U.S. patent application number 17/065208 was filed with the patent office on 2021-06-03 for novel lipids and compositions for the delivery of therapeutics.
This patent application is currently assigned to ARBUTUS BIOPHARMA CORPORATION. The applicant listed for this patent is ARBUTUS BIOPHARMA CORPORATION. Invention is credited to David Butler, Laxman Eltepu, Muthusamy Jayaraman, Martin Maier, Muthiah Manoharan, Jayaprakash K. Nair, Kallanthottathil G. Rajeev.
Application Number | 20210162053 17/065208 |
Document ID | / |
Family ID | 1000005389624 |
Filed Date | 2021-06-03 |
United States Patent
Application |
20210162053 |
Kind Code |
A1 |
Manoharan; Muthiah ; et
al. |
June 3, 2021 |
NOVEL LIPIDS AND COMPOSITIONS FOR THE DELIVERY OF THERAPEUTICS
Abstract
The present invention provides lipids that are advantageously
used in lipid particles for the in vivo delivery of therapeutic
agents to cells. In particular, the invention provides lipids
having the following structure (I) wherein R.sub.1 and R.sub.2 are
each independently for each occurrence optionally substituted
C.sub.10-C.sub.30 alkyl, optionally substituted C.sub.10-C.sub.30
alkenyl, optionally substituted C.sub.10-C.sub.30 alkynyl,
optionally substituted C.sub.10-C.sub.30 acyl, or -linker-ligand;
R.sub.3 is H, optionally substituted C.sub.1-C.sub.10 alkyl,
optionally substituted C.sub.2-C.sub.10 alkenyl, optionally
substituted C.sub.2-C.sub.10 alkynyl, alkylheterocycle,
alkylphosphate, alkylphosphorothioate, alkylphosphorodithioate,
alkylphosphonates, alkylamines, hydroxyalkyls, .omega.-aminoalkyls,
.omega.-(substituted)aminoalkyls, .omega.-phosphoalkyls,
.omega.-thiophosphoalkyls, optionally substituted polyethylene
glycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K),
heteroaryl, heterocycle, or linker-ligand; E is O, S, N(Q), C(O),
N(Q)C(O), C(O)N(Q), (Q)N(CO)O, O(CO)N(Q), S(O), NS(O)2N(Q), S(O)2,
N(Q)S(O)2, SS, O.dbd.N, aryl, heteroaryl, cyclic or heterocycle;
and, Q is H, alkyl, .omega.-aminoalkyl,
.omega.-(substituted)aminoalkyl, .omega.-phosphoalkyl or
.omega.-thiophosphoalkyl. ##STR00001##
Inventors: |
Manoharan; Muthiah;
(Cambridge, MA) ; Rajeev; Kallanthottathil G.;
(Cambridge, MA) ; Jayaraman; Muthusamy;
(Cambridge, MA) ; Butler; David; (Medford, MA)
; Nair; Jayaprakash K.; (Cambridge, MA) ; Maier;
Martin; (Cambridge, MA) ; Eltepu; Laxman;
(Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ARBUTUS BIOPHARMA CORPORATION |
Burnaby |
|
CA |
|
|
Assignee: |
ARBUTUS BIOPHARMA
CORPORATION
Burnaby
CA
|
Family ID: |
1000005389624 |
Appl. No.: |
17/065208 |
Filed: |
October 7, 2020 |
Related U.S. Patent Documents
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10821186 |
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17065208 |
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15603069 |
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61234098 |
Aug 14, 2009 |
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61225898 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 203/10 20130101;
A61K 47/10 20130101; C07C 271/20 20130101; C12N 2320/32 20130101;
Y02A 50/30 20180101; A61K 39/39 20130101; C07C 323/25 20130101;
A61K 2039/55555 20130101; A61K 47/20 20130101; A61K 47/18 20130101;
C07D 491/056 20130101; C07D 317/28 20130101; A61K 39/00 20130101;
C07D 319/06 20130101; C12N 2310/14 20130101; C07D 317/44 20130101;
C07D 317/46 20130101; A61K 31/713 20130101; A61K 9/1272 20130101;
C07D 491/113 20130101; A61K 2039/55561 20130101; C07C 237/16
20130101; A61K 47/28 20130101; C07C 271/12 20130101; C07C 229/08
20130101; A61K 31/7088 20130101; C07C 251/38 20130101; C12N
2310/3515 20130101; C07D 317/72 20130101; C07D 405/12 20130101;
C07C 251/78 20130101; A61K 47/44 20130101; C12N 15/111 20130101;
C12N 15/113 20130101; C07C 229/30 20130101 |
International
Class: |
A61K 47/44 20060101
A61K047/44; A61K 39/39 20060101 A61K039/39; C07C 229/08 20060101
C07C229/08; A61K 31/713 20060101 A61K031/713; C07D 319/06 20060101
C07D319/06; C07D 317/28 20060101 C07D317/28; C07D 203/10 20060101
C07D203/10; C07C 229/30 20060101 C07C229/30; C12N 15/113 20060101
C12N015/113; A61K 39/00 20060101 A61K039/00; C12N 15/11 20060101
C12N015/11; A61K 9/127 20060101 A61K009/127; A61K 47/10 20060101
A61K047/10; A61K 47/18 20060101 A61K047/18; A61K 47/20 20060101
A61K047/20; A61K 47/28 20060101 A61K047/28; C07C 237/16 20060101
C07C237/16; C07C 251/38 20060101 C07C251/38; C07C 251/78 20060101
C07C251/78; C07C 271/12 20060101 C07C271/12; C07C 271/20 20060101
C07C271/20; C07C 323/25 20060101 C07C323/25; A61K 31/7088 20060101
A61K031/7088; C07D 317/44 20060101 C07D317/44; C07D 317/46 20060101
C07D317/46; C07D 317/72 20060101 C07D317/72; C07D 405/12 20060101
C07D405/12; C07D 491/056 20060101 C07D491/056; C07D 491/113
20060101 C07D491/113 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The work described herein was carried out, at least in part,
using funds from the U.S. Government under grant number
HHSN266200600012C awarded by the National Institute of Allergy and
Infectious Diseases. The government may therefore have certain
rights in the invention.
Claims
1. (canceled)
2. A lipid having the structure ##STR00151## or a salt or isomer
thereof, wherein, E is O, S, N(Q), C(O), N(Q)C(O), C(O)N(Q),
(Q)N(CO)O, O(CO)N(Q), S(O), NS(O).sub.2N(Q), S(O).sub.2,
N(Q)S(O).sub.2, SS, O.dbd.N, aryl, heteroaryl, cyclic or
heterocycle; Q is H, alkyl, .omega.-aminoalkyl,
.omega.-(substituted)aminoalkyl, .omega.-phosphoalkyl or
.omega.-thiophosphoalkyl; R.sub.1 and R.sub.2 and R.sub.x are each
independently for each occurrence H, optionally substituted
C.sub.1-C.sub.10 alkyl, optionally substituted C.sub.10-C.sub.30
alkyl, optionally substituted C.sub.10-C.sub.30 alkenyl, optionally
substituted C.sub.10-C.sub.30 alkynyl, optionally substituted
C.sub.10-C.sub.30 acyl, or linker-ligand, provided that at least
one of R.sub.1, R.sub.2 and R.sub.x is not H; R.sub.3 is H,
optionally substituted C.sub.1-C.sub.10 alkyl, optionally
substituted C.sub.2-C.sub.10 alkenyl, optionally substituted
C.sub.2-C.sub.10 alkynyl, alkylheterocycle, alkylphosphate,
alkylphosphorothioate, alkylphosphorodithioate, alkylphosphonates,
alkylamines, hydroxyalkyls, .omega.-aminoalkyls,
.omega.-(substituted)aminoalkyls, .omega.-phosphoalkyls,
.omega.-thiophosphoalkyls, optionally substituted polyethylene
glycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K),
heteroaryl, heterocycle, or linker-ligand; n is 0, 1, 2, or 3.
3. A lipid particle comprising a lipid of claim 2.
4. (canceled)
5. The lipid particle of claim 3, wherein the particle further
comprises a neutral lipid and a lipid capable of reducing
aggregation.
6. The lipid particle of claim 3, wherein the lipid particle
consists essentially of a. a lipid of claim 2; b. a neutral lipid
selected from DSPC, DPPC, POPC, DOPE and SM; c. a sterol; and d.
PEG-DMG, in a molar ratio of about 20-60% lipid of claim 2:5-25%
neutral lipid:25-55% sterol:0.5-15% PEG-DMG.
7. The lipid particle of claim 3, further comprising a therapeutic
agent.
8. The lipid particle of claim 7, wherein the therapeutic agent is
a nucleic acid.
9. The lipid particle of claim 8, wherein the nucleic acid is a
plasmid.
10. The lipid particle of claim 8, wherein the nucleic acid is an
immunostimulatory oligonucleotide.
11. The lipid particle of claim 8, wherein the nucleic acid is
selected from the group consisting of an siRNA, an antisense
oligonucleotide, a microRNA, an antagomir, an aptamer, and a
ribozyme.
12. The lipid particle of claim 11, wherein the nucleic acid is an
siRNA.
13. A pharmaceutical composition comprising a lipid particle of
claim 7 and a pharmaceutically acceptable excipient, carrier, or
diluent.
14. A method of modulating the expression of a target gene in a
cell, comprising providing to a cell the lipid particle of claim
7.
15. The method of claim 14, wherein the therapeutic agent is
selected from an siRNA, an antagomir, an antisense oligonucleotide,
and a plasmid capable of expressing an siRNA, a ribozyme, an
aptamer or an antisense oligonucleotide.
16. The method of claim 14, wherein the nucleic acid is a plasmid
that encodes the polypeptide or a functional variant or fragment
thereof, such that expression of the polypeptide or the functional
variant or fragment thereof is increased.
17. A method of treating a disease or disorder characterized by
overexpression of a polypeptide in a subject, comprising providing
to the subject the pharmaceutical composition of claim 13, wherein
the therapeutic agent is selected from an siRNA, a microRNA, an
antisense oligonucleotide, and a plasmid capable of expressing an
siRNA, a microRNA, or an antisense oligonucleotide, and wherein the
siRNA, microRNA, or antisense RNA comprises a polynucleotide that
specifically binds to a polynucleotide that encodes the
polypeptide, or a complement thereof.
18. A method of treating a disease or disorder characterized by
underexpression of a polypeptide in a subject, comprising providing
to the subject the pharmaceutical composition of claim 13, wherein
the therapeutic agent mRNA or is a plasmid that encodes the
polypeptide or a functional variant or fragment thereof.
19. A method of inducing an immune response in a subject,
comprising providing to the subject the pharmaceutical composition
of claim 7.
20. The method of claim 19, wherein the pharmaceutical composition
is provided to the patient in combination with a vaccine or
antigen.
21. A vaccine comprising the lipid particle of claim 7 and an
antigen associated with a disease or pathogen.
22. The vaccine of claim 21, wherein said antigen is a tumor
antigen.
23. The vaccine of claim 22, wherein said antigen is a viral
antigen, a bacterial antigen, or a parasitic antigen.
24. The lipid particle of claim 7, wherein the molar ratio is 52%
lipid of claim 2:5% neutral lipid:30% sterol:13% PEG-DMG.
25. The method of claim 15, wherein the target gene is selected
from the group consisting of Factor VII, Eg5, PCSK9, TPX2, apoB,
SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2
gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1/2 gene,
PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, Cyclin D
gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1
gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3
gene, survivin gene, Her2/Neu gene, SORT1 gene, XBP1 gene,
topoisomerase I gene, topoisomerase II alpha gene, p73 gene,
p21(WAF1/CIP1) gene, p27(KIP1) gene, PPM1D gene, RAS gene, caveolin
I gene, MIB I gene, MTAI gene, M68 gene, tumor suppressor genes,
and p53 tumor suppressor gene.
26. The lipid particle of claim 8, wherein the nucleic acid is
mRNA.
Description
CLAIM OF PRIORITY
[0001] This application claims priority to U.S. Ser. No.
61/113,179, filed Nov. 10, 2008; U.S. Ser. No. 61/154,350, filed
Feb. 20, 2009; U.S. Ser. No. 61/171,439, filed Apr. 21, 2009; U.S.
Ser. No. 61/185,438, filed Jun. 9, 2009; U.S. Ser. No. 61/225,898,
filed Jul. 15, 2009; and U.S. Ser. No. 61/234,098, filed Aug. 14,
2009, the contents of each of which is incorporated herein by
reference in its entirety.
BACKGROUND
Technical Field
[0003] The present invention relates to the field of therapeutic
agent delivery using lipid particles. In particular, the present
invention provides cationic lipids and lipid particles comprising
these lipids, which are advantageous for the in vivo delivery of
nucleic acids, as well as nucleic acid-lipid particle compositions
suitable for in vivo therapeutic use. Additionally, the present
invention provides methods of making these compositions, as well as
methods of introducing nucleic acids into cells using these
compositions, e.g., for the treatment of various disease
conditions.
Description of the Related Art
[0004] Therapeutic nucleic acids include, e.g., small interfering
RNA (siRNA), micro RNA (miRNA), antisense oligonucleotides,
ribozymes, plasmids, immune stimulating nucleic acids, antisense,
antagomir, antimir, microRNA mimic, supermir, U1 adaptor, and
aptamer. These nucleic acids act via a variety of mechanisms. In
the case of siRNA or miRNA, these nucleic acids can down-regulate
intracellular levels of specific proteins through a process termed
RNA interference (RNAi). Following introduction of siRNA or miRNA
into the cell cytoplasm, these double-stranded RNA constructs can
bind to a protein termed RISC. The sense strand of the siRNA or
miRNA 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 siRNA or miRNA. 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.
[0005] The therapeutic applications of RNAi are extremely broad,
since siRNA and miRNA constructs can be synthesized with any
nucleotide sequence directed against a target protein. 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.
[0006] However, two problems currently faced by siRNA or miRNA
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 the free siRNA or miRNA. These
double-stranded constructs can be stabilized by incorporation of
chemically modified nucleotide linkers within the molecule, for
example, phosphothioate groups. However, these chemical
modifications provide only limited protection from nuclease
digestion and may decrease the activity of the construct.
Intracellular delivery of siRNA or miRNA can be facilitated by use
of carrier systems such as polymers, cationic liposomes or by
chemical modification of the construct, for example by the covalent
attachment of cholesterol molecules. However, improved delivery
systems are required to increase the potency of siRNA and miRNA
molecules and reduce or eliminate the requirement for chemical
modification.
[0007] Antisense oligonucleotides and ribozymes can also inhibit
mRNA translation into protein. In the case of antisense constructs,
these single stranded deoxynucleic acids have a complementary
sequence to that of the target protein mRNA and can bind to the
mRNA by Watson-Crick base pairing. This binding either prevents
translation of the target mRNA and/or triggers RNase H degradation
of the mRNA transcripts. Consequently, antisense oligonucleotides
have tremendous potential for specificity of action (i.e.,
down-regulation of a specific disease-related protein). To date,
these compounds have shown promise in several in vitro and in vivo
models, including models of inflammatory disease, cancer, and HIV
(reviewed in Agrawal, Trends in Biotech. 14:376-387 (1996)).
Antisense can also affect cellular activity by hybridizing
specifically with chromosomal DNA. Advanced human clinical
assessments of several antisense drugs are currently underway.
Targets for these drugs include the bcl2 and apolipoprotein B genes
and mRNA products.
[0008] Immune-stimulating nucleic acids include deoxyribonucleic
acids and ribonucleic acids. In the case of deoxyribonucleic acids,
certain sequences or motifs have been shown to illicit immune
stimulation in mammals. These sequences or motifs include the CpG
motif, pyrimidine-rich sequences and palindromic sequences. It is
believed that the CpG motif in deoxyribonucleic acids is
specifically recognized by an endosomal receptor, toll-like
receptor 9 (TLR-9), which then triggers both the innate and
acquired immune stimulation pathway. Certain immune stimulating
ribonucleic acid sequences have also been reported. It is believed
that these RNA sequences trigger immune activation by binding to
toll-like receptors 6 and 7 (TLR-6 and TLR-7). In addition,
double-stranded RNA is also reported to be immune stimulating and
is believe to activate via binding to TLR-3.
[0009] One well known problem with the use of therapeutic nucleic
acids relates to the stability of the phosphodiester
internucleotide linkage and the susceptibility of this linker to
nucleases. The presence of exonucleases and endonucleases in serum
results in the rapid digestion of nucleic acids possessing
phosphodiester linkers and, hence, therapeutic nucleic acids can
have very short half-lives in the presence of serum or within
cells. (Zelphati, O., et al., Antisense. Res. Dev. 3:323-338
(1993); and Thierry, A. R., et al., pp147-161 in Gene Regulation:
Biology of Antisense RNA and DNA (Eds. Erickson, R P and Izant, J
G; Raven Press, NY (1992)). Therapeutic nucleic acid being
currently being developed do not employ the basic phosphodiester
chemistry found in natural nucleic acids, because of these and
other known problems.
[0010] This problem has been partially overcome by chemical
modifications that reduce serum or intracellular degradation.
Modifications have been tested at the internucleotide
phosphodiester bridge (e.g., using phosphorothioate,
methylphosphonate or phosphoramidate linkages), at the nucleotide
base (e.g., 5-propynyl-pyrimidines), or at the sugar (e.g.,
2'-modified sugars) (Uhlmann E., et al. Antisense: Chemical
Modifications. Encyclopedia of Cancer, Vol. X., pp 64-81 Academic
Press Inc. (1997)). Others have attempted to improve stability
using 2'-5' sugar linkages (see, e.g., U.S. Pat. No. 5,532,130).
Other changes have been attempted. However, none of these solutions
have proven entirely satisfactory, and in vivo free therapeutic
nucleic acids still have only limited efficacy.
[0011] In addition, as noted above relating to siRNA and miRNA,
problems remain with the limited ability of therapeutic nucleic
acids 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 anaphylaxis,
altered coagulatory properties, and cytopenia (Galbraith, et al.,
Antisense Nucl. Acid Drug Des. 4:201-206 (1994)).
[0012] To attempt to improve efficacy, investigators have also
employed lipid-based carrier systems to deliver chemically modified
or unmodified therapeutic nucleic acids. In Zelphati, 0 and Szoka,
F. C., J. Contr. Rel. 41:99-119 (1996), the authors refer to the
use of anionic (conventional) liposomes, pH sensitive liposomes,
immunoliposomes, fusogenic liposomes, and cationic lipid/antisense
aggregates. Similarly siRNA has been administered 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)).
[0013] 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-efficiency,
have high drug:lipid ratios, protect the encapsulated nucleic acid
from degradation and clearance in serum, be suitable for systemic
delivery, and provide intracellular delivery of the encapsulated
nucleic acid. In addition, these lipid-nucleic acid 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 significant toxicity and/or risk to the
patient. The present invention 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
[0014] The present invention provides novel cationic lipids, as
well as lipid particles comprising the same. These lipid particles
may further comprise an active agent and be used according to
related methods of the invention to deliver the active agent to a
cell.
[0015] In one aspect, the invention provides lipids and the
corresponding salts and isomers thereof, having the structure,
##STR00002##
wherein:
[0016] R.sub.1 and R.sub.2 are each independently for each
occurrence optionally substituted C.sub.10-C.sub.30 alkyl,
optionally substituted C.sub.10-C.sub.30 alkenyl, optionally
substituted C.sub.10-C.sub.30 alkynyl, optionally substituted
C.sub.10-C.sub.30 acyl, or -linker-ligand;
[0017] R.sub.3 is H, optionally substituted C.sub.1-C.sub.10 alkyl,
optionally substituted C.sub.2-C.sub.10 alkenyl, optionally
substituted C.sub.2-C.sub.10 alkynyl, alkylheterocycle,
alkylphosphate, alkylphosphorothioate, alkylphosphorodithioate,
alkylphosphonates, alkylamines, hydroxyalkyls, .omega.-aminoalkyls,
.omega.-(substituted)aminoalkyls, .omega.-phosphoalkyls,
.omega.-thiophosphoalkyls, optionally substituted polyethylene
glycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K),
heteroaryl, heterocycle, or linker-ligand;
[0018] E is O, S, N(Q), C(O), N(Q)C(O), C(O)N(Q), (Q)N(CO)O,
O(CO)N(Q), S(O), NS(O).sub.2N(Q), S(O).sub.2, N(Q)S(O).sub.2, SS,
O.dbd.N, aryl, heteroaryl, cyclic or heterocycle; and,
[0019] Q is H, alkyl, .omega.-aminoalkyl,
.omega.-(substituted)aminoalkyl, .omega.-phosphoalkyl or
.omega.-thiophosphoalkyl.
[0020] In another aspect, the invention provides a lipid particle
comprising the lipids of the present invention. In certain
embodiments, the lipid particle further comprises a neutral lipid
and a lipid capable of reducing particle aggregation. In one
embodiment, the lipid particle consists essentially of (i) at least
one lipid of the present invention; (ii) a neutral lipid selected
from DSPC, DPPC, POPC, DOPE and SM; (iii) sterol, e.g. cholesterol;
and (iv) peg-lipid, e.g. PEG-DMG or PEG-DMA, in a molar ratio of
about 20-60% cationic lipid:5-25% neutral lipid:25-55% sterol;
0.5-15% PEG-lipid. In one embodiment, the lipid of the present
invention is optically pure.
[0021] In additional related embodiments, the present invention
includes lipid particles of the invention that further comprise
therapeutic agent. In one embodiment, the therapeutic agent is a
nucleic acid. In one embodiment, the nucleic acid is a plasmid, an
immunostimulatory oligonucleotide, a single stranded
oligonucleotide, e.g. an antisense oligonucleotide, an antagomir; a
double stranded oligonucleotide, e.g. a siRNA; an aptamer or a
ribozyme.
[0022] In yet another related embodiment, the present invention
includes a pharmaceutical composition comprising a lipid particle
of the present invention and a pharmaceutically acceptable
excipient, carrier of diluent.
[0023] The present invention further includes, in other related
embodiments, a method of modulating the expression of a target gene
in a cell, the method comprising providing to a cell a lipid
particle or pharmaceutical composition of the present invention.
The target gene can be a wild type gene. In another embodiment, the
target gene contains one or more mutations. In a particular
embodiment, the method comprises specifically modulating expression
of a target gene containing one or more mutations. In particular
embodiments, the lipid particle comprises a therapeutic agent
selected from an immunostimulatory oligonucleotide, a single
stranded oligonucleotide, e.g. an antisense oligonucleotide, an
antagomir; a double stranded oligonucleotide, e.g. a siRNA, an
aptamer, a ribozyme. In one embodiment, the nucleic acid is plasmid
that encodes a siRNA, an antisense oligonucleotide, an aptamer or a
ribozyme.
[0024] In one aspect of the invention, the target gene is selected
from the group consisting of Factor VII, Eg5, PCSK9, TPX2, apoB,
SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2
gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1/2 gene,
PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, Cyclin D
gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1
gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3
gene, survivin gene, Her2/Neu gene, SORT1 gene, XBP1 gene,
topoisomerase I gene, topoisomerase II alpha gene, p73 gene,
p21(WAF1/CIP1) gene, p27(KIP1) gene, PPM1D gene, RAS gene, caveolin
I gene, MIB I gene, MTAI gene, M68 gene, mutations in tumor
suppressor genes, p53 tumor suppressor gene, and combinations
thereof.
[0025] In another embodiment, the nucleic acid is a plasmid that
encodes a polypeptide or a functional variant or fragment thereof,
such that expression of the polypeptide or the functional variant
or fragment thereof is increased.
[0026] In yet a further related embodiment, the present invention
includes a method of treating a disease or disorder characterized
by overexpression of a polypeptide in a subject, comprising
providing to the subject a lipid particle or pharmaceutical
composition of the present invention, wherein the therapeutic agent
is selected from an siRNA, a microRNA, an antisense
oligonucleotide, and a plasmid capable of expressing an siRNA, a
microRNA, or an antisense oligonucleotide, and wherein the siRNA,
microRNA, or antisense RNA comprises a polynucleotide that
specifically binds to a polynucleotide that encodes the
polypeptide, or a complement thereof.
[0027] In another related embodiment, the present invention
includes a method of treating a disease or disorder characterized
by underexpression of a polypeptide in a subject, comprising
providing to the subject the pharmaceutical composition of the
present invention, wherein the therapeutic agent is a plasmid that
encodes the polypeptide or a functional variant or fragment
thereof.
[0028] In a further embodiment, the present invention includes a
method of inducing an immune response in a subject, comprising
providing to the subject a pharmaceutical composition of the
present invention, wherein the therapeutic agent is an
immunostimulatory oligonucleotide. In particular embodiments, the
pharmaceutical composition is provided to the patient in
combination with a vaccine or antigen.
[0029] In a related embodiment, the present invention includes a
vaccine comprising the lipid particle of the present invention and
an antigen associated with a disease or pathogen. In one
embodiment, the lipid particle comprises an immunostimulatory
nucleic acid or oligonucleotide. In a particular embodiment, the
antigen is a tumor antigen. In another embodiment, the antigen is a
viral antigen, a bacterial antigen, or a parasitic antigen.
[0030] The present invention further includes methods of preparing
the lipid particles and pharmaceutical compositions of the present
invention, as well as kits useful in the preparation of these lipid
particle and pharmaceutical compositions.
[0031] In another aspect, the invention provides a method of
evaluating a composition that includes an agent, e.g. a therapeutic
agent or diagnostic agent, and a lipid of the present
invention.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0032] FIG. 1. Schematic representation of an optically pure lipid
with conjugated targeting ligands.
[0033] FIG. 2. Schematic representation of features of the lipids
of the present invention.
[0034] FIG. 3. A graph illustrating the relative FVII protein
levels in animals administered with 0.05 or 0.005 mg/kg of lipid
particles containing different cationic lipids.
[0035] FIG. 4. A table depicting the EC50 and pKa values of
exemplary lipids tested using method described in the Examples.
DETAILED DESCRIPTION
[0036] The present invention is based, in part, upon the discovery
of cationic lipids that provide advantages when used in lipid
particles for the in vivo delivery of a therapeutic agent. In
particular, as illustrated by the accompanying Examples, the
present invention provides nucleic acid-lipid particle compositions
comprising a cationic lipid according to the present invention. In
some embodiments, a composition described herein provides increased
activity of the nucleic acid and/or improved tolerability of the
compositions in vivo, which can result in a significant increase in
therapeutic index as compared to lipid-nucleic acid particle
compositions previously described. Additionally compositions and
methods of use are disclosed that can provide for amelioration of
the toxicity observed with certain therapeutic nucleic acid-lipid
particles.
[0037] In certain embodiments, the present invention specifically
provides for improved compositions for the delivery of siRNA
molecules. It is shown herein that these compositions are effective
in down-regulating the protein levels and/or mRNA levels of target
proteins. Furthermore, it is shown that the activity of these
improved compositions is dependent on the presence of a certain
cationic lipids and that the molar ratio of cationic lipid in the
formulation can influence activity.
[0038] The lipid particles and compositions of the present
invention may be used for a variety of purposes, including the
delivery of associated or encapsulated therapeutic agents to cells,
both in vitro and in vivo. Accordingly, the present invention
provides methods of treating diseases or disorders in a subject in
need thereof, by contacting the subject with a lipid particle of
the present invention associated with a suitable therapeutic
agent.
[0039] As described herein, the lipid particles of the present
invention are particularly useful for the delivery of nucleic
acids, including, e.g., siRNA molecules and plasmids. Therefore,
the lipid particles and compositions of the present invention may
be used to modulate the expression of target genes and proteins
both in vitro and in vivo by contacting cells with a lipid particle
of the present invention associated with a nucleic acid that
reduces target gene expression (e.g., an siRNA) or a nucleic acid
that may be used to increase expression of a desired protein (e.g.,
a plasmid encoding the desired protein).
[0040] Various exemplary embodiments of the cationic lipids of the
present invention, as well as lipid particles and compositions
comprising the same, and their use to deliver therapeutic agents
and modulate gene and protein expression are described in further
detail below.
Lipids
[0041] The present invention provides novel lipids having certain
design features. As shown in FIG. 2, the lipid design features
include at least one of the following: a head group with varying
pKa, a cationic, 1.degree., 2.degree. and 3.degree., monoamine, Di
and triamine, Oligoamine/polyamine, a low pKa head
groups--imidazoles and pyridine, guanidinium, anionic, zwitterionic
and hydrophobic tails can include symmetric and/or unsymmetric
chains, long and shorter, saturated and unsaturated chain the back
bone includes Backbone glyceride and other acyclic analogs, cyclic,
spiro, bicyclic and polycyclic linkages with ethers, esters,
phosphate and analogs, sulfonate and analogs, disulfides, pH
sensitive linkages like acetals and ketals, imines and hydrazones,
and oximes.
[0042] In one embodiment, the lipid has one of the following
structures:
##STR00003##
[0043] wherein:
[0044] R.sub.1 and R.sub.2 are each independently for each
occurrence optionally substituted C.sub.10-C.sub.30 alkyl,
optionally substituted C.sub.10-C.sub.30 alkenyl, optionally
substituted C.sub.10-C.sub.30 alkynyl, optionally substituted
C.sub.10-C.sub.30 acyl, or -linker-ligand;
[0045] R.sub.3 is independently for each occurrence H, optionally
substituted C.sub.1-C.sub.10 alkyl, optionally substituted
C.sub.2-C.sub.10 alkenyl, optionally substituted C.sub.2-C.sub.10
alkynyl, alkylheterocycle, alkylphosphate, alkylphosphorothioate,
alkylphosphorodithioate, alkylphosphonates, alkylamines,
hydroxyalkyls, .omega.-aminoalkyls,
.omega.-(substituted)aminoalkyls, .omega.-phosphoalkyls,
.omega.-thiophosphoalkyls, optionally substituted polyethylene
glycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K),
heteroaryl, heterocycle, or linker-ligand;
[0046] X and Y are each independently O, S, alkyl or N(Q);
[0047] Q.sub.1 is independently for each occurrence O or S;
[0048] Q.sub.2 is independently for each occurrence O, S, N(Q),
alkyl or alkoxy;
A.sub.1, A.sub.2, A.sub.4 and A.sub.5 are each independently O, S,
CH.sub.2, CHF or CF.sub.2; and
[0049] i and j are 0-10; or
[0050] a salt or isomer thereof.
[0051] In one embodiment, X and Y can be independently (CO), O(CO),
O(CO)N, N(CO)O, (CO)O, O(CO)O, a sulfonate, or a phosphate.
[0052] It has been found that cationic lipids comprising
unsaturated alkyl chains are particularly useful for forming lipid
nucleic acid particles with increased membrane fluidity. In one
embodiment, at least one of R.sub.1 or R.sub.2 comprises at least
one, at least two or at least three sites of unsaturation, e.g.
double bond or triple bond.
[0053] In one embodiment, only one of R.sub.1 or R.sub.2 comprises
at least one, at least two or at least three sites of
unsaturation.
[0054] In one embodiment, R.sub.1 and R.sub.2 both comprise at
least one, at least two or at least three sites of
unsaturation.
[0055] In one embodiment, R.sub.1 and R.sub.2 comprise different
numbers of unsaturation, e.g., one of R.sub.1 and R.sub.2 has one
site of unsaturation and the other has two or three sites of
unsaturation.
[0056] In one embodiment, R.sub.1 and R.sub.2 both comprise the
same number of unsaturation sites.
[0057] In one embodiment, R.sub.1 and R.sub.2 comprise different
types of unsaturation, e.g. unsaturation in one of R.sub.1 and
R.sub.2 is double bond and in the other unsaturation is triple
bond.
[0058] In one embodiment, R.sub.1 and R.sub.2 both comprise the
same type of unsaturation, e.g. double bond or triple bond.
[0059] In one embodiment, at least one of R.sub.1 or R.sub.2
comprises at least one double bond and at least one triple
bond.
[0060] In one embodiment, only one of R.sub.1 or R.sub.2 comprises
at least one double bond and at least one triple bond.
[0061] In one embodiment, R.sub.1 and R.sub.2 both comprise at
least one double bond and at least one triple bond.
[0062] In one embodiment, R.sub.1 and R.sub.2 are both same, e.g.
R.sub.1 and R.sub.2 are both linoleyl (C18) or R.sub.1 and R.sub.2
are both heptadeca-9-enyl.
[0063] In one embodiment, R.sub.1 and R.sub.2 are different from
each other.
[0064] In one embodiment, at least one of R.sub.1 and R.sub.2 is
cholesterol.
[0065] In one embodiment, one of R.sub.1 and R.sub.2 is
-linker-ligand.
[0066] In one embodiment, one of R.sub.1 and R.sub.2 is
-linker-ligand and ligand is a lipophile.
[0067] In one embodiment, at least one of R.sub.1 or R.sub.2
comprises at least one CH.sub.2 group with one or both H replaced
by F, e.g. CHF or CF.sub.2. In one embodiment, both R.sub.1 and
R.sub.2 comprise at least one CH.sub.2 group with one or two H
replaced by F, e.g. CHF or CF.sub.2.
[0068] In one embodiment, only one of R.sub.1 and R.sub.2 comprises
at least one CH.sub.2 group with one or both H replaced by F.
[0069] In one embodiment, at least one of R.sub.1 or R.sub.2
terminates in CH.sub.2F, CHF.sub.2 or CF.sub.3. In one embodiment,
both R.sub.1 and R.sub.2 terminate in CH.sub.2F, CHF.sub.2 or
CF.sub.3.
[0070] In one embodiment, at least one of R.sub.1 or R.sub.2 is
--(CF.sub.2).sub.y--Z''--(CH.sub.2).sub.y--CH.sub.3, wherein each y
is independently 1-10 and Z'' is O, S or N(Q).
[0071] In one embodiment, both of R.sub.1 and R.sub.2 are
--(CF.sub.2).sub.y--Z''--(CH.sub.2).sub.y--CH.sub.3, wherein each y
is independently 1-10 and Z'' is O, S or N(Q).
[0072] In one embodiment, at least one of R.sub.1 or R.sub.2 is
--(CH.sub.2).sub.y--Z''--(CF.sub.2).sub.y--CF.sub.3, wherein each y
is independently 1-10 and Z'' is O, S or N(Q).
[0073] In one embodiment, both of R.sub.1 and R.sub.2 are
--(CH.sub.2).sub.y--Z''--(CF.sub.2).sub.y--CF.sub.3, wherein each y
is independently 1-10 and Z'' is O, S or N(Q).
[0074] In one embodiment, at least one of R.sub.1 or R.sub.2 is
--(CF.sub.2).sub.y--(CF.sub.2).sub.y--CF.sub.3, wherein each y is
independently 1-10.
[0075] In one embodiment, both of R.sub.1 and R.sub.2 are
--(CF.sub.2).sub.y--(CF.sub.2).sub.y--CF.sub.3, wherein each y is
independently 1-10.
[0076] In one embodiment, R.sub.3 is chosen from a group consisting
of methyl, ethyl, polyamine, --(CH.sub.2).sub.h-heteroaryl,
--(CH.sub.2).sub.h--N(Q).sub.2, --O--N(Q).sub.2,
--(CH.sub.2).sub.h--Z'--(CH.sub.2).sub.h-heteroaryl, linker-ligand,
--(CH.sub.2).sub.h-heterocycle, and
--(CH.sub.2).sub.h--Z''--(CH.sub.2).sub.h-heterocycle, wherein each
h is independently 0-13 and Z'' is O, S or N(Q).
[0077] In one embodiment, ligand is fusogenic peptide.
[0078] In one embodiment, the lipid is a racemic mixture.
[0079] In one embodiment, the lipid is enriched in one
diastereomer, e.g. the lipid has at least 95%, at least 90%, at
least 80% or at least 70% diastereomeric excess.
[0080] In one embodiment, the lipid is enriched in one enantiomer,
e.g. the lipid has at least 95%, at least 90%, at least 80% or at
least 70% enantiomer excess.
[0081] In one embodiment, the lipid is chirally pure, e.g. is a
single optical isomer.
[0082] In one embodiment, the lipid is enriched for one optical
isomer.
[0083] Where a double bond is present (e.g., a carbon-carbon double
bond or carbon-nitrogen double bond), there can be isomerism in the
configuration about the double bond (i.e. cis/trans or E/Z
isomerism). Where the configuration of a double bond is illustrated
in a chemical structure, it is understood that the corresponding
isomer can also be present. The amount of isomer present can vary,
depending on the relative stabilities of the isomers and the energy
required to convert between the isomers. Accordingly, some double
bonds are, for practical purposes, present in only a single
configuration, whereas others (e.g., where the relative stabilities
are similar and the energy of conversion low) may be present as
inseparable equilibrium mixture of configurations.
[0084] In one aspect, the lipid is a compound of formula
XXXIII,
##STR00004##
wherein:
[0085] R.sub.1 and R.sub.2 are each independently for each
occurrence optionally substituted C.sub.10-C.sub.30 alkyl,
optionally substituted C.sub.10-C.sub.30 alkenyl, optionally
substituted C.sub.10-C.sub.30 alkynyl, optionally substituted
C.sub.10-C.sub.30 acyl, or -linker-ligand;
[0086] R.sub.3 is H, optionally substituted C.sub.1-C.sub.10 alkyl,
optionally substituted C.sub.2-C.sub.10 alkenyl, optionally
substituted C.sub.2-C.sub.10 alkynyl, alkylheterocycle,
alkylphosphate, alkylphosphorothioate, alkylphosphorodithioate,
alkylphosphonates, alkylamines, hydroxyalkyls, .omega.-aminoalkyls,
.omega.-(substituted)aminoalkyls, .omega.-phosphoalkyls,
.omega.-thiophosphoalkyls, optionally substituted polyethylene
glycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K),
heteroaryl, heterocycle, or linker-ligand;
[0087] E is O, S, N(Q), C(O), N(Q)C(O), C(O)N(Q), (Q)N(CO)O,
O(CO)N(Q), S(O), NS(O).sub.2N(Q), S(O).sub.2, N(Q)S(O).sub.2, SS,
O.dbd.N, aryl, heteroaryl, cyclic or heterocycle; and,
[0088] Q is H, alkyl, .omega.-aminoalkyl,
.omega.-(substituted)aminoalkyl, .omega.-phosphoalkyl or
.omega.-thiophosphoalkyl; or
[0089] a salt or isomer thereof.
[0090] In one embodiment, R.sub.1 and R.sub.2 are each
independently for each occurrence optionally substituted
C.sub.10-C.sub.30 alkyl, optionally substituted C.sub.10-C.sub.30
alkoxy, optionally substituted C.sub.10-C.sub.30 alkenyl,
optionally substituted C.sub.10-C.sub.30 alkenyloxy, optionally
substituted C.sub.10-C.sub.30 alkynyl, optionally substituted
C.sub.10-C.sub.30 alkynyloxy, or optionally substituted
C.sub.10-C.sub.30 acyl.
[0091] In another embodiment, R.sub.3 is H, optionally substituted
C.sub.1-C.sub.10 alkyl, optionally substituted C.sub.2-C.sub.10
alkenyl, optionally substituted C.sub.2-C.sub.10 alkynyl,
optionally substituted alkylheterocycle, optionally substituted
heterocycloalkyl, optionally substituted alkylphosphate, optionally
substituted phosphoalkyl, optionally substituted
alkylphosphorothioate, optionally substituted phosphorothioalkyl,
optionally substituted alkylphosphorodithioate, optionally
substituted phosphorodithioalkyl, optionally substituted
alkylphosphonate, optionally substituted phosphonoalkyl, optionally
substituted amino, optionally substituted alkylamino, optionally
substituted di(alkyl)amino, optionally substituted aminoalkyl,
optionally substituted alkylaminoalkyl, optionally substituted
di(alkyl)aminoalkyl, optionally substituted hydroxyalkyl,
optionally substituted polyethylene glycol (PEG, mw 100-40K),
optionally substituted mPEG (mw 120-40K), optionally substituted
heteroaryl, optionally substituted heterocycle, or
linker-ligand.
[0092] In yet another embodiment, E is --O--, --S--, --N(Q)-,
--C(O)--, --N(Q)C(O)--, --C(O)N(Q)-, --N(Q)C(O)O--, --OC(O)N(Q)-,
S(O), --N(Q)S(O).sub.2N(Q)-, --S(O).sub.2--, --N(Q)S(O).sub.2--,
--SS--, --O--N.dbd., .dbd.N--O--, --C(O)--N(Q)-N.dbd.,
--N(Q)-N.dbd., --N(Q)-O--, --C(O)S--, arylene, heteroarylene,
cyclalkylene, or heterocyclylene; and
[0093] Q is H, alkyl, .omega.-aminoalkyl,
.omega.-(substituted)aminoalkyl, .omega.-phosphoalkyl or
.omega.-thiophosphoalkyl.
[0094] In another embodiment, the lipid is a compound of formula
XXXIII, wherein E is O, S, N(Q), C(O), N(Q)C(O), C(O)N(Q),
(Q)N(CO)O, O(CO)N(Q), S(O), NS(O).sub.2N(Q), S(O).sub.2,
N(Q)S(O).sub.2, SS, O.dbd.N, aryl, heteroaryl, cyclic or
heterocycle.
[0095] In one embodiment, the lipid is a compound of formula
XXXIII, wherein R.sub.3 is H, optionally substituted
C.sub.2-C.sub.10 alkenyl, optionally substituted C.sub.2-C.sub.10
alkynyl, alkylheterocycle, alkylphosphate, alkylphosphorothioate,
alkylphosphorodithioate, alkylphosphonates, alkylamines,
hydroxyalkyls, .omega.-aminoalkyls,
.omega.-(substituted)aminoalkyls, .omega.-phosphoalkyls,
.omega.-thiophosphoalkyls, optionally substituted polyethylene
glycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K),
heteroaryl, heterocycle, or linker-ligand.
[0096] In yet another embodiment, the lipid is a compound of
formula XXXIII, wherein R.sub.1 and R.sub.2 are each independently
for each occurrence optionally substituted C.sub.10-C.sub.30 alkyl,
optionally substituted C.sub.10-C.sub.30 alkynyl, optionally
substituted C.sub.10-C.sub.30 acyl, or -linker-ligand.
[0097] In one embodiment, the invention features a lipid of formula
XXXVIII:
##STR00005##
[0098] wherein
[0099] E is O, S, N(Q), C(O), N(Q)C(O), C(O)N(Q), (Q)N(CO)O,
O(CO)N(Q), S(O), NS(O).sub.2N(Q), S(O).sub.2, N(Q)S(O).sub.2, SS,
O.dbd.N, aryl, heteroaryl, cyclic or heterocycle;
[0100] Q is H, alkyl, .omega.-aminoalkyl,
.omega.-(substituted)aminoalkyl, .omega.-phosphoalkyl or
.omega.-thiophosphoalkyl;
[0101] R.sub.1 and R.sub.2 and R.sub.x are each independently for
each occurrence H, optionally substituted C.sub.1-C.sub.10 alkyl,
optionally substituted C.sub.10-C.sub.30 alkyl, optionally
substituted C.sub.10-C.sub.30 alkenyl, optionally substituted
C.sub.10-C.sub.30 alkynyl, optionally substituted C.sub.10-C.sub.30
acyl, or linker-ligand, provided that at least one of R.sub.1,
R.sub.2 and R.sub.x is not H;
[0102] R.sub.3 is H, optionally substituted C.sub.1-C.sub.10 alkyl,
optionally substituted C.sub.2-C.sub.10 alkenyl, optionally
substituted C.sub.2-C.sub.10 alkynyl, alkylheterocycle,
alkylphosphate, alkylphosphorothioate, alkylphosphorodithioate,
alkylphosphonates, alkylamines, hydroxyalkyls, .omega.-aminoalkyls,
.omega.-(substituted)aminoalkyls, .omega.-phosphoalkyls,
.omega.-thiophosphoalkyls, optionally substituted polyethylene
glycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K),
heteroaryl, heterocycle, or linker-ligand; and
[0103] n is 0, 1, 2, or 3;
[0104] or a salt or isomer thereof.
[0105] In some embodiments, each of R.sub.1 and R.sub.2 is
independently for each occurrence optionally substituted
C.sub.10-C.sub.30 alkyl, optionally substituted C.sub.10-C.sub.30
alkenyl, optionally substituted C.sub.10-C.sub.30 alkynyl,
optionally substituted C.sub.10-C.sub.30 acyl, or
linker-ligand.
[0106] In some embodiments, R.sub.x is H or optionally substituted
C.sub.1-C.sub.10 alkyl.
[0107] In some embodiments, R.sub.x is optionally substituted
C.sub.10-C.sub.30 alkyl, optionally substituted C.sub.10-C.sub.30
alkenyl, optionally substituted C.sub.10-C.sub.30 alkynyl,
optionally substituted C.sub.10-C.sub.30 acyl, or
linker-ligand.
[0108] In one embodiment, R.sub.1 and R.sub.2 are each
independently for each occurrence optionally substituted
C.sub.10-C.sub.30 alkyl, optionally substituted C.sub.10-C.sub.30
alkoxy, optionally substituted C.sub.10-C.sub.30 alkenyl,
optionally substituted C.sub.10-C.sub.30 alkenyloxy, optionally
substituted C.sub.10-C.sub.30 alkynyl, optionally substituted
C.sub.10-C.sub.30 alkynyloxy, or optionally substituted
C.sub.10-C.sub.30 acyl, or -linker-ligand.
[0109] In one embodiment, R.sub.3 is independently for each
occurrence H, optionally substituted C.sub.1-C.sub.10 alkyl,
optionally substituted C.sub.2-C.sub.10 alkenyl, optionally
substituted C.sub.2-C.sub.10 alkynyl, optionally substituted
alkylheterocycle, optionally substituted heterocycloalkyl,
optionally substituted alkylphosphate, optionally substituted
phosphoalkyl, optionally substituted alkylphosphorothioate,
optionally substituted phosphorothioalkyl, optionally substituted
alkylphosphorodithioate, optionally substituted
phosphorodithioalkyl, optionally substituted alkylphosphonate,
optionally substituted phosphonoalkyl, optionally substituted
amino, optionally substituted alkylamino, optionally substituted
di(alkyl)amino, optionally substituted aminoalkyl, optionally
substituted alkylaminoalkyl, optionally substituted
di(alkyl)aminoalkyl, optionally substituted hydroxyalkyl,
optionally substituted polyethylene glycol (PEG, mw 100-40K),
optionally substituted mPEG (mw 120-40K), optionally substituted
heteroaryl, or optionally substituted heterocycle, or
linker-ligand.
[0110] In one embodiment, X and Y are each independently --O--,
--S--, alkylene, --N(Q)-, --C(O)--, --O(CO)--, --OC(O)N(Q)-,
--N(Q)C(O)O--, --C(O)O, --OC(O)O--, --OS(O)(Q.sub.2)O--, or
--OP(O)(Q.sub.2)O--.
[0111] In one embodiment, Q is H, alkyl, .omega.-aminoalkyl,
.omega.-(substituted)aminoalkyl, .omega.-phosphoalkyl, or
.omega.-thiophosphoalkyl.
[0112] In one embodiment, Q.sub.1 is independently for each
occurrence O or S.
[0113] In one embodiment, Q.sub.2 is independently for each
occurrence O, S, N(Q)(Q), alkyl or alkoxy,
[0114] In one embodiment, A.sub.1, A.sub.2, A.sub.4, and A.sub.5
are each independently --O--, --5-, --CH.sub.2--, --CHR.sup.5--,
--CR.sup.5R.sup.5--, --CHF-- or --CF.sub.2--.
[0115] In one embodiment, E are is --O--, --S--, --N(Q)-, --C(O)--,
--C(O)N(Q)-, --N(Q)C(O)--, --S(O)--, --S(O).sub.2--, --SS--,
--O--N.dbd., .dbd.N--O--, arylene, heteroarylene, cycloalkylene, or
heterocyclylene.
[0116] In one embodiment, i and j are each independently 0-10.
[0117] In some circumstances, R.sub.3 is .omega.-aminoalkyl,
.omega.-(substituted)aminoalkyl, .omega.-phosphoalkyl, or
.omega.-thiophosphoalkyl; each of which is optionally substituted.
Examples of .omega.-(substituted)aminoalkyl groups include
2-(dimethylamino)ethyl, 3-(diisopropylamino)propyl, or
3-(N-ethyl-N-isopropylamino)-1-methylpropyl.
[0118] In one embodiment, X and Y can be independently --O--,
--S--, alkylene, or --N(Q)-. In one embodiment, the cationic lipid
is chosen from a group consisting of lipids shown in Table 1
below.
TABLE-US-00001 TABLE 1 Some cationic lipids of the present
invention. ##STR00006## n = 0-6 ##STR00007## n = 0-6 ##STR00008## n
= 0-6 ##STR00009## n = 0-6 ##STR00010## n = 0-6 ##STR00011##
##STR00012## ##STR00013## ##STR00014## ##STR00015## ##STR00016##
##STR00017## ##STR00018## ##STR00019## ##STR00020## ##STR00021##
##STR00022## ##STR00023## ##STR00024## ##STR00025## ##STR00026##
##STR00027## ##STR00028## ##STR00029## ##STR00030## ##STR00031##
##STR00032## ##STR00033## ##STR00034## ##STR00035## ##STR00036##
##STR00037## ##STR00038## ##STR00039## ##STR00040## ##STR00041##
##STR00042## ##STR00043## ##STR00044## ##STR00045## ##STR00046##
##STR00047## ##STR00048## ##STR00049## ##STR00050## ##STR00051##
##STR00052## ##STR00053## ##STR00054## ##STR00055## ##STR00056##
##STR00057## ##STR00058## ##STR00059## ##STR00060## ##STR00061##
##STR00062## ##STR00063## ##STR00064## ##STR00065## ##STR00066##
##STR00067## ##STR00068## ##STR00069## ##STR00070## ##STR00071##
##STR00072## Q is NH, NMe ##STR00073## Q is NH, NMe ##STR00074## Q
is NH, NMe ##STR00075## Q is NH, NMe ##STR00076## Q is NH, NMe
##STR00077## Q is NH, NMe ##STR00078## Q is NH, NMe ##STR00079## Q
is NH, NMe ##STR00080## Q is NH, NMe ##STR00081## Q is NH, NMe
##STR00082## Q is NH, NMe ##STR00083## Q is NH, NMe ##STR00084## Q
is NH, NMe ##STR00085## Q is NH, NMe ##STR00086## Q is NH, NMe
##STR00087## Q is NH, NMe ##STR00088## Q is NH, NMe ##STR00089## Q
is NH, NMe ##STR00090## Q is NH, NMe ##STR00091## Q is NH, NMe
##STR00092## Q is NH, NMe ##STR00093## Q is NH, NMe ##STR00094## Q
is NH, NMe ##STR00095## Q is NH, NMe ##STR00096## ##STR00097##
##STR00098## ##STR00099##
##STR00100##
[0119] Although not all diasteromers for a lipid are shown, one
aspect of the present invention is to provide all diastereomers and
as such chirally pure and diastereomerically enriched lipids are
also part of this invention.
[0120] In one embodiment, R.sub.3 is -linker-ligand.
[0121] In particular embodiments, the lipids of the present
invention are cationic lipids. As used herein, the term "cationic
lipid" is meant to include those lipids having one or two fatty
acid or fatty alkyl chains and an amino head group (including an
alkylamino or dialkylamino group) that may be protonated to form a
cationic lipid at physiological pH. In some embodiments, a cationic
lipid is referred to as an "amino lipid."
[0122] Other cationic lipids would include those having alternative
fatty acid groups and other dialkylamino groups, including those in
which the alkyl substituents are different (e.g.,
N-ethyl-N-methylamino-, N-propyl-N-ethylamino- and the like). For
those embodiments in which R.sub.1 and R.sub.2 are both long chain
alkyl or acyl groups, they can be the same or different. In
general, lipids (e.g., a cationic lipid) having less saturated acyl
chains are more easily sized, particularly when the complexes are
sized below about 0.3 microns, for purposes of filter
sterilization. Cationic lipids containing unsaturated fatty acids
with carbon chain lengths in the range of C.sub.10 to C.sub.20 are
typical. Other scaffolds can also be used to separate the amino
group (e.g., the amino group of the cationic lipid) and the fatty
acid or fatty alkyl portion of the cationic lipid. Suitable
scaffolds are known to those of skill in the art.
[0123] In certain embodiments, cationic lipids of the present
invention have at least one protonatable or deprotonatable group,
such that the lipid is positively charged at a pH at or below
physiological pH (e.g. pH 7.4), and neutral at a second pH,
preferably at or above physiological pH. Such lipids are also
referred to as cationic lipids. It will, of course, be understood
that the addition or removal of protons as a function of pH is an
equilibrium process, and that the reference to a charged or a
neutral lipid refers to the nature of the predominant species and
does not require that all of the lipid be present in the charged or
neutral form. Lipids that have more than one protonatable or
deprotonatable group, or which are zwiterrionic, are not excluded
from use in the invention.
[0124] In certain embodiments, protonatable lipids (i.e., cationic
lipids) according to the invention have a pKa of the protonatable
group in the range of about 4 to about 11. Typically, lipids will
have a pKa of about 4 to about 7, e.g., between about 5 and 7, such
as between about 5.5 and 6.8, when incorporated into lipid
particles. Such lipids will be cationic at a lower pH formulation
stage, while particles will be largely (though not completely)
surface neutralized at physiological pH around pH 7.4. One of the
benefits of a pKa in the range of between about 4 and 7 is that at
least some nucleic acid associated with the outside surface of the
particle will lose its electrostatic interaction at physiological
pH and be removed by simple dialysis; thus greatly reducing the
particle's susceptibility to clearance. pKa measurements of lipids
within lipid particles can be performed, for example, by using the
fluorescent probe 2-(p-toluidino)-6-napthalene sulfonic acid (TNS),
using methods described in Cullis et al., (1986) Chem Phys Lipids
40, 127-144.
[0125] In one embodiment, the formulations of the invention are
entrapped by at least 75%, at least 80% or at least 90%.
[0126] In one embodiment, the formulations of the invention further
comprise an apolipoprotein. As used herein, the term
"apolipoprotein" or "lipoprotein" refers to apolipoproteins known
to those of skill in the art and variants and fragments thereof and
to apolipoprotein agonists, analogues or fragments thereof
described below. Suitable apolipoproteins include, but are not
limited to, ApoA-I, ApoA-II, ApoA-IV, ApoA-V and ApoE, and active
polymorphic forms, isoforms, variants and mutants as well as
fragments or truncated forms thereof. In certain embodiments, the
apolipoprotein is a thiol containing apolipoprotein. "Thiol
containing apolipoprotein" refers to an apolipoprotein, variant,
fragment or isoform that contains at least one cysteine residue.
The most common thiol containing apolipoproteins are ApoA-I Milano
(ApoA-I.sub.M) and ApoA-I Paris (ApoA-Ip) which contain one
cysteine residue (Jia et al., 2002, Biochem. Biophys. Res. Comm.
297: 206-13; Bielicki and Oda, 2002, Biochemistry 41: 2089-96).
ApoA-II, ApoE2 and ApoE3 are also thiol containing apolipoproteins.
Isolated ApoE and/or active fragments and polypeptide analogues
thereof, including recombinantly produced forms thereof, are
described in U.S. Pat. Nos. 5,672,685; 5,525,472; 5,473,039;
5,182,364; 5,177,189; 5,168,045; 5,116,739; the disclosures of
which are herein incorporated by reference. ApoE3 is disclosed in
Weisgraber, et al., "Human E apoprotein heterogeneity:
cysteine-arginine interchanges in the amino acid sequence of the
apo-E isoforms," J. Biol. Chem. (1981) 256: 9077-9083; and Rall, et
al., "Structural basis for receptor binding heterogeneity of
apolipoprotein E from type III hyperlipoproteinemic subjects,"
Proc. Nat. Acad. Sci. (1982) 79: 4696-4700. See also GenBank
accession number K00396.
[0127] In certain embodiments, the apolipoprotein can be in its
mature form, in its preproapolipoprotein form or in its
proapolipoprotein form. Homo- and heterodimers (where feasible) of
pro- and mature ApoA-I (Duverger et al., 1996, Arterioscler.
Thromb. Vasc. Biol. 16(12):1424-29), ApoA-I Milano (Klon et al.,
2000, Biophys. J. 79:(3) 1679-87; Franceschini et al., 1985, J.
Biol. Chem. 260: 1632-35), ApoA-I Paris (Daum et al., 1999, J. Mol.
Med. 77:614-22), ApoA-II (Shelness et al., 1985, J. Biol. Chem.
260(14):8637-46; Shelness et al., 1984, J. Biol. Chem.
259(15):9929-35), ApoA-IV (Duverger et al., 1991, Euro. J. Biochem.
201(2):373-83), and ApoE (McLean et al., 1983, J. Biol. Chem.
258(14):8993-9000) can also be utilized within the scope of the
invention.
[0128] In certain embodiments, the apolipoprotein can be a
fragment, variant or isoform of the apolipoprotein. The term
"fragment" refers to any apolipoprotein having an amino acid
sequence shorter than that of a native apolipoprotein and which
fragment retains the activity of native apolipoprotein, including
lipid binding properties. By "variant" is meant substitutions or
alterations in the amino acid sequences of the apolipoprotein,
which substitutions or alterations, e.g., additions and deletions
of amino acid residues, do not abolish the activity of native
apolipoprotein, including lipid binding properties. Thus, a variant
can comprise a protein or peptide having a substantially identical
amino acid sequence to a native apolipoprotein provided herein in
which one or more amino acid residues have been conservatively
substituted with chemically similar amino acids. Examples of
conservative substitutions include the substitution of at least one
hydrophobic residue such as isoleucine, valine, leucine or
methionine for another. Likewise, the present invention
contemplates, for example, the substitution of at least one
hydrophilic residue such as, for example, between arginine and
lysine, between glutamine and asparagine, and between glycine and
serine (see U.S. Pat. Nos. 6,004,925, 6,037,323 and 6,046,166). The
term "isoform" refers to a protein having the same, greater or
partial function and similar, identical or partial sequence, and
may or may not be the product of the same gene and usually tissue
specific (see Weisgraber 1990, J. Lipid Res. 31(8):1503-11; Hixson
and Powers 1991, J. Lipid Res. 32(9):1529-35; Lackner et al., 1985,
J. Biol. Chem. 260(2):703-6; Hoeg et al., 1986, J. Biol. Chem.
261(9):3911-4; Gordon et al., 1984, J. Biol. Chem. 259(1):468-74;
Powell et al., 1987, Cell 50(6):831-40; Aviram et al., 1998,
Arterioscler. Thromb. Vase. Biol. 18(10):1617-24; Aviram et al.,
1998, J. Clin. Invest. 101(8):1581-90; Billecke et al., 2000, Drug
Metab. Dispos. 28(11):1335-42; Draganov et al., 2000, J. Biol.
Chem. 275(43):33435-42; Steinmetz and Utermann 1985, J. Biol. Chem.
260(4):2258-64; Widler et al., 1980, J. Biol. Chem.
255(21):10464-71; Dyer et al., 1995, J. Lipid Res. 36(1):80-8;
Sacre et al., 2003, FEBS Lett. 540(1-3):181-7; Weers, et al., 2003,
Biophys. Chem. 100(1-3):481-92; Gong et al., 2002, J. Biol. Chem.
277(33):29919-26; Ohta et al., 1984, J. Biol. Chem.
259(23):14888-93 and U.S. Pat. No. 6,372,886).
[0129] In certain embodiments, the methods and compositions of the
present invention include the use of a chimeric construction of an
apolipoprotein. For example, a chimeric construction of an
apolipoprotein can be comprised of an apolipoprotein domain with
high lipid binding capacity associated with an apolipoprotein
domain containing ischemia reperfusion protective properties. A
chimeric construction of an apolipoprotein can be a construction
that includes separate regions within an apolipoprotein (i.e.,
homologous construction) or a chimeric construction can be a
construction that includes separate regions between different
apolipoproteins (i.e., heterologous constructions). Compositions
comprising a chimeric construction can also include segments that
are apolipoprotein variants or segments designed to have a specific
character (e.g., lipid binding, receptor binding, enzymatic, enzyme
activating, antioxidant or reduction-oxidation property) (see
Weisgraber 1990, J. Lipid Res. 31(8):1503-11; Hixson and Powers
1991, J. Lipid Res. 32(9):1529-35; Lackner et al., 1985, J. Biol.
Chem. 260(2):703-6; Hoeg et al, 1986, J. Biol. Chem. 261(9):3911-4;
Gordon et al., 1984, J. Biol. Chem. 259(1):468-74; Powell et al.,
1987, Cell 50(6):831-40; Aviram et al., 1998, Arterioscler. Thromb.
Vasc. Biol. 18(10):1617-24; Aviram et al., 1998, J. Clin. Invest.
101(8):1581-90; Billecke et al., 2000, Drug Metab. Dispos.
28(11):1335-42; Draganov et al., 2000, J. Biol. Chem.
275(43):33435-42; Steinmetz and Utermann 1985, J. Biol. Chem.
260(4):2258-64; Widler et al., 1980, J. Biol. Chem.
255(21):10464-71; Dyer et al., 1995, J. Lipid Res. 36(1):80-8;
Sorenson et al., 1999, Arterioscler. Thromb. Vasc. Biol.
19(9):2214-25; Palgunachari 1996, Arterioscler. Throb. Vasc. Biol.
16(2):328-38: Thurberg et al., J. Biol. Chem. 271(11):6062-70; Dyer
1991, J. Biol. Chem. 266(23):150009-15; Hill 1998, J. Biol. Chem.
273(47):30979-84).
[0130] Apolipoproteins utilized in the invention also include
recombinant, synthetic, semi-synthetic or purified apolipoproteins.
Methods for obtaining apolipoproteins or equivalents thereof,
utilized by the invention are well-known in the art. For example,
apolipoproteins can be separated from plasma or natural products
by, for example, density gradient centrifugation or immunoaffinity
chromatography, or produced synthetically, semi-synthetically or
using recombinant DNA techniques known to those of the art (see,
e.g., Mulugeta et al., 1998, J. Chromatogr. 798(1-2): 83-90; Chung
et al., 1980, J. Lipid Res. 21(3):284-91; Cheung et al., 1987, J.
Lipid Res. 28(8):913-29; Persson, et al., 1998, J. Chromatogr.
711:97-109; U.S. Pat. Nos. 5,059,528, 5,834,596, 5,876,968 and
5,721,114; and PCT Publications WO 86/04920 and WO 87/02062).
[0131] Apolipoproteins utilized in the invention further include
apolipoprotein agonists such as peptides and peptide analogues that
mimic the activity of ApoA-I, ApoA-I Milano (ApoA-I.sub.M), ApoA-I
Paris (ApoA-Ip), ApoA-II, ApoA-IV, and ApoE. For example, the
apolipoprotein can be any of those described in U.S. Pat. Nos.
6,004,925, 6,037,323, 6,046,166, and 5,840,688, the contents of
which are incorporated herein by reference in their entireties.
[0132] Apolipoprotein agonist peptides or peptide analogues can be
synthesized or manufactured using any technique for peptide
synthesis known in the art including, e.g., the techniques
described in U.S. Pat. Nos. 6,004,925, 6,037,323 and 6,046,166. For
example, the peptides may be prepared using the solid-phase
synthetic technique initially described by Merrifield (1963, J. Am.
Chem. Soc. 85:2149-2154). Other peptide synthesis techniques may be
found in Bodanszky et al., Peptide Synthesis, John Wiley &
Sons, 2d Ed., (1976) and other references readily available to
those skilled in the art. A summary of polypeptide synthesis
techniques can be found in Stuart and Young, Solid Phase Peptide.
Synthesis, Pierce Chemical Company, Rockford, Ill., (1984).
Peptides may also be synthesized by solution methods as described
in The Proteins, Vol. II, 3d Ed., Neurath et. al., Eds., p.
105-237, Academic Press, New York, N.Y. (1976). Appropriate
protective groups for use in different peptide syntheses are
described in the above-mentioned texts as well as in McOmie,
Protective Groups in Organic Chemistry, Plenum Press, New York,
N.Y. (1973). The peptides of the present invention might also be
prepared by chemical or enzymatic cleavage from larger portions of,
for example, apolipoprotein A-I.
[0133] In certain embodiments, the apolipoprotein can be a mixture
of apolipoproteins. In one embodiment, the apolipoprotein can be a
homogeneous mixture, that is, a single type of apolipoprotein. In
another embodiment, the apolipoprotein can be a heterogeneous
mixture of apolipoproteins, that is, a mixture of two or more
different apolipoproteins. Embodiments of heterogenous mixtures of
apolipoproteins can comprise, for example, a mixture of an
apolipoprotein from an animal source and an apolipoprotein from a
semi-synthetic source. In certain embodiments, a heterogenous
mixture can comprise, for example, a mixture of ApoA-I and ApoA-I
Milano. In certain embodiments, a heterogeneous mixture can
comprise, for example, a mixture of ApoA-I Milano and ApoA-I Paris.
Suitable mixtures for use in the methods and compositions of the
invention will be apparent to one of skill in the art.
[0134] If the apolipoprotein is obtained from natural sources, it
can be obtained from a plant or animal source. If the
apolipoprotein is obtained from an animal source, the
apolipoprotein can be from any species. In certain embodiments, the
apolipoprotien can be obtained from an animal source. In certain
embodiments, the apolipoprotein can be obtained from a human
source. In preferred embodiments of the invention, the
apolipoprotein is derived from the same species as the individual
to which the apolipoprotein is administered.
Lipid Particles
[0135] The present invention also provides lipid particles
comprising one or more of the cationic lipids described above.
Lipid particles include, but are not limited to, liposomes. As used
herein, a liposome is a structure having lipid-containing membranes
enclosing an aqueous interior. Liposomes may have one or more lipid
membranes. The invention contemplates both single-layered
liposomes, which are referred to as unilamellar, and multi-layered
liposomes, which are referred to as multilamellar. When complexed
with nucleic acids, lipid particles may also be lipoplexes, which
are composed of cationic lipid bilayers sandwiched between DNA
layers, as described, e.g., in Feigner, Scientific American.
[0136] The lipid particles of the present invention may further
comprise one or more additional lipids and/or other components such
as cholesterol. Other lipids may be included in the liposome
compositions of the present invention for a variety of purposes,
such as to prevent lipid oxidation or to attach ligands onto the
liposome surface. Any of a number of lipids may be present in
liposomes of the present invention, including amphipathic, neutral,
cationic, and anionic lipids. Such lipids can be used alone or in
combination. Specific examples of additional lipid components that
may be present are described below.
[0137] Additional components that may be present in a lipid
particle of the present invention include bilayer stabilizing
components such as polyamide oligomers (see, e.g., U.S. Pat. No.
6,320,017), peptides, proteins, detergents, lipid-derivatives, such
as PEG coupled to phosphatidylethanolamine and PEG conjugated to
ceramides (see, U.S. Pat. No. 5,885,613).
[0138] In particular embodiments, the lipid particles include one
or more of a second amino lipid or cationic lipid, a neutral lipid,
a sterol, and a lipid selected to reduce aggregation of lipid
particles during formation, which may result from steric
stabilization of particles which prevents charge-induced
aggregation during formation.
[0139] Examples of lipids that reduce aggregation of particles
during formation include polyethylene glycol (PEG)-modified lipids,
monosialoganglioside Gm1, and polyamide oligomers ("PAO") such as
(described in U.S. Pat. No. 6,320,017). Other compounds with
uncharged, hydrophilic, steric-barrier moieties, which prevent
aggregation during formulation, like PEG, Gm1 or ATTA, can also be
coupled to lipids for use as in the methods and compositions of the
invention. ATTA-lipids are described, e.g., in U.S. Pat. No.
6,320,017, and PEG-lipid conjugates are described, e.g., in U.S.
Pat. Nos. 5,820,873, 5,534,499 and 5,885,613. Typically, the
concentration of the lipid component selected to reduce aggregation
is about 1 to 15% (by mole percent of lipids).
[0140] Specific examples of PEG-modified lipids (or
lipid-polyoxyethylene conjugates) that are useful in the present
invention can have a variety of "anchoring" lipid portions to
secure the PEG portion to the surface of the lipid vesicle.
Examples of suitable PEG-modified lipids include PEG-modified
phosphatidylethanolamine and phosphatidic acid, PEG-ceramide
conjugates (e.g., PEG-CerC14 or PEG-CerC20) which are described in
.omega.-pending U.S. Ser. No. 08/486,214, incorporated herein by
reference, PEG-modified dialkylamines and PEG-modified
1,2-diacyloxypropan-3-amines. Particularly preferred are
PEG-modified diacylglycerols and dialkylglycerols.
[0141] In embodiments where a sterically-large moiety such as PEG
or ATTA are conjugated to a lipid anchor, the selection of the
lipid anchor depends on what type of association the conjugate is
to have with the lipid particle. It is well known that mPEG
(mw2000)-diastearoylphosphatidylethanolamine (PEG-DSPE) will remain
associated with a liposome until the particle is cleared from the
circulation, possibly a matter of days. Other conjugates, such as
PEG-CerC20 have similar staying capacity. PEG-CerC14, however,
rapidly exchanges out of the formulation upon exposure to serum,
with a T.sub.1/2 less than 60 mins. in some assays. As illustrated
in U.S. patent application Ser. No. 08/486,214, at least three
characteristics influence the rate of exchange: length of acyl
chain, saturation of acyl chain, and size of the steric-barrier
head group. Compounds having suitable variations of these features
may be useful for the invention. For some therapeutic applications
it may be preferable for the PEG-modified lipid to be rapidly lost
from the nucleic acid-lipid particle in vivo and hence the
PEG-modified lipid will possess relatively short lipid anchors. In
other therapeutic applications it may be preferable for the nucleic
acid-lipid particle to exhibit a longer plasma circulation lifetime
and hence the PEG-modified lipid will possess relatively longer
lipid anchors.
[0142] It should be noted that aggregation preventing compounds do
not necessarily require lipid conjugation to function properly.
Free PEG or free ATTA in solution may be sufficient to prevent
aggregation. If the particles are stable after formulation, the PEG
or ATTA can be dialyzed away before administration to a
subject.
[0143] Neutral lipids, when present in the lipid particle, can be
any of a number of lipid species which exist either in an uncharged
or neutral zwitterionic form at physiological pH. Such lipids
include, for example diacylphosphatidylcholine,
diacylphosphatidylethanolamine, ceramide, sphingomyelin,
dihydrosphingomyelin, cephalin, and cerebrosides. The selection of
neutral lipids for use in the particles described herein is
generally guided by consideration of, e.g., liposome size and
stability of the liposomes in the bloodstream. Preferably, the
neutral lipid component is a lipid having two acyl groups, (i.e.,
diacylphosphatidylcholine and diacylphosphatidylethanolamine).
Lipids having a variety of acyl chain groups of varying chain
length and degree of saturation are available or may be isolated or
synthesized by well-known techniques. In one group of embodiments,
lipids containing saturated fatty acids with carbon chain lengths
in the range of C.sub.10 to C.sub.20 are preferred. In another
group of embodiments, lipids with mono or diunsaturated fatty acids
with carbon chain lengths in the range of C.sub.10 to C.sub.20 are
used. Additionally, lipids having mixtures of saturated and
unsaturated fatty acid chains can be used. Preferably, the neutral
lipids used in the present invention are DOPE, DSPC, POPC, DPPC or
any related phosphatidylcholine. The neutral lipids useful in the
present invention may also be composed of sphingomyelin,
dihydrosphingomyeline, or phospholipids with other head groups,
such as serine and inositol.
[0144] The sterol component of the lipid mixture, when present, can
be any of those sterols conventionally used in the field of
liposome, lipid vesicle or lipid particle preparation. A preferred
sterol is cholesterol.
[0145] Other cationic lipids, which carry a net positive charge at
about physiological pH, in addition to those specifically described
above, may also be included in lipid particles of the present
invention. Such cationic lipids include, but are not limited to,
N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC");
N-(2,3-dioleyloxy)propyl-N,N--N-triethylammonium chloride
("DOTMA"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB");
N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
("DOTAP"); 1,2-Dioleyloxy-3-trimethylaminopropane chloride salt
("DOTAP.C1");
3-(N--(N',N'-dimethylaminoethane)-carbamoyl)cholesterol
("DC-Chol"),
N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl-
ammonium trifluoracetate ("DOSPA"), dioctadecylamidoglycyl
carboxyspermine ("DOGS"), 1,2-dileoyl-sn-3-phosphoethanolamine
("DOPE"), 1,2-dioleoyl-3-dimethylammonium propane ("DODAP"), N,
N-dimethyl-2,3-dioleyloxy)propylamine ("DODMA"), and
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide ("DMRIE"). Additionally, a number of commercial
preparations of cationic lipids can be used, such as, e.g.,
LIPOFECTIN (including DOTMA and DOPE, available from GIBCO/BRL),
and LIPOFECTAMINE (comprising DOSPA and DOPE, available from
GIBCO/BRL). In particular embodiments, a cationic lipid is an amino
lipid.
[0146] Anionic lipids suitable for use in lipid particles of the
present invention include, but are not limited to,
phosphatidylglycerol, cardiolipin, diacylphosphatidylserine,
diacylphosphatidic acid, N-dodecanoyl phosphatidylethanoloamine,
N-succinyl phosphatidylethanolamine, N-glutaryl
phosphatidylethanolamine, lysylphosphatidylglycerol, and other
anionic modifying groups joined to neutral lipids.
[0147] In numerous embodiments, amphipathic lipids are included in
lipid particles of the present invention. "Amphipathic lipids"
refer to any suitable material, wherein the hydrophobic portion of
the lipid material orients into a hydrophobic phase, while the
hydrophilic portion orients toward the aqueous phase. Such
compounds include, but are not limited to, phospholipids,
aminolipids, and sphingolipids. Representative phospholipids
include sphingomyelin, phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,
phosphatidic acid, palmitoyloleoyl phosphatdylcholine,
lysophosphatidylcholine, lysophosphatidylethanolamine,
dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,
distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine.
Other phosphorus-lacking compounds, such as sphingolipids,
glycosphingolipid families, diacylglycerols, and
.delta.-acyloxyacids, can also be used. Additionally, such
amphipathic lipids can be readily mixed with other lipids, such as
triglycerides and sterols.
[0148] Also suitable for inclusion in the lipid particles of the
present invention are programmable fusion lipids. Such lipid
particles have little tendency to fuse with cell membranes and
deliver their payload until a given signal event occurs. This
allows the lipid particle to distribute more evenly after injection
into an organism or disease site before it starts fusing with
cells. The signal event can be, for example, a change in pH,
temperature, ionic environment, or time. In the latter case, a
fusion delaying or "cloaking" component, such as an ATTA-lipid
conjugate or a PEG-lipid conjugate, can simply exchange out of the
lipid particle membrane over time. By the time the lipid particle
is suitably distributed in the body, it has lost sufficient
cloaking agent so as to be fusogenic. With other signal events, it
is desirable to choose a signal that is associated with the disease
site or target cell, such as increased temperature at a site of
inflammation.
[0149] In certain embodiments, it is desirable to target the lipid
particles of this invention using targeting moieties that are
specific to a cell type or tissue. Targeting of lipid particles
using a variety of targeting moieties, such as ligands, cell
surface receptors, glycoproteins, vitamins (e.g., riboflavin) and
monoclonal antibodies, has been previously described (see, e.g.,
U.S. Pat. Nos. 4,957,773 and 4,603,044). The targeting moieties can
comprise the entire protein or fragments thereof. Targeting
mechanisms generally require that the targeting agents be
positioned on the surface of the lipid particle in such a manner
that the target moiety is available for interaction with the
target, for example, a cell surface receptor. A variety of
different targeting agents and methods are known and available in
the art, including those described, e.g., in Sapra, P. and Allen, T
M, Prog. Lipid Res. 42(5):439-62 (2003); and Abra, R M et al., J.
Liposome Res. 12:1-3, (2002).
[0150] The use of lipid particles, i.e., liposomes, with a surface
coating of hydrophilic polymer chains, such as polyethylene glycol
(PEG) chains, for targeting has been proposed (Allen, et al.,
Biochimica et Biophysica Acta 1237: 99-108 (1995); DeFrees, et al.,
Journal of the American Chemistry Society 118: 6101-6104 (1996);
Blume, et al., Biochimica et Biophysica Acta 1149: 180-184 (1993);
Klibanov, et al., Journal of Liposome Research 2: 321-334 (1992);
U.S. Pat. No. 5,013,556; Zalipsky, Bioconjugate Chemistry 4:
296-299 (1993); Zalipsky, FEBS Letters 353: 71-74 (1994); Zalipsky,
in Stealth Liposomes Chapter 9 (Lasic and Martin, Eds) CRC Press,
Boca Raton Fla. (1995). In one approach, a ligand, such as an
antibody, for targeting the lipid particle is linked to the polar
head group of lipids forming the lipid particle. In another
approach, the targeting ligand is attached to the distal ends of
the PEG chains forming the hydrophilic polymer coating (Klibanov,
et al., Journal of Liposome Research 2: 321-334 (1992); Kirpotin et
al., FEBS Letters 388: 115-118 (1996)).
[0151] Standard methods for coupling the target agents can be used.
For example, phosphatidylethanolamine, which can be activated for
attachment of target agents, or derivatized lipophilic compounds,
such as lipid-derivatized bleomycin, can be used. Antibody-targeted
liposomes can be constructed using, for instance, liposomes that
incorporate protein A (see, Renneisen, et al., J. Bio. Chem.,
265:16337-16342 (1990) and Leonetti, et al., Proc. Natl. Acad. Sci.
(USA), 87:2448-2451 (1990). Other examples of antibody conjugation
are disclosed in U.S. Pat. No. 6,027,726, the teachings of which
are incorporated herein by reference. Examples of targeting
moieties can also include other proteins, specific to cellular
components, including antigens associated with neoplasms or tumors.
Proteins used as targeting moieties can be attached to the
liposomes via covalent bonds (see, Heath, Covalent Attachment of
Proteins to Liposomes, 149 Methods in Enzymology 111-119 (Academic
Press, Inc. 1987)). Other targeting methods include the
biotin-avidin system.
[0152] In one exemplary embodiment, the lipid particle comprises a
mixture of a cationic lipid of the present invention, neutral
lipids (other than a cationic lipid), a sterol (e.g., cholesterol)
and a PEG-modified lipid (e.g., a PEG-DMG or PEG-DMA). In certain
embodiments, the lipid mixture consists of or consists essentially
of a cationic lipid of the present invention, a neutral lipid,
cholesterol, and a PEG-modified lipid. In further preferred
embodiments, the lipid particle consists of or consists essentially
of the above lipid mixture in molar ratios of about 20-70% amino
lipid:5-45% neutral lipid:20-55% cholesterol:0.5-15% PEG-modified
lipid.
[0153] In one embodiment, the lipid particle comprises at least two
lipids disclosed herein. For example, a mixture of cationic lipids
can be used in a lipid particle, such that the mixture comprises
20-60% of the total lipid content on a molar basis.
[0154] In particular embodiments, the lipid particle consists of or
consists essentially of a cationic lipid chosen from Table 1, DSPC,
Chol, and either PEG-DMG or PEG-DMA, e.g., in a molar ratio of
about 20-60% cationic lipid:5-25% DSPC:25-55% Chol:0.5-15% PEG-DMG
or PEG-DMA. In particular embodiments, the molar lipid ratio is
approximately 40/10/40/10 (mol % cationic lipid/DSPC/Chol/PEG-DMG
or PEG-DMA), 35/15/40/10 (mol % cationic lipid/DSPC/Chol/PEG-DMG or
PEG-DMA) or 52/13/30/5 (mol % cationic lipid/DSPC/Chol/PEG-DMG or
PEG-DMA). In another group of embodiments, the neutral lipid, DSPC,
in these compositions is replaced with POPC, DPPC, DOPE or SM.
Therapeutic Agent-Lipid Particle Compositions and Formulations
[0155] The present invention includes compositions comprising a
lipid particle of the present invention and an active agent,
wherein the active agent is associated with the lipid particle. In
particular embodiments, the active agent is a therapeutic agent. In
particular embodiments, the active agent is encapsulated within an
aqueous interior of the lipid particle. In other embodiments, the
active agent is present within one or more lipid layers of the
lipid particle. In other embodiments, the active agent is bound to
the exterior or interior lipid surface of a lipid particle.
[0156] "Fully encapsulated" as used herein indicates that the
nucleic acid in the particles is not significantly degraded after
exposure to serum or a nuclease assay that would significantly
degrade free nucleic acids. In a fully encapsulated system,
preferably less than 25% of particle nucleic acid is degraded in a
treatment that would normally degrade 100% of free nucleic acid,
more preferably less than 10% and most preferably less than 5% of
the particle nucleic acid is degraded. Alternatively, full
encapsulation may be determined by an Oligreen.RTM. assay.
Oligreen.RTM. is an ultra-sensitive fluorescent nucleic acid stain
for quantitating oligonucleotides and single-stranded DNA in
solution (available from Invitrogen Corporation, Carlsbad, Calif.).
Fully encapsulated also suggests that the particles are serum
stable, that is, that they do not rapidly decompose into their
component parts upon in vivo administration.
[0157] Active agents, as used herein, include any molecule or
compound capable of exerting a desired effect on a cell, tissue,
organ, or subject. Such effects may be biological, physiological,
or cosmetic, for example. Active agents may be any type of molecule
or compound, including e.g., nucleic acids, peptides and
polypeptides, including, e.g., antibodies, such as, e.g.,
polyclonal antibodies, monoclonal antibodies, antibody fragments;
humanized antibodies, recombinant antibodies, recombinant human
antibodies, and Primatized.TM. antibodies, cytokines, growth
factors, apoptotic factors, differentiation-inducing factors, cell
surface receptors and their ligands; hormones; and small molecules,
including small organic molecules or compounds.
[0158] In one embodiment, the active agent is a therapeutic agent,
or a salt or derivative thereof. Therapeutic agent derivatives may
be therapeutically active themselves or they may be prodrugs, which
become active upon further modification. Thus, in one embodiment, a
therapeutic agent derivative retains some or all of the therapeutic
activity as compared to the unmodified agent, while in another
embodiment, a therapeutic agent derivative lacks therapeutic
activity.
[0159] In various embodiments, therapeutic agents include any
therapeutically effective agent or drug, such as anti-inflammatory
compounds, anti-depressants, stimulants, analgesics, antibiotics,
birth control medication, antipyretics, vasodilators,
anti-angiogenics, cytovascular agents, signal transduction
inhibitors, cardiovascular drugs, e.g., anti-arrhythmic agents,
vasoconstrictors, hormones, and steroids.
[0160] In certain embodiments, the therapeutic agent is an oncology
drug, which may also be referred to as an anti-tumor drug, an
anti-cancer drug, a tumor drug, an antineoplastic agent, or the
like. Examples of oncology drugs that may be used according to the
invention include, but are not limited to, adriamycin, alkeran,
allopurinol, altretamine, amifostine, anastrozole, araC, arsenic
trioxide, azathioprine, bexarotene, biCNU, bleomycin, busulfan
intravenous, busulfan oral, capecitabine (Xeloda), carboplatin,
carmustine, CCNU, celecoxib, chlorambucil, cisplatin, cladribine,
cyclosporin A, cytarabine, cytosine arabinoside, daunorubicin,
cytoxan, daunorubicin, dexamethasone, dexrazoxane, dodetaxel,
doxorubicin, doxorubicin, DTIC, epirubicin, estramustine, etoposide
phosphate, etoposide and VP-16, exemestane, FK506, fludarabine,
fluorouracil, 5-FU, gemcitabine (Gemzar), gemtuzumab-ozogamicin,
goserelin acetate, hydrea, hydroxyurea, idarubicin, ifosfamide,
imatinib mesylate, interferon, irinotecan (Camptostar, CPT-111),
letrozole, leucovorin, leustatin, leuprolide, levamisole,
litretinoin, megastrol, melphalan, L-PAM, mesna, methotrexate,
methoxsalen, mithramycin, mitomycin, mitoxantrone, nitrogen
mustard, paclitaxel, pamidronate, Pegademase, pentostatin, porfimer
sodium, prednisone, rituxan, streptozocin, STI-571, tamoxifen,
taxotere, temozolamide, teniposide, VM-26, topotecan (Hycamtin),
toremifene, tretinoin, ATRA, valrubicin, velban, vinblastine,
vincristine, VP16, and vinorelbine. Other examples of oncology
drugs that may be used according to the invention are ellipticin
and ellipticin analogs or derivatives, epothilones, intracellular
kinase inhibitors and camptothecins.
Nucleic Acid-Lipid Particles
[0161] In certain embodiments, lipid particles of the present
invention are associated with a nucleic acid, resulting in a
nucleic acid-lipid particle. In particular embodiments, the nucleic
acid is fully encapsulated in the lipid particle. As used herein,
the term "nucleic acid" is meant to include any oligonucleotide or
polynucleotide. Fragments containing up to 50 nucleotides are
generally termed oligonucleotides, and longer fragments are called
polynucleotides. In particular embodiments, oligonucletoides of the
present invention are 15-50 nucleotides in length.
[0162] In the context of this invention, the terms "polynucleotide"
and "oligonucleotide" refer to a polymer or oligomer of nucleotide
or nucleoside monomers consisting of naturally occurring bases,
sugars and intersugar (backbone) linkages. The terms
"polynucleotide" and "oligonucleotide" also includes polymers or
oligomers comprising non-naturally occurring monomers, or portions
thereof, which function similarly. Such modified or substituted
oligonucleotides are often preferred over native forms because of
properties such as, for example, enhanced cellular uptake and
increased stability in the presence of nucleases.
[0163] The nucleic acid that is present in a lipid-nucleic acid
particle according to this invention includes any form of nucleic
acid that is known. The nucleic acids used herein can be
single-stranded DNA or RNA, or double-stranded DNA or RNA, or
DNA-RNA hybrids. Examples of double-stranded DNA include structural
genes, genes including control and termination regions, and
self-replicating systems such as viral or plasmid DNA. Examples of
double-stranded RNA include siRNA and other RNA interference
reagents. Single-stranded nucleic acids include, e.g., antisense
oligonucleotides, ribozymes, microRNA, and triplex-forming
oligonucleotides. The nucleic acid that is present in a
lipid-nucleic acid particle of this invention may include one or
more of the oligonucleotide modifications described below.
[0164] Nucleic acids of the present invention may be of various
lengths, generally dependent upon the particular form of nucleic
acid. For example, in particular embodiments, plasmids or genes may
be from about 1,000 to 100,000 nucleotide residues in length. In
particular embodiments, oligonucleotides may range from about 10 to
100 nucleotides in length. In various related embodiments,
oligonucleotides, single-stranded, double-stranded, and
triple-stranded, may range in length from about 10 to about 50
nucleotides, from about 20 o about 50 nucleotides, from about 15 to
about 30 nucleotides, from about 20 to about 30 nucleotides in
length.
[0165] In particular embodiments, the oligonucleotide (or a strand
thereof) of the present invention specifically hybridizes to or is
complementary to a target polynucleotide. "Specifically
hybridizable" and "complementary" are terms which are used to
indicate a sufficient degree of complementarity such that stable
and specific binding occurs between the DNA or RNA target and the
oligonucleotide. It is understood that an oligonucleotide need not
be 100% complementary to its target nucleic acid sequence to be
specifically hybridizable. An oligonucleotide is specifically
hybridizable when binding of the oligonucleotide to the target
interferes with the normal function of the target molecule to cause
a loss of utility or expression therefrom, and there is a
sufficient degree of complementarity to avoid non-specific binding
of the oligonucleotide to non-target sequences under conditions in
which specific binding is desired, i.e., under physiological
conditions in the case of in vivo assays or therapeutic treatment,
or, in the case of in vitro assays, under conditions in which the
assays are conducted. Thus, in other embodiments, this
oligonucleotide includes 1, 2, or 3 base substitutions, e.g.
mismatches, as compared to the region of a gene or mRNA sequence
that it is targeting or to which it specifically hybridizes.
RNA Interference Nucleic Acids
[0166] In particular embodiments, nucleic acid-lipid particles of
the present invention are associated with RNA interference (RNAi)
molecules. RNA interference methods using RNAi molecules may be
used to disrupt the expression of a gene or polynucleotide of
interest. Small interfering RNA (siRNA) has essentially replaced
antisense ODN and ribozymes as the next generation of targeted
oligonucleotide drugs under development.
[0167] SiRNAs are RNA duplexes normally 16-30 nucleotides long that
can associate with a cytoplasmic multi-protein complex known as
RNAi-induced silencing complex (RISC). RISC loaded with siRNA
mediates the degradation of homologous mRNA transcripts, therefore
siRNA can be designed to knock down protein expression with high
specificity. Unlike other antisense technologies, siRNA function
through a natural mechanism evolved to control gene expression
through non-coding RNA. This is generally considered to be the
reason why their activity is more potent in vitro and in vivo than
either antisense ODN or ribozymes. A variety of RNAi reagents,
including siRNAs targeting clinically relevant targets, are
currently under pharmaceutical development, as described, e.g., in
de Fougerolles, A. et al., Nature Reviews 6:443-453 (2007).
[0168] While the first described RNAi molecules were RNA:RNA
hybrids comprising both an RNA sense and an RNA antisense strand,
it has now been demonstrated that DNA sense:RNA antisense hybrids,
RNA sense:DNA antisense hybrids, and DNA:DNA hybrids are capable of
mediating RNAi (Lamberton, J. S. and Christian, A. T., (2003)
Molecular Biotechnology 24:111-119). Thus, the invention includes
the use of RNAi molecules comprising any of these different types
of double-stranded molecules. In addition, it is understood that
RNAi molecules may be used and introduced to cells in a variety of
forms. Accordingly, as used herein, RNAi molecules encompasses any
and all molecules capable of inducing an RNAi response in cells,
including, but not limited to, double-stranded oligonucleotides
comprising two separate strands, i.e. a sense strand and an
antisense strand, e.g., small interfering RNA (siRNA);
double-stranded oligonucleotide comprising two separate strands
that are linked together by non-nucleotidyl linker;
oligonucleotides comprising a hairpin loop of complementary
sequences, which forms a double-stranded region, e.g., shRNAi
molecules, and expression vectors that express one or more
polynucleotides capable of forming a double-stranded polynucleotide
alone or in combination with another polynucleotide.
[0169] A "single strand siRNA compound" as used herein, is an siRNA
compound which is made up of a single molecule. It may include a
duplexed region, formed by intra-strand pairing, e.g., it may be,
or include, a hairpin or pan-handle structure. Single strand siRNA
compounds may be antisense with regard to the target molecule.
[0170] A single strand siRNA compound may be sufficiently long that
it can enter the RISC and participate in RISC mediated cleavage of
a target mRNA. A single strand siRNA compound is at least 14, and
in other embodiments at least 15, 20, 25, 29, 35, 40, or 50
nucleotides in length. In certain embodiments, it is less than 200,
100, or 60 nucleotides in length.
[0171] Hairpin siRNA compounds will have a duplex region equal to
or at least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs.
The duplex region will may be equal to or less than 200, 100, or
50, in length. In certain embodiments, ranges for the duplex region
are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in
length. The hairpin may have a single strand overhang or terminal
unpaired region. In certain embodiments, the overhangs are 2-3
nucleotides in length. In some embodiments, the overhang is at the
sense side of the hairpin and in some embodiments on the antisense
side of the hairpin.
[0172] A "double stranded siRNA compound" as used herein, is an
siRNA compound which includes more than one, and in some cases two,
strands in which interchain hybridization can form a region of
duplex structure.
[0173] The antisense strand of a double stranded siRNA compound may
be equal to or at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 60
nucleotides in length. It may be equal to or less than 200, 100, or
50, nucleotides in length. Ranges may be 17 to 25, 19 to 23, and 19
to21 nucleotides in length. As used herein, term "antisense strand"
means the strand of an siRNA compound that is sufficiently
complementary to a target molecule, e.g. a target RNA.
[0174] The sense strand of a double stranded siRNA compound may be
equal to or at least 14, 15, 16 17, 18, 19, 25, 29, 40, or 60
nucleotides in length. It may be equal to or less than 200, 100, or
50, nucleotides in length. Ranges may be 17 to 25, 19 to 23, and 19
to 21 nucleotides in length.
[0175] The double strand portion of a double stranded siRNA
compound may be equal to or at least, 14, 15, 16 17, 18, 19, 20,
21, 22, 23, 24, 25, 29, 40, or 60 nucleotide pairs in length. It
may be equal to or less than 200, 100, or 50, nucleotides pairs in
length. Ranges may be 15-30, 17 to 23, 19 to 23, and 19 to 21
nucleotides pairs in length.
[0176] In many embodiments, the siRNA compound is sufficiently
large that it can be cleaved by an endogenous molecule, e.g., by
Dicer, to produce smaller siRNA compounds, e.g., siRNAs agents.
[0177] The sense and antisense strands may be chosen such that the
double-stranded siRNA compound includes a single strand or unpaired
region at one or both ends of the molecule. Thus, a double-stranded
siRNA compound may contain sense and antisense strands, paired to
contain an overhang, e.g., one or two 5' or 3' overhangs, or a 3'
overhang of 1-3 nucleotides. The overhangs can be the result of one
strand being longer than the other, or the result of two strands of
the same length being staggered. Some embodiments will have at
least one 3' overhang. In one embodiment, both ends of an siRNA
molecule will have a 3' overhang. In some embodiments, the overhang
is 2 nucleotides.
[0178] In certain embodiments, the length for the duplexed region
is between 15 and 30, or 18, 19, 20, 21, 22, and 23 nucleotides in
length, e.g., in the ssiRNA compound range discussed above. ssiRNA
compounds can resemble in length and structure the natural Dicer
processed products from long dsiRNAs. Embodiments in which the two
strands of the ssiRNA compound are linked, e.g., covalently linked
are also included. Hairpin, or other single strand structures which
provide the required double stranded region, and a 3' overhang are
also within the invention.
[0179] The siRNA compounds described herein, including
double-stranded siRNA compounds and single-stranded siRNA compounds
can mediate silencing of a target RNA, e.g., mRNA, e.g., a
transcript of a gene that encodes a protein. For convenience, such
mRNA is also referred to herein as mRNA to be silenced. Such a gene
is also referred to as a target gene. In general, the RNA to be
silenced is an endogenous gene or a pathogen gene. In addition,
RNAs other than mRNA, e.g., tRNAs, and viral RNAs, can also be
targeted.
[0180] As used herein, the phrase "mediates RNAi" refers to the
ability to silence, in a sequence specific manner, a target RNA.
While not wishing to be bound by theory, it is believed that
silencing uses the RNAi machinery or process and a guide RNA, e.g.,
an ssiRNA compound of 21 to 23 nucleotides.
[0181] In one embodiment, an siRNA compound is "sufficiently
complementary" to a target RNA, e.g., a target mRNA, such that the
siRNA compound silences production of protein encoded by the target
mRNA. In another embodiment, the siRNA compound is "exactly
complementary" to a target RNA, e.g., the target RNA and the siRNA
compound anneal, for example to form a hybrid made exclusively of
Watson-Crick base pairs in the region of exact complementarity. A
"sufficiently complementary" target RNA can include an internal
region (e.g., of at least 10 nucleotides) that is exactly
complementary to a target RNA. Moreover, in certain embodiments,
the siRNA compound specifically discriminates a single-nucleotide
difference. In this case, the siRNA compound only mediates RNAi if
exact complementary is found in the region (e.g., within 7
nucleotides of) the single-nucleotide difference.
MicroRNAs
[0182] Micro RNAs (miRNAs) are a highly conserved class of small
RNA molecules that are transcribed from DNA in the genomes of
plants and animals, but are not translated into protein. Processed
miRNAs are single stranded .about.17-25 nucleotide (nt) RNA
molecules that become incorporated into the RNA-induced silencing
complex (RISC) and have been identified as key regulators of
development, cell proliferation, apoptosis and differentiation.
They are believed to play a role in regulation of gene expression
by binding to the 3'-untranslated region of specific mRNAs. RISC
mediates down-regulation of gene expression through translational
inhibition, transcript cleavage, or both. RISC is also implicated
in transcriptional silencing in the nucleus of a wide range of
eukaryotes.
[0183] The number of miRNA sequences identified to date is large
and growing, illustrative examples of which can be found, for
example, in: "miRBase: microRNA sequences, targets and gene
nomenclature" Griffiths-Jones S, Grocock R J, van Dongen S, Bateman
A, Enright A J. NAR, 2006, 34, Database Issue, D140-D144; "The
microRNA Registry" Griffiths-Jones S. NAR, 2004, 32, Database
Issue, D109-D111; and also at
http://microrna.sanger.ac.uk/sequences/.
Antisense Oligonucleotides
[0184] In one embodiment, a nucleic acid is an antisense
oligonucleotide directed to a target polynucleotide. The term
"antisense oligonucleotide" or simply "antisense" is meant to
include oligonucleotides that are complementary to a targeted
polynucleotide sequence. Antisense oligonucleotides are single
strands of DNA or RNA that are complementary to a chosen sequence,
e.g. a target gene mRNA. Antisense oligonucleotides are thought to
inhibit gene expression by binding to a complementary mRNA. Binding
to the target mRNA can lead to inhibition of gene expression by
either preventing translation of complementary mRNA strands by
binding to it or by leading to degradation of the target mRNA.
Antisense DNA can be used to target a specific, complementary
(coding or non-coding) RNA. If binding takes places this DNA/RNA
hybrid can be degraded by the enzyme RNase H. In particular
embodiment, antisense oligonucleotides contain from about 10 to
about 50 nucleotides, more preferably about 15 to about 30
nucleotides. The term also encompasses antisense oligonucleotides
that may not be exactly complementary to the desired target gene.
Thus, the invention can be utilized in instances where non-target
specific-activities are found with antisense, or where an antisense
sequence containing one or more mismatches with the target sequence
is the most preferred for a particular use.
[0185] Antisense oligonucleotides have been demonstrated to be
effective and targeted inhibitors of protein synthesis, and,
consequently, can be used to specifically inhibit protein synthesis
by a targeted gene. The efficacy of antisense oligonucleotides for
inhibiting protein synthesis is well established. For example, the
synthesis of polygalactauronase and the muscarine type 2
acetylcholine receptor are inhibited by antisense oligonucleotides
directed to their respective mRNA sequences (U.S. Pat. Nos.
5,739,119 and 5,759,829). Further, examples of antisense inhibition
have been demonstrated with the nuclear protein cyclin, the
multiple drug resistance gene (MDG1), ICAM-1, E-selectin, STK-1,
striatal GABA.sub.A receptor and human EGF (Jaskulski et al.,
Science. 1988 Jun. 10; 240(4858):1544-6; Vasanthakumar and Ahmed,
Cancer Commun. 1989; 1(4):225-32; Penis et al., Brain Res Mol Brain
Res. 1998 Jun. 15; 57(2):310-20; U.S. Pat. Nos. 5,801,154;
5,789,573; 5,718,709 and 5,610,288). Furthermore, antisense
constructs have also been described that inhibit and can be used to
treat a variety of abnormal cellular proliferations, e.g. cancer
(U.S. Pat. Nos. 5,747,470; 5,591,317 and 5,783,683).
[0186] Methods of producing antisense oligonucleotides are known in
the art and can be readily adapted to produce an antisense
oligonucleotide that targets any polynucleotide sequence. Selection
of antisense oligonucleotide sequences specific for a given target
sequence is based upon analysis of the chosen target sequence and
determination of secondary structure, T.sub.m, binding energy, and
relative stability. Antisense oligonucleotides may be selected
based upon their relative inability to form dimers, hairpins, or
other secondary structures that would reduce or prohibit specific
binding to the target mRNA in a host cell. Highly preferred target
regions of the mRNA include those regions at or near the AUG
translation initiation codon and those sequences that are
substantially complementary to 5' regions of the mRNA. These
secondary structure analyses and target site selection
considerations can be performed, for example, using v.4 of the
OLIGO primer analysis software (Molecular Biology Insights) and/or
the BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids
Res. 1997, 25(17):3389-402).
Antagomirs
[0187] Antagomirs are RNA-like oligonucleotides that harbor various
modifications for RNAse protection and pharmacologic properties,
such as enhanced tissue and cellular uptake. They differ from
normal RNA by, for example, complete 2'-O-methylation of sugar,
phosphorothioate backbone and, for example, a cholesterol-moiety at
3'-end. Antagomirs may be used to efficiently silence endogenous
miRNAs by forming duplexes comprising the antagomir and endogenous
miRNA, thereby preventing miRNA-induced gene silencing. An example
of antagomir-mediated miRNA silencing is the silencing of miR-122,
described in Krutzfeldt et al, Nature, 2005, 438: 685-689, which is
expressly incorporated by reference herein in its entirety.
Antagomir RNAs may be synthesized using standard solid phase
oligonucleotide synthesis protocols. See U.S. patent application
Ser. Nos. 11/502,158 and 11/657,341 (the disclosure of each of
which are incorporated herein by reference).
[0188] An antagomir can include ligand-conjugated monomer subunits
and monomers for oligonucleotide synthesis. Exemplary monomers are
described in U.S. application Ser. No. 10/916,185, filed on Aug.
10, 2004. An antagomir can have a ZXY structure, such as is
described in PCT Application No. PCT/US2004/07070 filed on Mar. 8,
2004. An antagomir can be complexed with an amphipathic moiety.
Exemplary amphipathic moieties for use with oligonucleotide agents
are described in PCT Application No. PCT/US2004/07070, filed on
Mar. 8, 2004.
Aptamers
[0189] Aptamers are nucleic acid or peptide molecules that bind to
a particular molecule of interest with high affinity and
specificity (Tuerk and Gold, Science 249:505 (1990); Ellington and
Szostak, Nature 346:818 (1990)). DNA or RNA aptamers have been
successfully produced which bind many different entities from large
proteins to small organic molecules. See Eaton, Curr. Opin. Chem.
Biol. 1:10-16 (1997), Famulok, Curr. Opin. Struct. Biol.
9:324-9(1999), and Hermann and Patel, Science 287:820-5 (2000).
Aptamers may be RNA or DNA based, and may include a riboswitch. A
riboswitch is a part of an mRNA molecule that can directly bind a
small target molecule, and whose binding of the target affects the
gene's activity. Thus, an mRNA that contains a riboswitch is
directly involved in regulating its own activity, depending on the
presence or absence of its target molecule. Generally, aptamers are
engineered through repeated rounds of in vitro selection or
equivalently, SELEX (systematic evolution of ligands by exponential
enrichment) to bind to various molecular targets such as small
molecules, proteins, nucleic acids, and even cells, tissues and
organisms. The aptamer may be prepared by any known method,
including synthetic, recombinant, and purification methods, and may
be used alone or in combination with other aptamers specific for
the same target. Further, as described more fully herein, the term
"aptamer" specifically includes "secondary aptamers" containing a
consensus sequence derived from comparing two or more known
aptamers to a given target.
Ribozymes
[0190] According to another embodiment of the invention, nucleic
acid-lipid particles are associated with ribozymes. Ribozymes are
RNA molecules complexes having specific catalytic domains that
possess endonuclease activity (Kim and Cech, Proc Natl Acad Sci
USA. 1987 December; 84(24):8788-92; Forster and Symons, Cell. 1987
Apr. 24; 49(2):211-20). For example, a large number of ribozymes
accelerate phosphoester transfer reactions with a high degree of
specificity, often cleaving only one of several phosphoesters in an
oligonucleotide substrate (Cech et al., Cell. 1981 December; 27(3
Pt 2):487-96; Michel and Westhof, J Mol Biol. 1990 Dec. 5;
216(3):585-610; Reinhold-Hurek and Shub, Nature. 1992 May 14;
357(6374):173-6). This specificity has been attributed to the
requirement that the substrate bind via specific base-pairing
interactions to the internal guide sequence ("IGS") of the ribozyme
prior to chemical reaction.
[0191] At least six basic varieties of naturally-occurring
enzymatic RNAs are known presently. Each can catalyze the
hydrolysis of RNA phosphodiester bonds in trans (and thus can
cleave other RNA molecules) under physiological conditions. In
general, enzymatic nucleic acids act by first binding to a target
RNA. Such binding occurs through the target binding portion of an
enzymatic nucleic acid which is held in close proximity to an
enzymatic portion of the molecule that acts to cleave the target
RNA. Thus, the enzymatic nucleic acid first recognizes and then
binds a target RNA through complementary base-pairing, and once
bound to the correct site, acts enzymatically to cut the target
RNA. Strategic cleavage of such a target RNA will destroy its
ability to direct synthesis of an encoded protein. After an
enzymatic nucleic acid has bound and cleaved its RNA target, it is
released from that RNA to search for another target and can
repeatedly bind and cleave new targets.
[0192] The enzymatic nucleic acid molecule may be formed in a
hammerhead, hairpin, a hepatitis .delta. virus, group I intron or
RNaseP RNA (in association with an RNA guide sequence) or
Neurospora VS RNA motif, for example. Specific examples of
hammerhead motifs are described by Rossi et al. Nucleic Acids Res.
1992 Sep. 11; 20(17):4559-65. Examples of hairpin motifs are
described by Hampel et al. (Eur. Pat. Appl. Publ. No. EP 0360257),
Hampel and Tritz, Biochemistry 1989 Jun. 13; 28(12):4929-33; Hampel
et al., Nucleic Acids Res. 1990 Jan. 25; 18(2):299-304 and U.S.
Pat. No. 5,631,359. An example of the hepatitis .delta. virus motif
is described by Perrotta and Been, Biochemistry. 1992 Dec. 1;
31(47):11843-52; an example of the RNaseP motif is described by
Guerrier-Takada et al., Cell. 1983 December; 35(3 Pt 2):849-57;
Neurospora VS RNA ribozyme motif is described by Collins (Saville
and Collins, Cell. 1990 May 18; 61(4):685-96; Saville and Collins,
Proc Natl Acad Sci USA. 1991 Oct. 1; 88(19):8826-30; Collins and
Olive, Biochemistry. 1993 Mar. 23; 32(11):2795-9); and an example
of the Group I intron is described in U.S. Pat. No. 4,987,071.
Important characteristics of enzymatic nucleic acid molecules used
according to the invention are that they have a specific substrate
binding site which is complementary to one or more of the target
gene DNA or RNA regions, and that they have nucleotide sequences
within or surrounding that substrate binding site which impart an
RNA cleaving activity to the molecule. Thus the ribozyme constructs
need not be limited to specific motifs mentioned herein.
[0193] Methods of producing a ribozyme targeted to any
polynucleotide sequence are known in the art. Ribozymes may be
designed as described in Int. Pat. Appl. Publ. No. WO 93/23569 and
Int. Pat. Appl. Publ. No. WO 94/02595, each specifically
incorporated herein by reference, and synthesized to be tested in
vitro and in vivo, as described therein.
[0194] Ribozyme activity can be optimized by altering the length of
the ribozyme binding arms or chemically synthesizing ribozymes with
modifications that prevent their degradation by serum ribonucleases
(see e.g., Int. Pat. Appl. Publ. No. WO 92/07065; Int. Pat. Appl.
Publ. No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur.
Pat. Appl. Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int.
Pat. Appl. Publ. No. WO 94/13688, which describe various chemical
modifications that can be made to the sugar moieties of enzymatic
RNA molecules), modifications which enhance their efficacy in
cells, and removal of stem II bases to shorten RNA synthesis times
and reduce chemical requirements.
Immunostimulatory Oligonucleotides
[0195] Nucleic acids associated with lipid particles of the present
invention may be immunostimulatory, including immunostimulatory
oligonucleotides (ISS; single- or double-stranded) capable of
inducing an immune response when administered to a subject, which
may be a mammal or other patient. ISS include, e.g., certain
palindromes leading to hairpin secondary structures (see Yamamoto
S., et al. (1992) J. Immunol. 148: 4072-4076), or CpG motifs, as
well as other known ISS features (such as multi-G domains, see WO
96/11266).
[0196] The immune response may be an innate or an adaptive immune
response. The immune system is divided into a more innate immune
system, and acquired adaptive immune system of vertebrates, the
latter of which is further divided into humoral cellular
components. In particular embodiments, the immune response may be
mucosal.
[0197] In particular embodiments, an immunostimulatory nucleic acid
is only immunostimulatory when administered in combination with a
lipid particle, and is not immunostimulatory when administered in
its "free form." According to the present invention, such an
oligonucleotide is considered to be immunostimulatory.
[0198] Immunostimulatory nucleic acids are considered to be
non-sequence specific when it is not required that they
specifically bind to and reduce the expression of a target
polynucleotide in order to provoke an immune response. Thus,
certain immunostimulatory nucleic acids may comprise a sequence
corresponding to a region of a naturally occurring gene or mRNA,
but they may still be considered non-sequence specific
immunostimulatory nucleic acids.
[0199] In one embodiment, the immunostimulatory nucleic acid or
oligonucleotide comprises at least one CpG dinucleotide. The
oligonucleotide or CpG dinucleotide may be unmethylated or
methylated. In another embodiment, the immunostimulatory nucleic
acid comprises at least one CpG dinucleotide having a methylated
cytosine. In one embodiment, the nucleic acid comprises a single
CpG dinucleotide, wherein the cytosine in said CpG dinucleotide is
methylated. In a specific embodiment, the nucleic acid comprises
the sequence 5' TAACGTTGAGGGGCAT 3'. In an alternative embodiment,
the nucleic acid comprises at least two CpG dinucleotides, wherein
at least one cytosine in the CpG dinucleotides is methylated. In a
further embodiment, each cytosine in the CpG dinucleotides present
in the sequence is methylated. In another embodiment, the nucleic
acid comprises a plurality of CpG dinucleotides, wherein at least
one of said CpG dinucleotides comprises a methylated cytosine.
[0200] In one specific embodiment, the nucleic acid comprises the
sequence 5' TTCCATGACGTTCCTGACGT 3'. In another specific
embodiment, the nucleic acid sequence comprises the sequence 5'
TCCATGACGTTCCTGACGT 3', wherein the two cytosines indicated in bold
are methylated. In particular embodiments, the ODN is selected from
a group of ODNs consisting of ODN #1, ODN #2, ODN #3, ODN #4, ODN
#5, ODN #6, ODN #7, ODN #8, and ODN #9, as shown below.
TABLE-US-00002 TABLE 3 Exemplary Immunostimulatory Oligonucleotides
(ODNs) SEQ ODN NAME ID ODN SEQUENCE (5'-3'). ODN 1
5'-TAACGTTGAGGGGCAT-3 human c-myc * ODN 1m 5'-TAAZGTTGAGGGGCAT-3
ODN 2 5'-TCCATGACGTTCCTGACGTT-3 * ODN 2m 5'-TCCATGAZGTTCCTGAZGTT-3
ODN 3 5'-TAAGCATACGGGGTGT-3 ODN 5 5'-AACGTT-3 ODN 6
5'-GATGCTGTGTCGGGGTCTCCGGGC-3' ODN 7 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'
ODN 7m 5'-TZGTZGTTTTGTZGTTTTGTZGTT-3' ODN 8
5'-TCCAGGACTTCTCTCAGGTT-3' ODN 9 5'-TCTCCCAGCGTGCGCCAT-3' ODN 10
murine 5'-TGCATCCCCCAGGCCACCAT-3 Intracellular Adhesion Molecule-1
ODN 11 human 5'-GCCCAAGCTGGCATCCGTCA-3' Intracellular Adhesion
Molecule-1 ODN 12 human 5'-GCCCAAGCTGGCATCCGTCA-3' Intracellular
Adhesion Molecule-1 ODN 13 human erb-B-2 5'-GGT GCTCACTGC GGC-3'
ODN 14 human c-myc 5'-AACC GTT GAG GGG CAT-3' ODN 15 human c-myc
5'-TAT GCT GTG CCG GGG TCT TCG GGC-3' ODN 16 5'-GTGCCG
GGGTCTTCGGGC-3' ODN 17 human Insulin 5'-GGACCCTCCTCCGGAGCC-3'
Growth Factor 1 - Receptor ODN 18 human Insulin 5'-TCC TCC GGA GCC
AGA CTT-3' Growth Factor 1 - Receptor ODN 19 human Epidermal 5'-AAC
GTT GAG GGG CAT-3' Growth Factor - Receptor ODN 20 Epidermal Growth
5'-CCGTGGTCA TGCTCC-3' Factor - Receptor ODN 21 human Vascular
5'-CAG CCTGGCTCACCG CCTTGG-3' Endothelial Growth Factor ODN 22
murine 5'-CAG CCA TGG TTC CCC CCA AC-3' Phosphokinase C - alpha ODN
23 5'-GTT CTC GCT GGT GAG TTT CA-3' ODN 24 human Bcl-2 5'-TCT
CCCAGCGTGCGCCAT-3' ODN 25 human C-Raf-s 5'-GTG CTC CAT TGA TGC-3'
ODN #26 human Vascular 5'-GAGUUCUGAUGAGGCCGAAAGG- Endothelial
Growth CCGAAAGUCUG-3' Factor Receptor-1 ODN #27 5'-RRCGYY-3' ODN
#28 5'-AACGTTGAGGGGCAT-3' ODN #29 5'-CAACGTTATGGGGAGA-3' ODN #30
human c-myc 5'-TAACGTTGAGGGGCAT-3' "Z" represents a methylated
cytosine residue. ODN 14 is a 15-mer oligonucleotide and ODN 1 is
the same oligonucleotide having a thymidine added onto the 5' end
making ODN 1 into a 16-mer. No difference in biological activity
between ODN 14 and ODN 1 has been detected and both exhibit similar
immunostimulatory activity (Mui et at., 2001)
[0201] Additional specific nucleic acid sequences of
oligonucleotides (ODNs) suitable for use in the compositions and
methods of the invention are described in Raney et al., Journal of
Pharmacology and Experimental Therapeutics, 298:1185-1192 (2001).
In certain embodiments, ODNs used in the compositions and methods
of the present invention have a phosphodiester ("PO") backbone or a
phosphorothioate ("PS") backbone, and/or at least one methylated
cytosine residue in a CpG motif.
Decoy Oligonucleotides
[0202] Because transcription factors recognize their relatively
short binding sequences, even in the absence of surrounding genomic
DNA, short oligonucleotides bearing the consensus binding sequence
of a specific transcription factor can be used as tools for
manipulating gene expression in living cells. This strategy
involves the intracellular delivery of such "decoy
oligonucleotides", which are then recognized and bound by the
target factor. Occupation of the transcription factor's DNA-binding
site by the decoy renders the transcription factor incapable of
subsequently binding to the promoter regions of target genes.
Decoys can be used as therapeutic agents, either to inhibit the
expression of genes that are activated by a transcription factor,
or to upregulate genes that are suppressed by the binding of a
transcription factor. Examples of the utilization of decoy
oligonucleotides may be found in Mann et al., J. Clin. Invest.,
2000, 106: 1071-1075, which is expressly incorporated by reference
herein, in its entirety.
Supermir
[0203] A supermir refers to a single stranded, double stranded or
partially double stranded oligomer or polymer of ribonucleic acid
(RNA) or deoxyribonucleic acid (DNA) or both or modifications
thereof, which has a nucleotide sequence that is substantially
identical to an miRNA and that is antisense with respect to its
target. This term includes oligonucleotides composed of
naturally-occurring nucleobases, sugars and covalent
internucleoside (backbone) linkages and which contain at least one
non-naturally-occurring portion which functions similarly. Such
modified or substituted oligonucleotides are preferred over native
forms because of desirable properties such as, for example,
enhanced cellular uptake, enhanced affinity for nucleic acid target
and increased stability in the presence of nucleases. In a
preferred embodiment, the supermir does not include a sense strand,
and in another preferred embodiment, the supermir does not
self-hybridize to a significant extent. An supermir featured in the
invention can have secondary structure, but it is substantially
single-stranded under physiological conditions. An supermir that is
substantially single-stranded is single-stranded to the extent that
less than about 50% (e.g., less than about 40%, 30%, 20%, 10%, or
5%) of the supermir is duplexed with itself. The supermir can
include a hairpin segment, e.g., sequence, preferably at the 3' end
can self hybridize and form a duplex region, e.g., a duplex region
of at least 1, 2, 3, or 4 and preferably less than 8, 7, 6, or n
nucleotides, e.g., 5 nucleotides. The duplexed region can be
connected by a linker, e.g., a nucleotide linker, e.g., 3, 4, 5, or
6 dTs, e.g., modified dTs. In another embodiment the supermir is
duplexed with a shorter oligo, e.g., of 5, 6, 7, 8, 9, or 10
nucleotides in length, e.g., at one or both of the 3' and 5' end or
at one end and in the non-terminal or middle of the supermir.
miRNA Mimics
[0204] miRNA mimics represent a class of molecules that can be used
to imitate the gene silencing ability of one or more miRNAs. Thus,
the term "microRNA mimic" refers to synthetic non-coding RNAs (i.e.
the miRNA is not obtained by purification from a source of the
endogenous miRNA) that are capable of entering the RNAi pathway and
regulating gene expression. miRNA mimics can be designed as mature
molecules (e.g. single stranded) or mimic precursors (e.g., pri- or
pre-miRNAs). miRNA mimics can be comprised of nucleic acid
(modified or modified nucleic acids) including oligonucleotides
comprising, without limitation, RNA, modified RNA, DNA, modified
DNA, locked nucleic acids, or 2'-0,4'-C-ethylene-bridged nucleic
acids (ENA), or any combination of the above (including DNA-RNA
hybrids). In addition, miRNA mimics can comprise conjugates that
can affect delivery, intracellular compartmentalization, stability,
specificity, functionality, strand usage, and/or potency. In one
design, miRNA mimics are double stranded molecules (e.g., with a
duplex region of between about 16 and about 31 nucleotides in
length) and contain one or more sequences that have identity with
the mature strand of a given miRNA. Modifications can comprise 2'
modifications (including 2'-O methyl modifications and 2' F
modifications) on one or both strands of the molecule and
internucleotide modifications (e.g. phosphorthioate modifications)
that enhance nucleic acid stability and/or specificity. In
addition, miRNA mimics can include overhangs. The overhangs can
consist of 1-6 nucleotides on either the 3' or 5' end of either
strand and can be modified to enhance stability or functionality.
In one embodiment, a miRNA mimic comprises a duplex region of
between 16 and 31 nucleotides and one or more of the following
chemical modification patterns: the sense strand contains
2'-O-methyl modifications of nucleotides 1 and 2 (counting from the
5' end of the sense oligonucleotide), and all of the Cs and Us; the
antisense strand modifications can comprise 2' F modification of
all of the Cs and Us, phosphorylation of the 5' end of the
oligonucleotide, and stabilized internucleotide linkages associated
with a 2 nucleotide 3' overhang.
Antimir or miRNA Inhibitor.
[0205] The terms "antimir" "microRNA inhibitor", "miR inhibitor",
or "inhibitor" are synonymous and refer to oligonucleotides or
modified oligonucleotides that interfere with the ability of
specific miRNAs. In general, the inhibitors are nucleic acid or
modified nucleic acids in nature including oligonucleotides
comprising RNA, modified RNA, DNA, modified DNA, locked nucleic
acids (LNAs), or any combination of the above. Modifications
include 2' modifications (including 2'-0 alkyl modifications and 2'
F modifications) and internucleotide modifications (e.g.
phosphorothioate modifications) that can affect delivery,
stability, specificity, intracellular compartmentalization, or
potency. In addition, miRNA inhibitors can comprise conjugates that
can affect delivery, intracellular compartmentalization, stability,
and/or potency. Inhibitors can adopt a variety of configurations
including single stranded, double stranded (RNA/RNA or RNA/DNA
duplexes), and hairpin designs, in general, microRNA inhibitors
comprise contain one or more sequences or portions of sequences
that are complementary or partially complementary with the mature
strand (or strands) of the miRNA to be targeted, in addition, the
miRNA inhibitor may also comprise additional sequences located 5'
and 3' to the sequence that is the reverse complement of the mature
miRNA. The additional sequences may be the reverse complements of
the sequences that are adjacent to the mature miRNA in the
pri-miRNA from which the mature miRNA is derived, or the additional
sequences may be arbitrary sequences (having a mixture of A, G, C,
or U). In some embodiments, one or both of the additional sequences
are arbitrary sequences capable of forming hairpins. Thus, in some
embodiments, the sequence that is the reverse complement of the
miRNA is flanked on the 5' side and on the 3' side by hairpin
structures. Micro-RNA inhibitors, when double stranded, may include
mismatches between nucleotides on opposite strands. Furthermore,
micro-RNA inhibitors may be linked to conjugate moieties in order
to facilitate uptake of the inhibitor into a cell. For example, a
micro-RNA inhibitor may be linked to cholesteryl
5-(bis(4-methoxyphenyl)(phenyl)methoxy)-3 hydroxypentylcarbamate)
which allows passive uptake of a micro-RNA inhibitor into a cell.
Micro-RNA inhibitors, including hairpin miRNA inhibitors, are
described in detail in Vermeulen et al., "Double-Stranded Regions
Are Essential Design Components Of Potent Inhibitors of RISC
Function," RNA 13: 723-730 (2007) and in WO2007/095387 and WO
2008/036825 each of which is incorporated herein by reference in
its entirety. A person of ordinary skill in the art can select a
sequence from the database for a desired miRNA and design an
inhibitor useful for the methods disclosed herein.
U1 Adaptor
[0206] U1 adaptor inhibit polyA sites and are bifunctional
oligonucleotides with a target domain complementary to a site in
the target gene's terminal exon and a `U1 domain` that binds to the
U1 smaller nuclear RNA component of the U1 snRNP (Goraczniak, et
al., 2008, Nature Biotechnology, 27(3), 257-263, which is expressly
incorporated by reference herein, in its entirety). U1 snRNP is a
ribonucleoprotein complex that functions primarily to direct early
steps in spliceosome formation by binding to the pre-mRNA
exon-intron boundary (Brown and Simpson, 1998, Annu Rev Plant
Physiol Plant Mol Biol 49:77-95). Nucleotides 2-11 of the 5'end of
U1 snRNA base pair bind with the 5'ss of the pre mRNA. In one
embodiment, oligonucleotides of the invention are U1 adaptors. In
one embodiment, the U1 adaptor can be administered in combination
with at least one other iRNA agent.
Oligonucleotide Modifications
[0207] Unmodified oligonucleotides may be less than optimal in some
applications, e.g., unmodified oligonucleotides can be prone to
degradation by e.g., cellular nucleases. Nucleases can hydrolyze
nucleic acid phosphodiester bonds. However, chemical modifications
of oligonucleotides can confer improved properties, and, e.g., can
render oligonucleotides more stable to nucleases.
[0208] As oligonucleotides are polymers of subunits or monomers,
many of the modifications described below occur at a position which
is repeated within an oligonucleotide, e.g., a modification of a
base, a sugar, a phosphate moiety, or the non-bridging oxygen of a
phosphate moiety. It is not necessary for all positions in a given
oligonucleotide to be uniformly modified, and in fact more than one
of the aforementioned modifications may be incorporated in a single
oligonucleotide or even at a single nucleoside within an
oligonucleotide.
[0209] In some cases the modification will occur at all of the
subject positions in the oligonucleotide but in many, and in fact
in most cases it will not. By way of example, a modification may
only occur at a 3' or 5' terminal position, may only occur in the
internal region, may only occur in a terminal regions, e.g. at a
position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10
nucleotides of an oligonucleotide. A modification may occur in a
double strand region, a single strand region, or in both. A
modification may occur only in the double strand region of a
double-stranded oligonucleotide or may only occur in a single
strand region of a double-stranded oligonucleotide. E.g., a
phosphorothioate modification at a non-bridging oxygen position may
only occur at one or both termini, may only occur in a terminal
regions, e.g., at a position on a terminal nucleotide or in the
last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in
double strand and single strand regions, particularly at termini.
The 5' end or ends can be phosphorylated.
[0210] A modification described herein may be the sole
modification, or the sole type of modification included on multiple
nucleotides, or a modification can be combined with one or more
other modifications described herein. The modifications described
herein can also be combined onto an oligonucleotide, e.g. different
nucleotides of an oligonucleotide have different modifications
described herein.
[0211] In some embodiments it is particularly preferred, e.g., to
enhance stability, to include particular nucleobases in overhangs,
or to include modified nucleotides or nucleotide surrogates, in
single strand overhangs, e.g., in a 5' or 3' overhang, or in both.
E.g., it can be desirable to include purine nucleotides in
overhangs. In some embodiments all or some of the bases in a 3' or
5' overhang will be modified, e.g., with a modification described
herein. Modifications can include, e.g., the use of modifications
at the 2' OH group of the ribose sugar, e.g., the use of
deoxyribonucleotides, e.g., deoxythymidine, instead of
ribonucleotides, and modifications in the phosphate group, e.g.,
phosphothioate modifications. Overhangs need not be homologous with
the target sequence.
[0212] Specific modifications are discussed in more detail
below.
The Phosphate Group
[0213] The phosphate group is a negatively charged species. The
charge is distributed equally over the two non-bridging oxygen
atoms. However, the phosphate group can be modified by replacing
one of the oxygens with a different substituent. One result of this
modification to RNA phosphate backbones can be increased resistance
of the oligoribonucleotide to nucleolytic breakdown. Thus while not
wishing to be bound by theory, it can be desirable in some
embodiments to introduce alterations which result in either an
uncharged linker or a charged linker with unsymmetrical charge
distribution.
[0214] Examples of modified phosphate groups include
phosphorothioate, phosphoroselenates, borano phosphates, borano
phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl
or aryl phosphonates and phosphotriesters. In certain embodiments,
one of the non-bridging phosphate oxygen atoms in the phosphate
backbone moiety can be replaced by any of the following: S, Se,
BR.sub.3 (R is hydrogen, alkyl, aryl), C (i.e. an alkyl group, an
aryl group, etc. . . . ), H, NR.sub.2 (R is hydrogen, alkyl, aryl),
or OR (R is alkyl or aryl). The phosphorous atom in an unmodified
phosphate group is achiral. However, replacement of one of the
non-bridging oxygens with one of the above atoms or groups of atoms
renders the phosphorous atom chiral; in other words a phosphorous
atom in a phosphate group modified in this way is a stereogenic
center. The stereogenic phosphorous atom can possess either the "R"
configuration (herein Rp) or the "S" configuration (herein Sp).
[0215] Phosphorodithioates have both non-bridging oxygens replaced
by sulfur. The phosphorus center in the phosphorodithioates is
achiral which precludes the formation of oligoribonucleotides
diastereomers. Thus, while not wishing to be bound by theory,
modifications to both non-bridging oxygens, which eliminate the
chiral center, e.g. phosphorodithioate formation, may be desirable
in that they cannot produce diastereomer mixtures. Thus, the
non-bridging oxygens can be independently any one of S, Se, B, C,
H, N, or OR (R is alkyl or aryl).
[0216] The phosphate linker can also be modified by replacement of
bridging oxygen, (i.e. oxygen that links the phosphate to the
nucleoside), with nitrogen (bridged phosphoroamidates), sulfur
(bridged phosphorothioates) and carbon (bridged
methylenephosphonates). The replacement can occur at the either
linking oxygen or at both the linking oxygens. When the bridging
oxygen is the 3'-oxygen of a nucleoside, replacement with carbon is
preferred. When the bridging oxygen is the 5'-oxygen of a
nucleoside, replacement with nitrogen is preferred.
Replacement of the Phosphate Group
[0217] The phosphate group can be replaced by non-phosphorus
containing connectors. While not wishing to be bound by theory, it
is believed that since the charged phosphodiester group is the
reaction center in nucleolytic degradation, its replacement with
neutral structural mimics should impart enhanced nuclease
stability. Again, while not wishing to be bound by theory, it can
be desirable, in some embodiment, to introduce alterations in which
the charged phosphate group is replaced by a neutral moiety.
[0218] Examples of moieties which can replace the phosphate group
include methyl phosphonate, hydroxylamino, siloxane, carbonate,
carboxymethyl, carbamate, amide, thioether, ethylene oxide linker,
sulfonate, sulfonamide, thioformacetal, formacetal, oxime,
methyleneimino, methylenemethylimino, methylenehydrazo,
methylenedimethylhydrazo and methyleneoxymethylimino. Preferred
replacements include the methylenecarbonylamino and
methylenemethylimino groups.
[0219] Modified phosphate linkages where at least one of the
oxygens linked to the phosphate has been replaced or the phosphate
group has been replaced by a non-phosphorous group, are also
referred to as "non phosphodiester backbone linkage."
Replacement of Ribophosphate Backbone
[0220] Oligonucleotide-mimicking scaffolds can also be constructed
wherein the phosphate linker and ribose sugar are replaced by
nuclease resistant nucleoside or nucleotide surrogates. While not
wishing to be bound by theory, it is believed that the absence of a
repetitively charged backbone diminishes binding to proteins that
recognize polyanions (e.g. nucleases). Again, while not wishing to
be bound by theory, it can be desirable in some embodiment, to
introduce alterations in which the bases are tethered by a neutral
surrogate backbone. Examples include the morpholino, cyclobutyl,
pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. A
preferred surrogate is a PNA surrogate.
Sugar Modifications
[0221] A modified RNA can include modification of all or some of
the sugar groups of the ribonucleic acid. E.g., the 2' hydroxyl
group (OH) can be modified or replaced with a number of different
"oxy" or "deoxy" substituents. While not being bound by theory,
enhanced stability is expected since the hydroxyl can no longer be
deprotonated to form a 2'-alkoxide ion. The 2'-alkoxide can
catalyze degradation by intramolecular nucleophilic attack on the
linker phosphorus atom. Again, while not wishing to be bound by
theory, it can be desirable to some embodiments to introduce
alterations in which alkoxide formation at the 2' position is not
possible.
[0222] Examples of "oxy"-2' hydroxyl group modifications include
alkoxy or aryloxy (OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl,
heteroaryl or sugar); polyethyleneglycols (PEG),
O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2OR; "locked" nucleic
acids (LNA) in which the 2' hydroxyl is connected, e.g., by a
methylene bridge, to the 4' carbon of the same ribose sugar;
O-AMINE (AMINE=NH.sub.2; alkylamino, dialkylamino, heterocyclyl,
arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino,
ethylene diamine, polyamino) and aminoalkoxy,
O(CH.sub.2).sub.nAMINE, (e.g., AMINE=NH.sub.2; alkylamino,
dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl
amino, or diheteroaryl amino, ethylene diamine, polyamino). It is
noteworthy that oligonucleotides containing only the methoxyethyl
group (MOE), (OCH.sub.2CH.sub.2OCH.sub.3, a PEG derivative),
exhibit nuclease stabilities comparable to those modified with the
robust phosphorothioate modification.
[0223] "Deoxy" modifications include hydrogen (i.e. deoxyribose
sugars, which are of particular relevance to the overhang portions
of partially ds RNA); halo (e.g., fluoro); amino (e.g. NH.sub.2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, diheteroaryl amino, or amino acid);
NH(CH.sub.2CH.sub.2NH).sub.nCH.sub.2CH.sub.2-AMINE (AMINE=NH.sub.2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, or diheteroaryl amino), --NHC(O)R (R=alkyl,
cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto;
alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl
and alkynyl, which may be optionally substituted with e.g., an
amino functionality. Preferred substitutents are 2'-methoxyethyl,
2'-OCH3, 2'-O-allyl, 2'-C-allyl, and 2'-fluoro.
[0224] The sugar group can also contain one or more carbons that
possess the opposite stereochemical configuration than that of the
corresponding carbon in ribose. Thus, an oligonucleotide can
include nucleotides containing e.g., arabinose, as the sugar. The
monomer can have an alpha linkage at the 1' position on the sugar,
e.g., alpha-nucleosides. Oligonucleotides can also include "abasic"
sugars, which lack a nucleobase at C-1'. These abasic sugars can
also be further containing modifications at one or more of the
constituent sugar atoms. Oligonucleotides can also contain one or
more sugars that are in the L form, e.g. L-nucleosides.
Terminal Modifications
[0225] The 3' and 5' ends of an oligonucleotide can be modified.
Such modifications can be at the 3' end, 5' end or both ends of the
molecule. They can include modification or replacement of an entire
terminal phosphate or of one or more of the atoms of the phosphate
group. E.g., the 3' and 5' ends of an oligonucleotide can be
conjugated to other functional molecular entities such as labeling
moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3
or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon,
boron or ester). The functional molecular entities can be attached
to the sugar through a phosphate group and/or a linker. The
terminal atom of the linker can connect to or replace the linking
atom of the phosphate group or the C-3' or C-5' O, N, S or C group
of the sugar. Alternatively, the linker can connect to or replace
the terminal atom of a nucleotide surrogate (e.g., PNAs).
[0226] When a linker/phosphate-functional molecular
entity-linker/phosphate array is interposed between two strands of
a dsRNA, this array can substitute for a hairpin RNA loop in a
hairpin-type RNA agent.
[0227] Terminal modifications useful for modulating activity
include modification of the 5' end with phosphate or phosphate
analogs. E.g., in preferred embodiments antisense strands of
dsRNAs, are 5' phosphorylated or include a phosphoryl analog at the
5' prime terminus. 5'-phosphate modifications include those which
are compatible with RISC mediated gene silencing. Suitable
modifications include: 5'-monophosphate ((HO).sub.2(O)P--O-5');
5'-diphosphate ((HO).sub.2(O)P--O--P(HO)(O)--O-5'); 5'-triphosphate
((HO).sub.2(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-guanosine cap
(7-methylated or non-methylated)
(7m-G-O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-adenosine
cap (Appp), and any modified or unmodified nucleotide cap structure
(N--O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5');
5'-monothiophosphate (phosphorothioate; (HO).sub.2(S)P--O-5');
5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P--O-5'),
5'-phosphorothiolate ((HO).sub.2(O)P--S-5'); any additional
combination of oxygen/sulfur replaced monophosphate, diphosphate
and triphosphates (e.g. 5'-alpha-thiotriphosphate,
5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates
((HO).sub.2(O)P--NH-5', (HO)(NH2)(O)P--O-5'), 5'-alkylphosphonates
(R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g.
RP(OH)(O)--O-5'-, (OH).sub.2(O)P-5'-CH2-),
5'-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-),
ethoxymethyl, etc., e.g. RP(OH)(O)--O-5'-).
[0228] Terminal modifications can also be useful for monitoring
distribution, and in such cases the preferred groups to be added
include fluorophores, e.g., fluorescein or an Alexa dye, e.g.,
Alexa 488. Terminal modifications can also be useful for enhancing
uptake, useful modifications for this include cholesterol. Terminal
modifications can also be useful for cross-linking an RNA agent to
another moiety; modifications useful for this include mitomycin
C.
Nucleobases
[0229] Adenine, guanine, cytosine and uracil are the most common
bases found in RNA. These bases can be modified or replaced to
provide RNA's having improved properties. E.g., nuclease resistant
oligoribonucleotides can be prepared with these bases or with
synthetic and natural nucleobases (e.g., inosine, thymine,
xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine)
and any one of the above modifications. Alternatively, substituted
or modified analogs of any of the above bases, e.g., "unusual
bases", "modified bases", "non-natural bases" and "universal bases"
described herein, can be employed. Examples include without
limitation 2-aminoadenine, 6-methyl and other alkyl derivatives of
adenine and guanine, 2-propyl and other alkyl derivatives of
adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil
and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil
(pseudouracil), 4-thiouracil, 5-halouracil,
5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino,
thiol, thioalkyl, hydroxyl and other 8-substituted adenines and
guanines, 5-trifluoromethyl and other 5-substituted uracils and
cytosines, 7-methylguanine, 5-substituted pyrimidines,
6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,
including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine,
2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl
cytosine,7-deazaadenine, N6, N6-dimethyladenine, 2,6-diaminopurine,
5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles,
2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil,
uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,
5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,
5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil,
3-methylcytosine, 5-methylcytosine, N.sup.4-acetyl cytosine,
2-thiocytosine, N6-methyladenine, N6-isopentyladenine,
2-methylthio-N6-isopentenyladenine, N-methylguanines, or
O-alkylated bases. Further purines and pyrimidines include those
disclosed in U.S. Pat. No. 3,687,808, those disclosed in the
Concise Encyclopedia Of Polymer Science And Engineering, pages
858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and
those disclosed by Englisch et al., Angewandte Chemie,
International Edition, 1991, 30, 613.
Cationic Groups
[0230] Modifications to oligonucleotides can also include
attachment of one or more cationic groups to the sugar, base,
and/or the phosphorus atom of a phosphate or modified phosphate
backbone moiety. A cationic group can be attached to any atom
capable of substitution on a natural, unusual or universal base. A
preferred position is one that does not interfere with
hybridization, i.e., does not interfere with the hydrogen bonding
interactions needed for base pairing. A cationic group can be
attached e.g., through the C2' position of a sugar or analogous
position in a cyclic or acyclic sugar surrogate. Cationic groups
can include e.g., protonated amino groups, derived from e.g.,
O-AMINE (AMINE=NH.sub.2; alkylamino, dialkylamino, heterocyclyl,
arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino,
ethylene diamine, polyamino); aminoalkoxy, e.g.,
O(CH.sub.2).sub.nAMINE, (e.g., AMINE=NH.sub.2; alkylamino,
dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl
amino, or diheteroaryl amino, ethylene diamine, polyamino); amino
(e.g. NH.sub.2; alkylamino, dialkylamino, heterocyclyl, arylamino,
diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid);
or NH(CH.sub.2CH.sub.2NH).sub.nCH.sub.2CH.sub.2-AMINE
(AMINE=NH.sub.2; alkylamino, dialkylamino, heterocyclyl, arylamino,
diaryl amino, heteroaryl amino, or diheteroaryl amino).
Placement within an Oligonucleotide
[0231] Some modifications may preferably be included on an
oligonucleotide at a particular location, e.g., at an internal
position of a strand, or on the 5' or 3' end of an oligonucleotide.
A preferred location of a modification on an oligonucleotide, may
confer preferred properties on the agent. For example, preferred
locations of particular modifications may confer optimum gene
silencing properties, or increased resistance to endonuclease or
exonuclease activity.
[0232] One or more nucleotides of an oligonucleotide may have a
2'-5' linkage. One or more nucleotides of an oligonucleotide may
have inverted linkages, e.g. 3'-3', 5'-5', 2'-2' or 2'-3'
linkages.
[0233] A double-stranded oligonucleotide may include at least one
5'-uridine-adenine-3' (5'-UA-3') dinucleotide wherein the uridine
is a 2'-modified nucleotide, or a terminal 5'-uridine-guanine-3'
(5'-UG-3') dinucleotide, wherein the 5'-uridine is a 2'-modified
nucleotide, or a terminal 5'-cytidine-adenine-3' (5'-CA-3')
dinucleotide, wherein the 5'-cytidine is a 2'-modified nucleotide,
or a terminal 5'-uridine-uridine-3' (5'-UU-3') dinucleotide,
wherein the 5'-uridine is a 2'-modified nucleotide, or a terminal
5'-cytidine-cytidine-3' (5'-CC-3') dinucleotide, wherein the
5'-cytidine is a 2'-modified nucleotide, or a terminal
5'-cytidine-uridine-3' (5'-CU-3') dinucleotide, wherein the
5'-cytidine is a 2'-modified nucleotide, or a terminal
5'-uridine-cytidine-3' (5'-UC-3') dinucleotide, wherein the
5'-uridine is a 2'-modified nucleotide. Double-stranded
oligonucleotides including these modifications are particularly
stabilized against endonuclease activity.
General References
[0234] The oligoribonucleotides and oligoribonucleosides used in
accordance with this invention may be synthesized with solid phase
synthesis, see for example "Oligonucleotide synthesis, a practical
approach", Ed. M. J. Gait, IRL Press, 1984; "Oligonucleotides and
Analogues, A Practical Approach", Ed. F. Eckstein, IRL Press, 1991
(especially Chapter 1, Modern machine-aided methods of
oligodeoxyribonucleotide synthesis, Chapter 2, Oligoribonucleotide
synthesis, Chapter 3, 2'-O-Methyloligoribonucleotide-s: synthesis
and applications, Chapter 4, Phosphorothioate oligonucleotides,
Chapter 5, Synthesis of oligonucleotide phosphorodithioates,
Chapter 6, Synthesis of oligo-2'-deoxyribonucleoside
methylphosphonates, and. Chapter 7, Oligodeoxynucleotides
containing modified bases. Other particularly useful synthetic
procedures, reagents, blocking groups and reaction conditions are
described in Martin, P., Helv. Chian. Acta, 1995, 78, 486-504;
Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48, 2223-2311
and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49,
6123-6194, or references referred to therein. Modification
described in WO 00/44895, WO01/75164, or WO02/44321 can be used
herein. The disclosure of all publications, patents, and published
patent applications listed herein are hereby incorporated by
reference.
Phosphate Group References
[0235] The preparation of phosphinate oligoribonucleotides is
described in U.S. Pat. No. 5,508,270. The preparation of alkyl
phosphonate oligoribonucleotides is described in U.S. Pat. No.
4,469,863. The preparation of phosphoramidite oligoribonucleotides
is described in U.S. Pat. No. 5,256,775 or 5,366,878. The
preparation of phosphotriester oligoribonucleotides is described in
U.S. Pat. No. 5,023,243. The preparation of borano phosphate
oligoribonucleotide is described in U.S. Pat. Nos. 5,130,302 and
5,177,198. The preparation of 3'-Deoxy-3'-amino phosphoramidate
oligoribonucleotides is described in U.S. Pat. No. 5,476,925.
3'-Deoxy-3'-methylenephosphonate oligoribonucleotides is described
in An, H, et al. J. Org. Chem. 2001, 66, 2789-2801. Preparation of
sulfur bridged nucleotides is described in Sproat et al.
Nucleosides Nucleotides 1988, 7,651 and Crosstick et al.
Tetrahedron Lett. 1989, 30, 4693.
Sugar Group References
[0236] Modifications to the 2' modifications can be found in Verma,
S. et al. Annu. Rev. Biochem. 1998, 67, 99-134 and all references
therein. Specific modifications to the ribose can be found in the
following references: 2'-fluoro (Kawasaki et. al., J. Med. Chem.,
1993, 36, 831-841), 2'-MOE (Martin, P. Helv. Chim. Acta 1996, 79,
1930-1938), "LNA" (Wengel, J. Acc. Chem. Res. 1999, 32,
301-310).
Replacement of the Phosphate Group References
[0237] Methylenemethylimino linked oligoribonucleosides, also
identified herein as MMI linked oligoribonucleosides,
methylenedimethylhydrazo linked oligoribonucleosides, also
identified herein as MDH linked oligoribonucleosides, and
methylenecarbonylamino linked oligonucleosides, also identified
herein as amide-3 linked oligoribonucleosides, and
methyleneaminocarbonyl linked oligonucleosides, also identified
herein as amide-4 linked oligoribonucleosides as well as mixed
backbone compounds having, as for instance, alternating MMI and PO
or PS linkages can be prepared as is described in U.S. Pat. Nos.
5,378,825, 5,386,023, 5,489,677 and in published PCT applications
PCT/US92/04294 and PCT/US92/04305 (published as WO 92/20822 WO and
92/20823, respectively). Formacetal and thioformacetal linked
oligoribonucleosides can be prepared as is described in U.S. Pat.
Nos. 5,264,562 and 5,264,564. Ethylene oxide linked
oligoribonucleosides can be prepared as is described in U.S. Pat.
No. 5,223,618. Siloxane replacements are described in Cormier, J.
F. et al. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements
are described in Tittensor, J. R. J. Chem. Soc. C 1971, 1933.
Carboxymethyl replacements are described in Edge, M. D. et al. J.
Chem. Soc. Perkin Trans. 1 1972, 1991. Carbamate replacements are
described in Stirchak, E. P. Nucleic Acids Res. 1989, 17, 6129.
Replacement of the Phosphate-Ribose Backbone References
[0238] Cyclobutyl sugar surrogate compounds can be prepared as is
described in U.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate
can be prepared as is described in U.S. Pat. No. 5,519,134.
Morpholino sugar surrogates can be prepared as is described in U.S.
Pat. Nos. 5,142,047 and 5,235,033, and other related patent
disclosures. Peptide Nucleic Acids (PNAs) are known per se and can
be prepared in accordance with any of the various procedures
referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties
and Potential Applications, Bioorganic & Medicinal Chemistry,
1996, 4, 5-23. They may also be prepared in accordance with U.S.
Pat. No. 5,539,083.
Terminal Modification References
[0239] Terminal modifications are described in Manoharan, M. et al.
Antisense and Nucleic Acid Drug Development 12, 103-128 (2002) and
references therein.
Nucleobases References
[0240] N-2 substituted purine nucleoside amidites can be prepared
as is described in U.S. Pat. No. 5,459,255. 3-Deaza purine
nucleoside amidites can be prepared as is described in U.S. Pat.
No. 5,457,191. 5,6-Substituted pyrimidine nucleoside amidites can
be prepared as is described in U.S. Pat. No. 5,614,617. 5-Propynyl
pyrimidine nucleoside amidites can be prepared as is described in
U.S. Pat. No. 5,484,908.
Linkers
[0241] The term "linker" means an organic moiety that connects two
parts of a compound. Linkers typically comprise a direct bond or an
atom such as oxygen or sulfur, a unit such as NR.sup.1, C(O),
C(O)NH, SO, SO.sub.2, SO.sub.2NH or a chain of atoms, such as
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, arylalkyl,
arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl,
heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl,
heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl,
cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl,
alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl,
alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl,
alkylheteroarylalkyl, alkylheteroarylalkenyl,
alkylheteroarylalkynyl, alkenylheteroarylalkyl,
alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,
alkynylheteroarylalkyl, alkynylheteroarylalkenyl,
alkynylheteroarylalkynyl, alkylheterocyclylalkyl,
alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,
alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,
alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,
alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,
alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,
alkynylhereroaryl, where one or more methylenes can be interrupted
or terminated by O, S, S(O), SO.sub.2, N(R.sup.1).sub.2, C(O),
cleavable linking group, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heterocyclic; where R.sup.1 is hydrogen, acyl,
aliphatic or substituted aliphatic.
[0242] In one embodiment, the linker is
--[(P-Q-R).sub.q--X--(P'-Q'-R').sub.q'].sub.q''-T-, wherein:
[0243] P, R, T, P', R' and T are each independently for each
occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH.sub.2,
CH.sub.2NH, CH.sub.2O; NHCH(R.sup.a)C(O),
--C(O)--CH(R.sup.a)--NH--, CH.dbd.N--O,
##STR00101##
or heterocyclyl;
[0244] Q and Q' are each independently for each occurrence absent,
--(CH.sub.2).sub.n--, --C(R.sup.1)(R.sup.2)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nC(R.sup.1)(R.sup.2)--,
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2--, or
--(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2NH--;
[0245] X is absent or a cleavable linking group;
[0246] R.sup.a is H or an amino acid side chain;
[0247] R.sup.1 and R.sup.2 are each independently for each
occurrence H, CH.sub.3, OH, SH or N(R.sup.N).sub.2;
[0248] R.sup.N is independently for each occurrence H, methyl,
ethyl, propyl, isopropyl, butyl or benzyl;
[0249] q, q' and q'' are each independently for each occurrence
0-20 and wherein the repeating unit can be the same or
different;
[0250] n is independently for each occurrence 1-20; and
[0251] m is independently for each occurrence 0-50.
[0252] In one embodiment, the linker comprises at least one
cleavable linking group.
[0253] In certain embodiments, the linker is a branched linker. The
branchpoint of the branched linker may be at least trivalent, but
may be a tetravalent, pentavalent or hexavalent atom, or a group
presenting such multiple valencies. In certain embodiments, the
branchpoint is --N, --N(Q)-C, --O--C, --S--C, --SS--C,
--C(O)N(Q)-C, --OC(O)N(Q)-C, --N(Q)C(O)--C, or --N(Q)C(O)O--C;
wherein Q is independently for each occurrence H or optionally
substituted alkyl. In other embodiment, the branchpoint is glycerol
or glycerol derivative.
Cleavable Linking Groups
[0254] A cleavable linking group is one which is sufficiently
stable outside the cell, but which upon entry into a target cell is
cleaved to release the two parts the linker is holding together. In
a preferred embodiment, the cleavable linking group is cleaved at
least 10 times or more, preferably at least 100 times faster in the
target cell or under a first reference condition (which can, e.g.,
be selected to mimic or represent intracellular conditions) than in
the blood of a subject, or under a second reference condition
(which can, e.g., be selected to mimic or represent conditions
found in the blood or serum). Cleavable linking groups are
susceptible to cleavage agents, e.g., pH, redox potential or the
presence of degradative molecules. Generally, cleavage agents are
more prevalent or found at higher levels or activities inside cells
than in serum or blood. Examples of such degradative agents
include: redox agents which are selected for particular substrates
or which have no substrate specificity, including, e.g., oxidative
or reductive enzymes or reductive agents such as mercaptans,
present in cells, that can degrade a redox cleavable linking group
by reduction; esterases; endosomes or agents that can create an
acidic environment, e.g., those that result in a pH of five or
lower; enzymes that can hydrolyze or degrade an acid cleavable
linking group by acting as a general acid, peptidases (which can be
substrate specific), and phosphatases.
[0255] A cleavable linkage group, such as a disulfide bond can be
susceptible to pH. The pH of human serum is 7.4, while the average
intracellular pH is slightly lower, ranging from about 7.1-7.3.
Endosomes have a more acidic pH, in the range of 5.5-6.0, and
lysosomes have an even more acidic pH at around 5.0. Some linkers
will have a cleavable linking group that is cleaved at a preferred
pH, thereby releasing the cationic lipid from the ligand inside the
cell, or into the desired compartment of the cell.
[0256] A linker can include a cleavable linking group that is
cleavable by a particular enzyme. The type of cleavable linking
group incorporated into a linker can depend on the cell to be
targeted. For example, liver targeting ligands can be linked to the
cationic lipids through a linker that includes an ester group.
Liver cells are rich in esterases, and therefore the linker will be
cleaved more efficiently in liver cells than in cell types that are
not esterase-rich. Other cell-types rich in esterases include cells
of the lung, renal cortex, and testis.
[0257] Linkers that contain peptide bonds can be used when
targeting cell types rich in peptidases, such as liver cells and
synoviocytes.
[0258] In general, the suitability of a candidate cleavable linking
group can be evaluated by testing the ability of a degradative
agent (or condition) to cleave the candidate linking group. It will
also be desirable to also test the candidate cleavable linking
group for the ability to resist cleavage in the blood or when in
contact with other non-target tissue. Thus one can determine the
relative susceptibility to cleavage between a first and a second
condition, where the first is selected to be indicative of cleavage
in a target cell and the second is selected to be indicative of
cleavage in other tissues or biological fluids, e.g., blood or
serum. The evaluations can be carried out in cell free systems, in
cells, in cell culture, in organ or tissue culture, or in whole
animals. It may be useful to make initial evaluations in cell-free
or culture conditions and to confirm by further evaluations in
whole animals. In preferred embodiments, useful candidate compounds
are cleaved at least 2, 4, 10 or 100 times faster in the cell (or
under in vitro conditions selected to mimic intracellular
conditions) as compared to blood or serum (or under in vitro
conditions selected to mimic extracellular conditions).
Redox Cleavable Linking Groups
[0259] One class of cleavable linking groups are redox cleavable
linking groups that are cleaved upon reduction or oxidation. An
example of reductively cleavable linking group is a disulphide
linking group (--S--S--). To determine if a candidate cleavable
linking group is a suitable "reductively cleavable linking group,"
or for example is suitable for use with a particular iRNA moiety
and particular targeting agent one can look to methods described
herein. For example, a candidate can be evaluated by incubation
with dithiothreitol (DTT), or other reducing agent using reagents
know in the art, which mimic the rate of cleavage which would be
observed in a cell, e.g., a target cell. The candidates can also be
evaluated under conditions which are selected to mimic blood or
serum conditions. In a preferred embodiment, candidate compounds
are cleaved by at most 10% in the blood. In preferred embodiments,
useful candidate compounds are degraded at least 2, 4, 10 or 100
times faster in the cell (or under in vitro conditions selected to
mimic intracellular conditions) as compared to blood (or under in
vitro conditions selected to mimic extracellular conditions). The
rate of cleavage of candidate compounds can be determined using
standard enzyme kinetics assays under conditions chosen to mimic
intracellular media and compared to conditions chosen to mimic
extracellular media.
Phosphate-Based Cleavable Linking Groups
[0260] Phosphate-based cleavable linking groups are cleaved by
agents that degrade or hydrolyze the phosphate group. An example of
an agent that cleaves phosphate groups in cells are enzymes such as
phosphatases in cells. Examples of phosphate-based linking groups
are --O--P(O)(ORk)-O--, --O--P(S)(ORk)-O--, --O--P(S)(SRk)-O--,
--S--P(O)(ORk)-O--, --O--P(O)(ORk)-S--, --S--P(O)(ORk)-S--,
--O--P(S)(ORk)-S--, --S--P(S)(ORk)-O--, --O--P(O)(Rk)-O--,
--O--P(S)(Rk)-O--, --S--P(O)(Rk)-O--, --S--P(S)(Rk)-O--,
--S--P(O)(Rk)-S--, --O--P(S)(Rk)-S--. Preferred embodiments are
--O--P(O)(OH)--O--, --O--P(S)(OH)--O--, --O--P(S)(SH)--O--,
--S--P(O)(OH)--O--, --O--P(O)(OH)--S--, --S--P(O)(OH)--S--,
--O--P(S)(OH)--S--, --S--P(S)(OH)--O--, --O--P(O)(H)--O--,
--O--P(S)(H)--O--, --S--P(O)(H)--O--, --S--P(S)(H)--O--,
--S--P(O)(H)--S--, --O--P(S)(H)--S--. A preferred embodiment is
--O--P(O)(OH)--O--. These candidates can be evaluated using methods
analogous to those described above.
Acid Cleavable Linking Groups
[0261] Acid cleavable linking groups are linking groups that are
cleaved under acidic conditions. In preferred embodiments acid
cleavable linking groups are cleaved in an acidic environment with
a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower),
or by agents such as enzymes that can act as a general acid. In a
cell, specific low pH organelles, such as endosomes and lysosomes
can provide a cleaving environment for acid cleavable linking
groups. Examples of acid cleavable linking groups include but are
not limited to hydrazones, esters, and esters of amino acids. Acid
cleavable groups can have the general formula --C.dbd.NN--, C(O)O,
or --OC(O). A preferred embodiment is when the carbon attached to
the oxygen of the ester (the alkoxy group) is an aryl group,
substituted alkyl group, or tertiary alkyl group such as dimethyl
pentyl or t-butyl. These candidates can be evaluated using methods
analogous to those described above.
Ester-Based Linking Groups
[0262] Ester-based cleavable linking groups are cleaved by enzymes
such as esterases and amidases in cells. Examples of ester-based
cleavable linking groups include but are not limited to esters of
alkylene, alkenylene and alkynylene groups. Ester cleavable linking
groups have the general formula --C(O)O--, or --OC(O)--. These
candidates can be evaluated using methods analogous to those
described above.
Peptide-Based Cleaving Groups
[0263] Peptide-based cleavable linking groups are cleaved by
enzymes such as peptidases and proteases in cells. Peptide-based
cleavable linking groups are peptide bonds formed between amino
acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)
and polypeptides. Peptide-based cleavable groups do not include the
amide group (--C(O)NH--). The amide group can be formed between any
alkylene, alkenylene or alkynylene. A peptide bond is a special
type of amide bond formed between amino acids to yield peptides and
proteins. The peptide based cleavage group is generally limited to
the peptide bond (i.e., the amide bond) formed between amino acids
yielding peptides and proteins and does not include the entire
amide functional group. Peptide-based cleavable linking groups have
the general formula --NHCHR.sup.AC(O)NHCHR.sup.BC(O)--, where
R.sup.A and R.sup.B are the R groups of the two adjacent amino
acids. These candidates can be evaluated using methods analogous to
those described above.
Ligands
[0264] A wide variety of entities can be coupled to the
oligonucleotides and lipids of the present invention. Preferred
moieties are ligands, which are coupled, preferably covalently,
either directly or indirectly via an intervening tether.
[0265] In preferred embodiments, a ligand alters the distribution,
targeting or lifetime of the molecule into which it is
incorporated. In preferred embodiments a ligand provides an
enhanced affinity for a selected target, e.g., molecule, cell or
cell type, compartment, e.g., a cellular or organ compartment,
tissue, organ or region of the body, as, e.g., compared to a
species absent such a ligand. Ligands providing enhanced affinity
for a selected target are also termed targeting ligands. Preferred
ligands for conjugation to the lipids of the present invention are
targeting ligands.
[0266] Some ligands can have endosomolytic properties. The
endosomolytic ligands promote the lysis of the endosome and/or
transport of the composition of the invention, or its components,
from the endosome to the cytoplasm of the cell. The endosomolytic
ligand may be a polyanionic peptide or peptidomimetic which shows
pH-dependent membrane activity and fusogenicity. In certain
embodiments, the endosomolytic ligand assumes its active
conformation at endosomal pH. The "active" conformation is that
conformation in which the endosomolytic ligand promotes lysis of
the endosome and/or transport of the composition of the invention,
or its components, from the endosome to the cytoplasm of the cell.
Exemplary endosomolytic ligands include the GALA peptide (Subbarao
et al., Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel
et al., J. Am. Chem. Soc., 1996, 118: 1581-1586), and their
derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559:
56-68). In certain embodiments, the endosomolytic component may
contain a chemical group (e.g., an amino acid) which will undergo a
change in charge or protonation in response to a change in pH. The
endosomolytic component may be linear or branched. Exemplary
primary sequences of peptide based endosomolytic ligands are shown
in Table 4.
TABLE-US-00003 TABLE 4 List of peptides with endosomolytic
activity. Name Sequence (N to C) Ref. GALA
AALEALAEALEALAEALEALAEAAAAGGC 1 EALA AALAEALAEALAEALAEALAEALAAAAGGC
2 ALEALAEALEALAEA 3 INF-7 GLFEAIEGFIENGWEGMIWDYG 4 Inf HA-2
GLFGAIAGFIENGWEGMIDGWYG 5 diINF-7 GLF EAI EGFI ENGW EGMI DGWYGC 5
GLF EAI EGFI ENGW EGMI DGWYGC diINF3 GLF EAI EGFI ENGW EGMI DGGC 6
GLF EAI EGFI ENGW EGMI DGGC GLF GLFGALAEALAEALAEHLAEALAEALEALA 6
AGGSC GALA-INF3 GLFEAIEGFIENGWEGLAEALAEALEALAA 6 GGSC INF-5 GLF EAI
EGFI ENGW EGnI DG K 4 GLF EAI EGFI ENGW EGnI DG n, norleucine
References 1. Subbarao et al., Biochemistry, 1987, 26: 2964-2972.
2. Vogel etal., J. Am. Chem. Soc., 1996, 118: 1581-1586 3. Turk, M.
J., Reddy, J. A. et al. (2002). Characterization of a novel
pH-sensitive peptide that enhances drug release from
folate-targeted liposomes at endosomal pHs. Biochim. Biophys. Acta
1559, 56-68. 4. Plank, C. Oberhauser, B. Mechtler, K. Koch, C.
Wagner, E. (1994). The influence of endosome-disruptive peptides on
gene transfer using synthetic virus-like gene transfer systems, J.
Biol. Chem. 269 12918-12924. 5. Mastrobattista, E., Koning, G. A.
et al. (2002). Functional characterization of an
endosome-disruptive peptide and its application in cytosolic
delivery of immunoliposome-entrapped proteins. J. Biol. Chem. 277,
27135-43. 6. Oberhauser, B., Plank, C. et al. (1995). Enhancing
endosomal exit of nucleic acids using pH-sensitive viral fusion
peptides. Deliv. Strategies Antisense Oligonucleotide Ther.
247-66.
[0267] Preferred ligands can improve transport, hybridization, and
specificity properties and may also improve nuclease resistance of
the resultant natural or modified oligoribonucleotide, or a
polymeric molecule comprising any combination of monomers described
herein and/or natural or modified ribonucleotides.
[0268] Ligands in general can include therapeutic modifiers, e.g.,
for enhancing uptake; diagnostic compounds or reporter groups e.g.,
for monitoring distribution; cross-linking agents; and
nuclease-resistance conferring moieties. General examples include
lipids, steroids, vitamins, sugars, proteins, peptides, polyamines,
and peptide mimics.
[0269] Ligands can include a naturally occurring substance, such as
a protein (e.g., human serum albumin (HSA), low-density lipoprotein
(LDL), high-density lipoprotein (HDL), or globulin); an
carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin,
cyclodextrin or hyaluronic acid); or a lipid. The ligand may also
be a recombinant or synthetic molecule, such as a synthetic
polymer, e.g., a synthetic polyamino acid, an oligonucleotide (e.g.
an aptamer). Examples of polyamino acids include polyamino acid is
a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,
styrene-maleic acid anhydride copolymer,
poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic
anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer
(HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA),
polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide
polymers, or polyphosphazine. Example of polyamines include:
polyethylenimine, polylysine (PLL), spermine, spermidine,
polyamine, pseudopeptide-polyamine, peptidomimetic polyamine,
dendrimer polyamine, arginine, amidine, protamine, cationic lipid,
cationic porphyrin, quaternary salt of a polyamine, or an alpha
helical peptide.
[0270] Ligands can also include targeting groups, e.g., a cell or
tissue targeting agent, e.g., a lectin, glycoprotein, lipid or
protein, e.g., an antibody, that binds to a specified cell type
such as a kidney cell. A targeting group can be a thyrotropin,
melanotropin, lectin, glycoprotein, surfactant protein A, Mucin
carbohydrate, multivalent lactose, multivalent galactose,
N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose,
multivalent fucose, glycosylated polyaminoacids, multivalent
galactose, transferrin, bisphosphonate, polyglutamate,
polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate,
vitamin B12, biotin, an RGD peptide, an RGD peptide mimetic or an
aptamer. Table 5 shows some examples of targeting ligands and their
associated receptors.
TABLE-US-00004 TABLE 5 Targeting Ligands and their associated
receptors Liver Cells Ligand Receptor 1) Parenchymal Galactose
ASGP-R Cell (PC) (Asiologlycoprotein (Hepatocytes) receptor) Gal
NAc ASPG-R (n-acetyl-galactosamine) Gal NAc Receptor Lactose
Asialofetuin ASPG-r 2) Sinusoidal Hyaluronan Hyaluronan receptor
Endothelial Procollagen Procollagen receptor Cell (SEC) Negatively
charged molecules Scavenger receptors Mannose Mannose receptors
N-acetyl Glucosamine Scavenger receptors Immunoglobulins Fc
Receptor LPS CD14 Receptor Insulin Receptor mediated transcytosis
Transferrin Receptor mediated transcytosis Albumins Non-specific
Sugar-Albumin conjugates Mannose-6-phosphate Mannose-6-phosphate
receptor 3) Kupffer Mannose Mannose receptors Cell (KC) Fucose
Fucose receptors Albumins Non-specific Mannose-albumin
conjugates
[0271] Other examples of ligands include dyes, intercalating agents
(e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C),
porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic
hydrocarbons (e.g., phenazine, dihydrophenazine), artificial
endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol,
cholic acid, adamantane acetic acid, 1-pyrene butyric acid,
dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl
group, hexadecylglycerol, borneol, menthol, 1,3-propanediol,
heptadecyl group, palmitic acid, myristic acid,
O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,
dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,
antennapedia peptide, Tat peptide), alkylating agents, phosphate,
amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG].sub.2,
polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes,
haptens (e.g. biotin), transport/absorption facilitators (e.g.,
aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g.,
imidazole, bisimidazole, histamine, imidazole clusters,
acridine-imidazole conjugates, Eu3+ complexes of
tetraazamacrocycles), dinitrophenyl, HRP, or AP.
[0272] Ligands can be proteins, e.g., glycoproteins, or peptides,
e.g., molecules having a specific affinity for a co-ligand, or
antibodies e.g., an antibody, that binds to a specified cell type
such as a cancer cell, endothelial cell, or bone cell. Ligands may
also include hormones and hormone receptors. They can also include
non-peptidic species, such as lipids, lectins, carbohydrates,
vitamins, cofactors, multivalent lactose, multivalent galactose,
N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose,
multivalent fucose, or aptamers. The ligand can be, for example, a
lipopolysaccharide, an activator of p38 MAP kinase, or an activator
of NF-.kappa.B.
[0273] The ligand can be a substance, e.g., a drug, which can
increase the uptake of the iRNA agent into the cell, for example,
by disrupting the cell's cytoskeleton, e.g., by disrupting the
cell's microtubules, microfilaments, and/or intermediate filaments.
The drug can be, for example, taxon, vincristine, vinblastine,
cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin,
swinholide A, indanocine, or myoservin.
[0274] The ligand can increase the uptake of the iRNA agent into
the cell by activating an inflammatory response, for example.
Exemplary ligands that would have such an effect include tumor
necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma
interferon.
[0275] In one aspect, the ligand is a lipid or lipid-based
molecule. Such a lipid or lipid-based molecule preferably binds a
serum protein, e.g., human serum albumin (HSA). An HSA binding
ligand allows for distribution of the conjugate to a target tissue,
e.g., a non-kidney target tissue of the body. For example, the
target tissue can be the liver, including parenchymal cells of the
liver. Other molecules that can bind HSA can also be used as
ligands. For example, neproxin or aspirin can be used. A lipid or
lipid-based ligand can (a) increase resistance to degradation of
the conjugate, (b) increase targeting or transport into a target
cell or cell membrane, and/or (c) can be used to adjust binding to
a serum protein, e.g., HSA.
[0276] A lipid based ligand can be used to modulate, e.g., control
the binding of the conjugate to a target tissue. For example, a
lipid or lipid-based ligand that binds to HSA more strongly will be
less likely to be targeted to the kidney and therefore less likely
to be cleared from the body. A lipid or lipid-based ligand that
binds to HSA less strongly can be used to target the conjugate to
the kidney.
[0277] In a preferred embodiment, the lipid based ligand binds HSA.
Preferably, it binds HSA with a sufficient affinity such that the
conjugate will be preferably distributed to a non-kidney tissue.
However, it is preferred that the affinity not be so strong that
the HSA-ligand binding cannot be reversed.
[0278] In another preferred embodiment, the lipid based ligand
binds HSA weakly or not at all, such that the conjugate will be
preferably distributed to the kidney. Other moieties that target to
kidney cells can also be used in place of or in addition to the
lipid based ligand.
[0279] In another aspect, the ligand is a moiety, e.g., a vitamin,
which is taken up by a target cell, e.g., a proliferating cell.
These are particularly useful for treating disorders characterized
by unwanted cell proliferation, e.g., of the malignant or
non-malignant type, e.g., cancer cells. Exemplary vitamins include
vitamin A, E, and K. Other exemplary vitamins include are B
vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or
other vitamins or nutrients taken up by cancer cells. Also included
are HAS, low density lipoprotein (LDL) and high-density lipoprotein
(HDL).
[0280] In another aspect, the ligand is a cell-permeation agent,
preferably a helical cell-permeation agent. Preferably, the agent
is amphipathic. An exemplary agent is a peptide such as tat or
antennopedia. If the agent is a peptide, it can be modified,
including a peptidylmimetic, invertomers, non-peptide or
pseudo-peptide linkages, and use of D-amino acids. The helical
agent is preferably an alpha-helical agent, which preferably has a
lipophilic and a lipophobic phase.
[0281] The ligand can be a peptide or peptidomimetic. A
peptidomimetic (also referred to herein as an oligopeptidomimetic)
is a molecule capable of folding into a defined three-dimensional
structure similar to a natural peptide. The peptide or
peptidomimetic moiety can be about 5-50 amino acids long, e.g.,
about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long
(see Table 6, for example).
TABLE-US-00005 TABLE 6 Exemplary Cell Permeation Peptides. Cell
Permeation Peptide Amino acid Sequence Reference Penetratin
RQIKIWFQNRRMKWKK Derossi et al., J. Biol. Chem. 269:10444, 1994 Tat
fragment GRKKRRQRRRPPQC Vives et al., J. Biol. Chem., (48-60)
272:16010, 1997 Signal Sequence- GALFLGWLGAAGSTMGAWSQPKKKRKV
Chaloin et al., Biochem. based peptide Biophys. Res. Commun.,
243:601, 1998 PVEC LLIILRRRIRKQAHAHSK Elmquist et al., Exp. Cell
Res., 269:237, 2001 Transportan GWTLNSAGYLLKINLKALAALAKKIL Pooga et
al., FASEB J., 12:67, 1998 Amphiphilic KLALKLALKALKAALKLA Oehlke et
al., Mol. Ther., model peptide 2:339, 2000 Arg.sub.9 RRRRRRRRR
Mitchell et al., J. Pept. Res., 56:318, 2000 Bacterial cell
KFFKFFKFFK wall permeating LL-37 LLGDFFRKSKEKIGKEFKRIVQRIKDFL
RNLVPRTES Cecropin P1 SWLSKTAKKLENSAKKRISEGIAIAIQG GPR
.alpha.-defensin ACYCRIPACIAGERRYGTCIYQGRLWA FCC b-dcfensin
DHYNCVSSGGQCLYSACPIFTKIQGTC YRGKAKCCK Bactenecin RKCRIVVIRVCR PR-39
RRRPRPPYLPRPRPPPFFPPRLPPRIPPGF PPRFPPRFPGKR-NH2 Indolicidin
ILPWKWPWWPWRR-NH2
[0282] A peptide or peptidomimetic can be, for example, a cell
permeation peptide, cationic peptide, amphipathic peptide, or
hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or
Phe). The peptide moiety can be a dendrimer peptide, constrained
peptide or crosslinked peptide. In another alternative, the peptide
moiety can include a hydrophobic membrane translocation sequence
(MTS). An exemplary hydrophobic MTS-containing peptide is RFGF
having the amino acid sequence AAVALLPAVLLALLAP. An RFGF analogue
(e.g., amino acid sequence AALLPVLLAAP) containing a hydrophobic
MTS can also be a targeting moiety. The peptide moiety can be a
"delivery" peptide, which can carry large polar molecules including
peptides, oligonucleotides, and protein across cell membranes. For
example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ) and the
Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK) have been found
to be capable of functioning as delivery peptides. A peptide or
peptidomimetic can be encoded by a random sequence of DNA, such as
a peptide identified from a phage-display library, or
one-bead-one-compound (OBOC) combinatorial library (Lam et al.,
Nature, 354:82-84, 1991). Preferably the peptide or peptidomimetic
tethered to an iRNA agent via an incorporated monomer unit is a
cell targeting peptide such as an arginine-glycine-aspartic acid
(RGD)-peptide, or RGD mimic. A peptide moiety can range in length
from about 5 amino acids to about 40 amino acids. The peptide
moieties can have a structural modification, such as to increase
stability or direct conformational properties. Any of the
structural modifications described below can be utilized.
[0283] An RGD peptide moiety can be used to target a tumor cell,
such as an endothelial tumor cell or a breast cancer tumor cell
(Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide
can facilitate targeting of an iRNA agent to tumors of a variety of
other tissues, including the lung, kidney, spleen, or liver (Aoki
et al., Cancer Gene Therapy 8:783-787, 2001). Preferably, the RGD
peptide will facilitate targeting of an iRNA agent to the kidney.
The RGD peptide can be linear or cyclic, and can be modified, e.g.,
glycosylated or methylated to facilitate targeting to specific
tissues. For example, a glycosylated RGD peptide can deliver an
iRNA agent to a tumor cell expressing .alpha..sub.v.beta..sub.3
(Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).
[0284] Peptides that target markers enriched in proliferating cells
can be used. E.g., RGD containing peptides and peptidomimetics can
target cancer cells, in particular cells that exhibit an
.alpha.v.beta.3 integrin. Thus, one could use RGD peptides, cyclic
peptides containing RGD, RGD peptides that include D-amino acids,
as well as synthetic RGD mimics. In addition to RGD, one can use
other moieties that target the .alpha.v.beta.3 integrin ligand.
Generally, such ligands can be used to control proliferating cells
and angiogenesis. Preferred conjugates of this type ligands that
targets PECAM-1, VEGF, or other cancer gene, e.g., a cancer gene
described herein.
[0285] A "cell permeation peptide" is capable of permeating a cell,
e.g., a microbial cell, such as a bacterial or fungal cell, or a
mammalian cell, such as a human cell. A microbial cell-permeating
peptide can be, for example, an .alpha.-helical linear peptide
(e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide
(e.g., .alpha.-defensin, .beta.-defensin or bactenecin), or a
peptide containing only one or two dominating amino acids (e.g.,
PR-39 or indolicidin). A cell permeation peptide can also include a
nuclear localization signal (NLS). For example, a cell permeation
peptide can be a bipartite amphipathic peptide, such as MPG, which
is derived from the fusion peptide domain of HIV-1 gp41 and the NLS
of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.
31:2717-2724, 2003).
[0286] In one embodiment, a targeting peptide tethered to an iRNA
agent and/or the carrier oligomer can be an amphipathic
.alpha.-helical peptide. Exemplary amphipathic .alpha.-helical
peptides include, but are not limited to, cecropins, lycotoxins,
paradaxins, buforin, CPF, bombinin-like peptide (BLP),
cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal
antimicrobial peptides (HFIAPs), magainines, brevinins-2,
dermaseptins, melittins, pleurocidin, H.sub.2A peptides, Xenopus
peptides, esculentinis-1, and caerins. A number of factors will
preferably be considered to maintain the integrity of helix
stability. For example, a maximum number of helix stabilization
residues will be utilized (e.g., leu, ala, or lys), and a minimum
number helix destabilization residues will be utilized (e.g.,
proline, or cyclic monomeric units. The capping residue will be
considered (for example Gly is an exemplary N-capping residue
and/or C-terminal amidation can be used to provide an extra H-bond
to stabilize the helix. Formation of salt bridges between residues
with opposite charges, separated by i.+-.3, or i.+-.4 positions can
provide stability. For example, cationic residues such as lysine,
arginine, homo-arginine, ornithine or histidine can form salt
bridges with the anionic residues glutamate or aspartate.
[0287] Peptide and peptidomimetic ligands include those having
naturally occurring or modified peptides, e.g., D or L peptides; a,
13, or y peptides; N-methyl peptides; azapeptides; peptides having
one or more amide, i.e., peptide, linkages replaced with one or
more urea, thiourea, carbamate, or sulfonyl urea linkages; or
cyclic peptides.
[0288] The targeting ligand can be any ligand that is capable of
targeting a specific receptor. Examples are: folate, GalNAc,
galactose, mannose, mannose-6P, clusters of sugars such as GalNAc
cluster, mannose cluster, galactose cluster, or an aptamer. A
cluster is a combination of two or more sugar units. The targeting
ligands also include integrin receptor ligands, Chemokine receptor
ligands, transferrin, biotin, serotonin receptor ligands, PSMA,
endothelin, GCPII, somatostatin, LDL and HDL ligands. The ligands
can also be based on nucleic acid, e.g., an aptamer. The aptamer
can be unmodified or have any combination of modifications
disclosed herein.
[0289] Endosomal release agents include imidazoles, poly or
oligoimidazoles, PEIs, peptides, fusogenic peptides,
polycaboxylates, polyacations, masked oligo or poly cations or
anions, acetals, polyacetals, ketals/polyketyals, orthoesters,
polymers with masked or unmasked cationic or anionic charges,
dendrimers with masked or unmasked cationic or anionic charges.
[0290] PK modulator stands for pharmacokinetic modulator. PK
modulator include lipophiles, bile acids, steroids, phospholipid
analogues, peptides, protein binding agents, PEG, vitamins etc.
Exemplary PK modulator include, but are not limited to,
cholesterol, fatty acids, cholic acid, lithocholic acid,
dialkylglycerides, diacylglyceride, phospholipids, sphingolipids,
naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that
comprise a number of phosphorothioate linkages are also known to
bind to serum protein, thus short oligonucleotides, e.g.
oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases,
comprising multiple of phosphorothioate linkages in the backbone
are also amenable to the present invention as ligands (e.g. as PK
modulating ligands).
[0291] In addition, aptamers that bind serum components (e.g. serum
proteins) are also amenable to the present invention as PK
modulating ligands.
[0292] Other ligands amenable to the invention are described in
copending applications U.S. Ser. No. 10/916,185, filed Aug. 10,
2004; U.S. Ser. No. 10/946,873, filed Sep. 21, 2004; U.S. Ser. No.
10/833,934, filed Aug. 3, 2007; U.S. Ser. No. 11/115,989 filed Apr.
27, 2005 and U.S. Ser. No. 11/944,227 filed Nov. 21, 2007, which
are incorporated by reference in their entireties for all
purposes.
[0293] When two or more ligands are present, the ligands can all
have same properties, all have different properties or some ligands
have the same properties while others have different properties.
For example, a ligand can have targeting properties, have
endosomolytic activity or have PK modulating properties. In a
preferred embodiment, all the ligands have different
properties.
[0294] Ligands can be coupled to the oligonucleotides various
places, for example, 3'-end, 5'-end, and/or at an internal
position. In preferred embodiments, the ligand is attached to the
oligonucleotides via an intervening tether. The ligand or tethered
ligand may be present on a monomer when said monomer is
incorporated into the growing strand. In some embodiments, the
ligand may be incorporated via coupling to a "precursor" monomer
after said "precursor" monomer has been incorporated into the
growing strand. For example, a monomer having, e.g., an
amino-terminated tether (i.e., having no associated ligand), e.g.,
TAP-(CH.sub.2)--NH.sub.2 may be incorporated into a growing sense
or antisense strand. In a subsequent operation, i.e., after
incorporation of the precursor monomer into the strand, a ligand
having an electrophilic group, e.g., a pentafluorophenyl ester or
aldehyde group, can subsequently be attached to the precursor
monomer by coupling the electrophilic group of the ligand with the
terminal nucleophilic group of the precursor monomer's tether.
[0295] For double-stranded oligonucleotides, ligands can be
attached to one or both strands. In some embodiments, a
double-stranded iRNA agent contains a ligand conjugated to the
sense strand. In other embodiments, a double-stranded iRNA agent
contains a ligand conjugated to the antisense strand.
[0296] In some embodiments, ligands can be conjugated to
nucleobases, sugar moieties, or internucleosidic linkages of
nucleic acid molecules. Conjugation to purine nucleobases or
derivatives thereof can occur at any position including, endocyclic
and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or
8-positions of a purine nucleobase are attached to a conjugate
moiety. Conjugation to pyrimidine nucleobases or derivatives
thereof can also occur at any position. In some embodiments, the
2-, 5-, and 6-positions of a pyrimidine nucleobase can be
substituted with a conjugate moiety. Conjugation to sugar moieties
of nucleosides can occur at any carbon atom. Example carbon atoms
of a sugar moiety that can be attached to a conjugate moiety
include the 2', 3', and 5' carbon atoms. The 1' position can also
be attached to a conjugate moiety, such as in an abasic residue.
Internucleosidic linkages can also bear conjugate moieties. For
phosphorus-containing linkages (e.g., phosphodiester,
phosphorothioate, phosphorodithioate, phosphoroamidate, and the
like), the conjugate moiety can be attached directly to the
phosphorus atom or to an O, N, or S atom bound to the phosphorus
atom. For amine- or amide-containing internucleosidic linkages
(e.g., PNA), the conjugate moiety can be attached to the nitrogen
atom of the amine or amide or to an adjacent carbon atom.
[0297] There are numerous methods for preparing conjugates of
oligomeric compounds. Generally, an oligomeric compound is attached
to a conjugate moiety by contacting a reactive group (e.g., OH, SH,
amine, carboxyl, aldehyde, and the like) on the oligomeric compound
with a reactive group on the conjugate moiety. In some embodiments,
one reactive group is electrophilic and the other is
nucleophilic.
[0298] For example, an electrophilic group can be a
carbonyl-containing functionality and a nucleophilic group can be
an amine or thiol. Methods for conjugation of nucleic acids and
related oligomeric compounds with and without linking groups are
well described in the literature such as, for example, in Manoharan
in Antisense Research and Applications, Crooke and LeBleu, eds.,
CRC Press, Boca Raton, Fla., 1993, Chapter 17, which is
incorporated herein by reference in its entirety.
[0299] Representative United States patents that teach the
preparation of oligonucleotide conjugates include, but are not
limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;
5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;
5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;
5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;
4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;
4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;
5,112,963; 5,149,782; 5,214,136; 5,245,022; 5,254,469; 5,258,506;
5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;
5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;
5,567,810; 5,574, 142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;
5,599, 923; 5,599,928; 5,672,662; 5,688,941; 5,714,166; 6,153,737;
6,172,208; 6,300,319; 6,335,434; 6,335,437; 6,395,437; 6,444,806;
6,486,308; 6,525,031; 6,528,631; 6,559,279; each of which is herein
incorporated by reference.
Characteristic of Nucleic Acid-Lipid Particles
[0300] In certain embodiments, the present invention relates to
methods and compositions for producing lipid-encapsulated nucleic
acid particles in which nucleic acids are encapsulated within a
lipid layer. Such nucleic acid-lipid particles, incorporating siRNA
oligonucleotides, are characterized using a variety of biophysical
parameters including: (1)drug to lipid ratio; (2) encapsulation
efficiency; and (3) particle size. High drug to lipid rations, high
encapsulation efficiency, good nuclease resistance and serum
stability and controllable particle size, generally less than 200
nm in diameter are desirable. In addition, the nature of the
nucleic acid polymer is of significance, since the modification of
nucleic acids in an effort to impart nuclease resistance adds to
the cost of therapeutics while in many cases providing only limited
resistance. Unless stated otherwise, these criteria are calculated
in this specification as follows:
[0301] Nucleic acid to lipid ratio is the amount of nucleic acid in
a defined volume of preparation divided by the amount of lipid in
the same volume. This may be on a mole per mole basis or on a
weight per weight basis, or on a weight per mole basis. For final,
administration-ready formulations, the nucleic acid:lipid ratio is
calculated after dialysis, chromatography and/or enzyme (e.g.,
nuclease) digestion has been employed to remove as much of the
external nucleic acid as possible;
[0302] Encapsulation efficiency refers to the drug to lipid ratio
of the starting mixture divided by the drug to lipid ratio of the
final, administration competent formulation. This is a measure of
relative efficiency. For a measure of absolute efficiency, the
total amount of nucleic acid added to the starting mixture that
ends up in the administration competent formulation, can also be
calculated. The amount of lipid lost during the formulation process
may also be calculated. Efficiency is a measure of the wastage and
expense of the formulation; and
[0303] Size indicates the size (diameter) of the particles formed.
Size distribution may be determined using quasi-elastic light
scattering (QELS) on a Nicomp Model 370 sub-micron particle sizer.
Particles under 200 nm are preferred for distribution to
neo-vascularized (leaky) tissues, such as neoplasms and sites of
inflammation.
Pharmaceutical Compositions
[0304] The lipid particles of present invention, particularly when
associated with a therapeutic agent, may be formulated as a
pharmaceutical composition, e.g., which further comprises a
pharmaceutically acceptable diluent, excipient, or carrier, such as
physiological saline or phosphate buffer, selected in accordance
with the route of administration and standard pharmaceutical
practice.
[0305] In particular embodiments, pharmaceutical compositions
comprising the lipid-nucleic acid particles of the invention are
prepared according to standard techniques and further comprise a
pharmaceutically acceptable carrier. Generally, normal saline will
be employed as the pharmaceutically acceptable carrier. Other
suitable carriers include, e.g., water, buffered water, 0.9%
saline, 0.3% glycine, and the like, including glycoproteins for
enhanced stability, such as albumin, lipoprotein, globulin, etc. In
compositions comprising saline or other salt containing carriers,
the carrier is preferably added following lipid particle formation.
Thus, after the lipid-nucleic acid compositions are formed, the
compositions can be diluted into pharmaceutically acceptable
carriers such as normal saline.
[0306] The resulting pharmaceutical preparations may be sterilized
by conventional, well known sterilization techniques. The aqueous
solutions can then be packaged for use or filtered under aseptic
conditions and lyophilized, the lyophilized preparation being
combined with a sterile aqueous solution prior to administration.
The compositions may contain pharmaceutically acceptable auxiliary
substances as required to approximate physiological conditions,
such as pH adjusting and buffering agents, tonicity adjusting
agents and the like, for example, sodium acetate, sodium lactate,
sodium chloride, potassium chloride, calcium chloride, etc.
Additionally, the lipidic suspension may include lipid-protective
agents which protect lipids against free-radical and
lipid-peroxidative damages on storage. Lipophilic free-radical
quenchers, such as .alpha.-tocopherol and water-soluble
iron-specific chelators, such as ferrioxamine, are suitable.
[0307] The concentration of lipid particle or lipid-nucleic acid
particle in the pharmaceutical formulations can vary widely, i.e.,
from less than about 0.01%, usually at or at least about 0.05-5% to
as much as 10 to 30% by weight and will be selected primarily by
fluid volumes, viscosities, etc., in accordance with the particular
mode of administration selected. For example, the concentration may
be increased to lower the fluid load associated with treatment.
This may be particularly desirable in patients having
atherosclerosis-associated congestive heart failure or severe
hypertension. Alternatively, complexes composed of irritating
lipids may be diluted to low concentrations to lessen inflammation
at the site of administration. In one group of embodiments, the
nucleic acid will have an attached label and will be used for
diagnosis (by indicating the presence of complementary nucleic
acid). In this instance, the amount of complexes administered will
depend upon the particular label used, the disease state being
diagnosed and the judgement of the clinician but will generally be
between about 0.01 and about 50 mg per kilogram of body weight,
preferably between about 0.1 and about 5 mg/kg of body weight.
[0308] As noted above, the lipid-therapeutic agent (e.g., nucleic
acid) particles of the invention may include polyethylene glycol
(PEG)-modified phospholipids, PEG-ceramide, or ganglioside
G.sub.M1-modified lipids or other lipids effective to prevent or
limit aggregation. Addition of such components does not merely
prevent complex aggregation. Rather, it may also provide a means
for increasing circulation lifetime and increasing the delivery of
the lipid-nucleic acid composition to the target tissues.
[0309] The present invention also provides lipid-therapeutic agent
compositions in kit form. The kit will typically be comprised of a
container that is compartmentalized for holding the various
elements of the kit. The kit will contain the particles or
pharmaceutical compositions of the present invention, preferably in
dehydrated or concentrated form, with instructions for their
rehydration or dilution and administration. In certain embodiments,
the particles comprise the active agent, while in other
embodiments, they do not.
Methods of Manufacture
[0310] The methods and compositions of the invention make use of
certain cationic lipids, the synthesis, preparation and
characterization of which is described below and in the
accompanying Examples. In addition, the present invention provides
methods of preparing lipid particles, including those associated
with a therapeutic agent, e.g., a nucleic acid. In the methods
described herein, a mixture of lipids is combined with a buffered
aqueous solution of nucleic acid to produce an intermediate mixture
containing nucleic acid encapsulated in lipid particles wherein the
encapsulated nucleic acids are present in a nucleic acid/lipid
ratio of about 3 wt % to about 25 wt %, preferably 5 to 15 wt %.
The intermediate mixture may optionally be sized to obtain
lipid-encapsulated nucleic acid particles wherein the lipid
portions are unilamellar vesicles, preferably having a diameter of
30 to 150 nm, more preferably about 40 to 90 nm. The pH is then
raised to neutralize at least a portion of the surface charges on
the lipid-nucleic acid particles, thus providing an at least
partially surface-neutralized lipid-encapsulated nucleic acid
composition.
[0311] As described above, several of these cationic lipids are
amino lipids that are charged at a pH below the pK.sub.a of the
amino group and substantially neutral at a pH above the pK.sub.a.
These cationic lipids are termed titratable cationic lipids and can
be used in the formulations of the invention using a two-step
process. First, lipid vesicles can be formed at the lower pH with
titratable cationic lipids and other vesicle components in the
presence of nucleic acids. In this manner, the vesicles will
encapsulate and entrap the nucleic acids. Second, the surface
charge of the newly formed vesicles can be neutralized by
increasing the pH of the medium to a level above the pK.sub.a of
the titratable cationic lipids present, i.e., to physiological pH
or higher. Particularly advantageous aspects of this process
include both the facile removal of any surface adsorbed nucleic
acid and a resultant nucleic acid delivery vehicle which has a
neutral surface. Liposomes or lipid particles having a neutral
surface are expected to avoid rapid clearance from circulation and
to avoid certain toxicities which are associated with cationic
liposome preparations. Additional details concerning these uses of
such titratable cationic lipids in the formulation of nucleic
acid-lipid particles are provided in U.S. Pat. Nos. 6,287,591 and
6,858,225, incorporated herein by reference.
[0312] It is further noted that the vesicles formed in this manner
provide formulations of uniform vesicle size with high content of
nucleic acids. Additionally, the vesicles have a size range of from
about 30 to about 150 nm, more preferably about 30 to about 90
nm.
[0313] Without intending to be bound by any particular theory, it
is believed that the very high efficiency of nucleic acid
encapsulation is a result of electrostatic interaction at low pH.
At acidic pH (e.g. pH 4.0) the vesicle surface is charged and binds
a portion of the nucleic acids through electrostatic interactions.
When the external acidic buffer is exchanged for a more neutral
buffer (e.g., pH 7.5) the surface of the lipid particle or liposome
is neutralized, allowing any external nucleic acid to be removed.
More detailed information on the formulation process is provided in
various publications (e.g., U.S. Pat. Nos. 6,287,591 and
6,858,225).
[0314] In view of the above, the present invention provides methods
of preparing lipid/nucleic acid formulations. In the methods
described herein, a mixture of lipids is combined with a buffered
aqueous solution of nucleic acid to produce an intermediate mixture
containing nucleic acid encapsulated in lipid particles, e.g.,
wherein the encapsulated nucleic acids are present in a nucleic
acid/lipid ratio of about 10 wt % to about 20 wt %. The
intermediate mixture may optionally be sized to obtain
lipid-encapsulated nucleic acid particles wherein the lipid
portions are unilamellar vesicles, preferably having a diameter of
30 to 150 nm, more preferably about 40 to 90 nm. The pH is then
raised to neutralize at least a portion of the surface charges on
the lipid-nucleic acid particles, thus providing an at least
partially surface-neutralized lipid-encapsulated nucleic acid
composition.
[0315] In certain embodiments, the mixture of lipids includes at
least two lipid components: a first lipid component of the present
invention that is selected from among lipids which have a pKa such
that the lipid is cationic at pH below the pKa and neutral at pH
above the pKa, and a second lipid component that is selected from
among lipids that prevent particle aggregation during lipid-nucleic
acid particle formation. In particular embodiments, the amino lipid
is a novel cationic lipid of the present invention.
[0316] In preparing the nucleic acid-lipid particles of the
invention, the mixture of lipids is typically a solution of lipids
in an organic solvent. This mixture of lipids can then be dried to
form a thin film or lyophilized to form a powder before being
hydrated with an aqueous buffer to form liposomes. Alternatively,
in a preferred method, the lipid mixture can be solubilized in a
water miscible alcohol, such as ethanol, and this ethanolic
solution added to an aqueous buffer resulting in spontaneous
liposome formation. In most embodiments, the alcohol is used in the
form in which it is commercially available. For example, ethanol
can be used as absolute ethanol (100%), or as 95% ethanol, the
remainder being water. This method is described in more detail in
U.S. Pat. No. 5,976,567).
[0317] In one exemplary embodiment, the mixture of lipids is a
mixture of cationic lipids, neutral lipids (other than a cationic
lipid), a sterol (e.g., cholesterol) and a PEG-modified lipid
(e.g., a PEG-DMG or PEG-DMA) in an alcohol solvent. In preferred
embodiments, the lipid mixture consists essentially of a cationic
lipid, a neutral lipid, cholesterol and a PEG-modified lipid in
alcohol, more preferably ethanol. In further preferred embodiments,
the first solution consists of the above lipid mixture in molar
ratios of about 20-70% cationic lipid:5-45% neutral lipid:20-55%
cholesterol:0.5-15% PEG-modified lipid. In still further preferred
embodiments, the first solution consists essentially of a lipid
chosen from Table 1, DSPC, Chol and PEG-DMG or PEG-DMA, more
preferably in a molar ratio of about 20-60% cationic lipid:5-25%
DSPC:25-55% Chol:0.5-15% PEG-DMG or PEG-DMA. In particular
embodiments, the molar lipid ratio is approximately 40/10/40/10
(mol % cationic lipid/DSPC/Chol/PEG-DMG or PEG-DMA), 35/15/40/10
(mol % cationic lipid/DSPC/Chol/PEG-DMG or PEG-DMA) or 52/13/30/5
(mol % cationic lipid/DSPC/Chol/PEG-DMG or PEG-DMA). In another
group of preferred embodiments, the neutral lipid in these
compositions is replaced with POPC, DPPC, DOPE or SM.
[0318] In accordance with the invention, the lipid mixture is
combined with a buffered aqueous solution that may contain the
nucleic acids. The buffered aqueous solution of is typically a
solution in which the buffer has a pH of less than the pK.sub.a of
the protonatable lipid in the lipid mixture. Examples of suitable
buffers include citrate, phosphate, acetate, and MES. A
particularly preferred buffer is citrate buffer. Preferred buffers
will be in the range of 1-1000 mM of the anion, depending on the
chemistry of the nucleic acid being encapsulated, and optimization
of buffer concentration may be significant to achieving high
loading levels (see, e.g., U.S. Pat. Nos. 6,287,591 and 6,858,225).
Alternatively, pure water acidified to pH 5-6 with chloride,
sulfate or the like may be useful. In this case, it may be suitable
to add 5% glucose, or another non-ionic solute which will balance
the osmotic potential across the particle membrane when the
particles are dialyzed to remove ethanol, increase the pH, or mixed
with a pharmaceutically acceptable carrier such as normal saline.
The amount of nucleic acid in buffer can vary, but will typically
be from about 0.01 mg/mL to about 200 mg/mL, more preferably from
about 0.5 mg/mL to about 50 mg/mL.
[0319] The mixture of lipids and the buffered aqueous solution of
therapeutic nucleic acids is combined to provide an intermediate
mixture. The intermediate mixture is typically a mixture of lipid
particles having encapsulated nucleic acids. Additionally, the
intermediate mixture may also contain some portion of nucleic acids
which are attached to the surface of the lipid particles (liposomes
or lipid vesicles) due to the ionic attraction of the
negatively-charged nucleic acids and positively-charged lipids on
the lipid particle surface (the amino lipids or other lipid making
up the protonatable first lipid component are positively charged in
a buffer having a pH of less than the pK.sub.a of the protonatable
group on the lipid). In one group of preferred embodiments, the
mixture of lipids is an alcohol solution of lipids and the volumes
of each of the solutions is adjusted so that upon combination, the
resulting alcohol content is from about 20% by volume to about 45%
by volume. The method of combining the mixtures can include any of
a variety of processes, often depending upon the scale of
formulation produced. For example, when the total volume is about
10-20 mL or less, the solutions can be combined in a test tube and
stirred together using a vortex mixer. Large-scale processes can be
carried out in suitable production scale glassware.
[0320] Optionally, the lipid-encapsulated therapeutic agent (e.g.,
nucleic acid) complexes which are produced by combining the lipid
mixture and the buffered aqueous solution of therapeutic agents
(nucleic acids) can be sized to achieve a desired size range and
relatively narrow distribution of lipid particle sizes. Preferably,
the compositions provided herein will be sized to a mean diameter
of from about 70 to about 200 nm, more preferably about 90 to about
130 nm. Several techniques are available for sizing liposomes to a
desired size. One sizing method is described in U.S. Pat. No.
4,737,323, incorporated herein by reference. Sonicating a liposome
suspension either by bath or probe sonication produces a
progressive size reduction down to small unilamellar vesicles
(SUVs) less than about 0.05 microns in size. Homogenization is
another method which relies on shearing energy to fragment large
liposomes into smaller ones. In a typical homogenization procedure,
multilamellar vesicles are recirculated through a standard emulsion
homogenizer until selected liposome sizes, typically between about
0.1 and 0.5 microns, are observed. In both methods, the particle
size distribution can be monitored by conventional laser-beam
particle size determination. For certain methods herein, extrusion
is used to obtain a uniform vesicle size.
[0321] Extrusion of liposome compositions through a small-pore
polycarbonate membrane or an asymmetric ceramic membrane results in
a relatively well-defined size distribution. Typically, the
suspension is cycled through the membrane one or more times until
the desired liposome complex size distribution is achieved. The
liposomes may be extruded through successively smaller-pore
membranes, to achieve a gradual reduction in liposome size. In some
instances, the lipid-nucleic acid compositions which are formed can
be used without any sizing.
[0322] In particular embodiments, methods of the present invention
further comprise a step of neutralizing at least some of the
surface charges on the lipid portions of the lipid-nucleic acid
compositions. By at least partially neutralizing the surface
charges, unencapsulated nucleic acid is freed from the lipid
particle surface and can be removed from the composition using
conventional techniques. Preferably, unencapsulated and surface
adsorbed nucleic acids are removed from the resulting compositions
through exchange of buffer solutions. For example, replacement of a
citrate buffer (pH about 4.0, used for forming the compositions)
with a HEPES-buffered saline (HBS pH about 7.5) solution, results
in the neutralization of liposome surface and nucleic acid release
from the surface. The released nucleic acid can then be removed via
chromatography using standard methods, and then switched into a
buffer with a pH above the pKa of the lipid used.
[0323] Optionally the lipid vesicles (i.e., lipid particles) can be
formed by hydration in an aqueous buffer and sized using any of the
methods described above prior to addition of the nucleic acid. As
described above, the aqueous buffer should be of a pH below the pKa
of the amino lipid. A solution of the nucleic acids can then be
added to these sized, preformed vesicles. To allow encapsulation of
nucleic acids into such "pre-formed" vesicles the mixture should
contain an alcohol, such as ethanol. In the case of ethanol, it
should be present at a concentration of about 20% (w/w) to about
45% (w/w). In addition, it may be necessary to warm the mixture of
pre-formed vesicles and nucleic acid in the aqueous buffer-ethanol
mixture to a temperature of about 25.degree. C. to about 50.degree.
C. depending on the composition of the lipid vesicles and the
nature of the nucleic acid. It will be apparent to one of ordinary
skill in the art that optimization of the encapsulation process to
achieve a desired level of nucleic acid in the lipid vesicles will
require manipulation of variable such as ethanol concentration and
temperature. Examples of suitable conditions for nucleic acid
encapsulation are provided in the Examples. Once the nucleic acids
are encapsulated within the preformed vesicles, the external pH can
be increased to at least partially neutralize the surface charge.
Unencapsulated and surface adsorbed nucleic acids can then be
removed as described above.
Method of Use
[0324] The lipid particles of the present invention may be used to
deliver a therapeutic agent to a cell, in vitro or in vivo. In
particular embodiments, the therapeutic agent is a nucleic acid,
which is delivered to a cell using a nucleic acid-lipid particles
of the present invention. While the following description o various
methods of using the lipid particles and related pharmaceutical
compositions of the present invention are exemplified by
description related to nucleic acid-lipid particles, it is
understood that these methods and compositions may be readily
adapted for the delivery of any therapeutic agent for the treatment
of any disease or disorder that would benefit from such
treatment.
[0325] In certain embodiments, the present invention provides
methods for introducing a nucleic acid into a cell. Preferred
nucleic acids for introduction into cells are siRNA,
immune-stimulating oligonucleotides, plasmids, antisense and
ribozymes. These methods may be carried out by contacting the
particles or compositions of the present invention with the cells
for a period of time sufficient for intracellular delivery to
occur.
[0326] The compositions of the present invention can be adsorbed to
almost any cell type. Once adsorbed, the nucleic acid-lipid
particles can either be endocytosed by a portion of the cells,
exchange lipids with cell membranes, or fuse with the cells.
Transfer or incorporation of the nucleic acid portion of the
complex can take place via any one of these pathways. Without
intending to be limited with respect to the scope of the invention,
it is believed that in the case of particles taken up into the cell
by endocytosis the particles then interact with the endosomal
membrane, resulting in destabilization of the endosomal membrane,
possibly by the formation of non-bilayer phases, resulting in
introduction of the encapsulated nucleic acid into the cell
cytoplasm. Similarly in the case of direct fusion of the particles
with the cell plasma membrane, when fusion takes place, the
liposome membrane is integrated into the cell membrane and the
contents of the liposome combine with the intracellular fluid.
Contact between the cells and the lipid-nucleic acid compositions,
when carried out in vitro, will take place in a biologically
compatible medium. The concentration of compositions can vary
widely depending on the particular application, but is generally
between about 1 .mu.mol and about 10 mmol. In certain embodiments,
treatment of the cells with the lipid-nucleic acid compositions
will generally be carried out at physiological temperatures (about
37.degree. C.) for periods of time from about 1 to 24 hours,
preferably from about 2 to 8 hours. For in vitro applications, the
delivery of nucleic acids can be to any cell grown in culture,
whether of plant or animal origin, vertebrate or invertebrate, and
of any tissue or type. In preferred embodiments, the cells will be
animal cells, more preferably mammalian cells, and most preferably
human cells.
[0327] In one group of embodiments, a lipid-nucleic acid particle
suspension is added to 60-80% confluent plated cells having a cell
density of from about 10.sup.3 to about 10.sup.5 cells/mL, more
preferably about 2.times.10.sup.4 cells/mL. The concentration of
the suspension added to the cells is preferably of from about 0.01
to 20 .mu.g/mL, more preferably about 1 .mu.g/mL.
[0328] In another embodiment, the lipid particles of the invention
can be may be used to deliver a nucleic acid to a cell or cell line
(for example, a tumor cell line). Non-limiting examples of such
cell lines include: HELA (ATCC Cat N: CCL-2), KB (ATCC Cat N:
CCL-17), HEP3B (ATCC Cat N: HB-8064), SKOV-3 (ATCC Cat N: HTB-77),
HCT-116 (ATCC Cat N: CCL-247), HT-29 (ATCC Cat N: HTB-38), PC-3
(ATCC Cat N: CRL-1435), A549 (ATCC Cat N: CCL-185), MDA-MB-231
(ATCC Cat N: HTB-26).
[0329] Typical applications include using well known procedures to
provide intracellular delivery of siRNA to knock down or silence
specific cellular targets. Alternatively applications include
delivery of DNA or mRNA sequences that code for therapeutically
useful polypeptides. In this manner, therapy is provided for
genetic diseases by supplying deficient or absent gene products
(i.e., for Duchenne's dystrophy, see Kunkel, et al., Brit. Med.
Bull. 45(3):630-643 (1989), and for cystic fibrosis, see
Goodfellow, Nature 341:102-103 (1989)). Other uses for the
compositions of the present invention include introduction of
antisense oligonucleotides in cells (see, Bennett, et al., Mol.
Pharm. 41:1023-1033 (1992)).
[0330] Alternatively, the compositions of the present invention can
also be used for deliver of nucleic acids to cells in vivo, using
methods which are known to those of skill in the art. With respect
to delivery of DNA or mRNA sequences, Zhu, et al., Science
261:209-211 (1993), incorporated herein by reference, describes the
intravenous delivery of cytomegalovirus (CMV)-chloramphenicol
acetyltransferase (CAT) expression plasmid using DOTMA-DOPE
complexes. Hyde, et al., Nature 362:250-256 (1993), incorporated
herein by reference, describes the delivery of the cystic fibrosis
transmembrane conductance regulator (CFTR) gene to epithelia of the
airway and to alveoli in the lung of mice, using liposomes.
Brigham, et al., Am. J. Med. Sci. 298:278-281 (1989), incorporated
herein by reference, describes the in vivo transfection of lungs of
mice with a functioning prokaryotic gene encoding the intracellular
enzyme, chloramphenicol acetyltransferase (CAT). Thus, the
compositions of the invention can be used in the treatment of
infectious diseases.
[0331] For in vivo administration, the pharmaceutical compositions
are preferably administered parenterally, i.e., intraarticularly,
intravenously, intraperitoneally, subcutaneously, or
intramuscularly. In particular embodiments, the pharmaceutical
compositions are administered intravenously or intraperitoneally by
a bolus injection. For one example, see Stadler, et al., U.S. Pat.
No. 5,286,634, which is incorporated herein by reference.
Intracellular nucleic acid delivery has also been discussed in
Straubringer, et al., METHODS IN ENZYMOLOGY, Academic Press, New
York. 101:512-527 (1983); Mannino, et al., Biotechniques 6:682-690
(1988); Nicolau, et al., Crit. Rev. Ther. Drug Carrier Syst.
6:239-271 (1989), and Behr, Acc. Chem. Res. 26:274-278 (1993).
Still other methods of administering lipid-based therapeutics are
described in, for example, Rahman et al., U.S. Pat. No. 3,993,754;
Sears, U.S. Pat. No. 4,145,410; Papahadjopoulos et al., U.S. Pat.
No. 4,235,871; Schneider, U.S. Pat. No. 4,224,179; Lenk et al.,
U.S. Pat. No. 4,522,803; and Fountain et al., U.S. Pat. No.
4,588,578.
[0332] In other methods, the pharmaceutical preparations may be
contacted with the target tissue by direct application of the
preparation to the tissue. The application may be made by topical,
"open" or "closed" procedures. By "topical," it is meant the direct
application of the pharmaceutical preparation to a tissue exposed
to the environment, such as the skin, oropharynx, external auditory
canal, and the like. "Open" procedures are those procedures which
include incising the skin of a patient and directly visualizing the
underlying tissue to which the pharmaceutical preparations are
applied. This is generally accomplished by a surgical procedure,
such as a thoracotomy to access the lungs, abdominal laparotomy to
access abdominal viscera, or other direct surgical approach to the
target tissue. "Closed" procedures are invasive procedures in which
the internal target tissues are not directly visualized, but
accessed via inserting instruments through small wounds in the
skin. For example, the preparations may be administered to the
peritoneum by needle lavage. Likewise, the pharmaceutical
preparations may be administered to the meninges or spinal cord by
infusion during a lumbar puncture followed by appropriate
positioning of the patient as commonly practiced for spinal
anesthesia or metrazamide imaging of the spinal cord.
Alternatively, the preparations may be administered through
endoscopic devices.
[0333] The lipid-nucleic acid compositions can also be administered
in an aerosol inhaled into the lungs (see, Brigham, et al., Am. J.
Sci. 298(4):278-281 (1989)) or by direct injection at the site of
disease (Culver, Human Gene Therapy, MaryAnn Liebert, Inc.,
Publishers, New York. pp. 70-71 (1994)).
[0334] The methods of the present invention may be practiced in a
variety of hosts. Preferred hosts include mammalian species, such
as humans, non-human primates, dogs, cats, cattle, horses, sheep,
and the like.
[0335] Dosages for the lipid-therapeutic agent particles of the
present invention will depend on the ratio of therapeutic agent to
lipid and the administrating physician's opinion based on age,
weight, and condition of the patient.
[0336] In one embodiment, the present invention provides a method
of modulating the expression of a target polynucleotide or
polypeptide. These methods generally comprise contacting a cell
with a lipid particle of the present invention that is associated
with a nucleic acid capable of modulating the expression of a
target polynucleotide or polypeptide. As used herein, the term
"modulating" refers to altering the expression of a target
polynucleotide or polypeptide. In different embodiments, modulating
can mean increasing or enhancing, or it can mean decreasing or
reducing. Methods of measuring the level of expression of a target
polynucleotide or polypeptide are known and available in the arts
and include, e.g., methods employing reverse
transcription-polymerase chain reaction (RT-PCR) and
immunohistochemical techniques. In particular embodiments, the
level of expression of a target polynucleotide or polypeptide is
increased or reduced by at least 10%, 20%, 30%, 40%, 50%, or
greater than 50% as compared to an appropriate control value.
[0337] For example, if increased expression of a polypeptide
desired, the nucleic acid may be an expression vector that includes
a polynucleotide that encodes the desired polypeptide. On the other
hand, if reduced expression of a polynucleotide or polypeptide is
desired, then the nucleic acid may be, e.g., an antisense
oligonucleotide, siRNA, or microRNA that comprises a polynucleotide
sequence that specifically hybridizes to a polynucleotide that
encodes the target polypeptide, thereby disrupting expression of
the target polynucleotide or polypeptide. Alternatively, the
nucleic acid may be a plasmid that expresses such an antisense
oligonucleotide, siRNA, or microRNA.
[0338] In one particular embodiment, the present invention provides
a method of modulating the expression of a polypeptide by a cell,
comprising providing to a cell a lipid particle that consists of or
consists essentially of a lipid chosen from Table 1, DSPC, Chol and
PEG-DMG or PEG-DMA, e.g., in a molar ratio of about 20-60% cationic
lipid:5-25% DSPC:25-55% Chol:0.5-15% PEG-DMG or PEG-DMA, wherein
the lipid particle is associated with a nucleic acid capable of
modulating the expression of the polypeptide. In particular
embodiments, the molar lipid ratio is approximately 40/10/40/10
(mol % cationic lipid/DSPC/Chol/PEG-DMG or PEG-DMA), 35/15/40/10
(mol % cationic lipid/DSPC/Chol/PEG-DMG or PEG-DMA) or 52/13/30/5
(mol % cationic lipid/DSPC/Chol/PEG-DMG or PEG-DMA). In another
group of embodiments, the neutral lipid in these compositions is
replaced with POPC, DPPC, DOPE or SM.
[0339] In particular embodiments, the therapeutic agent is selected
from an siRNA, a microRNA, an antisense oligonucleotide, and a
plasmid capable of expressing an siRNA, a microRNA, or an antisense
oligonucleotide, and wherein the siRNA, microRNA, or antisense RNA
comprises a polynucleotide that specifically binds to a
polynucleotide that encodes the polypeptide, or a complement
thereof, such that the expression of the polypeptide is
reduced.
[0340] In other embodiments, the nucleic acid is a plasmid that
encodes the polypeptide or a functional variant or fragment
thereof, such that expression of the polypeptide or the functional
variant or fragment thereof is increased.
[0341] In related embodiments, the present invention provides a
method of treating a disease or disorder characterized by
overexpression of a polypeptide in a subject, comprising providing
to the subject a pharmaceutical composition of the present
invention, wherein the therapeutic agent is selected from an siRNA,
a microRNA, an antisense oligonucleotide, and a plasmid capable of
expressing an siRNA, a microRNA, or an antisense oligonucleotide,
and wherein the siRNA, microRNA, or antisense RNA comprises a
polynucleotide that specifically binds to a polynucleotide that
encodes the polypeptide, or a complement thereof.
[0342] In one embodiment, the pharmaceutical composition comprises
a lipid particle that consists of or consists essentially of a
lipid chosen from Table 1, DSPC, Chol and PEG-DMG or PEG-DMA, e.g.,
in a molar ratio of about 20-60% cationic lipid:5-25% DSPC:25-55%
Chol:0.5-15% PEG-DMG or PEG-DMA, wherein the lipid particle is
associated with the therapeutic nucleic acid. In particular
embodiments, the molar lipid ratio is approximately 40/10/40/10
(mol % cationic lipid/DSPC/Chol/PEG-DMG or PEG-DMA), 35/15/40/10
(mol % cationic lipid/DSPC/Chol/PEG-DMG or PEG-DMA) or 52/13/30/5
(mol % cationic lipid/DSPC/Chol/PEG-DMG or PEG-DMA). In another
group of embodiments, the neutral lipid in these compositions is
replaced with POPC, DPPC, DOPE or SM.
[0343] In another related embodiment, the present invention
includes a method of treating a disease or disorder characterized
by underexpression of a polypeptide in a subject, comprising
providing to the subject a pharmaceutical composition of the
present invention, wherein the therapeutic agent is a plasmid that
encodes the polypeptide or a functional variant or fragment
thereof.
[0344] In one embodiment, the pharmaceutical composition comprises
a lipid particle that consists of or consists essentially of a
lipid chosen from Table 1, DSPC, Chol and PEG-DMG or PEG-DMA, e.g.,
in a molar ratio of about 20-60% cationic lipid:5-25% DSPC:25-55%
Chol:0.5-15% PEG-DMG or PEG-DMA, wherein the lipid particle is
associated with the therapeutic nucleic acid. In particular
embodiments, the molar lipid ratio is approximately 40/10/40/10
(mol % cationic lipid/DSPC/Chol/PEG-DMG or PEG-DMA), 35/15/40/10
(mol % cationic lipid/DSPC/Chol/PEG-DMG or PEG-DMA) or 52/13/30/5
(mol % cationic lipid/DSPC/Chol/PEG-DMG or PEG-DMA). In another
group of embodiments, the neutral lipid in these compositions is
replaced with POPC, DPPC, DOPE or SM.
[0345] The present invention further provides a method of inducing
an immune response in a subject, comprising providing to the
subject the pharmaceutical composition of the present invention,
wherein the therapeutic agent is an immunostimulatory
oligonucleotide. In certain embodiments, the immune response is a
humoral or mucosal immune response. In one embodiment, the
pharmaceutical composition comprises a lipid particle that consists
of or consists essentially of a lipid chosen from Table 1, DSPC,
Chol and PEG-DMG or PEG-DMA, e.g., in a molar ratio of about 20-60%
cationic lipid:5-25% DSPC:25-55% Chol:0.5-15% PEG-DMG or PEG-DMA,
wherein the lipid particle is associated with the therapeutic
nucleic acid. In particular embodiments, the molar lipid ratio is
approximately 40/10/40/10 (mol % cationic lipid/DSPC/Chol/PEG-DMG
or PEG-DMA), 35/15/40/10 (mol % cationic lipid/DSPC/Chol/PEG-DMG or
PEG-DMA) or 52/13/30/5 (mol % cationic lipid/DSPC/Chol/PEG-DMG or
PEG-DMA). In another group of embodiments, the neutral lipid in
these compositions is replaced with POPC, DPPC, DOPE or SM.
[0346] In further embodiments, the pharmaceutical composition is
provided to the subject in combination with a vaccine or antigen.
Thus, the present invention itself provides vaccines comprising a
lipid particle of the present invention, which comprises an
immunostimulatory oligonucleotide, and is also associated with an
antigen to which an immune response is desired. In particular
embodiments, the antigen is a tumor antigen or is associated with
an infective agent, such as, e.g., a virus, bacteria, or
parasite.
[0347] A variety of tumor antigens, infections agent antigens, and
antigens associated with other disease are well known in the art
and examples of these are described in references cited herein.
Examples of antigens suitable for use in the present invention
include, but are not limited to, polypeptide antigens and DNA
antigens. Specific examples of antigens are Hepatitis A, Hepatitis
B, small pox, polio, anthrax, influenza, typhus, tetanus, measles,
rotavirus, diphtheria, pertussis, tuberculosis, and rubella
antigens. In a preferred embodiment, the antigen is a Hepatitis B
recombinant antigen. In other aspects, the antigen is a Hepatitis A
recombinant antigen. In another aspect, the antigen is a tumor
antigen. Examples of such tumor-associated antigens are MUC-1, EBV
antigen and antigens associated with Burkitt's lymphoma. In a
further aspect, the antigen is a tyrosinase-related protein tumor
antigen recombinant antigen. Those of skill in the art will know of
other antigens suitable for use in the present invention.
[0348] Tumor-associated antigens suitable for use in the subject
invention include both mutated and non-mutated molecules that may
be indicative of single tumor type, shared among several types of
tumors, and/or exclusively expressed or overexpressed in tumor
cells in comparison with normal cells. In addition to proteins and
glycoproteins, tumor-specific patterns of expression of
carbohydrates, gangliosides, glycolipids and mucins have also been
documented. Exemplary tumor-associated antigens for use in the
subject cancer vaccines include protein products of oncogenes,
tumor suppressor genes and other genes with mutations or
rearrangements unique to tumor cells, reactivated embryonic gene
products, oncofetal antigens, tissue-specific (but not
tumor-specific) differentiation antigens, growth factor receptors,
cell surface carbohydrate residues, foreign viral proteins and a
number of other self proteins.
[0349] Specific embodiments of tumor-associated antigens include,
e.g., mutated antigens such as the protein products of the Ras p21
protooncogenes, tumor suppressor p53 and BCR-abl oncogenes, as well
as CDK4, MUM1, Caspase 8, and Beta catenin; overexpressed antigens
such as galectin 4, galectin 9, carbonic anhydrase, Aldolase A,
PRAME, Her2/neu, ErbB-2 and KSA, oncofetal antigens such as alpha
fetoprotein (AFP), human chorionic gonadotropin (hCG); self
antigens such as carcinoembryonic antigen (CEA) and melanocyte
differentiation antigens such as Mart 1/Melan A, gp100, gp75,
Tyrosinase, TRP1 and TRP2; prostate associated antigens such as
PSA, PAP, PSMA, PSM-P1 and PSM-P2; reactivated embryonic gene
products such as MAGE 1, MAGE 3, MAGE 4, GAGE 1, GAGE 2, BAGE,
RAGE, and other cancer testis antigens such as NY-ESO1, SSX2 and
SCP1; mucins such as Muc-1 and Muc-2; gangliosides such as GM2, GD2
and GD3, neutral glycolipids and glycoproteins such as Lewis (y)
and globo-H; and glycoproteins such as Tn, Thompson-Freidenreich
antigen (TF) and sTn. Also included as tumor-associated antigens
herein are whole cell and tumor cell lysates as well as immunogenic
portions thereof, as well as immunoglobulin idiotypes expressed on
monoclonal proliferations of B lymphocytes for use against B cell
lymphomas.
[0350] Pathogens include, but are not limited to, infectious
agents, e.g., viruses, that infect mammals, and more particularly
humans. Examples of infectious virus include, but are not limited
to: Retroviridae (e.g., human immunodeficiency viruses, such as
HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or
HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g.,
polio viruses, hepatitis A virus; enteroviruses, human Coxsackie
viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains
that cause gastroenteritis); Togaviridae (e.g., equine encephalitis
viruses, rubella viruses); Flaviridae (e.g., dengue viruses,
encephalitis viruses, yellow fever viruses); Coronoviridae (e.g.,
coronaviruses); Rhabdoviradae (e.g., vesicular stomatitis viruses,
rabies viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae
(e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae
(e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza
viruses, mumps virus, measles virus, respiratory syncytial virus);
Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g.,
Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses);
Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g.,
reoviruses, orbiviurses and rotaviruses); Birnaviridae;
Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses);
Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae
(most adenoviruses); Herpesviridae herpes simplex virus (HSV) 1 and
2, varicella zoster virus, cytomegalovirus (CMV), herpes virus;
Poxviridae (variola viruses, vaccinia viruses, pox viruses); and
Iridoviridae (e.g., African swine fever virus); and unclassified
viruses (e.g., the etiological agents of Spongiform
encephalopathies, the agent of delta hepatitis (thought to be a
defective satellite of hepatitis B virus), the agents of non-A,
non-B hepatitis (class 1=internally transmitted; class
2=parenterally transmitted (i.e., Hepatitis C); Norwalk and related
viruses, and astroviruses).
[0351] Also, gram negative and gram positive bacteria serve as
antigens in vertebrate animals. Such gram positive bacteria
include, but are not limited to Pasteurella species, Staphylococci
species, and Streptococcus species. Gram negative bacteria include,
but are not limited to, Escherichia coli, Pseudomonas species, and
Salmonella species. Specific examples of infectious bacteria
include but are not limited to: Helicobacter pyloris, Borelia
burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g., M.
tuberculosis, M. avium, M. intracellulare, M. kansaii, M.
gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria
meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group
A Streptococcus), Streptococcus agalactiae (Group B Streptococcus),
Streptococcus (viridans group), Streptococcus faecalis,
Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus
pneumoniae, pathogenic Campylobacter sp., Enterococcus sp.,
Haemophilus infuenzae, Bacillus antracis, Corynebacterium
diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae,
Clostridium perfringers, Clostridium tetani, Enterobacter
aerogenes, Klebsiella pneumoniae, Pasteurella multocida,
Bacteroides sp., Fusobacterium nucleatum, Streptobacillus
moniliformis, Treponema pallidium, Treponema pertenue, Leptospira,
Rickettsia, and Actinomyces israelli.
[0352] Additional examples of pathogens include, but are not
limited to, infectious fungi that infect mammals, and more
particularly humans. Examples of infectious fingi include, but are
not limited to: Cryptococcus neoformans, Histoplasma capsulatum,
Coccidioides immitis, Blastomyces dermatitidis, Chlamydia
trachomatis, Candida albicans. Examples of infectious parasites
include Plasmodium such as Plasmodium falciparum, Plasmodium
malariae, Plasmodium ovale, and Plasmodium vivax. Other infectious
organisms (i.e., protists) include Toxoplasma gondii.
[0353] In one embodiment, the formulations of the invention can be
used to silence or modulate a target gene such as but not limited
to FVII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene,
Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK
gene, RAF gene, Erk1/2 gene, PCNA(p21) gene, MYB gene, JUN gene,
FOS gene, BCL-2 gene, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A
gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC
gene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu gene, SORT1
gene, XBP1 gene, topoisomerase I gene, topoisomerase II alpha gene,
p73 gene, p21(WAF1/CIP1) gene, p27(KIP1) gene, PPM1D gene, RAS
gene, caveolin I gene, MIB I gene, MTAI gene, M68 gene, tumor
suppressor genes, p53 tumor suppressor gene, p53 family member
DN-p63, pRb tumor suppressor gene, APC1 tumor suppressor gene,
BRCA1 tumor suppressor gene, PTEN tumor suppressor gene, mLL fusion
gene, BCR/ABL fusion gene, TEL/AML1 fusion gene, EWS/FLI1 fusion
gene, TLS/FUS1 fusion gene, PAX3/FKHR fusion gene, AML1/ETO fusion
gene, alpha v-integrin gene, Flt-1 receptor gene, tubulin gene,
Human Papilloma Virus gene, a gene required for Human Papilloma
Virus replication, Human Immunodeficiency Virus gene, a gene
required for Human Immunodeficiency Virus replication, Hepatitis A
Virus gene, a gene required for Hepatitis A Virus replication,
Hepatitis B Virus gene, a gene required for Hepatitis B Virus
replication, Hepatitis C Virus gene, a gene required for Hepatitis
C Virus replication, Hepatitis D Virus gene, a gene required for
Hepatitis D Virus replication, Hepatitis E Virus gene, a gene
required for Hepatitis E Virus replication, Hepatitis F Virus gene,
a gene required for Hepatitis F Virus replication, Hepatitis G
Virus gene, a gene required for Hepatitis G Virus replication,
Hepatitis H Virus gene, a gene required for Hepatitis H Virus
replication, Respiratory Syncytial Virus gene, a gene that is
required for Respiratory Syncytial Virus replication, Herpes
Simplex Virus gene, a gene that is required for Herpes Simplex
Virus replication, herpes Cytomegalovirus gene, a gene that is
required for herpes Cytomegalovirus replication, herpes Epstein
Barr Virus gene, a gene that is required for herpes Epstein Barr
Virus replication, Kaposi's Sarcoma-associated Herpes Virus gene, a
gene that is required for Kaposi's Sarcoma-associated Herpes Virus
replication, JC Virus gene, human gene that is required for JC
Virus replication, myxovirus gene, a gene that is required for
myxovirus gene replication, rhinovirus gene, a gene that is
required for rhinovirus replication, coronavirus gene, a gene that
is required for coronavirus replication, West Nile Virus gene, a
gene that is required for West Nile Virus replication, St. Louis
Encephalitis gene, a gene that is required for St. Louis
Encephalitis replication, Tick-borne encephalitis virus gene, a
gene that is required for Tick-borne encephalitis virus
replication, Murray Valley encephalitis virus gene, a gene that is
required for Murray Valley encephalitis virus replication, dengue
virus gene, a gene that is required for dengue virus gene
replication, Simian Virus 40 gene, a gene that is required for
Simian Virus 40 replication, Human T Cell Lymphotrophic Virus gene,
a gene that is required for Human T Cell Lymphotrophic Virus
replication, Moloney-Murine Leukemia Virus gene, a gene that is
required for Moloney-Murine Leukemia Virus replication,
encephalomyocarditis virus gene, a gene that is required for
encephalomyocarditis virus replication, measles virus gene, a gene
that is required for measles virus replication, Varicella zoster
virus gene, a gene that is required for Varicella zoster virus
replication, adenovirus gene, a gene that is required for
adenovirus replication, yellow fever virus gene, a gene that is
required for yellow fever virus replication, poliovirus gene, a
gene that is required for poliovirus replication, poxvirus gene, a
gene that is required for poxvirus replication, plasmodium gene, a
gene that is required for plasmodium gene replication,
Mycobacterium ulcerans gene, a gene that is required for
Mycobacterium ulcerans replication, Mycobacterium tuberculosis
gene, a gene that is required for Mycobacterium tuberculosis
replication, Mycobacterium leprae gene, a gene that is required for
Mycobacterium leprae replication, Staphylococcus aureus gene, a
gene that is required for Staphylococcus aureus replication,
Streptococcus pneumoniae gene, a gene that is required for
Streptococcus pneumoniae replication, Streptococcus pyogenes gene,
a gene that is required for Streptococcus pyogenes replication,
Chlamydia pneumoniae gene, a gene that is required for Chlamydia
pneumoniae replication, Mycoplasma pneumoniae gene, a gene that is
required for Mycoplasma pneumoniae replication, an integrin gene, a
selectin gene, complement system gene, chemokine gene, chemokine
receptor gene, GCSF gene, Gro1 gene, Gro2 gene, Gro3 gene, PF4
gene, MIG gene, Pro-Platelet Basic Protein gene, MIP-1I gene,
MIP-1J gene, RANTES gene, MCP-1 gene, MCP-2 gene, MCP-3 gene,
CMBKR1 gene, CMBKR2 gene, CMBKR3 gene, CMBKR5v, AIF-1 gene, 1-309
gene, a gene to a component of an ion channel, a gene to a
neurotransmitter receptor, a gene to a neurotransmitter ligand,
amyloid-family gene, presenilin gene, HD gene, DRPLA gene, SCA1
gene, SCA2 gene, MJD1 gene, CACNL1A4 gene, SCAT gene, SCA8 gene,
allele gene found in LOH cells, or one allele gene of a polymorphic
gene.
Definitions
[0354] "Alkyl" means a straight chain or branched, noncyclic or
cyclic, saturated aliphatic hydrocarbon containing from 1 to 24
carbon atoms. Representative saturated straight chain alkyls
include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and
the like; while saturated branched alkyls include isopropyl,
sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.
Representative saturated cyclic alkyls include cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, and the like; while
unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl,
and the like.
[0355] "Alkenyl" means an alkyl, as defined above, containing at
least one double bond between adjacent carbon atoms. Alkenyls
include both cis and trans isomers. Representative straight chain
and branched alkenyls include ethylenyl, propylenyl, 1-butenyl,
2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,
3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and
the like.
[0356] "Alkynyl" means any alkyl or alkenyl, as defined above,
which additionally contains at least one triple bond between
adjacent carbons. Representative straight chain and branched
alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl,
1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.
[0357] The term "acyl" refers to hydrogen, alkyl, partially
saturated or fully saturated cycloalkyl, partially saturated or
fully saturated heterocycle, aryl, and heteroaryl substituted
carbonyl groups. For example, acyl includes groups such as
(C1-C20)alkanoyl (e.g., formyl, acetyl, propionyl, butyryl,
valeryl, caproyl, t-butylacetyl, etc.), (C3-C20)cycloalkylcarbonyl
(e.g., cyclopropylcarbonyl, cyclobutylcarbonyl,
cyclopentylcarbonyl, cyclohexylcarbonyl, etc.), heterocyclic
carbonyl (e.g., pyrrolidinylcarbonyl, pyrrolid-2-one-5-carbonyl,
piperidinylcarbonyl, piperazinylcarbonyl,
tetrahydrofuranylcarbonyl, etc.), aroyl (e.g., benzoyl) and
heteroaroyl (e.g., thiophenyl-2-carbonyl, thiophenyl-3-carbonyl,
furanyl-2-carbonyl, furanyl-3-carbonyl, 1H-pyrroyl-2-carbonyl,
1H-pyrroyl-3-carbonyl, benzo[b]thiophenyl-2-carbonyl, etc.).
[0358] The term "aryl" refers to an aromatic monocyclic, bicyclic,
or tricyclic hydrocarbon ring system, wherein any ring atom can be
substituted. Examples of aryl moieties include, but are not limited
to, phenyl, naphthyl, anthracenyl, and pyrenyl.
[0359] "Heterocycle" means a 5- to 7-membered monocyclic, or 7- to
10-membered bicyclic, heterocyclic ring which is either saturated,
unsaturated, or aromatic, and which contains from 1 or 2
heteroatoms independently selected from nitrogen, oxygen and
sulfur, and wherein the nitrogen and sulfur heteroatoms may be
optionally oxidized, and the nitrogen heteroatom may be optionally
quaternized, including bicyclic rings in which any of the above
heterocycles are fused to a benzene ring. The heterocycle may be
attached via any heteroatom or carbon atom. Heterocycles include
heteroaryls as defined below. Heterocycles include morpholinyl,
pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,
hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,
tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,
tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,
tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
[0360] The term "heteroaryl" refers to an aromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein any ring atom can be substituted. The
heteroaryl groups herein described may also contain fused rings
that share a common carbon-carbon bond. The term "alkylheterocycle"
refers to a heteroaryl wherein at least one of the ring atoms is
substituted with alkyl, alkenyl or alkynyl
[0361] The term "substituted" refers to the replacement of one or
more hydrogen radicals in a given structure with the radical of a
specified substituent including, but not limited to: halo, alkyl,
alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, oxo,
thioxy, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl,
alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy,
aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,
alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl,
cyano, nitro, alkylamino, arylamino, alkylaminoalkyl,
arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl,
carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl,
aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl,
phosphonic acid, aryl, heteroaryl, heterocyclic, and aliphatic. It
is understood that the substituent may be further substituted.
Exemplary substituents include amino, alkylamino, dialkylamino, and
cyclic amino compounds.
[0362] "Halogen" means fluoro, chloro, bromo and iodo.
[0363] The terms "alkylamine" and "dialkylamine" refer to
--NH(alkyl) and --N (alkyl).sub.2 radicals respectively.
[0364] The term "alkylphosphate" refers to -O--P(Q')(Q'')-O--R,
wherein Q' and Q'' are each independently O, S, N(R).sub.2,
optionally substituted alkyl or alkoxy; and R is optionally
substituted alkyl, .omega.-aminoalkyl or
.omega.-(substituted)aminoalkyl.
[0365] The term "alkylphosphorothioate" refers to an alkylphosphate
wherein at least one of Q' or Q'' is S.
[0366] The term "alkylphosphonate" refers to an alkylphosphate
wherein at least one of Q' or Q'' is alkyl.
[0367] The term "hydroxyalkyl" means --O-alkyl radical.
[0368] The term "alkylheterocycle" refers to an alkyl where at
least one methylene has been replaced by a heterocycle.
[0369] The term ".omega.-aminoalkyl" refers to -alkyl-NH.sub.2
radical. And the term ".omega.-(substituted)aminoalkyl refers to an
.omega.-aminoalkyl wherein at least one of the H on N has been
replaced with alkyl.
[0370] The term ".omega.-phosphoalkyl" refers to
-alkyl-O--P(Q')(Q'')-O--R, wherein Q' and Q'' are each
independently O or S and R optionally substituted alkyl.
[0371] The term ".omega.-thiophosphoalkyl refers to
.omega.-phosphoalkyl wherein at least one of Q' or Q" is S.
[0372] In some embodiments, the methods of the invention may
require the use of protecting groups. Protecting group methodology
is well known to those skilled in the art (see, for example,
PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Green, T. W. et. al.,
Wiley-Interscience, New York City, 1999). Briefly, protecting
groups within the context of this invention are any group that
reduces or eliminates unwanted reactivity of a functional group. A
protecting group can be added to a functional group to mask its
reactivity during certain reactions and then removed to reveal the
original functional group. In some embodiments an "alcohol
protecting group" is used. An "alcohol protecting group" is any
group which decreases or eliminates unwanted reactivity of an
alcohol functional group. Protecting groups can be added and
removed using techniques well known in the art.
[0373] The compounds of the present invention may be prepared by
known organic synthesis techniques, including the methods described
in more detail in the Examples.
EXAMPLES
Example 1
Synthesis of Methanesulfonic Acid octadeca-9, 12-dienyl ester 2
##STR00102##
[0375] To a solution of the alcohol 1 (26.6 g, 100 mmol) in
dichloromethane (100 mL), triethylamine (13.13 g, 130 mmol) was
added and this solution was cooled in an ice-bath. To this cold
solution, a solution of mesyl chloride (12.6 g, 110 mmol) in
dichloromethane (60 mL) was added dropwise and after the completion
of the addition, the reaction mixture was allowed to warm to
ambient temperature and stirred overnight. The TLC of the reaction
mixture showed the completion of the reaction. The reaction mixture
was diluted with dichloromethane (200 mL), washed with water (200
mL), satd. NaHCO.sub.3 (200 mL), brine (100 mL) and dried
(NaSO.sub.4). The organic layer was concentrated to get the crude
product which was purified by column chromatography (silica gel)
using 0-10% Et.sub.2O in hexanes. The pure product fractions were
combined and concentrated to obtain the pure product 2 as colorless
oil (30.6 g, 89%). .sup.1H NMR (CDCl.sub.3, 400 MHz)
.delta.=5.42-5.21 (m, 4H), 4.20 (t, 2H), 3.06 (s, 3H), 2.79 (t,
2H), 2.19-2.00 (m, 4H), 1.90-1.70 (m, 2H), 1.06-1.18 (m, 18H), 0.88
(t, 3H). .sup.13C NMR (CDCl.sub.3) .delta.=130.76, 130.54, 128.6,
128.4, 70.67, 37.9, 32.05, 30.12, 29.87, 29.85, 29.68, 29.65,
29.53, 27.72, 27.71, 26.15, 25.94, 23.09, 14.60. MS. Molecular
weight calculated for C.sub.19H.sub.36O.sub.3S, Cal. 344.53, Found
343.52 (M-H.sup.-).
Synthesis of 18-Bromo-octadeca-6, 9-diene 3
[0376] The mesylate 2 (13.44 g, 39 mmol) was dissolved in anhydrous
ether (500 mL) and to it the MgBr.Et.sub.2O complex (30.7 g, 118
mmol) was added under argon and the mixture was refluxed under
argon for 26 h after which the TLC showed the completion of the
reaction. The reaction mixture was diluted with ether (200 mL) and
ice-cold water (200 mL) was added to this mixture and the layers
were separated. The organic layer was washed with 1% aqueous
K.sub.2CO.sub.3 (100 mL), brine (100 mL) and dried (Anhyd.
Na.sub.2SO.sub.4). Concentration of the organic layer provided the
crude product which was further purified by column chromatography
(silica gel) using 0-1% Et.sub.2O in hexanes to isolate the bromide
3 (12.6 g, 94%) as a colorless oil. .sup.1H NMR (CDCl.sub.3, 400
MHz) .delta.=5.41-5.29 (m, 4H), 4.20 (d, 2H), 3.40 (t, J=7 Hz, 2H),
2.77 (t, J=6.6 Hz, 2H), 2.09-2.02 (m, 4H), 1.88-1.00 (m, 2H),
1.46-1.27 (m, 18H), 0.88 (t, J=3.9 Hz, 3H). .sup.13C NMR
(CDCl.sub.3) .delta.=130.41, 130.25, 128.26, 128.12, 34.17, 33.05,
31.75, 29.82, 29.57, 29.54, 29.39, 28.95, 28.38, 27.42, 27.40,
25.84, 22.79, 14.28.
Synthesis of 18-Cyano-octadeca-6, 9-diene 4
[0377] To a solution of the mesylate (3.44 g, 10 mmol) in ethanol
(90 mL), a solution of KCN (1.32 g, 20 mmol) in water (10 mL) was
added and the mixture was refluxed for 30 min. after which, the TLC
of the reaction mixture showed the completion of the reaction after
which, ether (200 mL) was added to the reaction mixture followed by
the addition of water. The reaction mixture was extracted with
ether and the combined organic layers was washed with water (100
mL), brine (200 mL) and dried. Concentration of the organic layer
provided the crude product which was purified by column
chromatography (0-10% Et.sub.2O in hexanes). The pure product 4 was
isolated as colorless oil (2 g, 74%). .sup.1H NMR (CDCl.sub.3, 400
MHz) .delta.=5.33-5.22 (m, 4H), 2.70 (t, 2H), 2.27-2.23 (m, 2H),
2.00-1.95 (m, 4H), 1.61-1.54 (m, 2H), 1.39-1.20 (m, 18H), 0.82 (t,
3H). .sup.13C NMR (CDCl.sub.3) .delta.=130.20, 129.96, 128.08,
127.87, 119.78, 70.76, 66.02, 32.52, 29.82, 29.57, 29.33, 29.24,
29.19, 29.12, 28.73, 28.65, 27.20, 27.16, 25.62, 25.37, 22.56,
17.10, 14.06. MS. Molecular weight calculated for
C.sub.19H.sub.33N, Cal. 275.47, Found 276.6 (M-H.sup.-).
Synthesis of Heptatriaconta-6,9,28,31-tetraen-19-one 7
[0378] To a flame dried 500 mL 2NRB flask, freshly activated Mg
turnings (0.144 g, 6 mmol) were added and the flask was equipped
with a magnetic stir bar and a reflux condenser. This set-up was
degassed, flushed with argon and 10 mL of anhydrous ether was added
to the flask via syringe. The bromide 3 (1.65 g, 5 mmol) was
dissolved in anhydrous ether (10 mL) and added dropwise via syringe
to the flask. An exothermic reaction was noticed (to
confirm/accelerate the Grignard reagent formation, 2 mg of iodine
was added and immediate decolorization was observed confirming the
formation of the Grignard reagent) and the ether started refluxing.
After the completion of the addition the reaction mixture was kept
at 35.degree. C. for 1 h and then cooled in ice bath. The cyanide 4
(1.38 g, 5 mmol) was dissolved in anhydrous ether (20 mL) and added
dropwise to the reaction mixture with stirring. An exothermic
reaction was observed and the reaction mixture was stirred
overnight at ambient temperature. The reaction was quenched by
adding 10 mL of acetone dropwise followed by ice cold water (60
mL). The reaction mixture was treated with aq. H.sub.2SO.sub.4 (10%
by volume, 200 mL) until the solution became homogeneous and the
layers were separated. The aq. phase was extracted with ether
(2.times.100 mL). The combined ether layers were dried
(Na.sub.2SO.sub.4) and concentrated to get the crude product which
was purified by column (silica gel, 0-10% ether in hexanes)
chromatography. The pure product fractions were evaporated to
provide the pure ketone 7 as a colorless oil (2 g, 74%). .sup.1H
NMR (CDCl.sub.3, 400 MHz) .delta.=5.33-5.21 (m, 8H), 2.69 (t, 4H),
2.30 (t, 4H), 2.05-1.95 (m, 8H), 1.55-1.45 (m, 2H), 1.35-1.15 (m,
18H), 0.82 (t, 3H). .sup.13C NMR (CDCl.sub.3) .delta.=211.90,
130.63, 130.54, 128.47, 128.41, 43.27, 33.04, 32.01, 30.93, 29.89,
29.86, 29.75, 29.74, 27.69, 26.11, 24.35, 23.06, 14.05. MS.
Molecular weight calculated for C.sub.37H.sub.66O, Cal. 526.92,
Found 528.02 (M+H.sup.+).
Example 2: Alternative Synthesis of the Ketone 7
##STR00103##
[0379] Synthesis of Compound 6b
[0380] To a flame dried 500 mL RB flask, freshly activated Mg
turnings (2.4 g, 100 mmol) were added and the flask was equipped
with a magnetic stir bar, an addition funnel and a reflux
condenser. This set-up was degassed and flushed with argon and 10
mL of anhydrous ether was added to the flask via syringe. The
bromide 3 (26.5 g, 80.47 mmol) was dissolved in anhydrous ether (50
mL) and added to the addition funnel. About 5 mL of this ether
solution was added to the Mg turnings while stirring vigorously. An
exothermic reaction was noticed (to confirm/accelerate the Grignard
reagent formation, 5 mg of iodine was added and immediate
decolorization was observed confirming the formation of the
Grignard reagent) and the ether started refluxing. The rest of the
solution of the bromide was added dropwise while keeping the
reaction under gentle reflux by cooling the flask in water. After
the completion of the addition the reaction mixture was kept at
35.degree. C. for 1 h and then cooled in ice bath. Ethyl formate
(2.68 g, 36.2 mmol) was dissolved in anhydrous ether (40 mL) and
transferred to the addition funnel and added dropwise to the
reaction mixture with stirring. An exothermic reaction was observed
and the reaction mixture started refluxing. After the initiation of
the reaction the rest of the ethereal solution of formate was
quickly added as a stream and the reaction mixture was stirred for
a further period of 1 h at ambient temperature. The reaction was
quenched by adding 10 mL of acetone dropwise followed by ice cold
water (60 mL). The reaction mixture was treated with aq.
H.sub.2SO.sub.4 (10% by volume, 300 mL) until the solution became
homogeneous and the layers were separated. The aq. phase was
extracted with ether (2.times.100 mL). The combined ether layers
were dried (Na.sub.2SO.sub.4) and concentrated to get the crude
product which was purified by column (silica gel, 0-10% ether in
hexanes) chromatography. The slightly less polar fractions were
concentrated to get the formate 6a (1.9 g) and the pure product
fractions were evaporated to provide the pure product 6b as a
colorless oil (14.6 g, 78%).
Synthesis of Compound 7
[0381] To a solution of the alcohol 6b (3 g, 5.68 mmol) in
CH.sub.2Cl.sub.2 (60 mL), freshly activated 4 A molecular sieves
(50 g) were added and to this solution powdered PCC (4.9 g, 22.7
mmol) was added portion wise over a period of 20 minutes and the
mixture was further stirred for 1 hour (Note: careful monitoring of
the reaction is necessary in order to get good yields since
prolonged reaction times leads to lower yields) and the TLC of the
reaction mixture was followed every 10 minutes (5% ether in
hexanes) After completion of the reaction, the reaction mixture was
filtered through a pad of silica gel and the residue was washed
with CH.sub.2Cl.sub.2 (400 mL). The filtrate was concentrated and
the thus obtained crude product was further purified by column
chromatography (silica gel, 1% Et.sub.2O in hexanes) to isolate the
pure product 7 (2.9 g, 97%) as a colorless oil. .sup.1H NMR
(CDCl.sub.3, 400 MHz) .delta.=5.33-5.21 (m, 8H), 2.69 (t, 4H), 2.30
(t, 4H), 2.05-1.95 (m, 8H), 1.55-1.45 (m, 2H), 1.35-1.15 (m, 18H),
0.82 (t, 3H). .sup.13C NMR (CDCl.sub.3) .delta.=211.90, 130.63,
130.54, 128.47, 128.41, 43.27, 33.04, 32.01, 30.93, 29.89, 29.86,
29.75, 29.74, 27.69, 26.11, 24.35, 23.06, 14.05. MS. Molecular
weight calculated for C.sub.37H.sub.66O, Cal. 526.92, Found 528.02
(M+H.sup.+).
Example 3: Synthesis of Unsymmetric Ketones 25 and 27
##STR00104##
[0382] Synthesis of heptatriaconta-6,9,28-trien-19-one 25
[0383] To a dry 50 ml 2NRB flask, a freshly activated Mg turnings
(132 mg, 0.0054 mol) was added and the flask was equipped with a
magnetic stir bar and a reflux condenser. This setup was degassed
and flushed with nitrogen and 10 mL of anhydrous ether was added to
the flask via syringe. The bromide 24 (1.8 g, 0.0054 mol) was
dissolved in anhydrous ether (10 mL) and added dropwise via syringe
to the flask. An exothermic reaction was noticed (reaction
initiated with dibromoethane) and the ether started refluxing.
After completion of the addition the reaction mixture was kept at
35.degree. C. for 1 h and then cooled in ice bath to 10-15.degree.
C. The cyanide 4 (0.5 g, 0.0018 mol) was dissolved in dry THF (5
mL) and added dropwise to the reaction with stirring. An exothermic
reaction was observed and the reaction mixture was refluxed (at
70.degree. C.) for 12h and quenched with ammonium chloride
solution. It was then treated with 25% HCl solution until the
solution became homogenous and the layers were separated. The
aqueous phase was extracted with ether. The combined ether layers
were dried and concentrated to get the crude product which was
purified by column chromatography. The pure product fractions were
evaporated to provide the pure ketone 25 as colorless oil.
[0384] Yield: 0.230 g (24%). .sup.1H-NMR (CDCl.sub.3, 400 MHz):
.delta.=5.37-5.30 (m, 6H), 2.77-2.74 (t, 2H), 2.38-2.34 (t, 4H),
2.05-1.95 (m, 8H), 1.56-1.52 (m, 4H), 1.35-1.25 (m, aliphatic
protons), 0.89-0.85 (t, 6H). IR (cm-1):2924, 2854, 1717, 1465,
1049, 721.
Synthesis of heptatriaconta-6,9-dien-19-one 27
[0385] To a flame dried 500 mL 2NRB flask, a freshly activated Mg
turnings (0.144 g, 6 mmol) is added and the flask is equipped with
a magnetic stir bar and a reflux condenser. This set-up is degassed
and flushed with argon and 10 mL of anhydrous ether is added to the
flask via syringe. The commercially available bromide 26 (2.65 g, 5
mmol) is dissolved in anhydrous ether (10 mL) and added dropwise
via syringe to the flask. After the completion of the addition the
reaction mixture is kept at 35.degree. C. for 1 h and then cooled
in ice bath. The cyanide 4 (1.38 g, 5 mmol) is dissolved in
anhydrous ether (20 mL) and added dropwise to the reaction mixture
with stirring. An exothermic reaction is observed and the reaction
mixture is stirred overnight at ambient temperature. The reaction
is quenched by adding 10 mL of acetone dropwise followed by ice
cold water (60 mL). The reaction mixture is treated with aq.
H.sub.2SO.sub.4 (10% by volume, 200 mL) until the solution becomes
homogeneous and the layers are separated. The aq. phase is
extracted with ether (2.times.100 mL). The combined ether layers
are dried (Na.sub.2SO.sub.4) and concentrated to get the crude
product which is purified by column chromatography to provide the
pure ketone 27 as a colorless oil. 1H-NMR (CDCl.sub.3, 400 MHz):
.delta.=5.42-5.30 (m, 4H), 2.79-2.78 (t, 2H), 2.40-2.37 (t, 4H),
2.08-2.03 (m, 4H), 1.58-1.54 (m, 4H), 1.36-1.26 (br m, aliphatic
protons), 0.91-0.87 (t, 6H). IR (cm-1):2924, 2854, 1716, 1465,
1375, 721.
Example 4: Synthesis of Unsymmetrical Ketones with C.sub.12
Chain
##STR00105##
[0387] To a dry 50 ml 2NRB flask, a freshly activated Mg turnings
(175 mg, 0.0072 mol) was added and the flask was equipped with a
magnetic stir bar and a reflux condenser. This setup was degassed
and flushed with nitrogen and 10 mL of anhydrous ether was added to
the flask via syringe. The bromide 28 (1.5 g, 0.006 mol) was
dissolved in anhydrous ether (7 ml) and added dropwise via syringe
to the flask. An exothermic reaction was noticed (reaction
initiated with dibromoethane) and the ether started refluxing.
After completion of the addition the reaction mixture was kept at
35.degree. C. for 1 h and then cooled in ice bath to 10-15.degree.
C. The cyanide 4 (1 g, 0.0036 mol) was dissolved in anhydrous ether
(7 mL) and added dropwise to the reaction with stirring. An
exothermic reaction was observed and the reaction mixture was
refluxed for 12h and quenched with ammonium chloride solution. It
was then treated with 25% HCl solution until the solution becomes
homogenous and the layers were separated. The aq phase was
extracted with ether. The combined ether layers were dried and
concentrated to get the crude product which was purified by column
chromatography. The pure product fractions were evaporated to
provide the pure ketone 29 as colorless oil. Yield: 0.65 g (26%).
.sup.1H-NMR (.delta. ppm): 5.388-5.302 (m, 4H), 2.77-2.74 (t, 2H),
2.38-2.34 (t, 4H), 2.04-2.01 (m, 4H), 1.34-1.18 (m, 36H), 0.89-0.85
(m 6H). IR (cm.sup.-1): 3009, 2920, 2851, 1711 (C.dbd.O), 1466,
1376, 1261.
Example 5: Synthesis of Unsymmetrical Ketones with C.sub.10 Chain
31
##STR00106##
[0389] To a dry 50 ml 2NRB flask, a freshly activated Mg turnings
(266 mg, 0.0109 mol) was added and the flask was equipped with a
magnetic stir bar and a reflux condenser. This setup was degassed
and flushed with nitrogen and 10 mL of anhydrous ether was added to
the flask via syringe. The bromide (2.43 g, 0.0109 mol) was
dissolved in anhydrous ether (7 ml) and added dropwise via syringe
to the flask. An exothermic reaction was noticed (reaction
initiated with dibromoethane) and the ether started refluxing.
After completion of the addition the reaction mixture was kept at
35.degree. C. for 1 h and then cooled in ice bath to 10-15.degree.
C. The cyanide (1 g, 0.0036 mol) was dissolved in anhydrous ether
(7 mL) and added dropwise to the reaction with stirring. An
exothermic reaction was observed and the reaction mixture was
stirred at ambient temperature for 2 hr. THF (4 ml) was added to
the reaction mixture and it was warmed to 45-50.degree. C. for 4 hr
till the cyano derivative was complete consumed. The reaction was
quenched by adding 3 mL of acetone dropwise followed by ice cold
water. The reaction mixture was treated with 25% HCl solution until
the solution becomes homogenous and the layers were separated. The
aq. phase was extracted with ether. The combined ether layers were
dried and concentrated to get the crude product which was purified
by column chromatography. The pure product fractions were
evaporated to provide the pure ketone as colorless oil. Yield: 0.93
gms (61%). .sup.1H-NMR (.delta. ppm): 5.37-5.302 (m, 4H), 2.77-2.74
(t, 2H), 2.38-2.34 (t, 4H), 2.05-2.00 (m, 4H), 1.55-1.52 (m, 2H),
1.35-1.24 (m, 34H), 0.89-0.84 (m 6H). IR (cm.sup.-1): 3009, 2925,
2854, 1717 (C.dbd.O), 1465, 1376.
Example 6: Synthesis of Unsymmetrical Ketones with Cholesterol
33
##STR00107##
[0391] Using a similar procedure to that used for the synthesis of
ketone 31, the cholesteryl chloride on conversion to the
corresponding magnesium chloride followed by addition to the
linoleyl cyanide provided the ketone 33.
Example 7: Synthesis of Unsymmetrical Ketones with Cholesterol
35
##STR00108##
[0393] The treatment of the cholesterol chloroformate with
3-bromopropylamine provided the bromide 34 which is converted to
the corresponding Grignard reagent 34a which on treatment with the
linoleyl cyanide provided the corresponding unsymmetrical ketone 35
in good yield.
Example 8: Synthesis of Unsymmetric Ketone 40
##STR00109##
[0394] Synthesis of Compound 37
[0395] To a 500 ml two neck RBF containing LiAlH.sub.4 (1.02 g,
0.0269 mol) was added anhydrous THF (20 mL) at room temperature
under nitrogen atmosphere. The suspension was stirred for 1 h at
room temperature and then cooled to 0.degree. C. To this mixture
was added a solution of compound 1 (5 g, 0.01798 mol) in anhydrous
THF (50 mL) slowly maintaining the inside temperature 0.degree. C.
After completion of the addition, reaction mixture was warmed to
ambient temperature and stirred for 1 h. Progress of the reaction
was monitored by TLC. Upon completion of the reaction, mixture was
cooled to 0.degree. C. and quenched with sat. solution of aq.
Na.sub.2SO.sub.4. Reaction mixture was stirred for 30 minutes and
solid formed was filtered through celite bed and washed with ethyl
acetate (100 mL). Filtrate and washings were combined and
evaporated on rotary evaporator to afford the compound 37 as
colorless liquid, which was taken as such for the next stage
without any purification. Yield: (4.5 g, 95%); .sup.1H NMR (400
MHz, CDCl.sub.3) .delta.=5.39-5.28 (m, 6H), 3.64-3.61 (t, 2H),
2.81-2.78 (t, 4H), 2.10-2.01 (m, 4H), 1.59-1.51 (m, 2H), 1.29-1.22
(m, aliphatic protons), 0.98-0.94 (t, 3H).
Synthesis of Compound 38
[0396] Compound 37 (14 g, 0.0530 mol) was dissolved in DCM (300 ml)
in a 500 ml two neck RBF and cooled to 0.degree. C. To this
solution was added triethylamine (29.5 ml, 0.2121 mol) slowly under
inert atmosphere. Reaction mixture was then stirred for 10-15
minutes and to it mesyl chloride (6.17 mL, 0.0795 mol)) was added
slowly. After complete addition, the reaction mixture was allowed
to warm to ambient temperature and stirred for 20 h. Reaction was
monitored by TLC. Upon completion, the reaction mixture was diluted
with water (200 mL) stirred for few minutes and organic layer was
separated. Organic phase was further washed with brine (1.times.70
mL), dried over Na.sub.2SO.sub.4. and solvent was removed on rotary
evaporator to get the crude compound 38 as brown oil which was used
as such for next reaction. Yield: (17 g, 93%)'H NMR (400 MHz,
CDCl.sub.3) .delta.=5.39-5.31 (m, 6H), 4.22-4.19 (t, 2H), 2.99 (s,
3H), 2.81-2.78 (m, 4H), 2.08-2.01 (m, 4H), 1.75.1.69 (m, 2H),
1.39-1.29 (m, aliphatic protons), 0.98-0.94 (t, 3H).
Synthesis of Compound 39
[0397] The mesylate 38 (10 g, 0.2923 mol) was dissolved in (300 mL)
anhydrous ether in a 1000 mL two neck RBF and MgBr.sub.2.Et.sub.2O
complex (22.63 g, 0.0877 mol) was added into it under nitrogen
atmosphere. Resulting mixture was then heated to reflux for 26 h.
After completion of the reaction (by TLC), reaction mixture was
diluted with ether (300 mL) and ice cold water (200 mL) and ether
layer was separated out. Organic layer was then washed with 1% aq.
K.sub.2CO.sub.3 (100 mL) followed by brine (80 mL). Organic phase
was then dried over anhydrous Na.sub.2SO.sub.4 and solvent was
evaporated off under vacuum to give the crude material which was
chromatographed on silica gel (60-120 mesh) using 0-1% ethyl
acetate in hexane as eluting system to yield the desired compound
39 as oil. Yield: (7 g, 73%) .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta.=5.39-5.31 (m, 6H), 3.41-3.37 (t, 2H), 2.81-2.78 (m, 4H),
2.08-2.02 (m, 4H), 1.86-1.80 (m, 2H), 1.42-1.29 (m, aliphatic
protons), 0.98-0.94 (t, 3H).
Synthesis of Unsymmetric Ketone 40
[0398] To a flame dried 500 mL two neck RBF, equipped with magnetic
stir bar and a reflux condenser, freshly activated Mg turnings
(0.88 g, 0.03636 mol) were added. This set up was degassed, flushed
with argon and ether (150 mL) was added into it. Few drops of bromo
compound 4 (11.89 g, 0.03636 mol) in 50 mL ether was added at the
beginning to initiate the reaction (note: catalytic amount of 1,
2-dibromo ethane was also added to accelerate formation of Grignard
reagent). Upon initiation, the remaining solution of bromo compound
was added slowly to the refluxing ethereal solution. After complete
addition, the reaction mixture was refluxed at 40.degree. C. for
1.5 h. It was then cooled to 10.degree. C. and the linoleyl cyanide
4 (5 g, 0.01818 mol) in 30 mL of dry ether was added drop wise and
the resulting mixture was then heated to reflux for 20 h at
40.degree. C. Progress of the reaction was monitored by TLC. After
complete consumption of the cyano derivative 40 (by TLC), mixture
was cooled to room temperature and quenched with 30 mL of acetone
followed by (50 mL)ice water. This solution was further acidified
with 10% HCl solution and ether layer was separated out. Aqueous
phase was further extracted with diethyl ether (2.times.100 mL).
Removal of the solvent after drying over anhydrous Na.sub.2SO.sub.4
afforded the crude ketone which was purified by silica gel column
chromatography (100-200 mesh) using 0-5% ether in hexane as eluting
system to give the title compound 40 as pale yellow oil. Yield:
(4.8 g, 50.5%)'H NMR (400 MHz, CDCl.sub.3) .delta.=5.38-5.28 (m,
10H), 2.80-2.74 (m, 6H), 2.38-2.34 (t, 4H), 2.08-2.00 (m, 8H),
1.55-1.52 (m, 4H), 1.35-1.26 (m, aliphatic protons), 0.98-0.94 (t,
3H), 0.89-0.85 (t, 3H). HPLC--98.04%.
Example 9: Oligonucleotide Synthesis
[0399] All oligonucleotides were synthesized on an AKTAoligopilot
synthesizer. Commercially available controlled pore glass solid
support (dT-CPG, 500 {acute over (.ANG.)}, Prime Synthesis) and RNA
phosphoramidites with standard protecting groups,
5'-O-dimethoxytrityl
N6-benzoyl-2'-t-butyldimethylsilyl-adenosine-3'-O--N,N'-diisopropyl-2-cya-
noethylphosphoramidite,
5'-O-dimethoxytrityl-N4-acetyl-2'-t-butyldimethylsilyl-cytidine-3'-O--N,N-
'-diisopropyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N2-isobutyryl-2'-t-butyldimethylsilyl-guanosine-3'-O-
--N,N'-diisopropyl-2-cyanoethylphosphoramidite, and
5'-O-dimethoxytrityl-2'-t-butyldimethylsilyl-uridine-3'-0-N,N'-diisopropy-
l-2-cyanoethylphosphoramidite (Pierce Nucleic Acids Technologies)
were used for the oligonucleotide synthesis. The 2'-F
phosphoramidites,
5'-O-dimethoxytrityl-N4-acetyl-2'-fluoro-cytidine-3'-O--N,N'-diisopropyl--
2-cyanoethyl-phosphoramidite and
5'-O-dimethoxytrityl-2'-fluoro-uridine-3'-O--N,N'-diisopropyl-2-cyanoethy-
l-phosphoramidite were purchased from (Promega). All
phosphoramidites were used at a concentration of 0.2M in
acetonitrile (CH.sub.3CN) except for guanosine which was used at
0.2M concentration in 10% THF/ANC (v/v). Coupling/recycling time of
16 minutes was used. The activator was 5-ethyl thiotetrazole
(0.75M, American International Chemicals), for the PO-oxidation
Iodine/Water/Pyridine was used and the PS-oxidation PADS (2%) in
2,6-lutidine/ACN (1:1 v/v) was used.
[0400] 3'-ligand conjugated strands were synthesized using solid
support containing the corresponding ligand. For example, the
introduction of cholesterol unit in the sequence was performed from
a hydroxyprolinol-cholesterol phosphoramidite. Cholesterol was
tethered to trans-4-hydroxyprolinol via a 6-aminohexanoate linkage
to obtain a hydroxyprolinol-cholesterol moiety. 5'-end Cy-3 and
Cy-5.5 (fluorophore) labeled siRNAs were synthesized from the
corresponding Quasar-570 (Cy-3) phosphoramidite were purchased from
Biosearch Technologies. Conjugation of ligands to 5'-end and or
internal position is achieved by using appropriately protected
ligand-phosphoramidite building block An extended 15 min coupling
of 0.1M solution of phosphoramidite in anhydrous CH.sub.3CN in the
presence of 5-(ethylthio)-1H-tetrazole activator to a solid bound
oligonucleotide. Oxidation of the internucleotide phosphite to the
phosphate was carried out using standard iodine-water as reported
(1) or by treatment with tert-butyl
hydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation
wait time conjugated oligonucleotide. Phosphorothioate was
introduced by the oxidation of phosphite to phosphorothioate by
using a sulfur transfer reagent such as DDTT (purchased from AM
Chemicals), PADS and or Beaucage reagent The cholesterol
phosphoramidite was synthesized in house, and used at a
concentration of 0.1 M in dichloromethane. Coupling time for the
cholesterol phosphoramidite was 16 minutes.
[0401] After completion of synthesis, the support was transferred
to a 100 ml glass bottle (VWR). The oligonucleotide was cleaved
from the support with simultaneous deprotection of base and
phosphate groups with 80 mL of a mixture of ethanolic ammonia
[ammonia: ethanol (3:1)] for 6.5h at 55.degree. C. The bottle was
cooled briefly on ice and then the ethanolic ammonia mixture was
filtered into a new 250 ml bottle. The CPG was washed with
2.times.40 mL portions of ethanol/water (1:1 v/v). The volume of
the mixture was then reduced to .about.30 ml by roto-vap. The
mixture was then frozen on dyince and dried under vacuum on a speed
vac.
[0402] The dried residue was resuspended in 26 ml of triethylamine,
triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO
(3:4:6) and heated at 60.degree. C. for 90 minutes to remove the
tert-butyldimethylsilyl (TBDMS) groups at the 2' position. The
reaction was then quenched with 50 ml of 20 mM sodium acetate and
pH adjusted to 6.5, and stored in freezer until purification.
[0403] The oligonucleotides were analyzed by high-performance
liquid chromatography (HPLC) prior to purification and selection of
buffer and column depends on nature of the sequence and or
conjugated ligand.
[0404] The ligand conjugated oligonucleotides were purified reverse
phase preparative HPLC. The unconjugated oligonucleotides were
purified by anion-exchange HPLC on a TSK gel column packed in
house. The buffers were 20 mM sodium phosphate (pH 8.5) in 10%
CH.sub.3CN (buffer A) and 20 mM sodium phosphate (pH 8.5) in 10%
CH.sub.3CN, 1M NaBr (buffer B). Fractions containing full-length
oligonucleotides were pooled, desalted, and lyophilized.
Approximately 0.15 OD of desalted oligonucleotides were diluted in
water to 150 .mu.l and then pipetted in special vials for CGE and
LC/MS analysis. Compounds were finally analyzed by LC-ESMS and
CGE.
[0405] For the preparation of siRNA, equimolar amounts of sense and
antisense strand were heated in 1.times.PBS at 95.degree. C. for 5
min and slowly cooled to room temperature. Integrity of the duplex
was confirmed by HPLC analysis
TABLE-US-00006 TABLE 7 siRNA duplexes for Luc and FVII targeting.
SEQ ID Duplex Sense/Antisense Sequence 5'-3' NO: Target 1000/2434
CUU ACG CUG AGU ACU UCG AdTdT Luc U*CG AAG fUAC UCA GCG fUAA GdT*dT
2433/1001 C*UfU ACG CUG AGfU ACU UCG AdT*dT Luc UCG AAG UAC UCA GCG
UAA GdTdT 2433/2434 C*UfU ACG CUG AGfU ACU UCG AdT*dT Luc U*CG AAG
fUAC UCA GCG fUAA GdT*dT 1000/1001 CUU ACG CUG AGU ACU UCG AdTdT
Luc UCG AAG UAC UCA GCG UAA GdTdT AD- GGAUCAUCUCAAGUCUUACdTdT FVII
1596 GUAAGACUUGAGAUGAUCCdTdT AD-
GGAfUfCAfUfCfUfCAAGfUfCfUfUAfCdTsdT FVII 1661
GfUAAGAfCfUfUGAGAfUGAfUfCfCdT*dT Note: ##STR00110## lowercase is
2'-O-methyl modified nucleotide, * is phosphorothioate backbone
linkages, fN is a 2'-fluoro nucleotide, dN is 2'-deoxy
nucleotide.
Example 10: Serum Stability Assay for siRNA
[0406] A medium throughput assay for initial sequence-based
stability selection was performed by the "stains all" approach. To
perform the assay, an siRNA duplex was incubated in 90% human serum
at 37.degree. C. Samples of the reaction mix were quenched at
various time points (at 0 min., 15, 30, 60, 120, and 240 min.) and
subjected to electrophoretic analysis (FIG. 1). Cleavage of the RNA
over the time course provided information regarding the
susceptibility of the siRNA duplex to serum nuclease
degradation.
[0407] A radiolabeled dsRNA and serum stability assay was used to
further characterize siRNA cleavage events. First, a siRNA duplex
was 5'end-labeled with .sup.32P on either the sense or antisense
strand. The labeled siRNA duplex was incubated with 90% human serum
at 37.degree. C., and a sample of the solution was removed and
quenched at increasing time points. The samples were analyzed by
electrophoresis.
Example 11: FVII In Vivo Evaluation Using the Cationic Lipid
Derived Liposomes In Vivo Rodent Factor VII and ApoB Silencing
Experiments
[0408] C57BL/6 mice (Charles River Labs, MA) and Sprague-Dawley
rats (Charles River Labs, MA) received either saline or siRNA in
desired formulations via tail vein injection at a volume of 0.01
mL/g. At various time points post-administration, animals were
anesthetized by isofluorane inhalation and blood was collected into
serum separator tubes by retro orbital bleed. Serum levels of
Factor VII protein were determined in samples using a chromogenic
assay (Coaset Factor VII, DiaPharma Group, OH or Biophen FVII,
Aniara Corporation, OH) according to manufacturer protocols. A
standard curve was generated using serum collected from saline
treated animals. In experiments where liver mRNA levels were
assessed, at various time points post-administration, animals were
sacrificed and livers were harvested and snap frozen in liquid
nitrogen. Frozen liver tissue was ground into powder. Tissue
lysates were prepared and liver mRNA levels of Factor VII and apoB
were determined using a branched DNA assay (QuantiGene Assay,
Panomics, CA).
Example 12; Preparation of 1,2-Di-O-alkyl-sn3-Carbomoylglyceride
(PEG-DMG)
##STR00111##
[0409] Preparation of IVa
[0410] 1,2-Di-O-tetradecyl-sn-glyceride Ia (30 g, 61.80 mmol) and
N,N'-succinimidylcarboante (DSC, 23.76 g, 1.5 eq) were taken in
dichloromethane (DCM, 500 mL) and stirred over an ice water
mixture. Triethylamine (TEA, 25.30 mL, 3 eq) was added to the
stirring solution and subsequently the reaction mixture was allowed
to stir overnight at ambient temperature. Progress of the reaction
was monitored by TLC. The reaction mixture was diluted with DCM
(400 mL) and the organic layer was washed with water (2.times.500
mL), aqueous NaHCO.sub.3 solution (500 mL) followed by standard
work-up. The residue obtained was dried at ambient temperature
under high vacuum overnight. After drying the crude carbonate IIa
thus obtained was dissolved in dichloromethane (500 mL) and stirred
over an ice bath. To the stirring solution mPEG.sub.2000-NH.sub.2
(III, 103.00 g, 47.20 mmol, purchased from NOF Corporation, Japan)
and anhydrous pyridine (Py, 80 mL, excess) were added under argon.
The reaction mixture was then allowed to stir at ambient
temperature overnight. Solvents and volatiles were removed under
vacuum and the residue was dissolved in DCM (200 mL) and charged on
a column of silica gel packed in ethyl acetate. The column was
initially eluted with ethyl acetate and subsequently with gradient
of 5-10% methanol in dichloromethane to afford the desired
PEG-Lipid IVa as a white solid (105.30 g, 83%).'H NMR (CDCl.sub.3,
400 MHz)=5.20-5.12 (m, 1H), 4.18-4.01 (m, 2H), 3.80-3.70 (m, 2H),
3.70-3.20 (m, --O--CH.sub.2--CH.sub.2--O--, PEG-CH.sub.2),
2.10-2.01 (m, 2H), 1.70-1.60 (m, 2H), 1.56-1.45 (m, 4H), 1.31-1.15
(m, 48H), 0.84 (t, J=6.5 Hz, 6H). MS range found: 2660-2836.
Preparation of IVb
[0411] 1,2-Di-O-hexadecyl-sn-glyceride Ib (1.00 g, 1.848 mmol) and
DSC (0.710 g, 1.5 eq) were taken together in dichloromethane (20
mL) and cooled down to 0.degree. C. in an ice water mixture.
Triethylamine (1.00 mL, 3 eq) was added and the reaction was
stirred overnight. The reaction was followed by TLC, diluted with
DCM, washed with water (2 times), NaHCO.sub.3 solution and dried
over sodium sulfate. Solvents were removed under reduced pressure
and the resulting residue of IIb was maintained under high vacuum
overnight. This compound was directly used for the next reaction
without further purification. MPEG.sub.2000-NH.sub.2 III (1.50 g,
0.687 mmol, purchased from NOF Corporation, Japan) and IIb (0.702
g, 1.5 eq) were dissolved in dichloromethane (20 mL) under argon.
The reaction was cooled to 0.degree. C. Pyridine (1 mL, excess) was
added and the reaction stirred overnight. The reaction was
monitored by TLC. Solvents and volatiles were removed under vacuum
and the residue was purified by chromatography (first ethyl acetate
followed by 5-10% MeOH/DCM as a gradient elution) to obtain the
required compound IVb as a white solid (1.46 g, 76%). .sup.1H NMR
(CDCl.sub.3, 400 MHz) .delta.=5.17 (t, J=5.5 Hz, 1H), 4.13 (dd,
J=4.00 Hz, 11.00 Hz, 1H), 4.05 (dd, J=5.00 Hz, 11.00 Hz, 1H),
3.82-3.75 (m, 2H), 3.70-3.20 (m, --O--CH.sub.2--CH.sub.2--O--,
PEG-CH.sub.2), 2.05-1.90 (m, 2H), 1.80-1.70 (m, 2H), 1.61-1.45 (m,
6H), 1.35-1.17 (m, 56H), 0.85 (t, J=6.5 Hz, 6H). MS range found:
2716-2892.
Preparation of IVc
[0412] 1,2-Di-O-octadecyl-sn-glyceride Ic (4.00 g, 6.70 mmol) and
DSC (2.58 g, 1.5 eq) were taken together in dichloromethane (60 mL)
and cooled down to 0.degree. C. in an ice water mixture.
Triethylamine (2.75 mL, 3 eq) was added and the reaction was
stirred overnight. The reaction was followed by TLC, diluted with
DCM, washed with water (2 times), NaHCO.sub.3 solution, and dried
over sodium sulfate. Solvents were removed under reduced pressure
and the residue was maintained under high vacuum overnight. This
compound was directly used for the next reaction without further
purification. MPEG.sub.2000-NH.sub.2 III (1.50 g, 0.687 mmol,
purchased from NOF Corporation, Japan) and IIc (0.760 g, 1.5 eq)
were dissolved in dichloromethane (20 mL) under argon. The reaction
was cooled to 0.degree. C. Pyridine (1 mL, excess) was added and
the reaction was stirred overnight. The reaction was monitored by
TLC. Solvents and volatiles were removed under vacuum and the
residue was purified by chromatography (ethyl acetate followed by
5-10% MeOH/DCM as a gradient elution) to obtain the desired
compound We as a white solid (0.92 g, 48%). .sup.1H NMR
(CDCl.sub.3, 400 MHz) .delta.=5.22-5.15 (m, 1H), 4.16 (dd, J=4.00
Hz, 11.00 Hz, 1H), 4.06 (dd, J=5.00 Hz, 11.00 Hz, 1H), 3.81-3.75
(m, 2H), 3.70-3.20 (m, --O--CH.sub.2--CH.sub.2--O--, PEG-CH.sub.2),
1.80-1.70 (m, 2H), 1.60-1.48 (m, 4H), 1.31-1.15 (m, 64H), 0.85 (t,
J=6.5 Hz, 6H). MS range found: 2774-2948.
Example 13
##STR00112##
[0413] Synthesis of 2005
[0414] To a solution of 2004(50 g, 95 mmol) in DCM (400 ml) under
Ar atmosphere, was added TEA (53 mL, 378 mmol) and DMAP (1.2 g, 9.5
mmol) and stirred at room temperature under Ar atmosphere. Reaction
mass was cooled to -5.degree. C. and the solution of mesyl chloride
(15 mL, 190 mmol) in DCM (100 ml) was added slowly at temperature
below -5.degree. C. and allowed to warm to RT after addition. After
30 minutes (TLC), reaction mass was quenched with ice cold water
(20 ml). Organic layer was separated, washed with 1N HCl (30 ml),
water, brine, dried over sodium sulfate and evaporated at reduced
pressure to obtain pure product (55 g, 95.5%) as yellow liquid. 1H
NMR (400 MHz, CDCl.sub.3): .delta. 0.89 (t, 6H, J=6.8), 1.2-1.5 (m,
36H), 1.67 (m, 4H), 2.05 (q, 8H, J1=6.8, J2=6.8), 2.77 (t, 4H,
J=6.4), 2.99 (s, 3H), 4.71 (m, 1H) and 5.36 (m, 8H).
Synthesis 2006
[0415] To a solution of 2005 (50 g, 82 mmol) in DMF (500 mL) under
argon atmosphere, was added NaN.sub.3 (27 g, 410 mmol) and heated
to 70.degree. C. and maintained the temperature for four hours
(TLC). The mixture was diluted with water and extracted with ethyl
acetate (3.times.250 ml). The organic layer was washed with water,
brine, dried over Na.sub.2SO.sub.4 and evaporated at reduced
pressure to give crude product, which was purified by silica gel
chromatography using hexane/ether as eluent. The product was eluted
at 2% ether hexane to get 2006 (36 g, 86%) as pale yellow liquid.
.sup.1H NMR (400 MHz, CDCl3): .delta. 0.90 (t, 8H), 1.30 (m, 36H),
1.49 (t, 4H, J=6.4 Hz) 2.04 (q, 8H, J1=7.6, J2=14 Hz), 2.77 (t, 4H,
J=6.4 Hz), 3.22 (m, 1H), 5.34 (m, 8H). .sup.13C NMR (400 MHz,
CDCl.sub.3): .delta. 14.1, 22.5, 25.6, 26.1, 27.2, 29.2, 29.3,
29.45, 29.65, 31.5, 34.1, 63.1, 127.9, and 130.1. IR (KBr):
2098.
Example 14
##STR00113##
[0416] Synthesis of 2007
[0417] To a solution of 2005(76 g, 125 mmol) in dimethylformamide
(500 mL), was added sodium hydrosulfide hydrate (35 g, 625 mmol) at
room temperature. Reaction mixture was heated to 70.degree. C. for
2 hrs (TLC). It was then cooled to room temperature and diluted
with water (7V) and extracted with ether (3.times.5V). Combined
ether layer was washed with water (2.times.3V), brine solution
(2.times.3V), dried over sodium sulfate and evaporated at reduced
pressure to obtain the crude product, which was purified by silica
gel chromatography using a hexane as eluent to get the product 2007
(43.6 g, 64%). MS: Molecular weight calculated for
C.sub.37H.sub.68S 544.50, Found: 545.51 (M+H).
Synthesis of 2008
[0418] To a solution of aldrithiol (20.2 g, 92 mmol) in
dichloromethane (400 ml) was added benzyl bromide (11 mL, 92 mmol)
at 0.degree. C. After stirring at 0.degree. C. for 15 min, it was
warmed to room temperature and stirred for 15 minutes. Reaction
mixture was cooled back to 0.degree. C. and added a solution of
2007(50 g, 92 mmol) in dichloromethane (100 ml) followed by
diisopropylethylamine (16 mL, 92 mmol). After addition, it was
heated to reflux for 2 hrs (TLC). It was then diluted with
dichloromethane (10V), washed with water (2.times.10V), brine
solution (2.times.10V), dried over sodium sulfate and evaporated at
reduced pressure to obtain crude product, which was purified by
silica gel chromatography using 3% ether/hexane to afford pure
product as pale yellow liquid. (35 g, 58%).sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 0.89 (t, 6H, J1=6.4 Hz, J2=7.2 Hz), 1.25-1.42
(m, 38H), 1.56-1.63 (m, 2H), 2.05 (q, 8H, J1=6.4 Hz, J2=14 Hz),
2.78 (t, 5H, J1=6.4 Hz, J2=6 Hz), 5.30-5.42 (m, 8H), 7.06 (t, 1H,
J1=5.2 Hz, J2=6.8 Hz), 7.62 (t, 1H, J1=7.6 Hz, J2=7.6 Hz), 7.76 (d,
1H, J=8 Hz), 8.42 (d, 1H, J=4.4 Hz). .sup.13C NMR (100 MHz,
CDCl.sub.3): .delta. 22.6, 25.6, 26.7, 27.2, 29.2, 29.3, 29.5,
29.6, 31.5, 33.74, 52.9, 119.9, 120.4, 127.9, 128, 130.1, 130.2,
136.7, 149.3, 161.5. MS: Molecular weight calculated for
C.sub.42H.sub.71NS.sub.2 653.50, Found: 654.49 (M+H).
Example 15
##STR00114##
[0419] Synthesis of 2009 (ALNY-138)
[0420] A solution of 2005 (5 g, 8 mmol) in DMF and
dimethylamine--40% aqueous solution was taken in a seal tube. The
reaction mixture was heated at 90.degree. C. for 20 hours (TLC). It
was then cooled to room temperature, poured to water and extracted
with ethyl acetate (3.times.50 ml). The organic layer was washed
with water & brine, dried over Na.sub.z SO.sub.4 and evaporated
to afford pure product as pale brown liquid (2.00 g, 45%).sup.1H
NMR (400 MHz, CDCl.sub.3): .delta. 0.89 (t, 6H, j=6.8), 1.2-1.4 (m,
40H), 2.05 (q, 8H, J.sub.1=6.8 Hz, J.sub.2=6.8 Hz), 2.2 (s, 6H),
2.77 (t, 4H, j=6.4 Hz), 5.35 (m, 8H). .sup.13C NMR (400 MHz,
CDCl.sub.3): .delta. 14.1, 22.5, 22.6, 27.1, 27.2, 29.3, 29.5,
29.57, 29.63, 29.67, 30.0, 31.5, 32.5, 40.5, 64.0, 127.9 and 130.1
MS: Molecular weight calculated for C.sub.39H.sub.73N 555.57,
Found: 556.55 (M+H).
Example 16
##STR00115##
[0421] Synthesis of 2010
[0422] To a solution of 2004(30 g, 56.8 mmol) in toluene, was added
N-Hydroxyphthalimide (13.9 g, 85 mmol) and TPP (22.30 g, 85 mmol)
under argon. The reaction mass was cooled to -5.degree. C., to this
was added TEA (11.84 mL), followed by DEAD (13.14 ml). The reaction
mass was allowed to stir for 12 hrs at room temperature (TLC). It
was then filtered through celite pad. The filtrate was evaporated
at reduced pressure to obtain crude product, which was purified by
silica gel chromatography to afford pure product, which was eluted
at 3% diethyl ether and hexane to get the product 2010 (22.90 g,
60.50%) as pale yellow liquid .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 0.90 (6H, t, J=7.2 Hz), 1.2-1.4 (34H, m), 1.66-1.70 (4H,
m), 2.03-2.08 (8H, m), 2.78 (4H, t, J=12.8 Hz), 4.22 (1H, m),
5.29-5.43 (8H, m), 7.74-7.76 (2H, m), 7.83-7.85 (2H, m). .sup.13C
NMR (100 MHz, CDCl.sub.3): .delta. 14.3, 22.5, 24.9, 25.6, 27.2,
27.20, 29.3, 29.3, 29.5, 29.5, 29.6, 29.7, 31.5, 32.4, 88.3, 123.3,
127.9, 129.0, 130.1, 134.3, 164.3. MS: Molecular weight calculated
for C.sub.45H.sub.71NO.sub.3 673.54, Found: 674.55 (M+H).
Example 17
##STR00116##
[0423] Example 18
##STR00117##
[0424] Example 19: Synthesis of
3-(dimethylamino)-N-((11Z,14Z)-2-((9Z,12Z)-octadeca-9,12-dienyl)icosa-11,-
14-dienyl)propanamide (ALNY-201)
##STR00118##
[0426] To a stirred suspension of N, N-dimethylamino propionic acid
hydrochloride (1, 0.198 g, 1.3 mmol, 1.0 eq) in DCM was added HBTU
(0.59 g, 1.56 mmol, 1.2 eq) and DIPEA (0.71 mL, 3.9 mmol, 3.0 eq)
at room temperature. After stirred for 10 minutes, a solution of
amine (2, 0.7 g. 1.3 mmol, 1.0 eq) in DCM was added drop wise at
room temperature and continued the stirring until completion of the
reaction. Reaction mixture was diluted with DCM, washed with
saturated NaHCO.sub.3 solution followed by brine, organic layer was
separated and dried over MgSO.sub.4, concentrated and purified by
the silica gel column chromatography using DCM:MeOH (5%) as
gradients to get pure oily compound 3 in 70% yield. .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 7.18 (brs, 1H), 5.47-5.19 (m, 8H),
3.18-3.07 (m, 4H), 2.76 (t, J=6.5, 4H), 2.70 (s, 6H), 2.60 (t,
J=6.0, 2H), 2.04 (q, J=6.8, 9H), 1.48 (brs, 1H), 1.40-1.14 (m,
43H), 0.88 (t, J=6.8, 6H). Calc. mass for the
C.sub.43H.sub.80N.sub.2O: 640.6, found 641.5.
TABLE-US-00007 Synthesis of novel dilinoleyl derivatives No
compound Name 1 ##STR00119## ALNY-192 2 ##STR00120## ALNY-200 3
##STR00121## ALNY-175 4 ##STR00122## ALNY-187 5 ##STR00123##
ALNY-149 6 ##STR00124## ALNY-202 Compound 1 ##STR00125## Compound 2
##STR00126## Compound 3 ##STR00127## Compound 4 ##STR00128##
Compound 5 ##STR00129## Compound 6 ##STR00130##
Experimental Details
Compound 1 (ALNY-192)
[0427] To a solution of N,N'-disuccinimidyl carbonate (5.50 g, 21.5
mmol) in CH.sub.2Cl.sub.2 (200 mL), 3-dimethylamino-1-propanol
(2.43 g, 23.6 mmol) was added dropwise. The resulting mixture was
stirred at room temperature overnight. Taken up 50 mL of the
solution, Et.sub.3N (0.822 mL, 5.90 mmol) and ALN-SAN-30 (2.08 g,
3.93 mmol) were added and the reaction mixture was stirred at room
temperature overnight. The reaction mixture was diluted with
CH.sub.2Cl.sub.2 and washed with saturated NaHCO.sub.3 aq. The
organic layer was dried over anhydrous MgSO.sub.4, filtered and
concentrated. The crude was purified by silica gel column
chromatography (0-5% MeOH in CH.sub.2Cl.sub.2) to give compound 1
(1.66 g, 2.53 mmol, 64%, R.sub.f=0.22 with 5% MeOH in
CH.sub.2Cl.sub.2). .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta.
5.30-5.41 (m, 8H), 4.37 (d, J=8.0 Hz, 1H), 4.09 (t, J=6.0 Hz, 2H),
3.57 (brs, 1H), 2.78 (t, J=6.0 Hz, 4H), 2.33 (t, J=8.0 Hz, 2H),
2.23 (s, 6H), 2.02-2.06 (m, 8H), 1.76-1.80 (m, 2H), 1.27-1.45 (m,
40H), 0.89 (t, J=8.0 Hz, 6H). .sup.13C NMR (CDCl.sub.3, 100 MHz)
.delta. 156.5, 130.4, 130.3, 128.2, 128.1, 63.2, 56.6, 51.4, 45.7,
35.7, 31.7, 29.9, 29.8, 29.7, 29.6, 29.5, 27.7, 27.5, 27.4, 26.0,
25.8, 22.8, 14.3. Molecular weight for
C.sub.43H.sub.81N.sub.2O.sub.2 (M+H).sup.+ Calc. 657.63, Found
657.5.
Compound 2 (ALNY-200)
[0428] To a solution of N,N'-disuccinimidyl carbonate (5.50 g, 21.5
mmol) in CH.sub.2Cl.sub.2 (200 mL), 3-dimethylamino-1-propanol
(2.43 g, 23.6 mmol) was added dropwise. The resulting mixture was
stirred at room temperature overnight. Taken up 50 mL of the
solution, Et.sub.3N (0.697 mL, 5.00 mmol) and ALN-SAN-033 (1.71 g,
3.15 mmol) were added and the reaction mixture was stirred at room
temperature overnight. The reaction mixture was diluted with
CH.sub.2Cl.sub.2 and washed with saturated NaHCO.sub.3 aq. The
organic layer was dried over anhydrous MgSO.sub.4, filtered and
concentrated. The crude was purified by silica gel column
chromatography (0-5% MeOH in CH.sub.2Cl.sub.2) to give compound 2
(1.14 g, 1.70 mmol, 54%, R.sub.f=0.13 with 5% MeOH in
CH.sub.2Cl.sub.2). Molecular weight for
C.sub.44H.sub.83N.sub.2O.sub.2 (M+H).sup.+ Calc. 671.65, Found
671.5.
Compound 3 (ALNY-175)
[0429] To a flask containing EtOH (50 mL) was added
dimethylaminoethyl hydrazine dihydrochloride (1.00 g, 5.70 mmol)
and ALNY-SAN-003 (2.00 g, 3.80 mmol). The mixture was heated at
60.degree. C. for 16 hours. After addition of Et.sub.3N (0.5 mL),
the reaction mixture was evaporated. The residue was extracted with
Et.sub.2O and saturated NaHCO.sub.3 aq., and the organic layer was
dried over MgSO.sub.4, filtered and concentrated. The crude was
purified by silica gel column chromatography
(CH.sub.2Cl.sub.2:MeOH:NH.sub.3 aq.=95:5:0.5, R.sub.f=0.29) to give
compound 3 (1.78 g, 2.91 mmol, 76%). Molecular weight for
C.sub.41H.sub.78N.sub.3 (M+H).sup.+ Calc. 612.62, Found 612.5.
Compound 4 (ALNY-187)
[0430] 3-Dimethylamino-propionic acid hydrazide (Ryan Scientific,
500 mg, 3.89 mmol) in EtOH (10 mL) and the dilinoleyl ketone (1.74
g, 3.31 mmol) in EtOH (20 mL) were mixed together. To the solution
was added acetic acid (0.038 mL, 0.662 mmol), and the reaction
mixture was heated at 65.degree. C. for 5 hours. After addition of
Et.sub.3N (0.5 mL), the reaction mixture was evaporated. The
residue was extracted with CH.sub.2Cl.sub.2 and saturated
NaHCO.sub.3 aq., and the organic layer was dried over MgSO.sub.4,
filtered and concentrated. The crude was purified by silica gel
column chromatography (CH.sub.2Cl.sub.2:MeOH:NH.sub.3 aq.=95:5:0.5,
R.sub.f=0.30) to give compound 4 (1.40 g, 2.19 mmol, 66%).
Molecular weight for C.sub.42H.sub.78N.sub.3O (M+H).sup.+ Calc.
640.61, Found 640.5.
Compound 5 (ALNY-149)
[0431] ALY-SAN-031 (2.36 g, 3.50 mmol) was treated with hydrazine
monohydrate (0.424 mL, 5.60 mmol) in CH.sub.2Cl.sub.2 (36 mL) and
EtOH (4 mL) for 2 hours. After filtration of the resulting white
precipitation, the filtrate was concentrated. The residue was
extracted with Et.sub.2O and saturated NaHCO.sub.3 aq., and the
organic layer was dried over MgSO.sub.4, filtered and concentrated.
The crude material was used for next step without further
purification. R.sub.f: 0.44 (10% EtOAC in Hexane). Molecular weight
for C.sub.37H.sub.70NO (M+H).sup.+ Calc. 544.55, Found 544.2.
[0432] The aminooxy compound was dissolved in EtOH (30 mL), and
4-(dimethylamino)butan-2-one (Matrix Scientific, 500 mg, 4.34 mmol)
and acetic acid (0.040 mL, 0.70 mmol) was added to the solution.
The reaction mixture was stirred at room temperature for 14 hours.
After addition of Et.sub.3N (0.5 mL), the reaction mixture was
evaporated. The residue was extracted with Et.sub.2O and saturated
NaHCO.sub.3 aq., and the organic layer was dried over MgSO.sub.4,
filtered and concentrated. The crude was purified by silica gel
column chromatography (Hexane:EtOAc=1:1) to give compound 5 as a
mixture of E/Z-isomers (1.90 g, 2.96 mmol, 85%, 2 steps,
R.sub.f=0.39, 0.21 developed with Hexane:EtOAc=1:1). Molecular
weight for C.sub.43H.sub.81N.sub.2O (M+H).sup.+ Calc. 641.63, Found
641.5.
Compound 6 (ALNY-202)
[0433] To a solution of N,N'-disuccinimidyl carbonate (5.50 g, 21.5
mmol) in CH.sub.2Cl.sub.2 (200 mL), 3-dimethylamino-1-propanol
(2.37 mL, 23.6 mmol) was added dropwise. The resulting mixture was
stirred at room temperature overnight. Taken up 50 mL of the
solution, Et.sub.3N (0.822 mL, 5.90 mmol) and ALN-SAN-30 (2.07 g,
3.93 mmol) were added and the reaction mixture was stirred at room
temperature overnight. The reaction mixture was diluted with
CH.sub.2Cl.sub.2 and washed with saturated NaHCO.sub.3 aq. The
organic layer was dried over anhydrous MgSO.sub.4, filtered and
concentrated. The crude was purified by silica gel column
chromatography (0-5% MeOH in CH.sub.2Cl.sub.2) to give compound 6.
Molecular weight for C.sub.42H.sub.79N.sub.2O.sub.2 (M+H).sup.+
Calc. 643.61, Found 643.5.
[0434] Compounds of the present invention can be further
synthesized by the procedures described in the following papers,
which are hereby incorporated by their entirety: [0435] 1.
Schlueter, Urs; Lu, Jun; Fraser-Reid, Bert. Synthetic Approaches To
Heavily Lipidated Phosphoglyceroinositides. Organic Letters (2003),
5(3), 255-257 [0436] 2. King, J. F.; Allbutt, A. D. Can. J. Chem.
1970, 48, 1754-1769 [0437] 3. Mach, Mateusz; Schlueter, Urs;
Mathew, Felix; Fraser-Reid, Bert; Hazen, Kevin C. Comparing
n-pentenyl orthoesters and n-pentenyl glycosides as alternative
glycosyl donors. Tetrahedron (2002), 58(36), 7345-7354.
Example 20: Determination of Efficacy of Lipid Particle
Formulations Containing Various Cationic Lipids Using an In Vivo
Rodent Factor VII Silencing Model
[0438] Factor VII (FVII), a prominent protein in the coagulation
cascade, is synthesized in the liver (hepatocytes) and secreted
into the plasma. FVII levels in plasma can be determined by a
simple, plate-based colorimetric assay. As such, FVII represents a
convenient model for determining sirna-mediated downregulation of
hepatocyte-derived proteins, as well as monitoring plasma
concentrations and tissue distribution of the nucleic acid lipid
particles and siRNA.
TABLE-US-00008 SEQ Duplex Sequence 5'-3' ID NO: Target AD-1661
GGAfUfCAfUfCfUfCAAGfUfCfUfUA FVII fCdTdT
GfUAAGAfCfUfUGAGAfUGAfUfCfCd TsdT Lower case is 2'OMe modification
and Nf is a 2'F modified nucleobase, dT is deoxythymidine, s is
phosphothioate
[0439] The following cationic lipids were tested:
TABLE-US-00009 Com- Molecular pound Compound Structure data A
##STR00131## C.sub.42H.sub.77N.sub.3O Mol Wt: 640.08 B ##STR00132##
C.sub.42H.sub.78N.sub.2O.sub.2 Mol Wt: 643.08 C ##STR00133##
C.sub.41H.sub.77NS.sub.2 Mol Wt: 648.19 D ##STR00134##
C.sub.41H.sub.77N.sub.3 Mol Wt: 612.07 E ##STR00135##
C.sub.43H.sub.80N.sub.2O.sub.2 Mol Wt: 657.11 F ##STR00136##
C.sub.43H.sub.80N.sub.2O.sub.2 Mol Wt: 657.11 G ##STR00137##
C.sub.44H.sub.82N.sub.2O.sub.2 Mol Wt: 671.134 H ##STR00138##
C.sub.43H.sub.80N.sub.2O Mol Wt: 641.108 I ##STR00139##
C.sub.43H.sub.80N.sub.2O Mol Wt: 641.11 J ##STR00140##
C.sub.42H.sub.78N.sub.2O.sub.2 Mol Wt: 643.081 K ##STR00141##
C.sub.43H.sub.80N.sub.2O.sub.2 Mol Wt: 657.107
[0440] The cationic lipids shown above were used to formulate
liposomes containing the AD-1661 duplex using an in-line mixing
method, as described in U.S. provisional patent application
61/228,373. Lipid particles were formulated using the following
molar ratio: 50% Cationic lipid/10% distearoylphosphatidylcholine
(DSPC)/38.5% Cholesterol/1.5% PEG-DMG
(1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol, with
an average PEG molecular weight of 2000).
[0441] C57BL/6 mice (Charles River Labs, MA) received either saline
or formulated siRNA via tail vein injection. At various time points
after administration, serum samples were collected by retroorbital
bleed. Serum levels of Factor VII protein were determined in
samples using a chromogenic assay (Biophen FVII, Aniara
Corporation, OH). To determine liver mRNA levels of Factor VII,
animals were sacrificed and livers were harvested and snap frozen
in liquid nitrogen. Tissue lysates were prepared from the frozen
tissues and liver mRNA levels of Factor VII were quantified using a
branched DNA assay (QuantiGene Assay, Panomics, CA).
[0442] FVII activity was evaluated in FVII siRNA-treated animals at
48 hours after intravenous (bolus) injection in C57BL/6 mice. FVII
was measured using a commercially available kit for determining
protein levels in serum or tissue, following the manufacturer's
instructions at a microplate scale. FVII reduction was determined
against untreated control mice, and the results were expressed as %
Residual FVII. Two dose levels (0.05 and 0.005 mg/kg FVII siRNA)
were used in the screen of each novel liposome composition. FIG. 3
shows a graph illustrating the relative FVII protein levels in
animals administered with 0.05 or 0.005 mg/kg of lipid particles
containing different cationic lipids.
Example 21; siRNA Formulation Using Preformed Vesicles
[0443] Cationic lipid containing particles were made using the
preformed vesicle method. Cationic lipid, DSPC, cholesterol and
PEG-lipid were solubilised in ethanol at a molar ratio of
40/10/40/10, respectively. The lipid mixture was added to an
aqueous buffer (50 mM citrate, pH 4) with mixing to a final ethanol
and lipid concentration of 30% (vol/vol) and 6.1 mg/mL respectively
and allowed to equilibrate at room temperature for 2 min before
extrusion. The hydrated lipids were extruded through two stacked 80
nm pore-sized filters (Nuclepore) at 22.degree. C. using a Lipex
Extruder (Northern Lipids, Vancouver, BC) until a vesicle diameter
of 70-90 nm, as determined by Nicomp analysis, was obtained. This
generally required 1-3 passes. For some cationic lipid mixtures
which did not form small vesicles hydrating the lipid mixture with
a lower pH buffer (50 mM citrate, pH 3) to protonate the phosphate
group on the DSPC headgroup helped form stable 70-90 nm
vesicles.
[0444] The FVII siRNA (solubilised in a 50 mM citrate, pH 4 aqueous
solution containing 30% ethanol) was added to the vesicles,
pre-equilibrated to 35.degree. C., at a rate of -5 mL/min with
mixing. After a final target siRNA/lipid ratio of 0.06 (wt/wt) was
achieved, the mixture was incubated for a further 30 min at
35.degree. C. to allow vesicle re-organization and encapsulation of
the FVII siRNA. The ethanol was then removed and the external
buffer replaced with PBS (155 mM NaCl, 3 mM Na2HPO4, 1 mM KH2PO4,
pH 7.5) by either dialysis or tangential flow diafiltration. The
final encapsulated siRNA-to-lipid ratio was determined after
removal of unencapsulated siRNA using size-exclusion spin columns
or ion exchange spin columns.
Example 22: In Vivo Determination of Efficacy of Novel Lipid
Formulations
[0445] Test formulations were initially assessed for their FVII
knockdown in female 7-9 week old, 15-25 g, female C57Bl/6 mice at
0.1, 0.3, 1.0 and 5.0 mg/kg with 3 mice per treatment group. All
studies included animals receiving either phosphate-buffered saline
(PBS, Control group) or a benchmark formulation. Formulations were
diluted to the appropriate concentration in PBS immediately prior
to testing. Mice were weighed and the appropriate dosing volumes
calculated (10 .mu.l/g body weight). Test and benchmark
formulations as well as PBS (for Control animals) were administered
intravenously via the lateral tail vein. Animals were anesthetised
24 h later with an intraperitoneal injection of Ketamine/Xylazine
and 500-700 .mu.l of blood was collected by cardiac puncture into
serum separator tubes (BD Microtainer). Blood was centrifuged at
2,000.times.g for 10 min at 15.degree. C. and serum was collected
and stored at -70.degree. C. until analysis. Serum samples were
thawed at 37.degree. C. for 30 min, diluted in PBS and aliquoted
into 96-well assay plates. Factor VII levels were assessed using a
chromogenic assay (Biophen FVII kit, Hyphen BioMed) according to
manufacturer's instructions and absorbance measured in microplate
reader equipped with a 405 nm wavelength filter. Plasma FVII levels
were quantified and ED50s (dose resulting in a 50% reduction in
plasma FVII levels compared to control animals) calculated using a
standard curve generated from a pooled sample of serum from Control
animals. Those formulations of interest showing high levels of FVII
knockdown (ED50<<0.1 mg/kg) were re-tested in independent
studies at a lower dose range to confirm potency and establish
ED50.
[0446] FIG. 4 provides a Table depicting the EC50 of exemplary
compounds tested using this method.
Example 22A: Determination of pKa of Formulated Lipids
[0447] The pKa's of the different ionisable cationic lipids were
determined essentially as described (Eastman et al 1992
Biochemistry 31:4262-4268) using the fluorescent probe
2-(p-toluidino)-6-naphthalenesulfonic acid (TNS), which is
non-fluorescent in water but becomes appreciably fluorescent when
bound to membranes. Vesicles composed of cationic
lipid/DSPC/CH/PEG-c-DOMG (40:10:40:10 mole ratio) were diluted to
0.1 mM in buffers (130 mM NaCl, 10 mM CH.sub.3COONH.sub.4, 10 mM
MES, 10 mM HEPES) of various pH's, ranging from 2 to 11. An aliquot
of the TNS aqueous solution (1 .mu.M final) was added to the
diluted vesicles and after a 30 second equilibration period the
fluorescent of the TNS-containing solution was measured at
excitation and emission wavelengths of 321 nm and 445 nm,
respectively. The pKa of the cationic lipid-containing vesicles was
determined by plotting the measured fluorescence against the pH of
the solutions and fitting the data to a Sigmodial curve using the
commercial graphing program IgorPro.
[0448] FIG. 4 provides a Table depicting the pKa of exemplary
compounds tested using this method.
Example 23: Synthesis of Amide Linked Lipid
##STR00142##
[0450] To a stirred suspension of N,N-dimethylamino propionic acid
hydrochloride (1, 0.198 g, 1.3 mmol, 1.0 eq) in DCM was added HBTU
(0.59 g, 1.56 mmol, 1.2 eq) and DIPEA (0.71 mL, 3.9 mmol, 3.0 eq)
at room temperature. After stirred for 10 minutes, a solution of
amine (2, 0.7 g. 1.3 mmol, 1.0 eq) in DCM was added drop wise at
room temperature and continued the stirring until completion of the
reaction. Reaction mixture was diluted with DCM, washed with
saturated NaHCO.sub.3 solution followed by brine, organic layer was
separated and dried over MgSO.sub.4, concentrated and purified by
the silica gel column chromatography using DCM:MeOH (5%) as
gradients to get pure oily compound 3 (ALNY-201) in 70% yield.
[0451] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.18 (brs, 1H),
5.47-5.19 (m, 8H), 3.18-3.07 (m, 4H), 2.76 (t, J=6.5, 4H), 2.70 (s,
6H), 2.60 (t, J=6.0, 2H), 2.04 (q, J=6.8, 9H), 1.48 (brs, 1H),
1.40-1.14 (m, 43H), 0.88 (t, J=6.8, 6H). .sup.13C NMR (101 MHz,
CDCl.sub.3) .delta. 172.26, 130.41, 130.36, 128.17, 128.15, 77.54,
77.22, 76.90, 55.70, 43.85, 43.02, 37.90, 31.99, 31.74, 30.25,
29.92, 29.86, 29.81, 29.57, 27.47, 27.42, 26.84, 25.85, 22.79,
14.29. Calc. mass for the C.sub.43H.sub.80N.sub.2O: 640.6, found
641.5.
Example 24: Synthesis of Carbamate and Urea Linked Lipids
Compound 1033
##STR00143##
[0452] Stage-1
TABLE-US-00010 [0453] Chemicals/Reagents S. No & solvents M.
Wt. Mol. Eq. Qty. 1 Alcohol 1030 528 0.095 1 50 g 2 DCM 500 ml 3
Triethylamine 101.2 0.378 4 53 ml (TEA) 4 DMAP 122.17 0.0095 0.1
1.2 g 5 Mesyl chloride 114.55 0.19 2 15 ml
[0454] To a solution of Alcohol 1030 in DCM (400 ml) under Ar
atmosphere, was added TEA and DMAP and stirred at room temperature
under Ar atmosphere. Reaction mass was cooled to -5.degree. C. and
the solution of mesyl chloride in DCM (100 ml) was added slowly at
temperature below -5.degree. C. and allowed to warm to RT after
addition. After 30 minutes (TLC), reaction mass was quenched with
ice cold water (20 ml). Organic layer was separated, washed with 1N
HCl (30 ml), water, brine, dried over sodium sulfate and evaporated
at reduced pressure to obtain pure product 1031 (55 g, yield 95.5%)
as an yellow liquid. HPLC: 99.8%; .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 0.89 (t, 6H, J=6.8 Hz), 1.2-1.5 (m, 36H), 1.67
(m, 4H), 2.05 (q, 8H, J=6.8 Hz), 2.77 (t, 4H, J=6.4 Hz), 2.99 (s,
3H), 4.71 (m, 1H) and 5.36 (m, 8H). .sup.13C NMR (100 MHz,
CDCl.sub.3): .delta. 14.0, 22.5, 24.9, 25.6, 27.2, 29.2, 29.3,
29.4, 29.5, 29.6, 31.5, 34.4, 38.6, 45.9, 84.3, 127.9, 128.0,
130.0, 130.1.
Stage-2
TABLE-US-00011 [0455] S. No Chemicals/Reagents & solvents M.
Wt. Mol. Eq. Qty. 1 Mesylate 1031 606 0.0165 1 l0 g 2
Dimethylformamide (DMF) 100 ml 3 Sodium cyanide 49 0.0330 2 1.617
g
[0456] To a solution of sodium cyanide in DMF under Ar atmosphere,
was added stage-1 product in DMF slowly and then heated to
55.degree. C. for 24 hrs (HPLC). It was then cooled to room
temperature, diluted with water and extracted with ethyl acetate
(several times). The combined organic layer was washed with water,
brine, dried over sodium sulfate and evaporated at reduced pressure
to obtain crude product, which was purified silica gel
chromatography using 1% ether/hexane as eluent to afford pure
product 1032 (5.8 g, yield: (62%) as a pale yellow liquid. .sup.1H
NMR (400 MHz, CDCl.sub.3): .delta. 0.87 (t, 6H, J=6.8 Hz), 1.25 (m,
38H), 1.52 (m, 4H), 2.03 (q, 8H, J=6.8 Hz), 2.47 (m, 1H), 2.76 (t,
4H, J=6.4 Hz), 5.32 (m, 8H).
Stage-3
TABLE-US-00012 [0457] S. No Chemicals/Reagents & solvents M.
Wt. Mol. Eq. Qty. 1 Nitrile 1032 538 0.0097 1 5.2 g 2 Lithium
aluminiumhydride 38 0.0387 4 1.5 g 3 Tetrahydrofuran (THF) 52
ml
[0458] To a suspension of lithium aluminiumhydride in dry THF at Ar
atmosphere, was added stage-2 product in THF at 0.degree. C.
drop-wise. It was then allowed to warm to room temperature (RT) and
stirred for 20 hrs at RT (TLC). It was cooled to 0.degree. C. and
quenched with saturated solution of sodium sulfate. The quenched
mass was filtered through celite bed and washed with ethyl acetate.
The combined filtrate was evaporated at reduced pressure to obtain
crude product, which was purified by silica gel chromatography
using 10% ethyl acetate in hexane to afford pure product 1033 (3.7
g, yield: 71%) as pale brown liquid, HPLC: 93.8%. .sup.1H NMR (400
MHz, CDCl.sub.3): .delta. 0.87 (t, 6H, J=6.8 Hz), 1.27 (m, 48H),
2.03 (q, 8H, J=6.8 Hz), 2.60 (d, 2H, J=4.0 Hz), 2.76 (t, 4H, J=6.4
Hz), 5.31 (m, 8H). .sup.13C NMR (100 MHz, CDCl.sub.3): .delta.
14.1, 22.6, 25.6, 26.8, 27.1, 27.2, 29.3, 29.5, 29.6, 30.1, 31.5,
40.9, 45.2, 128.0, 130.1. LC-MS: 543(M+).
##STR00144##
Compound 1003 (ALNY-192)
[0459] To a solution of N,N'-disuccinimidyl carbonate (5.50 g, 21.5
mmol) in CH.sub.2Cl.sub.2 (200 mL), 3-dimethylamino-1-propanol
(1001, 2.43 g, 23.6 mmol) was added dropwise. The resulting mixture
was stirred at room temperature overnight. Taken up 50 mL of the
solution, Et.sub.3N (0.822 mL, 5.90 mmol) and ALN-SAN-30 (2.08 g,
3.93 mmol) were added and the reaction mixture was stirred at room
temperature overnight. The reaction mixture was diluted with
CH.sub.2Cl.sub.2 and washed with saturated NaHCO.sub.3 aq. The
organic layer was dried over anhydrous MgSO.sub.4, filtered and
concentrated. The crude was purified by silica gel column
chromatography (0-5% MeOH in CH.sub.2Cl.sub.2) to give compound
1003 (1.66 g, 2.53 mmol, 64%, R.sub.f=0.22 with 5% MeOH in
CH.sub.2Cl.sub.2).'H NMR (CDCl.sub.3, 400 MHz) .delta. 5.30-5.41
(m, 8H), 4.37 (d, J=8.0 Hz, 1H), 4.09 (t, J=6.0 Hz, 2H), 3.57 (brs,
1H), 2.78 (t, J=6.0 Hz, 4H), 2.33 (t, J=8.0 Hz, 2H), 2.23 (s, 6H),
2.02-2.06 (m, 8H), 1.76-1.80 (m, 2H), 1.27-1.45 (m, 40H), 0.89 (t,
J=8.0 Hz, 6H). .sup.13C NMR (CDCl.sub.3, 100 MHz) .delta. 156.5,
130.4, 130.3, 128.2, 128.1, 63.2, 56.6, 51.4, 45.7, 35.7, 31.7,
29.9, 29.8, 29.7, 29.6, 29.5, 27.7, 27.5, 27.4, 26.0, 25.8, 22.8,
14.3. Molecular weight for C.sub.43H.sub.81N.sub.2O.sub.2
(M+H).sup.+ Calc. 657.63, Found 657.5.
Compound 1004 (ALNY-200)
[0460] To a solution of N,N'-disuccinimidyl carbonate (5.50 g, 21.5
mmol) in CH.sub.2Cl.sub.2 (200 mL), 3-dimethylamino-1-propanol
(1001, 2.43 g, 23.6 mmol) was added dropwise. The resulting mixture
was stirred at room temperature overnight. Taken up 50 mL of the
solution, Et.sub.3N (0.697 mL, 5.00 mmol) and amine 1033 (1.71 g,
3.15 mmol) were added and the reaction mixture was stirred at room
temperature overnight. The reaction mixture was diluted with
CH.sub.2Cl.sub.2 and washed with saturated NaHCO.sub.3 aq. The
organic layer was dried over anhydrous MgSO.sub.4, filtered and
concentrated. The crude was purified by silica gel column
chromatography (0-5% MeOH in CH.sub.2Cl.sub.2) to give compound
1004 (1.14 g, 1.70 mmol, 54%, R.sub.f=0.13 with 5% MeOH in
CH.sub.2Cl.sub.2). Molecular weight for
C.sub.44H.sub.83N.sub.2O.sub.2 (M+H).sup.+ Calc. 671.65, Found
671.5.
##STR00145##
Compound 1007
[0461] To a solution of N,N'-disuccinimidyl carbonate (5.50 g, 21.5
mmol) in CH.sub.2Cl.sub.2 (200 mL), 2-dimethylaminoethanol (1005,
2.37 mL, 23.6 mmol) was added dropwise. The resulting mixture was
stirred at room temperature overnight. Taken up 50 mL of the
solution, Et.sub.3N (0.822 mL, 5.90 mmol) and ALN-SAN-30 (2.07 g,
3.92 mmol) were added and the reaction mixture was stirred at room
temperature overnight. The reaction mixture was diluted with
CH.sub.2Cl.sub.2 and washed with saturated NaHCO.sub.3 aq. The
organic layer was dried over anhydrous MgSO.sub.4, filtered and
concentrated. The crude was purified by silica gel column
chromatography (0-5% MeOH in CH.sub.2Cl.sub.2) to give compound
1007 (1.78 g, 2.77 mmol, 71%, 2 steps, R.sub.f=0.26 developed with
5% MeOH in CH.sub.2Cl.sub.2). Molecular weight for
C.sub.42H.sub.79N.sub.2O.sub.2 (M+H).sup.+ Calc. 643.61, Found
643.5.
Compound 1008
[0462] To a solution of N,N'-disuccinimidyl carbonate (5.50 g, 21.5
mmol) in CH.sub.2Cl.sub.2 (200 mL), 2-dimethylaminoethanol (1005,
2.37 mL, 23.6 mmol) was added dropwise. The resulting mixture was
stirred at room temperature overnight. Taken up 50 mL of the
solution, Et.sub.3N (0.697 mL, 5.00 mmol) and 1033 (440 mg, 0.812
mmol) were added and the reaction mixture was stirred at room
temperature overnight. The reaction mixture was diluted with
CH.sub.2Cl.sub.2 and washed with saturated NaHCO.sub.3 aq. The
organic layer was dried over anhydrous MgSO.sub.4, filtered and
concentrated. The crude was purified by silica gel column
chromatography (0-5% MeOH in CH.sub.2Cl.sub.2) to give compound 8
(332 mg, 0.505 mmol, 62%, R.sub.f=0.30 with 5% MeOH in
CH.sub.2Cl.sub.2). Molecular weight for
C.sub.43H.sub.81N.sub.2O.sub.2 (M+H).sup.+ Calc. 657.63, Found
657.5.
Example 25: Synthesis of Guanidinium Linked Lipids
Guanidinium Analogs
##STR00146##
[0463] Synthesis of 2063
[0464] To a solution of 2058 (6.7 g, 0.0112 mol) in DMF/Ethyl
acetate mixture was added Bis-Boc-S-methylisothiourea (3.4 g,
0.0118 mol) and triethylamine (3.5 mL, 0.246 mol) at 0.degree. C.
To the homogeneous solution was added HgCl.sub.2 (3.3 g, 0.0123
mol) at 0.degree. C. and stirred at RT for 1 hr. TLC showed the
absence of starting material. The reaction mass was then diluted
with ethyl acetate (100 ml). Filtered through a pad of celite and
washed with ethyl acetate. The filtrate was given water wash
(2.times.150 ml) and brine wash (200 ml). The hazy organic layer
was again filtered through a pad of celite/230-400 mesh silica
gel/celite. The filtrate was evaporated at reduced pressure to
obtain the crude product, which was purified by neutral alumina
chromatography using and DCM/Hexane as eluent. The product got
eluted at 40% DCM in Hexane as yellow liquid (Yield 5.2 g, 55%).
.sup.1H NMR (400 MHz, CDCl.sub.3): 0.89 (t, 6H, J=6.8 Hz),
1.27-1.46 (m, 43H), 1.49 (s, 9H), 1.50 (s, 9H), 2.02 (q, 8H,
J.sub.1=6.8 Hz, J.sub.2=6.8 Hz), 2.12 (d, 2H, J=7.2 Hz), 2.16 (s,
3H), 2.46 (t, 2H, J=5.6 Hz), 2.77 (t, 4H, J=6 Hz), 3.47 (m, 2H),
5.30 (m, 8H), 8.67 (s, 1H), 11.48 (9s, 1H).
Synthesis of 2064 (ALNY-139)
[0465] To a solution of 2063 (5.2 g, 0.0062 mol) in 10 ml of DCM at
0.degree. C., was added 10 ml of TFA in 60 ml of DCM slowly. After
addition the reaction mass was stirred at RT for 3 hrs. The TLC
showed the absence of starting material. Excess TFA was removed
under vacuum, to obtain the required product as brown viscous
liquid (5.3 g, 78%). .sup.1H NMR (400 MHz, CDCl.sub.3): 0.89 (t,
6H, J=6.8 Hz), 1.27-1.46 (m, 44H), 1.78 (s, 1H), 2.02 (q, 8H,
J.sub.1=6.4 Hz, J.sub.2=6.8 Hz), 2.77 (t, 4H, J=6.4 Hz), 2.86 (s,
3H), 2.92-3.01 (m, 2H), 3.27-3.39 (m, 2H), 3.76-3.9 (m, 2H), 5.30
(m, 8H), 7.12 (m, 2H), 8.41 (m, 1H), 10.02 (m, 3H). .sup.13C NMR
(100 MHz, CDCl.sub.3): 14.0, 22.5, 25.6, 25.8, 26.0, 27.17, 27.19,
27.6, 29.3, 29.33, 29.5, 29.6, 31.0, 31.5, 33.9, 36.3, 41.0, 54.1,
55.2, 62.0, 62.19, 111.4, 114.3, 117.1, 119.9, 127.9, 127.95,
130.1, 130.2, 152.1, 155.0, 157.4, 161.2, 161.6, 161.96, 162.3. MS:
1093 (tetra TFA salt).
Example 26: Synthesis of Oxime- and Hydrazone Linked Lipids
##STR00147##
[0466] Experimental Details
Compound 5006 (ALNY-175)
[0467] To a flask containing EtOH (50 mL) was added
dimethylaminoethyl hydrazine dihydrochloride (1.00 g, 5.70 mmol)
and the ketone 5005 (2.00 g, 3.80 mmol). The mixture was heated at
60.degree. C. for 16 hours. After addition of Et.sub.3N (0.5 mL),
the reaction mixture was evaporated. The residue was extracted with
Et.sub.2O and saturated NaHCO.sub.3 aq., and the organic layer was
dried over MgSO.sub.4, filtered and concentrated. The crude was
purified by silica gel column chromatography
(CH.sub.2Cl.sub.2:MeOH:NH.sub.3 aq.=95:5:0.5, R.sub.f=0.29) to give
compound 3 (1.78 g, 2.91 mmol, 76%). Molecular weight for
C.sub.41H.sub.78N.sub.3 (M+H).sup.+ Calc. 612.62, Found 612.5.
Compound 5007 (ALNY-187)
[0468] 3-Dimethylamino-propionic acid hydrazide (Ryan Scientific,
500 mg, 3.89 mmol) in EtOH (10 mL) and the dilinoleyl ketone 5005
(1.74 g, 3.31 mmol) in EtOH (20 mL) were mixed together. To the
solution was added acetic acid (0.038 mL, 0.662 mmol), and the
reaction mixture was heated at 65.degree. C. for 5 hours. After
addition of Et.sub.3N (0.5 mL), the reaction mixture was
evaporated. The residue was extracted with CH.sub.2Cl.sub.2 and
saturated NaHCO.sub.3 aq., and the organic layer was dried over
MgSO.sub.4, filtered and concentrated. The crude was purified by
silica gel column chromatography (CH.sub.2Cl.sub.2:MeOH:NH.sub.3
aq.=95:5:0.5, R.sub.f=0.30) to give compound 5007 (1.40 g, 2.19
mmol, 66%). Molecular weight for C.sub.42H.sub.78N.sub.3O
(M+H).sup.+ Calc. 640.61, Found 640.5.
##STR00148##
Compound 5008b
[0469] To a solution of 5008a (30 g, 56.8 mmol) in toluene, was
added N-Hydroxyphthalimide (13.9 g, 85 mmol) and TPP (22.30 g, 85
mmol) under argon. The reaction mass was cooled to -5.degree. C.,
to this was added TEA (11.84 mL), followed by DEAD (13.14 ml). The
reaction mass was allowed to stir for 12 hrs at room temperature
(TLC). It was then filtered through celite pad. The filtrate was
evaporated at reduced pressure to obtain crude product, which was
purified by silica gel chromatography to afford pure product, which
was eluted at 3% diethyl ether and hexane to get the product 5008b
(22.90 g, 60.50%) as pale yellow liquid .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 0.90 (6H, t, J=7.2 Hz), 1.2-1.4 (34H, m),
1.66-1.70 (4H, m), 2.03-2.08 (8H, m), 2.78 (4H, t, J=12.8 Hz), 4.22
(1H, m), 5.29-5.43 (8H, m), 7.74-7.76 (2H, m), 7.83-7.85 (2H, m).
.sup.13C NMR (100 MHz, CDCl.sub.3): .delta. 14.3, 22.5, 24.9, 25.6,
27.2, 27.20, 29.3, 29.3, 29.5, 29.5, 29.6, 29.7, 31.5, 32.4, 88.3,
123.3, 127.9, 129.0, 130.1, 134.3, 164.3. MS: Molecular weight
calculated for C.sub.45H.sub.71NO.sub.3 673.54, Found: 674.55
(M+H).
Compound 5010 (ALY-SAN-031)
[0470] (2.36 g, 3.50 mmol) was treated with hydrazine monohydrate
(0.424 mL, 5.60 mmol) in CH.sub.2Cl.sub.2 (36 mL) and EtOH (4 mL)
for 2 hours. After filtration of the resulting white precipitation,
the filtrate was concentrated. The residue was extracted with
Et.sub.2O and saturated NaHCO.sub.3 aq., and the organic layer was
dried over MgSO.sub.4, filtered and concentrated. The crude 5008
was used for next step without further purification. R.sub.f=0.44
(10% EtOAC in Hexane). Molecular weight for C.sub.37H.sub.70N0
(M+H).sup.+ Calc. 544.55, Found 544.2.
[0471] The compound 5008 was dissolved in EtOH (30 mL), and
4-(dimethylamino)butan-2-one (Matrix Scientific, 500 mg, 4.34 mmol)
and acetic acid (0.040 mL, 0.70 mmol) was added to the solution.
The reaction mixture was stirred at room temperature for 14 hours.
After addition of Et.sub.3N (0.5 mL), the reaction mixture was
evaporated. The residue was extracted with Et.sub.2O and saturated
NaHCO.sub.3 aq., and the organic layer was dried over MgSO.sub.4,
filtered and concentrated. The crude was purified by silica gel
column chromatography (Hexane:EtOAc=1:1) to give compound 5010 as a
mixture of E/Z-isomers (1.90 g, 2.96 mmol, 85%, 2 steps,
R.sub.f=0.39, 0.21 developed with Hexane:EtOAc=1:1). Molecular
weight for C.sub.43H.sub.81N.sub.2O (M+H).sup.+ Calc. 641.63, Found
641.5.
Compound 5009
[0472] Compound 5006 (800 mg, 1.47 mmol) was dissolved in EtOH (15
mL), (Dimethylamino)acetone (Aldrich, 0.220 mL, 1.91 mmol) and
acetic acid (0.017 mL, 0.294 mmol) were added to the solution then
the reaction mixture was stirred at room temperature for 14 hours.
After addition of Et.sub.3N (0.5 mL), the reaction mixture was
evaporated. The residue was extracted with Et.sub.2O and saturated
NaHCO.sub.3 aq., and the organic layer was dried over MgSO.sub.4,
filtered and concentrated. The crude was purified by silica gel
column chromatography (Hexane:EtOAc=9:1) to give compound 5009 (868
mg, 1.38 mmol, 94%, R.sub.f=0.22 developed with Hexane:EtOAc=9:1).
Molecular weight for C.sub.42H.sub.79N.sub.2O (M+H).sup.+ Calc.
627.62, Found 627.5.
Compound 5011
[0473] Compound 5006 (1.09 g, 2.00 mmol) was dissolved in EtOH (20
mL). 1-Methyl-4-piperidone (Aldrich, 0.320 mL, 2.60 mmol) and
acetic acid (0.40 mL, 0.400 mmol) were added to the solution then
the reaction mixture was stirred at room temperature for 14 hours.
After addition of Et.sub.3N (0.5 mL), the reaction mixture was
evaporated. The residue was extracted with Et.sub.2O and saturated
NaHCO.sub.3 aq., and the organic layer was dried over MgSO.sub.4,
filtered and concentrated. The crude was purified by silica gel
column chromatography (CH.sub.2Cl.sub.2:MeOH:NH.sub.4OH=97:3:0.3)
to give compound 5011 (1.11 g, 1.74 mmol, 87%, R.sub.f=0.20
developed with CH.sub.2Cl.sub.2:MeOH:NH.sub.4OH=97:3:0.3).
Molecular weight for C.sub.43H.sub.79N.sub.2O (M+H).sup.+ Calc.
639.62, Found 639.5.
Example 27: Synthesis of Other Lipids
Synthesis of Compound 2056 (ALNY-181)
##STR00149##
[0474] Synthesis of 2051
[0475] To a solution of 2004(50 g, 95 mmol) in DCM (400 ml) under
Ar atmosphere, was added TEA (53 mL, 378 mmol) and DMAP (1.2 g, 9.5
mmol) and stirred at room temperature under Ar atmosphere. Reaction
mass was cooled to -5.degree. C. and the solution of mesyl
chloride(15 mL, 190 mmol) in DCM (100 ml) was added slowly at
temperature below -5.degree. C. and allowed to warm to RT after
addition. After 30 minutes (TLC), reaction mass was quenched with
ice cold water (20 ml). Organic layer was separated, washed with 1N
HCl (30 ml), water, brine, dried over sodium sulfate and evaporated
at reduced pressure to obtain pure product (55 g, 95.5%) as yellow
liquid. 1H NMR (400 MHz, CDCl.sub.3): .delta. 0.89 (t, 6H, J=6.8),
1.2-1.5 (m, 36H), 1.67 (m, 4H), 2.05 (q, 8H, J1=6.8, J2=6.8), 2.77
(t, 4H, J=6.4), 2.99 (s, 3H), 4.71 (m, 1H) and 5.36 (m, 8H).
Synthesis of 2052
[0476] To a stirred solution of sodium cyanide (1.70 g, 0.0330 mol)
in DMF, was added compound 2051 (10 g, 0.0165 mol) in DMF (100 mL)
slowly and heated to 55.degree. C. for 24 hrs (TLC). It was then
cooled to room temperature, diluted with water and extracted with
ethyl acetate several times. The combined organic layers were
washed with water, brine, dried over sodium sulfate and evaporated
at reduced pressure to obtain crude product, which was purified
silica gel chromatography using 1% ether/hexane to get the product
as a pale yellow liquid (5.80 g, 62%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 0.87 (t, 6H, J=6.8 Hz), 1.25 (m, 38H), 1.52
(m, 4H), 2.03 (q, 8H, J=6.8 Hz, J=6.8 Hz), 2.47 (m, 1H), 2.76 (t,
4H, J=6.4 Hz), 5.32 (m, 8H).
Synthesis of 2053
[0477] To a cooled suspension of LAH (1.50 g, 0.0387 mol) in THF
(52 ml) at 0.degree. C. under argon atmosphere, was added compound
2052 (5.2 g, 0.0097 mol) in THF drop-wise. After addition, it was
allowed to warm to RT and stirred for 20 hrs (TLC). It was cooled
to 0.degree. C. and quenched with saturated solution of sodium
sulfate (10 ml) followed by ethyl acetate. It was filtered through
celite bed and washed with ethyl acetate. The combined organic
filtrate was evaporated at reduced pressure to obtain crude
product, which was purified by silica gel chromatography using 10%
ethyl acetate in hexane to get the product as pale brown liquid
(3.70 g, 71%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 0.87 (t,
6H, J=6.8 Hz), 1.27 (m, 48H), 2.03 (q, 8H, 6.8 Hz, 6.8 Hz), 2.60
(d, 2H, J=4.0 Hz), 2.76 (t, 4H, J=6.4 Hz), 5.31 (m, 8H). .sup.13C
NMR (100 MHz, CDCl.sub.3): .delta. 14.1, 22.6, 25.6, 26.8, 27.1,
27.2, 29.3, 29.5, 29.6, 30.1, 31.5, 40.9, 45.2, 128.0, 130.1. Mass
543 (M+).
Synthesis of 2054
[0478] To a solution of compound 2053 (45 g, 0.083 mol) in DCM (450
mL) under argon atmosphere at 0.degree. C., was added 2,6-Lutidine
(19.3 mL, 0.166 mol) followed by benzyl chloroformate (12.1 mL,
0.0847 mol) drop-wise. It was then warmed to 20.degree. C. and
stirred for one hour at that temperature (TLC). Then it was diluted
with DCM (200 ml), washed with 10% citric acid (2.times.200 ml),
water, brine and dried over anhydrous sodium sulfate, evaporated at
reduced pressure to obtain crude product, which was purified by
silica gel chromatography using 3% ether/hexane to get the final
product as pale brown liquid (36 g, 64%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 0.87 (t, 6H, J=6 Hz), 1.28 (m, 44H), 2.02 (q,
8H, J.sub.1=6.8 Hz, J.sub.2=6.8 Hz), 2.76 (t, 4H, J=6.4 Hz), 3.11
(t, 2H, J=5.6 Hz), 4.67 (s, 1H), 5.18 (s, 2H), 5.30 (m, 8H), 7.31
(m, 4H).
Synthesis of 2055
[0479] To a suspension of lithium aluminiumhydride (4.05 g, 0.1066
mol) in THF (360 mL) under argon atmosphere at 0.degree. C., was
added a solution of 2054 (36 g, 0.0533 mol) in THF drop-wise. After
addition, it was allowed to warm to room temperature and stirred
for 15 hours (TLC). The reaction mass was cooled to 0.degree. C.
and quenched with saturated solution of sodium sulfate followed by
ethyl acetate. It was filtered through celite bed and washed with
ethyl acetate. Combined filtrates were evaporated and purified by
silica gel using 100% methanol to get the final product 26 g, 87%).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 0.87 (t, 6H, J=6.8 Hz),
1.27 (m, 42H), 2.03 (q, 8H, J.sub.1=6.8 Hz, J.sub.2=6.8 Hz), 2.45
(s, 3H), 2.49 (d, 2H, J=6 Hz), 2.76 (t, 4H, J=6.4 Hz), 5.30 (m,
8H).
Synthesis of 2055a
[0480] Compound 2055 (4 g, 0.0072 mol) was dissolved in DCM (40 mL)
under argon atmosphere and cooled to 0.degree. C. To this solution
2,6-Lutidine (1.7 mL, 0.0144 mol) was added drop-wise followed by
benzyl chloroformate (1.0 mL, 0.0074 mol). It was then allowed to
warm to 20.degree. C. and stirred for one hour (TLC). Then it was
diluted with DCM (200 ml), washed with 10% citric acid (2.times.200
mL), water and brine. The organic layer was dried over anhydrous
sodium sulfate and evaporated at reduced pressure to obtain crude
product, which was purified by silica gel using 3% ether/hexane to
get the final product (3.80 g, 76%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 0.87 (t, 6H, J=6.8 Hz), 1.20 (m, 44H), 2.02
(q, 8H, J.sub.1=6.8 Hz, J.sub.2=6.8 Hz), 2.76 (t, 4H, J=6.4 Hz),
2.89 (d, 3H, J=6 Hz), 3.14 (m, 2H), 5.12 (s, 2H), 5.30 (m, 8H),
7.26 (m, 4H).
Synthesis of 2056
[0481] To a suspension of lithium aluminiumhydride (0.52 g, 0.0138
mol) in THF under argon atmosphere at 0.degree. C., was added a
solution of 2055a (3.80 g, 0.0055 mol) in THF (38 mL) dropwise.
After addition, it was allowed to warm to room temperature and
stirred for 15 hours (TLC). The reaction mass was cooled to
0.degree. C. and quenched with saturated solution of sodium sulfate
followed by ethyl acetate. Whole mass was filtered through celite
bed and washed with ethyl acetate. Combined filtrates were
evaporated at reduced pressure to obtain crude product, which was
purified silica gel chromatography using 100% methanol to get the
final product as colorless liquid(2.20 g, 70%) 1H NMR (400 MHz,
CDCl.sub.3): .delta. 0.87 (t, 6H, J=6.8 Hz), 1.21 (m, 44H), 2.03
(q, 8H, J=6.8 Hz, J=6.4 Hz), 2.18 (s, 6H), 2.76 (t, 4H, J=6.4 Hz),
5.30 (m, 8H). .sup.13C NMR (100 MHz, CDCl.sub.3): .delta. 14.0,
22.4, 25.5, 26.5, 27.0, 27.1, 29.2, 29.4, 29.6, 30.0, 31.4, 32.1,
35.6, 45.9, 64.8, 127.8, 130.0. ELSD: 99.0% Mass: 570.2 (M+).
Synthesis of 2062 (ALNY-141)
##STR00150##
[0482] Synthesis of 2061
[0483] To a solution of 2055 (5 g, 0.0089 mol) in 100 ml of DCM
under argon at 0.degree. C. was added NaBH(OAc).sub.3 (2.30 g,
0.0106 mol) Stirred for 20 minutes. Aldehyde(1.70 g, 0.0082 mol) in
700 ml of DCM was added slowly to the reaction mass over a period
of 45 minutes. After addition the reaction mass was allowed to stir
at RT for 15-20 minutes. TLC showed the absence of starting
material. The reaction mass was washed with sat. NaHCO.sub.3
(2.times.500 ml) and water (500 ml). The aqueous layer was
re-extracted with DCM (500 ml). The combined organic layer was
washed with brine (500 ml). The organic layer was dried over
Na.sub.2SO.sub.4, filtered and concentrated. The crude obtained was
purified by silica gel chromatography and Hexane/Diethyl ether as
eluent. The product got eluted at 8% of ether in hexane as brown
liquid (yield, 6.40 g, 96%). .sup.1H NMR: (400 MHz,
CDCl.sub.3):_0.89 (t, 6H, J=7.2 Hz), 1.26-1.43 (m, 40H), 1.85 (m,
1H), 2.06 (q, 8H, J1=6.8 Hz, J2=6.8 Hz), 2.15 (s, 2H), 2.20 (s,
3H), 2.45 (m, 2H), 2.77 (t, 4H, J=6 Hz), 2.95 (s, 3H), 3.35 (m,
2H), 5.12 (s, 2H), 5.32 (m, 8H), 7.35 (m, 5H).
Synthesis of 2062
[0484] To a suspension of lithium aluminiumhydride (0.751 g, 0.0198
mol) in THF under argon atmosphere at 0.degree. C., was added a
solution of 2061 (5.7 g, 0.0076 mol) in THF drop-wise. After
addition, it was allowed to warm to room temperature and stirred
for 15 hours (TLC). The reaction mass was cooled to 0.degree. C.
and quenched with saturated solution of sodium sulfate (50 ml)
followed by ethyl acetate (100 ml). It was filtered through celite
bed and washed with ethyl acetate. Combined filtrates were
evaporated at reduced pressure to obtain crude product, which was
purified silica gel chromatography using
DCM/Ethylacetate/Chloroform/Methanol as eluent. The product eluted
at 3% chloroform in methanol as brown liquid (3.80 g, 80%).sup.1H
NMR: (400 MHz, CDCl.sub.3): 0.89 (t, 6H, J=6.8 Hz), 1.26-1.37 (m,
40H), 1.42 (m, 1H), 2.06 (q, 8H, J1=6.8 Hz, J2=6.8 Hz), 2.15 (d,
2H, J=7.2 Hz), 2.20 (s, 3H), 2.29 (s, 6H), 2.45 (s, 4H), 2.78 (t,
4H, J=6.4 Hz), 5.36 (m, 8H). .sup.13C NMR: (100 MHz, CDCl.sub.3):
14.1, 22.6, 25.6, 26.6, 27.2, 27.22, 28.9, 29.3, 29.6, 29.7, 30.1,
31.5, 32.2, 35.8, 43.2, 45.7, 56.2, 57.2, 63.3, 127.9, 130.2. HPLC
ELSD: 100% Mass: 627.53
[0485] The various embodiments described above can be combined to
provide further embodiments. All of the U.S. patents, U.S. patent
application publications, foreign patents, foreign patent
applications and non-patent publications referred to in this
specification and/or listed in the Application Data Sheet, are
incorporated herein by reference, in their entirety. Aspects of the
embodiments can be modified, if necessary to employ concepts of the
various patents, applications and publications to provide yet
further embodiments.
[0486] These and other changes can be made to the embodiments in
light of the above-detailed description. In general, in the
following claims, the terms used should not be construed to limit
the claims to the specific embodiments disclosed in the
specification and the claims, but should be construed to include
all possible embodiments along with the full scope of equivalents
to which such claims are entitled. Accordingly, the claims are not
limited by the disclosure.
Sequence CWU 1
1
71116DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1taacgttgag gggcat 16216DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 2taacgttgag gggcat 16320DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 3tccatgacgt tcctgacgtt 20420DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 4tccatgacgt tcctgacgtt 20516DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 5taagcatacg gggtgt 16624DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 6gatgctgtgt cggggtctcc gggc 24724DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 7tcgtcgtttt gtcgttttgt cgtt 24824DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 8tcgtcgtttt gtcgttttgt cgtt 24920DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 9tccaggactt ctctcaggtt 201018DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 10tctcccagcg tgcgccat 181120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 11tgcatccccc aggccaccat 201220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 12gcccaagctg gcatccgtca 201320DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 13gcccaagctg gcatccgtca 201415DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 14ggtgctcact gcggc 151516DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 15aaccgttgag gggcat 161624DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 16tatgctgtgc cggggtcttc gggc 241718DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 17gtgccggggt cttcgggc 181818DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 18ggaccctcct ccggagcc 181918DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 19tcctccggag ccagactt 182015DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 20aacgttgagg ggcat 152115DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 21ccgtggtcat gctcc 152221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 22cagcctggct caccgccttg g 212320DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 23cagccatggt tccccccaac 202420DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 24gttctcgctg gtgagtttca 202518DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 25tctcccagcg tgcgccat 182615DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 26gtgctccatt gatgc 152733RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 27gaguucugau gaggccgaaa ggccgaaagu cug
332815DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 28aacgttgagg ggcat 152916DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 29caacgttatg gggaga 163016DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 30taacgttgag gggcat 163129PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 31Ala
Ala Leu Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Glu Ala1 5 10
15Leu Glu Ala Leu Ala Glu Ala Ala Ala Ala Gly Gly Cys 20
253230PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 32Ala Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu
Ala Glu Ala Leu Ala1 5 10 15Glu Ala Leu Ala Glu Ala Leu Ala Ala Ala
Ala Gly Gly Cys 20 25 303315PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 33Ala Leu Glu Ala Leu Ala Glu
Ala Leu Glu Ala Leu Ala Glu Ala1 5 10 153422PRTInfluenza A virus
34Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly1
5 10 15Met Ile Trp Asp Tyr Gly 203523PRTInfluenza A virus 35Gly Leu
Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly1 5 10 15Met
Ile Asp Gly Trp Tyr Gly 203624PRTInfluenza A virus 36Gly Leu Phe
Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly1 5 10 15Met Ile
Asp Gly Trp Tyr Gly Cys 203722PRTInfluenza A virus 37Gly Leu Phe
Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly1 5 10 15Met Ile
Asp Gly Gly Cys 203835PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 38Gly Leu Phe Gly Ala Leu
Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu1 5 10 15His Leu Ala Glu Ala
Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala Gly 20 25 30Gly Ser Cys
353934PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 39Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu
Asn Gly Trp Glu Gly1 5 10 15Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu
Ala Leu Ala Ala Gly Gly 20 25 30Ser Cys4021PRTInfluenza A
virusMOD_RES(17)..(17)Norleucine 40Gly Leu Phe Glu Ala Ile Glu Gly
Phe Ile Glu Asn Gly Trp Glu Gly1 5 10 15Leu Ile Asp Gly Lys
204120PRTInfluenza A virusMOD_RES(17)..(17)Norleucine 41Gly Leu Phe
Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly1 5 10 15Leu Ile
Asp Gly 204216PRTDrosophila sp. 42Arg Gln Ile Lys Ile Trp Phe Gln
Asn Arg Arg Met Lys Trp Lys Lys1 5 10 154314PRTHuman
immunodeficiency virus 43Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
Pro Pro Gln Cys1 5 104427PRTHuman immunodeficiency virus 44Gly Ala
Leu Phe Leu Gly Trp Leu Gly Ala Ala Gly Ser Thr Met Gly1 5 10 15Ala
Trp Ser Gln Pro Lys Lys Lys Arg Lys Val 20
254518PRTUnknownDescription of Unknown PVEC peptide 45Leu Leu Ile
Ile Leu Arg Arg Arg Ile Arg Lys Gln Ala His Ala His1 5 10 15Ser
Lys4626PRTUnknownDescription of Unknown Transportan peptide 46Gly
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Lys Ile Asn Leu Lys1 5 10
15Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu 20
254718PRTUnknownDescription of Unknown Amphiphilic model peptide
47Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys1
5 10 15Leu Ala489PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 48Arg Arg Arg Arg Arg Arg Arg Arg Arg1
54910PRTUnknownDescription of Unknown Bacterial cell wall
permeating peptide 49Lys Phe Phe Lys Phe Phe Lys Phe Phe Lys1 5
105037PRTUnknownDescription of Unknown LL-37 polypeptide 50Leu Leu
Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu1 5 10 15Phe
Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val 20 25
30Pro Arg Thr Glu Ser 355131PRTUnknownDescription of Unknown
Cecropin P1 polypeptide 51Ser Trp Leu Ser Lys Thr Ala Lys Lys Leu
Glu Asn Ser Ala Lys Lys1 5 10 15Arg Ile Ser Glu Gly Ile Ala Ile Ala
Ile Gln Gly Gly Pro Arg 20 25 305230PRTUnknownDescription of
Unknown Alpha-defensin polypeptide 52Ala Cys Tyr Cys Arg Ile Pro
Ala Cys Ile Ala Gly Glu Arg Arg Tyr1 5 10 15Gly Thr Cys Ile Tyr Gln
Gly Arg Leu Trp Ala Phe Cys Cys 20 25 305336PRTUnknownDescription
of Unknown B-defensin polypeptide 53Asp His Tyr Asn Cys Val Ser Ser
Gly Gly Gln Cys Leu Tyr Ser Ala1 5 10 15Cys Pro Ile Phe Thr Lys Ile
Gln Gly Thr Cys Tyr Arg Gly Lys Ala 20 25 30Lys Cys Cys Lys
355412PRTUnknownDescription of Unknown Bactenecin peptide 54Arg Lys
Cys Arg Ile Val Val Ile Arg Val Cys Arg1 5
105542PRTUnknownDescription of Unknown PR-39 polypeptide 55Arg Arg
Arg Pro Arg Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Pro1 5 10 15Phe
Phe Pro Pro Arg Leu Pro Pro Arg Ile Pro Pro Gly Phe Pro Pro 20 25
30Arg Phe Pro Pro Arg Phe Pro Gly Lys Arg 35
405613PRTUnknownDescription of Unknown Indolicidin peptide 56Ile
Leu Pro Trp Lys Trp Pro Trp Trp Pro Trp Arg Arg1 5
105716PRTUnknownDescription of Unknown RFGF peptide 57Ala Ala Val
Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro1 5 10
155811PRTUnknownDescription of Unknown RFGF analogue peptide 58Ala
Ala Leu Leu Pro Val Leu Leu Ala Ala Pro1 5 105913PRTHuman
immunodeficiency virus 59Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
Pro Pro Gln1 5 106016PRTDrosophila sp. 60Arg Gln Ile Lys Ile Trp
Phe Gln Asn Arg Arg Met Lys Trp Lys Lys1 5 10 156121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 61cuuacgcuga guacuucgat t 216221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 62ucgaaguacu cagcguaagt t 216321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 63cuuacgcuga guacuucgat t 216421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 64ucgaaguacu cagcguaagt t 216521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 65ggaucaucuc aagucuuact t 216621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 66guaagacuug agaugaucct t 216721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 67ggaucaucuc aagucuuact t 216821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 68guaagacuug agaugaucct t 216920DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 69ttccatgacg ttcctgacgt 207019DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 70tccatgacgt tcctgacgt 19714PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 71Glu
Ala Leu Ala1
* * * * *
References