionally include various other steps, including heating the aqueous solution above the phase transition temperature of the lipid molecules before adding it to the dry lipid mixture, wherein a specific temperature range includes about 40°C to about 70°C; incubating the combination of the dry lipid mixture and the aqueous solution, wherein a specific time range includes about 30 minutes to about 2 hours; mixing the dry lipid mixture and the aqueous solution during incubation, for example by vortex mixing, shaking, stirring or agitation; adding non-electrolytes to the aqueous solution to ensure physiological osmotic pressure, such as a solution of 0.9% saline, 5% dextrose and 10% sucrose; disrupting large multilamellar vesicles, for example by extrusion or sonication; and additionally incubating preformed liposomes with the polypeptide-lipid conjugate, wherein the dry lipid mixture does not contain lipid molecules. One skilled in the art will be able to identify specific temperatures and incubation times in this hydrogenation step to ensure that the derivatized lipid molecules are incorporated into the liposomes or to obtain stable liposomes.

The compounds of this invention can be added at any point in the process of forming liposomes. In one example, the compound is added to lipids and lipid components during the formation of a dry lipid mixture. In another example, the compound is added to liposomes preformed with a dry lipid mixture containing lipids and lipid components. And in another example, micelles are formed with the compound, liposomes are formed with a dry lipid mixture containing lipids and lipid components, and then micelles and liposomes are hatched together. The aqueous solution can include other components with stabilizers or liposomes, such as buffer, salt, chelating agent, saline, dextrose, sucrose, etc.

In one example of this procedure, a dry film composed of a lipid mixture is hydrated with an aqueous solution containing an agent. The mixture is first heated to 50°C for 30 minutes and then cooled to room temperature. Next, the mixture is transferred to a dry film containing a polypeptide-lipid conjugate. The mixture is then incubated at 37°C for 2 hours to incorporate the polypeptide-lipid conjugate into the liposomes containing the agent. See, for example, Zhang et al., J. Control. Release 112: 229-239 (2006).

Lipid particles with vesicular structure

In certain embodiments, the lipid particle includes a cationic lipid (e.g., DODMA, DOTMA and/or an amino-amine lipid, an amino-amide lipid, or other lipids of the invention) and a RNAi agent, and a neutral or zwitterionic lipid, a PEG-lipid conjugate, and optionally cholesterol.

Lipid particles with one or more RNA binders and one or more transfection lipids

Lipid granules also include those with one or more RNA binding agents and one or more transfection lipids. In one embodiment, one or more RNA binding agents form internal aggregates, and one or more transfection lipids form external aggregate surfaces. In specific embodiments, external aggregate surfaces are not membranes, lipid bilayers and/or multilayers. In certain embodiments, one or more RNA binding agents (e.g., lipids) represent about 10-90% of total lipids. In other embodiments, one or more RNA binding agents (e.g., lipids) represent about 50% of total lipids. In other embodiments, one or more RNA binding agents (e.g., lipids) represent about 30% of total lipids. In certain embodiments, the complex/aggregate of one or more RNA binding agents of nucleic acid payload and lipid granules includes cationic lipids (e.g., DODMA, DOTMA and/or amino-amine lipids or amino-amides of the present invention) and RNAi agents; and one or more transfection lipids of lipid granules include neutral or zwitterionic lipids, PEG-lipid conjugates and optional cholesterol. In other embodiments, the one or more transfection lipids of the particle include a cationic lipid (e.g., DODMA, DOTMA, an amino-amine lipid, and/or an amino-amide lipid), a neutral lipid, a PEG-lipid conjugate, and optionally cholesterol.

RNAi agents

RNA interference (RNAi) is a mechanism that inhibits gene expression by causing the degradation of specific RNA molecules or hindering the transcription of specific genes. In nature, RNAi targets are usually RNA molecules from viruses and transposons (a form of innate immune response), although it also plays a role in regulating development and genome maintenance. The key to the RNAi mechanism is a small interfering RNA chain (siRNA), which has a nucleotide sequence that is sufficiently complementary to the target messenger RNA (mRNA) molecule. siRNA directs proteins within the RNAi pathway to the target mRNA and degrades them, breaking them down into smaller parts that can no longer be translated into proteins.

The RNAi pathway is initiated by the enzyme Dicer, which cracks long double-stranded RNA (dsRNA) molecules into siRNA molecules, which are generally about 21 to about 23 nucleotides in length and contain about 19 base pair duplexes. One of the two chains of each fragment, called the guide strand, is then incorporated into the RNA-induced silencing complex (RISC) and paired with a complementary sequence. RISC mediates the cracking of single-stranded RNA with a sequence complementary to the antisense strand of the siRNA duplex. The cracking of the target RNA occurs in the middle of the region complementary to the antisense strand of the siRNA duplex. The result of this recognition event is post-transcriptional gene silencing. This occurs when the guide strand specifically pairs the mRNA molecule and induces degradation by Argonaute (the catalytic component of the RISC complex).

The compounds of the present invention can be used to deliver one or more RNAi agents to cells in vitro or in vivo (e.g., in a subject). RNAi agents can include different types of double-stranded molecules, including RNA: RNA or RNA: DNA chains. These agents can be introduced into cells with various structures, including duplexes (e.g., with or without overhangs at the 3' end), hairpin loops, or expression vectors that can form one or more polynucleotides of double-stranded polynucleotides that are combined with another polynucleotide. Exemplary RNAi agents include siRNA, shRNA, DsiRNA, and miRNA agents described herein. Generally speaking, the length of these agents is about 10 to about 40 nucleotides, and preferred lengths are described below for specific RNAi agents.

Functional gene silencing of RNAi agents does not necessarily include complete inhibition of the target gene product. In some cases, the marginal decrease in gene product expression caused by the RNAi agent can translate into significant functional or phenotypic changes in the host cell, tissue, organ, or animal. Therefore, it is understood that gene silencing is functionally equivalent, and the degree of gene product degradation that achieves silencing can vary between gene targets or host cell types.

siRNA

Small interfering RNA (siRNA) is generally a double-stranded RNA molecule of 16 to 30 nucleotides (e.g., 18 to 25 nucleotides, such as 21 nucleotides) in length, with one or two nucleotide overhangs or without any overhangs at the 3' end. The skilled person can vary the sequence length (e.g., to increase or decrease the overall level of gene silencing). In certain embodiments, the overhang is UU or dTdT at the 3' end. Generally speaking, the siRNA molecule is fully complementary to one strand of the target DNA molecule, as even single base pair mismatches have been shown to reduce silencing. In other embodiments, the siRNA can have a modified backbone composition, such as a 2'-deoxy- or 2'-O-methyl modification, or any modification described herein.

siRNA refers to a nucleic acid molecule that can inhibit or downregulate gene expression in a sequence-specific manner; see, for example, Zamore et al., Cell 101: 25-33 (2000); Bass, Nature 411: 428-429 (2001); Elbashir et al., Nature 411: 494-498 (2001); and PCT Publication Nos. WO 00/44895, WO 01/36646, WO 99/32619, WO 00/01846, WO 01/29058, WO 99/07409, and WO 00/44914. Methods for preparing siRNA molecules for gene silencing are described in U.S. Pat. No. 7,078,196, which is incorporated herein by reference.

shRNA

Short hairpin RNA (shRNA) is a single-stranded RNA molecule in which there is a hairpin loop structure, and the hairpin loop structure allows the complementary nucleotides in the same chain to form an intermolecular bond. shRNA can show a sensitivity to nuclease degradation reduced compared with siRNA. In certain embodiments, shRNA has a stem length of 19 to 29 nucleotides (for example, 19 to 21 nucleotides or 25 to 29 nucleotides). In some embodiments, the ring size is 4 to 23 nucleotides in length. shRNA generally can contain one or more mispairings, for example, the G-U mispairing between the two chains of the shRNA stem, and does not reduce titer.

DsiRNA

Dicer-substrate RNA (DsiRNA) is a double-stranded RNA agent of 25 to 35 nucleotides. It is believed that agents of this length are processed by the Dicer enzyme of the RNA interference (RNAi) pathway, while agents shorter than 25 nucleotides generally mimic Dicer products and escape Dicer processing. In some embodiments, the DsiRNA has a single-stranded nucleotide overhang at the 3' end of the antisense or sense strand of 1 to 4 nucleotides (e.g., 1 or 2 nucleotides).

Certain modified structures of DsiRNA agents were previously described in US Patent Publication No. 2007/0265220, which is incorporated herein by reference. Other DsiRNA structures and specific compositions suitable for use in the formulations of the present invention are described in U.S. Patent Application No. 12/586,283; U.S. Patent Publication Nos. 2005/0244858, 2005/0277610, 2007/0265220, 2011/0021604, 2010/0173974, 2010/0184841, 2010/0249214, 2010/0331389, 2011/0003881, 2011/0059187, 2011/0111056; and PCT Publication Nos. WO 2010/080129, WO 2010/093788, WO 2010/115202, WO 2010/115206, WO 2010/141718, WO 2010/141724, WO 2010/141933, WO 2011/072292, WO 2011/075188, which are incorporated herein by reference. In general, DsiRNA constructs are synthesized using solid phase oligonucleotide synthesis methods as described for 19-23mer siRNAs (see U.S. Patent Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117; 6,469,158; 6,111,086; 6,008,400; and 6,111,086).

miRNA

MicroRNA (miRNA) is a single-stranded RNA molecule with a length of 17 to 25 nucleotides (for example, 21 to 23 nucleotides). Technicians can change the sequence length to increase or reduce the overall level of gene silencing. These agents silence the target gene by binding to the complementary sequence on the target messenger RNA. As used herein, the term "miRNA precursor" is used to cover but not limited to primary RNA transcripts, pri-miRNA and pre-miRNA. "miRNA payload" of the present invention can include pri-miRNA, pre-miRNA and/or miRNA (or mature miRNA). In certain embodiments, siRNA of the present invention (for example, DsiRNA) can provide a guide strand (making this siRNA become "miRNA mimics") that has been incorporated into a miRNA sequence or is sufficiently homologous to a miRNA sequence to give play to the miRNA function.

Antisense compounds

Exemplary antisense compounds include a continuous nucleotide length range, wherein the upper limit of the scope is 50 nucleotides, and wherein the lower limit of the scope is 8 nucleotides. In certain embodiments, the upper limit of the scope is 35 nucleotides, and the lower limit of the scope is 14 nucleotides. In other embodiments, the upper limit of the scope is 24 nucleotides, and the lower limit of the scope is 17 nucleotides. In other embodiments, the antisense compound is 20 continuous nucleotides. One skilled in the art will readily understand that the upper end of the range as disclosed herein includes 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive nucleotides, and that the upper end of the range includes 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides.

Exemplary antisense compounds include a stretch of at least 8, optionally at least 12, optionally at least 15 contiguous nucleotides that are sufficiently complementary to a target sequence to interfere with the transcription, translation, promote degradation (optionally nuclease-mediated degradation) of the target sequence, and/or otherwise disrupt the function of the target sequence (e.g., interfere with the function of other functional target sequences, e.g., disrupt a promoter, enhancer, or other functional nucleic acid target sequence by an antisense compound-mediated mechanism).

Modifications can be made to the antisense compounds and can include conjugated groups attached to one of the ends selected from the nucleobase positions, sugar positions, or nucleotide linkages. Possible modifications include, but are not limited to, 2'-fluoro (2'-F), 2'-O methyl (2'-OMe), 2'-O-(2-methoxyethyl) (2'-MOE) high-affinity sugar modifications, inverted abasic caps, deoxynucleobases, and bicyclic nucleobase analogs such as locked nucleic acids (LNA) and ethylene-bridged nucleic acids (ENA).

Methods for preparing RNAi agents

RNAi agent includes at least one antisense nucleotide sequence of a target nucleic acid (e.g., a target gene). Antisense nucleotides are single-stranded DNA or RNA complementary to a selected target sequence. In the case of antisense RNA, they prevent the translation of a complementary RNA chain by binding to it. Antisense DNA can be used for targeting specific complementary (coding or non-coding) RNA. In a specific embodiment, antisense nucleotides contain about 10 to about 40 nucleotides, more preferably about 15 to about 30 nucleotides. Antisense nucleotides can have 80%, 85%, 90%, 95%, 99% or even 100% complementarity to the expected target gene.

Methods for preparing antisense and sense nucleotides and corresponding duplexes or hairpin loops are known in the art and can be readily adapted to prepare antisense oligonucleotides targeting any target nucleic acid sequence. Antisense nucleotide sequences can be selected to optimize target specificity, for example by analyzing the target sequence and determining secondary structure, Tm, binding energy, and relative stability; and/or to reduce the formation of secondary structures, such as dimers, hairpins, or other secondary structures that will reduce or prevent specific binding to the target mRNA in the host cell. In some embodiments, highly preferred target regions of mRNA include those at or near the AUG translation start codon and those sequences that are substantially complementary to the 5' region of the mRNA. These secondary structure analyses and target site selection considerations can be performed, for example, using OLIGO primer analysis software (Molecular Biology Insights, version 4) and/or BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res. 25(17): 3389-3402, 1997). Non-limiting methods of making RNAi agents are described in U.S. Patent Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117; 6,469,158; 6,111,086; 6,008,400; and 6,111,086, which are incorporated herein by reference.

RNAi agents can have any useful form, such as single-stranded, double-stranded, linear, circular (e.g., plasmid), nicked circular, helical, supercoiled, concatemerized, or charged. In addition, the nucleotides can contain 5' and 3' sense and antisense strand end modifications and can have blunt or overhanging terminal nucleotides (e.g., UU or TT at the 3' end) or a combination thereof.

Modified nucleic acids include modified DNA or RNA molecules that can be used to replace naturally occurring nucleic acids in the polynucleotides described herein (e.g., RNAi agents). Modified nucleic acids can improve the half-life, stability, specificity, delivery, solubility, and nuclease resistance of the polynucleotides described herein. For example, siRNA agents can be composed in part or entirely of nucleotide analogs that confer the above-mentioned beneficial properties. As described in Elmén et al. (Nucleic Acids Res. 33: 439-447 (2005)), synthetic RNA-like nucleotide analogs (e.g., locked nucleic acids (LNA)) can be used to construct siRNA molecules that exhibit silencing activity against target gene products.

Phosphorothioate (PS) backbone modifications, in which the non-bridging oxygen in the phosphodiester bond is replaced by sulfur, are one of the earliest and most common approaches for stabilizing nucleic acid drugs against nuclease degradation. In general, it appears that PS modifications can be performed broadly on both siRNA strands without significantly affecting activity (Kurreck, Eur. J. Biochem. 270: 1628-44 (2003)). In specific embodiments, PS modifications are typically limited to one or two bases at the 3' and 5' ends. Boranophosphate linkers can be used to enhance siRNA activity while having low toxicity (Hall et al., Nucleic Acids Res. 32: 5991-6000 (2004)). Other exemplary modifications to the oligonucleotide backbone include methyl phosphonates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkyl phosphonates (e.g., 3'-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-aminophosphoramidates), aminoalkylphosphoramidates, thionylphosphoramidates, thionylalkylphosphonates, thionylalkylphosphotriesters, and protein nucleotide (PNA) backbones having repeating N-(2-aminoethyl)-glycine units linked by peptide bonds, representative PNA compounds include, but are not limited to, those described in U.S. Pat. Nos. 5,539,082, 5,714,331, and 5,719,262, and Nielsen et al., Science 254:1497-1500 (1991).

Other modifications to the backbone include those in which the phosphorus atom is replaced with a short chain alkyl or cycloalkyl internucleotide linkage, a mixed heteroatom and alkyl or cycloalkyl internucleotide linkage, or one or more short chain heteroatom or heterocyclic internucleotide linkages (e.g., morpholino linkages; siloxane backbones; sulfide, sulfoxide and sulfone backbones; formyl and thioformyl backbones; methyleneformyl and thioformyl backbones; olefin-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazinyl backbones; sulfonate and sulfonamide backbones; amide backbones; and others with mixed N, O, S and _CH2 components).

Certain modified nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention, such as 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines (e.g., 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, and 5-methylcytosine). Exemplary modified nucleobases include 5-methylcytosine (5-me-C or m5c); 5-hydroxymethylcytosine, xanthine, and hypoxanthine; 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil; 2-thiothymine; 2-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azouracil, cytosine, and thymine. ; 5-uracil (pseudouracil); 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines; 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines; 7-methylguanine; 7-methyladenine; 8-azaguanine; 8-azaadenine; 7-deazaguanine; 7-deazaadenine; 3-deazaguanine; and 3-deazaadenine. In certain embodiments, these modified nucleobases can be combined with other modifications, such as any sugar modification described herein.

Modified oligonucleotides may also contain one or more substituted sugar moieties, where the modification may be made at any active site of the ribose ring (eg, the 2'-OH of the ribose ring) or at one or more universal bases. Exemplary modifications include 2'-halo, such as F, Br, or Cl; 2'-O-alkyl, 2'-S-alkyl, or 2'-N-alkyl, such as 2'-OMe;2'-O-(alkyl-O) _n -alkyl, such as 2'-O-methoxyethyl (2'-O- _MOE ), 2'-O _[ (CH2) _{nO ]mCH3, 2'-O(CH2)nOCH3 , 2' -} O( _CH2 ) _{2ON ( CH3} ) _2O (CH2 _{) nNH2} , _O ( _CH2 ) _nCH3 , 2'- _O (CH2) _nONH2 , _and 2'-O( _CH2 ) _nON [( _CH2 ) _nCH3 ) _{] 2} , wherein n and m are 1 to about 10; 2'-O-alkenyl, 2'-S-alkenyl or 2'-N-alkenyl;2'-O-alkynyl,2'-S-alkynyl or 2'-N-alkynyl, wherein the alkyl, alkenyl and alkynyl groups can be substituted or unsubstituted C _1-10 alkyl or C _2-10 alkenyl and alkynyl, and a bridge modification between the 2' and 4' positions of the ribose to form a locked nucleic acid (LNA). Exemplary universal bases include heterocyclic moieties located at the 1' position of the nucleotide sugar portion of the modified nucleotide or at the equivalent position in the nucleotide sugar portion substitution, such as 1-β-D-ribofurano-5-nitroindole and 1-β-D-ribofurano-3-nitropyrrole.

In certain embodiments, nucleic acids having the described modifications and/or modification patterns can be used. Additional details on exemplary modifications and modification patterns of nucleic acids can be found, for example, in at least the following references: US 2010/0240734; WO 2010/080129; WO 2010/033225; US 2011/0021604; WO 2011/075188; WO 2011/072292; WO 2010/141724; WO 2010/141726; WO 2010/141933; WO 2010/115202; WO 2008/136902; WO/2011/109294; WO/2011/075188; PCT/US11/42810; PCT/US11/42820; U.S. Serial No. 61/435,304; U.S. Serial No. 61/478,093; U.S. Serial No. 61/497,387; U.S. Serial No. 61/529,422; U.S. Patent No. 7,893,245; WO 2007/051303; and US 2010/0184209. Each of the foregoing documents is incorporated herein by reference in its entirety.

RNAi gene targets

The present invention is characterized by silencing target genes in diseased tissues or organs by combining RNAi agent treatment with a compound or formulation. The therapeutic potential of the present invention is realized when the mRNA molecules of specific targeted genes known or believed to be involved in establishing or maintaining a disease state (e.g., cancer) are degraded by RNAi agents.

Examples of RNAi targets for use in the present invention include developmental proteins, such as adhesion molecules, cell cycle protein kinase inhibitors, Wnt family members, Pax family members, Winged helix family members, Hox family members, cytokines/lymphokines and their receptors, growth/differentiation factors and their receptors, neurotransmitters and their receptors; proteins encoded by oncogenes (e.g., ABL1 (UniProt Accession No. P00519, NCBI Gene ID: 25), AR (UniProt Accession No. P10275, NCBI Gene ID: 3647), β-catenin (CTNNB1, UniProt Accession No. P35222, NCBI Gene ID: 1499), BCL1 (UniProt Accession No. P24385, NCBI Gene ID: 595), BCL2 (UniProt Accession No. P10415, NCBI Gene ID: ID: 596), BCL6 (UniProt accession number P41182), CBFA2 (UniProt accession number Q01196, NCBI Gene ID: 861), CBL (UniProt accession number P22681, NCBI Gene ID: 687), CSF1R (UniProt accession number P07333, NCBI Gene ID: 1436), ERBA1 (UniProt accession number P10827, NCBI Gene ID: 7067), ERBA2 (UniProt accession number P10828, NCBI Gene ID: 7068), ERBB (UniProt accession number P00533, NCBI Gene ID: 1956), ERBB2 (UniProt accession number P04626, NCBI Gene ID: 2064), ERBB3 (UniProt accession number P21860, NCBI Gene ID: 190151), ERBB4 (UniProt accession number Q15303, NCBI Gene ID: 600543), ETS1 (UniProt accession number P14921, NCBI Gene ID: 2113), ETS2 (UniProt accession number P15036, NCBI Gene ID: 2114), ETV6 (UniProt accession number 41212, NCBI Gene ID: 2120), FGR (UniProt accession number P09769, NCBI Gene ID: 2268), FOS (UniProt accession number P0110, NCBI Gene ID: 2353), FYN (UniProt accession number P06241, NCBI Gene ID: 2534), HCR (UniProt accession number Q8TD31, NCBI Gene ID: 54535), HRAS (UniProt accession number P01112, NCBI Gene ID: 3265), JUN (UniProt accession number P05412, NCBI Gene ID: 3725), KRAS (UniProt accession number P01116, NCBI Gene ID: 3845), LCK (UniProt accession number P06239, NCBI Gene ID: 3932), LYN (UniProt accession number P07948, NCBI Gene ID: 4067), MDM2 (UniProt accession number Q00987, NCBI Gene ID: 4193), MLL1 (UniProt accession number Q03164, NCBI Gene ID: 4297), MLL2 (UniProt accession number O14686, NCBI Gene ID: 8085), MLL3 (UniProt accession number Q8NEZ4, NCBI Gene ID: 58508), MYB (UniProt accession number P10242, NCBI Gene ID: 4602), MYC (UniProt accession number P01106, NCBI Gene ID: 4609), MYCL1 (UniProt accession number P12524, NCBI Gene ID: 4610), MYCN (UniProt accession number P04198, NCBI Gene ID: 4613), NRAS (UniProt accession number P01111, NCBI Gene ID: 4893), PIM1 (UniProt accession number P11309, NCBI Gene ID: 5292), PML (UniProt accession number P29890, NCBI Gene ID: 5371), RET (UniProt accession number P07949, NCBI Gene ID: 5979), SRC (UniProt accession number P12931, NCBI Gene ID: 6714), TAL1 (UniProt accession number P17542, NCBI Gene ID: 6886), TAL2 (UniProt accession number Q16559, NCBI Gene ID: 6887), TCL3 (UniProt accession number P31314, NCBI Gene ID: 3195), TCL5 (UniProt accession number P17542, NCBI Gene ID: 6886), and YES (UniProt accession number P07947, NCBI Gene ID: 7525); tumor suppressor proteins (e.g., BRCA1 (UniProt accession number P38398, NCBI Gene ID: 672), BRCA2 (UniProt accession number P51587, NCBI Gene ID: 675), MADH4 (UniProt accession number Q13485, NCBI Gene ID: 4089), MCC (UniProt accession number P23508, NCBI Gene ID: 4163), NF1 (UniProt accession number P21359, NCBI Gene ID: 4164), ID: 4763), NF2 (UniProt accession number P35240, NCBI Gene ID: 4771), RB1 (UniProt accession number P06400, NCBI Gene ID: 5925), TP53 (UniProt accession number P04637, NCBI Gene ID: 7157), PLK1 (UniProt accession number P53350, NCBI Gene ID: 9606), KIF1-binding protein (UniProt accession number Q96EK5, NCBI Gene ID: 9606), and WT1 (UniProt accession number P19544, NCBI Gene ID: 4790)); lipoproteins (e.g., apolipoprotein B (ApoB100, UniProt accession number P04114, NCBI Gene ID: 338)); enzymes (e.g., ACC synthases and oxidases, ACP dehydrogenases and hydroxylases, ADP-glucose pyrophosphorylases, ATPases, alcohol dehydrogenases, amylases, amyloglucosidases, catalases, cellulases, chalcone synthases, chitinases, cyclooxygenases, decarboxylases, dextrinases, DNA and RNA polymerases, galactosidases, glucanases, glucose oxidases, granule-bound starch synthases, GTPases, helicases, hemicellulases, integrases, inulinases, invertases, isomerases, kinases (e.g., PLK1 (UniProt accession number P53350, NCBI Gene ID: 9606)), lactase, ligase (e.g., RING finger- and WD repeat-containing protein 2 (RFWD2), also known as COP1), lipase, lipoxygenase, lysozyme, nopaline synthase, octopine synthase, pectin methylesterase, peroxidase, phosphatase, phospholipase, phosphorylase, phytase, plant growth regulator synthase, polygalacturonase, protease and peptidase, pullulanases, recombinase, reverse transcriptase, ribonuclease-1, 5-bisphosphate carboxylase oxygenase (RuBisCos), topoisomerase, transferase, such as hypoxanthine guanine phosphoribosyltransferase 1 (HPRT1), and xylanase).

Consider its important role in metabolism (for example, lipoprotein metabolism in various hypercholesterolemias) and secretion of circulating proteins (for example, coagulation factors in hemophilia), liver is one of the most important target tissues of nucleic acid therapy. In addition, acquired conditions, such as chronic hepatitis and cirrhosis are common, and may also be treated by liver therapy based on polynucleotides. Many diseases or conditions that affect the liver or are affected by the liver may be treated by knocking out (inhibition) of gene expression in the liver. Exemplary liver diseases and conditions can be selected from the list comprising: liver cancer (including hepatocellular carcinoma, HCC), viral infection (including hepatitis), metabolic disorders (including hyperlipidemia and diabetes), fibrosis and acute liver injury. Exemplary molecular targets for liver therapeutics (e.g., including, in particular, therapeutics targeting HCC)—and optionally exemplary molecular targets for therapeutics addressing other targets, diseases, and/or conditions, including other cancers—include CSN5 (UniProt Accession No. Q92905, NCBI Gene ID: 10987), CDK6 (UniProt Accession No. Q00534, NCBI Gene ID: 1021), ITGB1 (UniProt Accession No. P05556, NCBI Gene ID: 3688), MYC (UniProt Accession No. P01106, NCBI Gene ID: 4609), TGFβ1 (UniProt Accession No. P01137, NCBI Gene ID: 7040), cyclin D1 (UniProt Accession No. Q9H014, NCBI Gene ID: 595), hepcidin (UniProt Accession No. P81172, NCBI Gene ID: 596), IL-67 (UniProt Accession No. P81173, NCBI Gene ID: 596), IL-77 (UniProt Accession No. P81174, NCBI Gene ID: 596), IL-67 (UniProt Accession No. P81175, NCBI Gene ID: 596), IL-67 (UniProt Accession No. P81177, NCBI Gene ID: 596), IL-77 (UniProt Accession No. P81178, NCBI Gene ID: ID: 57817), PCSK9 (UniProt accession number Q8NBP7, NCBI Gene ID: 255738) and transthyretin (TTR, UniProt accession number P02766, NCBI Gene ID: 7276), etc.

The formulations of the present invention can optionally be targeted to normal tissues (eg, normal liver tissue) as well as various models (eg, orthotopic liver models, subcutaneous liver models, etc.).

An exemplary target of the formulation of the present invention is apolipoprotein B (ApoB), which is present in different classes of lipoproteins: chylomicrons, very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL) and low density lipoproteins (LDL). ApoB acts as a recognition signal for cellular binding and internalization of LDL particles through the ApoB/E receptor. Accumulation or excess of lipoproteins containing apolipoprotein B can lead to lipid-related disorders, such as atherosclerosis. Customized therapies that reduce ApoB can be used to treat lipid-related disorders. A nucleic acid-based therapy in the form of antisense therapy has been shown to reduce ApoB levels in mice, and treatment subsequently reduced serum cholesterol and triglyceride levels (U.S. Publication No. 2003/0215943). These results illustrate the moderate downregulation of ApoB and its use as a target for treating lipid-related disorders.

Another exemplary target for the formulations of the invention is protein C, which can be targeted, for example, for the treatment of hemophilia.

Delivery of therapeutic agents

Preparations of the present invention can be used for delivering therapeutic agents (e.g., polyanionic agents, nucleic acids, or RNAi agents) to cells. The agent delivered by the preparation can be used for gene silencing (e.g., in vitro or in a subject) or treatment or preventive treatment of a subject's disease (e.g., cancer).

Can be by using any useful method to assess the delivery of therapeutic agent.For example, can be by following assessment using the delivery of the preparation containing the compounds of this invention: 1) knockout of target gene or 2) toxicity or tolerance compared with equivalent dose control.These assessments can use any useful lipid combination in preparation to determine, for example with the compounds of this invention (for example, any compound in formula (I) or table 1) any cationic lipid described herein (for example, DOTAP, DODMA, DLinDMA and/or DLin-KC2-DMA).In specific embodiments, when using the compounds of this invention, observe the improvement that therapeutic agent sends, wherein said improvement is compared with control and exceeds 25% (for example, more than 2 times, 5 times, 10 times, 100 times or 1000 times delivery improvement).

Delivery of RNAi agents

RNAi silencing can be used for many kinds of cells, wherein HeLa S3, COS7, 293, NIH/3T3, A549, HT-29, CHO-KI and MCF-7 cell lines are susceptible to a certain level of siRNA silencing. Moreover, the inhibition in mammalian cells can occur under the specific RNA level to the targeted gene, wherein the strong association between RNA and protein inhibition has been observed. Accordingly, the compounds of this invention and its preparation can be used for delivery RNAi agent to one or more cells (for example, in vitro or in vivo). Exemplary RNAi agents include siRNA, shRNA, dsRNA, miRNA and DsiRNA agents as described herein.

In vitro target knockout

The delivery of RNAi agents can be assessed by any useful method. For example, a preparation comprising a therapeutic agent can be transfected in vitro in a cell culture model (e.g., HeLa cells), wherein endpoint measurements include but are not limited to one or more of the following: (i) mRNA quantification using qPCR; (ii) protein quantification using Western blots; (iii) cellular internalization of the labeling of an agent and/or the amino-amine or amino-amide cationic lipid of the present invention. Uptake or delivery can be evaluated for the degree and duration of the above-mentioned endpoints. Prior to delivery, the preparation can be diluted in a cell culture fluid for about 30 minutes at room temperature, and the final concentration can be varied from 0 to 50 nM therapeutic agent or amino-amine or amino-amide cationic lipid in a dose-response experiment. For time-course experiments, the optimal concentration of a dosage experiment can be studied for various incubation times, such as 30 minutes to 7 days.

The functionality of the polycationic payload and lipid formulation can also be tested by differentially labeling the lipid compound and therapeutic agent with a fluorescent tag and performing fluorescence colocalization studies. The ability of the compounds of the invention to deliver polycationic payloads and/or attached fluorescent labels can be assessed by measuring total fluorescence within the cell and by measuring fluorescence that is unstable in the endosomal or lysosomal compartments (in order to function, the therapeutic agent that triggers RNAi is required to reach not only the interior of the cell but also the cytoplasm of the cell). The performance of fluorescence localization and cellular trafficking studies has been described in the prior art (Lu, et al., Mol. Pharm. 6(3): 763, 2009; McNaughton et al., Proc. Natl. Acad. Sci. U.S.A. 106(15): 6111, 2009).

Delivery to specific target cell types and tissues

The compounds of this invention can be used to deliver therapeutic agents to various organs and tissues to treat various diseases. Exemplary target tissues or organs include but are not limited to liver, pancreas, lung, prostate, kidney, bone marrow, spleen, thymus, lymph nodes, brain, spinal cord, heart, skeletal muscle, skin, oral mucosa, esophagus, stomach, ileum, small intestine, colon, bladder, cervix uteri, ovary, testis, mammary gland, adrenal gland, adipose tissue (white and/or brown), blood (such as hematopoietic stem cells, such as human hematopoietic progenitor cells, human hematopoietic stem cells, CD34+ cells, CD4+ cells), lymphocytes and other blood lineage cells.

cancer therapy

The compounds of the present invention can be used to deliver one or more therapeutic agents (e.g., RNAi agents) to subjects with cancer or at risk of developing cancer (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% increased risk). Exemplary cancers include liver cancer (e.g., hepatocellular carcinoma, hepatoblastoma, cholangiocarcinoma, angiosarcoma or malignant hemangioma) or neuroblastoma. Exemplary tumor diseases and related complications include, but are not limited to, cancer (e.g., in situ lung, breast, pancreas, colon, hepatocellular, kidney, female reproductive tract, squamous cell carcinoma), lymphoma (e.g., histiocytic lymphoma, non-Hodgkin lymphoma), MEN2 syndrome, multiple neurofibromas (including Schwann cell tumors), myelodysplastic syndrome, leukemia, tumor angiogenesis, thyroid cancer, liver, bone, skin, brain, central nervous system, pancreas, lung (e.g., small cell lung cancer, non-small cell lung cancer (NSCLC)), breast, colon, Bladder, prostate, gastrointestinal tract, endometrium, fallopian tube, testicle and ovary, gastrointestinal stromal tumors (GIST), prostate tumors, mast cell tumors (including canine mast cell tumors), acute myeloid myelofibrosis, leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, melanoma, mastocytosis, glioma, glioblastoma, astrocytoma, neuroblastoma, sarcoma (e.g., sarcoma of neuroectodermal origin or leiomyosarcoma), metastasis to other tissues, and chemotherapy-induced hypoxia.

Administration and dosage

The present invention also relates to pharmaceutical compositions containing a compound or a therapeutically effective amount of a composition, such as a formulation comprising a therapeutic agent (e.g., an RNAi agent). The composition can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the composition of an appropriate formulation. Suitable formulations for use in the present invention are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th edition, 1985. For a brief review of drug delivery methods, see, for example, Langer, Science 249: 1527-1533, 1990.

The pharmaceutical composition is intended to be used for parenteral, intranasal, surface, oral or topical administration, for example, by transdermal means, for preventive and/or therapeutic treatment. The pharmaceutical composition can be administered parenterally (for example, by intravenous, intramuscular or subcutaneous injection), or by oral administration, or by surface application or intraarticular injection in the area affected by blood vessels or cancer symptoms. Other routes of administration include intravascular, intraarterial, intratumoral, intraperitoneal, intraventricular, intradural, and nose, eye, intrasclera, intraorbital, rectal, surface or aerosol inhalation administration. Sustained release administration is also particularly included in the present invention, by means of such as long-acting injections or erodible implants or components. Therefore, the invention provides compositions for parenteral administration, comprising the above-mentioned agents dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, for example water, buffered water, saline, PBS, etc. The composition can contain pharmaceutically acceptable auxiliary substances required for appropriate physiological conditions, such as pH adjusting agents and buffers, osmotic pressure regulators, wetting agents, detergents, etc. The present invention also provides compositions for oral delivery, which may contain inert ingredients, such as binders or fillers for tablets, capsules, etc. Furthermore, the present invention provides compositions for topical administration, which may contain inert ingredients, such as solvents or emulsifiers for creams, ointments, etc.

These compositions can be sterilized by conventional sterilization techniques, or can be aseptically filtered. The aqueous solution obtained can be packaged for original use or lyophilized, and the lyophilized product is combined with a sterile aqueous carrier before application. The pH of the product is generally 3 to 11, more preferably 5 to 9 or 6 to 8, and most preferably 7 to 8, for example 7 to 7.5. The composition in solid form obtained can be packaged in multiple single-dose units, each containing a fixed amount of the above-mentioned agent or multiple agents, for example in a sealed tablet or capsule. The composition in solid form can also be packaged in a flexible number of containers, for example, in a squeezable tube designed for a cream or ointment for topical application.

Compositions containing an effective amount can be administered for preventive or therapeutic treatment. In preventive applications, compositions can be administered to patients with clinically determined susceptibility or increased susceptibility to the development of tumors or cancer. Patients (e.g., people) can be administered a compound of the present invention in an amount sufficient to delay, reduce, or preferably prevent clinical disease or tumorigenesis. In therapeutic applications, patients (e.g., people) who have already suffered from cancer are administered a composition sufficient to cure or at least partially stop the symptomatic amount of the condition and its complications. An amount sufficient to achieve this purpose is defined as a "therapeutically effective dose," which is the amount of a compound sufficient to substantially improve some symptoms associated with a disease or medical condition. For example, in cancer treatment, an agent or compound that reduces, prevents, delays, suppresses, or stops any symptom of a disease or condition will be therapeutically effective. A therapeutically effective amount of an agent or compound is not necessary to cure a disease or condition, but will provide treatment for a disease or condition so that the occurrence of a disease or condition in an individual is delayed, hindered, or prevented, or the disease or condition symptoms are alleviated, or the duration of the disease or condition is changed, or, for example, is less severe or accelerates recovery.

The effective amount of this purpose can be based on the severity of the disease or condition and the patient's weight and general state, but generally ranges from about 0.5 mg per dose of each patient to about 3000 mg of the agent or multiple agents. Suitable schemes for initial administration and booster administration are typically: initial administration, followed by subsequent administration of repeated doses once or multiple times at hourly, daily, weekly or monthly intervals. The total effective amount of the agent present in the compositions of the present invention can be administered to mammals in a relatively short period of time with a single dose (as a rapid bolus injection or infusion), or a fractionated treatment regimen can be used, wherein multiple doses (e.g., every 4-6, 8-12, 14-16 or 18-24 hours or every 2-4 days, 1-2 weeks, monthly doses) are administered in a longer period of time. Alternatively, continuous intravenous infusion sufficient to maintain a therapeutically effective concentration in the blood is contemplated.

The therapeutically effective amount of one or more agents present in the compositions of the present invention and applied to a mammal (e.g., a person) in the inventive method can be determined by one of ordinary skill in the art considering individual differences in mammal age, body weight, and symptom. The agent of the present invention is administered to a subject (e.g., a mammal, e.g., a person) in an effective amount that produces the desired result (e.g., slowing down or eliminating cancer or neurodegenerative disorders) in the treated subject. This therapeutically effective amount can be empirically determined by those skilled in the art.

Patients can also receive a dose of about 0.1 to 3,000 mg per dose once or more per week (e.g., 2, 3, 4, 5, 6, or 7 or more times per week), 0.1 to 2,500 (e.g., 2,000, 1,500, 1,000, 500, 100, 10, 1, 0.5, or 0.1) mg per dose. Patients can also receive a dose of the composition in the range of 0.1 to 3,000 mg per dose once every two weeks or every three weeks.

The amount (dosage) of the preparation to be administered and the payload (e.g., DsiRNA) can be determined empirically. In certain embodiments, effective knockout of gene expression is observed using a nucleic acid payload of 0.0001-10 mg/kg animal body weight and a delivery formulation of 0.001-200 mg/kg animal body weight. An exemplary amount in mice is 0.1-5 mg/kg nucleic acid payload and 0.7-100 mg/kg delivery formulation. Optionally, about 1-50 mg/kg delivery formulation is administered. Because it is generally non-toxic at larger doses, the amount of payload (e.g., DsiRNA) is easily increased.

In certain embodiments, the agent may be administered daily, or only once, or at other intervals over days, weeks, or longer (e.g., 1 to 28 days or longer), depending on, for example, acute versus chronic indications.

Single or multiple administrations of a composition of the invention comprising an effective amount can be performed using a dosage level and pattern selected by the treating physician. The dosage and administration schedule can be determined and adjusted based on the severity of the patient's disease or condition, which can be monitored throughout the treatment process using methods commonly used by physicians or as described herein.

The compounds of the present invention and preparations can be used in combination with conventional treatment methods or therapy, or can be used separately from conventional treatment methods or therapy. When the compounds of the present invention and preparations are used in combination therapy with other agents, they can be administered to individuals continuously or concurrently. Alternatively, the pharmaceutical composition according to the present invention includes a combination of the compounds of the present invention or preparations combined with a pharmaceutically acceptable excipient as described herein and another therapeutic agent or preventive known in the art.

The prepared agent can be packaged together as a medicine box. Non-limiting examples include containing, for example, two pills, a pill and a powder, a suppository and a liquid in a vial, two surface creams, etc. The medicine box can include an auxiliary unit dose administered to the patient's optional component, such as a vial for reconstructing a powder form, a syringe for injection, a customized IV delivery system, an inhaler, etc. In addition, the unit dose medicine box can contain instructions for preparing and administering the composition. The medicine box can be produced as a single-use unit dose for a patient, for multiple uses (with a constant dose, or wherein individual compounds can change with therapeutic progress in potency) for a specific patient; or the medicine box can contain a plurality of agents ("batch packaging") that are suitable for administering to a plurality of patients. The medicine box components can be assembled in cardboard boxes, blister packs, bottles, tubes, and the like.

Measurement of pKa values of lipids in assembled nanoparticles

In the case of lipids, the different physiochemical properties of lipids greatly determine the behavior of lipids. One of these important properties is the ionization constant (Ka) of lipids. When present in the nanoparticles of assembly, the intrinsic pKa of lipids may not be the correct representation of their behavior. When present in aqueous environments, lipids have experienced an environment with a high dielectric constant, and when present in the nanoparticles/capsules of assembly, they are surrounded by lipids with a low dielectric constant. In addition, the lipids of lipids, cholesterol, and PEGylation all affect the apparent pKa of preparations. The interaction properties between cationic lipids and nucleic acids are electrostatic, and the apparent pKa of preparations determines the encapsulation of nucleic acids in the nanoparticles and their subsequent intracellular release.

The TNS fluorescence method can be used to determine the apparent pKa of lipids in a formulation. TNS (2-(p-toluidinyl)-6-naphthalenesulfonic acid) is a negatively charged fluorescent dye whose fluorescence is quenched in the presence of water. TNS partitions into positively charged membranes, and this results in an increase in fluorescence due to water removal. Therefore, the increase in fluorescence can be used to estimate the ionization of cationic lipids when present in different pH environments. Methods for determining pKa using TNS are known in the art, for example, as described in the Examples.

Example

Example 1: Synthesis of amino-amine lipid L-1 from ketone and primary amine

Completely under N ₂ atmosphere, ketone a (1 equivalent) and amine b (1.1 equivalents) are dissolved in the dichloroethane in a dry flask and stirred at room temperature (RT) for 30 minutes. Triacetoxyborohydride (1.5 equivalents) is added and the mixture is stirred at room temperature overnight. The reaction is quenched with 1N NaOH. The quenched reaction is diluted with DCM and extracted once with water, once with brine, and the organic phase is dried over Na ₂ SO _4. The dried solution is filtered and concentrated on a rotary evaporator. The residue is purified by silica column (step gradient starting from 1% MeOH/DCM to 5% MeOH/DCM, yield variation from 60% to 90%) to produce compound L-1. H ¹ NMR (CDCl ₃ ): 5.41-5.30 (m, 8H), 3.12 (t, 2H), 2.91 (m, 1H), 2.77 (t, 6H), 2.48 (bs, 6H), 2.20 (m, 2H), 2.05 (q, 8H), 1.80-1.69 (m, 4H), 1.38-1.25 (m, 40H), 0.89 (t, 3H); MS: electrospray: [M+1] theory: 613, found: 613.

By adapting the synthetic steps of this example, other amino-amine lipids were prepared, such as those provided in Figures 2A, 2B, and 3.

Example 2: Synthesis of amino-amine lipid L-2 from ketone and secondary amine

Ketone a (1 equivalent) was dissolved in dry MeOH in a dry flask completely under _N2 atmosphere. Amine b (1.1 equivalents) was added, followed by triacetoxyborohydride (1.5 equivalents) and AcOH (1 equivalent), and the reaction was stirred at room temperature overnight. The reaction was diluted with DCM and extracted once with water, once with brine, and _the organic phase was dried over _Na2SO4 . The dried solution was filtered and concentrated on a rotary evaporator. The residue was purified by silica column (step gradient starting from 1% MeOH/DCM to 5% MeOH/DCM, yield varying from 60% to 90%) to produce compound L-2. H ¹ NMR: (CD ₃ OD) 5.39-5.30 (m, 8H), 2.78 (t, 4H), 2.59-2.52 (m, 10H), 2.33 (bs, 8H), 2.07 (q, 8H), 1.25 (m, 2H), 1.40-1.26 (m, 40H), 0.914 (t, 6H); MS: electrospray pos. [M+1] theory 668, found 668.

By adapting the synthetic steps of this example, other amino-amine lipids, such as the L-2 and L-6 analogs provided in Figures 4 and 5, were prepared.

Example 3: Synthesis of lipid L-46 from ketone and morpholine

To a mixture of ketone a (2.66 g; 5.05 mmol), morpholine b (1.34 ml; 15 mmol) and AcOH (1.77 ml; 30 mmol) in DCE (12 ml) was added NaBH(AcO) ( _1.6 g; 7.5 mmol). The reaction mixture was stirred at room temperature for 72 hours. TLC analysis (silica gel; eluted with hexane:EtAc- _Et3N 95:5) indicated approximately 45% conversion. The reaction mixture was diluted with 5% _{aqueous K2CO3} and extracted with DCM. The solvent was dried _{over K2CO3} and evaporated on a rotary evaporator. The residue was separated by LC on silica gel (eluted with hexane:EtAc 90:10). The desired product L-46 was obtained in 39% yield (1.18 g) and was pure by NMR analysis.

Example 4: Synthesis of lipid L-47 from ketone and piperidine

To a mixture of dilinoleyl ketone a (3.99 g; 7.58 mmol), piperidine b (2.25 ml; 22 mmol) and AcOH (1.33 ml; 23 mmol) in DCE (24 ml) was added NaBH(AcO) ₍ 2.4 g; 11.3 mmol). The reaction mixture was stirred at room temperature for 96 hours. TLC analysis (silica gel; eluted with hexane:EtAc- _Et3N 95:5) indicated approximately 35% conversion. The reaction mixture was diluted with 5% _{aqueous K2CO3} and extracted with DCM. The solvent was dried _{over K2CO3} and evaporated on a rotary evaporator. The residue was separated by LC on silica gel (eluted with hexane:EtAc 90:10). The desired product L-47 was obtained in 29% yield (1.30 g) and was pure by NMR analysis.

By using the methods provided in this example and Example 3, cationic lipids with a variety of head groups, such as those provided in Figure 9, can be prepared.

Example 5: Synthesis of amide cationic lipids from primary amines and carboxylic acids

The following dilinoleylamide derivatives were prepared using the following general procedure. To a solution of dilinoleylamine (338 mg; 0.64 mmol), HOBt (65 mg; 0.5 mmol), an amino acid (1 mmol) and DIPEA (1 equiv) in DCM (15 g/mL) was combined, followed by the addition of EDC (1.2 mmol). The reaction mixture was stirred at room temperature overnight. TLC indicated the reaction was complete. The reaction mixture was diluted with 0.5% _{K2CO3 aqueous} solution and extracted with DCM. After concentration on a rotary evaporator, the crude product was purified by silica gel chromatography (gradient from hexane: _Et3N 95:5 to hexane: _CHCl3 : _Et3N 46:44:5). The yield obtained was 80-85%.

Dioleyl derivatives were also prepared based on the scheme provided below:

Synthesis of lipid L-30 from primary amines and carboxylic acids

Synthesis of lipid L-31 from primary amines and carboxylic acids

Synthesis of lipid L-32 from primary amines and carboxylic acids

Synthesis of lipid L-42 from primary amines and carboxylic acids

By adapting the synthetic steps of this example, other amidoamine lipids were prepared, such as those provided in Figures 6-8.

Example 6: Preparation of amine lipid formulations

To test the efficacy of lipids L-1 and L-2, formulations were prepared using a cationic lipid (DODMA), a neutral lipid (DSPC), PEG-lipid conjugates (PEG-DMPE and PEG-DMG), and cholesterol with an RNAi agent (DsiRNA for HPRT1) having the following structure:

5′-GCCAGACUUUGUUGGAUUUGAAAtt (SEQ ID NO: 1)

3′- UUC G G U C U G A A A C A A C C UAAACUUUAA (SEQ ID NO: 2),

Wherein capital letters represent RNA nucleotides, underlined capital letters represent 2'-O-methyl-RNA nucleotides, and lowercase letters represent DNA nucleotides.

Preparation of DsiRNA strands: oligonucleotide synthesis and purification

Individual RNA strands were synthesized and HPLC purified according to standard methods (Integrated DNA Technologies, Coralville, Iowa). For example, RNA oligonucleotides were synthesized using solid-phase phosphoramidite chemistry, deprotected, and desalted on a NAP-5 column (Amersham Pharmacia Biotech, Piscataway, N.J.) using standard techniques (Damha and Olgivie, Methods Mol. Biol. 20:81, 1993; Wincott et al., Nucleic Acids Res. 23:2677, 1995). Oligomers were purified by ion exchange high performance liquid chromatography (IE-HPLC) on an Amersham Source 15Q column (1.0 cm x 25 cm; Amersham Pharmacia Biotech, Piscataway, N.J.) using a 15-minute stepwise linear gradient. The gradient was from 90:10 buffer A:B to 52:48 buffer A:B, where buffer A was 100 mM Tris pH 8.5 and buffer B was 100 mM Tris pH 8.5, 1 M NaCl. Samples were monitored at 260 nm, and peaks corresponding to full-length oligonucleotide species were collected, pooled, desalted on a NAP-5 column, and lyophilized.

The purity of each oligomer was determined by capillary electrophoresis (CE) on a Beckman PACE 5000 (Beckman Coulter, Inc., Fullerton, Calif.). The CE capillary had an inner diameter of 100 μm and contained ssDNA 100R Gel (Beckman-Coulter). Typically, approximately 0.6 nanomoles of oligonucleotide were injected into the capillary, run in an electric field of 444 V/cm, and detected by UV absorbance at 260 nm. Denatured Tris-Borate-7M-urea running buffer was purchased from Beckman-Coulter. Oligoribonucleotides that were at least 90% pure, as assessed by CE, were obtained and used in the following experiments. Compound identity was confirmed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry on a Voyager DE™ Biospectometry Workstation (Applied Biosystems, Foster City, Calif.), according to the manufacturer's recommended protocol. Relative molecular weights were obtained for all oligomers and were generally within 0.2% of the expected molecular weight.

Preparation of DsiRNA duplexes

Single-stranded RNA (ssRNA) oligomers are resuspended in a buffer consisting of 100 mM potassium acetate, 30 mM HEPES, pH 7.5 at a concentration of, for example, 100 μM. Complementary sense and antisense strands are mixed in equimolar amounts to produce, for example, a 50 μM final solution of duplexes. The sample is heated to 100° C. for 5 minutes in RNA buffer (IDT) and allowed to cool to room temperature before use. Double-stranded RNA (dsRNA) oligomers are stored at -20° C. Single-stranded RNA oligomers are stored lyophilized or in nuclease-free water at -80° C.

Preparation of vesicle-based lipid formulations

Lipid particles were prepared using the mol% provided in Table 5. The total lipid to DsiRNA ratio was approximately 1:7.

Table 5

Preparation of RNA binders and transfection lipid formulations

Lipid particles were prepared using the mol% provided in Table 6. The total lipid to DsiRNA ratio was approximately 1:20.

Table 6

In Tables 5 and 6, PEG-DMPE is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], and PEG-DMG is (R)-3-[(ω-methoxy-PEG2000-carbamoyl)]-1,2-di-O-tetradecyl-sn-glyceride.

Example 7: In vitro performance of amine lipid formulations

To evaluate the efficacy of various lipid formulations, in vitro assays were performed using DsiRNA molecules targeting HPRT1. As described in Example 6 above, lipid formulations were prepared using DsiRNA against HPRT1.

Cell culture and RNA transfection

HeLa cells were obtained from ATCC and maintained in Dulbecco's modified Eagle's medium (HyClone) supplemented with 10% fetal bovine serum (HyClone) at 37°C, 5% _CO . The dsRNA-cationic lipid formulation of the present invention was transfected into HeLa cells by incubation with a final concentration of 1 nM, 5 nM, or 25 nM of the formulation of the present invention. 0.1 nM or 1 nM Lipofectamine ^™ RNAiMAX (Invitrogen) dsRNA was used as a positive control. In brief, 2.5 μL of 0.2 μM or 0.02 μM stock solutions of each dsRNA were mixed with 47.5 μL Opti-MEM I (Invitrogen). For Lipofectamine ^™ controls, 2.5 μL of 0.2 μM or 0.02 μM stock solutions of each dsRNA were mixed with 46.5 μL Opti-MEM I (Invitrogen) and 1 μL Lipofectamine ^™ RNAiMAX. 50 μL of the resulting mixture was added to a single well of a 12-well plate and incubated at room temperature for 20 minutes to allow the formation of dsRNA:Lipofectamine ^™ RNAiMAX complexes.

At the same time, HeLa cells were trypsinized and resuspended in culture medium at a final concentration of approximately 367 cells/μL. Finally, 450 μL of cell suspension was added to each well (final volume 500 μL) and the plate was placed in an incubator for 24 hours. For dose response studies, the concentration of dsRNA was varied from an initial 10 pM to 100 nM. For time course studies, incubation times of approximately 4 hours to approximately 72 hours were studied.

Assessment of inhibition

Target gene knockdown was determined by qRT-PCR, and values were normalized to HPRT expression control treatments, including Lipofectamine ^™ RNAiMAX alone (vehicle control) or no treatment.

RNA isolation and analysis

Cells were washed once with 2 mL of PBS, and total RNA was extracted using the RNeasy Mini Kit ^™ (Qiagen) and eluted in a total volume of 30 μL. 1 μg of total RNA was reverse transcribed using the Transcriptor ^1st Strand cDNA Kit ^™ (Roche) and random hexamers according to the manufacturer's instructions. One-thirtieth of the resulting cDNA (0.66 μL) was mixed with 5 μL of IQ Multiplex Powermix (Bio-Rad), 3.33 μL of _H₂O , and 1 μL of a 3 μM mixture of primers and probes specific for the target sequence of the human gene HPRT-1 (Accession No. NM_000194).

Quantitative RT-PCR

A CFX96 Real-Time System with a C1000 Thermal Cycler (Bio-Rad) was used for amplification reactions. PCR conditions were: 95°C for 3 minutes, followed by 40 cycles at 95°C for 10 seconds, and 55°C for 1 minute. Each sample was tested in triplicate. Relative HPRT mRNA levels were normalized to target mRNA levels and compared to mRNA levels obtained in control samples treated with transfection agent alone or without treatment. Data were analyzed using Bio-Rad CFX Manager version 1.0 software. Expression data are presented as a comparison of expression under treatment with an amino-amine cationic lipid formulation of dsRNA versus a dsRNA formulation without an amino-amine cationic lipid.

result

Figure 10 provides the result of the in vitro knockout using the lipid granules containing amino-amine lipid L-1 or L-2.In general, when applied to HeLa cells, L-1 and L-2 effectively suppressed target mRNA levels.Particularly, L-1 provides about 70% residual mRNA levels at minimum concentration 1nM. Accordingly, when applied to HeLa cells by transfection, amino-amine lipid provides the effective delivery of RNAi agent.Therefore, any one of the compounds of this invention, such as any lipid or preparation thereof, will be used to deliver a polycationic payload, such as RNAi agent or antisense payload.

Example 8: In vivo performance of amine lipid formulations

To further evaluate the performance of lipids, in vivo experiments were performed using formulations with DsiRNA against HPRT1.

Formulations were prepared using the following appropriate percentages: 20 mol% of one of L-1, L-2, L-5, L-6, L-7, L-8, L-22, or L-30; 26 mol% DODMA; 3 mol% PEG2000-DMPE; 3 mol% PEG2000-DMG; 13 mol% DSPC; and 33 mol% cholesterol. The formulation also included a DsiRNA:total lipid ratio of approximately 1:20 (w/w).

Approximately 4-week-old CD1 female mice were administered a single dose (1 mg/kg or 5 mg/kg) of the lipid particle formulation via intravenous administration via the tail vein at a volume of 10 μL/g body weight. 48 hours after administration, tissues were collected in RNALater (Qiagen). In endpoint analysis, total RNA was isolated from mouse livers for RT-qPCR. RNA samples were heated at 70°C with Oligo (dT) primers for 5 minutes before room temperature. In the PCR reaction, mHPRT expression was normalized with RPL23 (a housekeeping gene, used as a control here). Figures 11 and 12 show data with error bars, mean ± SD of n = 5 animals/group.

In the first group of experiments, the dosage of lipid formulations was single dose 5mg/kg (Figure 11). With this dosage, compounds L-1 and L-7 provided approximately 80% to approximately 90% residual mRNA levels. As demonstrated by approximately 15% residual mRNA levels, the addition of oxygen groups in the head group, such as L-30, provides a significant increase in gene silencing. In addition, compounds with heterocyclic groups in the head group (e.g., L-2, L-5, L-6, L-8, and L-22) provide compounds with approximately 15% to approximately 45% residual mRNA levels. The % mRNA knockouts of various preparations are shown in Table 7. Table 7 also shows the pKa values of each lipid, as measured by the TNS fluorescence method.

To determine the pKa values of the cationic lipids of the present invention, the formulations were incubated in phosphate buffer at different pH values (at a concentration of 1 mM), to which TNS dissolved in DMSO was added (resulting in a final concentration of 6 μM TNS). The fluorescence of the resulting solutions was measured on an M3 fluorescence plate reader with an excitation wavelength of 325 nm and an emission wavelength of 435 nm. The measured TNS fluorescence was fitted with a three-parameter sigmoid function as shown in Equation 1.

The pH at which half-maximal fluorescence is achieved is reported as the apparent pKa of the formulation, where a and b are dimensionless parameters reflecting the maximum observed fluorescence and the slope of the sigmoidal function, respectively.

Table 7

In the second set of experiments, compounds L-2, L-5, L-6, and L-30 (Figure 12) were evaluated at a single dose of 1 mg/kg or 5 mg/kg. Specifically, L-5, L-6, and L-30 provided effective gene silencing at a lower dose of 1 mg/kg. Overall, these data provide various lipid compounds and dosages that are effective inhibitors of target RNA levels in an in vivo model.

In order to evaluate the tolerance of lipid formulations containing amino-amine or amino-amide cationic lipids and dsRNA of the present invention, female CD-1 mice were injected with L-6 and L-30 formulations [administered (qod) at 2 doses of 10 mg/kg DsiRNA dosage, each approximately 200 mg/kg total lipid dosage], and serum samples were collected 48 hours after the second dose. A group of clinical chemistry evaluations of the test serum samples were performed, including liver function tests (LFTs) by measuring enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Phosphate buffered saline (PBS) was used as the vehicle control group. The ALT and AST elevations of formulations L-6 and L-30 were <3x of the PBS group. For L-6 and L-30 formulations, no changes in body weight or liver were observed. Therefore, L-6 and L-30 formulations are well tolerated. Therefore, any lipid described herein and its formulation can be used to deliver one or more agents, such as polyanions or antisense payloads.

Example 9: Use of lipid formulations containing dsRNA to reduce target gene expression in a subcutaneous animal tumor model

In order to evaluate the delivery efficiency and subsequent function of lipid formulations containing amino-amine or amino-amide cationic lipids and dsRNA, a subcutaneous (sc) tumor model with certain adjustments was used (Judge et al., J. Clin. Invest. 119: 661, 2009). Hep3B tumors were established in male nu/nu mice by sc injection of 3×10 ⁶ cells in 50 μL PBS into the left flank. 10-17 days after tumor seeding became palpable, mice were randomly divided into treatment groups. Lipid formulations of dsRNA or vehicle control were administered by standard intravenous (iv) injection via the lateral tail vein, calculated based on mg dsRNA/kg body weight according to individual animal weight. Tumors were measured using a digital caliper at 2 scales (width × length) to assess tumor growth. Tumor volume was calculated using the equation x*y*y/2, where x=maximum diameter and y=minimum diameter, and expressed as group mean ± SD. Tumor tissue was also removed from animals in different treatment groups and gene knockout was confirmed. Tumor volume, survival, and RNA expression data are presented as a comparison between treatment with dsRNA lipid formulations and treatment with dsRNA formulations without amino-amine or amino-amide cationic lipids.

Example 10: Reduction of target gene expression in the Hep3B orthotopic liver tumor model using lipid formulations containing dsRNA

To evaluate the targeting efficiency and subsequent function of amino-amine or amino-amide cationic lipid formulations of dsRNA, an intrahepatic tumor model with certain modifications was used (Judge et al., J. Clin. Invest. 119: 661, 2009). Liver tumors were established in mice by direct intrahepatic injection of Hep3B tumor cells. Male nu/nu mice were used as hosts for Hep3B tumors. Mice were maintained anesthetized using 2,2,2-tribromoethanol (Sigma), a single 1-cm incision across the midline was made below the sternum, and the left hepatic lobe was removed. Approximately 2×10 ⁶ Hep3B cells suspended in 40 μL of 50% PBS/50% Matrigel ^™ (BD) were slowly injected into the lobe at a shallow angle using a Hamilton syringe and a 30-gauge needle. A swab was then applied to the puncture wound to stop any bleeding before suturing. Mice were allowed to recover from anesthesia in a sterile cage and closely monitored for 2-4 hours before returning to regular housing. Approximately three weeks after tumor implantation, mice were randomized into treatment groups. Mice (n=7 per group) received: (1) an amino-amine or amino-amide lipid formulation of dsRNA; (2) a dsRNA formulation without an amino-amine or amino-amide cationic lipid; or (3) a vehicle control, as administered by standard intravenous (iv) injection via the lateral tail vein. Doses were calculated based on mg dsRNA/kg body weight according to the weight of the individual animal.

For the experiments that produced the results shown in Figure 13, animals were administered 5 mg/kg DsiRNA containing L-6- or L-30-formulated lipid particles. Table 8 provides the specific composition of the lipid formulations containing L-6 and L-30 lipids used in this study.

Table 8

Body weight was monitored throughout the study as an indicator of developing tumor burden and treatment tolerance. For efficacy studies, defined humanized endpoints were determined as a surrogate for survival. A combination of clinical signs, weight loss, and abdominal distension was assessed to determine the date of euthanasia due to tumor burden. Tumor tissue was removed from animals in the different treatment groups and gene knockout was confirmed.

As shown in Figure 13, the L-6 and L-30 formulations tested were very effective in delivering the formulated anti-HPRT1 DsiRNA payload to liver and orthotopic Hep3B tumor tissue. Specifically, greater than 50% knockout (and in some cases, 60-80% knockout) of HPRT1 target mRNA was observed in liver and orthotopic Hep3B tumor tissue compared to the PBS control. Therefore, any lipid or its formulation will be useful for reducing the expression of a target gene (e.g., a target gene associated with cancer).

Example 11: Reduction of target gene expression in the HepG2 orthotopic liver tumor model using lipid formulations containing dsRNA

In order to evaluate the targeting efficiency and subsequent function of the amino-amine or amino-amide cationic lipid formulations of dsRNA, a second intrahepatic tumor model was used. Liver tumors were established in mice by direct intrahepatic injection of HepG2 tumor cells. Female nu/nu mice were used as hosts for HepG2 tumors. Mice were maintained under anesthesia using Avertin (Sigma), a single 1-cm incision across the midline was made below the sternum, and the left hepatic lobe was removed. Approximately 3×10 ⁶ HepG2 cells suspended in 60 μL 50% PBS/50% Matrigel ^™ (BD) were slowly injected into the lobe at a shallow angle using a Hamilton syringe and a 30-gauge needle. A swab was then applied to the puncture wound to stop any bleeding before suturing. Mice were allowed to recover from anesthesia in a sterile cage and closely monitored for 2-4 hours before returning to conventional housing. About three weeks after tumor implantation, mice were randomly divided into treatment groups. Mice (n=7 per group) received: (1) an amino-amine or amino-amide lipid formulation of dsRNA; (2) a dsRNA formulation without an amino-amine or amino-amide cationic lipid; or (3) a vehicle control, as administered by standard intravenous (iv) injection via the lateral tail vein. Dosage was calculated based on mg dsRNA/kg body weight according to the weight of the individual animal. The experiments that produced the results shown in Figure 14 administered 5 mg/kg DsiRNA in L-6- or L-30-formulated lipid particles. Table 8 provides the specific composition of the lipid formulations containing L-6 and L-30 lipids used in this study. Body weight was monitored throughout the study as an indicator of tumor burden and treatment tolerance. For efficacy studies, defined humanized endpoints were determined as a surrogate for survival. A combination of clinical signs, weight loss, and abdominal distension were assessed to determine the date of euthanasia due to tumor burden. Tumor tissue was removed from animals in the different treatment groups and gene knockout was confirmed.

As shown in Figure 14, both the L-6 and L-30 formulations tested were very effective in delivering formulated anti-HPRT1 DsiRNA payloads to liver tissue. Simultaneously, 20-50% knockdown of the HPRT1 target mRNA was observed for both formulations in orthotopic HepG2 tumor tissue. The above results confirm that the L-6 and L-30 formulations tested here are effective dsRNA delivery vehicles for delivery to normal liver and at least some tumor tissues (e.g., orthotopic Hep3B tumors, and secondarily, orthotopic HepG2 tumors). Therefore, any lipid or formulation thereof will be useful for reducing target gene expression (e.g., a target gene associated with cancer).

The ability of lipid formulations of dsRNA to be taken up by tumor cells can also be tested by labeling the lipids and/or dsRNA with fluorescent tags and performing fluorescent biodistribution studies using a live animal imaging system (Xenogen or BioRad) (Eguchi et al., Nat. Biotechnol. 27: 567, 2009). Using this method, and by comparison with a separate dsRNA formulation, the ability of amino-amine or amino-amide cationic lipids to promote dsRNA internalization by tumor cells was demonstrated. In contrast, the separate dsRNA formulations used as controls in this study were not taken up and delivered to the tumor surface to the same extent. Efficacy endpoints, RNA expression, and biodistribution data are presented as a comparison between treatments with dsRNA lipid formulations and treatments with dsRNA formulations without amino-amine or amino-amide cationic lipids.

Example 12: Antitumor efficacy of lipid formulations against hepatocellular carcinoma

Liver tumors were established in mice by direct intrahepatic injection of Hep3B tumor cells as described in Example 10. Approximately 2 weeks after tumor implantation, mice were randomized into treatment groups. Mice (n=6 per group) received: (1) an amino-amine or amino-amide lipid formulation of a control dsRNA; (2) an amino-amine or amino-amide lipid formulation of an active dsRNA; or (3) a vehicle control, as administered by standard intravenous (i.v.) injection via the lateral tail vein. Doses were calculated based on a mg dsRNA/kg body weight basis according to the weight of the individual animal. In the experiments that produced the results shown in Figures 15 and 16, animals were given 5 mg/kg DsiRNA containing L-6- or L-30-formulated lipid particles. Table 8 above provides the specific composition of the lipid formulations containing L-6 and L-30 lipids used in this study.

Body weight was monitored throughout the study as an indicator of tumor burden and treatment tolerance. For efficiency studies, defined humanized endpoints were used as a surrogate for survival. Assessment was performed based on a combination of clinical signs, weight loss, and abdominal distension to determine the date of euthanasia due to tumor burden. Tumor tissue was taken out from the animals in the different treatment groups and tumor weight was measured to determine the effectiveness of the different treatment groups. Serum alpha-fetoprotein (AFP) levels were also measured as a biomarker of tumor burden.

L-6 and L-30 formulations with active payloads were highly effective in reducing serum AFP ( FIG. 15 ) and tumor weight ( FIG. 16 ) compared to L-6 and L-30 formulations with control payload and PBS control.

Example 13: Using different L-30 lipid formulations with dsRNA to reduce the expression of various target genes in multiple orthotopic liver cancer models

To evaluate whether different L-30 formulations could modulate HPRT1 knockdown in tumors relative to liver, the PEG-lipid content was adjusted. Table 9 provides the specific composition of the lipid formulations containing L-30 lipids used in this study. The experiments that produced the results shown in Figure 18 administered 1, 3, and 10 mg/kg DsiRNA in lipid particles formulated with L-30[1] and 10 mg/kg DsiRNA in lipid particles formulated with L-30[2]. Any useful solvent or solvent system can be used to introduce the RNA binder and DsiRNA into the formulation, including solvents and solvent systems that are the same or different than the solvent used for transfection lipids (e.g., aqueous and/or non-aqueous solvents).

As shown in Figure 18, both L-30[1] and L-30[2] formulations were effective in delivering the formulated anti-HPRT1 DsiRNA payload to liver tissue and in orthotopic Hep3B tumor tissue. Furthermore, L-30[2] containing 10 mg/kg DsiRNA significantly reduced liver knockout compared to the L-30[1] formulation without adversely affecting tumor knockout. These results indicate that increasing the PEG-lipid content in lipid formulations can affect the delivery of lipid particles to certain tissues and subsequent knockout of target genes.

The efficacy of the L-30[1] formulation as a dsRNA delivery vehicle was tested in various orthotopic liver cancer models using different dsRNAs. Figure 19 shows the results generated from experiments using different liver cancer models, showing that L-30[1] was effective in delivering formulated anti-HPRT1 DsiRNA payloads to all tested cancer models compared to controls. Figure 20 shows the results generated from experiments using L-30[1] formulations containing multiple independent DsiRNAs and knockdown of the corresponding genes in an orthotopic Hep3B HCC tumor model. Accordingly, any lipid described herein can be used to replace L-30 in the specific composition of the lipid formulations in Table 9, and any dsRNA can be used to reduce the expression of a target gene (e.g., a target gene associated with a cancer or disease described herein).

Table 9

¹ Before purification; ² Lipid molar percentage

Example 14: Reduction of target gene expression in Hep3B HCC tumor tissue using different L-30 lipid formulations with dsRNA

To evaluate the targeting efficiency and subsequent function of the L-30 formulations of dsRNA, L-30 formulations with varying lipid molar percentages were tested. Table 10 provides the specific composition of lipid formulations comprising L-30 as a transfection lipid. Specifically, L-30[E] and L-30[G] formulations contain L-48 as an RNA binder in place of DODMA. L-48 includes an H-5 head group and a dioleyl tail group (Figure 17). Any useful solvent or solvent system can be used to introduce the RNA binder and DsiRNA into the formulation, including solvents and solvent systems (e.g., aqueous and/or non-aqueous solvents) that are the same or different from the solvent used for the transfection lipid.

The results of hHPRT1 knockdown in Hep3B HCC tumor tissue are shown in Figure 21. All L-30 formulations (i.e., [A] to [G]) resulted in decreased hHPRT1 expression in tumor tissue compared to the PBS control. Specifically, L-30 [A] provided the greatest decrease in hHPRT1 expression, followed by L-30 [D], L-30 [G], and L-30 [E].

Table 10

¹ Before purification; ² Lipid molar percentage; ³ The same preparation containing 8% ethanol was also successfully prepared

Example 15: Reduction of target gene expression in lung and prostate tumor tissue using different L-6 and L-30 lipid formulations with dsRNA

To evaluate the targeting efficiency and subsequent function of different L-6 and L-30 formulations, HPRT1 mRNA knockdown was tested in different tumor tissues. Tables 9, 10, and 11 provide the specific compositions of the lipid formulations containing L-6 and L-30 lipids used in this study. Any useful solvent or solvent system can be used to introduce the RNA binder and nucleic acid payload (e.g., DsiRNA) into the formulation, including solvents and solvent systems (e.g., aqueous and/or non-aqueous solvents) that are the same or different than the solvent used for transfection lipids.

The experiment generating Figure 22 involved administration of 10 mg/kg DsiRNA in lipid particles formulated with L-6[2] and L-30[2] and administered on days 1 and 3 of the experiment. Tumors were harvested on day 5. Knockdown of hHPRT1 mRNA was measured in H1975 NSCLC lung tumor tissue. Greater knockdown of the HPRT1 target mRNA was observed with the L-30[2] formulation compared to the L-6[2] formulation.

The experiment that generated Figure 23 involved administration of 10 mg/kg DsiRNA in lipid particles formulated with L-6[2] and L-30[3] and administered on days 1 and 3 of the experiment. Tumors were harvested on day 5. Knockdown of hHPRT1 mRNA was measured in 22Rv1 prostate cancer SC xenograft tumor tissue. A greater level of knockdown of the HPRT1 target mRNA was observed with the L-30[3] formulation compared to the L-6[2] formulation. Figure 24 shows the results of hHPRT1 knockdown in 22Rv1 prostate cancer implanted in the liver. An experiment similar to that performed in Figure 23 was set up. In the specific experiment of Figure 24, a greater level of knockdown of the HPRT1 target mRNA was observed with the L-6[1] formulation compared to both the L-30[A] and L-30[E] formulations. Accordingly, any lipid described herein can be used to replace L-6 or L-30 in the specific composition of the lipid formulations in Table 9, and any dsRNA can be used to reduce the expression of a target gene associated with cancer (e.g., any cancer described herein).

Table 11

¹ Before purification; ² Lipid molar percentage

Example 16: Lipid formulation containing L-30 and dsRNA

Table 12 provides specific components of lipid formulations comprising L-30 as a transfection lipid. Any useful solvent or solvent system can be used to introduce the RNA binding agent and DsiRNA into the formulation, including solvents and solvent systems (e.g., aqueous and/or non-aqueous solvents) that are the same or different from the solvent used for the transfection lipid. Moreover, any lipid described herein can be used to replace L-30 (e.g., any one described herein, such as Table 1) as a transfection lipid in Table 12, and any dsRNA can be used to reduce the expression of a target gene (e.g., a target gene associated with a cancer or disease described herein).

Table 12

¹ Combination of DODMA and L-30

Other implementation plans

While the invention has been described with reference to particular embodiments thereof, it will be understood that it is capable of further modifications, and that this application is intended to cover any variations, uses, or adaptations of the invention which generally follow from the principles of the invention and include such departures from the present disclosure as come within the known or customary practice in the art to which the invention pertains, and to which the basic features hereinbefore set forth may be applied.

All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Sequence Listing

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Claims (23)

1. A compound having the following formula: Each of ^R1 and ^R2 is independently a _C11-24 alkenyl group; and Selected from: 2. The compound of claim 1, wherein the compound is selected from L-2, L-5, L-6, L-22, L-24, L-25, L-26 and L-48 as shown below: 3. A formulation comprising the compound of claim 1 or 2, the formulation further comprising cationic lipids, neutral lipids, sterol derivatives and dsRNA. 4. The formulation of claim 3, wherein the cationic lipid is selected from N,N-dimethyl-(2,3-dioleenyloxy)propylamine, 1,2-di-O-octadecenyl-3-trimethylammonium propane, 1,2-dipalmitoyl-sn-glycero-O-ethyl-3-choline phosphate, 1,2-dioleoyl-3-dimethylammonium propane, and 1,2-dioleoyl-3-trimethylammonium-propane; and the neutral lipid is selected from 1,2-distearate-sn-glycero-3-choline phosphate, 1-palmitoyl-2-oleoyl-sn-glycero-3-choline phosphate, 1,2-dioleoyl-glycero-sn-3-phosphate ethanolamine, and sphingomyelin. 5. The formulation of claim 3, wherein the cationic lipid is N,N-dimethyl-(2,3-dioletenyloxy)propylamine, and the neutral lipid is 1,2-distearate-sn-glycero-3-choline phosphate. 6. The formulation of claim 3, wherein the formulation further comprises a PEG-lipoconjugate. 7. The formulation of claim 6, wherein the PEG-lipid conjugate is selected from 1,2-dimyristoyl-sn-glycerol-3-(methoxy-polyethylene glycol), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine-N-(carbonyl-methoxy-polyethylene glycol), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-(carbonyl-methoxy-polyethylene glycol), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-(carbonyl-methoxy-polyethylene glycol), 1,2-dipalmitoyl-sn-glycerol-3-(methoxy-polyethylene glycol), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine-N-(carbonyl-methoxy-polyethylene glycol), and 1,2-dioleoyl-sn-glycerol-3-(methoxy-polyethylene glycol). 8. The formulation of claim 6, wherein the PEG-lipid conjugate is 1,2-dimyristoyl-sn-glycero-3-phosphate ethanolamine-N-(carbonyl-methoxy-polyethylene glycol) or 1,2-distearate-sn-glycero-3-phosphate ethanolamine-N-(carbonyl-methoxy-polyethylene glycol). 9. The formulation of claim 3, wherein the sterol derivative is selected from cholesterol; cholesterol ketones; cholesterolenones; coprosterol; 3β-[-(N-(N’,N’-dimethylaminoethane)-carbamoyl]cholesterol; biguanide-triaminoethylamine-cholesterol; (2S,3S)-2-(((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3, 4,7,8,9,10,11,12,13,14,15,16,17-Tetradecano-1H-cyclopentadieno[a]phenanthrene-3-yloxy)carbonylamino)ethyl 2,3,4,4-tetrahydroxybutyrate; (2S,3S)-((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12 ,13,14,15,16,17-Tetradecano[a]phenanthrene-3-yl)2,3,4,4-tetrahydroxybutyrate; bis((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecano[a]phenanthrene -3-yl)2,3,4-trihydroxyglutarate; or 6-(((3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecano-1H-cyclopentadienyl[a]phenanthrene-3-yloxy)oxophosphoryloxy)-2,3,4,5-tetrahydroxyhexanoate. 10. The formulation of claim 4, wherein the sterol derivative is cholesterol. 11. The formulation of claim 6, comprising 20 mol% to 25 mol% of the compound of claim 1, 20 mol% to 30 mol% of cationic lipids, 2 mol% to 8 mol% of PEG-lipid conjugates, 10 mol% to 20 mol% of neutral lipids, and 25 mol% to 35 mol% of sterol derivatives. 12. The formulation of claim 6, comprising 22 mol% of the compound of claim 1, 26 mol% of cationic lipid, 5 mol% to 9 mol% of PEG-lipid conjugate, 14 mol% of neutral lipid, and 29 mol% to 33 mol% of sterol derivative. 13. The formulation of claim 3, further comprising lipid particles, said lipid particles comprising transfected lipids. 14. The formulation of claim 3, wherein the dsRNA comprises 10 mol% to 40 mol% of one or more cationic lipids and 0.5 mol% to 10 mol% of one or more PEG-lipid conjugates. 15. The formulation of claim 13, wherein the transfected lipid comprises 5 mol% to 20 mol% of neutral lipid, 0.5 mol% to 10 mol% of PEG-lipid conjugate, and 20 mol% to 40 mol% of sterol derivative. 16. The formulation of claim 3, wherein the dsRNA has a length selected from the following: 10 to 40 nucleotides, 16 to 30 nucleotides, 19 to 29 nucleotides, 25 to 35 nucleotides, and 8 to 50 nucleotides. 17. The formulation of claim 3, wherein the formulation comprises dsRNA in a ratio of 1:10 (w/w) to 1:100 (w/w) with total lipids present in the formulation. 18. The formulation of claim 3, further comprising liposomes, liposome complexes or micelles. 19. The formulation of claim 18, wherein the liposomes are lipid nanoparticles. 20. A formulation comprising the compound of claim 1, wherein the formulation further comprises one or more components selected from cationic lipids, neutral lipids, sterol derivatives, PEG-lipoconjugates, lipid particles containing one or more RNA binding agents, transfected lipids, dsRNA, liposomes, liposome complexes, and micelles. 21. A pharmaceutical composition comprising the formulation of claim 3 and a pharmaceutically acceptable excipient. 22. Use of the pharmaceutical composition of claim 21 in the preparation of a medicament for treating or preventing a disease in a subject, wherein the disease is selected from hepatocellular carcinoma, lung cancer, prostate cancer, or neuroblastoma. 23. Use of the pharmaceutical composition of claim 21 in the preparation of a medicament, wherein the medicament is used by a method for regulating the expression of a target nucleic acid in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 21 sufficient to reduce the expression of the target gene in the subject. The target genes are selected from the following groups: ABL1, AR, β-catenin, BCL1, BCL2, BCL6, CBFA2, CBL, CSF1R, ERBA1, ERBA2, ERBB1, ERBB2, ERBB3, ERBB4, ETS1, ETS2, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MET, MDM2, MLL1, MLL2, MLL3, MYB The expression of the target genes in the subjects was reduced, including MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET, SRC, TAL1, TAL2, TCL3, TCL5, YES, BRCA1, BRCA2, MADH4, MCC, NF1, NF2, RB1, TP53, WT1, ApoB100, CSN5, CDK6, ITGB1, TGFβ1, cyclin D1, PLK1, and KIF1-binding protein.