(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2013/0116307 A1
`(43) Pub. Date:
`May 9, 2013
`Heyes et al.
`
`US 2013 0116307A1
`
`(54)
`
`NOVELCYCLIC CATIONIC LIPIDS AND
`METHODS OF USE
`
`(75)
`
`Inventors: James Heyes, Vancouver (CA); Mark
`Wood, Port Moody (CA); Alan Martin,
`Vancouver (CA)
`
`Assignee: PROTIVA BIOTHERAPEUTICS
`INC., Burnaby (CA)
`13/696,999
`May 12, 2011
`
`Appl. No.:
`PCT Filed:
`
`(73)
`
`(21)
`(22)
`(86)
`
`PCT/GB2O11AOOOT22
`
`PCT NO.:
`S371 (c)(1),
`Jan. 17, 2013
`(2), (4) Date:
`Foreign Application Priority Data
`
`(30)
`May 12, 2010 (US) .................................... 61334096
`
`Publication Classification
`
`(51) Int. Cl.
`A6IR 48/00
`CD7C 229/06
`A638/17
`(52) U.S. Cl.
`USPC ........ 514/44A: 560/173: 514/785: 514/44 R:
`435/375
`
`(2006.01)
`(2006.01)
`(2006.01)
`
`ABSTRACT
`(57)
`The present invention provides compositions and methods for
`the delivery of therapeutic agents to cells. In particular, these
`include novel cationic lipids and nucleic acid-lipid particles
`that provide efficient encapsulation of nucleic acids and effi
`cient delivery of the encapsulated nucleic acid to cells in vivo.
`The compositions of the present invention are highly potent,
`thereby allowing effective knock-down of a specific target
`protein at relatively low doses. In addition, the compositions
`and methods of the present invention are less toxic and pro
`vide a greater therapeutic index compared to compositions
`and methods previously known in the art.
`
`ARBUTUS - EXHIBIT 2011
`Moderna Therapeutics, Inc. v. Arbutus Biopharma Corporation
`IPR2019-00554
`
`

`

`Patent Application Publication
`
`May 9, 2013 Sheet 1 of 7
`
`US 2013/011 6307 A1
`
`
`
`

`

`Patent Application Publication
`
`May 9, 2013 Sheet 2 of 7
`
`US 2013/011 6307 A1
`
`
`
`

`

`Patent Application Publication
`
`May 9, 2013 Sheet 3 of 7
`
`US 2013/011 6307 A1
`
`%99
`
`
`
`O
`cN
`Oley WNYu OdVO:godw JeM)
`
`

`

`Patent Application Publication
`
`May 9, 2013
`
`Sheet 4 of 7
`
`US 2013/0116307 A1
`
`
`
`

`

`Patent Application Publication May 9, 2013 Sheet 5 of 7
`
`US 2013/0116307 A1
`
`s? eg
`c?
`
`S5
`CM
`CM
`
`sS
`to
`
`5?
`5
`
`<D in i
`
`£ <7> T
`
`r" ^ v;.,",rw''
`; ^ Lu
`I
`
`- -
`|
`
`10
`TJ-
`
`CO
`CO
`
`5?
`
`I
`
`s?
`CO 9
`
`5?
`?:
`1
`
`in
`CM
`
`CM
`
`in
`0!iey vNyw adVO:aodv JSAn
`
`5?
`§
`
`CO
`I
`
`2?
`eg
`CO
`
`0>
`00
`I
`
`H
`
`s?
`(D
`CO
`
`)
`
`CO
`CO
`I
`
`s I
`
`in
`o
`
`5 8
`5
`S 3
`
`I
`
`O
`° fe
`
`£
`
`o
`
`CO o
`tn T
`
`f) c o 0) d — s
`5 d
`
`»o
`CD
`
`T—
`
`S'^
`
`0' ?
`~ s
`<0 _l
`O J.
`q ^
`o Q-
`O
`
`s
`
`O
`
`fO O
`?
`q -iJ
`o>
`o
`in
`
`o
`
`S O
`0. r-o m
`5 d
`
`o
`
`CO
`
`s S
`
`O
`
`ID
`
`5 d
`
`(/)
`03
`0.
`
`o
`o
`
`

`

`Patent Application Publication
`
`May 9, 2013 Sheet 6 of 7
`
`US 2013/011 6307 A1
`
`
`
`| 9200 || 80W-vºrdo Zººl
`
`
`
`(~3700 || cow-uêT-6-do 29:1
`
`S s
`s
`d
`WNNs ua pesee
`fuego)
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`%100],
`
`IOLO3) So
`
`

`

`Patent Application Publication May 9, 2013 Sheet 7 of 7
`
`US 2013/0116307 A1
`
`p
`O to
`in
`
`p o
`h-
`
`p o
`CD
`T
`
`p
`o
`
`0s
`h- H
`CD
`
`p o
`00
`m
`
`p d-
`IT)
`
`:
`
`•
`
`o
`w
`
`c^. O
`c^. O,
`o-. o
`\p.
`o
`cP.
`O'. O
`r; sP.
`o
`cP.
`C-<S>
`O'
`v.
`\P.
`
`<P. o.
`
`c^.
`O
`\P.
`
`\P.
`
`o.
`cA
`U
`
`'sP.
`
`(!)
`It
`
`lO
`CM
`
`lO
`
`in
`o
`o
`o
`CM
`ouey VN^W adV9:aodv JQAH
`
`o
`o
`
`

`

`US 2013/011 6307 A1
`
`May 9, 2013
`
`NOVEL CYCLIC CATIONIC LIPIDS AND
`METHODS OF USE
`
`CROSS-REFERENCE TO RELATED
`APPLICATION
`0001. The present application claims priority to U.S. Pro
`visional Application No. 61/334,096, filed May 12, 2010, the
`disclosure of which is hereby incorporated by reference in its
`entirety for all purposes.
`
`BACKGROUND OF THE INVENTION
`0002 Therapeutic nucleic acids include, e.g., small inter
`fering RNA (siRNA), microRNA (miRNA), antisense oligo
`nucleotides, ribozymes, plasmids, and immune-stimulating
`nucleic acids. These nucleic acids act via a variety of mecha
`nisms. In the case of interfering RNA molecules such as
`siRNA and miRNA, these nucleic acids can down-regulate
`intracellular levels of specific proteins through a process
`termed RNA interference (RNAi). Following introduction of
`interfering RNA into the cell cytoplasm, these double
`stranded RNA constructs can bind to a protein termed RISC.
`The sense strand of the interfering RNA is displaced from the
`RISC complex, providing a template within RISC that can
`recognize and bind mRNA with a complementary sequence
`to that of the bound interfering RNA. Having bound the
`complementary mRNA, the RISC complex cleaves the
`mRNA and releases the cleaved strands. RNAi can provide
`down-regulation of specific proteins by targeting specific
`destruction of the corresponding mRNA that encodes for
`protein synthesis.
`0003) The therapeutic applications of RNAi are extremely
`broad, since interfering RNA constructs can be synthesized
`with any nucleotide sequence directed against a target pro
`tein. To date, siRNA constructs have shown the ability to
`specifically down-regulate target proteins in both in vitro and
`in vivo models. In addition, siRNA constructs are currently
`being evaluated in clinical studies.
`0004. However, two problems currently faced by interfer
`ing RNA constructs are, first, their susceptibility to nuclease
`digestion in plasma and, second, their limited ability to gain
`access to the intracellular compartment where they can bind
`RISC when administered systemically as free interfering
`RNA molecules. These double-stranded constructs can be
`stabilized by the incorporation of chemically modified nucle
`otide linkers within the molecule, e.g., phosphothioate
`groups. However, such chemically modified linkers provide
`only limited protection from nuclease digestion and may
`decrease the activity of the construct. Intracellular delivery of
`interfering RNA can be facilitated by the use of carrier sys
`tems such as polymers, cationic liposomes, or by the covalent
`attachment of a cholesterol moiety to the molecule. However,
`improved delivery systems are required to increase the
`potency of interfering RNA molecules such as siRNA and
`miRNA and to reduce or eliminate the requirement for chemi
`cally modified nucleotide linkers.
`0005. In addition, problems remain with the limited ability
`of therapeutic nucleic acids such as interfering RNA to cross
`cellular membranes (see, Vlassov et al., Biochim. Biophys.
`Acta, 1197:95-1082 (1994)) and in the problems associated
`with Systemic toxicity, such as complement-mediated ana
`phylaxis, altered coagulatory properties, and cytopenia (Gal
`braith et al., Antisense Nucl. Acid Drug Des., 4:201-206
`(1994)).
`
`0006 To attempt to improve efficacy, investigators have
`also employed lipid-based carrier systems to deliver chemi
`cally modified or unmodified therapeutic nucleic acids. Zel
`phati et al. (J. Contr. Rel, 41:99-119 (1996)) describes the use
`ofanionic (conventional) liposomes, pH sensitive liposomes,
`immunoliposomes, fusogenic liposomes, and cationic lipid/
`antisense aggregates. Similarly, siRNA has been adminis
`tered systemically in cationic liposomes, and these nucleic
`acid-lipid particles have been reported to provide improved
`down-regulation of target proteins in mammals including
`non-human primates (Zimmermann et al., Nature, 441: 111
`114 (2006)).
`0007. In spite of this progress, there remains a need in the
`art for improved lipid-therapeutic nucleic acid compositions
`that are suitable for general therapeutic use. Preferably, these
`compositions would encapsulate nucleic acids with high effi
`ciency, have high drug:lipid ratios, protect the encapsulated
`nucleic acid from degradation and clearance in serum, be
`suitable for systemic delivery, and provide intracellular deliv
`ery of the encapsulated nucleic acid. In addition, these nucleic
`acid-lipid particles should be well-tolerated and provide an
`adequate therapeutic index, such that patient treatment at an
`effective dose of the nucleic acid is not associated with sig
`nificant toxicity and/or risk to the patient. The present inven
`tion provides such compositions, methods of making the
`compositions, and methods of using the compositions to
`introduce nucleic acids into cells, including for the treatment
`of diseases.
`
`BRIEF SUMMARY OF THE INVENTION
`0008. The present invention provides novel cationic
`(amino) lipids and lipid particles comprising these lipids,
`which are advantageous for the in vivo delivery of active
`agents or therapeutic agents such as nucleic acids, as well as
`lipid particles such as nucleic acid-lipid particle compositions
`suitable for in vivo therapeutic use. The present invention also
`provides methods of making these lipid compositions, as well
`as methods of introducing active agents or therapeutic agents
`Such as nucleic acids into cells using these lipid compositions,
`e.g., for the treatment of various disease conditions.
`I0009. In one aspect, the present invention provides a cat
`ionic lipid of Formula I having the following structure:
`
`(I)
`
`R1 R3
`V /
`N-Z
`
`-R
`
`R{ Nr.
`X
`
`or salts thereof, wherein:
`(0010) R' and Rare either the same or different and are
`independently hydrogen (H) or an optionally substituted
`C-C alkyl, C-C alkenyl, or C-C alkynyl, or R' and
`R may join to form an optionally substituted heterocy
`clic ring;
`I0011 R is either absent or, if present, is hydrogen (H)
`or a C-C alkyl to provide a quaternary amine;
`I0012) R' and Rare either the same or different and are
`independently an optionally substituted Co-C alkyl,
`Co-C24 alkenyl, Co-C24 alkynyl, or Co-C2a acyl,
`
`

`

`US 2013/011 6307 A1
`
`May 9, 2013
`
`wherein at least one of RandR comprises at least one
`optionally substituted cyclic alkyl group;
`0013 X and Y are either the same or different and are
`independently O, S, N(R), C(O), C(O)O, OC(O), C(O)
`N(R), N(R)C(O), OC(O)N(R), N(R)C(O)O, C(O)S,
`C(S)O, S(O), S(O)(O), C(S), oran optionally substituted
`heterocyclic ring, wherein R is hydrogen (H) or an
`optionally substituted C-Co alkyl, C-Coalkenyl, or
`C-C alkynyl; and
`0014 Z is either absent or, if present, is an optionally
`Substituted C-C alkyl, C-C alkenyl, or C-C alky
`nyl.
`0015. In one embodiment, R' and R are independently
`selected from the group consisting of a methyl group and an
`ethyl group, i.e., RandR are both methyl groups, RandR
`are both ethyl groups, or R' and Rare a combination of one
`methyl group and one ethyl group. In another embodiment,
`R" and Rarejoined to forman optionally substituted hetero
`cyclic ring having from 2 to 5 carbon atoms (e.g., 2, 3, 4, or 5
`carbon atoms, or from 2-5, 2-4, 2-3, 3-5, 3-4, or 4-5 carbon
`atoms) and from 1 to 3 heteroatoms (e.g., 1, 2, or 3 heteroa
`toms, or from 1-3, 1-2, or 2-3 heteroatoms) selected from the
`group consisting of nitrogen (N), oxygen (O), Sulfur (S), and
`combinations thereof. In certain instances, R' and R are
`joined to form an optionally Substituted diazole (e.g., an
`optionally substituted imidazole) or an optionally substituted
`triazole.
`0016. In yet another embodiment, X and Y are either the
`same or different and are independently O, C(O)O, C(O)N
`(R), N(R)C(O)O, or C(O)S. In certain instances, R is
`hydrogen (H), an optionally Substituted methyl group, an
`optionally Substituted ethyl group, or an optionally Substi
`tuted C-Co alkyl, alkenyl, or alkynyl group (e.g., an option
`ally Substituted Cs, Ca, Cs, Co., C7, Cs, Co., or Co alkyl,
`alkenyl, or alkynyl group). In a further embodiment, X and Y
`are independently an optionally Substituted heterocyclic ring
`having from 2 to 5 carbon atoms (e.g., 2, 3, 4, or 5 carbon
`atoms, or from 2-5, 2-4, 2-3, 3-5, 3-4, or 4-5 carbon atoms)
`and from 1 to 3 heteroatoms (e.g., 1, 2, or 3 heteroatoms, or
`from 1-3, 1-2, or 2-3 heteroatoms) selected from the group
`consisting of nitrogen (N), oxygen (O), Sulfur (S), and com
`binations thereof. In certain instances, X and Y are indepen
`dently an optionally Substituted diazole (e.g., an optionally
`substituted imidazole) or an optionally substituted triazole. In
`preferred embodiments, X and Y are both O. In other embodi
`ments, Z is (CH), and n is 0, 1, 2, 3, 4, 5, or 6. In particular
`embodiments, n is 1,2,3, or 4 (e.g., n is 1-4, 1-3, 1-2, 2-4, 2-3,
`or 3-4).
`0017. In certain embodiments, at least one of R and R'
`comprises at least one, two, three, or more optionally Substi
`
`tuted cyclic alkyl groups. In particular embodiments, both R'
`and R independently comprise at least one, two, three, or
`more optionally Substituted cyclic alkyl groups. In some
`instances, both RandR comprise the same number of (e.g.,
`1, 2, 3, 4, 5, 6, or more) optionally substituted cyclic alkyl
`groups. In other instances, R and R comprise a different
`number of optionally Substituted cyclic alkyl groups. In one
`embodiment, each of the optionally substituted cyclic alkyl
`groups in R and/or R comprises an independently selected
`optionally Substituted Saturated cyclic alkyl group or an
`optionally Substituted unsaturated cyclic alkyl group. In cer
`tain instances, at least one, two, three, or more of the option
`ally Substituted cyclic alkyl groups present in one or both of
`RandR independently comprises an optionally substituted
`C-8 cycloalkyl group Such as, e.g., a cyclopropyl group, an
`optionally substituted Cls cycloalkenyl group, and combina
`tions thereof. In some embodiments, one of R' or R com
`prises at least one, two, three, or more optionally substituted
`cyclic alkyl groups and the other side-chain comprises an
`optionally Substituted Co-C alkyl, Co-C alkenyl, Co
`C2 alkynyl, or Co-C2a acyl group (e.g., a side-chain com
`prising at least one, two, or three sites of unsaturation). In
`some instances, R and R both comprise the same type or
`types of optionally substituted cyclic alkyl groups. In other
`instances, R and R comprise different types of optionally
`Substituted cyclic alkyl groups.
`10018. In particular embodiments, Rand Rare both C
`Coalkyl groups (e.g., C.s alkyl groups) having at least one,
`two, three, or more optionally Substituted cyclic alkyl groups.
`In preferred embodiments, Rand Rare both C-C (e.g.,
`Cs) alkyl groups having the same number of (e.g., at least
`one, two, three, or more) optionally Substituted cyclic alkyl
`groups. In certain embodiments, the at least one, two, three, or
`more optionally Substituted cyclic alkyl groups present in
`both R and R independently comprises an optionally sub
`stituted Saturated cyclic alkyl group (e.g., a Cs cycloalkyl
`group Such as a cyclopropyl group) or an optionally Substi
`tuted unsaturated cyclic alkyl group (e.g., a Cs cycloalkenyl
`group).
`0019. In some embodiments, at least one, two, three, or
`more optionally substituted cyclic alkyl groups are present on
`each of R and/or R in combination with at least one, two,
`three, or more sites of unsaturation and/or branched alkyl
`and/or acyl groups. For example, R* may comprise one, two,
`or three Css cycloalkyl groups such as cyclopropyl groups
`and one, two, or three sites of unsaturation, while R may
`comprise the same or different number and type of substitu
`entS.
`0020. In particular embodiments, the cationic lipid of For
`mula I has one of the following structures:
`
`CP-DODMA
`
`CP-DPetroDMA
`
`

`

`US 2013/011 6307 A1
`
`May 9, 2013
`
`-continued
`
`CP-DLIDMA
`
`N ~/N-A-->
`r
`s
`N-n-n-n-n-n-n-n-
`--~As-As-As
`N
`r
`>~~~~v-n-r/-r
`in Czy?yzy
`
`CP-DLeDMA
`
`CP-YDLenDMA
`
`0021. In another aspect, the present invention provides a
`cationic lipid of Formula II having the following structure:
`
`(II)
`
`)
`
`(1,
`
`R4
`\-z C
`A
`2 R
`x- M. R.
`
`or salts thereof, wherein:
`(0022) R' and Rare either the same or different and are
`independently an optionally Substituted C-C alkyl,
`C-C alkenyl, or C-C alkynyl, or R' and R may join
`to form an optionally Substituted heterocyclic ring;
`I0023 R is either absent or, if present, is hydrogen (H)
`or a C-C alkyl to provide a quaternary amine;
`0024 Rand Rare either the same or different and are
`independently an optionally Substituted Co-C alkyl,
`Co-C24 alkenyl, Co-C24 alkynyl, or Co-C24 acyl,
`wherein at least one of R and R comprises at least one
`optionally substituted cyclic alkyl group;
`0025 m, n, and pare either the same or different and are
`independently either 0, 1, or 2, with the proviso that m,
`n, and pare not simultaneously 0.
`0026 X and Y are either the same or different and are
`independently O, S, N(R), C(O), C(O)C, OC(O), C(O)
`N(R), N(R)C(O), OC(O)N(R), N(R)C(O)O, C(O)S,
`C(S)O, S(O), S(O)(O), or C(S), wherein R is hydrogen
`(H) or an optionally substituted C-C alkyl, C-Co
`alkenyl, or C-Co alkynyl; and
`0027 Z is either absent or, if present, is an optionally
`Substituted C-C alkyl, C-C alkenyl, or C-C alky
`nyl.
`0028. In one embodiment, R' and R are independently
`selected from the group consisting of a methyl group and an
`ethyl group, i.e., R and Rare both methyl groups, RandR
`are both ethyl groups, or R' and Rare a combination of one
`methyl group and one ethyl group. In another embodiment,
`R" and Rarejoined to forman optionally substituted hetero
`cyclic ring having from 2 to 5 carbon atoms (e.g., 2, 3, 4, or 5
`
`carbon atoms, or from 2-5, 2-4, 2-3, 3-5, 3-4, or 4-5 carbon
`atoms) and from 1 to 3 heteroatoms (e.g., 1, 2, or 3 heteroa
`toms, or from 1-3, 1-2, or 2-3 heteroatoms) selected from the
`group consisting of nitrogen (N), oxygen (O), Sulfur (S), and
`combinations thereof. In certain instances, R' and R are
`joined to form an optionally Substituted diazole (e.g., an
`optionally substituted imidazole) or an optionally substituted
`triazole.
`0029. In yet another embodiment, X and Y are either the
`same or different and are independently O, C(O)O, C(O)N
`(R), N(R)C(O)C), or C(O)S. In certain instances, R is
`hydrogen (H), an optionally Substituted methyl group, an
`optionally Substituted ethyl group, or an optionally Substi
`tuted C-Co alkyl, alkenyl, or alkynyl group (e.g., an option
`ally substituted C. C4, Cs, C, C7, Cs, Co, or Co alkyl,
`alkenyl, or alkynyl group). In preferred embodiments, X and
`Y are both O. In other embodiments, Zis (CH), and q is 0, 1,
`2, 3, 4, 5, or 6. In particular embodiments, q is 1, 2, 3, or 4
`(e.g., q is 1-4, 1-3, 1-2, 2-4, 2-3, or 3-4).
`10030. In certain embodiments, at least one of R and R'
`comprises at least one, two, three, or more optionally Substi
`tuted cyclic alkyl groups. In particular embodiments, both R'
`and R independently comprise at least one, two, three, or
`more optionally Substituted cyclic alkyl groups. In some
`instances, both RandR comprise the same number of (e.g.,
`1, 2, 3, 4, 5, 6, or more) optionally substituted cyclic alkyl
`groups. In other instances, R and R comprise a different
`number of optionally Substituted cyclic alkyl groups. In one
`embodiment, each of the optionally substituted cyclic alkyl
`groups in R and/or R comprises an independently selected
`optionally Substituted Saturated cyclic alkyl group or an
`optionally Substituted unsaturated cyclic alkyl group. In cer
`tain instances, at least one, two, three, or more of the option
`ally Substituted cyclic alkyl groups present in one or both of
`RandR independently comprises an optionally substituted
`C-8 cycloalkyl group Such as, e.g., a cyclopropyl group, an
`optionally substituted Cls cycloalkenyl group, and combina
`tions thereof. In some embodiments, one of R' or R com
`prises at least one, two, three, or more optionally substituted
`cyclic alkyl groups and the other side-chain comprises an
`optionally Substituted Co-C alkyl, Co-C alkenyl, Co
`
`

`

`US 2013/011 6307 A1
`
`May 9, 2013
`
`C24 alkynyl, or Co-C2a acyl group (e.g., a side-chain com
`prising at least one, two, or three sites of unsaturation). In
`some instances, R and R both comprise the same type or
`types of optionally substituted cyclic alkyl groups. In other
`instances, R and R comprise different types of optionally
`Substituted cyclic alkyl groups.
`I0031. In particular embodiments, Rand Rare both C
`Coalkyl groups (e.g., C.s alkyl groups) having at least one,
`two, three, or more optionally Substituted cyclic alkyl groups.
`In preferred embodiments, Rand Rare both Co-Co (e.g.,
`Cs) alkyl groups having the same number of (e.g., at least
`one, two, three, or more) optionally Substituted cyclic alkyl
`groups. In certain embodiments, the at least one, two, three, or
`more optionally Substituted cyclic alkyl groups present in
`both R" and Rindependently comprises an optionally sub
`stituted Saturated cyclic alkyl group (e.g., a C-s cycloalkyl
`group Such as a cyclopropyl group) or an optionally Substi
`tuted unsaturated cyclic alkyl group (e.g., a Cs cycloalkenyl
`group).
`0032. In some embodiments, at least one, two, three, or
`more optionally Substituted cyclic alkyl groups are present on
`each of R and/or R in combination with at least one, two,
`three, or more sites of unsaturation and/or branched alkyl
`and/or acyl groups. For example, R may comprise one, two,
`or three Cs cycloalkyl groups such as cyclopropyl groups
`and one, two, or three sites of unsaturation, while R may
`comprise the same or different number and type of substitu
`entS.
`0033. In particular embodiments, the cationic lipid of For
`mula II has one of the following structures:
`
`or salts thereof, wherein:
`0035) R' and Rare either the same or different and are
`independently hydrogen (H) or an optionally substituted
`C-C alkyl, C-C alkenyl, or C-C alkynyl, or R' and
`R may join to form an optionally substituted heterocy
`clic ring:
`I0036) R is either absent or, if present, is hydrogen (H)
`or a C-C alkyl to provide a quaternary amine;
`0037 R and Rare either the same or different and are
`independently an optionally substituted C-C alkyl,
`Co-C24 alkenyl, Co-C24 alkynyl, or Co-C24 acyl,
`wherein at least one of R and R comprises at least one
`optionally Substituted cyclic alkyl group;
`0038 X is O, S, N(R), C(O), C(O)O, OC(O), C(O)N
`(R), N(R)C(O), OC(O)N(R), N(R)C(O)O, C(O)S,
`C(S)O, S(O), S(O)(O), C(S), or an optionally substituted
`heterocyclic ring, wherein R is hydrogen (H) or an
`optionally substituted C-C alkyl, C-C alkenyl, or
`C-Co alkynyl; and
`0.039 Y is either absent or, if present, is an optionally
`Substituted C-C alkyl, C-C alkenyl, or C-C alky
`nyl.
`I0040. In one embodiment, R' and R are independently
`selected from the group consisting of a methyl group and an
`ethyl group, i.e., RandR are both methyl groups, RandR
`are both ethyl groups, or R' and Rare a combination of one
`methyl group and one ethyl group. In another embodiment,
`R" and Rarejoined to forman optionally substituted hetero
`
`rC, , ,
`-N-n() O
`
`,
`
`,
`
`or
`
`CP-DLen-C2K-DMA
`
`rC, , c.
`
`0034. In yet another aspect, the present invention provides
`a cationic lipid of Formula III having the following structure:
`
`(III)
`
`cyclic ring having from 2 to 5 carbon atoms (e.g., 2, 3, 4, or 5
`carbon atoms, or from 2-5, 2-4, 2-3, 3-5, 3-4, or 4-5 carbon
`atoms) and from 1 to 3 heteroatoms (e.g., 1, 2, or 3 heteroa
`toms, or from 1-3, 1-2, or 2-3 heteroatoms) selected from the
`group consisting of nitrogen (N), oxygen (O), Sulfur (S), and
`combinations thereof. In certain instances, R' and R are
`joined to form an optionally Substituted diazole (e.g., an
`optionally substituted imidazole) or an optionally substituted
`triazole.
`
`

`

`US 2013/011 6307 A1
`
`May 9, 2013
`
`0041. In yet another embodiment, X is O, C(O)O, C(O)N
`(R), N(R)C(O)O, or C(O)S. In certain instances, R is
`hydrogen (H), an optionally Substituted methyl group, an
`optionally Substituted ethyl group, or an optionally Substi
`tuted C-Co alkyl, alkenyl, or alkynyl group (e.g., an option
`ally Substituted C, C, Cs, C, C7, Cs Co., or Co alkyl,
`alkenyl, or alkynyl group). In a further embodiment, X is an
`optionally Substituted heterocyclic ring having from 2 to 5
`carbonatoms (e.g., 2, 3, 4, or 5 carbonatoms, or from 2-5, 2-4,
`2-3, 3-5, 3-4, or 4-5 carbon atoms) and from 1 to 3 heteroat
`oms (e.g., 1, 2, or 3 heteroatoms, or from 1-3, 1-2, or 2-3
`heteroatoms) selected from the group consisting of nitrogen
`(N), oxygen (O), sulfur (S), and combinations thereof. In
`certain instances, X is an optionally substituted diazole (e.g.,
`an optionally Substituted imidazole) or an optionally Substi
`tuted triazole. In preferred embodiments, X is C(O)O. In
`other embodiments, Y is (CH), and n is 0, 1, 2, 3, 4, 5, or 6.
`In particular embodiments, n is 1, 2, 3, or 4 (e.g., n is 1-4, 1-3,
`1-2, 2-4, 2-3, or 3-4).
`0042. In certain embodiments, at least one of R and R'
`comprises at least one, two, three, or more optionally Substi
`tuted cyclic alkyl groups. In particular embodiments, both R'
`and R independently comprise at least one, two, three, or
`more optionally Substituted cyclic alkyl groups. In some
`instances, both RandR comprise the same number of (e.g.,
`1, 2, 3, 4, 5, 6, or more) optionally substituted cyclic alkyl
`groups. In other instances, R and R comprise a different
`number of optionally Substituted cyclic alkyl groups. In one
`embodiment, each of the optionally substituted cyclic alkyl
`groups in R* and/or R comprises an independently selected
`optionally Substituted Saturated cyclic alkyl group or an
`optionally Substituted unsaturated cyclic alkyl group. In cer
`tain instances, at least one, two, three, or more of the option
`ally Substituted cyclic alkyl groups present in one or both of
`RandR independently comprises an optionally substituted
`
`C-8 cycloalkyl group Such as, e.g., a cyclopropyl group, an
`optionally substituted Cls cycloalkenyl group, and combina
`tions thereof. In some embodiments, one of R' or R com
`prises at least one, two, three, or more optionally substituted
`cyclic alkyl groups and the other side-chain comprises an
`optionally Substituted Co-C alkyl, Co-C alkenyl, Co
`C2 alkynyl, or Co-C2a acyl group (e.g., a side-chain com
`prising at least one, two, or three sites of unsaturation). In
`some instances, R and R both comprise the same type or
`types of optionally substituted cyclic alkyl groups. In other
`instances, R and R comprise different types of optionally
`Substituted cyclic alkyl groups.
`10043. In particular embodiments, Rand Rare both C
`Coalkyl groups (e.g., C.s alkyl groups) having at least one,
`two, three, or more optionally Substituted cyclic alkyl groups.
`In preferred embodiments, Rand Rare both C-C (e.g.,
`Cs) alkyl groups having the same number of (e.g., at least
`one, two, three, or more) optionally Substituted cyclic alkyl
`groups. In certain embodiments, the at least one, two, three, or
`more optionally Substituted cyclic alkyl groups present in
`both R and R independently comprises an optionally sub
`stituted Saturated cyclic alkyl group (e.g., a Cs cycloalkyl
`group Such as a cyclopropyl group) or an optionally Substi
`tuted unsaturated cyclic alkyl group (e.g., a Cs cycloalkenyl
`group).
`0044. In some embodiments, at least one, two, three, or
`more optionally substituted cyclic alkyl groups are present on
`each of R and/or R in combination with at least one, two,
`three, or more sites of unsaturation and/or branched alkyl
`and/or acyl groups. For example, R* may comprise one, two,
`or three Css cycloalkyl groups such as cyclopropyl groups
`and one, two, or three sites of unsaturation, while R may
`comprise the same or different number and type of substitu
`entS.
`0045. In particular embodiments, the cationic lipid of For
`mula III has one of the following structures:
`
`CP-LenMC3
`
`s
`
`,
`ric, ,
`rcycycyc
`ric, , or
`
`CP-Y-LenMC3
`
`, or
`
`

`

`US 2013/011 6307 A1
`
`May 9, 2013
`
`0046. In a further aspect, the present invention provides a
`lipid particle comprising one or more of the above cationic
`lipids of Formulas I-III or salts thereof. In certain embodi
`ments, the lipid particle further comprises one or more non
`cationic lipids such as neutral lipids. In certain other embodi
`ments, the lipid particle further comprises one or more
`conjugated lipids capable of reducing or inhibiting particle
`aggregation. In additional embodiments, the lipid particle
`further comprises one or more active agents or therapeutic
`agents.
`0047. In certain embodiments, the non-cationic lipid com
`ponent of the lipid particle may comprise a phospholipid,
`cholesterol (or cholesterol derivative), or a mixture thereof. In
`one particular embodiment, the phospholipid comprises
`dipalmitoylphosphatidylcholine (DPPC), distearoylphos
`phatidylcholine (DSPC), or a mixture thereof. In some
`embodiments, the conjugated lipid component of the lipid
`particle comprises a polyethyleneglycol (PEG)-lipid conju
`gate. In certain instances, the PEG-lipid conjugate comprises
`a PEG-diacylglycerol (PEG-DAG) conjugate, a PEG-dialky
`loxypropyl (PEG-DAA) conjugate, or a mixture thereof. In
`other embodiments, the lipid conjugate comprises a polyox
`azoline (POZ)-lipid conjugate such as a POZ-DAA conju
`gate.
`0048. In some embodiments, the active agent or therapeu
`tic agent comprises a nucleic acid. In certain instances, the
`nucleic acid comprises an interfering RNA molecule which
`includes any double-stranded RNA capable of mediating
`RNAi, such as, e.g., an siRNA, Dicer-substrate dsRNA,
`shRNA, aiRNA, pre-miRNA, or mixtures thereof. In certain
`other instances, the nucleic acid comprises single-stranded or
`double-stranded DNA, RNA, or a DNA/RNA hybrid such as,
`e.g., an antisense oligonucleotide, a ribozyme, a plasmid, an
`immunostimulatory oligonucleotide, or mixtures thereof.
`0049. In other embodiments, the active agent or therapeu
`tic agent is fully encapsulated within the lipid portion of the
`lipid particle Such that the active agent or therapeutic agent in
`the lipid particle is resistant in aqueous solution to enzymatic
`degradation, e.g., by a nuclease or protease. In further
`embodiments, the lipid particle is Substantially non-toxic to
`mammals such as humans.
`0050. In preferred embodiments, the present invention
`provides serum-stable nucleic acid-lipid particles (SNALP)
`comprising: (a) one or more nucleic acids such as interfering
`RNA molecules; (b) one or more cationic lipids of Formulas
`I-III or salts thereof (c) one or more non-cationic lipids; and
`(d) one or more conjugated lipids that inhibit aggregation of
`particles.
`0051. In some embodiments, the present invention pro
`vides nucleic acid-lipid particles (e.g., SNALP) comprising:
`(a) one or more nucleic acids; (b) one or more cationic lipids
`of Formulas I-III or salts thereof comprising from about 50
`mol % to about 85 mol % of the total lipid present in the
`particle; (c) one or more non-cationic lipids comprising from
`about 13 mol % to about 49.5 mol% of the total lipid present
`in the particle; and (d) one or more conjugated lipids that
`inhibit aggregation of particles comprising from about 0.5
`mol % to about 2 mol % of the total lipid present in the
`particle.
`0052. In one aspect of this embodiment, the nucleic acid
`lipid particle comprises: (a) one or more nucleic acids; (b) one
`or more cationic lipids of Formulas I-III or a salt thereof
`comprising from about 50 mol % to about 65 mol % of the
`total lipid present in the particle; (c) one or more non-cationic
`
`lipids comprising a mixture of one or more phospholipids and
`cholesterol or a derivative thereof, wherein the one or more
`phospholipids comprises from about 4 mol% to about 10 mol
`% of the total lipid present in the particle and the cholesterol
`orderivative thereof comprises from about 30 mol% to about
`40 mol% of the total lipid present in the particle; and (d) one
`or more PEG-lipid conjugates comprising from about 0.5 mol
`% to about 2 mol % of the total lipid present in the particle.
`This embodiment of nucleic acid-lipid particle is generally
`referred to herein as the “1:57 formulation.
`0053. In certain instances, the 1:57 formulation com
`prises: (a) one or more nucleic acids; (b) one or more cationic
`lipids of Formulas I-III or a salt thereof comprising from
`about 52 mol% to about 62 mol% of the total lipid present in
`the particle; (c) a mixture of one or more phospholipids and
`cholesterol or a derivative thereof comprising from about 36
`mol % to about 47 mol % of the total lipid present in the
`particle; and (d) one or more PEG-lipid conjugates compris
`ing from about 1 mol % to about 2 mol % of the total lipid
`present in the particle. In one particular embodiment, the 1:57
`formulation is a four-component system comprising about
`1.4 mol % PEG-lipid conjugate (e.g., PEG2000-C-DMA),
`about 57.1 mol % cationic lipid of Formulas I-III or a salt
`thereof, about 7.1 mol % DPPC (or DSPC), and about 34.3
`mol % cholesterol (or derivative thereof).
`0054. In another aspect of this embodiment, the nucleic
`acid-lipid particle comprises: (a) one or more nucleic acids;
`(b) one or more ca