U.S. patent application number 12/974906 was filed with the patent office on 2011-08-18 for lipids, lipid compositions, and methods of using them.
This patent application is currently assigned to NOVARTIS AG. Invention is credited to Jeremy BARYZA, Keith BOWMAN, Andrew GEALL, Tanzina LABONTE, Cameron LEE, Chandra VARGEESE, Laura WEST, Junping ZHAO.
Application Number | 20110200582 12/974906 |
Document ID | / |
Family ID | 43857652 |
Filed Date | 2011-08-18 |
United States Patent
Application |
20110200582 |
Kind Code |
A1 |
BARYZA; Jeremy ; et
al. |
August 18, 2011 |
LIPIDS, LIPID COMPOSITIONS, AND METHODS OF USING THEM
Abstract
Disclosed are formulation and optimization protocols for
delivery of therapeutically effective amounts of biologically
active agents to liver, tumors, and/or other cells or tissues. Also
provided are compositions and uses for cationic lipid compounds of
formula (I). ##STR00001## The invention also relates to
compositions and uses for stealth lipids of formula (XI).
##STR00002## Also provided are processes for making such compounds,
compositions, and formulations, plus methods and uses of such
compounds, compositions, and formulations to deliver biologically
active agents to cells and/or tissues.
Inventors: |
BARYZA; Jeremy; (Quincy,
MA) ; BOWMAN; Keith; (Cambridge, MA) ; GEALL;
Andrew; (Littleton, MA) ; LABONTE; Tanzina;
(Boston, MA) ; LEE; Cameron; (Cambridge, MA)
; VARGEESE; Chandra; (US) ; WEST; Laura;
(Weymouth, MA) ; ZHAO; Junping; (Waltham,
MA) |
Assignee: |
NOVARTIS AG
BASEL
CH
|
Family ID: |
43857652 |
Appl. No.: |
12/974906 |
Filed: |
December 21, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61284787 |
Dec 23, 2009 |
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Current U.S.
Class: |
424/130.1 ;
424/94.1; 514/169; 514/19.3; 514/44A; 514/772; 514/785; 540/113;
549/555; 560/160; 568/675 |
Current CPC
Class: |
A61K 47/30 20130101;
A61P 1/16 20180101; A61P 43/00 20180101; A61P 35/00 20180101; A61K
2300/00 20130101; C07J 43/003 20130101; C07J 41/0055 20130101; A61K
31/7088 20130101; A61K 47/18 20130101; C07J 31/006 20130101; A61K
47/28 20130101; A61K 9/127 20130101; A61K 9/1272 20130101; C07J
9/00 20130101; A61K 47/10 20130101; A61K 47/14 20130101; A61K 47/18
20130101; A61K 2300/00 20130101; A61K 47/30 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/130.1 ;
514/772; 540/113; 514/785; 549/555; 568/675; 560/160; 514/169;
514/19.3; 514/44.A; 424/94.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 47/06 20060101 A61K047/06; C07J 43/00 20060101
C07J043/00; A61P 35/00 20060101 A61P035/00; A61K 47/28 20060101
A61K047/28; C07D 303/24 20060101 C07D303/24; C07C 43/178 20060101
C07C043/178; C07C 271/16 20060101 C07C271/16; A61K 31/575 20060101
A61K031/575; A61K 38/02 20060101 A61K038/02; A61K 31/7088 20060101
A61K031/7088; A61K 38/43 20060101 A61K038/43 |
Claims
1. A composition comprising at least one cationic lipid, at least
one helper lipid and at least one stealth lipid for delivery of a
biologically active agent, wherein the biologically active agent is
for delivery to a tissue or cell selected from: a) a liver or liver
cells, wherein the composition has a cationic lipid with a pKa of
from about 6.2 or above; b) a tumor or tumor cell, wherein the
composition has a cationic lipid with a pKa of from about 6.2 or
below; c) a liver or liver cells, wherein the composition has a
cationic lipid with a pKa of from about 5.1 to about 7.4; and d) a
tumor or tumor cell, wherein the composition has a cationic lipid
with a pKa of from about 5.0 to about 6.7.
2. A compound of formula (I): ##STR00346## or a salt or
pharmaceutically acceptable derivative thereof, wherein: R.sup.1
and R.sup.2 together with the nitrogen atom to which they are
attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group; a is
absent or optionally substituted C.sub.1-4 alkylene; b is absent or
optionally substituted C.sub.1-4 alkylene; c is absent or
optionally substituted C.sub.1-4 alkylene; X.sup.1 is O or S;
X.sup.2 is O or S; Y.sup.1 is optionally substituted
C.sub.10-30alkenyl, C.sub.10-30alkynyl, C.sub.10-30heteroalkenyl or
C.sub.10-30heteroalkynyl; L is absent or
-(L.sup.b).sub.d-(L.sup.b).sub.e-(L.sup.c).sub.f-, wherein L.sup.a
is optionally substituted C.sub.1-15alkylene, C.sub.1-15
alkenylene, C.sub.1-15alkynylene, C.sub.1-15heteroalkylene,
C.sub.1-15heteroalkenylene or C.sub.1-15heteroalkynylene; L.sup.b
is optionally substituted C.sub.6-14arylene or
C.sub.5-13heteroarylene; L.sup.c is optionally substituted
C.sub.1-15alkylene, C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; d is 0 or 1; e is 0 or 1; and f is 0 or
1; and Y.sup.2 is an optionally substituted steroid.
3. The compound of claim 2, wherein one or more substituents are
selected from one or more the following: (a) a is selected from
optionally substituted C.sub.1-2 alkylene and optionally
substituted C.sub.1 alkylene; (b) b is selected from optionally
substituted C.sub.0-2 alkylene and optionally substituted C.sub.1
alkylene; (c) c is absent or is optionally substituted C.sub.1
alkylene; and/or (d) a, b and c are unsubstituted. (e) R.sup.1 and
R.sup.2 together with the nitrogen atom to which they are attached
form an optionally substituted C.sub.3-20-heterocycloalkyl,
C.sub.3-20-heterocycloalkenyl or C.sub.3-20-heterocycloalkynyl
group; (f) wherein R.sup.1 and R.sup.2 together with the nitrogen
atom to which they are attached form a compound selected from a
cyclic optionally substituted C.sub.5-16 group and a cyclic
optionally substituted C.sub.5-12 group; (g) R.sup.1 and R.sup.2
together with the nitrogen atom to which they are attached form a
cyclic optionally substituted C.sub.5 group, C.sub.6 group or
C.sub.7 group; and (h) R.sup.1 and R.sup.2 together with the
nitrogen atom to which they are attached are selected from at least
one of the head groups H.sup.1 to H.sup.52. (i) wherein X.sup.1 is
O; (j) wherein X.sup.2 is O; (k) wherein both X.sup.1 and X.sup.2
is O; (l) wherein L comprises at least one heteroatom; (m) wherein
L comprises at least one O atom; (n) wherein L.sup.c is selected
from one of formulae L.sup.c-i to L.sup.c-xxxxiii. (o) wherein d is
0; e is 0, and f is 1; (p) wherein Y.sup.1 is a C.sub.12-25 group;
(q) wherein Y.sup.1 has at least one alkene group; (r) wherein
Y.sup.1 has at least one cis unsaturated alkene group; (s) wherein
Y.sup.1 is selected from Y.sup.1-i to Y.sup.1-vii; (t) wherein
Y.sup.2 is linked to L via an oxygen atom on the optionally
substituted steroid; (u) wherein Y.sup.2 is a sterol in which the
hydrogen atom of the hydroxy group at the 3-position of the A
steroid ring has been removed; (v) wherein Y.sup.2 is the sterol of
part (u), and the sterol is selected from the group consisting of:
annasterol; avenasterol; beta-sitosterol; brassicasterol;
calciferol; campesterol; chalinosterol; chinasterol; cholestanol;
cholesterol; coprostanol; cycloartenol; dehydrocholesterol;
desmosterol; dihydrocalciferol; dihydrocholesterol;
dihydroergosterol; dinosterol; epicholesterol; ergosterol;
fucosterol; hexahydrolumisterol; hexaol; hydroxycholesterol;
lanosterol; lumisterol; parkeol; poriferasterol; saringosterol;
sitostanol; sitosterol; stigmastanol; stigmasterol; weinbersterol;
zymosterol; sterol bile acids (including one or more selected from
cholic acid; chenodeoxycholic acid; glycocholic acid; taurocholic
acid; deoxycholic acid, and lithocholic acid); and/or a salt or a
pharmaceutically acceptable derivative thereof; and (w) wherein
Y.sup.2 is a cholesterol.
4. A stealth lipid of formula (XI) ##STR00347## or a salt or
pharmaceutically acceptable derivative thereof, wherein Z is a
hydrophilic head group component selected from PEG and polymers
based on poly(oxazoline), poly(ethylene oxide), poly(vinyl
alcohol), poly(glycerol), poly(N-vinylpyrrolidone),
poly[N-(2-hydroxypropyl)methacrylamide] and poly(amino acid)s,
wherein the polymer may be linear or branched, and wherein the
polymer may be optionally substituted; wherein Z is polymerized by
n subunits; n is a number-averaged degree of polymerization between
10 and 200 units of Z, wherein n is optimized for different polymer
types; L.sub.1 is an optionally substituted C.sub.1-10 alkylene or
C.sub.1-10heteroalkylene linker including zero, one, two or more of
an ether (e.g., --O--), ester (e.g., --C(O)O--), succinate (e.g.,
--O(O)C--CH.sub.2--CH.sub.2--C(O)O--)), carbamate (e.g.,
--OC(O)--NR'--), carbonate (e.g., --OC(O)O--), ketone (e.g.,
--C--C(O)--C--), carbonyl (e.g., --C(O)--), urea (e.g.,
--NRC(O)NR'--), amine (e.g., --NR'--), amide (e.g., --C(O)NR'--),
imine (e.g., --C(NR')--), thioether (e.g., --S--), xanthate (e.g.,
--OC(S)S--), and phosphodiester (e.g., --OP(O).sub.2O--); any of
which may be substituted by zero, one or more Z groups; wherein R'
is independently selected from H, --NH--, --O--, --S--, a phosphate
or an optionally substituted C.sub.1-10 alkylene; X.sub.1 and
X.sub.2 are independently selected from a carbon or a heteroatom
selected from --NH--, --O--, --S-- or a phosphate; A.sub.1 and
A.sub.2 are independently selected from a C.sub.6-30 alkyl,
C.sub.6-30 alkenyl, and C.sub.6-30 alkynyl, wherein A.sub.1 and
A.sub.2 may be the same or different, or wherein A.sub.1 and
A.sub.2 together with the carbon atom to which they are attached
form an optionally substituted steroid.
5. The composition of claim 1 comprising one or more of the
compounds selected from: a) the compound of formula (I); and/or b)
the compound of formulat (XI).
6. The composition of claim 1, wherein the biologically active
agent is for delivery to the liver or a liver cell and the cationic
lipid has a pKa selected from at least: (a) about 5.1 to about 7.4;
(b) about 5.3 to about 7.3; (c) about 5.9 to about 7.0; (d) about
6.2 to about 6.8; and (e) about 6.1 or above
7. The composition of claim 1, wherein the biologically active
agent is for delivery to a tumor or tumor cells and the cationic
lipid has a pKa selected from at least: (a) about 5.0 to about 6.7;
(b) about 5.2 to about 6.3; (c) about 5.4 to about 6.2; (d) about
5.8 to about 6.1; and (e) about 6.1 or below.
8. The composition of claim 1 further comprising at least one
neutral lipid.
9. The composition of claim 1, wherein the composition is optimized
by selection of at least one of a stealth lipid, formulation
method, N/P ratio, particle size, and molar ratio of the cationic
lipid, an optional neutral lipid, helper lipid, stealth lipid and
an optional alkyl resorcinol based lipid.
10. The composition of claim 5, wherein the cationic lipid is
selected from E0001-E0171 and E0175-E0180.
11. The composition of claim 5, wherein the stealth lipid is
selected from S001-S009 and S012-S026.
12. The composition of claim 1, wherein the composition is
optimized for at least one parameter including but not limited to
individual selection of the pKa of the cationic lipid optimized for
the type of cell or organ being targeted, the cationic lipid used,
the stealth lipid used, the helper lipid, the neutral lipid used,
whether the neutral lipid is present or absent, the ratio of the
selected helper lipid, optional neutral lipid, stealth lipid and
cationic lipid, the N/P ratio, the particle size, the dosage
regiment, the dose given, the formulation method, and the like.
13. The composition of claim 1, further comprising a biologically
active agent.
14. The composition of claim 13, wherein the composition comprises
a biologically active agent in an amount effective for therapeutic
treatment of a disease or disorder.
15. The composition of claim 13, wherein said biologically active
agent is selected from the group consisting of antibodies,
cholesterol, hormones, antivirals, peptides, polypeptides,
proteins, nucleoproteins, chemotherapeutics, low molecular weight
drugs, vitamins, co-factors, nucleosides, nucleoside derivatives,
nucleotides, oligonucleotides, enzymatic nucleic acids, antisense
nucleic acids, triplex forming oligonucleotides, 2,5-A antisense
chimeras, allozymes, aptamers, decoy RNA molecules and analogs
thereof, and small nucleic acid molecules, such as an RNA
interfering agent (RNAi), short interfering nucleic acid (siNA),
short interfering RNA (siRNA), double-stranded RNA (dsRNA),
micro-RNA (miRNA), and short hairpin RNA (shRNA).
16. The composition of claim 15 wherein said biologically active
agent is selected from one or more of a nucleoside or nucleoside
derivative, an RNAi, an siNA, an RNAi inhibitor, a miRNA, an siRNA
and a shRNA.
17. The composition of claim 1 that further comprises a
pharmaceutically acceptable carrier.
18. A kit comprising any one or more components of the composition
of claim 1, and instructions for use.
19. A method for the treatment of a disease or condition comprising
the step of administering a therapeutically effective amount of a
composition of claim 1 to a patient in need thereof.
20. The method of claim 19 wherein the disease or condition is a
cancer, a disease of the liver, or a disease that is responsive to
treatment with an RNAi construct.
21. A method for the treatment of a disease or condition in a
subject in need thereof, the method comprising the step of
administering a therapeutically effective amount of a biologically
active agent in a formulation comprising one or more compositions
of claim 1.
22. The method of claim 21 wherein the disease or condition is a
tumor, a disease of the liver, or a disease that is responsive to
treatment with an RNAi construct.
Description
[0001] This application claims benefit under 35 U.S.C. .sctn.119(e)
of U.S. Provisional Application 61/284,787, filed 23 Dec. 2009, the
contents of which are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0002] This invention relates to cationic lipid compounds, stealth
lipid compounds and to compositions comprising such compounds. The
invention also relates to processes for making such compounds and
compositions, and to methods and uses of such compounds and
compositions, e.g., to deliver biologically active agents to cells
and tissues. The invention describes optimized pKa ranges for
cationic lipids for use in lipid formulations to deliver
biologically active agents to specific cell types, including
especially liver and tumors, and methods for optimizing the
formulations.
BACKGROUND TO THE INVENTION
[0003] The delivery of biologically active agents (including
therapeutically relevant compounds) to subjects is often hindered
by difficulties in the compounds reaching the target cell or
tissue. In particular, the trafficking of many biologically active
agents into living cells is highly restricted by the complex
membrane systems of the cells. These restrictions can result in the
need to use much higher concentrations of biologically active
agents than is desirable to achieve a result, which increases the
risk of toxic effects and side effects. One solution to this
problem is to utilise specific carrier molecules which are allowed
selective entry into the cell. Lipid carriers, biodegradable
polymers and various conjugate systems can be used to improve
delivery of biologically active agents to cells.
[0004] One class of biologically active agents that is particularly
difficult to deliver to cells is a biotherapeutic (including
nucleosides, nucleotides, polynucleotides, nucleic acids and
derivatives). In general, nucleic acids are stable for only a
limited duration in cells or plasma. The development of RNA
interference, RNAi therapy, RNA drugs, antisense therapy and gene
therapy, among others, has increased the need for effective means
of introducing active nucleic acid agents into cells. For these
reasons, compositions that can stabilise and deliver nucleic
acid-based agents into cells are of particular interest.
[0005] The most well-studied approaches for improving the transport
of foreign nucleic acids into cells involve the use of viral
vectors or cationic lipids. Viral vectors can be used to transfer
genes efficiently into some cell types, but they generally cannot
be used to introduce chemically synthesized molecules into
cells.
[0006] An alternative approach is to use delivery compositions
incorporating cationic lipids which interact with a biologically
active agent at one part and interact with a membrane system at
another part (for a review, see Feigner, 1990, Advanced Drug
Delivery Reviews, 5, 162-187 and Feigner, 1993, J. Liposome Res.,
3, 3-16). Such compositions are reported to contain liposomes.
[0007] Since the first description of liposomes in 1965 by Bangham
(J. Mol. Biol. 13, 238-252), there has been a sustained interest
and effort in developing lipid-based carrier systems for the
delivery of biologically active agents. The process of introducing
functional nucleic acids into cultured cells by using positively
charged liposomes is first described by Philip Feigner et al. Proc.
Natl. Acad. Sci., USA, 84, 7413-7417 (1987). The process was later
demonstrated in vivo by K. L. Brigham et al., Am. J. Med. Sci.,
298, 278-281 (1989).
[0008] Liposomes are attractive carriers since they protect
biological molecules from degradation while improving their
cellular uptake. Out of the various classes of liposome, liposomes
which contain cationic lipids are commonly used for delivering
polyanions (e.g. nucleic acids). Such liposomes can be formed using
cationic lipids alone and optionally including other lipids and
amphiphiles such as phosphatidylethanolamine. It is well known in
the art that both the composition of the lipid formulation as well
as its method of preparation affect the structure and size of the
resultant aggregate.
[0009] The use of cationic lipids for cellular delivery of
biologically active agents has several advantages. The
encapsulation of anionic compounds using cationic lipids is
essentially quantitative due to electrostatic interaction. In
addition, it is believed that the cationic lipids interact with the
negatively charged cell membranes initiating cellular membrane
transport (Akhtar et al., 1992, Trends Cell Bio., 2, 139; Xu et
al., 1996, Biochemistry 35, 5616).
[0010] Following Feigner's early work on introducing functional
nucleic acids into cultured cells by using positively charged
liposomes, cationic lipid compounds based on general formula I have
been disclosed in patent application EP 0 187 702.
##STR00003##
[0011] These cationic lipid compounds generally consist of two
alkyl or alkenyl chains linked to the nitrogen containing "head"
group. There is also the disclosure of two or three of R.sup.3,
R.sup.4 and R.sup.5 taken together being quinuclidino, piperidino,
pyrrolidino or morpholino.
[0012] Since EP 0 187 702, various other researchers have disclosed
cationic lipid compounds. A relevant patent application is
WO00/030444 which describes synthetic cationic lipids and
liposomes. The application discloses cationic lipid compounds with
a variety of different head groups; some examples feature more than
one head group. Amongst the compounds disclosed are compounds of
formula (II).
##STR00004##
[0013] WO 2005/121348 discloses lipid-based formulations. The
nucleic acid-lipid particles disclosed therein comprise an
interference RNA molecule, a cationic lipid with alkyl side chains
from about 10 to 20 carbon atoms having more than a single site of
unsaturation, a noncationic lipid and a conjugated lipid that
inhibits aggregation of the particle such as a polyethyleneglycol
(PEG)-lipid conjugate or a polyamide (ATTA)-conjugate. Specific
cationic lipid compounds disclosed in this patent application
include DSDMA, DODMA, DLinDMA, DLenDMA.
##STR00005##
[0014] Cullis and Bailey, Biochemistry 1994, 33, 12573-12580,
reports liposomes which comprise amino lipids such as the
following.
##STR00006##
[0015] Another recent publication concerning new cationic lipid
compounds is WO2008/137758 which describes a range of amino acid
lipid compounds reported as being useful for drug delivery and
diagnosis.
[0016] US 2006/0240554 and related applications US 2008/0020058 and
US 2009/0048197 also relate to cationic lipids. These lipids are
reported to be capable of delivering biologically active agents,
including small nucleic acid molecules such as short interfering
nucleic acids (siNA) to cells and/or tissues.
[0017] US2006/0240554, US2008/0020058 and US2009/0048197 describe
the use of the cationic lipid CLinDMA, in combination with
cholesterol and PEG-DAG for delivering siRNA to cells. The
structure of CLinDMA is set out below; at the "head" of the
molecule is the --N(Me).sub.2 group. ClinDMA is also referenced
herein as E0173.
##STR00007##
[0018] WO2009/086558 discloses the following compounds:
##STR00008##
[0019] It also discloses amino lipids having the following general
formula (In which R.sup.3 and R.sup.4 may join to form an
optionally substituted heterocyclic ring of 4 to 6 carbon atoms and
1 or 2 heteroatoms chosen from nitrogen and oxygen):
##STR00009##
[0020] There is a need for further cationic lipids which
facilitiate the systemic and local delivery of biologically active
agents to cells. There is also a need for cationic lipids which,
relative to those cationic lipids that are known in the art,
improve the systemic and local delivery of biologically active
agents to cells. There is a further need for lipid formulations
that have optimized physical characteristics for improved systemic
and local delivery of biologically active agents to specific organs
and to tumors, especially tumors outside the liver.
SUMMARY OF THE INVENTION
[0021] The invention provides novel cationic lipids and stealth
lipids, and formulations containing them, and their methods of use.
Also provided are formulations for use of such lipids for delivery
of therapeutically effective amounts of drugs, including especially
RNAi constructs, for delivery to subject in need thereof.
Particular formulations containing cationic lipids with a pKa
within specific ranges are provided for administering
therapeutically effective amounts of drugs to the liver and/or to
tumor in the somatic tissues of a subject. As a general rule (to
which there are exceptions), formulations that are the most
effective for delivery to tumors (as described in greater detail
below) contain cationic lipids with a pKa of about 6.1 or below,
although particular ranges include from about 5.0 to about 6.7,
including especially from about 5.2 to about 6.3, or from about 5.4
to about 6.2, or from about 5.8 to about 6.1, depending on tumor
type; whereas formulations that are the most effective for delivery
to liver (as described in greater detail below) contain cationic
lipids with a pKa of about 6.1 or above, although particular ranges
include from about 5.1 to about 7.4, including from about 5.3 to
about 7.3, and including especially from about 5.9 to about 7.0,
and in one embodiment is from about 6.2 to about 6.8.
[0022] Formulations may be further optimized by one skilled in the
art by adjusting other aspects of the formulation, including but
not limited to individual selection of, e.g., the pKa of the
cationic lipid optimized for the type of cell or organ being
targeted, the cationic lipid used, the stealth lipid used, the
helper lipid, the neutral lipid used, including whether the neutral
lipid is present or absent, the ratio of the selected helper lipid,
optional neutral lipid, stealth lipid and cationic lipid, the N/P
ratio, the particle size, the dosage regimen, the dose given, the
formulation method, and the like.
[0023] This invention provides cationic lipids (also referred to
herein as "compounds") and compositions comprising such lipids. The
invention also provides processes for making such compounds and
compositions, and methods and uses of such compounds and
compositions to deliver biologically active (including therapeutic)
agents to cells (including in vivo delivery) and for optimizing
such formulations for delivery in vivo to specific cell types and
tissues. This invention also provides stealth lipids.
[0024] In one embodiment, the invention provides a compound of
formula (I):
##STR00010##
or a pharmaceutically acceptable derivative thereof, wherein:
[0025] R.sup.1 and R.sup.2 together with the nitrogen atom to which
they are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group;
[0026] a is absent or optionally substituted C.sub.1-4
alkylene;
[0027] b is absent or optionally substituted C.sub.1-4
alkylene;
[0028] c is absent or optionally substituted C.sub.1-4
alkylene;
[0029] X.sup.1 is O or S;
[0030] X.sup.2 is O or S;
[0031] Y.sup.1 is optionally substituted C.sub.10-30alkenyl,
C.sub.10-30alkynyl, C.sub.10-30heteroalkenyl or
C.sub.10-30heteroalkynyl;
[0032] L is absent or
-(L.sup.a).sub.d-(L.sup.b).sub.e-(L.sup.c).sub.f-, wherein [0033]
L.sup.a is optionally substituted C.sub.1-15alkylene,
C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [0034] L.sup.b is optionally
substituted C.sub.6-14arylene or C.sub.5-13heteroarylene; [0035]
L.sup.c is optionally substituted C.sub.1-15alkylene,
C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [0036] d is 0 or 1; [0037] e is 0 or 1;
and [0038] f is 0 or 1; and
[0039] Y.sup.2 is an optionally substituted steroid.
[0040] The present invention also provides a pharmaceutical
composition comprising a compound of formula (I). These
compositions may comprise a biologically active agent, optionally
in combination with other lipid components.
[0041] The present invention also provides a pharmaceutical
composition comprising a compound of formula (XI). These
compositions may comprise a biologically active agent, optionally
in combination with other lipid components.
[0042] In one embodiment, the invention provides a compound of
formula (XI):
##STR00011##
or a salt or pharmaceutically acceptable derivative thereof,
wherein:
[0043] Z is a hydrophilic head group component selected from PEG
and polymers based on poly(oxazoline), poly(ethylene oxide),
poly(vinyl alcohol), poly(glycerol), poly(N-vinyl pyrrolidone),
poly[N-(2-hydroxypropyl)methacrylamide] and poly(amino acid)s,
wherein the polymer may be linear or branched, and wherein the
polymer may be optionally substituted;
[0044] wherein Z is polymerized by n subunits;
[0045] n is a number-averaged degree of polymerization between 10
and 200 units of Z, wherein n is optimized for different polymer
types;
[0046] L.sub.1 is an optionally substituted C.sub.1-10 alkylene or
C.sub.1-10 heteroalkylene linker including zero, one, two or more
of an ether (e.g., --O--), ester (e.g., --C(O)O--), succinate
(e.g., --O(O)C--CH.sub.2--CH.sub.2--C(O)O--)), carbamate (e.g.,
--OC(O)--NR'--), carbonate (e.g., --OC(O)O--), ketone (e.g.,
--C--C(O)--C--); carbonyl (e.g., --C(O)--); urea (e.g.,
--NRC(O)NR'--), amine (e.g., --NR'--), amide (e.g., --C(O)NR'--),
imine (e.g., --C(NR')--), thioether (e.g., --S--), xanthate (e.g.,
--OC(S)S--), and phosphodiester (e.g., --OP(O).sub.2O--); any of
which may be substituted by zero, one or more Z groups;
[0047] wherein R' is independently selected from --H, --NH--,
--NH.sub.2, --O--, --S--, a phosphate or an optionally substituted
C.sub.1-10 alkylene;
[0048] X.sub.1 and X.sub.2 are independently selected from a carbon
or a heteroatom selected from --NH--, --O--, --S-- or a
phosphate;
[0049] A.sub.1 and A.sub.2 are independently selected from a
C.sub.6-30 alkyl, C.sub.6-30 alkenyl, and C.sub.6-30 alkynyl,
wherein A.sub.1 and A.sub.2 may be the same or different,
[0050] or wherein A.sub.1 and A.sub.2 together with the carbon atom
to which they are attached form an optionally substituted
steroid.
[0051] Compositions containing lipids of the invention are useful,
e.g., in delivering therapeutic compounds (e.g., one or more
biologically active agents) for the treatment of disorders or
diseases, including especially those disorders or diseases that
respond to modulation of gene expression in a patient or
administration of a therapeutic to a targeted cell or tissue. As
such, the compounds and compositions of the present invention can
be used to treat diseases and conditions in a patient. In
particular, the compounds can be utilised in liposomes and/or lipid
nanoparticle formulation compositions to deliver biologically
active agents, including, e.g., antibodies, low molecular weight
compositions, protein therapeutics and nucleic acid compositions
such as siRNA for RNAi, to cells or tissues.
[0052] In a method of the invention the biologically active agents
are delivered, utilising the described cationic lipids, to cells,
during which process they may crass epithelial and endothelial
tissues, such as skin, mucous membranes, vascular tissues,
gastrointestinal tissues, blood brain barrier tissues,
opthalmological tissues, pulmonary tissues, liver tissues, cardiac
tissues, kidney tissues, tumor tissues, etc. The compounds and
compositions can be used for both local and systemic delivery of
the biologically active agents.
DETAILED DESCRIPTION OF THE INVENTION
[0053] To date, the therapeutic potential of the RNAi field has not
been met because of issues with delivery of therapeutically
effective amounts of RNAi composition to most somatic tissues. RNAi
therapeutics have some effectiveness when directed to tissues in
the eye, skin, lungs and liver. A need remains for compositions and
methods for delivery of therapeutically effective amounts of RNAi
for the treatment of all other somatic tissues and for cancer,
including metastatic cancers.
[0054] A discovery described herein is that the optimal pKa range
of cationic lipids in formulations for delivery to tumors is lower
than that for the liver. As a general rule (to which there are
exceptions), formulations with the most effective lipids for
delivery to tumors (as described in greater detail below) contain
cationic lipids with a pKa of from about 5.0 to about 6.7,
including especially from about 5.8 to about 6.1, depending on
tumor type, whereas formulations with the most effective lipids for
delivery to liver (as described in greater detail below) contain
cationic lipids with a pKa of from about 5.1 to about 7.4,
including especially from about 5.9 to about 7.0. In one
embodiment, a cationic lipid with a pKa of about 6.1 or below is
more effective in a formulation for delivery of a biologically
active agent to a tumor or tumor cell; whereas a cationic lipid
with a pKa of about 6.1 or above is more effective in a formulation
for delivery of a biologically active agent to the liver or a liver
cell.
[0055] Provided herein are novel cationic lipids, stealth lipids,
and formulations containing them; plus methods of use. Exemplary
cationic lipids of a particular pKa range are described, wherein
formulations containing these cationic lipids may deliver
therapeutically effective amounts of RNAi compositions to tumors
when administered to a subject in vivo. Other formulations made
with cationic lipids of another pKa range are described, wherein
formulations containing these cationic lipids may deliver
therapeutically effective amounts of RNAi compositions to liver
when administered to a subject in vivo.
Cationic Lipids of the Invention
[0056] In one embodiment, the invention provides a compound of
formula (I):
##STR00012##
or a pharmaceutically acceptable derivative thereof, wherein:
[0057] R.sup.1 and R.sup.2 together with the nitrogen atom to which
they are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group;
[0058] a is absent or optionally substituted C.sub.1-4
alkylene;
[0059] b is absent or optionally substituted C.sub.1-4
alkylene;
[0060] c is absent or optionally substituted C.sub.1-4
alkylene;
[0061] X.sup.1 is O or S;
[0062] X.sup.2 is O or S;
[0063] Y.sup.1 is optionally substituted C.sub.10-30alkenyl,
C.sub.10-30alkynyl, C.sub.10-30heteroalkenyl or
C.sub.10-30heteroalkynyl;
[0064] L is absent or
-(L.sup.a).sub.d-(L.sup.b).sub.e-(L.sup.c).sub.f-, wherein [0065]
L.sup.a is optionally substituted C.sub.1-15alkylene,
C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [0066] L.sup.b is optionally
substituted C.sub.6-14arylene or C.sub.5-13heteroarylene; [0067]
L.sup.c is optionally substituted C.sub.1-15alkylene,
C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [0068] d is 0 or 1; [0069] e is 0 or 1;
and [0070] f is 0 or 1; and
[0071] Y.sup.2 is an optionally substituted steroid.
[0072] In one embodiment of formula I, a is an optionally
substituted C.sub.1-2 alkylene. In one embodiment of formula I, a
is an optionally substituted C.sub.1 alkylene. In one embodiment of
formula I, b is an optionally substituted C.sub.0-2 alkylene. In
one embodiment of formula I, b is an optionally substituted C.sub.1
alkylene. In one embodiment of formula I, c is absent or is an
optionally substituted C.sub.1 alkylene. In one embodiment of
formula I, a, b and c are unsubstituted. In one embodiment of
formula I, c is absent.
[0073] In one embodiment of formula I, R.sup.1 and R.sup.2 together
with the nitrogen atom to which they are attached form an
optionally substituted C.sub.3-20-heterocycloalkyl,
C.sub.3-20-heterocycloalkenyl or C.sub.3-20-heterocycloalkynyl
group. In one embodiment of formula I, R.sup.1 and R.sup.2 together
with the nitrogen atom to which they are attached form an
optionally substituted C.sub.3-20-heterocycloalkyl group. In one
embodiment of formula I, R.sup.1 and R.sup.2 together with the
nitrogen atom to which they are attached form an optionally
substituted C.sub.5-16 group. In one embodiment of formula I,
R.sup.1 and R.sup.2 together with the nitrogen atom to which they
are attached form an optionally substituted C.sub.5-12 group.
[0074] In one embodiment of formula I, R.sup.1 and R.sup.2 together
with the nitrogen atom to which they are attached form an
optionally substituted C.sub.5 group, C.sub.6 group or C.sub.7
group. In one embodiment of formula I, R.sup.1 and R.sup.2 together
with the nitrogen atom to which they are attached form an
optionally substituted C.sub.5 group or C.sub.6 group.
[0075] In one embodiment of formula I, R.sup.1 and R.sup.2 together
with the nitrogen atom to which they are attached are selected from
H.sup.1 to H.sup.52.
[0076] In one embodiment of formula I, X.sup.1 is O. In one
embodiment of formula I, X.sup.2 is O.
[0077] In one embodiment of formula I, L comprises at least one
heteroatom. In one embodiment of formula I, L comprises at least
one O atom. In one embodiment of formula I, L comprises at least
two heteroatoms. In one embodiment of formula I, L comprises at
least two substitutions of O atoms. In one embodiment of formula I,
L.sup.c is an optionally substituted C.sub.1-15alkylene or
C.sub.1-15heteroalkylene. In one embodiment of formula I, L.sup.c
is selected from any one or more of formulae to L.sup.c-i to
L.sup.c-xxxxiii.
[0078] In one embodiment of formula I, L.sup.c is an optionally
substituted C.sub.1-15heteroalkylene. In one embodiment of formula
I, L.sup.c is an optionally substituted C.sub.1-11 group. In one
embodiment of formula I, L.sup.c is an optionally substituted
C.sub.1-9 group. In one embodiment of formula I, L.sup.c is an
optionally substituted C.sub.3-8 group. In one embodiment of
formula I, L.sup.c is an optionally substituted C.sub.4-7 group. In
one embodiment of formula I, L.sup.c is an optionally substituted
C.sub.5, C.sub.6 or C.sub.7 group.
[0079] In one embodiment of formula I, d is 0; e is 0, and f is
1.
[0080] In one embodiment of formula I, Y.sup.1 is a C.sub.12-28
group. In one embodiment of formula I, Y.sup.1 is a C.sub.14-26
group. In one embodiment of formula I, Y.sup.1 is a C.sub.16-24
group. In one embodiment of formula I, Y.sup.1 is a C.sub.16-22
group. In one embodiment of formula I, Y.sup.1 has at least one
alkene group. In one embodiment of formula I, Y.sup.1 has 1, 2 or 3
alkene groups. In one embodiment of formula I, Y.sup.1 has an
alkene group at the omega-3 position. In one embodiment of formula
I, Y.sup.1 has an alkene group at the omega-6 position. In one
embodiment of formula I, Y.sup.1 has an alkene group at the omega-9
position.
[0081] In one embodiment of formula I, Y.sup.1 has at least one cis
unsaturated alkene group. In one embodiment of formula I, Y.sup.1
has at least two cis unsaturated alkene groups. In one embodiment
of formula I, Y.sup.1 has at least three cis unsaturated alkene
groups. In one embodiment of formula I, Y.sup.1 is selected from
to
[0082] In one embodiment of formula I, Y.sup.2 is linked to L via
an oxygen atom on the optionally substituted steroid. In one
embodiment of formula I, Y.sup.2 is linked to L via an oxygen atom
on the 3-position of the A steroid ring. In one embodiment of
formula I, Y.sup.2 is a sterol in which the hydrogen atom of the
hydroxy group at the 3-position of the A steroid ring has been
removed. In one embodiment of formula I, the sterol is
cholesterol.
[0083] A second embodiment of the invention is represented by a
compound of formula (II):
##STR00013##
or a pharmaceutically acceptable derivative thereof, wherein:
[0084] R.sup.1 and R.sup.2 together with the nitrogen atom to which
they are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group;
[0085] a is absent or optionally substituted C.sub.1-4
alkylene;
[0086] b is absent or optionally substituted C.sub.1-4
alkylene;
[0087] c is absent or optionally substituted C.sub.1-4
alkylene;
[0088] X.sup.1 is O or S;
[0089] X.sup.2 is O or S;
[0090] Y.sup.1 is optionally substituted C.sub.10-30alkenyl,
C.sub.10-30alkynyl, C.sub.10-30heteroalkenyl or
C.sub.10-30heteroalkynyl;
[0091] L is -(L.sup.a).sub.d-(L.sup.b).sub.e-(L.sup.c).sub.f-,
wherein [0092] L.sup.a is optionally substituted
C.sub.1-15alkylene, C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [0093] L.sup.b is optionally
substituted C.sub.6-14arylene or C.sub.5-13heteroarylene; [0094]
L.sup.c is optionally substituted C.sub.1-15alkylene,
C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [0095] d is 0 or 1; [0096] e is 0 or 1;
and [0097] f is 0 or 1;
[0098] provided that L comprises one or more heteroatoms, and
[0099] Y.sup.2 is an optionally substituted steroid.
[0100] A third embodiment of the invention is represented by a
compound of formula (III):
##STR00014##
or a pharmaceutically acceptable derivative thereof, wherein:
[0101] R.sup.1 and R.sup.2 together with the nitrogen atom to which
they are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group;
[0102] a is methylene;
[0103] b is methylene;
[0104] c is absent;
[0105] X.sup.1 is O or S;
[0106] X.sup.2 is O or S;
[0107] Y.sup.1 is optionally substituted C.sub.10-30alkenyl,
C.sub.10-30alkynyl, C.sub.10-30heteroalkenyl or
C.sub.10-30heteroalkynyl;
[0108] L is -(L.sup.a).sub.d-(L.sup.b).sub.e-(L.sup.c).sub.f-,
wherein [0109] L.sup.a is optionally substituted
C.sub.1-15alkylene, C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [0110] L.sup.b is optionally
substituted C.sub.6-14arylene or C.sub.5-13heteroarylene; [0111]
L.sup.c is optionally substituted C.sub.1-15alkylene,
C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [0112] d is 0 or 1; [0113] e is 0 or 1;
and [0114] f is 0 or 1; and
[0115] Y.sup.2 is an optionally substituted steroid.
[0116] A fourth embodiment of the invention is represented by a
compound of formula (IV):
##STR00015##
or a pharmaceutically acceptable derivative thereof, wherein:
[0117] R.sup.1 and R.sup.2 together with the nitrogen atom to which
they are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group;
[0118] a is methylene;
[0119] b is methylene;
[0120] c is absent;
[0121] X.sup.1 is O or S;
[0122] X.sup.2 is O or S;
[0123] Y.sup.1 is optionally substituted C.sub.10-30alkenyl,
C.sub.10-30alkynyl, C.sub.10-30heteroalkenyl or
C.sub.10-30heteroalkynyl;
[0124] L is -(L.sub.a).sub.d-(L.sup.b).sub.e-(L.sup.c).sub.f-,
wherein [0125] L.sup.a is optionally substituted
C.sub.1-15alkylene, C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [0126] L.sup.b is optionally
substituted C.sub.6-14arylene or C.sub.5-13heteroarylene; [0127]
L.sup.c is optionally substituted C.sub.1-15alkylene,
C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [0128] d is 0 or 1; [0129] e is 0 or 1;
and [0130] f is 0 or 1;
[0131] provided that L comprises one or more heteroatoms, and
[0132] Y.sup.2 is an optionally substituted steroid.
[0133] A fifth embodiment of the invention is represented by a
compound of formula (V):
##STR00016##
or a pharmaceutically acceptable derivative thereof, wherein:
[0134] R.sup.1 and R.sup.2 together with the nitrogen atom to which
they are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group;
[0135] a is methylene;
[0136] b is methylene;
[0137] c is absent;
[0138] X.sup.1 is O;
[0139] X.sup.2 is O;
[0140] Y.sup.1 is optionally substituted C.sub.10-30alkenyl,
C.sub.10-30alkynyl, C.sub.10-30heteroalkenyl or
C.sub.10-30heteroalkynyl;
[0141] L is -(L.sup.a).sub.d-(L.sup.b).sub.e-(L.sup.c).sub.f-,
wherein [0142] L.sup.a is optionally substituted
C.sub.1-15alkylene, C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [0143] L.sup.b is optionally
substituted C.sub.6-14arylene or C.sub.5-13heteroarylene; [0144]
L.sup.e is optionally substituted C.sub.1-15alkylene,
C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [0145] d is 0 or 1; [0146] e is 0 or 1;
and [0147] f is 0 or 1;
[0148] provided that L comprises one or more heteroatoms, and
[0149] Y.sup.2 is an optionally substituted steroid.
[0150] A sixth embodiment of the invention is represented by a
compound of formula (VI):
##STR00017##
or a pharmaceutically acceptable derivative thereof, wherein:
[0151] R.sup.1 and R.sup.2 together with the nitrogen atom to which
they are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group;
[0152] a is methylene;
[0153] b is methylene;
[0154] c is absent;
[0155] X.sup.1 is O;
[0156] X.sup.2 is O;
[0157] Y.sup.1 is optionally substituted C.sub.10-30alkenyl,
C.sub.10-30alkynyl, C.sub.10-30heteroalkenyl or
C.sub.10-30heteroalkynyl;
[0158] L is -L.sup.c-, wherein [0159] L.sup.c is optionally
substituted C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; and
[0160] Y.sup.2 is an optionally substituted steroid.
[0161] A seventh embodiment of the invention is represented by a
compound of formula (VII):
##STR00018##
or a pharmaceutically acceptable derivative thereof, wherein:
[0162] R.sup.1 and R.sup.2 together with the nitrogen atom to which
they are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group;
[0163] a is methylene;
[0164] b is methylene;
[0165] c is absent;
[0166] X.sup.1 is O;
[0167] X.sup.2 is O;
[0168] Y.sup.1 is an optionally substituted C.sub.16-22 alkenyl
group;
[0169] L is -L.sup.c-, wherein [0170] L.sup.c is optionally
substituted C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; and
[0171] Y.sup.2 is an optionally substituted steroid.
[0172] An eighth embodiment of the invention is represented by a
compound of formula (VIII):
##STR00019##
or a pharmaceutically acceptable derivative thereof, wherein:
[0173] R.sup.1 and R.sup.2 together with the nitrogen atom to which
they are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group;
[0174] a is methylene;
[0175] b is methylene;
[0176] c is absent;
[0177] X.sup.1 is O;
[0178] X.sup.2 is O;
[0179] Y.sup.1 is an optionally substituted C.sub.16-22 alkenyl
group;
[0180] L is -L.sup.c-, wherein [0181] L.sup.c is optionally
substituted C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; and
[0182] Y.sup.2 is cholesterol connected through the hydroxy group
at the 3-position of the A steroid ring, the hydrogen atom of said
hydroxy group being absent.
[0183] A ninth embodiment of the invention is represented by a
compound of formula (IX):
##STR00020##
or a pharmaceutically acceptable derivative thereof, wherein:
[0184] R.sup.1 and R.sup.2 together with the nitrogen atom to which
they are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group;
[0185] a is methylene;
[0186] b is methylene;
[0187] c is absent;
[0188] X.sup.1 is O or S;
[0189] X.sup.2 is O or S;
[0190] Y.sup.1 is optionally substituted C.sub.10-30alkenyl,
C.sub.10-30alkynyl, C.sub.10-30heteroalkenyl or
C.sub.10-30heteroalkynyl;
[0191] L is -(L.sup.a).sub.d-(L.sup.b).sub.e-(L.sup.c).sub.f-,
wherein [0192] L.sup.a is optionally substituted
C.sub.1-15alkylene, C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [0193] L.sup.b is optionally
substituted C.sub.6-14arylene or C.sub.5-13heteroarylene; [0194]
L.sup.c is optionally substituted C.sub.1-15alkylene,
C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [0195] d is 0 or 1; [0196] e is 0 or 1;
and [0197] f is 0 or 1;
[0198] provided that L comprises one or more heteroatoms, and
[0199] Y.sup.2 is an optionally substituted steroid; and wherein
the pKa of the compound is from about 5.1 to about 7.4.
[0200] A tenth embodiment of the invention is represented by a
compound of formula (X):
##STR00021##
or a pharmaceutically acceptable derivative thereof, wherein:
[0201] R.sup.1 and R.sup.2 together with the nitrogen atom to which
they are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group;
[0202] a is methylene;
[0203] b is methylene;
[0204] c is absent;
[0205] X.sup.1 is O or S;
[0206] X.sup.2 is O or S;
[0207] Y.sup.1 is optionally substituted C.sub.10-30alkenyl,
C.sub.10-30alkynyl, C.sub.10-30heteroalkenyl or
C.sub.10-30heteroalkynyl;
[0208] L is -(L.sup.a).sub.d-(L.sup.b).sub.e-(L.sup.b).sub.f-,
wherein [0209] L.sup.a is optionally substituted
C.sub.1-15alkylene, C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [0210] L.sup.b is optionally
substituted C.sub.6-14arylene or C.sub.5-13heteroarylene; [0211]
L.sup.c is optionally substituted C.sub.1-15alkylene,
C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [0212] d is 0 or 1; [0213] e is 0 or 1;
and [0214] f is 0 or 1;
[0215] provided that L comprises one or more heteroatoms, and
[0216] Y.sup.2 is an optionally substituted steroid; and wherein
the pKa of the compound is from about 5.0 to about 6.7.
Stealth Lipids
[0217] Included in the present invention are "stealth lipids"
containing a hydrophilic head group linked to a lipid moiety.
Further characterization of stealth lipids is provided below.
[0218] In one embodiment is provided a stealth lipid composition of
formula (XI):
##STR00022##
or a salt or pharmaceutically acceptable derivative thereof,
wherein:
[0219] Z is a hydrophilic head group component selected from PEG
and polymers based on poly(oxazoline), poly(ethyleneoxide),
poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone),
poly[N-(2-hydroxypropyl)methacrylamide] and poly(amino acid)s,
wherein the polymer may be linear or branched, and wherein the
polymer may be optionally substituted; [0220] wherein Z is
polymerized by n subunits;
[0221] n is a number-averaged degree of polymerization between 10
and 200 units of Z, wherein n is optimized for different polymer
types;
[0222] L.sub.1 is an optionally substituted C.sub.1-10 alkylene or
C.sub.1-10 heteroalkylene linker including zero, one, two or more
of an ether (e.g., --O--), ester (e.g., --C(O)O--), succinate
(e.g., --O(O)C--CH.sub.2--CH.sub.2--C(O)O--)), carbamate (e.g.,
--OC(O)--NR'--), carbonate (e.g., --OC(O)O--), ketone (e.g.,
--C--C(O)--C--), carbonyl (e.g., --C(O)--), urea (e.g.,
--NRC(O)NR'--), amine (e.g., --NR'--), amide (e.g., --C(O)NR'--),
imine (e.g., --C(NR')--), thioether (e.g., --S--), xanthate (e.g.,
--OC(S)S--), and phosphodiester (e.g., --OP(O).sub.2O--); any of
which may be substituted by zero, one or more Z groups;
[0223] wherein R' is independently selected from --H, --NH--,
--NH.sub.2, --O--, --S--, a phosphate or an optionally substituted
C.sub.1-10 alkylene;
[0224] X.sub.1 and X.sub.2 are independently selected from a carbon
or a heteroatom selected from --NH--, --O--, --S-- or a
phosphate;
[0225] A.sub.1 and A.sub.2 are independently selected from a
C.sub.6-30 alkyl, C.sub.6-30 alkenyl, and C.sub.6-30 alkynyl,
wherein A.sub.1 and A.sub.2 may be the same or different,
[0226] or wherein A.sub.1 and A.sub.2 together with the carbon atom
to which they are attached form an optionally substituted
steroid.
[0227] In one embodiment, the invention provides a stealth lipid of
formula (XII)
##STR00023##
or a salt or pharmaceutically acceptable derivative thereof,
wherein
[0228] PEG is a polyethylene glycol) subunit, wherein the PEG may
be linear or branched;
[0229] n is a number-averaged degree of polymerization between 10
and 200 units of PEG, preferably about 23 units, about 45 units or
about 68 units;
[0230] L.sub.1 is an optionally substituted C.sub.1-10 alkylene or
C.sub.1-10heteroalkylene linker containing one, two or more of an
ether, ester, succinate, carbamate, carbonate, ketone, carbonyl,
urea, amine, amide, imine, thioether, xanthate, and phosphodiester;
any of which may be substituted by zero, one or more PEG
groups;
[0231] X.sub.1 and X.sub.2 are independently selected from carbon
or oxygen;
[0232] A.sub.1 and A.sub.2 are independently selected from a
C.sub.6-30 alkyl, C.sub.6-30 alkenyl, and C.sub.6-30 alkynyl,
wherein A.sub.1 and A.sub.2 may be the same or different,
[0233] or wherein A.sub.1 and A.sub.2 together with the carbon atom
to which they are attached form an optionally substituted
steroid.
[0234] The stealth lipids of formulae (XI) and (XII), when
formulated with e.g., the cationic lipids of formula (I), provide
lipid nanoparticles with increased in vivo potency compared to
previous comparable stealth lipids. Therefore the invention
provides stealth lipids having the potential to improve efficacy
and toxicity. Provided herewith is a composition containing these
stealth lipids and the use of these stealth lipids to deliver
biologically active agents to cells.
[0235] As provided in Table 8, two exemplary lipid nanoparticles
formulated using the same process and with otherwise identical
compositions, but differing in the stealth lipids, are delivered to
the liver. The lipid nanoparticle containing the prior art stealth
lipid S010 and delivering an siRNA construct specific to factor VII
("FVII") demonstrates an in vivo inhibition of 72.2% when
administered to the liver, while the lipid nanoparticle containing
the stealth lipid S006 in comparison demonstrated an in vivo Factor
VII inhibition of 83.8%.
[0236] In another example provided in Table 9, for delivery in vivo
to subcutaneous tumors, six lipid nanoparticles with otherwise
identical compositions except for the PEG/stealth lipid are
compared for effective delivery of an siRNA specific to Polo-Like
Kinase 1 ("PLK1"). The lipid nanoparticle containing the prior art
stealth lipid S011 demonstrates an in vivo PLK1 inhibition of 46%
in the tumor tissue, while lipid nanoparticles containing the
stealth lipids S004, S007, S009, S008, and S005 demonstrate in vivo
PLK1 inhibitions of 56%, 65%, 64%, 60%, and 52%, respectively, in
the tumor tissue.
[0237] The stealth lipids S001 through S009 and S012 through S026
individually and as a class thereby demonstrate improved
characteristics when used in formulations and therapeutic
composition for use in delivery of biologically active agents, in
this case for one or more siRNA.
[0238] Novel stealth lipids are provided in the invention. In one
embodiment of the invention, the stealth lipid is S001. In one
embodiment, the stealth lipid is S002. In one embodiment, the
stealth lipid is S003. In one embodiment, the stealth lipid is
S004. In one embodiment, the stealth lipid is S005. In one
embodiment, the stealth lipid is S006. In one embodiment, the
stealth lipid is S007. In one embodiment, the stealth lipid is
S008. In one embodiment, the stealth lipid is S009. In one
embodiment, the stealth lipid is S012. In one embodiment, the
stealth lipid is S013. In one embodiment, the stealth lipid is
S014. In one embodiment, the stealth lipid is S015. In one
embodiment, the stealth lipid is S016. In one embodiment, the
stealth lipid is S017. In one embodiment, the stealth lipid is
S018. In one embodiment, the stealth lipid is S019. In one
embodiment, the stealth lipid is S020. In one embodiment, the
stealth lipid is S021. In one embodiment, the stealth lipid is
S022. In one embodiment, the stealth lipid is S023. In one
embodiment, the stealth lipid is S024. In one embodiment, the
stealth lipid is S025. In one embodiment, the stealth lipid is
S026.
Formulations for Delivery of Biologically Active Agents
[0239] In general, whereas in the prior art the tissue dependent
efficacy was controlled by varying the stealth lipid alone, we have
found that efficacy with respect to a particular tissue can
surprisingly be controlled by varying the cationic lipid. As
discussed below, it has been discovered that lipid formulations for
delivery of biologically active agents can be adjusted to
preferrentially target one cell type or organ over another by
alterring only the cationic lipid included in the formulations. For
example, cationic lipids whose pKa is about 6.1 or above are much
more effective in formulations targeting the liver compared to
formulations containing cationic lipids whose pKa is about 6.1 or
lower, which are comparatively more effective in formulation
targeting tumors in vivo. As a general rule (to which there are
exceptions), formulations with the most effective lipids for
delivery to tumors (as described in greater detail below) contain
cationic lipids with a pKa of from about 5.0 to about 6.7,
including especially from about 5.2 to about 6.3, or from about 5.4
to about 6.2, or from about 5.8 to about 6.1, depending on tumor
type; whereas formulations with the most effective lipids for
delivery to liver (as described in greater detail below) contain
cationic lipids with a pKa of from about 5.1 to about 7.4,
including from about 5.3 to about 7.3, including from about 5.9 to
about 7.0, and in one embodiment including from about 6.2 to about
6.8.
[0240] In one embodiment, further optimization is possible by one
skilled in the art by combining cationic lipids with the desired
pKa range, stealth lipids, helper lipid, optional alkyl resorcinol
based lipids and optional neutral lipids into formulations,
including, e.g., liposome formulations, liponanoparticle (LNP)
formulations, and the like for delivery to specific cells and
tissues in vivo. In one embodment, further optimization is obtained
by adjusting the lipid molar ratio between these varioustypes of
lipids. In one embodment, further optimization is obtained by
adjusting one or more of: the desired particle size, N/P ratio,
formulation methods and/or dosing regimen (e.g., number of doses
administered over time, actual dose in mg/kg, timing of the doses,
combinations with other therapeutics, etc.). The various
optimization techniques known to those of skill in the art
pertaining to the above listed embodiments are considered as part
of this invention.
[0241] In one embodiment, cationic lipids of the invention are
provided wherein formulation for delivery of therapeutically
effective amounts of biologically active agents comprise at least
one each of a cationic lipid, a helper lipid, and a stealth lipid.
In one embodiment, such a formulation further comprises at least
one neutral lipid. In one embodiment the formulation is optimized
for delivery of a biologically active agent for delivery to a
tumor. In one embodiment the formulation is optimized for delivery
of a biologically active agent for delivery to liver. In one
embodiment the formulation is optimized for delivery of a
particular type of biologically active agent. Exemplary types of
biologically active agents include, but are not limited to, e.g.,
antibodies, cholesterol, hormones, antivirals, peptides,
polypeptides, proteins, nucleoproteins, chemotherapeutics, low
molecular weight drugs, vitamins, co-factors, nucleosides,
nucleoside derivatives, nucleotides, oligonucleotides, enzymatic
nucleic acids, antisense nucleic acids, triplex forming
oligonucleotides, 2,5-A antisense chimeras, allozymes, aptamers,
ribozyme, decoy RNA molecules and analogs thereof, and small
nucleic acid molecules, such as short interfering nucleic acid
(siRNA), short interfering RNA (siRNA), double-stranded RNA
(dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA). Such
biologically active agents may be optionally optimized with one or
more further chemical and biologic agents to increase their
therapeutic value, e.g., modifications that modulate biological
properties such as, e.g., stability, half-life, potency, and/or
immunogenicity.
[0242] For delivery of therapeutic agents to tumors, preferred
formulations are selected from those that deliver sufficient
amounts of a biologically active agent to effectively modulate the
activity of the therapeutic target in a subject in need of such
administration.
[0243] Where the biologically active agent is an RNAi construct, an
effective amount of an RNAi, siRNA, siNA, or shRNA is the amount
that provides a knock down (KD) at least 20% or greater, 50% or
greater, 60% or greater, 70% or greater, 75% or greater, 80% or
greater, 85% or greater, 90% or greater, 95% or greater, or up to
100% of the target mRNA expressed in the target cell. In general,
choice of which therapeutically relevant KD range is needed for
effective treatment may vary by the pathway being targetted, by
cell type or tissue, and/or by the disease or disorder being
treated.
[0244] Cationic lipids of the type described above, wherein R.sup.1
and R.sup.2 together with the nitrogen atom to which they are
attached form a cyclic "headgroup", are reported herein to be
effective cationic lipids for use in lipid formulations.
Furthermore, it is now reported that the presence of the cyclic
headgroup unexpectedly alters the behaviour of a lipid formulation
and, in particular, that it changes the influence of the other
substituents.
[0245] The headgroup (i.e., R.sup.1--N--R.sup.2) of the cationic
lipid compounds of the invention contains a tertiary amine group.
This feature causes the compounds to behave differently, e.g., from
if they had, e.g., quaternary (cationic) amine groups because
quaternization of the nitrogen puts a fixed charge on the atom,
removing its pH responsiveness and causing a compound to behave
very differently.
[0246] In one specific instance, the presence of a cyclic head
group in inventive compounds E0027 and E0014 altered the ability of
the cationic lipid as a whole to act as an effective delivery agent
in a formulation as compared to lipids E0173 and E0172, which
differs only in the head group. For example, as described in
greater detail below, a formulation containing a particular
cationic lipid (E0173, CLinDMA) with the --N(Me).sub.2 headgroup,
when used in a formulation for delivery of an RNAi construct
specific to Factor VII, demonstrates an in vivo Factor VII
inhibition of 98.5%. When that compound is modified by replacing
the L alkylene substituent with an L heteroalkylene substituent
(marked with an arrow below), the activity of the compound (E0172)
is found to decrease: an in vivo Factor VII inhibition of 40.8% is
found.
##STR00024##
[0247] In contrast, the present inventors have found that, when a
cyclic headgroup is present, changing the L alkylene to an L
heteroalkylene substituent has the opposite effect: the efficacy of
the compound increases. For example:
##STR00025##
[0248] Thus, one embodiment of the invention comprises those
compounds wherein L comprises one or more heteroatoms. The skilled
person would not arrive at such compounds starting from the
disclosure of CLinDMA because, as noted above, the skilled person
starting from compounds with a CLinDMA headgroup would have
discovered that an L group comprising two or more heteroatoms
reduces the efficacy of the final compounds, and so would not have
provided a compound which comprises such a group.
[0249] Without wishing to be bound by any theory, one possibility
is that the efficacy of the lipid formulations of the invention is
related to pKa. The pKa of a cationic lipid can be adjusted by
altering the structure, e.g. by varying the number of heteroatoms
in L or by varying the nature of the headgroup.
[0250] In one example, referring to the in vivo siRNA experiments,
e.g., in Table 8, using the formulations mentioned above
(containing cationic lipids E0172, E0171, E0027 and E0014) which
resulted in Factor-VII inhibitions in the liver of 98.5%, 40.8%,
30.3% and 97.4% respectively, their pKa values are found to be 6.7,
8.5, 5.7 and 6.4, respectively.
[0251] With respect to delivery of agents to the liver, for one
embodiment of the invention, it is thought that, as a general rule
(to which there are exceptions), the most effective cationic lipids
for use in such formulations have a pKa of from about 5.1 to about
7.4. In one embodiment, cationic lipids with a pKa of from about
5.3 to about 7.3 are provided for formulation of this invention for
liver delivery. In one embodiment, cationic lipids with a pKa of
from about 5.9 to about 7.0 are provided for formulation of this
invention for liver delivery. In one embodiment, preferred lipids
have a pKa range of about 6.2 to about 6.8 for use in formulation
for delivery of biologically active agents to the liver.
[0252] A surprising discovery of the invention is that tumor
tissues have different optimal pKa ranges for efficacy. Thus, the
pKa ranges in the previous paragraph apply to the extent that the
lipids are intended to deliver biologically active agents to liver
cells.
[0253] With respect to delivery of biologically active agents to a
tumor, it is thought that, as a general rule (to which there are
exceptions), the most effective cationic lipids of the invention
for use in such formulations have a pKa of from about 5.0 to about
6.7, and thus are preferred lipids for delivery to tumors in one
embodiment.
[0254] In one general embodiment, cationic lipids with a pKa of
from about 5.0 to about 6.7 are provided for formulation of this
invention for use in delivery of biologically active agents to one
or more tumors. In one embodiment, cationic lipids with a pKa of
from about 5.2 to about 6.3 are provided for formulation of this
invention for use in delivery of biologically active agents to one
or or more tumors. In one embodiment, cationic lipids with a pKa of
from about 5.4 to about 6.2 are provided for formulation of this
invention for use in delivery of biologically active agents to one
or or more tumors. In one embodiment, cationic lipids with a pKa of
from about 5.8 to about 6.1 are provided for formulation of this
invention for use in delivery of biologically active agents to one
or or more tumors.
[0255] In one embodiment, the cationic lipid used in the
formulation has a pKa optimized for delivery of a biologically
active agent to a particular tumor or cell type. Tumor types may be
primary tumors or may be metastatic.
[0256] In one specific embodiment, formulations optimized for
delivery to Hep3B-like tumors contain cationic lipids with a pKa of
from about 5.0 to about 6.7. In one specific embodiment,
formulations optimized for delivery to Hep3B-like tumors contain
cationic lipids with a pKa of from about 5.3 to about 6.3. In one
specific embodiment, formulations optimized for delivery to
Hep3B-like tumors contain cationic lipids with a pKa of from about
5.4 to about 5.9. In one specific embodiment, formulations
optimized for delivery to Hep3B-like tumors contain cationic lipids
with a pKa of from about 5.8 to about 5.9.
[0257] In one specific embodiment, formulations optimized for
delivery to HepG2-like tumors contain cationic lipids with a pKa of
from about 5.2 to about 6.2. In one specific embodiment,
formulations optimized for delivery to Hep3B-like tumors contain
cationic lipids with a pKa of from about 5.3 to about 6.2. In one
specific embodiment, formulations optimized for delivery to
Hep3B-like tumors contain cationic lipids with a pKa of from about
5.6 to about 6.1. In one specific embodiment, formulations
optimized for delivery to HepG2-like tumors contain cationic lipids
with a pKa of about 6.1.
[0258] In one specific embodiment, formulations optimized for
delivery to 786-O-like renal tumors, or their metastases, contain
cationic lipids with a pKa of about 6.1.
[0259] It is reasonable to postulate that other tissues,
indications, tumor types or administration routes may possess
preferred lipid pKa ranges. For liposome or LNP formulations, it is
also reasonable to postulate that various tissues, indications,
tumor types or administration routes may possess preferred cationic
lipid pKa ranges, N/P ratios, particle size, cationic lipid used,
stealth lipid used, helper lipid used, optional use of a selected
neutral lipid, relative molar ratios of each lipid component,
formulation method, biologically active agent to be delivered, and
dosage regimen including dose given. Optimizing each of these
aspects, either independently or in a coordinated manner, is
described below, and many specific aspects of such optimization is
believed to be within the ability of one skilled in the art without
requiring undue experimentation.
[0260] Formulations may be optimized by one skilled in the art by
adjusting other aspects of the formulation, including but not
limited to individual selection of, e.g., the pKa of the cationic
lipid optimized for the type of cell or organ being targeted; the
cationic lipid used; the stealth lipid used; the helper lipid used;
whether a neutral lipid is present or absent; the choice of neutral
lipid used if present; the molar ratio of the selected helper
lipid, optional neutral lipid, stealth lipid and cationic lipid;
the N/P ratio; the particle size; the dosage regimen; the dose
given; the formulation method; and the like.
Embodiments of the Compounds of Formulae (I) Through (X)
[0261] a, b and c
[0262] In one embodiment, a is optionally substituted C.sub.1-2
alkylene. In one embodiment, a is optionally substituted C.sub.1
alkylene.
[0263] In one embodiment, b is optionally substituted C.sub.0-2
alkylene. In one embodiment, b is optionally substituted C.sub.1
alkylene.
[0264] In one embodiment, c is absent or is optionally substituted
C.sub.1 alkylene. In one embodiment, c is absent.
[0265] In one embodiment, a, b and c are, if present,
unsubstituted.
The Headgroup for Cationic Lipids
[0266] In one embodiment, R.sup.1 and R.sup.2 together with the
nitrogen atom to which they are attached form an optionally
substituted C.sub.3-20-heterocycloalkyl,
C.sub.3-20-heterocycloalkenyl, C.sub.3-20-heterocycloalkynyl group,
C.sub.5-heteroaryl or C.sub.6-heteroaryl group. In one embodiment,
R.sup.1 and R.sup.2 together with the nitrogen atom to which they
are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl or
C.sub.3-20-heterocycloalkynyl group. In one embodiment, R.sup.1 and
R.sup.2 together with the nitrogen atom to which they are attached
form an optionally substituted C.sub.3-20-heterocycloalkyl
group.
[0267] In one embodiment, R.sup.1 and R.sup.2 together with the
nitrogen atom to which they are attached form an optionally
substituted cyclic C.sub.5-16 group. In one embodiment, R.sup.1 and
R.sup.2 together with the nitrogen atom to which they are attached
form an optionally substituted cyclic C.sub.5-12 group. In one
embodiment, R.sup.1 and R.sup.2 together with the nitrogen atom to
which they are attached form an optionally substituted cyclic
C.sub.5 group, cyclic C.sub.6 group or cyclic C.sub.7 group. In one
embodiment, R.sup.1 and R.sup.2 together with the nitrogen atom to
which they are attached form an optionally substituted cyclic
C.sub.5 group or cyclic C.sub.6 group.
[0268] In one embodiment of this invention, R.sup.1 and R.sup.2
together with the nitrogen atom to which they are attached forms a
cyclic species which comprises at least one oxygen atom.
[0269] In one embodiment, R.sup.1 and R.sup.2 together with the
nitrogen atom to which they are attached are selected from at least
one of the headgroups H.sup.1 to H.sup.52 as provided in Table
1.
TABLE-US-00001 TABLE 1 Moieties named H.sup.1 to H.sup.52 Structure
H.sup.1 ##STR00026## H.sup.2 ##STR00027## H.sup.3 ##STR00028##
H.sup.4 ##STR00029## H.sup.5 ##STR00030## H.sup.6 ##STR00031##
H.sup.7 ##STR00032## H.sup.8 ##STR00033## H.sup.9 ##STR00034##
H.sup.10 ##STR00035## H.sup.11 ##STR00036## H.sup.12 ##STR00037##
H.sup.13 ##STR00038## H.sup.14 ##STR00039## H.sup.15 ##STR00040##
H.sup.16 ##STR00041## H.sup.17 ##STR00042## H.sup.18 ##STR00043##
H.sup.19 ##STR00044## H.sup.20 ##STR00045## H.sup.21 ##STR00046##
H.sup.22 ##STR00047## H.sup.23 ##STR00048## H.sup.24 ##STR00049##
H.sup.25 ##STR00050## H.sup.26 ##STR00051## H.sup.27 ##STR00052##
H.sup.28 ##STR00053## H.sup.29 ##STR00054## H.sup.30 ##STR00055##
H.sup.31 ##STR00056## H.sup.32 ##STR00057## H.sup.33 ##STR00058##
H.sup.34 ##STR00059## H.sup.35 ##STR00060## H.sup.36 ##STR00061##
H.sup.37 ##STR00062## H.sup.38 ##STR00063## H.sup.39 ##STR00064##
H.sup.40 ##STR00065## H.sup.41 ##STR00066## H.sup.42 ##STR00067##
H.sup.43 ##STR00068## H.sup.44 ##STR00069## H.sup.45 ##STR00070##
H.sup.46 ##STR00071## H.sup.47 ##STR00072## H.sup.48 ##STR00073##
H.sup.49 ##STR00074## H.sup.50 ##STR00075## H.sup.51 ##STR00076##
H.sup.52 ##STR00077##
pKa for Cationic Lipids
[0270] In one embodiment, cationic lipids herein with pKa ranges in
the desired range are preferred, including especially for
formulations for delivery of biologically active agents.
[0271] As mentioned above, and shown below, a cationic lipid with a
pKa of from about 5.1 to about 7.4 are generally effective when
used in a formulation for targetting liver. In one embodiment, the
pKa of a cationic lipid is from about 5.1 to about 7.4 for delivery
to liver. In one embodiment, a cationic lipid with a pKa from about
5.3 to about 7.3 for use in formulations specific for targeting the
liver. Thus, in one embodiment, the pKa of a cationic lipid is from
about 5.3 to about 7.3 for delivery to liver. In one embodiment,
the pKa of a cationic lipid is from about 5.9 to about 7.0 for
delivery to liver. In one embodiment, the pKa of the cationic lipid
is from about 6.2 to about 6.8 for delivery to liver.
[0272] As mentioned above and illustrated experimentally below, a
cationic lipid with a pKa of from about 5.0 to about 6.7 is
particularly effective when used in a formulation for delivery of a
biologically active agent to a tumor. Thus, in one embodiment, the
pKa of a cationic lipid is from about 5.0 to about 6.7 for delivery
to tumors. In one embodiment, the pKa of a cationic lipid is from
about 5.2 to about 6.3 for delivery to tumors. In one embodiment,
the pKa of a cationic lipid is from about 5.4 to about 6.2 for
delivery to tumors. In one embodiment, the pKa of the cationic
lipid is from about 5.8 to about 6.1 for delivery to tumors. In a
general embodiment, the pKa of a cationic lipid is from about 6.1
or below for delivery of a biologically active agent to a tumor or
tumor cell.
[0273] The pKa of the cationic lipid for use in a formulation for
delivery of a biologically active agent may be further optimized
depending on tumor type. For example, as provided in Tables 9, 10
and 11, RNAi constructs specific to PLK1 mRNA are differentially
delivered to Hep3B, HepG2 and 786-0 renal tumors injected into the
flank of a mouse, in a manner that correlates with the pKa of the
cationic lipid in the LNP formulation. Upon further analysis, it is
apparent that the optimal pKa range for knockdown of PLK1 in Hep3B
tumors in vivo differs from the optimal pKa range for HepG2 and
786-0 tumors, although both ranges fall within the general range of
5.0 to 6.7 as listed in the above paragraph, and all ranges in
general include cationic lipids with a lower pKa than is optimal
for delivery to liver.
[0274] Therefore, in one embodiment for delivery of a biologically
active agent to Hep3B-like tumors in vivo, the cationic lipids of
the invention have pKa ranges from about 5.0 to about 6.7. In one
embodiment, the pKa of a cationic lipid is from about 5.3 to about
6.3 for delivery to Hep3B-like tumors. In one embodiment, the pKa
of a cationic lipid is from about 5.4 to about 5.9 for delivery to
Hep3B-like tumors. In one embodiment, the pKa of a cationic lipid
is from about 5.8 to about 5.9 for delivery to Hep3B-like
tumors.
[0275] Furthermore, in one embodiment for delivery of a
biologically active agent to HepG2-like tumors in vivo, the
cationic lipids of the invention have pKa ranges from about 5.2 to
about 6.2. In one embodiment, the pKa of a cationic lipid is from
about 5.3 to about 6.2 for delivery to HepG2-like tumors. In one
embodiment, the pKa of a cationic lipid is from about 5.6 to about
6.1 for delivery to HepG2-like tumors. In one embodiment, the pKa
of a cationic lipid is about 6.1 to HepG2-like tumors or to 786-0
renal tumor-like tumors.
X.sup.1 and X.sup.2
[0276] In one embodiment, X.sup.1 is O. In another embodiment,
X.sup.2 is O. In one embodiment, both X.sup.1 and X.sup.2 are
O.
Linker
[0277] In one embodiment, L comprises at least one heteroatom. This
means that the chain which provides a direct link between X.sup.2
and Y.sup.2 has at least one heteroatom. In other words, any
heteroatom in a substituent on L does not count for these purposes.
In one embodiment, L comprises at least one O atom.
[0278] In one embodiment, L comprises at least two heteroatoms. In
one embodiment, L comprises at least two O atoms.
[0279] In one embodiment, L.sup.c is optionally substituted
C.sub.1-15alkylene or C.sub.1-15heteroalkylene. In one embodiment,
L.sup.c is optionally substituted C.sub.1-15alkylene or
C.sub.1-15heteroalkylene and d and e are both zero (0).
[0280] In one embodiment, L.sup.c is selected from one of formulae
L.sup.c-i to L.sup.c-xxxxiii. In one embodiment, L.sup.c is
selected from one of formulae L.sup.c-i to L.sup.c-xxxxiii and d
and e are both zero (0). [0281] L.sup.c-i
--(CH.sub.2).sub.2O(CH.sub.2).sub.2-- [0282] L.sup.c-ii
--(CH.sub.2).sub.4-- [0283] L.sup.c-iii --CO(CH.sub.2).sub.2CO--
[0284] L.sup.c-iv --CO-- [0285] L.sup.c-v --COCH.sub.2OCH.sub.2CO--
[0286] L.sup.c-vi --(CH.sub.2).sub.2O(CH.sub.2).sub.2NHCO-- [0287]
L.sup.c-vii --(CH.sub.2).sub.3O(CH.sub.2).sub.3-- [0288]
L.sup.c-viii --(CH.sub.2).sub.2-- [0289] L.sup.c-ix
--(CH.sub.2).sub.2O(CH.sub.2).sub.2O(CH.sub.2).sub.2O(CH.sub.2).sub.2--
[0290] L.sup.c-x
--(CH.sub.2).sub.2O(CH.sub.2).sub.2O(CH.sub.2).sub.2--
[0290] ##STR00078## [0291] L.sup.c-xv
--(CH.sub.2).sub.2O(CH.sub.2).sub.2OCH(CH.sub.3)-- [0292]
L.sup.c-xvi --(CH.sub.2).sub.2O(CH.sub.2).sub.2OC
(.dbd.O)(CH.sub.2).sub.2CO-- [0293] L.sup.c-xvii
--(CH.sub.2).sub.2OC(.dbd.O)(CH.sub.2).sub.2CO-- [0294]
L.sup.c-xviii --(CH.sub.2).sub.2O(CH.sub.2).sub.2OCO-- [0295]
L.sup.c-xix
--(CH.sub.2).sub.2NHC(.dbd.O)CH.sub.2OCH.sub.2C(.dbd.O)-- [0296]
L.sup.c-xx --(CH.sub.2).sub.2NHC(.dbd.O)(CH.sub.2).sub.2C(.dbd.O)--
[0297] L.sup.c-xxi --(CH.sub.2).sub.2NHC(.dbd.O)-- [0298]
L.sup.c-xxii --(CH.sub.2).sub.2NHC(.dbd.O)CH.sub.2NHC(.dbd.O)--
[0299] L.sup.c-xxiii
--(CH.sub.2).sub.2NHC(.dbd.O)CH(side-chain-1)NHC(.dbd.O)--, wherein
side-chain-1 represents the group
[0299] ##STR00079## the dashed line representing the bond to the
rest of the molecule; [0300] L.sup.c-xxiv
--(CH.sub.2).sub.2OC(.dbd.O)-- [0301] L.sup.c-xxv
--(CH.sub.2).sub.2O(CH.sub.2).sub.2OC(.dbd.O)CH.sub.2-- [0302]
L.sup.c-xxvi --(CH.sub.2).sub.2OC(.dbd.O)CH.sub.2-- [0303]
L.sup.c-xxvii --(CH.sub.2).sub.2OC(.dbd.O)CH.sub.2NHC(.dbd.O)--
[0304] L.sup.c-xxviii
--(CH.sub.2).sub.2OC(.dbd.O)(CH.sub.2).sub.2NHC(.dbd.O)--
[0304] ##STR00080## [0305] L.sup.c-xxxvi
--(CH.sub.2).sub.2OCO.sub.2(CH.sub.2).sub.2-- [0306] L.sup.c-xxxvii
--(CH.sub.2).sub.2OC(.dbd.O)CH.sub.2OCH.sub.2C(.dbd.O)-- [0307]
L.sup.c-xxxviii
--(CH.sub.2).sub.2OC(.dbd.O)(CH.sub.2).sub.3C(.dbd.O)-- [0308]
L.sup.c-xxxix
--(CH.sub.2).sub.3OC(.dbd.O)(CH.sub.2).sub.2C(.dbd.O)--
[0308] ##STR00081## [0309] L.sup.c-xxxxii
--(CH.sub.2).sub.2OCH.sub.2C(.dbd.O)--; and
##STR00082##
[0310] Since groups in which L comprises at least one heteroatom
are preferred, L.sup.c is preferably selected from L.sup.c-i,
L.sup.c-iv to L.sup.c-vii and L.sup.c-ix to L.sup.c-xxxxiii.
[0311] In one embodiment, L.sup.c is optionally substituted
C.sub.1-15heteroalkylene.
[0312] In one embodiment, L.sup.c is an optionally substituted
C.sub.1-11 group. In one embodiment, L.sup.c is an optionally
substituted C.sub.1-9 group. In one embodiment, L.sup.c is an
optionally substituted C.sub.3-8 group. In one embodiment, wherein
L.sup.c is an optionally substituted C.sub.4-7 group. In one
embodiment, L.sup.c is an optionally substituted C.sub.5, C.sub.6
or C.sub.7 group.
[0313] In one embodiment, d is 0; e is 0, and f is 1. In one
embodiment, d is 0; e is 0, and f is 1 and L.sup.c is, within the
chain lengths set out above, heteroalkylene.
Y.sup.1 for Cationic Lipids
[0314] In one embodiment, Y.sup.1 is a C.sub.12-28 group. In one
embodiment, Y.sup.1 is an optionally substituted C.sub.14-26 group.
In one embodiment, Y.sup.1 is an optionally substituted C.sub.16-24
group. In one embodiment, Y.sup.1 is an optionally substituted
C.sub.16-22 group. In one embodiment, the optionally substituted
Y.sup.1 chain is 18, 19, 20 or 21 atoms long.
[0315] Within the carbon ranges set out above, Y.sup.1 is
preferably alkenyl or heteroalkenyl.
[0316] In one embodiment, Y.sup.1 has at least one alkene group. In
one embodiment, Y.sup.1 has 1, 2 or 3 alkene groups.
[0317] In one embodiment, Y.sup.1 has an alkene group at the
omega-3 position. In another embodiment, Y.sup.1 has an alkene
group at the omega-6 position. In another embodiment, Y.sup.1 has
an alkene group at the omega-9 position. In one embodiment, Y.sup.1
has an alkene group at two or three of the omega-3, omega-6 and
omega-9 positions. In one embodiment, Y.sup.1 is unsaturated at the
omega-6 and omega-9 positions. In another embodiment, Y.sup.1 is
unsaturated at the omega-3, omega-6 and omega-9 positions. In one
embodiment, Y.sup.1 is unsaturated at the omega-9 position.
[0318] In one embodiment, Y.sup.1 has at least one cis unsaturated
alkene group. In one embodiment, Y.sup.1 has at least two cis
unsaturated alkene groups. In one embodiment, Y.sup.1 has at least
three cis unsaturated alkene groups. The at least one cis
unsaturated alkene group may be at one, two or three of the
omega-3, omega-6 and omega-9 positions. Unsaturation in lipid
chains is discussed in MacLachlan et al., Journal of Controlled
Release 107 (2005) 276-287.
[0319] In one embodiment Y.sup.1 is selected from Y.sup.1-i to
Y.sup.1-vii as provided in Table 2.
TABLE-US-00002 TABLE 2 Y.sup.1 related Moieties named Y.sup.1 to
Y.sup.1-vii Name Structure Y.sup.1-i ##STR00083## Y.sup.1-ii
##STR00084## Y.sup.1-iii ##STR00085## Y.sup.1-iv ##STR00086##
Y.sup.1-v ##STR00087## Y.sup.1-vi ##STR00088## Y.sup.1-vii
##STR00089## ##STR00090##
Y.sup.2
[0320] In one embodiment, Y.sup.2 is linked to L via an oxygen atom
on the optionally substituted steroid. In one embodiment, Y.sup.2
is linked to L via an oxygen atom on the 3-position of the A
steroid ring. In one embodiment Y.sup.2 is a sterol in which the
hydrogen atom of the hydroxy group at the 3-position of the A
steroid ring has been removed (and the connection to L is through
the oxygen atom of said hydroxy group).
[0321] In one embodiment said sterol is selected from the group
consisting of:
TABLE-US-00003 annasterol; avenasterol; beta-sitosterol;
brassicasterol; calciferol; campesterol; chalinosterol;
chinasterol; cholestanol; cholesterol; coprostanol; cycloartenol;
dehydrocholesterol; desmosterol; dihydrocalciferol;
dihydrocholesterol; dihydroergosterol; dinosterol; epicholesterol;
ergosterol; fucosterol; hexahydrolumisterol; hexaol;
hydroxycholesterol; lanosterol; lumisterol; parkeol;
poriferasterol; saringosterol; sitostanol; sitosterol;
stigmastanol; stigmasterol; weinbersterol: zymosterol;
a sterol bile acid (such as cholic acid; chenodeoxycholic acid;
glycocholic acid; taurocholic acid; deoxycholic acid, and
lithocholic acid); and/or a pharmaceutically relevant salt or a
pharmaceutically acceptable derivative thereof.
[0322] In one embodiment, the sterol is cholesterol.
[0323] Specific Lipids for Use in Delivery of a Biologically Active
Agent
[0324] The novel cationic lipids and stealth lipids of the
invention may be used for the delivery of therapeutically
acceptable agents including, e.g., biologically active agents.
Formulations containing cationic lipids, stealth lipids, and other
types of lipids are described throughout this disclosure. Whereas
the lipids disclosed herein are believed novel and useful, certain
characteristics are preferred over others for therapeutic use, as
detailed further in the exemplary and nonbinding disclosure
provided below. In one embodiment, the liposome, lipid nanoparticle
or other such lipid formulation further comprises a biological
effective agent. In one embodiment, the liposome, lipid
nanoparticle or other such lipid formulation is empty.
[0325] In one embodiment, the separate lipid components for use in
a formulation are provided in a kit. In one embodiment, the kit
contains instructions for generation of the lipid formulation. The
kit may comprise a ready-made formulation or separate or partial
components that require mixing prior to administration. A kit may
further provide additional components such as, but not limited to,
controls, buffers, containers, and delivery components, or may be
limited to those inventive lipids and components as described
herein.
[0326] In one embodiment, the kit contains at least a liposome or
liposome components including but not limited to one or more of a
cationic lipid, a stealth lipid, a helper lipid, and/or an optional
neutral lipid. In one embodiment, the kit further comprises a
biologically active agent. In one embodiment, the kit further
comprises one or more control lipids, or control agents, a control
liposome formulation, stains, buffers, instructions for use, and
the like. In one alternative, the liposome formulation is premixed.
One or more of the kit's chemical components may be provided in a
dehydrated form or in a hydrated form. Any of the various methods
known in the art for dehydration on lyophilization of the various
compounds and compositions described herein may be used.
[0327] In one aspect of the invention, there is provided any one of
the specific compounds exemplified below or a pharmaceutically
acceptable derivative thereof.
[0328] In one embodiment, the compound is for delivery of a
biologically active agent to the liver and the compound is selected
from one or more of E0024, E0014, E0052, E0118, E0175, E0177 or
E0083. In one embodiment, a composition for delivery of a
biologically active agent to the liver comprises one or more of
compounds selected from E0024, E0014, E0052, E0118 or E0083. In one
embodiment, a composition for delivery of a biologically active
agent to the liver comprises compound E0024. In one embodiment, a
composition for delivery of a biologically active agent to the
liver comprises compound E0014. In one embodiment, a composition
for delivery of a biologically active agent to the liver comprises
compound E0052. In one embodiment, a composition for delivery of a
biologically active agent to the liver comprises compound E0118. In
one embodiment, a composition for delivery of a biologically active
agent to the liver comprises compound E0083.
[0329] In one embodiment, the compound is for delivery of a
biologically active agent to a tumor and the compound is selected
from one or more of E0011, E0008, E0025, E0026, E0076, E0077, E0085
or E0088. In one embodiment, a composition for delivery of a
biologically active agent to a tumor comprises one or more of
compounds selected from E0011, E0008, E0025, E0026, E0076, E0077,
E0085 or E0088. In one embodiment, a composition for delivery of a
biologically active agent to a tumor comprises compound E0011. In
one embodiment, a composition for delivery of a biologically active
agent to a tumor comprises compound E0008. In one embodiment, a
composition for delivery of a biologically active agent to a tumor
comprises compound E0025. In one embodiment, a composition for
delivery of a biologically active agent to a tumor comprises
compound E0026. In one embodiment, a composition for delivery of a
biologically active agent to a tumor comprises compound E0076. In
one embodiment, a composition for delivery of a biologically active
agent to a tumor comprises compound E0077. In one embodiment, a
composition for delivery of a biologically active agent to a tumor
comprises compound E0085. In one embodiment, a composition for
delivery of a biologically active agent to a tumor comprises
compound E0088.
[0330] Liposomes, lipid nanoparticles and other such lipid
formulations containing one or more of the lipids described herein
are useful for delivery of nucleic acid compositions to a cell or
tissue, either in vitro or in vivo. Therapeutically relevant
nucleic acid compositions include RNAi agents that are specific to
one or more genes associated with a disease or disorder, wherein
targetting the endogenous sequence in the cell or tissue with the
RNAi agent leasts to a therapeutic or prophylactic effect.
[0331] As a general non-binding rule, therapeutically effective
amounts of target inhibition for delivery of biologically active
agents, including especially RNAi agents, result in target
inhibition of at least 70% where the target is expressed in the
liver. In one embodiment, cationic lipids of the invention provide
at least 70% target inhibition when provided in a formulation for
delivery of an RNAi to the liver. Cationic lipids that provide at
least 70% KD when formulated for liver include, but are not limited
to, E0007, E0008, E0011, E0014, E0015, E0016, E0017, E0018, E0019,
E0022, E0024, E0025, E0026, E0032, E0034, E0040, E0042, E0043,
E0045, E0048, E0049, E0051, E0052, E0053, E0054, E0055 and E0118.
In one embodiment, cationic lipids of the invention provide at
least 80% target inhibition when provided in a formulation for
delivery of an RNAi to the liver. Cationic lipids that provide at
least 80% KD when formulated for liver include, but are not limited
to, E0008, E0011, E0014, E0016, E0017, E0018, E0019, E0022, E0024,
E0025, E0026, E0032, E0034, E0040, E0042, E0043, E0045, E0048,
E0052, E0053, E0054, E0055 and E0118. In one embodiment, cationic
lipids of the invention provide at least 90% target inhibition when
provided in a formulation for delivery of an RNAi to the liver.
Cationic lipids that provide at least 90% KD when formulated for
liver include, but are not limited to, E0011, E0014, E0017, E0018,
E0024, E0025, E0026, E0040, E0043, E0045, E0052, E0053, E0054,
E0055 and E0118. In one embodiment, cationic lipids of the
invention provide at least 95% target inhibition when provided in a
formulation for delivery of an RNAi to the liver. Cationic lipids
that provide at least 95% KD when formulated for liver include, but
are not limited to, E0014, E0017, E0018, E0024, E0026, E0040,
E0043, E0052, E0054, E0055 and E0118. In one embodiment, cationic
lipids of the invention provide at least 98% target inhibition when
provided in a formulation for delivery of an RNAi to the liver.
Cationic lipids that provide at least 98% KD when formulated for
liver include, but are not limited to, E0014, E0017, E0018, E0024,
E0052, E0054 and E0118.
[0332] As a general non-binding rule, therapeutically effective
amounts of target inhibition for delivery of biologically active
agents, including especially RNAi agents, result in target
inhibition of at least 50% where the target is a tumor. In one
embodiment, cationic lipids of the invention provide at least 50%
target inhibition when provided in a formulation for delivery of an
RNAi to a tumor or tumor cells. Cationic lipids that provide at
least 50% KD when formulated for tumors or tumor cells include, but
are not limited to, E0008, E0011, E0025, E0026, E0075, E0076,
E0077, E0085, E0088, E0095, E0104, E0178 and E0179. In one
embodiment, cationic lipids of the invention provide at least 60%
target inhibition when provided in a formulation for delivery of an
RNAi to a tumor or tumor cells. Cationic lipids that provide at
least 60% KD when formulated for tumors or tumor cells include, but
are not limited to, E0008, E0011, E0025, E0026, E0075, E0076,
E0077, E0085 and E0088. In one embodiment, cationic lipids of the
invention provide at least 70% target inhibition when provided in a
formulation for delivery of an RNAi to a tumor or tumor cells.
Cationic lipids that provide at least 70% KD when formulated for
tumors or tumor cells include, but are not limited to, E0011,
E0025, E0026, E0075, E0076, E0077 and E0088. In one embodiment,
cationic lipids of the invention provide at least 80% target
inhibition when provided in a formulation for delivery of an RNAi
to the tumor to a tumor or tumor cells. Cationic lipids that
provide at least 80% KD when formulated for tumors or tumor cells
include, but are not limited to, E0008, E0025 and E0076.
[0333] In a specific embodiment, therapeutically effective amounts
of target inhibition for delivery of biologically active agents,
including especially RNAi agents, result in target inhibition of at
least 30% where the target is a HepG2-like tumor or a 786-0-like
tumor. In one specific embodiment, cationic lipids of the invention
provide at least 30% target inhibition when provided in a
formulation for delivery of an RNAi to a HepG2-like tumor or a
786-0-like tumor, and include E0056, E0076, E0085, E0104, E0175,
E0176 and E0177. In one specific embodiment, cationic lipids of the
invention provide at least 30% target inhibition when provided in a
formulation for delivery of an RNAi to a HepG2-like tumor or a
786-O-like tumor, and include E0085, E0175 and E0177.
[0334] Specific Cationic Lipids for Delivery to Liver
[0335] In one embodiment, a preferred cationic lipid is E0014. In
one embodiment, a preferred cationic lipid is E0017. In one
embodiment, a preferred cationic lipid is E0018. In one embodiment,
a preferred cationic lipid is E0024. In one embodiment, a preferred
cationic lipid is E0052. In one embodiment, a preferred cationic
lipid is E0054. In one embodiment, a preferred cationic lipid is
E0118.
[0336] In one embodiment, a preferred formulation for delivery of a
biologically active agent to liver contains a cationic lipid with a
pKa of from about 5.1 to about 7.4. In one embodiment, a preferred
formulation for delivery of a biologically active agent to liver
contains a cationic lipid with a pKa of from about 5.3 to about
7.3. In one embodiment, a preferred formulation for delivery of a
biologically active agent to liver contains a cationic lipid with a
pKa of from about 5.9 to about 7.0. In one embodiment, a preferred
formulation for delivery of a biologically active agent to liver
contains a cationic lipid with a pKa of from about 6.2 to about
6.8. In one embodiment, a preferred formulation for delivery of a
biologically active agent to liver contains a cationic lipid with a
pKa of about 6.1 or higher.
[0337] Specific Cationic Lipids for Delivery to Tumors
[0338] In one embodiment, a preferred cationic lipid is E0008. In
one embodiment, a preferred cationic lipid is E0025. In one
embodiment, a preferred cationic lipid is E0076. In one embodiment,
a preferred cationic lipid is E0085. In one embodiment, a preferred
cationic lipid is E0175. In one embodiment, a preferred cationic
lipid is E0177.
[0339] In one embodiment, a preferred formulation for delivery of a
biologically active agent to a tumor in vivo contains a cationic
lipid with a pKa of from about 5.0 to about 6.7. In one embodiment,
a preferred formulation for delivery of a biologically active agent
to a tumor in vivo contains a cationic lipid with a pKa of from
about 5.2 to about 6.3. In one embodiment, a preferred formulation
for delivery of a biologically active agent to a tumor in vivo
contains a cationic lipid with a pKa of from about 5.4 to about
6.2. In one embodiment, a preferred formulation for delivery of a
biologically active agent to a tumor in vivo contains a cationic
lipid with a pKa of from about 5.8 to about 6.1. In one embodiment,
a preferred formulation for delivery of a biologically active agent
to a tumor or tumor cell contains a cationic lipid with a pKa of
about 6.1 or lower.
Pharmaceutical Compositions and Formulations
[0340] The present invention provides a pharmaceutical composition
comprising at least one cationic lipid compound of the invention.
The present invention provides a pharmaceutical composition
comprising at least one stealth lipid compound of the invention. In
one embodiment, at least one other lipid component is present. Such
compositions may also contain a biologically active agent,
optionally in combination with one or more other lipid components.
In one embodiment, the one or more components, compositions and/or
agents are provided in a kit. Compositions containing lipids of the
invention in combination with one or more biologically active
agents in one embodiment are provided as formulations for use,
e.g., in the delivery of therapeutically effective amounts of one
or more biologically active agents to a cell or tissue. In one
embodiment, the cell or tissue is in a subject in need of treatment
or prophylaxis. In one embodiment, the subject is a patient in need
of therapeutically effective amounts of the biologically active
agent. As used herein, subjects include both humans and non-human
animals.
[0341] The other lipid component(s) may be one or more selected
from the group consisting of cationic lipids, (optional) neutral
lipids, helper lipids, stealth lipids and alkyl resorcinol based
lipids. In one embodiment, the invention provides a composition
comprising: (a) a cationic lipid, e.g., compounds of any one of
Formulas I through X, and/or E0001-E0171 and E0175-E0180 of the
invention; (b) an optional neutral lipid, e.g. DSPC; (c) a helper
lipid, e.g. one containing cholesterol; (d) a stealth lipid, e.g.,
one of either Formula XI or XII or any one or more of S001-S009 and
S012-S026, or
1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy
(polyethylene glycol)-2000] (catalog #880150P from Avanti Polar
Lipids). In one embodiment, the lipid components are in a liposome
formulation, e.g., a nanoparticle or the like. In one embodiment,
the liposome formulation further comprises a biologically active
agent. In one embodiment, the liposome formulation further
comprises a therapeutically effectve amount of a biologically
active agent.
[0342] The other lipid component(s) may, e.g., be one or more
selected from the group of known cationic lipids consisting of
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),
N,N-distearyl-N,N-dimethylammonium bromide (DDAB),
N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium
chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),
1,2-Dioleoyl-3-Dimethylammonium-propane (DODAP),
1,2-Dioleoylcarbamyl-3-Dimethylammonium-propane (DOCDAP),
1,2-Dilineoyl-3-Dimethylammonium-propane (DLINDAP),
dilauryl(C.sub.12:0) trimethyl ammonium propane (DLTAP),
Dioleoyloxy-N-[2-sperminecarboxamido)ethyl}-N,N-dimethyl-1-propanaminiumt-
rifluoroacetate (DOSPA), Dioctadecylamidoglycyl spermine (DOGS),
DC-Chol, 1,2-Dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium
bromide (DMRIE),
3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-
tadecadienoxy)propane (CLinDMA),
2-[5'-(cholest-5-en-3[beta]-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',-
12'-octadecadienoxy)propane (CpLinDMA) and
N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA), dioleoyl
phosphatidylethanolamine (DOPE),
1,2-N,N'-Dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP). In one
embodiment the other lipid component(s) is DOTAP or DLTAP.
[0343] In one embodiment, the cationic lipid is selected from a
lipid of Formula I. In one embodiment, the cationic lipid is
selected from a lipid of Formula II. In one embodiment, the
cationic lipid is selected from a lipid of Formula III. In one
embodiment, the cationic lipid is selected from a lipid of Formula
IV. In one embodiment, the cationic lipid is selected from a lipid
of Formula V. In one embodiment, the cationic lipid is selected
from a lipid of Formula VI. In one embodiment, the cationic lipid
is selected from a lipid of Formula VII. In one embodiment, the
cationic lipid is selected from a lipid of Formula VIII. In one
embodiment, the cationic lipid is selected from a lipid of Formula
IX. In one embodiment, the cationic lipid is selected from a lipid
of Formula X. In one embodiment, the cationic lipid is selected
from the list of E0001 through E0171 (E0001-E0171) and E0175
through E0180 (E0175-E0180).
[0344] The other lipid component(s) may, e.g., be (a) neutral
lipid(s). The neutral lipid(s) may, in one embodiment, be one or
more selected from any of a variety of neutral uncharged or
zwitterionic lipids. Examples of neutral phospholipids for the
present invention include: 5-heptadecylbenzene-1,3-diol
(resorcinol), cholesterol hemisuccinate (CHEMS),
dipalmitoylphosphatidylcholine (DPPC),
distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC),
dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC),
phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC),
dilauryloylphosphatidylcholine (DLPC),
dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl
phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl
phosphatidylcholine (PMPC),
1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC),
1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC),
1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC),
1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC),
1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC),
palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl
choline, dilinoleoylphosphatidylcholine
distearoylphophatidylethanolamine (DSPE), dimyristoyl
phosphatidylethanolamine (DMPE), dipalmitoyl
phosphatidylethanolamine (DPPE), palmitoyloleoyl
phosphatidylethanolamine (POPE), lysophosphatidylethanolamine or a
combination thereof. In one embodiment, the neutral phospholipid is
selected from the group consisting of distearoylphosphatidylcholine
(DSPC) and dimyristoyl phosphatidyl ethanolamine (DMPE).
[0345] The other lipid component(s) may, e.g., be (a) anionic
lipid(s), e.g. anionic lipids capable of producing a stable
complex. Examples of anionic lipids are phosphatidylglycerol,
cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid,
N-dodecanoyl phosphatidyl ethanoloamine, N-succinyl
phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine and
lysylphosphatidylglycerol.
[0346] Suitable neutral and anionic lipids also include those
described in US 2009/0048197, paragraph [0119].
[0347] The total amount of lipid in the composition being
administered is, in one embodiment, from about 5 to about 30 mg
lipid per mg biologically active agent (e.g. siRNA), in another
embodiment from about 5 to about 25 mg lipid per mg biologically
active agent (e.g. siRNA), in another embodiment from about 7 to
about 25 mg lipid per mg biologically active agent (e.g. siRNA) and
in one embodiment from about 7 to about 15 mg lipid per mg
biologically active agent (e.g. siRNA).
[0348] Various methods for loading biologically active agents into
lipid compositions, such as liposomes and liponanoparticles are
available in the art, including both passive and active loading
methods. Either are contemplated as being within the scope of the
invention. The exact method used may be chosen based one multiple
factors that include, but are not limited to, e.g., the
biologically active agent to be loaded, the storage method to be
used once loaded, the size of the resulting particle, and the
dosage regimen contemplated. Methods include, e.g., mechanical
mixing of the drug and lipids at the time the liposomes are formed
or reconstituted, dissolving all components in an organic solvent
and concentrating them into a dry film, forming a pH or ion
gradient to draw the active agent into the interior of the
liposome, creating a transmembrane potential, and ionophore
mediated loading. See, e.g., at least Examples 68, 69 and 77 below,
PCT Publication No. WO 95/08986, U.S. Pat. No. 5,837,282, U.S. Pat.
No. 5,837,282, and U.S. Pat. No. 7,811,602.
[0349] The dose of biologically active agent administered will
depend on a number of factors such as the identity of the
biologically active agent and the target patient (e.g. species of
animal). The concentration of biologically active agent will be
adjusted accordingly but, when siRNA is being administered to an
animal, a concentration of from 0.1 mg/ml to 10 mg/ml is typical
per dose.
[0350] The total amount of siRNA can be measured by several
methods. HPLC methods include anion exchange, reverse phase (RP) or
size exclusion (SEC). Fluorescent methods may also be used. In all
of these methods the nanoparticles must be lysed to release the
siRNA from the nanoparticle prior to measuring the total siRNA
content.
[0351] In one embodiment, the composition comprises a cationic
lipid component which forms from about 10% to about 80%, from about
20% to about 70% or from about 30% to about 60% of the total lipid
present in the composition. These percentages are mole percentages
relative to the total moles of lipid components in the final lipid
particle.
[0352] In one embodiment, the composition comprises a neutral lipid
component which forms from about 0% to about 50%, from about 0% to
about 30% or from about 10% to about 20% of the total lipid present
in the composition. In one embodiment, the neutral lipid component
of the composition is optional. In one embodiment, the composition
has no neutral lipid component. These percentages are mole
percentages relative to the total moles of lipid components in the
final lipid particle.
[0353] In one embodiment, the composition comprises a helper lipid
component which forms from about 5% to about 80%, from about 20% to
about 70% or from about 30% to about 50% of the total lipid present
in the composition. These percentages are mole percentages relative
to the total moles of lipid components in the final lipid
particle.
[0354] In one embodiment, the composition comprises a stealth lipid
component which forms from about 0% to about 10%, from about 1% to
about 6%, or from about 2% to about 5% of the total lipid present
in the composition. These percentages are mole percentages relative
to the total moles of lipid components in the final lipid
particle.
[0355] In one embodiment, the composition comprises a cationic
lipid component forming from about 30 to about 60% of the total
lipid present in the formulation, a neutral lipid comprising
forming from about 0 to about 30% of the total lipid present in the
formulation, a helper lipid forming from about 18 to about 46% of
the total lipid present in the formulation and a stealth lipid
forming from about 2 to about 4% of the total lipid present in the
formulation. These percentages are mole percentages relative to the
total moles of lipid components in the final lipid particle.
[0356] Liposomal compositions of the invention are administered in
any of a number of ways, including parenteral, intravenous,
systemic, local, oral, intratumoral, intramuscular, subcutaneous,
intraperitoneal, inhalation, or any such method of delivery. In one
embodiment, the compositions are administered parenterally, i.e.,
intraarticularly, intravenously, intraperitoneally, subcutaneously,
or intramuscularly. In a specific embodiment, the liposomal
compositions are administered by intravenous infusion or
intraperitoneally by a bolus injection.
[0357] Liposomal compositions of the invention may be formulated as
pharmaceutical compositions suitable for delivery to a subject. The
pharmaceutical compositions of the invention will often further
comprise one or more buffers (e.g., neutral buffered saline or
phosphate buffered saline), carbohydrates (e.g., glucose, mannose,
sucrose, dextrose or dextrans), mannitol, proteins, polypeptides or
amino acids such as glycine, antioxidants, bacteriostats, chelating
agents such as EDTA or glutathione, adjuvants (e.g., aluminum
hydroxide), solutes that render the formulation isotonic, hypotonic
or weakly hypertonic with the blood of a recipient, suspending
agents, thickening agents and/or preservatives. Alternatively,
compositions of the present invention may be formulated as a
lyophilizate.
[0358] Suitable formulations for use in the present invention can
be found, e.g., in Remington's Pharmaceutical Sciences, Mack
Publishing Company, Philadelphia, Pa., 17.sup.th Ed. (1985). Often,
intravenous compositions will comprise a solution of the liposomes
suspended in an acceptable carrier, such as an aqueous carrier.
Method for Delivering Biologically Active Agents and Related
Uses
[0359] The cationic and stealth lipids of the invention are useful
for formulations used for delivery of biologically active agents.
Formulations containing the novel lipids of the invention may be in
various forms, including but not limited to particle forming
delivery agents including microparticles, nanoparticles and
trasfection agents that are useful for delivering various molecules
to cells. Specific formulations are effective at transfecting or
delivering biologically active agents, such as antibodies (e.g.,
monoclonal, chimeric, humanized, nanobodies, and fragments thereof
etc.), cholesterol, hormones, peptides, proteins, chemotherapeutics
and other types of antineoplastic agents, low molecular weight
drugs, vitamins, co-factors, nucleosides, nucleotides,
oligonucleotides, enzymatic nucleic acids, antisense nucleic acids,
triplex forming oligonucleotides, antisense DNA or RNA
compositions, chimeric DNA:RNA compositions, allozymes, aptamers,
ribozyme, decoys and analogs thereof, plasmids and other types of
expression vectors, and small nucleic acid molecules, RNAi agents,
short interfering nucleic acid (siNA), short interfering RNA
(sRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short
hairpin RNA (shRNA) molecules, to relevant cells and/or tissues,
such as in a cell culture, subject or organism. The above list of
biologically active agents is exemplary only, and is not intended
to be limiting. Such compounds may be purified or partially
purified, and may be naturally occurring or synthetic, and may be
chemically modified.
[0360] Such formulations containing biologically active agents are
useful, e.g., in providing compositions to prevent, inhibit, or
treat diseases, conditions, or traits in a cell, subject or
organism. Diseases, conditions or traits include, but are not
limited to, proliferative diseases, including cancer, inflammatory
disease, transplant and/or tissue rejection, autoimmune diseases or
conditions, age-related disease, neurological or neurodegenerative
disease, respiratory disease, cardiovacular disease, ocular
disease, metabolic disease, dermatological disease, auditory
disease, a liver disease, a kidney or renal disease, etc.
[0361] The amount of active agent administered per dose is an
amount above the minimal therapeutic dose but below a toxic dose.
The actual amount per dose may be determined by a physician
depending on a number of factors, such as the medical history of
the patient, the use of other therapies, the biologically active
agent to be provided, and the nature of the disease. The amount of
biologically active agent administered may be adjusted throughout
treatment, depending on the patient's response to treatment and the
presence or severity of any treatment-associated side effects.
Exemplary dosages and treatment for compounds that have been
approved by an appropriate regulatory agency are known and
available to those skilled in the art. See, e.g., Physician's Desk
Reference, 64th ed., Physician's Desk Reference Inc. (2010),
Remington's Pharmaceutical Sciences, Mack Publishing Company,
Philadelphia, Pa. (1985), and Remington The Science and Practice of
Pharmacy, 21st ed., Lippincott Williams & Williams Publishers
(2005).
[0362] In one embodiment, a single dose is administered of a
biologically active agent to a patient in need thereof. In one
embodiment, multiple doses are administered, wherein the multiple
doses may be administered concurrently, sequentially or
alternating. In one embodiment, the same formulation is
administered over multiple doses. In one embodiment, the
formulations differ over multiple doses. In various embodiments,
the doses may be administered once a day, or for one, two, three,
four or more consecutive days. In one embodiment, the doses are
administered once a week. In one embodiment, the doses are
administered once every other week. In one embodiment, patients
receive at least two courses of a treatment regimen, and
potentially more, depending on the response of the patient to the
treatment. In single agent regimens, total courses of treatment are
determined by the patient and physician based on observed responses
and toxicity. The above dosage regimens are to be considered as
exemplary. Other dosage regimens are contemplated as being within
the scope of the invention, and depend on the therapeutic effect
desired.
[0363] In one embodiment, the invention provides a method for
delivering a biologically active agent to a cell comprising
administering a composition, which comprises the biologically
active agent and a compound of the present invention, to the
cell.
[0364] The cell may be in vitro or in vivo.
[0365] The invention provides a compound of formula (I) for use in
therapy. It also provides subsets of compounds of formula I that
are further distinguished in formulas II to X. Compounds of
formulas I through X are generally referred to herein as cationic
lipids.
[0366] The invention further provides a compound of formula (XI)
for use in therapy. It also provides a subset of compound of
formula XI that are further distinguished in formula XII. Compounds
of formulas XI and XII are generally referred to herein as stealth
lipids.
[0367] The invention further provides a method for the treatment of
a disease or condition, comprising the step of administering a
therapeutically effective amount of a composition containing at
least one compound of formula (I) to a patient in combination with
a biologically active agent that treats the disease or condition.
The invention also provides a composition containing at least one
compound of formula (XI) for use in treating a disease or
condition.
[0368] The invention also provides the use of a compound of formula
(I) in the manufacture of a medicament for the treatment of a
disease or condition. In one embodiment, the medicament contains a
biologically active agent that treats the disease or condition. The
invention also provides the use of a biologically active agent
which treats a disease or condition in the manufacture of a
medicament for the treatment of the disease or condition, wherein
the medicament also contains a compound of formula (I) or formula
XI.
[0369] The invention also provides a method for the treatment of a
disease or condition comprising the step of administering a
therapeutically effective amount of a biologically active agent in
a formulation containing at least one composition of the invention
to a patient. In one embodiment, the disease or condition is a
disease of the liver, a tumor or a disease. In one embodiment, the
disease or condition is treatable by administering an siRNA
agent.
[0370] The invention also provides a composition of the invention
for use in treating a disease or condition in a patient. In one
embodiment, the disease or condition is a disease of the liver, a
tumor or a disease mediated by a protein encoded by a mRNA.
[0371] The invention also provides a product containing a compound
of formula (I) and/or formula XI. In one embodiment, the product
further comprises a biologically active agent as a combined
preparation for simultaneous, separate or sequential use in
therapy.
Administration & Formulation
General
[0372] For pharmaceutical use, the compounds and compositions of
the invention may be administered as at least one portion of a
medicament by enteral or parenteral routes, including intravenous,
intramuscular, subcutaneous, transdermal, airway (aerosol), oral,
intranasal, rectal, vaginal, buccal, nasopharangeal,
gastrointestinal or sublingual administration. The administration
may be systemic or topical. Topical administration may involve,
e.g., catheterization, implantation, osmotic pumping, direct
injection, dermal/transdermal application, stenting, ear/eye drops
or portal vein administration. The compounds of formula (I) and/or
formula XI should be assessed for their biopharmaceutical
properties, such as solubility and solution stability (across pH),
permeability, etc., in order to select the most appropriate dosage
form and route of administration for treatment of the proposed
indication.
[0373] The compounds and compositions of the invention will
generally, but not necessarily, be administered as a formulation in
association with one or more pharmaceutically acceptable
excipients. The term "excipient" includes any ingredient other than
the compound(s) of the invention, the other lipid component(s) and
the biologically active agent. An excipient may impart either a
functional (e.g drug release rate controlling) and/or a
non-functional (e.g. processing aid or diluent) characteristic to
the formulations. The choice of excipient will to a large extent
depend on factors such as the particular mode of administration,
the effect of the excipient on solubility and stability, and the
nature of the dosage form.
[0374] Typical pharmaceutically acceptable excipients include:
[0375] diluents, e.g. lactose, dextrose, sucrose, mannitol,
sorbitol, cellulose and/or glycine; [0376] lubricants, e.g. silica,
talcum, stearic acid, its magnesium or calcium salt and/or
polyethyleneglycol; [0377] binders, e.g. magnesium aluminum
silicate, starch paste, gelatin, tragacanth, methylcellulose,
sodium carboxymethylcellulose and/or polyvinylpyrrolidone; [0378]
disintegrants, e.g. starches, agar, alginic acid or its sodium
salt, or effervescent mixtures; and/or [0379] absorbants,
colorants, flavors and/or sweeteners.
[0380] The excipient may be an aqueous solution carrier which may
optionally contain a buffer (e.g. a PBS buffer) and/or a sugar.
[0381] A thorough discussion of pharmaceutically acceptable
excipients is available in Gennaro, Remington: The Science and
Practice of Pharmacy 2000, 20th edition (ISBN: 0683306472).
Oral Administration
[0382] The compounds and compositions of the invention may be
administered orally. Oral administration may involve swallowing, so
that the compound enters the gastrointestinal tract, and/or buccal,
lingual, or sublingual administration by which the compound enters
the blood stream directly from the mouth.
Parenteral Administration
[0383] The compounds and compositions of the invention can be
administered parenterally. The compounds and compositions of the
invention may be administered directly into the blood stream, into
subcutaneous tissue, into muscle, or into an internal organ.
Suitable means for administration include intravenous,
intraarterial, intrathecal, intraventricular, intraurethral,
intrasternal, intracranial, intramuscular, intrasynovial and
subcutaneous. Suitable devices for administration include needle
(including microneedle) injectors, needle-free injectors and
infusion techniques.
[0384] Parenteral formulations are typically aqueous or oily
solutions. Where the solution is aqueous, excipients such as sugars
(including but restricted to glucose, mannitol, sorbitol, etc.)
salts, carbohydrates and buffering agents (preferably to a pH of
from 3 to 9), but, for some applications, they may be more suitably
formulated as a sterile non-aqueous solution or as a dried form to
be used in conjunction with a suitable vehicle such as sterile,
pyrogen-free water (WFI).
[0385] Parenteral formulations may include implants derived from
degradable polymers such as polyesters (i.e. polylactic acid,
polylactide, polylactide-co-glycolide, polycapro-lactone,
polyhydroxybutyrate), polyorthoesters and polyanhydrides. These
formulations may be administered via surgical incision into the
subcutaneous tissue, muscular tissue or directly into specific
organs.
[0386] The preparation of parenteral formulations under sterile
conditions, e.g., by lyophilisation, may readily be accomplished
using standard pharmaceutical techniques well known to the skilled
person.
[0387] The solubility of the compounds and compositions used in the
preparation of parenteral solutions may be increased by the use of
appropriate formulation techniques, such as the incorporation of
co-solvents and/or solubility-enhancing agents such as surfactants,
micelle structures and cyclodextrins.
Inhalation & Intranasal Administration
[0388] The compounds and compositions of the invention can be
administered intranasally or by inhalation, typically in the form
of a dry powder (either alone, as a mixture, e.g., in a dry blend
with lactose, or as a mixed component particle, e.g., mixed with
phospholipids, such as phosphatidylcholine) from a dry powder
inhaler, as an aerosol spray from a pressurised container, pump,
spray, atomiser (preferably an atomiser using electrohydrodynamics
to produce a fine mist), or nebuliser, with or without the use of a
suitable propellant, such as 1,1,1,2-tetrafluoroethane or
1,1,1,2,3,3,3-heptafluoropropane, or as nasal drops. For intranasal
use, the powder may comprise a bioadhesive agent, e.g., chitosan or
cyclodextrin.
[0389] The pressurised container, pump, spray, atomizer, or
nebuliser contains a solution or suspension of the compound(s) of
the invention comprising, e.g., ethanol, aqueous ethanol, or a
suitable alternative agent for dispersing, solubilising, or
extending release of the compositions of the invention, a
propellant(s) as solvent and an optional surfactant, such as
sorbitan trioleate, oleic acid, or an oligolactic acid.
[0390] Prior to use in a dry powder or suspension formulation, the
compound or composition is micronised to a size suitable for
delivery by inhalation (typically less than 5 microns). This may be
achieved by any appropriate comminuting method, such as spiral jet
milling, fluid bed jet milling, supercritical fluid processing to
form nanoparticles, high pressure homogenisation, or spray
drying.
[0391] Capsules (made, e.g., from gelatin or
hydroxypropylmethylcellulose), blisters and cartridges for use in
an inhaler or insufflator may be formulated to contain a powder mix
of the compound or composition of the invention, a suitable powder
base such as lactose or starch and a performance modifier such as
l-leucine, mannitol, or magnesium stearate. The lactose may be
anhydrous or in the form of the monohydrate, preferably the latter.
Other suitable excipients include dextran, glucose, maltose,
sorbitol, xylitol, fructose, sucrose and trehalose.
[0392] Formulations for inhaled/intranasal administration may be
formulated to be immediate and/or modified release using, e.g.,
PGLA. Modified release formulations include delayed-, sustained-,
pulsed-, controlled-, targeted and programmed release.
Transdermal Administration
[0393] Suitable formulations for transdermal application include a
therapeutically effective amount of a compound or composition of
the invention with carrier. Advantageous carriers include
absorbable pharmacologically acceptable solvents to assist passage
through the skin of the host. Characteristically, transdermal
devices are in the form of a bandage comprising a backing member, a
reservoir containing the compound optionally with carriers,
optionally a rate controlling barrier to deliver the compound of
the skin of the host at a controlled and predetermined rate over a
prolonged period of time, and means to secure the device to the
skin.
Cells and Organs Targeted by the Invention
[0394] The compounds, compositions, methods and uses of the
invention can be used to deliver a biologically active agent to one
or more of the following in a patient:
[0395] the liver or liver cells (e.g. hepatocytes);
[0396] a kidney or kidney cells;
[0397] a tumor or tumor cells;
[0398] the CNS or CNS cells (Central Nervous System, e.g. brain
and/or spinal cord);
[0399] the PNS or PNS cells (Peripheral Nervous System);
[0400] a lung or lung cells;
[0401] the vasculature or vascular cells;
[0402] the skin or skin cells (e.g. dermis cells and/or follicular
cells);
[0403] an eye or ocular cells (e.g. macula, fovea, cornea, retina),
and
[0404] an ear or cells of the ear (e.g. cells of the inner ear,
middle ear and/or outer ear).
[0405] In one embodiment, the invention the compounds,
compositions, methods and uses of the invention are for delivering
a biologically active agent to liver cells (e.g. hepatocytes). In
one embodiment, the invention the compounds, compositions, methods
and uses of the invention are for delivering a biologically active
agent to a tumor or to tumor cells (e.g. a primary tumor or
metastatic cancer cells).
[0406] For delivery of a biologically active agent to the liver or
liver cells, in one embodiment a compound or composition of the
invention is contacted with the liver or liver cells of the patient
as is generally known in the art, such as via parental
administration (e.g. intravenous, intramuscular, subcutaneous
administration) or local administration (e.g. direct injection,
portal vein injection, catheterization, stenting), to facilitate
delivery.
[0407] For delivery of a biologically active agent to the kidney or
kidney cells, in one embodiment a compound or composition of the
invention is contacted with the kidney or kidney cells of the
patient as is generally known in the art, such as via parental
administration (e.g. intravenous, intramuscular, subcutaneous
administration) or local administration (e.g. direct injection,
catheterization, stenting), to facilitate delivery.
[0408] For delivery of a biologically active agent to a tumor or
tumor cells, in one embodiment a compound or composition of the
invention is contacted with the tumor or tumor cells of the patient
as is generally known in the art, such as via parental
administration (e.g. intravenous, intramuscular, subcutaneous
administration) or local administration (e.g. direct injection,
catheterization, stenting), to facilitate delivery.
[0409] For delivery of a biologically active agent to the CNS or
CNS cells (e.g. brain cells and/or spinal cord cells), in one
embodiment a compound or composition of the invention is contacted
with the CNS or CNS cells (e.g. brain cells and/or spinal cord
cells) of the patient as is generally known in the art, such as via
parental administration (e.g. intravenous, intramuscular,
subcutaneous administration) or local administration (e.g. direct
injection, catheterization, stenting, osmotic pump administration
(e.g. intrathecal or ventricular)), to facilitate delivery.
[0410] For delivery of a biologically active agent to the PNS or
PNS cells, in one embodiment a compound or composition of the
invention is contacted with the PNS or PNS cells of the patient as
is generally known in the art, such as via parental administration
(e.g. intravenous, intramuscular, subcutaneous administration) or
local administration (e.g. direct injection), to facilitate
delivery.
[0411] For delivery of a biologically active agent to a lung or
lung cells, in one embodiment a compound or composition of the
invention is contacted with the lung or lung cells of the patient
as is generally known in the art, such as via parental
administration (e.g. intravenous, intramuscular, subcutaneous
administration) or local administration (e.g. pulmonary
administration directly to lung tissues and cells), to facilitate
delivery.
[0412] For delivery of a biologically active agent to the
vasculature or vascular cells, in one embodiment a compound or
composition of the invention is contacted with the vasculature or
vascular cells of the patient as is generally known in the art,
such as via parental administration (e.g. intravenous,
intramuscular, subcutaneous administration) or local administration
(e.g. clamping, catheterization, stenting), to facilitate
delivery.
[0413] For delivery of a biologically active agent to the skin or
skin cells (e.g. dermis cells and/or follicular cells), in one
embodiment a compound or composition of the invention is contacted
with the skin or skin cells (e.g. dermis cells and/or follicular
cells) of the patient as is generally known in the art, such as via
parental administration (e.g. intravenous, intramuscular,
subcutaneous administration) or local administration (e.g. direct
dermal application, iontophoresis), to facilitate delivery.
[0414] For delivery of a biologically active agent to an eye or
ocular cells (e.g. macula, fovea, cornea, retina), in one
embodiment a compound or composition of the invention is contacted
with the eye or ocular cells (e.g. macula, fovea, cornea, retina)
of the patient as is generally known in the art, such as via
parental administration (e.g. intravenous, intramuscular,
subcutaneous administration) or local administration (e.g. direct
injection, intraocular injection, periocular injection,
iontophoresis, use of eyedrops, implants), to facilitate
delivery.
[0415] For delivery of a biologically active agent to an ear or
cells of the ear (e.g. cells of the inner ear, middle ear and/or
outer ear), in one embodiment a compound or composition of the
invention is contacted with the ear or cells of the ear (e.g. cells
of the inner ear, middle ear and/or outer ear) of the patient as is
generally known in the art, such as via parental administration
(e.g. intravenous, intramuscular, subcutaneous administration) or
local administration (e.g. direct injection), to facilitate
delivery.
Treatment of Diseases or Conditions
[0416] The diseases or conditions which may be treated by this
invention include those related to modulation in a patient of a
gene, gene expression, protein, protein activity, cellular pathway,
and the like. The disease or condition treated by this invention
may be one or more selected from the group consisting of: a
proliferative disease (e.g. a tumor); an inflammatory disease;
transplant and/or tissue rejection (allograft rejection); an
autoimmune disease; an infectious disease; an age-related disease;
a neurologic or neurodegenerative disease (e.g. Huntington's
disease); a metabolic disease; a cardiovascular disease; a
respiratory disease; an ocular disease; a dermatological disease;
an auditory disease (e.g. hearing loss, deafness); a liver disease
(e.g. hepatitis, HCV, HBV, diabetis, cirrhosis, hepatocellular
carcinoma), and a kidney/renal disease (e.g. polycystic kidney
disease). In one particular embodiment, the invention treats a
proliferative disease, e.g. a tumor or tumor cell. In one
particular embodiment, the invention treats a liver disease, e.g.
hepatitis, HCV, HBV, diabetis, cirrhosis and certain hepatocellular
carcinomas.
[0417] The skilled person would be able to select a biologically
active agent which in combination with a compound of the present
invention delivers a therapeutically effective amount of the agent.
Where the agent is a RNAi therapeutic, the desired therapeutic
effect is modulating expression of a target gene implicated in the
disease or condition of interest. In one embodiment, the reduction
of gene expression and thus reduction in the level of the
respective protein/RNA relieves, to some extent, the symptoms of
the disease or condition.
Efficacy
[0418] The compounds, compositions, methods and uses may involve
administration conditions suitable for reducing or inhibiting, or
ameliorating a disease or disorder. In one embodiment, a
therapeutically effective amount of an RNAi agent is administered
to a patient in need thereof, wherein the level of target gene
expression is reduced in the patient compared to an untreated
patient.
[0419] In one embodiment, the expression of a target gene
implicated in the disease or condition of interest is reduced by
about 10%, more preferably about 20%, more preferably about 30%,
more preferably about 40%, more preferably about 50%, more
preferably about 60%, more preferably about 70%, more preferably
about 80%, more preferably about 90%, more preferably about 95%,
more preferably about 98%, and most preferably about 100% relative
to an untreated patient.
DEFINITIONS
[0420] As used throughout this disclosure, articles such as "a" and
"an" refer to one or more than one (at least one) of the
grammatical object of the article.
[0421] Compounds of formulae (I), (II), (III), (IV), (V), (VI),
(VII), (VIII), (IX), (X), (XI) and (XII) and derivatives
thereof.
[0422] As used herein, the terms "(lipid) compound of the
invention", "(lipid) compound of formula (I)", "(lipid) compound",
"cationic lipid" etc. (i.e. all references to the cationic lipids
of the invention, and/or "stealth lipids" include pharmaceutically
acceptable derivatives thereof and polymorphs, isomers and
isotopically labelled variants thereof. Furthermore, said terms
include compounds of formula (II), (III), (IV), (V), (VI), (VII),
(VIII), (IX) and (X) for cationic lipids; and formulas (XI) and
(XII) for stealth lipids; and embodiments thereof disclosed
herein.
[0423] Pharmaceutically Acceptable Derivatives
[0424] The term "pharmaceutically acceptable derivative" includes
any pharmaceutically acceptable salt, solvate or hydrate of a
compound of formula (I). In one embodiment, the pharmaceutically
acceptable derivatives are pharmaceutically acceptable salts,
solvates or hydrates of a compound of formula (I).
[0425] Pharmaceutically Acceptable Salts
[0426] The term "pharmaceutically acceptable salt" includes a salt
prepared from pharmaceutically acceptable non-toxic acids or bases
including inorganic or organic acids and bases. For a review of
pharmaceutically acceptable salts, see Stahl and Wermuth, Handbook
of Pharmaceutical Salts: Properties, Selection and Use (Wiley-VCH,
Weinheim, Germany, 2002).
[0427] Solvates & Hydrates
[0428] The compounds of the invention may exist in both unsolvated
and solvated forms. The term "solvate" includes molecular complexes
comprising a compound of the invention and one or more
pharmaceutically acceptable solvent molecules such as water or
C.sub.1-6 alcohols, e.g. ethanol. The term "hydrate" means a
"solvate" where the solvent is water.
[0429] Isomeric Forms
[0430] Compounds of the invention may exist in one or more
geometrical, optical, enantiomeric, diastereomeric and tautomeric
forms, including but not limited to cis- and trans-forms, E- and
Z-forms, R-, S- and meso-forms, keto-, and enol-forms. All such
isomeric forms are included within the invention. The isomeric
forms may be in isomerically pure or enriched form, as well as in
mixtures of isomers (e.g. racemic or diastereomeric mixtures).
[0431] Accordingly, the invention provides at least, e.g.: [0432]
stereoisomeric mixtures of compounds of formula (I); [0433] a
diastereomerically enriched or diastereomerically pure isomer of a
compound of formula (I); or [0434] an enantiomerically enriched or
enantiomerically pure isomer of a compound of formula (I).
[0435] Where appropriate isomers can be separated from their
mixtures by the application or adaptation of known methods (e.g.
chromatographic techniques and recrystallisation techniques). Where
appropriate isomers can be prepared by the application or
adaptation of known methods (e.g. asymmetric synthesis).
[0436] Isotopic Labeling
[0437] The invention includes pharmaceutically acceptable
isotopically-labelled compounds of formula (I) wherein one or more
atoms are replaced by atoms having the same atomic number, but an
atomic mass or mass number different from the atomic mass or mass
number usually found in nature.
[0438] Examples of isotopes suitable for inclusion in the compounds
of the invention include isotopes of hydrogen, such as .sup.2H and
.sup.3H, carbon, such as .sup.11C, .sup.13C and .sup.14C, chlorine,
such as .sup.36Cl, fluorine, such as .sup.18F, iodine, such as
.sup.123I and .sup.125I, nitrogen, such as .sup.13N and .sup.15N,
oxygen, such as .sup.15O, .sup.17O and .sup.18O, phosphorus, such
as .sup.32P, and sulphur, such as .sup.35S. Certain
isotopically-labelled compounds of formula (I), e.g., those
incorporating a radioactive isotope, are useful in drug and/or
substrate tissue distribution studies. The radioactive isotopes
.sup.3H and .sup.14C are particularly useful for this purpose in
view of their ease of incorporation and ready means of
detection.
[0439] Substitution with positron emitting isotopes, such as
.sup.11C, .sup.18F, .sup.15O and .sup.13N, can be useful in
Positron Emission Topography (PET) studies.
[0440] Isotopically-labelled compounds of formula (I) can generally
be prepared by conventional techniques known to the skilled person
or by processes analogous to those described herein using an
appropriate isotopically-labelled reagent in place of the
non-labelled reagent previously employed.
[0441] Therapeutic Definitions
[0442] As used herein, "treatment" includes ameliorative, curative
and prophylactic treatment. As used herein, a "patient" means an
animal, preferably a mammal, preferably a human, in need of
treatment.
[0443] The "biologically active agent" is preferably a therapeutic
compound, i.e. a compound that is useful for the treatment or
prevention of a disease or a condition.
[0444] The biologically active agent includes but are not limited
to, e.g., antibodies, cholesterol, hormones, antivirals, peptides,
polypeptides, proteins, nucleoproteins, chemotherapeutics, low
molecular weight drugs, vitamins, co-factors, nucleosides,
nucleoside derivatives, nucleotides, oligonucleotides, enzymatic
nucleic acids, antisense nucleic acids, triplex forming
oligonucleotides, 2,5-A antisense chimeras, allozymes, aptamers,
ribozyme, decoy RNA molecules and analogs thereof, and small
nucleic acid molecules, such as an RNA inhibitor (RNAi) including,
e.g., short interfering nucleic acid (siNA), short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short
hairpin RNA (shRNA). In one embodiment the biologically active
agent is preferably a nucleoside or nucleoside derivative, e.g. a
nucleic acid, an oligonucleotide, a polynucleotide (e.g. siNA,
miRNA, RNAi, antisense, aptamer, ribozyme, decoy, ribozyme, 2-5A,
triplex forming oligonucleotide), and preferably an siRNA, miRNA,
siRNA inhibitor or an miRNA inhibitor.
[0445] In one embodiment, the biologically active agent is an siNA
(short interfering nucleic acid) molecule. In one embodiment, the
siNA is siDNA. In one embodiment, the siNA is siRNA. In one
embodiment, the siNA is miRNA.
[0446] In one embodiment, the biologically active agent is a small
nucleic acid molecule, referred to below for convenience purposes
only as a "siNA" molecule, down-regulates expression of a target
gene, e.g. wherein the target gene comprises a target encoding
sequence or wherein the target gene comprises a target non-coding
sequence or regulatory elements involved in target gene
expression.
[0447] The siNA can be single, double, or multiple stranded. In one
embodiment, it is double stranded.
[0448] In one embodiment, the siNA comprises unmodified nucleotides
and/or non-nucleotides. In one embodiment, the siNA comprises at
least one, or more than one, modified nucleotides and/or
non-nucleotides. In one embodiment, the modified nucleotide
comprises a modified base portion. In one embodiment, the modified
nucleotide comprises a modified sugar portion. In one embodiment,
the modified nucleotide comprises a modified backbone portion. In
one embodiment, the siNA comprises one or more of a modified base
portion, a modified sugar portion, and/or a modified backbone
portion.
[0449] In one embodiment, the siNA molecule comprises about 15 to
about 40 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) base
pairs, in a sub-embodiment about 15 to about 30 (e.g. about 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base
pairs, in a sub-embodiment about 15 to about 28 base pairs, in a
sub-embodiment about 17 to about 25 base pairs, in a sub-embodiment
about 18 to about 23 base pairs, in a further sub-embodiment about
19 to about 22 base pairs. In one embodiment the siNA comprises
about 17 base pairs. In one embodiment the siNA comprises about 18
base pairs. In one embodiment the siNA comprises about 19 base
pairs. In one embodiment the siNA comprises about 20 base pairs. In
one embodiment the siNA comprises about 21 base pairs.
[0450] In one embodiment, each of the two strands of the siNA
molecule independently comprises about 15 to about 40 (e.g. about
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides, in a
sub-embodiment about 15 to about 30 (e.g. about 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, in a
sub-embodiment about 15 to about 28 nucleotides, in a
sub-embodiment about 17 to about 25 nucleotides, in a
sub-embodiment about 18 to about 23 nucleotides, in a further
sub-embodiment about 19 to about 22 nucleotides. In one embodiment
each strand is about 17 nucleotides long. In one embodiment each
strand is about 18 nucleotides long. In one embodiment each strand
is about 19 nucleotides long. In one embodiment each strand is
about 20 nucleotides long. In one embodiment each strand is about
21 nucleotides.
[0451] In one embodiment, the siNA molecule directs cleavage of a
target RNA via the RISC complex, i.e., RNA interference (RNAi).
[0452] In one embodiment, the siNA molecule comprises a first and a
second strand, the first strand of the siNA comprising a nucleotide
sequence having sufficient complementarity to the target RNA for
the siNA molecule to direct cleavage of the target RNA via RNA
interference, and the second strand of said siNA molecule
comprising a nucleotide sequence that is complementary to the first
strand.
[0453] In one embodiment, the short interfering nucleic acid (siNA)
molecule is a chemically synthesized double stranded molecule.
[0454] In one embodiment, the siNA inhibits the expression of
target genes or a target gene family, wherein the genes or gene
family sequences share sequence homology. Such homologous sequences
can be identified as is known in the art, e.g., using sequence
alignments. Such siNA molecules can be designed to target such
homologous sequences, e.g., using perfectly complementary sequences
or by incorporating non-canonical base pairs, e.g., mismatches
and/or wobble base pairs that can provide additional target
sequences.
[0455] In one embodiment, the double-stranded siNA molecule does
not contain any ribonucleotides. In another embodiment, the
double-stranded siNA molecule comprises one or more
ribonucleotides.
[0456] In one embodiment, the siNA molecule which down-regulates
expression of a target gene comprises an antisense region, wherein
the antisense region comprises a nucleotide sequence that is
complementary to a nucleotide sequence of the target gene or a
portion thereof, and a sense region, wherein the sense region
comprises a nucleotide sequence substantially similar to the
nucleotide sequence of the target gene or a portion thereof. In one
embodiment, the antisense region and the sense region independently
comprise about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In one
embodiment, the antisense region comprises a nucleotide sequence
that is complementary to a nucleotide sequence of RNA encoded by
the target gene or a portion thereof, and the sense region
comprises a nucleotide sequence that is complementary to the
antisense region.
[0457] In one embodiment, the siNA molecule comprises one blunt
end. In one embodiment, the siNA molecule comprises two blunt ends,
i.e., a symmetric terminus without any overhanging unpaired
nucleotides.
[0458] In one embodiment, all nucleotides of each fragment of the
siNA molecule are base-paired to complementary nucleotides on the
other strand of the siNA molecule.
[0459] In one embodiment, the siNA molecule comprises one or more
of the following features: a mismatch, a bulge, a loop and a wobble
base pair, each of which may modulate the activity of the siNA
molecule to mediate RNA interference.
[0460] In one embodiment the sense region is connected to the
antisense region via a linker molecule, such as a polynucleotide
linker or a non-nucleotide linker.
[0461] In one embodiment, the siNA molecule has one or more
modified pyrimidine and/or purine nucleotides. In one embodiment,
the pyrimidine nucleotides in the sense region are
2'-O-methylpyrimidine nucleotides or 2'-deoxy-2'-fluoro pyrimidine
nucleotides and the purine nucleotides present in the sense region
are 2'-deoxy purine nucleotides. In another, embodiment, the
pyrimidine nucleotides in the sense region are 2'-deoxy-2'-fluoro
pyrimidine nucleotides and the purine nucleotides present in the
sense region are 2'-O-methyl purine nucleotides. In another
embodiment, the pyrimidine nucleotides in the sense region are
2'-deoxy-2'-fluoro pyrimidine nucleotides and the purine
nucleotides present in the sense region are 2'-deoxy purine
nucleotides. In one embodiment, the pyrimidine nucleotides in the
antisense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides and
the purine nucleotides present in the antisense region are
2'-O-methyl or 2'-deoxy purine nucleotides.
[0462] In one embodiment, the sense strand has a non-complementary
region. Optionally, the nucleotides present in said
non-complementary region are all 2'-deoxy nucleotides.
[0463] In one embodiment, the polynucleotide comprising the sense
region includes a terminal cap moiety at the 5'-end, the 3'-end, or
both of the 5' and 3' ends of the strand. In one embodiment, the
polynucleotide comprising the antisense strand includes a terminal
cap moiety at the 5'-end, the 3'-end, or both of the 5' and 3' ends
of the strand. In one embodiment, the terminal cap moiety is an
inverted deoxy abasic moiety or glyceryl moiety. Other examples of
terminal cap moieties are known in the art, e.g., WO 2005/021749
and WO 2007/128477.
[0464] In one embodiment, the siNA has phosphite, phosphodiester,
phosphorothioate and/or phosphorodithioate linkages in the
polynucleotide backbone. In one embodiment, the siNA has at least
one phosphorothioate linkage. In one embodiment, the siNA has at
least one phosphorodithioate linkage.
[0465] In one embodiment, the nucleotide modification(s) are
present at specifically selected locations in the siNA that are
sensitive to cleavage by ribonucleases, such as locations having
pyrimidine nucleotides.
[0466] In one embodiment, each of the two 3' terminal nucleotides
of each strand of the siNA molecule is a 2'-deoxy-pyrimidine
nucleotide, such as a 2'-deoxy-thymidine.
[0467] The amount of the compound of the invention and the
biologically active agent (e.g. the therapeutic compound)
administered should be a therapeutically effective amount where
used for the treatment of a disease or condition, and a
prophylactically effective amount where used for the prevention of
a disease or condition.
[0468] The term "therapeutically effective amount" refers to the
amount of the compound of the invention and the biologically active
agent (e.g. the therapeutic compound) needed to treat or ameliorate
a targeted disease or condition. The term "prophylactically
effective amount" used herein refers to the amount of the compound
of the invention and the biologically active agent (e.g. the
therapeutic compound) needed to prevent a targeted disease or
condition. The exact dosage will generally be dependent on the
patient's status at the time of administration. Factors that may be
taken into consideration when determining dosage include the
severity of the disease state in the patient, the general health of
the patient, the age, weight, gender, diet, time, frequency and
route of administration, identity of the biologically active agent
(e.g. the therapeutic compound), reaction sensitivities and the
patient's tolerance or response to therapy. The precise amount can
be determined by routine experimentation, but may ultimately lie
with the judgement of the clinician. Generally, an effective dose
will be from 0.01 mg/kg/day (mass of drug compared to mass of
patient) to 1000 mg/kg/day, e.g. 1 mg/kg/day to 100 mg/kg/day.
Compositions may be administered individually to a patient or may
be administered in combination with other agents, drugs or
hormones.
[0469] In any of the methods of treatment or associated uses, the
compound or composition of the invention can be administered to the
patient as a course of treatment, e.g., administration at various
time intervals, such as once per day over the course of treatment,
once every two days over the course of treatment, once every three
days over the course of treatment, once every four days over the
course of treatment, once every five days over the course of
treatment, once every six days over the course of treatment, once
per week over the course of treatment, once every other week over
the course of treatment, once per month over the course of
treatment, etc. In one embodiment, the course of treatment is once
every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In one embodiment,
the course of treatment is from about one to about 52 weeks or
longer (e.g. indefinitely). In one embodiment, the course of
treatment is from about one to about 48 months or longer (e.g.
indefinitely).
[0470] In one embodiment, a course of treatment involves an initial
course of treatment, such as once every 1, 2, 3, 4, 5, 6, 7, 8, 9,
10 or more weeks for a fixed interval (e.g. 1.times., 2.times.,
3.times., 4.times., 5.times., 6.times., 7.times., 8.times.,
9.times., 10.times. or more) followed by a maintenance course of
treatment, such as once every 4, 6, 8, 10, 15, 20, 25, 30, 35, 40,
or more weeks for an additional fixed interval (e.g. 1.times.,
2.times., 3.times., 4.times., 5.times., 6.times., 7.times.,
8.times., 9.times., 10.times. or more).
[0471] By "proliferative disease" as used herein is meant any
disease, condition, trait, genotype or phenotype characterized by
unregulated cell growth or replication as is known in the art. In
one embodiment, the proliferative disease is cancer. In one
embodiment, the proliferative disease is a tumor. In one
embodiment, the proliferative disease includes, but are not limited
to, e.g., liquid tumors such as, e.g., leukemias, e.g., acute
myelogenous leukemia (AML), chronic myelogenous leukemia (CML),
acute lymphocytic leukemia (ALL), multiple myeloma, and chronic
lymphocytic leukemia; and solid tumors, e.g., AIDS related cancers
such as Kaposi's sarcoma; breast cancers; bone cancers; brain
cancers; cancers of the head and neck, non-Hodgkins lymphoma,
adenoma, squamous cell carcinoma, laryngeal carcinoma, gallbladder
and bile duct cancers, cancers of the retina, cancers of the
esophagus, gastrointestinal cancers, ovarian cancer, uterine
cancer, thyroid cancer, testicular cancer, endometrial cancer,
melanoma, colorectal cancer, lung cancer, bladder cancer, prostate
cancer, lung cancer (including non-small cell lung carcinoma),
pancreatic cancer, sarcomas, Wilms' tumor, cervical cancer, head
and neck cancer, skin cancers, nasopharyngeal carcinoma,
liposarcoma, epithelial carcinoma, renal cell carcinoma,
gallbladder adeno carcinoma, endometrial sarcoma, multidrug
resistant cancers. In one embodiment, the proliferative disease
includes neovascularization associated with tumor angiogenesis,
macular degeneration (e.g. wet/dry age related macular
degeneration), corneal neovascularization, diabetic retinopathy,
neovascular glaucoma, myopic degeneration. In one embodiment, the
proliferative disease includes restenosis and polycystic kidney
disease.
[0472] By "inflammatory disease" as used herein is meant any
disease, condition, trait, genotype or phenotype characterized by
an inflammatory or allergic process as is known in the art.
Inflammatory diseases include, but are not limited to, e.g.,
inflammation (e.g. acute and/or chronic inflammation), respiratory
disease, atherosclerosis, psoriasis, dermatitis, restenosis,
asthma, allergic rhinitis, atopic dermatitis, septic shock,
rheumatoid arthritis, inflammatory bowl disease, inflammatory
pelvic disease, pain, ocular inflammatory disease, celiac disease,
tuberculosis, silicosis and other pneumoconioses.
[0473] By "autoimmune disease" as used herein is meant any disease,
condition, trait, genotype or phenotype characterized by
autoimmunity as is known in the art. Autoimmune diseases include,
but are not limited to, e.g., multiple sclerosis, diabetes
mellitus, lupus, scleroderms, fibromyalgia, transplantation
rejection (e.g. prevention of allograft rejection), pernicious
anemia, rheumatoid arthritis, systemic lupus erythematosus,
dermatomyositis, myasthenia gravis, lupus erythematosus, multiple
sclerosis, and Grave's disease.
[0474] By "infectious disease" is meant any disease, disorder or
condition associated with an infectious agent, such as a virus,
bacteria, fungus, prion or parasite.
[0475] By "neurologic disease" is meant any disease, disorder, or
condition affecting the central or peripheral nervous system.
Neurologic diseases include, but are not limited to, diseases or
disorders of either the peripheral or the central nervous system
including, e.g., Alzheimer's Disease, Aneurysm, Brain Injury,
Carpal Tunnel Syndrome, Cerebral Aneurysm, Chronic Pain,
Creutzfeldt-Jakob Disease, Epilepsy, Huntington's Disease,
Meningitis, Seizure Disorders, and other neurologic diseases,
disorders and syndromes.
[0476] By "respiratory disease" is meant any disease or condition
affecting the respiratory tract. Respiratory diseases include, but
are not limited to, e.g., asthma, chronic obstructive pulmonary
disease (COPD), allergic rhinitis, sinusitis, allergies, impeded
respiration, respiratory distress syndrome, cystic fibrosis,
pulmonary hypertension or vasoconstriction and emphysema.
[0477] By "cardiovascular disease" is meant and disease or
condition affecting the heart and vasculature. Cardiovascular
diseases include, but are not limited to, e.g., coronary heart
disease (CHD), cerebrovascular disease (CVD), aortic stenosis,
peripheral vascular disease, myocardial infarction (heart attack),
arrhythmia, and congestive heart failure.
[0478] By "ocular disease" as used herein is meant any disease,
condition, trait, genotype or phenotype of the eye and related
structures. Ocular diseases include, but are not limited to, e.g.,
cystoid macular edema, diabetic retinopathy, lattice degeneration,
retinal vein occlusion, retinal artery occlusion, macular
degeneration (e.g. age related macular degeneration such as wet AMD
or dry AMD), toxoplasmosis, retinitis pigmentosa, conjunctival
laceration, corneal laceration, glaucoma, and the like.
[0479] By "metabolic disease" is meant any disease or condition
affecting metabolic pathways. Metabolic disease can result in an
abnormal metabolic process, either congenital due to inherited
enzyme abnormality (inborn errors of metabolism) or acquired due to
disease of an endocrine organ or failure of a metabolically
important organ such as the liver. In one embodiment, metabolic
disease includes obesity, insulin resistance, and diabetes (e.g.
type I and/or type II diabetes).
[0480] By "dermatological disease" is meant any disease or
condition of the skin, dermis, or any substructure therein such as
a hair, a follicle, etc. Dermatological diseases, disorders,
conditions, and traits can include psoriasis, ectopic dermatitis,
skin cancers such as melanoma and basal cell carcinoma, hair loss,
hair removal and alterations in pigmentation.
[0481] By "auditory disease" is meant any disease or condition of
the auditory system, including the ear, such as the inner ear,
middle ear, outer ear, auditory nerve, and any substructures
therein. Auditory diseases, disorders, conditions, and traits can
include hearing loss, deafness, tinnitus, vertigo, balance and
motion disorders.
[0482] In one embodiment, the disease or condition is a disease of
the liver, a tumor, a disease mediated by FVII, and/or a disease
mediated by PLK1. Diseases mediated by FVII include abnormal blood
coagulation and tumors; such diseases thus include thrombosis (e.g.
venous thromboembolisms, pulmonary embolisms and strokes).
Biochemical Terms and Definitions
[0483] The term "lipid" refers to a group of organic compounds that
includes, but is not limited to, esters of fatty acids and are
characterised by being insoluble in water, but soluble in many
organic solvents. Lipids can be divided into at least three classes
(1) "simple lipids" which include fats and oils as well as waxes;
(2) "compound lipids" which include phospholipids and glycolipids,
and (3) "derived lipids" such as steroids.
[0484] The term "cationic lipid" as used herein is meant any
lipophilic compound having a cationic charge, such as a compound
having formula (I)
##STR00091##
wherein the definitions are as set out elsewhere herein. Other
examples of cationic lipids are set out above under the heading
"compositions".
[0485] Helper Lipids
[0486] The term "helper lipid" as used herein is meant a lipid that
enhances transfection (e.g. transfection of the nanoparticle
including the biologically active agent) to some extent. The
mechanism by which the helper lipid enhances transfection may
include, e.g., enhancing particle stability and/or enhancing
membrane fusogenicity. Helper lipids include steroids and alkyl
resorcinols. Examples of helper lipids are cholesterol,
5-heptadecylresorcinol, and cholesterol hemisuccinate
[0487] Stealth Lipids
[0488] The term "stealth lipid" as used herein is meant a lipid
that increases the length of time for which the nanoparticles can
exist in vivo (e.g. in the blood). In one embodiment, a stealth
lipid comprises a hydrophilic polymer head group operably linked to
a lipid moiety. In one embodiment stealth lipids in a liposome
formulation shield the nanoparticle surface and thereby reduce
opsonisation by blood proteins and uptake by the macrophages of the
mononuclear phagocyte system. Structures of stealth lipids suitable
for use in the present invention include but are not limited to,
e.g., compounds as provided in formula XI and formula XII. Other
contemplated stealth lipids and information about the biochemistry
of such lipids can be found in Romberg et al., Pharmaceutical
Research, Vol. 25, No. 1, 2008, p. 55-71 and Hoekstra et al.,
Biochimica et Biophysica Acta 1660 (2004) 41-52.
[0489] In one embodiment, the stealth lipid comprises a group
selected from PEG (sometimes referred to as poly(ethylene oxide)
and polymers based on poly(oxazoline), poly(vinyl alcohol),
poly(glycerol), poly(N-vinylpyrrolidone), polyaminoacids and
poly[N-(2-hydroxypropyl)methacrylamide]. In one embodiment the
helper lipid is able to "shed" as described in Romberg et al.
Specific stealth lipids of the invention are provided, e.g., in
formula XI and formula XII, which may be further substituted by one
skilled in the art. Additional suitable PEG lipids are disclosed,
e.g., in WO 2006/007712.
[0490] Specific suitable stealth lipids include
polyethyleneglycol-diacylglycerol or
polyethyleneglycol-diacylglycamide (PEG-DAG) conjugates including
those comprising a dialkylglycerol or dialkylglycamide group having
alkyl chain length independently comprising from about C.sub.4 to
about C.sub.40 saturated or unsaturated carbon atoms. The
dialkylglycerol or dialkylglycamide group can further comprise one
or more substituted alkyl groups. In any of the embodiments
described herein, the PEG conjugate can be selected from
PEG-dilaurylglycerol, PEG-dimyristylglycerol (catalog #GM-020 from
NOF), PEG-dipalmitoylglycerol, PEG-disterylglycerol,
PEG-dilaurylglycamide, PEG-dimyristylglycamide,
PEG-dipalmitoylglycamide, and PEG-disterylglycamide,
PEG-cholesterol
(1-[8'-(Cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-
-[omega]-methyl-poly(ethylene glycol), PEG-DMB
(3,4-Ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol)
ether), S001, S002, S003, S004, S005, S006, S007, S008, S009, S010,
S011, S012, S013, S014, S015, S016, S017, S018, S019, SO.sub.20,
S021, S022, S023, S024, S025, S026,
1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (catalog #880150P from Avanti Polar Lipids). S010 and
S011 are disclosed in WO 2009/086558 under the labels IVa and IVc,
respectively.
[0491] Unless otherwise indicated, the term "PEG" as used herein
means any polyethylene glycol or other polyalkylene ether polymer.
In one embodiment, PEG is an optionally substituted linear or
branched polymer of ethylene glycol or ethylene oxide. In one
embodiment PEG is unsubstituted. In one embodiment the PEG is
substituted, e.g., by one or more alkyl, alkoxy, acyl or aryl
groups. In one embodiment, the term includes PEG copolymers such as
PEG-polyurethane or PEG-polypropylene (see, e.g., J. Milton Harris,
Poly(ethylene glycol) chemistry: biotechnical and biomedical
applications (1992)); in another embodiment, the term does not
include PEG copolymers. In one embodiment, the PEG has a molecular
weight of from about 130 to about 50,000, in a sub-embodiment about
150 to about 30,000, in a sub-embodiment about 150 to about 20,000,
in a sub-embodiment about 150 to about 15,000, in a sub-embodiment
about 150 to about 10,000, in a sub-embodiment about 150 to about
6000, in a sub-embodiment about 150 to about 5000, in a
sub-embodiment about 150 to about 4000, in a sub-embodiment about
150 to about 3000, in a sub-embodiment about 300 to about 3000, in
a sub-embodiment about 1000 to about 3000, and in a sub-embodiment
about 1500 to about 2500.
[0492] In certain embodiments the PEG is a "PEG-2K", also termed
"PEG 2000", which has an average molecular weight of about 2000
daltons. PEG-2K is represented herein by the following formula
(Xlla), wherein n is 45, meaning that the number-averaged degree of
polymerization comprises about 45 subunits. However, other PEG
embodiment known in the art may be used, including, e.g., those
where the number-averaged degree of polymerization comprises about
23 subunits (n=23) and/or 68 subunits (n=68).
##STR00092##
Definitions for RNA Interference and RNAi Formulations
[0493] By "lipid nanoparticle" is meant a particle that comprises a
plurality of (i.e. more than one) lipid molecules physically
associated with each other by intermolecular forces. The lipid
nanoparticles may be, e.g., microspheres (including unilamellar and
multlamellar vesicles, e.g. liposomes), a dispersed phase in an
emulsion, micelles or an internal phase in a suspension.
[0494] The lipid nanoparticles have a size of about 1 to about
2,500 nm, about 1 to about 1,500 nm, about 1 to about 1,000 nm, in
a sub-embodiment about 50 to about 600 nm, in a sub-embodiment
about 50 to about 400 nm, in a sub-embodiment about 50 to about 250
nm, and in a sub-embodiment about 50 to about 150 nm. Unless
indicated otherwise, all sizes referred to herein are the average
sizes (diameters) of the fully formed nanoparticle, as measured by
dynamic light scattering on a Malvern Zetasizer. The nanoparticle
sample is diluted in phosphate buffered saline (PBS) so that the
count rate is approximately 200-400 kcts. The data is presented as
a weighted average of the intensity measure.
[0495] In one embodiment, the biologically active agent is
associated with the lipid nanoparticle (e.g. vesicle), and is
preferably encapsulated thereby.
[0496] In one embodiment, the lipid nanoparticle comprises a
biologically active agent, a compound of the invention, a neutral
lipid, a helper lipid and a stealth lipid.
[0497] In one embodiment, the liposome particles are stable in
serum.
[0498] The term "short interfering nucleic acid" (siNA) as used
herein refers to any nucleic acid molecule capable of inhibiting or
down regulating gene expression or viral replication by mediating
RNA interference (RNAi) or gene silencing in a sequence-specific
manner. It includes short interfering RNA (siRNA), microRNA
(miRNA), short interfering oligonucleotides and chemically-modified
short interfering nucleic acid molecules. siRNAs are responsible
for RNA interference, the process of sequence-specific
post-transcriptional gene silencing in animals and plants. siRNAs
are generated by ribonuclease III cleavage from longer
double-stranded RNA (dsRNA) which are homologous to, or specific
to, the silenced gene target.
[0499] By "RNA interference" (RNAi) is meant a biological process
of inhibiting or down regulating gene expression in a cell as is
generally known in the art, see e.g., Zamore and Haley, 2005,
Science, 309, 1519-1524; Zamore et al., 2000, Cell, 101, 25-33;
Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al.,
PCT Publication WO 00/44895; Fire, PCT Publication WO 99/32619;
Mello and Fire, PCT Publication WO 01/29058; and the like.
[0500] As used herein, RNAi is equivalent to other terms used to
describe sequence specific RNA interference, such as post
transcriptional gene silencing, translational inhibition,
transcriptional inhibition, or epigenetics. For example, the
formulations containing lipids of the invention can be used in
conjunction with siNA molecules to epigenetically silence genes at
both the post-transcriptional level and/or the pre-transcriptional
level. In a non-limiting example, modulation of gene expression by
siNA molecules can result from siNA mediated cleavage of RNA
(either coding or non-coding RNA) via RISC, or alternately,
translational inhibition as is known in the art. In another
embodiment, modulation of gene expression by siNA can result from
transcriptional inhibition such as is reported e.g., in Janowski et
al., 2005, Nature Chemical Biology, 1, 216-222.
[0501] By "RNAi inhibitor" is meant any molecule that can down
modulate (e.g. reduce or inhibit) RNA interference function or
activity in a cell or patient. An RNAi inhibitor can down regulate,
reduce or inhibit RNAi (e.g. RNAi mediated cleavage of a target
polynucleotide, translational inhibition, or transcriptional
silencing) by interaction with or interfering with the function of
any component of the RNAi pathway, including protein components
such as RISC, or nucleic acid components such as miRNAs or siRNAs.
An RNAi inhibitor can be a siNA molecule, an antisense molecule, an
aptamer, or a small molecule that interacts with or interferes with
the function of RISC, a miRNA, or a siRNA or any other component of
the RNAi pathway in a cell or patient. By inhibiting RNAi (e.g.
RNAi mediated cleavage of a target polynucleotide, translational
inhibition, or transcriptional silencing), an RNAi inhibitor can be
used to modulate (e.g, up-regulate or down regulate) the expression
of a target gene. In one embodiment, an RNA inhibitor is used to
up-regulate gene expression by interfering with (e.g. reducing or
preventing) endogenous down-regulation or inhibition of gene
expression through translational inhibition, transcriptional
silencing, or RISC mediated cleavage of a polynucleotide (e.g.
mRNA). By interfering with mechanisms of endogenous repression,
silencing, or inhibition of gene expression, RNAi inhibitors of the
invention can therefore be used to up-regulate gene expression for
the treatment of diseases or conditions resulting from a loss of
function. The term "RNAi inhibitor" is used in interchangeably with
the term "siNA" in various embodiments herein.
[0502] The term "enzymatic nucleic acid" as used herein refers to a
nucleic acid molecule that has complementarity in a substrate
binding region to a specified gene target, and also has an
enzymatic activity that acts to specifically cleave a target RNA,
thereby inactivating the target RNA molecule. The complementary
regions allow sufficient hybridization of the enzymatic nucleic
acid molecule to the target RNA and thus permit cleavage.
Complementarity of 100% is preferred, but complementarity as low as
50-75% can also be useful in this invention (see e.g., Werner and
Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et
al., 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31). The
nucleic acids can be modified at the base, sugar, and/or phosphate
groups. The term enzymatic nucleic acid is used interchangeably
with phrases such as ribozymes, catalytic RNA, enzymatic RNA,
catalytic DNA, aptazyme or aptamer-binding ribozyme, regulatable
ribozyme, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA
enzyme, endoribonuclease, endonuclease, minizyme, leadzyme,
oligozyme or DNA enzyme. All of these terminologies describe
nucleic acid molecules with enzymatic activity. The key features of
an enzymatic nucleic acid molecule are that it has a specific
substrate binding site that is complementary to one or more of the
target nucleic acid regions, and that it has nucleotide sequences
within or surrounding that substrate binding site that impart a
nucleic acid cleaving and/or ligation activity to the molecule
(see, e.g., Cech et al., U.S. Pat. No. 4,987,071; Cech et al.,
1988, 260 JAMA 3030). Ribozymes and enzymatic nucleic acid
molecules of the invention can be chemically modified, e.g., as
described in the art and elsewhere herein.
[0503] The term "antisense nucleic acid", as used herein, refers to
a non-enzymatic nucleic acid molecule that binds to target RNA by
means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid;
Egholm et al., 1993 Nature 365, 566) interactions and alters the
activity of the target RNA (for a review, see Stein and Cheng, 1993
Science 261, 1004 and Woolf et al., U.S. Pat. No. 5,849,902).
Antisense DNA can be synthesized chemically or expressed via the
use of a single stranded DNA expression vector or equivalent
thereof. Antisense molecules of the invention can be chemically
modified, e.g. as described in the art.
[0504] The term "RNase H activating region" as used herein, refers
to a region (generally greater than or equal to 4-25 nucleotides in
length, preferably from 5-11 nucleotides in length) of a nucleic
acid molecule capable of binding to a target RNA to form a
non-covalent complex that is recognized by cellular RNase H enzyme
(see e.g., Arrow et al., U.S. Pat. No. 5,849,902; Arrow et al.,
U.S. Pat. No. 5,989,912). The RNase H enzyme binds to the nucleic
acid molecule-target RNA complex and cleaves the target RNA
sequence.
[0505] The term "2-5A antisense chimera" as used herein, refers to
an antisense oligonucleotide containing a 5'-phosphorylated
2'-5'-linked adenylate residue. These chimeras bind to target RNA
in a sequence-specific manner and activate a cellular
2-5A-dependent ribonuclease that, in turn, cleaves the target RNA
(Torrence et al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300;
Silverman et al., 2000, Methods Enzymol., 313, 522-533; Player and
Torrence, 1998, Pharmacol. Ther., 78, 55-113). 2-5A antisense
chimera molecules can be chemically modified, e.g. as described in
the art.
[0506] The term "triplex forming oligonucleotides" as used herein,
refers to an oligonucleotide that can bind to a double-stranded DNA
in a sequence-specific manner to form a triple-strand helix.
Formation of such triple helix structure has been shown to inhibit
transcription of the targeted gene (Duval-Valentin et al., 1992
Proc. Natl. Acad. Sci. USA 89, 504; Fox, 2000, Curr. Med. Chem., 7,
17-37; Praseuth et. al., 2000, Biochim. Biophys. Acta, 1489,
181-206). Triplex forming oligonucleotide molecules of the
invention can be chemically modified, e.g. as described in the
art.
[0507] The term "decoy RNA" as used herein, refers to an RNA
molecule or aptamer that is designed to preferentially bind to a
predetermined ligand. Such binding can result in the inhibition or
activation of a target molecule. The decoy RNA or aptamer can
compete with a naturally occurring binding target for the binding
of a specific ligand. Similarly, a decoy RNA can be designed to
bind to a receptor and block the binding of an effector molecule,
or can be designed to bind to receptor of interest and prevent
interaction with the receptor. Decoy molecules of the invention can
be chemically modified, e.g. as described in the art.
[0508] The term "single stranded DNA" (ssDNA) as used herein refers
to a naturally occurring or synthetic deoxyribonucleic acid
molecule comprising a linear single strand, e.g., a ssDNA can be a
sense or antisense gene sequence or EST (Expressed Sequence
Tag).
[0509] The term "allozyme" as used herein refers to an allosteric
enzymatic nucleic acid molecule, including e.g., U.S. Pat. Nos.
5,834,186, 5,741,679, 5,589,332, 5,871,914, and PCT publication
Nos. WO 00/24931, WO 00/26226, WO 98/27104, and WO 99/29842.
[0510] By "aptamer" as used herein is meant a polynucleotide
composition that binds specifically to a target molecule, wherein
the polynucleotide has a sequence that differs from a sequence
normally recognized by the target molecule in a cell. Alternately,
an aptamer can be a nucleic acid molecule that binds to a target
molecule where the target molecule does not naturally bind to a
nucleic acid. The target molecule can be any molecule of interest.
Aptamer molecules of the invention can be chemically modified, e.g.
as described in the art.
[0511] By "modulate" is meant that the expression of the gene, or
level of RNA molecule or equivalent RNA molecules encoding one or
more proteins or protein subunits, or activity of one or more
proteins or protein subunits is up regulated or down regulated, or
is absent, such that expression, level, or activity is greater than
or less than that observed without the modulator. For example, in
one embodiment, the term "modulate" means "inhibit". In one
embodiment, modulation of a pathway denotes, within the terms of
the invention, an up-regulation or a down-regulation of a
therapeutically meaningful component and/or endpoint of a
biological pathway that contains, or is regulated by, e.g., the
protein, enzyme, or substance being targetted or encoded by the
target mRNA.
[0512] By "inhibit", "down-regulate", or "reduce", it is meant that
the expression of the gene, or level of RNA molecules or equivalent
RNA molecules encoding one or more proteins or protein subunits, or
activity of one or more proteins or protein subunits, is reduced
below that observed in a natural environment, e.g. in the absence
of the nucleic acid molecules (e.g. siNA). In one embodiment,
inhibition, down-regulation or reduction with a siNA molecule is
below that level observed in the presence of an inactive or
attenuated molecule. In one embodiment, inhibition,
down-regulation, or reduction with siNA molecules is below that
level observed in the presence of, e.g., a siNA molecule with
scrambled sequence or with mismatches. In one embodiment,
inhibition, down-regulation, or reduction of gene expression with a
nucleic acid molecule is greater in the presence of the nucleic
acid molecule than in its absence. In one embodiment, inhibition,
down regulation, or reduction of gene expression is associated with
post transcriptional silencing, such as RNAi mediated cleavage of a
target nucleic acid molecule (e.g. RNA) or inhibition of
translation. In one embodiment, inhibition, down regulation, or
reduction of gene expression is associated with pretranscriptional
silencing.
[0513] By "up-regulate", or "promote", it is meant that the
expression of the gene, or level of RNA molecules or equivalent RNA
molecules encoding one or more proteins or protein subunits, or
activity of one or more proteins or protein subunits, is increased
above that observed in a natural environment, e.g. in the absence
of the nucleic acid molecules (e.g. siNA). In one embodiment,
up-regulation or promotion of gene expression with an siNA molecule
is above that level observed in the presence of an inactive or
attenuated molecule. In one embodiment, up-regulation or promotion
of gene expression with siNA molecules is above that level observed
in the presence of, e.g., an siNA molecule with scrambled sequence
or with mismatches. In one embodiment, up-regulation or promotion
of gene expression with a nucleic acid molecule is greater in the
presence of the nucleic acid molecule than in its absence. In one
embodiment, up-regulation or promotion of gene expression is
associated with inhibition of RNA mediated gene silencing, such as
RNAi mediated cleavage or silencing of a coding or non-coding RNA
target that down regulates, inhibits, or silences the expression of
the gene of interest to be up-regulated.
[0514] By "gene", or "target gene", is meant a nucleic acid that
encodes RNA, e.g., nucleic acid sequences including, but not
limited to, structural genes encoding a polypeptide. A gene or
target gene can also encode a functional RNA (FRNA) or non-coding
RNA (ncRNA), such as small temporal RNA (stRNA), micro RNA (miRNA),
small nuclear RNA (snRNA), short interfering RNA (siRNA), small
nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA)
and precursor RNAs thereof. Such non-coding RNAs can serve as
target nucleic acid molecules for siNA mediated RNA interference in
modulating the activity of FRNA or ncRNA involved in functional or
regulatory cellular processes. Abberant FRNA or ncRNA activity
leading to disease can therefore be modulated by siNA
molecules.
[0515] By "target" as used herein is meant any target protein,
peptide, or polypeptide encoded by a target gene. The term "target"
also refers to nucleic acid sequences encoding any target protein,
peptide, or polypeptide having target activity, such as encoded by
target RNA. The term "target" is also meant to include other target
encoding sequence, such as other target isoforms, mutant target
genes, splice variants of target genes, and target gene
polymorphisms. By "target nucleic acid" is meant any nucleic acid
sequence whose expression or activity is to be modulated. The
target nucleic acid can be DNA or RNA.
[0516] By "non-canonical base pair" is meant any non-Watson Crick
base pair, such as mismatches and/or wobble base.
[0517] By "sense region" is meant a nucleotide sequence of a siNA
molecule having complementarity to an antisense region of the siNA
molecule. In addition, the sense region of a siNA molecule can
comprise a nucleic acid sequence having homology with a target
nucleic acid sequence. In one embodiment, the sense region of the
siNA molecule is referred to as the sense strand or passenger
strand.
[0518] By "antisense region" is meant a nucleotide sequence of a
siNA molecule having complementarity to a target nucleic acid
sequence. In addition, the antisense region of a siNA molecule can
optionally comprise a nucleic acid sequence having complementarity
to a sense region of the siNA molecule. In one embodiment, the
antisense region of the siNA molecule is referred to as the
antisense strand or guide strand.
[0519] By "target nucleic acid" or "target polynucleotide" is meant
any nucleic acid sequence whose expression or activity is to be
modulated. The target nucleic acid can be DNA or RNA. In one
embodiment, a target nucleic acid of the invention is target RNA.
In one embodiment, a target nucleic acid of the invention is target
DNA.
[0520] By "complementarity" is meant that a nucleic acid can form
hydrogen bond(s) with another nucleic acid sequence by either
traditional Watson-Crick or other non-traditional types such as
Hoogsteen base pairing. In one embodiment, a double stranded
nucleic acid molecule of the invention, such as an siNA molecule,
wherein each strand is between 15 and 40 nucleotides in length,
comprises between about 10% and about 100% (e.g. about 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100%)
complementarity between the two strands of the double stranded
nucleic acid molecule. A percent complementarity indicates the
percentage of contiguous residues in a nucleic acid molecule that
can pair through the formation of hydrogen bonds (e.g. Watson-Crick
base pairing) with a second nucleic acid sequence.
Chemical Terms and Definitions
[0521] For purposes of interpreting this specification, the
following definitions will apply and whenever appropriate, terms
used in the singular will also include the plural and vice
versa.
[0522] Halo
[0523] As used herein, the term "halogen" or "halo" refers to
fluoro, chloro, bromo, and iodo. The term "halogen" (or "halo")
includes fluorine, chlorine, bromine and iodine.
[0524] As used herein, the term "haloalkyl" refers to an alkyl as
defined herein that is substituted by one or more halo groups as
defined herein. The haloalkyl can be monohaloalkyl, dihaloalkyl or
polyhaloalkyl including perhaloalkyl. A monohaloalkyl can have one
iodo, bromo, chloro or fluoro within the alkyl group. Dihaloalky
and polyhaloalkyl groups can have two or more of the same halo
atoms or a combination of different halo groups within the alkyl.
Typically the polyhaloalkyl contains up to 12, or 10, or 8, or 6,
or 4, or 3, or 2 halo groups. Non-limiting examples of haloalkyl
include fluoromethyl, difluoromethyl, trifluoromethyl,
chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl,
heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl,
difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl. A
perhaloalkyl refers to an alkyl having all hydrogen atoms replaced
with halo atoms.
[0525] Alkyl, Alkylene, Alkenyl, Alkynyl, Cycloalkyl Etc.
[0526] The terms "alkyl", "alkylene", "alkenyl" and "alkynyl" are
used herein to refer to both straight and branched chain acyclic
forms. Cyclic analogs thereof are referred to as cycloalkyl,
cycloalkenyl, etc.
[0527] The term "alkyl" includes monovalent, straight or branched,
saturated, acyclic hydrocarbyl groups. As used herein, the term
"alkyl" refers to a fully saturated branched or unbranched
hydrocarbon moiety having up to 50 carbon atoms. Unless otherwise
provided, alkyl refers to hydrocarbon moieties having 1 to 50
carbon atoms, 1 to 40 carbon atoms, 1 to 30 carbon atoms, 1 to 20
carbon atoms, 1 to 16 carbon atoms, 1 to 16 carbon atoms, 1 to 10
carbon atoms, 1 to 7 carbon atoms, or 1 to 4 carbon atoms.
Representative examples of alkyl include, but are not limited to,
methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,
tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl,
2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl,
n-decyl and the like. In one embodiment alkyl is C.sub.1-10alkyl,
in another embodiment C.sub.1-6alkyl, in another embodiment
C.sub.1-4alkyl, such as methyl, ethyl, n-propyl, i-propyl or
t-butyl groups.
[0528] The term "cycloalkyl" includes monovalent, saturated, cyclic
hydrocarbyl groups. In one embodiment cycloalkyl is
C.sub.3-10cycloalkyl, in another embodiment C.sub.3-6cycloalkyl
such as cyclopentyl and cyclohexyl.
[0529] The term "alkoxy" means alkyl-O--, wherein alkyl is defined
herein above. Representative examples of alkoxy include, but are
not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy,
tert-butoxy, pentyloxy, hexyloxy, cyclopropyloxy-, cyclohexyloxy-
and the like. Typically, alkoxy groups have about 1-7, more
preferably about 1-4 carbons.
[0530] The term "alkenyl" includes monovalent, straight or
branched, unsaturated, acyclic hydrocarbyl groups having at least
one carbon-carbon double bond and, in one embodiment, no
carbon-carbon triple bonds. As used herein, the term "alkylene"
refers to divalent alkyl group as defined herein above having 1 to
50 carbon atoms. It comprises 1 to 50 carbon atoms, Unless
otherwise provided, alkylene refers to moieties having 1 to 50
carbon atoms, 1 to 40 carbon atoms, 1 to 30 carbon atoms, 1 to 20
carbon atoms, 1 to 16 carbon atoms, 1 to 10 carbon atoms, 1 to 7
carbon atoms, or 1 to 4 carbon atoms. Representative examples of
alkylene include, but are not limited to, methylene, ethylene,
n-propylene, iso-propylene, n-butylene, sec-butylene, iso-butylene,
tert-butylene, n-pentylene, isopentylene, neopentylene, n-hexylene,
3-methylhexylene, 2,2-dimethylpentylene, 2,3-dimethylpentylene,
n-heptylene, n-octylene, n-nonylene, n-decylene and the like. In
one embodiment alkenyl is C.sub.2-10alkenyl, in another embodiment
C.sub.2-6alkenyl, in another embodiment C.sub.2-4alkenyl.
[0531] The term "cycloalkenyl" includes monovalent, partially
unsaturated, cyclic hydrocarbyl groups having at least one
carbon-carbon double bond. In one embodiment cycloalkenyl is
C.sub.3-10cycloalkenyl, in another embodiment
C.sub.5-10cycloalkenyl, e.g. cyclohexenyl.
[0532] The term "alkynyl" includes monovalent, straight or
branched, unsaturated, acyclic hydrocarbyl groups having at least
one carbon-carbon triple bond and, in one embodiment, no
carbon-carbon double bonds. In one embodiment, alkynyl is
C.sub.2-10alkynyl, in another embodiment C.sub.2-6alkynyl, in
another embodiment C.sub.2-4alkynyl.
[0533] The term "cycloalkynyl" includes monovalent, partially
unsaturated, cyclic hydrocarbyl groups having at least one
carbon-carbon triple bond. In one embodiment cycloalkynyl is
C.sub.6-10cycloalkenyl, in another embodiment
C.sub.8-10cycloalkynyl.
[0534] The term "alkylene" includes divalent, straight or branched,
saturated, acyclic hydrocarbyl groups. In one embodiment alkylene
is C.sub.1-10alkylene, in another embodiment C.sub.1-6alkylene, in
another embodiment C.sub.1-4alkylene, such as methylene, ethylene,
n-propylene, i-propylene or t-butylene groups.
[0535] The term "alkenylene" includes divalent, straight or
branched, unsaturated, acyclic hydrocarbyl groups having at least
one carbon-carbon double bond and, in one embodiment, no
carbon-carbon triple bonds. In one embodiment alkenylene is
C.sub.2-10alkenylene, in another embodiment C.sub.2-6alkenylene, in
another embodiment C.sub.2-4alkenylene.
[0536] The term "alkynylene" includes divalent, straight or
branched, unsaturated, acyclic hydrocarbyl groups having at least
one carbon-carbon triple bond. In one embodiment alkynylene is
C.sub.2-10alkynylene, in another embodiment C.sub.2-6alkynylene, in
another embodiment C.sub.2-4alkynylene.
[0537] Heteroalkyl Etc.
[0538] As used herein, the term "hetero atoms" refers to nitrogen
(N), oxygen (O), phosphorus (P) or sulfur (S) atoms, in particular
nitrogen or oxygen.
[0539] The term "heteroalkyl" includes alkyl groups in which up to
six carbon atoms, in one embodiment up to five carbon atoms, in
another embodiment up to four carbon atoms, in another embodiment
up to three carbon atoms, in another embodiment up to two carbon
atoms, in another embodiment one carbon atom, are each replaced
independently by O, S(O).sub.q, N, P(O).sub.r or Si (and preferably
O, S(O).sub.q or N), provided at least one of the alkyl carbon
atoms remains. The heteroalkyl group may be C-linked or
hetero-linked, i.e. it may be linked to the remainder of the
molecule through a carbon atom or through O, S(O).sub.q, N,
P(O).sub.r or Si. Note that S(O).sub.q and P(O).sub.r are defined
below.
[0540] The term "heterocycloalkyl" includes cycloalkyl groups in
which up to six carbon atoms, in one embodiment up to five carbon
atoms, in another embodiment up to four carbon atoms, in another
embodiment up to three carbon atoms, in another embodiment up to
two carbon atoms, in another embodiment one carbon atom, are each
replaced independently by O, S(O).sub.q or N, provided at least one
of the cycloalkyl carbon atoms remains. Examples of
heterocycloalkyl groups include oxiranyl, thiaranyl, aziridinyl,
oxetanyl, thiatanyl, azetidinyl, tetrahydrofuranyl,
tetrahydrothiophenyl, pyrrolidinyl, tetrahydropyranyl,
tetrahydrothiopyranyl, piperidinyl, 1,4-dioxanyl, 1,4-oxathianyl,
morpholinyl, 1,4-dithianyl, piperazinyl, 1,4-azathianyl, oxepanyl,
thiepanyl, azepanyl, 1,4-dioxepanyl, 1,4-oxathiepanyl,
1,4-oxaazepanyl, 1,4-dithiepanyl, 1,4-thieazepanyl and
1,4-diazepanyl. The heterocycloalkyl group may be C-linked or
N-linked, i.e. it may be linked to the remainder of the molecule
through a carbon atom or through a nitrogen atom.
[0541] The term "heteroalkenyl" includes alkenyl groups in which up
to three carbon atoms, in one embodiment up to two carbon atoms, in
another embodiment one carbon atom, are each replaced independently
by O, S(O).sub.q or N, provided at least one alkenyl carbon-carbon
double bond remains. The heteroalkenyl group may be C-linked or
hetero-linked, i.e. it may be linked to the remainder of the
molecule through a carbon atom or through O, S(O).sub.q or N.
[0542] The term "heterocycloalkenyl" includes cycloalkenyl groups
in which up to three carbon atoms, in one embodiment up to two
carbon atoms, in another embodiment one carbon atom, are each
replaced independently by O, S(O).sub.q or N, provided at least one
cycloalkenyl carbon-carbon double bond remains. Examples of
heterocycloalkenyl groups include 3,4-dihydro-2H-pyranyl,
5-6-dihydro-2H-pyranyl, 2H-pyranyl, 1,2,3,4-tetrahydropyridinyl and
1,2,5,6-tetrahydropyridinyl. The heterocycloalkenyl group may be
C-linked or N-linked, i.e. it may be linked to the remainder of the
molecule through a carbon atom or through a nitrogen atom. In one
embodiment, heterocycloalkenyl groups of the invention include
C.sub.3-C.sub.10 cycloalkenyl groups. In one embodiment,
heterocycloalkenyl groups of the invention include C.sub.5-C.sub.10
cycloalkenyl groups.
[0543] The term "heteroalkynyl" includes alkynyl groups in which up
to three carbon atoms, in one embodiment up to two carbon atoms, in
another embodiment one carbon atom, are each replaced independently
by O, S(O).sub.q or N, provided at least one alkynyl carbon-carbon
triple bond remains. The heteroalkynyl group may be C-linked or
hetero-linked, i.e. it may be linked to the remainder of the
molecule through a carbon atom or through O, S(O).sub.q or N.
[0544] The term "heterocycloalkynyl" includes cycloalkynyl groups
in which up to three carbon atoms, in one embodiment up to two
carbon atoms, in another embodiment one carbon atom, are each
replaced independently by O, S(O).sub.q or N, provided at least one
of the cycloalkynyl carbon-carbon triple bonds remains. The
heterocycloalkenyl group may be C-linked or N-linked, i.e. it may
be linked to the remainder of the molecule through a carbon atom or
through a nitrogen atom. An example of a heterocycloalkynyl group
includes azacyclooct-4-yne. In one embodiment, the invention
includes C.sub.3-C.sub.10 heterocycloalkynyl groups. In one
embodiment, the invention includes C.sub.5-C.sub.10
heterocycloalkynyl groups.
[0545] The term "heteroalkylene" includes alkylene groups in which
up to three carbon atoms, in one embodiment up to two carbon atoms,
in another embodiment one carbon atom, are each replaced
independently by O, S(O).sub.q or N, provided at least one alkylene
carbon-carbon bond remains.
[0546] The term "heteroalkenylene" includes alkenylene groups in
which up to three carbon atoms, in one embodiment up to two carbon
atoms, in another embodiment one carbon atom, are each replaced
independently by O, S(O).sub.q or N, provided at least one
alkenylene carbon-carbon double bond remains.
[0547] The term "heteroalkynylene" includes alkynylene groups in
which up to three carbon atoms, in one embodiment up to two carbon
atoms, in another embodiment one carbon atom, are each replaced
independently by O, S(O).sub.q or N, provided at least one
alkynylene carbon-carbon triple bond remains.
[0548] Aryl
[0549] The term "aryl" includes monovalent, aromatic, cyclic
hydrocarbyl groups, such as phenyl or naphthyl (e.g. 1-naphthyl or
2-naphthyl). In general, the aryl groups may be monocyclic or
polycyclic fused ring aromatic groups. Preferred aryl are
C.sub.6-C.sub.14aryl. As used herein the term "aryl" refers to an
aromatic hydrocarbon group having 6-20 carbon atoms in the ring
portion. Typically, aryl is monocyclic, bicyclic or tricyclic aryl
having 6-20 carbon atoms and includes one or more aromatic rings
fused to one or more non-aromatic hydrocarbon rings. Non-limiting
examples include phenyl, naphthyl or tetrahydronaphthyl.
[0550] Other examples of aryl groups are monovalent derivatives of
aceanthrylene, acenaphthylene, acephenanthrylene, anthracene,
azulene, chrysene, coronene, fluoranthene, fluorene, as-indacene,
s-indacene, indene, naphthalene, ovalene, perylene, phenalene,
phenanthrene, picene, pleiadene, pyrene, pyranthrene and
rubicene.
[0551] The term "arylalkyl" means alkyl substituted with an aryl
group, e.g. benzyl.
[0552] The term "arylene" includes divalent aromatic, cyclic
hydrocarbyl groups, such as phenylene. In general, the arylene
groups may be monocyclic or polycyclic fused ring aromatic groups.
Preferred arylene are C.sub.6-C.sub.14arylene. Other examples of
arylene groups are divalent derivatives of aceanthrylene,
acenaphthylene, acephenanthrylene, anthracene, azulene, chrysene,
coronene, fluoranthene, fluorene, as-indacene, s-indacene, indene,
naphthalene, ovalene, perylene, phenalene, phenanthrene, picene,
pleiadene, pyrene, pyranthrene and rubicene.
[0553] Heteroaryl
[0554] The term "heteroaryl" includes monovalent, heteroaromatic,
cyclic hydrocarbyl groups additionally containing one or more
heteroatoms independently selected from O, S, N and NR.sup.N, where
R.sup.N is defined below (and in one embodiment is H or alkyl
(e.g., C.sub.1-6alkyl)). As used herein, the term "heteroaryl"
refers to a 5-14 membered monocyclic- or bicyclic- or
tricyclic-aromatic ring system, having 1 to 8 heteroatoms selected
from N, O or S. Typically, the heteroaryl is a 5-10 membered ring
system (e.g., 5-7 membered monocycle or an 8-10 membered bicycle)
or a 5-7 membered ring system. Typical heteroaryl groups include 2-
or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl, 2-, 4-, or
5-imidazolyl, 3-, 4-, or 5-pyrazolyl, 2-, 4-, or 5-thiazolyl, 3-,
4-, or 5-isothiazolyl, 2-, 4-, or 5-oxazolyl, 3-, 4-, or
5-isoxazolyl, 3- or 5-1,2,4-triazolyl, 4- or 5-1,2,3-triazolyl,
tetrazolyl, 2-, 3-, or 4-pyridyl, 3- or 4-pyridazinyl, 3-, 4-, or
5-pyrazinyl, 2-pyrazinyl, and 2-, 4-, or 5-pyrimidinyl.
[0555] The term "heteroaryl" also refers to a group in which a
heteroaromatic ring is fused to one or more aryl, cycloaliphatic,
or heterocyclyl rings, where the radical or point of attachment is
on the heteroaromatic ring. Nonlimiting examples include those head
groups provided herein as H.sup.15 and H.sup.29.
[0556] In general, the heteroaryl groups may be monocyclic or
polycyclic (e.g., bicyclic) fused ring heteroaromatic groups. In
one embodiment, heteroaryl groups contain 5-13 ring members (e.g.,
5-10 members) and 1, 2, 3 or 4 ring heteroatoms independently
selected from O, S, N and NR.sup.N. In one embodiment, a heteroaryl
group may be 5, 6, 9 or 10 membered, e.g., 5-membered monocyclic,
6-membered monocyclic, 9-membered fused-ring bicyclic or
10-membered fused-ring bicyclic.
[0557] Monocyclic heteroaromatic groups include heteroaromatic
groups containing 5-6 ring members and 1, 2, 3 or 4 heteroatoms
selected from O, S, N or NR.sup.N.
[0558] In one embodiment, 5-membered monocyclic heteroaryl groups
contain 1 ring member which is a --NR.sup.N-- group, an --O-- atom
or an --S-- atom and, optionally, 1-3 ring members (e.g. 1 or 2
ring members) which are .dbd.N-- atoms (where the remainder of the
5 ring members are carbon atoms).
[0559] Examples of 5-membered monocyclic heteroaryl groups are
pyrrolyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, isoxazolyl,
oxazolyl, isothiazolyl, thiazolyl, 1,2,3 triazolyl, 1,2,4
triazolyl, 1,2,3 oxadiazolyl, 1,2,4 oxadiazolyl, 1,2,5 oxadiazolyl;
1,3,4 oxadiazolyl, 1,3,4 thiadiazolyl, pyridyl, pyrimidinyl,
pyridazinyl, pyrazinyl, 1,3,5 triazinyl, 1,2,4 triazinyl, 1,2,3
triazinyl and tetrazolyl.
[0560] Examples of 6-membered monocyclic heteroaryl groups are
pyridinyl, pyridazinyl, pyrimidinyl and pyrazinyl.
[0561] In one embodiment, 6-membered monocyclic heteroaryl groups
contain 1 or 2 ring members which are .dbd.N-- atoms (where the
remainder of the 6 ring members are carbon atoms).
[0562] Bicyclic heteroaromatic groups include fused-ring
heteroaromatic groups containing 9-13 ring members and 1, 2, 3, 4
or more heteroatoms selected from O, S, N or NR.sup.N.
[0563] In one embodiment, 9-membered bicyclic heteroaryl groups
contain 1 ring member which is a --NR.sup.N-- group, an --O-- atom
or an --S-- atom and, optionally, 1-3 ring members (e.g. 1 or 2
ring members) which are .dbd.N-- atoms (where the remainder of the
9 ring members are carbon atoms).
[0564] Examples of 9-membered fused-ring bicyclic heteroaryl groups
are benzofuranyl, benzothiophenyl, indolyl, benzimidazolyl,
indazolyl, benzotriazolyl, pyrrolo[2,3-b]pyridinyl,
pyrrolo[2,3-c]pyridinyl, pyrrolo[3,2-c]pyridinyl,
pyrrolo[3,2-b]pyridinyl, imidazo[4,5-b]pyridinyl,
imidazo[4,5-c]pyridinyl, pyrazolo[4,3-d]pyridinyl,
pyrazolo[4,3-c]pyridinyl, pyrazolo[3,4-c]pyridinyl,
pyrazolo[3,4-b]pyridinyl, isoindolyl, indazolyl, purinyl,
indolininyl, imidazo[1,2-a]pyridinyl, imidazo[1,5-a]pyridinyl,
pyrazolo[1,2-a]pyridinyl, pyrrolo[1,2-b]pyridazinyl and
imidazo[1,2-c]pyrimidinyl.
[0565] The term "heteroarylalkyl" means alkyl substituted with a
heteroaryl group.
[0566] The term "heteroarylene" includes divalent heteroaromatic,
cyclic hydrocarbyl groups additionally containing one or more
heteroatoms independently selected from O, S, N and NR.sup.N, where
R.sup.N is defined below (and in one embodiment is H or alkyl (e.g.
C.sub.1-6alkyl)). In general, the heteroarylene groups may be
monocyclic or polycyclic (e.g. bicyclic) fused ring heteroaromatic
groups. In one embodiment, heteroarylene groups contain 5-13 ring
members (preferably 5-10 members) and 1, 2, 3 or 4 ring heteroatoms
independently selected from O, S, N and NR.sup.N. In one
embodiment, a heteroarylene group may be 5, 6, 9 or 10 membered,
e.g. 5-membered monocyclic, 6-membered monocyclic, 9-membered
fused-ring bicyclic or 10-membered fused-ring bicyclic. The term
"heteroarylene" includes divalent derivatives of each of the
heteroaryl groups discussed above.
[0567] The terms "aryl", "aromatic", "heteroaryl" and
"heteroaromatic" also include groups that are partially reduced.
Thus, e.g., "heteroaryl" includes fused species in which one of the
rings has been reduced to a saturated ring (e.g.,
1,2,3,4-tetrahydro-1,8-naphthyridin-2-yl).
[0568] General
[0569] Unless indicated explicitly otherwise, where combinations of
groups are referred to herein as one moiety, e.g. arylalkyl, the
last mentioned group contains the atom by which the moiety is
attached to the rest of the molecule.
[0570] Where reference is made to a carbon atom of an alkyl group
or other group being replaced by O, S(O).sub.q, N or P(O).sub.r,
what is intended is that:
##STR00093##
is replaced by
##STR00094##
(wherein E cannot be H); --CH.dbd. is replaced by --N.dbd. or
--P(O).sub.r.dbd.; .ident.C--H is replaced by .ident.N or
.ident.P(O).sub.r; or --CH.sub.2-- is replaced by --O--,
--S(O).sub.q--, --NR.sup.N-- or --P(O).sub.rR.sup.N--, where
R.sup.N is H or optionally substituted C.sub.1-6alkyl,
C.sub.1-6heteroalkyl, C.sub.3-6cycloalkyl,
C.sub.3-6heterocycloalkyl, C.sub.2-6alkenyl,
C.sub.2-6heteroalkenyl, C.sub.3-6cycloalkenyl,
C.sub.3-6heterocycloalkenyl, phenyl, or heteroaryl containing 5 or
6 ring members. R.sup.N is preferably H, C.sub.1-6alkyl or
C.sub.3-6cycloalkyl. q is independently 0, 1 or 2. In one
embodiment, q is 0. r is independently 0 or 1. In one embodiment, r
is 0.
[0571] Where reference is made to a carbon atom being replaced by
Si, what is intended is that the carbon atom is swapped for a
silicon atom but that the bonds otherwise remain the same. Thus,
e.g., --CH.sub.2-- is replaced by --SiH.sub.2--; --CH.dbd. is
replaced by --SiH.dbd.; and .ident.C--H is replaced by
.ident.Si--H.
[0572] By way of clarification, in relation to the above mentioned
heteroatom containing groups (such as heteroalkyl etc.), where a
numerical of carbon atoms is given, for instance
C.sub.3-6heteroalkyl, what is intended is a group based on
C.sub.3-6alkyl in which one or more of the 3-6 chain carbon atoms
is replaced by O, S(O).sub.q or N. Accordingly, a
C.sub.3-6heteroalkyl group would, e.g., contain less than 3-6 chain
carbon atoms. As another example, a pyridyl group would be classed
as a C.sub.6 heteroaryl group even though it contains 5 carbon
atoms.
[0573] Wherein reference is made to a specific functional group or
chemical moiety provided below, the intended chemical entity is as
follows:
ether (e.g., --O--); ester (e.g., --C(O)O--); succinate (e.g.,
--O(O)C--CH.sub.2--CH.sub.2--C(O)O--)); carbamate (e.g.,
--OC(O)--NR'--); carbonate (e.g., --OC(O)O--); ketone (e.g.,
--C--C(O)--C--); carbonyl (e.g., --C(O)--); urea (e.g.,
--NRC(O)NR'--); amine (e.g., --NR'--); amide (e.g., --C(O)NR'--);
imine (e.g., --C(NR')--); thioether (e.g., --S--); xanthate (e.g.,
--OC(S)S--); phosphodiester (e.g., --OP(O).sub.2O--); wherein R'
may be independently selected from H, --NH--, --O--, --S--, a
phosphate or an optionally substituted C.sub.1-10 alkylene.
[0574] pKa
[0575] Unless explicitly indicated otherwise, all pKas referred to
herein are measured in water at standard temperature and pressure.
Also, unless otherwise indicated, all references to pKa are
references to pKa measured using the following technique.
[0576] 2 mM solution of lipid in ethanol are prepared by weighing
the lipid and then dissolving in ethanol. 0.3 mM solution of
fluorescent probe TNS in ethanol:methanol 9:1 is prepared by first
making 3 mM solution of TNS in methanol and then diluting to 0.3 mM
with ethanol.
[0577] An aqueous buffer containing sodium phosphate, sodium
citrate sodium acetate and sodium chloride, at the concentrations
20 mM, 25 mM, 20 mM and 150 mM, respectively, is prepared. The
buffer is split into eight parts and the pH adjusted either with
12N HCl or 6N NaOH to 4.44-4.52, 5.27, 6.15-6.21, 6.57, 7.10-7.20,
7.72-7.80, 8.27-8.33 and 10.47-11.12. 400 uL of 2 mM lipid solution
and 800 uL of 0.3 mM TNS solution are mixed.
[0578] Using the Tecan Genesis RSP150 high throughput liquid
handler and Gemini Software, 7.5 uL of probe/lipid mix are added to
242.5 uL of buffer in a 1 mL 96 well plate (model NUNC 260252,
Nalgae Nunc International). This is done with all eight
buffers.
[0579] After mixing in 1 mL 96 well plate, 100 uL of each
probe/lipid/buffer mixture is transferred to a 250 uL black with
clear bottom 96 well plate (model COSTAR 3904, Corning).
[0580] The fluorescence measurements are carried out on the
SpectraMax M5 spectrophotometer using software SoftMax pro 5.2 and
following parameters:
[0581] Read Mode: Fluorescence, Top read
[0582] Wavelengths: Ex 322 nm, Em 431 nm, Auto Cutoff On 420 nm
[0583] Sensitivity: Readings 6, PMT: Auto
[0584] Automix: Before: Off
[0585] Autocalibrate: On
[0586] Assay plate type: 96 Well Standard clrbtm
[0587] Wells to read: Read entire plate
[0588] Settling time: Off
[0589] Column Way. Priority: Column priority
[0590] Carriage Speed: Normal
[0591] Auto read: Off
[0592] After the measurement, the background fluorescence value of
an empty well on the 96 well plate is subtracted from each
probe/lipid/buffer mixture. The fluorescence intensity values are
then normalized to the value at lowest pH. The normalized
fluorescence intensity vs. pH chart is then plotted in the
Microsoft Excel software. The eight points are connected with a
smooth line.
[0593] The point on the line at which the normalized fluorescence
intensity is equal to 0.5 is found. The pH corresponding to
normalized fluorescence intensity equal to 0.5 is found and is
considered the pKa of the lipid.
[0594] The pKa determined using this method is precise to about 0.2
pKa units.
[0595] Absent Groups
[0596] When group a, b or c in formula (I) is "absent", what is
meant is that a single bond is present instead, i.e. that the two
groups either side of group a, b or c are directly bonded to each
other.
[0597] Substitution
[0598] As used herein, the term "optionally substituted" as applied
to any of an aryl, heteroaryl, cycloalkyl or heterocyclyl group,
unless otherwise specified, refers to such a group that is
unsubstituted or is substituted by one or more, typically 1, 2, 3,
4 or 5 suitable non-hydrogen substituents, each of which is
independently selected from the group consisting of:
(a) alkyl; (b) hydroxy (or protected hydroxy); (c) halo; (d) oxo,
i.e., .dbd.O or alkylimino, i.e. .dbd.N-alkyl; (e) amino,
alkylamino or dialkylamino; (f) alkoxy; (g) cycloalkyl; (h)
carboxyl; (i) heterocyclooxy, wherein heterocyclooxy denotes a
heterocyclic group bonded through an oxygen bridge; (j)
alkyl-O--C(O)--; (k) mercapto; (l) nitro; (m) cyano; (n) sulfamoyl
or sulfonamido; (o) aryl; (p) alkyl-C(O)--O--; (q) aryl-C(O)--O--;
(r) aryl-S--; (s) aryloxy; (t) alkyl-S--; (u) formyl, i.e.,
HC(O)--; (v) carbamoyl; (w) aryl-alkyl-; and (x) aryl substituted
with alkyl, cycloalkyl, alkoxy, hydroxy, amino, alkyl-C(O)--NH--,
alkylamino, dialkylamino or halogen. As used herein, the term
"optionally substituted" as applied to any of an alkyl or a group
containing an alkyl, unless otherwise specified, refers to such a
group that is unsubstituted or is substituted by one or more,
typically 1, 2 or 3 suitable non-hydrogen substituents, each of
which is independently selected from the group consisting of: halo,
hydroxy (or protected hydroxy) or alkoxy groups.
[0599] Groups of the compounds of the invention (e.g. alkyl,
cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkyriyl, alkylene,
alkenylene, heteroalkyl, heterocycloalkyl, heteroalkenyl,
heterocycloalkenyl, heteroalkynyl, heteroalkylene, heteroalkenylene
aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl or
heteroarylheteroalkyl groups etc.) may be substituted or
unsubstituted, in one embodiment unsubstituted. Typically,
substitution involves the notional replacement of a hydrogen atom
with a substituent group, or two hydrogen atoms in the case of
substitution by .dbd.O.
[0600] Where substituted, there will generally be 1 to 5
substituents on each group, in one embodiment 1 to 3 substituents,
in one embodiment 1 or 2 substituents, in one embodiment 1
substituent. One embodiment includes more than one substituent on
the same atom, e.g. an acetal group.
[0601] In one embodiment, the substituent(s) is/are independently
Sub.sup.1 or Sub.sup.2 (in one embodiment Sub.sup.2) wherein:
[0602] Sub.sup.1 is independently Sub.sup.1 is independently
halogen, trihalomethyl, trihaloethyl, --NO.sub.2, --CN,
--N.sup.+(R.sup.s).sub.2O.sup.-, --CO.sub.2H, --CO.sub.2R.sup.s,
--SO.sub.3H, --SOR.sup.s, --SO.sub.2R.sup.s, --SO.sub.3R.sup.s,
--OC(.dbd.O)OR.sup.s, --C(.dbd.O)H, --C(.dbd.O)R.sup.s,
--OC(.dbd.O)R.sup.s, .dbd.O, --NR.sup.s.sub.2, --C(.dbd.O)NH.sub.2,
--C(.dbd.O)NR.sup.s.sub.2, --N(R.sup.s)C(.dbd.O)OR.sup.s,
--N(R.sup.s)C(.dbd.O)NR.sup.s.sub.2, --OC(.dbd.O)NR.sup.s.sub.2,
--N(R.sup.s)C(.dbd.O)R.sup.s, --C(.dbd.S)NR.sup.s.sub.2,
--NR.sup.sC(.dbd.S)R.sup.s, --SO.sub.2NR.sup.s.sub.2,
--NR.sup.sSO.sub.2R.sup.s, --N(R.sup.s)C(.dbd.S)NR.sup.s.sub.2,
--N(R.sup.s)SO.sub.2NR.sup.s.sub.2, --R.sup.s or --Z.sup.sR.sup.s,
wherein; [0603] Z.sup.s is independently O, S or NR.sup.s; [0604]
R.sup.s is independently H or C.sub.1-6alkyl, C.sub.1-6heteroalkyl,
-(Alk.sup.a).sub.f-C.sub.3-6cycloalkyl,
-(Alk.sup.a).sub.f-C.sub.3-6heterocycloalkyl, C.sub.2-6alkenyl,
C.sub.2-6heteroalkenyl, -(Alk.sup.a).sub.f-C.sub.3-6cycloalkenyl,
-(Alk.sup.a).sub.f-C.sub.3-6heterocycloalkenyl, C.sub.2-6alkynyl,
C.sub.2-6heteroalkynyl, -(Alk.sup.a).sub.f-C.sub.6-14aryl,
-(Alk.sup.a).sub.f-C.sub.6-14aryl or -(Alk.sup.a).sub.f-heteroaryl
(where heteroaryl contains 5-13 ring members), where [0605] f is 0
or 1; [0606] Alk.sup.a is C.sub.1-6alkylene or
C.sub.1-6heteroalkylene; and [0607] R.sup.s is optionally
substituted itself (in one embodiment unsubstituted) by 1 to 3
substituents Sub.sup.2;
[0608] Sub.sup.2 is independently halogen, trihalomethyl,
trihaloethyl, --NO.sub.2, --CN,
--N.sup.+(C.sub.1-6alkyl).sub.2O.sup.-, --CO.sub.2H,
--CO.sub.2C.sub.1-6alkyl, --SO.sub.3H, --SOC.sub.1-6alkyl,
--SO.sub.2C.sub.1-6alkyl, --SO.sub.3C.sub.1-6alkyl,
--OC(.dbd.O)OC.sub.1-6alkyl, --C(.dbd.O)H,
--C(.dbd.O)C.sub.1-6alkyl, --OC(.dbd.O)C.sub.1-6alkyl, .dbd.O,
--N(C.sub.1-6alkyl).sub.2, --C(.dbd.O)NH.sub.2,
--C(.dbd.O)N(C.sub.1-6alkyl).sub.2,
--N(C.sub.1-6alkyl)C(.dbd.O)C(C.sub.1-6alkyl),
--N(C.sub.1-6alkyl)C(.dbd.O)N(C.sub.1-6alkyl).sub.2,
--OC(.dbd.O)N(C.sub.1-6alkyl).sub.2,
--N(C.sub.1-6alkyl)C(.dbd.O)C.sub.1-6alkyl,
--C(.dbd.S)N(C.sub.1-6alkyl).sub.2,
--N(C.sub.1-6alkyl)C(.dbd.S)C.sub.1-6alkyl,
--SO.sub.2N(C.sub.1-6alkyl).sub.2,
--N(C.sub.1-6alkyl)SO.sub.2C.sub.1-6alkyl,
--N(C.sub.1-6alkyl)C(.dbd.S)N(C.sub.1-6alkyl).sub.2,
--N(C.sub.1-6alkyl)SO.sub.2N(C.sub.1-6alkyl).sub.2,
--C.sub.1-6heteroalkyl, --C.sub.3-6cycloalkyl,
--C.sub.3-6heterocycloalkyl, --C.sub.2-6alkenyl,
--C.sub.2-6heteroalkenyl, --C.sub.3-6cycloalkenyl,
--C.sub.3-6heterocycloalkenyl, --C.sub.2-6alkynyl,
--C.sub.2-6heteroalkynyl, --C.sub.6-14aryl, --C.sub.5-13heteroaryl,
--Z.sup.t--C.sub.1-6alkyl, --Z.sup.t--C.sub.3-6cycloalkyl,
--Z.sup.t--C.sub.2-6alkenyl, --Z.sup.t-C.sub.3-6cycloalkenyl, or
--Z.sup.t--C.sub.2-6alkynyl; and [0609] Z.sup.t is independently O,
S, NH or N(C.sub.1-6alkyl).
[0610] While R.sup.s in Sub.sup.1 can be optionally substituted by
1 to 3 substituents Sub.sup.2, Sub.sup.2 is unsubstituted. However,
in one embodiment, R.sup.s is unsubstituted.
[0611] In one embodiment, R.sup.s is H or C.sub.1-6alkyl,
optionally substituted by 1 to 3 substituents Sub.sup.2.
[0612] In one embodiment, Sub.sup.2 is independently halogen,
trihalomethyl, trihaloethyl, --NO.sub.2, --CN,
--N.sup.+(C.sub.1-6alkyl).sub.2O.sup.-, --CO.sub.2H, --SO.sub.3H,
--SOC.sub.1-6alkyl, --SO.sub.2C.sub.1-6alkyl, --C(.dbd.O)H,
--C(.dbd.O)C.sub.1-6alkyl, .dbd.O, --N(C.sub.1-6alkyl).sub.2,
--C(.dbd.O)NH.sub.2, --C.sub.1-6alkyl, --C.sub.3-6cycloalkyl,
--C.sub.3-6heterocycloalkyl, --Z.sup.t--C.sub.1-6alkyl or
--Z.sup.t--C.sub.3-6cycloalkyl.
[0613] In one embodiment, where the substituted group is acyclic
(e.g. alkyl, heteroalkyl, alkenyl etc.), Sub.sup.1 is not --R.sup.s
and Sub.sup.2 is not --C.sub.1-6alkyl, --C.sub.1-6heteroalkyl,
--C.sub.2-6alkenyl, --C.sub.2-6heteroalkenyl, --C.sub.2-6alkynyl or
--C.sub.2-6heteroalkynyl.
[0614] Where a group other than Sub.sup.2 has at least 2 positions
which may be substituted, the group may be substituted by both ends
of an alkylene, alkenylene, alkynylene, heteroalkylene,
heteroalkenylene or heteroalkynylene chain (in one embodiment
containing 1 to 6 atoms, in one embodiment 3 to 6 atoms, and in one
embodiment 3 or 4 atoms) to form a cyclic moiety. That chain is
optionally substituted by 1 to 3 substituents Sub.sup.2. In one
embodiment that chain is not substituted. Thus, the terms
optionally substituted "cycloalkyl", "cycloalkenyl",
"cycloalkynyl", "heterocycloalkyl", "heterocycloalkenyl",
"heterocycloalkynyl", "aryl" and "heteroaryl" include fused
species. E.g. "optionally substituted cycloalkyl" includes a
species in which two cycloalkyl rings are fused, and "optionally
substituted heteroaryl" includes a species in which a
heterocycloalkyl ring is fused to the aromatic ring (e.g.
5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl).
[0615] Where a group other than Sub.sup.2 has an atom which may be
substituted twice, that atom may be substituted by both ends of an
alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene
or heteroalkynylene chain (in one embodiment containing 2 to 8
atoms, in one embodiment 3 to 6 atoms, and in one embodiment 4 or 5
atoms) to form a cyclic moiety. That chain is optionally
substituted by 1 to 3 substituents Sub.sup.2. In one embodiment
that chain is not substituted. Thus, the terms optionally
substituted "cycloalkyl", "cycloalkenyl", "cycloalkynyl",
"heterocycloalkyl", "heterocycloalkenyl", "heterocycloalkynyl",
"aryl" and "heteroaryl" include spiro species.
[0616] By way of clarification, when a group has a heteroatom, a
substituent may be bonded to the heteroatom. Thus, e.g.,
"optionally substituted heteroalkyl" includes
--CH.sub.2--N(Sub.sup.1)-CH.sub.2--, --CH(Sub.sup.1)-NH--CH.sub.2--
and --CH(Sub.sup.1)-N(Sub.sup.1)-CH.sub.2-- etc.
[0617] Modifier Terms
[0618] When a list is preceded by a modifier, it is intended that
the modifier is to be understood as applying to each of the items
in the list. For example, the phrase "optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group" means
that each of the four items in the list, namely the
C.sub.3-20-heterocycloalkyl group, the
C.sub.3-20-heterocycloalkenyl group, the
C.sub.3-20-heterocycloalkynyl group and the C.sub.6-20-heteroaryl
group, may be optionally substituted.
[0619] When a group is characterised by a first modifier and then,
later on, the same group is characterised by a subsequent modifier,
what is meant is that the group is characterised by both modifiers
simultaneously. For example, if a group is described as a
"C.sub.3-20-heterocycloalkynyl" (the first modifier) group and then
later the same group is described as a "C.sub.5-16" (the subsequent
modifier) group, what is meant is a C.sub.5-16 heterocycloalkynyl
group.
[0620] Steroids
[0621] As used herein, the term "steroid" refers to any group
comprising the following structure (which structure is referred to
herein as the "steroid skeleton").
##STR00095##
[0622] Purely for the purposes of illustration, the steroid
skeleton has been drawn, above as fully saturated. The term
steroid, however, is also intended to cover instances where there
is unsaturation in the steroid skeleton. For example, the term
steroid covers a group which comprises the fully unsaturated
(mancude) basic skeleton, 15H-cyclopenta[a]phenanthrene:
##STR00096##
[0623] The term steroid also covers a group which comprises a
partially unsaturated steroid skeleton.
[0624] The term steroid also covers "seco" derivatives of the
steroid skeleton, i.e. groups in which ring cleavage has been
effected; "nor" and "homo" derivatives of the steroid skeleton
which involve ring contraction and expansion, respectively (see
Systemic Nomenclature of Organic Chemistry, by D. Hellwinkel,
published by Springer, 2001, ISBN: 3-540-41138-0, page 203 for
"seco" and page 204 for "nor" and "homo"). In one embodiment,
however, such "seco" derivatives are not encompassed by the term
"steroid". In another embodiment, such "nor" derivatives are not
encompassed by the term "steroid". In another embodiment, such
"homo" derivatives are not encompassed by the term "steroid". Thus
in one embodiment, such seco, nor and homo derivatives are not
encompassed by the term "steroid".
[0625] The term steroid also covers instances where one or more of
the carbon atoms in the structure labelled steroid skeleton is
replaced by a heteroatom. In one such embodiment, up to six carbon
atoms, in one embodiment up to five carbon atoms, in another
embodiment up to four carbon atoms, in another embodiment up to
three carbon atoms, in another embodiment up to two carbon atoms,
in another embodiment one carbon atom, are each replaced
independently by O, S(O).sub.q, N, P(O).sub.r or Si (and preferably
O, S(O).sub.q or N). In one embodiment, however, the term "steroid"
comprises species in which the "steroid basic skeleton" contains no
heteroatoms.
[0626] A steroid ring system is numbered according to the
convention set out below.
##STR00097##
[0627] The term steroid encompasses sterols, steroid hormones, bile
acids and salts of bile acids. A sterol is any steroid with a
hydroxyl group at the 3-position of the A-ring.
[0628] Unsaturation
[0629] In accordance with standard use, the omega-3 position refers
to the third bond from the (methyl) terminal of the chain; the
omega-6 position refers to the sixth bond from the (methyl)
terminal of the chain and the omega-9 position refers to the ninth
bond from the (methyl) terminal of the chain.
[0630] PDI
[0631] The acronym PDI stands for polydispersity index. Unless
indicated otherwise, all PDIs referred to herein are the PDI of the
fully formed nanoparticle, as measured by dynamic light scattering
on a Malvern Zetasizer. The nanoparticle sample is diluted in
phosphate buffered saline (PBS) so that the count rate is
approximately 200-400 kcts.
[0632] General Definitions
[0633] The term "comprising" encompasses "including" as well as
"consisting" e.g. a composition "comprising" X may consist
exclusively of X or may include something additional e.g. X+Y.
[0634] The word "substantially" does not exclude "completely" e.g.
a composition which is "substantially free" from Y may be
completely free from Y. Where necessary, the word "substantially"
may be omitted from the definition of the invention.
[0635] The term "about" in relation to a numerical value x means,
e.g., x.+-.10%. In one embodiment, in relation to all numerical
values disclosed, the term "about" is absent.
[0636] For the avoidance of doubt any feature that is explicitly
disclosed in the context of a compound, composition, method or use
is also hereby implicitly disclosed in the context of compounds,
compositions, methods and uses. For example, if it is herein
explicitly disclosed that an inventive compound has feature "A",
then there is herein implicitly disclosed a method of treatment
according to the invention which involves the compound of the
invention with feature "A".
[0637] Moreover, various embodiments of the invention have been
described herein. It will be recognised that features specified in
each embodiment may be combined with other specified features to
provide further embodiments.
[0638] Chemical Synthesis of Cationic Lipids of the Invention
[0639] Route A sets out a general method which can be used to
synthesise compounds of the invention. Route CDT illustrates how
the cholesterol diglycol tosylate reagent might be prepared.
##STR00098##
##STR00099##
Epoxide
Example 1
[0640] Linoleyl alcohol (48.7 g, 183 mmol) is added to a round
bottom flask and dissolve in THF (400 mL). The resulting solution
is cooled using an ice bath and sodium hydride (13.16 g, 329 mmol)
is added. The resulting slurry is stirred for 1 h at rt.
Epibromohydrin is added in one portion and the reaction is
continued at rt. After 4 h stirring, additional sodium hydride
(13.16 g, 329 mmol) is added. After an additional h stirring at rt,
another aliquot of epibromohydrin (32.5 g, 238 mmol) is added. The
reaction is then heated to 50.degree. C. overnight. The reaction is
then cooled to rt and quenched with water. EtOAc is added and the
resulting organic layer collected, washed with brine and dried over
sodium sulfate. The volatiles are removed by rotary evaporation and
the resulting residue purified by chromatography on silica in
EtOAc/heptanes to yield the desired epoxide.
Cholesterol Tosylate
Example 2
[0641] Cholesterol (50 g, 129 mmol) is dissolved in DCM (55 mL) and
pyridine (150 mL). The resulting solution is cooled to 0.degree. C.
and tosyl chloride is added in one portion as a solid. The reaction
is allowed to slowly warm to rt overnight. The reaction is
concentrated by rotary evaporation and MeOH (500 mL) is added to
produce a white solid. Stirring is continued for 30 min and the
precipitate collected by filtration, washed with MeOH and dried
under vacuum to yield the desired tosylate.
Cholesterol Diglycol
Example 3
[0642] Cholesterol tosylate (73 g, 128 mmol) is dissolved in
1,4-dioxane (750 mL). Diethylene glycol (294 mL, 3077 mmol) is
added and the reaction is heated to a gentle reflux overnight. The
resulting solution is cooled to rt and concentrated by rotary
evaporation. The resulting gel is taken up in DCM and stirred with
water. The resulting organic layer is collected and the aqueous
layer extracted once with DCM. The organic layers are combined,
dried over sodium sulfate, and concentrated by rotary evaporation.
The crude product is purified on silica in EtOAc/heptane to yield
the desired cholesterol diglycol.
Cholesterol Diglycol Tosylate
Example 4
[0643] Cholesterol diglycol (51.5 g, 103 mmol) is stirred in DCM
(160 mL) and pyridine (60 mL). Tosylanhydride (38.7 g, 119 mmol) is
added and the resulting solution is stirred overnight at rt. The
reaction is concentrated by rotary evaporation and the resulting
residue purified on silica in EtOAc/heptane to yield the desired
product.
3-piperidinyl-1,2-propanediol 1-linoleylether
Example 5
[0644] The epoxide from Example 1 (1.0 g, 3.1 mmol) is dissolved in
EtOH (17 mL). Piperidine (0.45 mL, 4.65 mmol) is added and the
mixture is heated in a microwave reactor to 140.degree. C. for 5
min. After cooling to rt, the mixture is concentrated by rotary
evaporation and purified on silica in MeOH/DCM to yield the desired
amino alcohol.
Final Compound 1
Example 6
[0645] The amino alcohol from Example 5 (0.447 mg, 1.09 mmol) is
stirred in toluene (15 mL) and NaH (0.102 g, 4.24 mmol) is added in
one portion. The resulting mixture is stirred at rt for 30 min and
the tosylate from Example 4 is added in one portion. The reaction
is heated to reflux overnight. After cooling to room temperature
("rt"), the reaction is quenched by the addition of saturated
aqueous sodium bicarbonate. The resulting mixture is stirred for 5
min and then concentrated by rotary evaporation. The resulting
residue is purified directly on silica in MeOH/DCM to yield a crude
product that is repurified on silica in EtOAc/heptane to yield the
desired compound.
[0646] The following compounds (the structures of which are set out
below) can be manufactured by the Route A methodology.
TABLE-US-00004 E0011; E0002; E0003; E0013; E0015; E0006; E0008;
E0001; E0022; E0026; E0030; E0037; E0038; E0039; E0042; E0050;
E0055; E0061 E0062; E0063; E0064; E0065; E0066; E0068; E0069;
E0070; E0071; E0072; E0073; E0074; E0075; E0076; E0077; E0078;
E0079; E0080; E0081; E0082; E0083; E0084; E0085; E0086; E0087;
E0088; E0089; E0090; E0091; E0092; E0093; E0094; E0095; E0096;
E0107; E0109; E0023; E0024; E0025; E0031; E0033; E0034; E0043;
E0046; E0059; E0067; E0014; E0119; E0016; E0004; E0005; E0017;
E0018; E0019; E0120; E0007; E0020; E0010; E0021; E0027; E0028;
E0029; E0032; E0035; E0009; E0040; E0041; E0044; E0048; E0049;
E0052; E0053; E0057; E0119; E0120; E0121; E0124; E0126; E0127;
E0147; E0149; E0158; E0051: E0067: E0112; E0113; E0114; E0118;
E0159; E0170; and E0171.
[0647] Route B also represents a general method which can be used
to synthesise compounds of the invention.
##STR00100##
Example 7
[0648] Cholesterol (3.33 g, 8.62 mmol), diglycolic anhydride (1 g,
8.62 mmol), DMAP (0.263 g, 2.154 mmol), CH.sub.2Cl.sub.2 (35 ml),
and a stirbar is added to a round bottomed flask. The reaction is
stirred for three days at room temperature.
[0649] The reaction mixture is concentrated, and the residue is
purified by flash chromatography on an IntelliFlash 280 (AnaLogix)
using a SF40-80G column: 0-2 CV, 100% CH.sub.2Cl.sub.2; 2-10 CV,
linear gradient of 100:0 CH.sub.2Cl.sub.2:(MeOH 10% AcOH) to 90:10
CH.sub.2Cl.sub.2:(MeOH 10% AcOH); 10-25 CV, linear gradient of
90:10 CH.sub.2Cl.sub.2:(MeOH 10% AcOH) to 85:15
CH.sub.2Cl.sub.2:(MeOH 10% AcOH). The product co-elutes with
cholesterol. The product-containing fractions are combined and
concentrated in vacuo to a white slurry, then diluted with heptanes
(cholesterol is soluble in heptanes) and chilled in an ice bath.
The white solid is filtered off, washed with heptanes, and placed
under vacuum, yielding 1.50 g (35%) of pure product.
Example 8
[0650] The amino alcohol from example 5 (105.3 mg, 0.258 mmol),
DMAP (22 mg, 0.180 mmol), and the carboxylic acid from example 7
(135.6 mg, 0.270 mmol) are weighed into a small vial.
Dichloromethane (2.5 mL) is added, followed by EDC.HCl (67.1 mg,
0.350 mmol). The reaction mixture is stirred at rt overnight.
[0651] The crude reaction mixture is purified via flash
chromatography on an IntelliFlash 280 (AnaLogix) using a SF15-24G
column: 0-3 CV, 100% CH.sub.2Cl.sub.2; 3-25 CV, linear gradient of
100:0 CH.sub.2Cl.sub.2:MeOH to 95:5 CH.sub.2Cl.sub.2:MeOH. The
product is still impure. The residue is purified again via flash
chromatography on an IntelliFlash 280 (AnaLogix) using a SF15-24G
column. 0-3 CV, 100% heptanes; 3-30 CV, linear gradient of 100:0
heptanes:ethyl acetate to 65:35 heptanes:ethyl acetate. The
product-containing fractions are identified by TLC, combined, and
concentrated in vacuo to yield 38 mg (13%) of pure product as a
clear liquid.
[0652] The following compounds (the structures of which are set out
below) can be manufactured by the Route B methodology: E0036 and
E0047.
[0653] Route C also represents a general method which can be used
to synthesise compounds of the invention.
##STR00101##
Example 9
[0654] 2-(2-Chloroethoxyethanol) (5.01 g, 40.2 mmol) is weighed
into a round bottomed flask and dissolve in DMF (100 ml). The
solution is stirred, NaN.sub.3 (2.89 g, 44.5 mmol) is added, and
the temperature is increased to 80.degree. C. The solution became
cloudy. The reaction is stirred overnight.
[0655] The next morning, the reaction is removed from the heat and
diluted with 300 mL water. This solution is washed with ethyl
acetate to extract the product (4.times.100 mL). The organic phase
is dried with sodium sulfate and concentrated to a liquid. By
.sup.1H NMR, this liquid contained 44% by weight of DMF. The
product--5.19 g (98%)--is taken to the next step without further
purification.
Example 10
[0656] The azidoalcohol from example 9 (5.25 g, 22.42 mmol) and
pyridine (3 mL, 37.1 mmol) are dissolved in CH.sub.2Cl.sub.2 (45
ml) in a round bottomed flask and stirred in an ice bath. Once
cold, tosyl chloride (4.70 g, 24.66 mmol) is added in one portion.
The reaction is stored in a refrigerator overnight.
[0657] The next day, the reaction is diluted with CH.sub.2Cl.sub.2
(50 mL) and extracted with 1M HCl (2.times.40 mL) to remove the
pyridine. The organic phase is washed once with brine (40 mL) and
concentrated. The residue is purified via flash chromatography on
InternFlash 280 (AnaLogix) using a SF40-115G column: 0-20 CV,
linear gradient of 100:0 heptanes:ethyl acetate to 50:50
heptanes:ethyl acetate. The product containing fractions are
identified by TLC, combined, and concentrated in vacuo to yield the
product with an estimated 90% purity: 0.61 g (10%).
Example 11
[0658] Performed as in example 6 but using the amino alcohol from
example 5 and the tosylate from example 10.
Example 12
[0659] The alkylazide from example 11 (334 mg, 0.641 mmol) is
dissolved in THF (4 mL) and water (0.400 mL) in a small vial. To
this solution is added a solution of trimethylphosphine in THF (2.5
mL, 2.500 mmol trimethylphosphine) and the reaction is stirred
overnight. The reaction appears complete by TLC the next morning.
The solvent is evaporated and the residue is dissolved in 10 mL
MeOH. The solution is loaded onto a SCX (10 g) column
preequilibrated with MeOH, washed with 50 mL MeOH, then elutes with
4.times.10 mL 7M NH.sub.3 in MeOH. The product elutes in the
second, third and fourth fractions; these fractions are pooled and
concentrated. Trimethyphosphine is still present by TLC (and by
smell), so the residue is taken up in 50 mL EtOAc and washed with
3.times.15 mL water made slightly basic with NaHCO.sub.3. The
organic phase is dried with Na.sub.2SO.sub.4 and concentrated to a
colorless liquid: 232 mg (73%).
Example 13
[0660] Dissolve the amine from example 12 (232 mg, 0.469 mmol) in
CH.sub.2Cl.sub.2 (5 mL) in a round bottomed flask. To this solution
is added cholesterol chloroformate (316 mg, 0.703 mmol) and DMAP
(86 mg, 0.703 mmol). The solution is stirred at rt overnight.
[0661] The crude reaction mixture is purified directly via flash
chromatography on an IntelliFlash 280 (AnaLogix) using a SF15-24G
column: 0-5 CV, 100% CH.sub.2Cl.sub.2; 5-15 CV, linear gradient of
100:0 CH.sub.2Cl.sub.2:MeOH to 95:5 CH.sub.2Cl.sub.2:MeOH; 15-30,
95:5 CH.sub.2Cl.sub.2:MeOH. The product containing fractions are
identified by TLC, combined, and concentrated to a pale yellow oil:
339 mg (80%).
[0662] The following compounds (the structures of which are set out
below) can be manufactured by the Route C methodology:
TABLE-US-00005 E0054; E0097; E0099; E0103; E0148; E0169; E0175; and
E0176.
[0663] Route D also represents a general method which can be used
to synthesize compounds of the invention
##STR00102##
Example 14
[0664] To a solution of diethylene glycol vinyl ether (3.96 g, 30
mmol) in CH.sub.2Cl.sub.2 at 0.degree. C., pyridine (4.85 mL) is
added, followed by tosyl chloride (6.86 g, 36 mmol). After 10 min
at 0.degree. C., the reaction is warmed to rt and stirred
overnight.
[0665] The reaction is extracted between a saturated aqueous
solution of NaHCO.sub.3 and ethyl acetate. The organic extracts are
combined, dried and concentrated. The residue is purified via flash
chromatography eluting with 30% ethyl acetate/70% heptane: 5.45 g
(63%).
Example 15
[0666] To a solution of the vinyl ether from example 14 (1.0 g,
3.49 mmol) in CH.sub.2Cl.sub.2 at rt, cholesterol (0.675 g, 1.746
mmol) is added, followed by PPTS (0.878 g, 3.49 mmol). The reaction
is stirred at rt for 5 hrs. By TLC, the product elutes very close
to cholesterol and is slightly UV-active. The reaction is extracted
between a saturated aqueous solution of NaHCO.sub.3 and ethyl
acetate. The organic extracts are combined, dried and concentrated.
The crude residue is purified via flash chromatography with 30%
ethyl acetate/70% heptane: 700 mg (60%).
Example 16
[0667] Performed as in example 6 but using the amino alcohol from
example 5 and the tosylate from example 15.
[0668] The following compounds (the structures of which are set out
below) can be manufactured by the Route D methodology: E0058.
[0669] Route E also represents a general method which can be used
to synthesise compounds of the invention.
##STR00103##
Example 17
[0670] Performed as in example 6 but using the amino alcohol from
example 5 and the tosylate from example 14.
Example 18
[0671] A 1N solution of aqueous HCl (0.38 mL, 0.38 mmol) is added
dropwise to a solution of the vinyl ether from example 17 (100 mg,
0.19 mmol) in 4 mL of 1:1 ethanol/THF at rt. After 1 h, the
reaction is extracted between a saturated aqueous solution of
NaHCO.sub.3 and ethyl acetate. The organic extracts are combined,
dried and concentrated to an oil that is used in the next step
without further purification: 85 mg (90%).
Example 19
[0672] Performed as in example 8 but using the amino alcohol from
example 18 and cholesterol hemisuccinate as the carboxylic
acid.
[0673] The following compounds (the structures of which are set out
below) can be manufactured by the Route E methodology:
TABLE-US-00006 E0060; E0104; E0129; E0130; E0143; E0150; E0151;
E0152; E0161; E0162; E0163; E0164; E0165; E0177; E0178; and
E0179.
[0674] Route F also represents a general method which can be used
to synthesise compounds of the invention.
##STR00104##
Example 20
[0675] To a solution of the amino alcohol from example 18 (600 mg,
1.21 mmol) in toluene (12 mL) at rt, 60% NaH (97 mg, 2.42 mmol) is
added (reaction became yellow). After 10 min, cholesterol
chloroformate (815 mg, 1.815 mmol) is added. The reaction is then
heated to 80.degree. C. (reaction became orange) and stirred
overnight.
[0676] The reaction mixture is cooled and extracted between brine
and ethyl acetate. The organic extracts are combined, dried with
Na.sub.2SO.sub.4, and concentrated to an oil. The crude oil is
purified via flash chromatography with 5% MeOH/95%
CH.sub.2Cl.sub.2: 720 mg (66%).
[0677] The following compounds (the structures of which are set out
below) can be manufactured by the Route F methodology.
TABLE-US-00007 E0056; E0122; E0123; E0138; and E0139.
[0678] Route G also represents a general method which can be used
to synthesise compounds of the invention.
##STR00105##
Example 21
[0679] A solution of the amino alcohol from example 5 (12.4 mmol)
in THF (10 mL) and a solution of DIAD (3 mL) in THF (10 mL) are
added simultaneously to a solution of PPh.sub.3 (2.5 g) and
phthalimide (2.5 g) in THF (50 mL). The resulting mixture is
stirred overnight at rt. The reaction is concentrated to dryness
and used directly in the next step.
Example 22
[0680] The material from example 21 is stirred in ethanol (25 mL)
and a solution of methylamine in THF is added. The reaction is
stirred at rt for 16 h and then concentrated to dryness. The crude
material is purified by chromatography on silica.
Example 23
[0681] The amine from example 22 (0.22 mmol) is stirred in DMF (5
mL) along with the cholesterol hemisuccinate (0.25 mmol), HATU
(0.26 mmol). N-methylmorpholine (0.55 mmol) is added and the
reaction is stirred overnight at rt. The resulting solution is
concentrated under vacuum and diluted with EtOAc. The resulting
organic layer is washed with water then brine and concentrated to a
yellow liquid. The resulting residue is purified on neutral alumina
to yield a pale yellow liquid.
[0682] The following compounds can be manufactured by the Route G
methodology:
TABLE-US-00008 E0098; E0100; E0101; E0105; E0106; and E0108.
[0683] Route H also represents a general method which can be used
to synthesise compounds of the invention.
##STR00106##
Example 24
[0684] To a solution of the amine from example 22 (3.55 mmol) in
DCM (10 mL) is added DIEA (5.7 mmol) and cholesterol chloroformate
(6.0 mmol). The resulting reaction is stirred at rt overnight. The
reaction is diluted with EtOAc (50 mL) and washed with water then
brine. The resulting organic layer is concentrated to a residue and
purified by chromatography on silica to yield a pale yellow
liquid.
[0685] E0102 can be prepared using the Route H methodology.
[0686] Route I also represents a general method which can be used
to synthesise compounds of the invention.
##STR00107##
Example 25
[0687] To a solution of amine from example 22 (0.33 mmol) in DCM
(25 mL) is added to BOC glycine (0.36 mmol), HATU (0.36 mmol) and
DIEA (0.66 mmol). The resulting mixture is stirred at rt overnight.
The resulting reaction mixture is washed with saturated aqueous
NH.sub.4Cl, dried over sodium sulfate and concentrated to a crude
solid that is used without further purification.
Example 26
[0688] The material from the previous step is stirred in DCM (15
mL) and TFA (5 mL) is added. The resulting solution is stirred
overnight at rt. The resulting mixture is concentrated to dryness
and 1.5N HCl (20 mL) is added. The solution is washed with EtOAc
and then reduced to pH >7 with solid NaHCO.sub.3. The resulting
mixture is extracted with DCM and the resulting organic layer dried
over sodium sulfate and concentrated to a crude solid that is used
without further purification.
Example 27
[0689] The material from the previous step is stirred in DCM (20
mL) and DIEA (0.53 mmol) is added. To this solution is added
cholesterol chloroformate (0.53 mmol) and the reaction is stirred
overnight at rt. The reaction is then diluted with EtOAc (50 mL)
and the resulting solution washed with 1.5 N HCl, 10% aq.
NaHCO.sub.3, and brine. The resulting organic layer is dried over
sodium sulfate and concentrated to solid.
[0690] E0110 and E0111 can be prepared using the Route I
methodology.
[0691] Examples 28 to 32 also represent routes which can be used to
synthesise compounds of the invention.
##STR00108##
Example 28
[0692] BOC protected cationic lipid prepared using Route A
methodology (150 mg) is stirred with triethylsilane (0.5 mL) and
TFA (10 mL) is added in one portion. After 2 h, the reaction is
concentrated to dryness. The resulting residue is purified on
silica on 0 to 15% MeOH in DCM and concentrated to a glassy
oil.
[0693] E0012 can be made using this process.
##STR00109##
Example 29
[0694] TBDMS protected cationic lipid prepared using Route A
methodology (135 mg, 0.138 mmol) is stirred in THF (2 mL) and a
solution of TBAF (0.28 mL, 1.0 M in THF, 0.28 mmol) is added. The
reaction is stirred at rt overnight. The reaction is purified
directly on silica in 0 to 20% MeOH in DCM to yield a clear
oil.
[0695] E0045 can be made using this process.
[0696] Examples 30 to 31 also represent routes which can be used to
synthesise compounds of the invention.
##STR00110##
Example 30
[0697] To a stirred suspension of paraformaldehyde (536 mg) in
TMSCl (9.4 mL) is added linoleyl alcohol (5.0 g) dropwise over 15
min. Once the reaction turns clear it is concentrated under reduced
pressure and used immediately in the next step.
Example 31
[0698] To a stirred solution of glycidol (1.5 mL) in THF (50 mL) is
added the compound from Example 30 (5.8 g), DIEA (9.5 mL),
tetrabutylammonium iodide (6.8 g). The reaction is stirred at rt
for 5 h. The solids are removed by filtration and washed with
diethyl ether. The filtrate is collected and washed with water and
brine. The resulting organic layer is dried over sodium sulfate and
concentrated under reduced pressure to a crude oil. The crude
material is purified by chromatography on silica that had been
pretreated with 3% triethylamine containing mobile phase. The
material is elutes using EtOAc in heptanes to yield 1.65 g pure
product as a clear liquid.
[0699] The starting material for making E0115, E0116, E0117 and
E0160 can be made using steps from Example 30 and this Example
31.
##STR00111##
Example 32
[0700] Ethylene glycol (0.30 g) is stirred in THF (20 mL) and 60 wt
% sodium hydride is added (0.19 g). The resulting mixture is
stirred for 20 min. Linoleyl mesylate (1.64 g) is added and the
reaction is heated to 50.degree. C. for 2 h and then to reflux
overnight. The reaction is cooled to rt and stirred for an
additional 24 h. To the reaction is added saturated aqueous
ammonium chloride and the resulting mixture is extracted with DCM.
The resulting organic layer is dried over sodium sulfate and
concentrated to a crude oil. The crude material is purified on
silica using EtOAc in heptanes to yield 640 mg of the desired
product.
[0701] The methods of examples 30 to 32 may be used in the
synthesis of lipids (e.g. E0055) having a spacer between the
linoleyl chains and the core of the molecule.
[0702] Route J also represents a general method which can be used
to synthesise compounds of the invention.
##STR00112##
Example 33
[0703] To a solution of cholesterol (10 g, 26 mmol) and borane-THF
(5 drops) in DCM (30 mL) is added ethyldiazoacetate (3.5 mL, 52
mmol). After stirring for 5 min the reaction mixture is
concentrated and purified by chromatography on silica in
hexane/EtOAc to yield 9 g of the desired product.
Example 34
[0704] To a solution of the compound from Example 33 (9 g, 19 mmol)
in EtOH (110 mL) is added NaOH (3 g, 76 mmol). The resulting
mixture is heated to 70.degree. C. for 3 h. The reaction is cooled
to rt and concentrated. The residue is taken up in water and
acidified with 2 N HCl and extracted with DCM (2.times.). The
resulting organic layers are combined and concentrated to yield 8 g
of the desired product.
Example 35
[0705] EDC (1.8 g, 9.8 mmol) and DMAP (80 mg, 0.66 mmol) is added
to a solution of the compound from Example 34 (2.2 g, 4.9 mmol) in
DCM (5 mL). The reaction is stirred for 10 min and then the alcohol
(1.5 g) is added and the reaction is allowed to stir overnight at
rt. The reaction is diluted with DCM and washed with water. The
resulting organic layer is concentrated to a residue and purified
on silica in chloroform/MeOH to yield the desired product as an
oil.
[0706] The following compounds can be manufactured by the Route J
methodology:
TABLE-US-00009 E0125; E0128; E0131; E0140; and E0144.
[0707] Route K also represents a general method which can be used
to synthesise compounds of the invention.
##STR00113##
Example 36
[0708] To a solution of N-Boc-betaalanine (1.25 g, 6.61 mmol) in
DCM (6 mL) is added EDC (2.5 g, 13 mmol) HOBt (0.3 g, 2 mmol) and
TEA (2 mL, 13 mmol). The resulting mixture is stirred for 30
minutes. A solution of the amino alcohol (2 g) in DCM (4 mL) is
added and the reaction stirred for 10 h. The reaction is diluted
with DCM and washed with saturated sodium bicarbonate and brine.
The resulting organic layer is dried over sodium sulfate and
concentrated to an oil that is purified on silica in MeOH/DCM. The
product is concentrated to 2.65 g of an oil.
Example 37
[0709] The compound from Example 36 (2.6 g) is stirred in DCM (10
mL) and TFA (10 mL) is added. After 3 h, the reaction is
concentrated to dryness and used directly in the next step.
Example 38
[0710] To a solution of the compound from Example 37 (2 g) in DCM
(20 mL) is added DIEA (2.7 mL, 15.7 mmol) and DMAP (80 mg, 0.6
mmol) followed by cholesterol chloroformate (2.1 g, 4.7 mmol). The
reaction is stirred for 3 h at rt. The reaction is diluted with DCM
and washed with water. The resulting organic layer is concentrated
and purified on silica in MeOH/chloroform to yield 2.1 g of the
desired product.
[0711] The following compounds can be manufactured by the Route K
methodology:
TABLE-US-00010 E0133; E0134; E0135; E0136; E0137; E0141; E0132;
E0142; E0145; E0166; and E0168.
[0712] Route L also represents a general method which can be used
to synthesise compounds of the invention.
##STR00114##
Example 39
[0713] To a solution of the alcohol (2.5 g, 5.8 mmol) in DCM (10
mL) is added p-nitrochloroformate and TEA. The reaction is stirred
at rt overnight. The reaction is diluted with DCM and washed with
water. The resulting organic layer is dried over sodium sulfate and
concentrated to an oil and purified on silica in EtOAc/hexane to
yield the desired product.
Example 40
[0714] The alcohol (1.3 g, 2.9 mmol) is stirred in MePh (10 mL).
NaH is added (0.46 g, 11.4 mmol) and the reaction is stirred at rt
for 30 min. The compound from Example 39 (1.7 g, 1.2 mmol) is added
and the reaction is stirred for 16 h at rt. The reaction is cooled
in an ice bath and quenched with water. The resulting mixture is
extracted with EtOAc. The organic layers are combined, washed with
brine, dried over sodium sulfate and concentrated to a crude oil.
The material is purified on silica in EtOAc/hexane to yield 1 g of
the desired product.
[0715] E0146 can be made using Route L technology.
Examples 41, 42 and 43
[0716] Examples 41, 42 and 43 are reserved and are purposefully
left blank.
[0717] Route X also represents a general method that can be used to
synthesise compounds of the invention.
##STR00115##
Example 44
[0718] To a solution of 2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethanol
(800 mg, 5.47 mmol), triethylamine (0.915 mL, 6.57 mmol) and
N,N-dimethylaminopyridine (134 mg, 1.10 mmol) in dichloromethane
(25 mL) at rt is added to tosyl chloride (1.10 g, 5.75 mmol). The
reaction is stirred at rt for 3 h. The reaction is diluted with
saturated aqueous NaHCO.sub.3, and extracted with ethyl acetate.
The organic phase is dried, concentrated and the crude product is
purified by flash chromatography on silica with an ethyl
acetate/heptane gradient: 1.24 g, 75%.
Example 45
[0719] The tosylate from example 44 (1.03 g, 3.43 mmol) and
morpholine (2.97 mL, 34.3 mmol) are combined in a flask. The flask
is sealed and heated at 60.degree. C. overnight. The reaction
mixture is diluted with ethylacetate and washed with saturated
aqueous NaHCO.sub.3. The organic phase is dried, concentrated and
the crude product is purified by flash chromatography on silica
with a dichloromethane/methanol gradient: 670 mg, 91%.
Example 46
[0720] The amine from example 45 (530 mg, 2.46 mmol) is dissolved
in dioxane (15 mL) at 0.degree. C. To the cooled solution is added
concentrated HCl (aqueous, 7.39 mmol). The reaction is stirred at
rt overnight. Excess solid K.sub.2CO.sub.3 is added to neutralize
the acid. The solids are removed by filtration and washed with
acetone. The filtrate is concentrated and the crude product is
purified by flash chromatography on silica with a
dichloromethane/methanol gradient: 320 mg, 74%.
Example 47
[0721] The diol from the previous step in Example 46 (320 mg, 1.8
mmol) is stirred in DCM (12 mL) with imidazole (249 mg, 3.65 mmol).
To this solution is added TBDPSCl (0.475 mL), 1.8 mmol). The
resulting mixture is stirred at rt overnight. The reaction is
diluted with DCM and ished with saturated sodium bicarbonate. The
resulting organic layer is dried over sodium sulfate, concentrated
to an oil and purified on silica in DCM/MeOH to yield 650 mg of the
desired product.
Example 48
[0722] To the material from the previous step (640 mg, 1.5 mmol) in
toluene (15 mL) is added sodium hydride (124 mg, 60 wt % in oil,
3.1 mmol). After 20 min, the cholesterol reagent (1.17 g, 1.86
mmol) is added and the reaction heated to reflux overnight. After
cooling to rt, the reaction is quenched with brine and extracted
with ethyl acetate. The resulting organic layer is dried,
concentreated, and purified on silica in EtOAc/heptanes to yield
560 mg of the desired product.
Example 49
[0723] To the material from the previous step (660 mg, 0.76 mmol)
in THF (6 mL) is added a 1 M solution of TBAF in THF (1.5 mL0, 1.5
mmol). The reaction is stirred at rt for 2 h and then diluted with
EtOAc. The resulting organic layer is ished with brine, dried, and
concentrated to a crude product that is purified on silica in
MeOH/DCM.
Example 50
[0724] The amino alcohol from example 49 (280 mg, 0.443 mmol) is
dissolved in toluene (4 mL) at rt, and then 60% NaH is added (44
mg, 1.1 mmol). After 20 minutes, oleyl tosylate (prepared from
oleyl alcohol in a method analagous to that described in example
10) (187 mg, 0.443 mmol) is added. The reaction is refluxed for 3
h. The reaction is cooled to rt diluted with ethyl acetate, and
ished with brine. The organic phase is dried, concentrated and the
crude product is purified by flash chromatography on silica with an
ethyl acetate/heptane gradient: 183 mg, 47%.
[0725] E0180 can be manufactured by the Route X methodology
[0726] Route Y also represents a general method that can be used to
synthesise compounds of the invention.
##STR00116##
Example 51
[0727] To a solution of cholesterol (700 mg, 1.8 mmol) in DCM (20
mL) is added potassium carbonate (750 g, 5.4 mmol) and DMAP (22 mg,
0.18 mmol) bromoacetyl bromide (0.19 mL, 2.2 mmol) is added
dropwise. After stirring for 90 min in an ice bath the reaction is
filtered, concentrated and purified on silica in DCM/heptane to
yield 850 mg of the desired product.
Example 52
[0728] To a solution of the amino alcohol (1.08 g, 2.37 mmol) in
MePh (20 mL) is added NaH (190 mg, 60 wt % in oil, 4.74 mmol). The
mixture is heated at reflux for 10 min and then the bromoacetyl
cholesterol (1.20 g, 2.37 mmol) is added. The reaction is stirred
for 2 h at reflux and the cooled to rt. To the reaction is added
water and EtOAc and brine. The resulting organic layer is
collected, dried, and concentrated to a crude material that is
purified on silica in EtOAc/heptane after the column is
equilibrated with 1% acetic acid in DCM. After purification, the
fractions containing product are combined and washed with saturated
aqueous sodium bicarbonate before concentrating to 700 mg of the
desired product.
[0729] E0167 can be manufactured using route Y methodology.
Example 53
Stealth Lipid Structures and Syntheses
[0730] The structures of the stealth lipids S001 through S026 are
provided in Table 3. The following examples 54 to 67 illustrate the
synthesis of stealth lipids.
TABLE-US-00011 TABLE 3 Stealth lipid structures Stealth Lipid Lipid
S001 ##STR00117## S002 ##STR00118## S003 ##STR00119## S004
##STR00120## S005 ##STR00121## S006 ##STR00122## S007 ##STR00123##
S008 ##STR00124## S009 ##STR00125## S010 ##STR00126## S011
##STR00127## S012 ##STR00128## S013 ##STR00129## S014 ##STR00130##
S015 ##STR00131## S016 ##STR00132## S017 ##STR00133## S018
##STR00134## S019 ##STR00135## S020 ##STR00136## S021 ##STR00137##
S022 ##STR00138## S023 ##STR00139## S024 ##STR00140## S025
##STR00141## S026 ##STR00142##
Example 54
S001
##STR00143##
[0732] Cholesterol hemisuccinate (608 mg, 1.25 mmol) and
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (240
mg, 1.25 mmol, EDC.HCl) are dissolved in anhydrous dichloromethane
(4 mL), and then N,N-dimethylaminopyridine (305 mg, 2.50 mmol) and
poly(ethylene glycol) methyl ether (500 mg, 0.250 mmol,
M.sub.n.about.2,000 g/mol, Sigma-Aldrich) are added. The reaction
mixture is stirred at rt. After 72 h, the entire reaction mixture
is loaded onto a 10 g Bond Elut SCX column (from Varian;
pre-equilibrated with 50:50 dichloromethane:methanol) and elutes
with 50:50 dichloromethane:methanol. The product containing
fractions are identified by TLC, combined, and concentrated. The
crude product is further purified by flash chromatography on silica
with a dichloromethane/methanol gradient. The product containing
fractions are identified by TLC, combined and concentrated to a
white solid: 304 mg, 48.6%.
[0733] Size exclusion chromatography in tetrahydrofuran shows a
single narrow peak. The peaks and integral values observed in the
.sup.1H NMR spectrum are consistent with the expected product.
Example 55
S002
##STR00144##
[0735] Step 1
[0736] .beta.-alanine hydrochloride (1.00 g, 7.16 mmol),
cholesterol chloroformate (3.06 g, 6.81 mmol) and triethylamine
(2.0 ml, 14 mmol) are dissolved in anhydrous chloroform (25 ml).
The solution is stirred at rt overnight. The next morning, the
solvent is evaporated and the residue is dissolved in ethyl acetate
(100 mL) and washed with 1 M HCl, brine, and dried with
Na.sub.2SO.sub.4. The product is concentrated to a white solid and
used in the next step without further purification: 3.31 g,
90.0%.
[0737] Step 2
[0738] The product (methyl ester) from the previous step (298 mg,
0.578 mmol) is dissolved in tetrahydrofuran (2 mL) and 1 M NaOH
(2.0 mL, 2.0 mmol) is added, resulting in a biphasic solution. The
solution is stirred at rt, forming an emulsion. After 2 h, the
reaction mixture is diluted with 10 mL water and acidified with 1 M
HCl. The product is extracted into ethyl acetate (100 mL). The
organic phase is washed with brine, dried with Na.sub.2SO.sub.4,
and concentrated to a white solid. .sup.1H NMR indicated the
presence of a small amount of methyl ester starting material
(.about.5 mol %) but is deemed pure enough for use in the next step
without further purification: 252 mg, 83.0%.
[0739] Step 3
[0740] N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride
(96 mg, 0.50 mmol), the product (carboxylic acid) from the previous
step (252 mg, 0.477 mmol), N,N-dimethylaminopyridine (122 mg, 1.00
mmol) and poly(ethylene glycol) methyl ether (200 mg, 0.100 mmol,
M.sub.n.about.2,000 g/mol, Sigma-Aldrich) are dissolved in
anhydrous dichloromethane (5.0 mL) and stirred at rt. After 24 h
the reaction mixture is loaded onto a 10 g Bond Elut SCX column
(from Varian; pre-equilibrated with 50:50 dichloromethane:methanol)
and elutes with 50:50 dichloromethane:methanol. The product
containing fractions are identified by TLC, combined, and
concentrated. The crude product is further purified by flash
chromatography on silica with a dichloromethane/methanol gradient.
The product containing fractions are identified by TLC, combined
and concentrated to a white solid: 151 mg, 60.3%.
[0741] Size exclusion chromatography in tetrahydrofuran shows a
single narrow peak. The peaks and integral values observed in the
.sup.1H NMR spectrum are consistent with the expected product.
Example 56
S003
##STR00145##
[0743] PEG-NH.sub.2 (500 mg, 0.250 mmol, M.sub.n.about.2000 g/mol,
"Sunbright MEPA-20H", NOF Corp.), cholesterol chloroformate (449
mg, 1.00 mmol), N,N-dimethylaminopyridine (122 mg, 1.00 mmol), and
N,N-diisopropylethylamine (148 mg, 1.15 mmol) are dissolved in 5 mL
of 1:1 toluene:dichloromethane and stirred at rt. After 72 h,
N,N-dimethylethylenediamine (0.2 mL) is added to quench excess
cholesterol chloroformate. After stirring 30 min, the reaction
mixture is loaded onto a 10 g Bond Elut SCX column (from Varian;
pre-equilibrated with 50:50 dichloromethane:methanol) and elutes
with 50:50 dichloromethane:methanol. The product containing
fractions are identified by TLC, combined, and concentrated. The
crude product is further purified by flash chromatography on silica
with a dichloromethane/methanol gradient. The product containing
fractions are identified by TLC, combined and concentrated to a
white solid: 376 mg, 57.8%.
[0744] Size exclusion chromatography in tetrahydrofuran shows a
single narrow peak. The peaks and integral values observed in the
.sup.1H NMR spectrum are consistent with the expected product.
Example 57
S004
##STR00146##
[0746] Step 1
[0747] To a heatgun-dried round bottomed flask and under nitrogen
is added octadecyl aldehyde (500 mg, 1.86 mmol) and tetrahydrofuran
(10 mL). A solution of 0.5 M octadecyl magnesium chloride in
tetrahydrofuran (7.5 mL, 3.8 mmol) is added via syringe and the
reaction is warmed to 40.degree. C. When the addition is complete,
the reaction is stirred for 1 h. The reaction is removed from the
heat and quenched with 1 mL of acetic acid. After reaching rt, the
solution is diluted with dichloromethane and washed with water, 0.1
M NaOH, 1 M HCl, and brine. The organic phase is dried with sodium
sulfate and filtered. The crude material is crystallized twice from
hot heptane, yielding the product as a white powder: 195 mg,
20.0%.
[0748] Step 2
[0749] The product (alcohol) from the previous step (194 mg, 0.371
mmol) is dissolved in dichloromethane (4 ml) at 40.degree. C. and
then pyridine (100 .mu.l, 1.24 mmol) and 4-nitrophenylchloroformate
(93 mg, 0.46 mmol) are added. The reaction is stirred at 40.degree.
C. overnight and then cooled to rt. The crude product is purified
by flash chromatography on silica with a heptane/dichloromethane
gradient. The product containing fractions are identified by TLC,
combined and concentrated to a white solid: 219 mg, 86.0%.
[0750] Step 3
[0751] The product (4-nitrophenyl carbonate) from the previous step
is dissolved in toluene (5 ml) at rt. PEG-NH.sub.2 (800 mg, 0.400
mmol, M.sub.n.about.2000 g/mol, "Sunbright MEPA-20H", NOF Corp.),
N,N-dimethylaminopyridine (50 mg, 0.409 mmol), and
N,N-diisopropylethylamine (200 .mu.l, 1.145 mmol) are added and the
solution is stirred at rt. After 72 h the reaction is loaded onto a
10 g Bond Elut SCX column (from Varian; pre-equilibrated with 50:50
dichloromethane:methanol) and elutes with 50:50
dichloromethane:methanol. The product containing fractions are
identified by TLC, combined, and concentrated. The crude product is
further purified by flash chromatography on silica with a
dichloromethane/methanol gradient. The product containing fractions
are identified by TLC, combined and concentrated. To remove
4-nitrophenol impurities, the product is dissolved in
dichloromethane (10 mL) and Si-Amine scavenging resin from
Silicycle (2.0 g, catalog number R52030B) is added. The solution is
agitated at rt for 1 h, filtered to remove the resin, and
concentrated to a pale yellow solid: 806 mg, 97.0%.
[0752] Size exclusion chromatography in N,N-dimethylformamide shows
a single narrow peak. The peaks and integral values observed in the
.sup.1H NMR spectrum are consistent with the expected product.
Example 58
S005
##STR00147##
[0754] Step 1
[0755] In a round bottomed flask, pentatriacontan-18-one (750 mg,
1.48 mmol) is dissolved in tetrahydrofuran (40 mL) with gentle
heating. After the ketone-containing solution cooled to rt, a 4 M
solution of lithium aluminum hydride in diethyl ether (0.74 mL,
2.96 mmol) is added dropwise. The reaction is stirred for 30 min at
rt. Solid sodium sulfate decahydrate is added and the slurry is
stirred for 20 min to quench excess lithium aluminum hydride. The
solids are filtered off, and the filtrate is diluted with heptane
and washed with 1 M HCl. The organic phase is dried with sodium
sulfate and concentrated to a white solid. The crude product is
pure enough to use in the next step without further purification:
650 mg, 86.0%.
[0756] Step 2
[0757] To a solution of the product (alcohol) from the previous
step (200 mg, 0.393 mmol) and pyridine (78 mg, 0.98 mmol) in
dichloromethane (10 mL) at rt is added 4-nitrophenyl chloroformate
(99 mg, 0.49 mmol). The reaction mixture is heated at 35.degree. C.
for 4 h. The reaction mixture is then diluted with heptane,
extracted with 1 M HCl, and then saturated sodium bicarbonate. The
organic phase is dried with sodium sulfate, concentrated, and
purified by flash chromatography on silica with a heptane:ethyl
acetate gradient. The product containing fractions are identified
by TLC, combined, and concentrated to a white solid: 200 mg,
76%.
[0758] Step 3
[0759] The product (4-nitrophenyl carbonate) from the previous step
(260 mg, 0.386 mmol) is dissolved in toluene (4 mL), followed by
PEG2k (620 mg, 0.310 mmol, M.sub.n.about.2000 g/mol, "Sunbright
MEPA-20H", NOF Corp.), N,N-dimethylaminopyridine (40 mg, 0.33
mmol), and N,N-diisopropylethylamine (200 .mu.l, 1.15 mmol). The
solution is stirred at rt overnight. The reaction mixture is loaded
onto a 10 g Bond Elut SCX column (from Varian; pre-equilibrated
with 50:50 dichloromethane:methanol) and elutes with 50:50
dichloromethane:methanol. The product containing fractions are
identified by TLC, combined, and concentrated. The crude product is
further purified by flash chromatography on silica with a
dichloromethane/methanol gradient. The product containing fractions
are identified by TLC, combined and concentrated. To remove
4-nitrophenol impurities, the crude product is dissolved in 1:1
dichloromethane:methanol and elutes through a 10 g Bond Elut NH2
column (from Varian; pre-equilibrated with 50:50
dichloromethane:methanol). The product containing fractions are
identified by TLC, combined, and concentrated to a pale yellow
solid: 631 mg, 78.0%.
[0760] Size exclusion chromatography in N,N-dimethylformamide shows
a single narrow peak. The peaks and integral values observed in the
.sup.1H NMR spectrum are consistent with the expected product.
Example 59
S007
##STR00148##
[0762] Step 1
[0763] To a round bottomed flask is added magnesium metal (0.201 g,
8.27 mmol) and a catalytic amount of iodine, followed by
tetrahydrofuran (30 mL) and 1-bromotetradecane (2.17 g, 7.82 mmol).
The mixture is refluxed for 2 h and then cooled to rt. A solution
of octadecyl aldehyde (0.600 g, 2.24 mmol) in tetrahydrofuran (5
mL) is added, and the reaction mixture is stirred for 30 min at rt.
The reaction is diluted with ethyl acetate, washed with 1 M HCl,
dried with sodium sulfate and concentrated. The crude product is
further purified by flash chromatography on silica with a
heptane/ethyl acetate gradient. The product containing fractions
are identified by TLC, combined and concentrated: 310 mg,
29.7%.
[0764] Step 2
[0765] The product (alcohol) from the previous step (310 mg, 0.664
mmol) is dissolved in dichloromethane (15 mL). Pyridine (0.134 mL,
1.66 mmol) is then added, followed by 4-nitrophenyl chloroformate
(167 mg, 0.830 mmol). The reaction is stirred overnight at rt. The
reaction is diluted with ethyl acetate, washed with a saturated
aqueous solution of NaHCO.sub.3, dried with sodium sulfate, and
concentrated. The crude product is further purified by flash
chromatography on silica with a heptane/ethyl acetate gradient. The
product containing fractions are identified by TLC, combined and
concentrated: 315 mg, 75.0%.
[0766] Step 3
[0767] To a solution of the product (4-nitrophenyl carbonate) from
the previous step (315 mg, 0.498 mmol) in toluene (10 mL) at rt are
added N,N-dimethylaminopyridine (48.7 mg, 0.399 mmol), and
N,N-diisopropylethylamine (0.174 ml, 0.997 mmol), followed by
PEG-NH.sub.2 (798 mg, 0.399 mmol, M.sub.n.about.2000 g/mol,
"Sunbright MEPA-20H", NOF Corp.). The yellow solution is stirred at
rt overnight. The reaction mixture is loaded onto a 10 g Bond Elut
SCX column (from Varian; pre-equilibrated with 50:50
dichloromethane:methanol) and elutes with 50:50
dichloromethane:methanol. The product containing fractions are
identified by TLC, combined, and concentrated. The crude product is
further purified by flash chromatography on silica with a
dichloromethane/methanol gradient. The product containing fractions
are identified by TLC, combined and concentrated: 540 mg,
54.3%.
[0768] The peaks and integral values observed in the .sup.1H NMR
spectrum are consistent with the expected product.
Example 60
S008
##STR00149##
[0770] Step 1
[0771] To a dried round bottomed flask under nitrogen is added
1-iodohexadecane (0.705 g, 2.00 mmol) and diethyl ether (15 mL).
The solution is cooled to -78.degree. C. (solution becomes a white
slurry) and a 1.7 M solution of t-butyl lithium in heptane (2.59
mL, 4.40 mmol) is added drop-wise. After stirring for 20 min, the
reaction mixture is warmed to rt and stirred for an additional 2 h.
To the reaction mixture is added a solution of octadecyl aldehyde
(0.268 g, 1.00 mmol) in diethyl ether (3 mL) drop-wise
(exothermic). The reaction is quenched with ice cold 1 M HCl,
extracted with dichloromethane, dried with sodium sulfate, and
concentrated. The crude product is further purified by flash
chromatography on silica with a heptane/dichloromethane gradient.
The product containing fractions are identified by TLC, combined
and concentrated: 150 mg, 30.3%.
[0772] Step 2
[0773] The product (alcohol) from the previous step (200 mg, 0.404
mmol) is dissolved in dichloromethane (6 mL). Pyridine (0.082 mL,
1.01 mmol) is then added, followed by 4-nitrophenyl chloroformate
(102 mg, 0.505 mmol). The reaction is stirred overnight at rt. The
reaction is diluted with ethyl acetate, washed with a saturated
aqueous solution of NaHCO.sub.3, dried with sodium sulfate, and
concentrated. The crude product is further purified by flash
chromatography on silica with a heptane/ethyl acetate gradient. The
product containing fractions are identified by TLC, combined and
concentrated: 220 mg, 82.0%.
[0774] Step 3
[0775] To a solution of the product (4-nitrophenyl carbonate) from
the previous step (230 mg, 0.348 mmol) in toluene (6 mL) at rt are
added N,N-dimethylaminopyridine (42.6 mg, 0.348 mmol), and
N,N-diisopropylethylamine (0.152 ml, 0.871 mmol), followed by
PEG-NH.sub.2 (697 mg, 0.348 mmol, M.sub.n.about.2000 g/mol,
"Sunbright MEPA-20H", NOF Corp.). The yellow solution is stirred at
rt overnight. The reaction mixture is loaded onto a 10 g Bond Elut
SCX column (from Varian; pre-equilibrated with 50:50
dichloromethane:methanol) and elutes with 50:50
dichloromethane:methanol. The product containing fractions are
identified by TLC, combined, and concentrated. The crude product is
further purified by flash chromatography on silica with a
dichloromethane/methanol gradient. The product containing fractions
are identified by TLC, combined and concentrated: 600 mg,
68.3%.
[0776] The peaks and integral values observed in the .sup.1H NMR
spectrum are consistent with the expected product.
[0777] S009 may be prepared in a manner analogous to that described
for S008.
Example 61
S010 and S011
[0778] S010 and S011 may be prepared, e.g., as provided in PCT
publication WO2009086558 compounds IVa and IVc, respectively. These
compounds may be synthesized as provided in Example 19 of
WO2009086558.
Example 62
S012
[0779] S012 may be prepared in a manner analogous to that described
for S001, utilizing PEG-NH.sub.2 ("Sunbright MEPA-20H", NOF Corp.)
instead of poly(ethylene glycol) methyl ether.
Example 63
S006 and S013 through S024
[0780] S006, S013, S014, S015, S016, S017, S018, S019, S020, S021,
S022, S023, and S024 may be prepared in a manner analogous to that
described for S004.
Example 64
S025
##STR00150##
[0782] Step 1
[0783] The product of Step 2 in the synthesis of compound S004
described herein (4-nitrophenyl carbonate) (285 mg, 0.414 mmol) is
suspended in N,N-dimethylformamide (5 mL), followed by D-glutamic
acid dimethyl ester (145 mg, 0.828 mmol), N,N-diisopropylethylamine
(0.145 ml, 0.828 mmol), and N,N-dimethylaminopyridine (101 mg,
0.828 mmol). The solution is heated at 60.degree. C. overnight. The
reaction mixture is loaded onto a 10 g Bond Elut SCX column (from
Varian; pre-equilibrated with 50:50 dichloromethane:methanol) and
elutes with 50:50 dichloromethane:methanol. The product containing
fractions are identified by TLC, combined, and concentrated. The
crude product is further purified by flash chromatography on silica
with an ethyl acetate/heptane gradient. The product containing
fractions are identified by TLC, combined and concentrated to a
white solid: 131 mg, 44%.
[0784] Step 2
[0785] The product from the previous step (di-methyl ester) (0.160
g, 0.221 mmol) is dissolved in tetrahydrofuran (5 mL), and a
solution of LiOH (52.9 mg, 2.21 mmol) in water (5 mL) is added. The
reaction is stirred at rt for 72 h. The solution is diluted with
chloroform and washed with 1N HCl (aqueous) and then brine. The
organic phase is dried with sodium sulfate and filtered. The
filtrate is concentrated to a white solid: 130 mg, 85%.
[0786] Step 3
[0787] The product from the previous step (di-acid) (130 mg, 0.187
mmol) is suspended in dichloromethane (9 mL) and heptane (1.5 mL)
at rt. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride
(107 mg, 0.560 mmol) is added to the suspension, followed by
hydroxybenzotriazole (86 mg, 0.56 mmol). After 30 min., PEG-NH2
(411 mg, 0.411 mmol, Mn.about.1000 g/mol, "mPEG-Amine, 1 k",
Creative PEGWorks) and N,N-diisopropylethylamine (65 .mu.L, 0.374
mmol) are added. The reaction is stirred overnight at rt. The
reaction mixture is loaded onto a 10 g Bond Elut SCX column (from
Varian; pre-equilibrated with 50:50 dichloromethane:methanol) and
elutes with 50:50 dichloromethane:methanol. The product containing
fractions are identified by TLC, combined, and concentrated. The
crude product is further purified by flash chromatography on silica
with a dichloromethane/methanol gradient. The product containing
fractions are identified by TLC, combined and concentrated to a
white solid: 376 mg, 57.8%.
Example 65
S026
##STR00151##
[0789] Step 1
[0790] The product of Step 1 in the synthesis of compound S004
described herein (alcohol) (200 mg, 0.382 mmol), succinic anhydride
(38 mg, 0.38 mmol), and N,N-dimethylaminopyridine (12 mg, 0.096
mmol) are weighed into a flask and suspended in chloroform (3.5
mL). The reaction mixture is stirred at 70.degree. C. overnight.
The reaction mixture is loaded onto a 1 g Bond Elut SCX column
(from Varian; pre-equilibrated with dichloromethane) and elutes
with dichloromethane. The product containing fractions are
identified by TLC, combined, and concentrated. The crude product is
further purified by flash chromatography on silica with an ethyl
acetate/heptane gradient. The product containing fractions are
identified by TLC, combined and concentrated to a white solid: 134
mg, 56%.
[0791] Step 2
[0792] The product from the previous step (carboxylic acid) (75 mg,
0.12 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide
hydrochloride (35 mg, 0.18 mmol) and hydroxybenzotriazole (28 mg,
0.18 mmol) is dissolved in anhydrous chloroform (1 mL) and stirred
for 0.5 h at rt. Then PEG-NH2 (265 mg, 0.132 mmol, Mn.about.2000
g/mol, "Sunbright MEPA-20H", NOF Corp.) and
N,N-diisopropylethylamine (32 .mu.L, 0.18 mmol) is added and the
solution is stirred overnight at rt. The reaction mixture is loaded
onto a 2 g Bond Elut SCX column (from Varian; pre-equilibrated with
50:50 dichloromethane:methanol) and elutes with dichloromethane.
The product containing fractions are identified by TLC, combined,
and concentrated to a white solid: 240 mg, 76%.
[0793] Characterisation data for these stealth lipids is as follows
in Tables 4 and 5.
TABLE-US-00012 TABLE 4 .sup.1H NMR for Stealth Lipids S001 through
S026 S001 0.68 (s, 3H), 0.78-1.63 (m, 33H), 1.76-1.91 (m, 3H),
1.91-2.07 (m, 2H), 2.32 (d, 2H), 2.56-2.70 (m, 4H), 3.39 (s, 3H),
3.43-3.89 (m, 210H), 4.25 (t, 2H), 4.64 (m, 1H), 5.33-5.41 (m, 1H)
S002 0.67 (s, 3H), 0.77-1.66 (m, 33H), 1.72-2.10 (m, 7H), 2.17-2.44
(m, 2H), 2.57 (t, 2H), 3.38 (s, 3H), 3.41-3.94 (m, 196H), 4.26 (t,
2H), 4.38-4.61 (m, 1H), 5.19-5.30 (m, 1H), 5.34-5.43 (m, 1H) S003
0.68 (s, 3H), 0.78-1.67 (m, 33H), 1.68-1.92 (m, 5), 1.92-2.07 (m,
2H), 2.15-2.46 (m, 2H), 3.22-3.34 (m, 2H), 3.38 (s, 3H), 3.41-3.90
(m, 200H), 4.40-4.57 (m, 1H), 5.14 (br, 1H), 5.33-5.43 (m, 1H) S004
0.88 (t, 6H), 1.25 (m, 62H), 1.48 (m, 4H), 1.77 (m, 2H), 3.18-3.34
(m, 2H), 3.38 (s, 3H), 3.42-3.88 (m, 205H), 4.71 (m, 1H), 5.06 (m,
1H) S005 0.89 (t, 6H), 1.26 (m, 60H), 1.48 (m, 4H), 1.78 (m, 2H),
3.17-3.33 (m, 2H), 3.39 (s, 3H), 3.42-3.88 (m, 203H), 4.71 (m, 1H),
5.07 (m, 1H) S006 0.88 (t, 6H), 1.25 (m, 46H), 1.48 (m, 4H), 1.77
(m, 2H), 3.14-3.34 (m, 2H), 3.38 (s, 3H), 3.41-3.91 (m, 209H), 4.71
(m, 1H), 5.06 (m, 1H) S007 0.87 (m, 6H), 1.25 (m, 54H), 1.48 (m,
4H), 1.76 (m, 2H), 3.15-3.32 (m, 2H), 3.37 (s, 3H), 3.41-3.89 (m,
206H), 4.70 (m, 1H), 5.06 (m, 1H) S008 0.84 (t, 6H), 1.21 (m, 58H),
1.43 (m, 4H), 1.74 (m, 2H), 2.99-3.29 (m, 2H), 3.34 (s, 3H),
3.37-4.08 (m, 187H), 4.66 (m, 1H), 5.08 (m, 1H) S009 0.88 (t, 6H),
1.25 (m, 54H), 1.47 (m, 4H), 1.77 (m, 2H), 3.16-3.34 (m, 2H), 3.38
(s, 3H), 3.41-3.87 (m, 207H), 4.70 (m, 1H), 5.06 (m, 1H) S010 0.88
(t, 6H), 1.25 (m, 44H), 1.55 (m, 4H), 1.78 (m, 2H), 3.15-3.33 (m,
2H), 3.38 (s, 3H), 3.40-3.95 (m, 207H), 3.97-4.30 (m, 2H),
4.96-5.27 (br m, 1H) S011 0.89 (t, 6H), 1.26 (m, 60H), 1.56 (m,
4H), 1.78 (m, 2H), 3.20-3.34 (m, 2H), 3.39 (s, 3H), 3.40-3.89 (m,
219H), 4.02-4.26 (m, 2H), 5.09-5.23 (m, 1H) S013 0.89 (t, 6H), 1.26
(m, 54H), 1.48 (m, 4H), 1.77 (m, 2H), 3.16-3.34 (m, 2H), 3.38 (s,
3H), 3.41-3.87 (m, 187H), 4.71 (m, 1H), 5.05 (m, 1H) S014 0.89 (t,
6H), 1.26 (m, 54H), 1.49 (m, 4H), 1.78 (m, 2H), 3.16-3.34 (m, 2H),
3.39 (s, 3H), 3.41-3.87 (m, 210H), 4.72 (m, 1H), 5.05 (m, 1H) S015
0.89 (t, 6H), 1.26 (m, 50H), 1.48 (m, 4H), 1.78 (m, 2H), 3.16-3.34
(m, 2H), 3.39 (s, 3H), 3.41-3.87 (m, 209H), 4.71 (m, 1H), 5.06 (m,
1H) S016 0.88 (t, 6H), 1.26 (m, 50H), 1.47 (m, 4H), 1.77 (m, 2H),
3.16-3.34 (m, 2H), 3.38 (s, 3H), 3.41-3.87 (m, 202H), 4.71 (m, 1H),
5.06 (m, 1H) S017 0.89 (t, 6H), 1.26 (m, 46H), 1.49 (m, 4H), 1.78
(m, 2H), 3.16-3.34 (m, 2H), 3.39 (s, 3H), 3.41-3.87 (m, 203H), 4.71
(m, 1H), 5.06 (m, 1H) S018 0.88 (t, 6H), 1.25 (m, 48H), 1.48 (m,
4H), 1.77 (m, 2H), 2.05 (m, 4H), 2.77 (m, 2H), 3.16-3.34 (m, 2H),
3.39 (s, 3H), 3.41-3.87 (m, 180H), 4.71 (m, 1H), 5.05 (m, 1H),
5.25-5.50 (m, 4H) S019 0.88 (t, 6H), 1.26 (m, 54H), 1.48 (m, 4H),
1.77 (m, 2H), 2.01 (m, 4H), 3.16-3.34 (m, 2H), 3.38 (s, 3H),
3.41-3.87 (m, 200H), 4.71 (m, 1H), 5.05 (m, 1H), 5.31-5.49 (m, 2H)
S020 0.88 (t, 6H), 1.26 (m, 62H), 1.49 (m, 4H), 3.38 (s, 3H),
3.4-3.9 (m, 299H), 4.72 (m, 1H), 5.11 (m, 1H) S021 0.89 (t, 6H),
1.26 (m, 36H), 1.48 (m, 4H), 1.78 (m, 2H), 3.20-3.35 (m, 2H), 3.39
(s, 3H), 3.41-3.87 (m, 196H), 4.71 (m, 1H), 5.07 (m, 1H) S022 0.89
(t, 6H), 1.26 (m, 62H), 1.49 (m, 4H), 3.36 (s, 3H), 3.41-3.87 (m,
161H), 4.72 (m, 1H), 5.14 (m, 1H) S023 0.88 (t, 6H), 1.26 (m, 64H),
1.47 (m, 4H), 1.77 (m, 2H), 3.20-3.35 (m, 2H), 3.39 (s, 3H),
3.42-3.88 (m, 194H), 4.71 (m, 1H), 5.06 (m, 1H) S024 0.88 (t, 6H),
1.25 (m, 48H), 1.47 (m, 4H), 1.77 (m, 2H), 3.20-3.35 (m, 2H), 3.39
(s, 3H), 3.42-3.88 (m, 194H), 4.70 (m, 1H), 5.08 (m, 1H) S025 0.87
(t, 6H), 1.24 (m, 62H), 1.47 (m, 4H), 2.01 (m, 2H), 2.2-2.4 (m,
2H), 3.36 (s, 3H), 3.41-3.87 (m, 229H), 4.13 (m, 1H), 4.65 (m, 1H),
5.78 (m, 1H), 6.65 (m, 1H), 7.07 (m, 1H) S026 0.87 (t, 6H), 1.25
(m, 62H), 1.49 (m, 4H), 1.76 (m, 2H), 2.44 (t, J = 8 Hz, 2H), 2.63
(t, J = 8 Hz, 2H), 3.37 (s, 3H), 3.41-3.87 (m, 213H), 4.84 (m, 1H),
6.32 (m, 1H)
TABLE-US-00013 TABLE 5 Other characteristics for Stealth Lipids
S001 through S026 TLC SEC MALDI TLC conditions TLC solvent S001
single peak (THF) ~2550 S002 single peak (THF) ~2550 S003 single
peak (THF) ~2600 S004 single peak (DMF) S005 single peak (DMF) S006
single peak (DMF) S007 0.39 1:9 MeOH: CH.sub.2Cl.sub.2 S008 1.39
1:9 MeOH: CH.sub.2Cl.sub.2 S010 single peak (THF) ~2800 S011 single
peak (DMF) S013 single peak (THF) ~2650 S014 single peak (THF)
~2750 S015 single peak (THF) ~2750 S016 single peak (THF) ~2700
S017 single peak (THF) ~2650 S018 single peak (THF) ~2750 S019
single peak (THF) ~2750 S020 single peak (THF) ~3600 S021 single
peak (DMF) ~2700 S022 single peak (DMF) 2364.1 (exact) S023 ~2900
S024 ~2700 S025 single peak (THF) ~3000 S026 single peak (THF)
~3050
Example 66
Summary Table of Results
[0794] The synthesised compounds are represented in the following
tables. For the avoidance of doubt, some of the substituent groups
have been drawn in such a way that they are overlapping, but the
true structure of the compound is nonetheless perfectly clear. For
example, the notation
##STR00152##
does not represent a chemically-impossible 3-membered
hydrogen-containing ring but instead represents the following.
##STR00153##
[0795] Table 6 provides the structures of the cationic lipids of
the invention.
TABLE-US-00014 TABLE 6 Characterization and structures for Cationic
Lipids Lipid Structure R.sup.1--N--R.sup.2 Y.sup.1 L E0001
##STR00154## H.sup.15 Y.sup.1-i L.sup.c-i E0002 ##STR00155##
H.sup.2 Y.sup.1-i L.sup.c-i E0003 ##STR00156## H.sup.1 Y.sup.1-ii
L.sup.c-i E0004 ##STR00157## H.sup.10 Y.sup.1-i L.sup.c-i E0005
##STR00158## H.sup.11 Y.sup.1-i L.sup.c-i E0006 ##STR00159##
H.sup.13 Y.sup.1-i L.sup.c-i E0007 ##STR00160## H.sup.19 Y.sup.1-i
L.sup.c-i E0008 ##STR00161## H.sup.43 Y.sup.1-i L.sup.c-i E0009
##STR00162## H.sup.34 Y.sup.1-ii L.sup.c-i E0010 ##STR00163##
H.sup.22 Y.sup.1-i L.sup.c-i E0011 ##STR00164## H.sup.1 Y.sup.1-i
L.sup.c-i E0012 ##STR00165## H.sup.3 Y.sup.1-i L.sup.c-i E0013
##STR00166## H.sup.4 Y.sup.1-i L.sup.c-i E0014 ##STR00167## H.sup.5
Y.sup.1-i L.sup.c-i E0015 ##STR00168## H.sup.7 Y.sup.1-i L.sup.c-i
E0016 ##STR00169## H.sup.8 Y.sup.1-i L.sup.c-i E0017 ##STR00170##
H.sup.14 Y.sup.1-i L.sup.c-i E0018 ##STR00171## H.sup.16 Y.sup.1-i
L.sup.c-i E0019 ##STR00172## H.sup.17 Y.sup.1-i L.sup.c-i E0020
##STR00173## H.sup.21 Y.sup.1-i L.sup.c-i E0021 ##STR00174##
H.sup.23 Y.sup.1-i L.sup.c-i E0022 ##STR00175## H.sup.24 Y.sup.1-i
L.sup.c-i E0023 ##STR00176## H.sup.25 Y.sup.1-i L.sup.c-i E0024
##STR00177## H.sup.35 Y.sup.1-i L.sup.c-i E0025 ##STR00178##
H.sup.27 Y.sup.1-i L.sup.c-i E0026 ##STR00179## H.sup.28 Y.sup.1-i
L.sup.c-i E0027 ##STR00180## H.sup.5 Y.sup.1-i L.sup.c-ii E0028
##STR00181## H.sup.1 Y.sup.1-i L.sup.c-ii E0029 ##STR00182##
H.sup.29 Y.sup.1-i L.sup.c-i E0030 ##STR00183## H.sup.30 Y.sup.1-i
L.sup.c-i E0031 ##STR00184## H.sup.31 Y.sup.1-i L.sup.c-i E0032
##STR00185## H.sup.20 Y.sup.1-i L.sup.c-i E0033 ##STR00186##
H.sup.33 Y.sup.1-i L.sup.c-i E0034 ##STR00187## H.sup.5 Y.sup.1-i
L.sup.c-i E0035 ##STR00188## H.sup.35 Y.sup.1-i L.sup.c-ii E0036
##STR00189## H.sup.5 Y.sup.1-i L.sup.c-iii E0037 ##STR00190##
H.sup.44 Y.sup.1-i L.sup.c-i E0038 ##STR00191## H.sup.36 Y.sup.1-i
L.sup.c-i E0039 ##STR00192## H.sup.37 Y.sup.1-i L.sup.c-i E0040
Mixture of the isomers E0053 and E0052 H.sup.12/26 Y.sup.1-i
L.sup.c-i E0041 ##STR00193## H.sup.32 Y.sup.1-i L.sup.c-i E0042
##STR00194## H.sup.39 Y.sup.1-i L.sup.c-i E0043 ##STR00195##
H.sup.5 Y.sup.1-i L.sup.c-i E0044 ##STR00196## H.sup.48 Y.sup.1-i
L.sup.c-i E0045 ##STR00197## H.sup.41 Y.sup.1-i L.sup.c-i E0046
##STR00198## H.sup.42 Y.sup.1-i L.sup.c-i E0047 ##STR00199##
H.sup.5 Y.sup.1-i L.sup.c-v E0048 ##STR00200## H.sup.26 Y.sup.1-i
L.sup.c-i E0049 ##STR00201## H.sup.12 Y.sup.1-i L.sup.c-i E0050
##STR00202## H.sup.9 Y.sup.1-i L.sup.c-i E0051 ##STR00203##
H.sup.40 Y.sup.1-i L.sup.c-i E0052 ##STR00204## H.sup.12 Y.sup.1-i
L.sup.c-i E0053 ##STR00205## H.sup.26 Y.sup.1-i L.sup.c-i E0054
##STR00206## H.sup.5 Y.sup.1-i L.sup.c-vi E0055 ##STR00207##
H.sup.5 Y.sup.1-iii L.sup.c-i E0056 ##STR00208## H.sup.5 Y.sup.1-i
L.sup.c-xviii E0057 ##STR00209## H.sup.47 Y.sup.1-i L.sup.c-i E0058
##STR00210## H.sup.5 Y.sup.1-i L.sup.c-xv E0059 ##STR00211##
H.sup.45 Y.sup.1-i L.sup.c-i E0060 ##STR00212## H.sup.5 Y.sup.1-i
L.sup.c-xvi E0061 ##STR00213## H.sup.5 Y.sup.1-iv L.sup.c-viii
E0062 ##STR00214## H.sup.35 Y.sup.1-iv L.sup.c-viii E0063
##STR00215## H.sup.5 Y.sup.1-iv L.sup.c-i E0064 ##STR00216##
H.sup.35 Y.sup.1-iv L.sup.c-i E0065 ##STR00217## H.sup.12/26
Y.sup.1-iv L.sup.c-i E0066 ##STR00218## H.sup.12/26 Y.sup.1-iv
L.sup.c-viii E0067 ##STR00219## H.sup.46 Y.sup.1-i L.sup.c-i E0068
##STR00220## H.sup.1 Y.sup.1-v L.sup.c-viii E0069 ##STR00221##
H.sup.5 Y.sup.1-v L.sup.c-viii E0070 ##STR00222## H.sup.12/26
Y.sup.1-v L.sup.c-viii E0071 ##STR00223## H.sup.5 Y.sup.1-v
L.sup.c-i E0072 ##STR00224## H.sup.12/26 Y.sup.1-v L.sup.c-i E0073
##STR00225## H.sup.1 Y.sup.1-v L.sup.c-i E0074 ##STR00226## H.sup.1
Y.sup.1-v L.sup.c-vii E0075 ##STR00227## H.sup.12/26 Y.sup.1-i
L.sup.c-vii E0076 ##STR00228## H.sup.5 Y.sup.1-i L.sup.c-viii E0077
##STR00229## H.sup.5 Y.sup.1-i L.sup.c-vii E0078 ##STR00230##
H.sup.5 Y.sup.1-iv L.sup.c-vii E0079 ##STR00231## H.sup.5 Y.sup.1-i
L.sup.c-ix E0080 ##STR00232## H.sup.1 Y.sup.1-iv L.sup.c-viii E0081
##STR00233## H.sup.35 Y.sup.1-iv L.sup.c-vii E0082 ##STR00234##
H.sup.12/26 Y.sup.1-i L.sup.c-x E0083 ##STR00235## H.sup.12/26
Y.sup.1-i L.sup.c-i E0084 ##STR00236## H.sup.12/26 Y.sup.1-i
L.sup.c-ix E0085 ##STR00237## H.sup.1 Y.sup.1-iv L.sup.c-i E0086
##STR00238## H.sup.1 Y.sup.1-iv L.sup.c-vii E0087 ##STR00239##
H.sup.35 Y.sup.1-i L.sup.c-viii E0088 ##STR00240## H.sup.35
Y.sup.1-i L.sup.c-vii E0089 ##STR00241## H.sup.35 Y.sup.1-v
L.sup.c-viii E0090 ##STR00242## H.sup.35 Y.sup.1-v L.sup.c-i E0091
##STR00243## H.sup.35 Y.sup.1-v L.sup.c-vii E0092 ##STR00244##
H.sup.1 Y.sup.1-i L.sup.c-viii E0093 ##STR00245## H.sup.1 Y.sup.1-i
L.sup.c-vii E0094 ##STR00246## H.sup.12/26 Y.sup.1-iv L.sup.c-vii
E0095 ##STR00247## H.sup.12/26 Y.sup.1-v L.sup.c-vii E0096
##STR00248## H.sup.5 Y.sup.1-v L.sup.c-vii E0097 ##STR00249##
H.sup.5 Y.sup.1-i L.sup.c-xiv E0098 ##STR00250## H.sup.5 Y.sup.1-i
L.sup.c-xi E0099 ##STR00251## H.sup.12/26 Y.sup.1-i L.sup.c-xiv
E0100 ##STR00252## H.sup.5 Y.sup.1-i L.sup.c-xii E0101 ##STR00253##
H.sup.5 Y.sup.1-i L.sup.c-xiii E0102 ##STR00254## H.sup.5 Y.sup.1-i
L.sup.c-xxi E0103 ##STR00255## H.sup.12/26 Y.sup.1-i L.sup.c-vi
E0104 ##STR00256## H.sup.5 Y.sup.1-i L.sup.c-xvii E0105
##STR00257## H.sup.5 Y.sup.1-i L.sup.c-xix E0106 ##STR00258##
H.sup.5 Y.sup.1-i L.sup.c-xx E0107 ##STR00259## H.sup.16 Y.sup.1-i
L.sup.c-ix E0108 ##STR00260## H.sup.12/26 Y.sup.1-i L.sup.c-xi
E0109 ##STR00261## H.sup.1 Y.sup.1-i L.sup.c-ix E0110 ##STR00262##
H.sup.5 Y.sup.1-i L.sup.c-xxii E0111 ##STR00263## H.sup.5 Y.sup.1-i
L.sup.c-xxiii E0112 ##STR00264## H.sup.7 Y.sup.1-i L.sup.c-x E0113
##STR00265## H.sup.7 Y.sup.1-i L.sup.c-ix E0114 ##STR00266##
H.sup.1 Y.sup.1-i L.sup.c-x E0115 ##STR00267## H.sup.5 Y.sup.1-vi
L.sup.c-i E0116 ##STR00268## H.sup.35 Y.sup.1-vi L.sup.c-i E0117
##STR00269## H.sup.12/26 Y.sup.1-vi L.sup.c-i E0118 Another mixture
of the isomers E0053 and E0052 H.sup.12/26 Y.sup.1-i L.sup.c-i
E0119 ##STR00270## H.sup.25 Y.sup.1-vi L.sup.c-i E0120 ##STR00271##
H.sup.21 Y.sup.1-i L.sup.c-i E0121 ##STR00272## H.sup.14 Y.sup.1-iv
L.sup.c-i E0122 ##STR00273## H.sup.1 Y.sup.1-iv L.sup.c-xviii
E0123 ##STR00274## H.sup.1 Y.sup.1-iv L.sup.c-xxiv E0124
##STR00275## H.sup.21 Y.sup.1-iv L.sup.c-vii E0125 ##STR00276##
H.sup.1 Y.sup.1-iv L.sup.c-xxv E0126 ##STR00277## H.sup.14
Y.sup.1-iv L.sup.c-vii E0127 ##STR00278## H.sup.25 Y.sup.1-iv
L.sup.c-vii E0128 ##STR00279## H.sup.1 Y.sup.1-iv L.sup.c-xxvi
E0129 ##STR00280## H.sup.49 Y.sup.1-iv L.sup.c-xvii E0130
##STR00281## H.sup.8 Y.sup.1-iv L.sup.c-xvii E0131 ##STR00282##
H.sup.5 Y.sup.1-iv L.sup.c-xxvi E0132 ##STR00283## H.sup.5
Y.sup.1-iv L.sup.c-xxvii E0133 ##STR00284## H.sup.5 Y.sup.1-i
L.sup.c-xxviii E0134 ##STR00285## H.sup.5 Y.sup.1-i L.sup.c-xxix
E0135 ##STR00286## H.sup.5 Y.sup.1-iv L.sup.c-xxx E0136
##STR00287## H.sup.5 Y.sup.1-iv L.sup.c-xxxi E0137 ##STR00288##
H.sup.5 Y.sup.1-iv L.sup.c-xxxii E0138 ##STR00289## H.sup.5
Y.sup.1-iv L.sup.c-xviii E0139 ##STR00290## H.sup.5 Y.sup.1-iv
L.sup.c-xxiv E0140 ##STR00291## H.sup.5 Y.sup.1-iv L.sup.c-xxv
E0141 ##STR00292## H.sup.5 Y.sup.1-iv L.sup.c-xxxiii E0142
##STR00293## H.sup.5 Y.sup.1-iv L.sup.c-xxxiv E0143 ##STR00294##
H.sup.50 Y.sup.1-iv L.sup.c-xvii E0144 ##STR00295## H.sup.5
Y.sup.1-iv L.sup.c-xxv E0145 ##STR00296## H.sup.5 Y.sup.1-iv
L.sup.c-xxxv E0146 ##STR00297## H.sup.5 Y.sup.1-iv L.sup.c-xxxvi
E0147 ##STR00298## H.sup.27 Y.sup.1-iv L.sup.c-i E0148 ##STR00299##
H.sup.25 Y.sup.1-iv L.sup.c-vi E0149 ##STR00300## H.sup.51
Y.sup.1-iv L.sup.c-i E0150 ##STR00301## H.sup.5 Y.sup.1-iv L.sup.c-
.sup.xxxvii E0151 ##STR00302## H.sup.5 Y.sup.1-iv L.sup.c-
.sup.xxxviii E0152 ##STR00303## H.sup.1 Y.sup.1-iv L.sup.c-xvi
E0158 ##STR00304## H.sup.52 Y.sup.1-i L.sup.c-i E0159 ##STR00305##
H.sup.52 Y.sup.1-i L.sup.c-i E0160 ##STR00306## H.sup.5 Y.sup.1-vii
L.sup.c-xxxix E0161 ##STR00307## H.sup.26 Y.sup.1-i L.sup.c-xxxix
E0162 ##STR00308## H.sup.12 Y.sup.1-i L.sup.c-xxxix E0163
##STR00309## H.sup.5 Y.sup.1-iv L.sup.c-xxxx E0164 ##STR00310##
H.sup.26 Y.sup.1-iv L.sup.c-xvii E0165 ##STR00311## H.sup.12
Y.sup.1-iv L.sup.c-xvii E0166 ##STR00312## H.sup.5 Y.sup.1-i
L.sup.c-xxxxi E0167 ##STR00313## H.sup.1 Y.sup.1-iv L.sup.c-
.sup.xxxxii E0168 ##STR00314## H.sup.5 Y.sup.1-i L.sup.c-
.sup.xxxxiii E0169 ##STR00315## H.sup.27 Y.sup.1-i L.sup.c-vi E0170
##STR00316## H.sup.6 Y.sup.1-i L.sup.c-i E0171 ##STR00317##
H.sup.18 Y.sup.1-i L.sup.c-i E0172 (comp- arative) ##STR00318##
##STR00319## Y.sup.1-i L.sup.c-i E0173 ##STR00320## Y.sup.1-i
L.sup.c-ii E0174 (comp- arative) ##STR00321## ##STR00322##
Y.sup.1-i L.sup.c-iii E0175 ##STR00323## H.sup.1 Y.sup.1-iv
L.sup.c-vi E0176 ##STR00324## H.sup.1 Y.sup.1-i L.sup.c-vi E0177
##STR00325## H.sup.5 Y.sup.1-iv L.sup.c-xvii E0178 ##STR00326##
H.sup.5 Y.sup.1-iv L.sup.c-xxxix E0179 ##STR00327## H.sup.5
Y.sup.1-i L.sup.c-xxxiv E0180 ##STR00328## H.sup.1 Y.sup.1-iv
L.sup.c-i indicates data missing or illegible when filed
[0796] For each of the above compounds except E0180, Y.sup.2 is
cholesterol linked to L via an oxygen atom on the 3-position of the
A steroid ring (the hydrogen atom on said hydroxy group being
absent); X.sup.1 and X.sup.2 are O; a=methylene; b=methylene and c
is absent; whereas for E0180, a=ethylene.
[0797] In addition to the characterisation data below, .sup.1H NMR
is taken of all lipids to assess purity and any olefin
isomerization that may have occurred in the synthesis.
Specifically, the integrals for the cholesterol derived singlet,
usually at or close to 0.68 ppm, is compared to the olefin derived
signals in the 5.2 to 5.5 ppm range. The olefin integral for the
desired cis, unconjugated olefins and the cholesterol olefinic
hydrogen is compared to any new signals above 5.5 ppm which
corresponded to isomerized products. In all cases, the degree of
isomerization is less than 10% as determined by comparing the
integrated signals in the .sup.1H NMR.
Example 67
[0798] NMR characterization of the various lipids is provided
below.
[0799] E0008
[0800] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 5.25-5.48 (m, 5H),
3.69-3.86 (m, 2H), 3.55-3.69 (m, 7H), 3.34-3.55 (m, 4H), 3.07-3.25
(m, 1H), 2.70-3.01 (m, 4H), 2.30-2.53 (m, 3H), 2.15-2.30 (m, 1H),
1.78-2.14 (m, 11H), 1.42-1.66 (m, 12H), 0.97-1.42 (m, 34H),
0.81-0.97 (m, 16H), 0.68 (s, 3H) ppm.
[0801] E0006
[0802] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 5.25-5.47 (m, 5H),
3.56-3.85 (m, 11H), 3.48-3.56 (m, 1H), 3.36-3.48 (m, 3H), 3.11-3.27
(m, 1H), 2.66-2.83 (m, 4H), 2.41-2.49 (m, 2H), 2.30-2.41 (m, 1H),
2.15-2.28 (m, 1H), 1.72-2.12 (m, 11H), 0.96-1.70 (m, 60H), 0.68 (s,
3H) ppm.
[0803] .sup.13C NMR (400 MHz, CDCl.sub.3) .delta. 140.9, 130.2,
130.1, 128.0, 127.9, 121.6, 79.5, 72.0, 71.7, 71.6, 70.9, 69.4,
67.3, 60.2, 60.1, 59.7, 56.8, 56.1, 50.2, 42.3, 39.8, 39.5, 39.0,
37.2, 36.8, 36.2, 35.8, 31.9, 31.9, 31.5, 29.7, 29.5, 29.5, 29.3,
29.3, 29.0, 28.3, 28.2, 28.0, 27.2, 27.2, 26.2, 25.6, 24.3, 23.8,
22.8, 22.7, 22.6, 22.6, 21.0, 19.4, 18.7, 14.1, 11.8 ppm.
[0804] E0003
[0805] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 5.22-5.46 (m, 7H),
3.66-3.93 (m, 5H), 3.54-3.66 (m, 6H), 3.35-3.54 (m, 4H), 3.07-3.23
(m, 1H), 2.73-2.89 (m, 4H), 2.39-2.73 (m, 4H), 2.28-2.39 (m, 1H),
2.28-2.39 (m, 1H), 1.70-2.12 (m, 10H), 1.20-1.65 (m, 24H),
0.94-1.19 (m, 13H), 0.77-0.94 (m, 14H), 0.66 (s, 3H) ppm.
[0806] .sup.13C NMR (400 MHz, CDCl.sub.3) .delta. 140.9, 130.4,
130.0, 128.3, 128.1, 127.8, 127.6, 121.6, 79.5, 71.6, 70.8, 70.7,
69.2, 67.3, 60.1, 56.7, 56.1, 54.2, 50.1, 42.3, 39.7, 39.5, 39.0,
37.2, 36.8, 36.2, 35.8, 31.9, 31.8, 31.5, 29.5, 29.4, 29.3, 28.3,
28.2, 28.0, 27.2, 27.2, 25.8, 25.6, 24.3, 23.8, 22.8, 22.6, 22.5,
21.0, 19.4, 18.7, 14.1, 11.8 ppm.
[0807] E0002
[0808] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 5.26-5.44 (m, 5H),
3.68-3.84 (m, 3H), 5.54-3.68 (m, 7H), 3.32-3.54 (m, 6H), 3.09-3.24
(m, 1H), 2.68-2.80 (m, 2H), 2.27-2.58 (m, 3H), 2.12-2.27 (m, 1H),
1.69-2.12 (m, 9H), 1.41-1.69 (m, 20H), 1.19-1.40 (m, 20H),
0.95-1.19 (m, 11H), 0.78-0.95 (m, 12H), 0.66 (s, 3H) ppm.
[0809] E0001
[0810] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.10-7.24 (m, 3H),
6.96-7.10 (m, 1H), 5.21-5.48 (m, 5H), 3.73-3.90 (m, 3H), 3.37-3.72
(m, 11H), 3.10-3.25 (m, 1H), 2.72-2.86 (m, 2H), 2.28-2.42 (m, 1H),
2.14-2.28 (m, 1H), 1.92-2.14 (m, 6H), 1.78-1.92 (m, 4H), 1.22-1.78
(m, 34H), 0.95-1.22 (m, 13H), 0.84-0.95 (m, 13H), 0.68 (s, 3H)
ppm.
[0811] E0004
[0812] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 5.24-5.44 (m, 5H),
3.66-3.83 (m, 2H), 3.54-3.65 (m, 7H), 3.34-3.51 (m, 4H), 3.11-3.23
(m, 1H), 2.94-3.10 (m, 2H), 2.56-2.87 (m, 6.5H), 2.41-2.50 (d,
J=5.02 Hz, 2H), 2.29-2.40 (m, 2.5H), 1.96-2.26 (m, 8H), 1.62-1.96
(m, 8H), 1.62-1.96 (m, 11H), 1.40-1.61 (m, 11H), 1.22-1.40 (m,
20H), 0.81-1.21 (m, 26H), 0.66 (s, 3H) ppm.
[0813] .sup.13C NMR CDCl.sub.3, 400 MHz) .delta. 142.5, 140.9,
139.7, 130.1, 130.1, 128.7, 127.9, 127.9, 125.8, 121.5, 79.4, 77.2,
71.8, 71.6, 70.8, 70.8, 69.3, 67.3, 63.1, 59.3, 56.7, 56.1, 53.6,
53.3, 50.1, 49.9, 42.2, 39.7, 39.4, 39.0, 37.2, 36.8, 36.1, 35.7,
31.9, 31.8, 31.5, 29.6, 29.5, 29.4, 29.3, 29.3, 28.3, 28.2, 27.9,
27.2, 27.1, 27.0, 26.9, 26.1, 25.6, 24.8, 24.2, 23.8, 22.8, 22.5,
21.3, 21.0, 19.3, 18.6, 14.0, 11.8 ppm.
[0814] E0005
[0815] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 5.28-5.44, (m,
3H), 3.69-3.82 (m, 2H), 3.57-3.67 (m, 7H), 3.48-3.64 (m, 1H),
3.39-3.48 (m, 3H), 3.33 (s, 3H), 3.12-3.24 (m, 2H), 2.70-2.86 (m,
4H), 2.42-2.47 (d, J=6.02 Hz, 2H), 2.33-2.42 (m, 3H), 1.97-2.10 (m,
5H), 1.76-1.97 (m, 7H), 1.42-1.64 (m, 12H), 1.23-1.42 (m, 20H),
0.97-1.23 (m, 12H), 0.81-0.97 (m, 13H), 0.68 (s, 3H) ppm.
[0816] .sup.13C NMR (400 MHz, CDCl.sub.3) .delta. 140.9, 130.2,
130.1, 127.9, 127.9, 121.5, 79.5, 77.5, 72.5, 72.1, 70.9, 70.8,
69.3, 67.3, 63.1, 59.3, 56.7, 56.1, 55.5, 52.0, 51.7, 50.1, 42.3,
39.8, 39.5, 39.0, 37.2, 36.8, 36.2, 35.8, 31.9, 31.9, 31.5, 31.0,
29.7, 29.5, 29.5, 29.3, 29.3, 28.3, 28.2, 28.0, 27.2, 27.2, 26.1,
25.6, 24.3, 23.8, 22.8, 22.6, 21.0, 19.4, 18.7, 14.1, 11.8 ppm.
[0817] E0007
[0818] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 5.26-5.44 (m, 5H),
3.84-3.94 (t, J=5.56 Hz, 4H), 3.68-3.87 (m, 2H), 3.56-3.67 (m, 7H),
3.47-3.54 (m, 1H), 3.38-3.47 (m, 3H), 3.11-3.23 (m, 1H), 2.73-2.82
(t, J=6.44 Hz, 2H), 2.42-2.57 (m, 5H), 2.32-2.41 (m, 1H), 2.11-2.27
(m, 2H), 1.97-2.10 (m, 6H), 1.76-1.96 (m, 7H), 1.66-1.75 (m, 2H),
1.41-1.62 (m, 9H), 1.22-1.41 (m, 20H), 0.81-1.22 (m, 25H), 0.67 (s,
3H) ppm.
[0819] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 141.0, 130.1,
130.1, 127.9, 127.9, 121.5, 96.2, 79.5, 77.6, 72.2, 71.6, 70.9,
70.8, 69.4, 67.3, 63.1, 59.3, 59.1, 56.8, 56.1, 50.5, 50.2, 42.3,
39.8, 39.5, 39.1, 37.2, 36.8, 36.2, 35.7, 32.8, 31.9, 31.9, 31.5,
29.7, 29.5, 29.4, 29.3, 29.3, 28.3, 28.2, 28.0, 27.2, 27.2, 26.1,
25.6, 25.6, 24.3, 23.8, 22.8, 22.5, 21.0, 19.3, 18.7, 14.0, 11.8
ppm.
[0820] E0009
[0821] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 5.27-5.44 (m, 5H),
4.07-4.22 (m, 2H), 3.67-3.83 (m, 2H), 3.55-3.67 (m, 6H), 3.37-3.52
(m, 4H), 3.12-3.25 (m, 1H), 2.81-2.90 (m, 1H), 2.73-2.81 (t, J=6.53
Hz, 2H), 2.42-2.64 (m, 3H), 2.15-2.41 (m, 4H), 1.76-2.11 (m, 11H),
1.64-1.72 (m, 4H), 1.42-1.63 (m, 12H), 1.21-1.41 (m, 22H),
0.81-1.22 (m, 26H), 0.68 (s, 3H) ppm.
[0822] .sup.13C NMR (400 MHz, CDCl.sub.3) .delta. 140.9, 130.2,
130.1, 127.9, 127.9, 121.5, 79.5, 77.2, 72.7, 72.0, 71.6, 71.5,
70.8, 69.4, 67.3, 61.9, 59.5, 56.7, 56.5, 56.1, 50.1, 49.3, 49.3,
42.3, 39.7, 39.5, 39.0, 37.2, 36.8, 36.2, 35.8, 31.9, 31.9, 31.5,
29.7, 29.5, 29.5, 29.3, 29.3, 28.4, 28.3, 28.2, 28.0, 27.4, 27.4,
27.2, 27.2, 26.5, 26.1, 25.6, 24.3, 23.8, 22.8, 22.6, 22.6, 21.0,
19.4, 18.7, 14.1, 11.8 ppm.
[0823] E0170
[0824] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 5.26-5.45 (m, 5H),
3.68-3.82 (m, 2H), 3.56-3.68 (m, 7H), 3.38-3.53 m, 4H), 3.11-3.24
(m, 1H), 2.73-2.82 (t, J=6.53 Hz, 2H), 2.56-2.69 (m, 4H), 2.50-2.56
(m, 2H), 2.32-2.41 (m, 1H), 2.09-2.27 (m, 2H), 1.77-2.09 (m, 13H),
1.42-1.63 (m, 9H), 1.21-1.42 (m, 20H), 0.83-1.21 (m, 25H), 0.68 (s,
3H) ppm.
[0825] .sup.13C NMR (400 MHz, CDCl.sub.3) .delta. 140.9, 130.2,
130.1, 128.0, 127.9, 124.4, 122.0, 121.5, 119.6, 79.5, 77.6, 77.2,
71.8, 71.6, 70.9, 70.8, 69.4, 67.3, 58.7, 56.7, 56.1, 50.7, 50.1,
42.3, 39.7, 39.5, 39.0, 37.2, 36.8, 36.1, 35.8, 34.0, 31.9, 31.8,
31.5, 29.6, 29.5, 29.4, 29.3, 28.3, 28.2, 28.0, 27.2, 27.2, 26.1,
25.6, 24.3, 23.8, 22.8, 22.6, 21.0, 19.3, 18.7, 14.1, 11.8 ppm.
[0826] E0171
[0827] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 5.27-5.46 (m, 5H),
3.58-3.82 (m, 9H), 3.38-3.54 (m, 4H), 3.13-3.26 (m, 1H), 2.74-2.82
(m, 2H), 2.31-2.50 (m, 6H), 2.16-2.2 (m, 1H), 1.70-2.10 (m, 12H),
1.24-1.65 (m, 33H), 0.97-1.23 (m, 13H), 0.82-0.97 (m, 19H), 0.68
(s, 3H) ppm.
[0828] .sup.13C NMR (400 MHz, CDCl.sub.3) .delta. 141.0, 130.2,
130.1, 127.9, 127.9, 121.5, 79.5, 77.2, 73.2, 72.5, 72.3, 71.6,
70.9, 70.8, 69.3, 67.3, 66.0, 61.9, 60.9, 60.2, 56.7, 56.1, 50.7,
50.1, 42.3, 39.8, 39.5, 39.0, 38.7, 37.2, 36.8, 36.2, 35.8, 31.9,
31.9, 31.5, 29.7, 29.5, 29.5, 29.3, 29.3, 28.3, 28.2, 28.0, 27.2,
27.2, 26.1, 25.6, 24.3, 23.8, 22.8, 22.6, 21.0, 19.4, 18.7, 14.1,
11.8 ppm.
[0829] E0010
[0830] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 5.26-5.45 (m, 5H),
3.53-3.84 (m, 9H), 3.37-3.53 (m, 4H), 3.13-3.24 (m, 1H), 2.91-3.00
(m, 1H), 2.74-2.86 (m, 2H), 2.28-2.43 (m, 2H), 2.10-2.28 (m, 2H),
1.42-2.10 (m, 32H), 0.82-1.41 (m, 50H), 0.68 (s, 3H) ppm.
[0831] .sup.13C NMR (400 MHz, CDCl.sub.3) .delta. 141.0, 130.2,
130.1, 127.9, 127.9, 121.5, 79.5, 77.2, 71.9, 71.6, 70.9, 70.8,
69.1, 67.3, 66.7, 56.8, 56.1, 55.5, 54.6, 54.0, 50.1, 42.3, 42.1,
42.0, 39.8, 39.5, 39.0, 37.2, 36.8, 36.2, 35.8, 33.2, 32.6, 31.9,
31.9, 31.5, 30.7, 29.7, 29.5, 29.5, 29.3, 29.3, 28.3, 28.2, 28.0,
27.2, 27.2, 26.2, 26.1, 25.8, 25.6, 24.3, 23.8, 22.8, 22.6, 21.0,
19.4, 18.7, 14.1, 11.8 ppm.
[0832] Other characterisation data for the compounds is set out in
the following Table 7.
TABLE-US-00015 TABLE 7 Cationic Lipid Characterization Data
Observed HPLC Expected MW TLC RT HPLC % Lipid MW (M + H) TLC Rf
Ratio TLC Solvents (min) purity Method E0001 0.77 4:6 EtOAc:Heptane
E0002 0.67 1:0 EtOAc:Heptane E0003 0.45 1:0 EtOAc:Heptane E0004
0.23 1:9 MeOH:DCM E0005 0.52 1:9 MeOH:DCM E0006 0.64 1:0
EtOAc:Heptane E0007 0.80 1:9 MeOH:DCM E0008 877.8 879.3 E0009 935.8
937.3 E0010 917.8 919.3 0.59 1:9 MeOH:DCM E0011 865.8 867.2 0.51
1:19 MeOH:DCM E0012 864.8 865.9 0.10 1:9 MeOH:DCM E0013 878.8 880.0
0.40 1:9 MeOH:DCM E0014 863.8 864.5 19.3 98.9 *1 E0015 849.8 850.9
0.62 1:9 MeOH:DCM E0016 921.8 923.0 0.49 1:9 MeOH:DCM E0017 877.8
879.0 0.51 1:9 MeOH:DCM E0018 861.8 863.3 0.54 1:9 MeOH:DCM E0019
877.8 879.0 0.54 1:9 MeOH:DCM E0020 917.8 919.3 0.15 2:1
heptane:EtOAc E0021 917.8 919.3 0.46 1:9 MeOH:DCM E0022 889.8 891.3
0.54 1:9 MeOH:DCM E0023 878.4 879.2 0.44 1:10 MeOH:EtOAc E0024
879.8 880.7 0.62 1:9 MeOH:DCM 15.3 99.4 *1 E0025 892.5 893.2 0.31
1:10 MeOH:EtOAc E0026 877.8 879.3 0.51 1:9 MeOH:DCM E0027 847.8
849.3 0.48 1:9 MeOH:DCM E0028 849.8 851.0 0.75 1:9 MeOH:DCM E0029
897.8 899.2 0.78 1:9 MeOH:DCM E0030 953.8 955.3 0.65 1:9 MeOH:DCM
E0031 882.5 882.7 0.31 1:3 EtOAc:Heptane E0032 847.7 849.2 0.43 1:9
MeOH:DCM E0033 922.5 923.2 0.53 1:10 MeOH:DCM E0034 864.4 865.1
0.30 1:10 MeOH:EtOAc E0035 863.8 865.2 0.64 1:9 MeOH:DCM E0036
876.4 877.0 0.54 1:10 MeOH:DCM E0037 893.8 895.2 0.55 1:9 MeOH:DCM
E0038 934.8 936.3 0.41 1:9 MeOH:DCM E0039 892.8 894.2 0.37 1:9
MeOH:DCM E0040 863.8 865.2 0.50 1:9 MeOH:DCM E0041 1004.8 1006.3
0.79 1:9 MeOH:DCM E0042 979.8 981.1 0.59 1:9 MeOH:DCM E0043 864.4
865.2 0.40 1:9 MeOH:DCM E0044 892.8 894.1 0.22 1:9 MeOH:DCM E0045
865.8 867.1 0.37 1:9 MeOH:DCM E0046 878.4 879.1 0.42 1:9 MeOH:DCM
E0047 892.4 893.0 0.60 1:10 MeOH:DCM E0048 863.8 865.2 0.45 1:9
MeOH:DCM E0049 863.8 865.1 0.45 1:9 MeOH:DCM E0050 925.8 927.0
0.33, 1:19 MeOH:DCM 0.40.sup.a E0051 847.4 847.9 0.39 1:3:6
MeOH:EtOAc:Heptane E0052 863.8 865.1 0.47 1:9 MeOH:DCM E0053 863.8
865.1 0.34 1:9 MeOH:DCM E0054 907.4 908.0 0.38 1:10 MeOH:DCM E0055
907.8 909.0 0.35 1:9 MeOH:DCM E0056 908.4 909.1 0.37 1:19 MeOH:DCM
E0057 878.4 880.0 0.37 1:10 MeOH:DCM E0058 908.5 909.0 0.37 1:19
MeOH:DCM E0059 864.4 864.9 0.37 1:19 MeOH:DCM E0060 964.5 965.0
0.56 4:96 MeOH:DCM 15.3 99.7 *1 E0061 821.8 822.8 5.3 97.3 *2 E0062
837.8 838.6 5.8 99.8 *2 E0063 865.8 866.7 14.2 97.4 *1 E0064 881.8
882.6 6.8 99.7 *3 E0065 865.7 866.7 15.2 97.8 *1 E0066 821.8 822.9
15.2 98.9 *1 E0067 864.4 866.0 0.37 1:19 MeOH:DCM E0068 849.8 850.5
15.7 99.5 *1 E0069 847.8 848.5 15.9 100.0 *1 E0070 847.8 848.5 15.9
99.7 *1 E0071 891.8 892.5 15.7 97.8 *1 E0072 891.8 892.5 15.7 99.6
*1 E0073 891.8 892.5 15.7 97.8 *1 E0074 921.8 922.7 20.7 92.2 *1
E0075 891.8 892.5 20.2 99.9 *1 E0076 819.7 820.5 14.9 99.7 *1 E0077
891.8 892.5 15.5 99.7 *1 E0078 893.8 894.7 20.4 97.7 *1 E0079 951.8
952.4 20.1 99.4 *1 E0080 823.7 824.7 15.2 99.8 *1 E0081 909.8 911.5
20.4 97.6 *1 E0082 907.7 909.3 15.5 99.9 *1 E0083 863.8 864.5 15.5
99.8 *1 E0084 951.8 953.5 15.3 97.4 *1 E0085 867.8 868.4 15.2 99.6
*1 E0086 895.8 897.5 15.7 99.3 *1 E0087 835.7 836.5 15.2 99.4 *1
E0088 907.8 908.7 18.0 99.7 *1 E0089 863.8 864.5 15.6 98.2 *1 E0090
907.8 908.7 15.3 99.5 *1 E0091 935.8 936.7 15.7 99.3 *1 E0092 821.7
822.5 17.5 99.8 *1 E0093 893.8 894.7 17.9 99.8 *1 E0094 893.8 894.7
18.0 99.9 *1 E0095 919.8 920.5 21.1 99.8 *1 E0096 919.8 920.5 20.6
96.8 *1 E0097 950.8 951.7 15.4 95.4 *1 E0098 1006.8 1008.6 15.1
97.8 *1 E0099 950.8 951.5 14.7 98.8 *1 E0100 978.8 980.8 14.8 97.7
*1 E0101 962.8 963.6 14.7 98.3 *1 E0102 862.7 863.7 15.1 99.5 *1
E0103 906.8 907.8 14.8 97.6 *1 E0104 919.8 920.5 15.3 99.8 *1 E0105
934.8 935.5 15.0 98.6 *1 E0106 918.8 919.7 15.3 98.5 *1 E0107 949.8
950.8 14.9 99.9 *1 E0108 1006.8 1008.5 15.0 97.9 *1 E0109 953.8
954.7 15.1 99.8 *1 E0110 919.8 920.7 15.0 98.7 *1 E0111 1013.8
1015.6 16.9 95.8 *4 E0112 893.8 894.8 15.2 99.8 *1 E0113 937.8
938.5 15.2 99.6 *1 E0114 909.8 910.5 14.5 99.0 *1 E0115 893.8 894.7
15.4 98.9 *1 E0116 909.8 910.6 15.5 99.2 *1 E0117 893.7 894.5 15.5
99.4 *1 E0118 863.8 865.2 0.47 1:9 MeOH:DCM E0119 880.5 880.6 0.84
85:5 Chloroform:MeOH E0120 920.5 920.7 0.85 9:1 Chloroform:MeOH
E0121 880.5 880.1 0.83 95:5 Chloroform:MeOH E0122 912.4 912.6 0.66
95:5 DCM:MeOH E0123 867.9 868.0 0.36 95:5 DCM:MeOH E0124 947.8
948.5 0.84 9:1 Chloroform:MeOH E0125 925.8 926.3 0.60 95:5 DCM:MeOH
E0126 907.8 908.5 0.83 95:5 DCM:MeOH E0127 907.8 908.1 0.83 85:15
Chloroform:MeOH E0128 881.8 882.5 0.61 95:5 DCM:MeOH E0129 993.8
994.4 0.29 95:5 DCM:MeOH E0130 979.8 980.1 0.60 9:1 Chloroform:MeOH
17.0 99.5 *5 E0131 879.8 880.3 0.43 9:1 Chloroform:MeOH E0132 922.8
923.0 0.43 9:1 Chloroform:MeOH E0133 934.8 935.5 0.44 9:1
Chloroform:MeOH E0134 1010.8 1012.2 0.40 95:5 DCM:MeOH E0135 936.7
937.4 0.45 9:1 Chloroform:MeOH 16.0 98.4 *6 E0136 964.8 965.5 0.46
9:1 DCM:MeOH 16.3 98.8 *6 E0137 962.8 963.8 0.43 9:1
Chloroform:MeOH 16.8 97.3 *6 E0138 909.8 910.4 0.43 95:5 DCM:MeOH
16.7 99.2 *6 E0139 865.7 866.4 0.43 95:5 DCM:MeOH 11.8 99.1 *7
E0140 923.8 924.5 0.42 95:5 DCM:MeOH 16.2 96.2 *6 E0141 950.8 951.0
0.38 95:5 DCM:MeOH 10.8 99.5 *7 E0142 1008.8 1009.7 0.38 95:5
DCM:MeOH E0143 979.8 980.9 0.46 95:5 DCM:MeOH 10.9 95.5 *7 E0144
923.8 925.0 0.47 95:5 DCM:MeOH 10.9 96.1 *7 E0145 1008.8 1010.2
0.38 95:5 DCM:MeOH 10.5 92.1 *7 E0146 909.8 910.8 0.18 50:50
EtOAc:hexane E0147 893.8 895.0 0.68 9:1 DCM:MeOH E0148 922.8 923.7
0.50 9:1 DCM:MeOH E0149 907.8 908.9 0.47 9:1 DCM:MeOH E0150 937.8
939.0 0.61 9:1 DCM:MeOH E0151 935.8 937.0 0.65 9:1 DCM:MeOH E0152
967.8 968.3 0.43 95:5 DCM:MeOH E0158 879.8 881.1 0.47 8:2
EtOAc:heptane E0159 879.8 881.0 0.41 8:2 EtOAc:heptane E0160 965.8
967.0 6.9 100.0 *8 E0161 933.8 935.0 0.43 9:1 DCM:MeOH E0162 933.8
935.0 0.60 9:1 DCM:MeOH E0163 933.8 934.9 4.2 100.0 *8 E0164 921.8
923.0 0.41 9:1 DCM:MeOH E0165 921.8 923.0 0.48 9:1 DCM:MeOH E0166
948.8 949.5 0.58 9:1 DCM:MeOH E0167 881.7 882.9 4.3 100.0 *8 E0168
974.8 975.4 0.58 9:1 DCM:MeOH E0169 936.8 938.0 0.46 9:1 DCM:MeOH
E0170 E0171 E0175 910.8 911.5 0.50 9:1 DCM:MeOH E0176 908.8 909.5
0.28 95:5 DCM:MeOH E0177 921.8 922.9 0.48 9:1 DCM:MeOH E0178 935.8
937.0 0.54 9:1 DCM:MeOH E0179 933.8 935.1 0.35 95:5 DCM:MeOH E0180
881.8 882.9 0.49 95:5 DCM:MeOH .sup.aE0050 is a mixture of
diastereomers whose two diastereomeric components have different Rf
values on silica. Method used is as follows where indicated: *1 -
Zorbax 300SB C3 150 .times. 4.6, 1 mL/min, water:MeCN w/0.1% TFA,
40 to 100% over 15 min, 100% for 5 min, ELSD detection. *2 - Zorbax
RX-SIL 250 .times. 4.6, 0.7 mL/min, hexane:EtOH 4:6, ELSD
detection. *3 - Zorbax NH2 250 .times. 4.6, 1 mL/min, hexane:IPA,
30 to 60% over 10 min, 60% for 5 min, ELSD detector. *4 - Xbridge
C8, 150 .times. 4.6 mm, 1 mL/min, water:MeCN w/0.1% TFA, 30 to 100%
over 15 min, 100% for 5 min, ELSD detection. *5 - Zorbax Eclipse
XDB-C18 250 .times. 4.6, 1 mL/min, water:MeCN w/0.1% TFA, 50% for 5
min, then 50 to 100% over 5 min, then hold at 100% for 12 min, ELSD
detector *6 - Zorbax Eclipse XDB-C18 250 .times. 4.6, 1 mL/min,
water:MeOH w/0.1% TFA, 50% for 5 min, then 50 to 100% over 5 min,
then hold at 100% for 5 min, ELSD detector. *7 - Zorbax Eclipse
XDB-C18 250 .times. 4.6, 1 mL/min, water:MeOH w/0.1% TFA, 50 to 70%
over 2 min, 70 to 100% over 3 min, then hold at 100% for 5 min,
ELSD detector. *8 - Acquity BEH Shield RP 18 50 .times. 2.1, 0.5
mL/min, 65.degree. C., water:IPA w/0.0125% TFA, 30 to 50% over 1.5
min, 50 to 75% over 10.5 min, 75 to 90% over 0.6 min, then hold at
90% for 0.4 min, CAD detector.
Example 68
Preparation of the Compositions
[0833] It is preferred that the compounds of the invention are
administered in the form of lipid nanoparticles. Thus it is
preferred that the compositions of the invention comprise lipid
nanoparticles which comprise the compounds of the invention and
optionally one or more other lipid components.
[0834] To achieve size reduction and/or to increase the homogeneity
of size in the particles, the skilled person may use the method
steps set out below, experimenting with different combinations.
Additionally, the skilled person could employ sonication,
filtration or other sizing techniques which are used in liposomal
formulations.
[0835] The process for making a composition of the invention
typically comprises providing an aqueous solution comprising a
biologically active agent in a first reservoir, providing a second
reservoir comprising an organic solution of the lipid(s) and then
mixing the aqueous solution with the organic lipid solution. The
first reservoir is optionally in fluid communication with the
second reservoir. The mixing step is optionally followed by an
incubation step, a filtration step, and a dilution and/or
concentration step.
[0836] In one embodiment, the biologically active agent(s) and/or
the lipid(s) is/are in a suitable buffer. In one embodiment, the
biologically active agent(s) is in an aqueous buffer such as a
citrate buffer. In one embodiment, the lipid(s) is in an organic
alcohol such as ethanol.
[0837] In one embodiment, the incubation step comprises allowing
the solution from the mixing step to stand in a vessel for about 0
to about 100 hours (preferably about 0 to about 24 hours) at about
rt and optionally protected from light.
[0838] In one embodiment, a dilution step follows the incubation
step. The dilution step may involve dilution with aqueous buffer
(e.g. citrate buffer) e.g., using a pumping apparatus (e.g. a
peristaltic pump).
[0839] In one embodiment, the filtration step is ultrafiltration.
In one embodiment, the ultrafiltration comprises concentration of
the diluted solution followed by diafiltration, e.g., using a
suitable pumping system (e.g. pumping apparatus such as a
peristaltic pump or equivalent thereof) in conjunction with a
suitable ultrafiltration membrane (e.g. GE Hollow fiber cartridges
or equivalent).
[0840] The process should result in the formation of lipid
nanoparticles. In one embodiment, the lipid nanoparticles comprise
the biologically active agent.
[0841] In one embodiment, the mixing step provides a clear single
phase.
[0842] In one embodiment, after the mixing step, the organic
solvent is removed to provide a suspension of particles, wherein
the biologically active agent is encapsulated by the lipid(s), e.g.
in a lipid bilayer.
[0843] The selection of an organic solvent will typically involve
consideration of solvent polarity and the ease with which the
solvent can be removed at the later stages of particle
formation.
[0844] The organic solvent, which is also used as a solubilizing
agent, is preferably in an amount sufficient to provide a clear
single phase mixture of biologically active agents and lipids.
[0845] The organic solvent may be selected from one or more (e.g.
two) of chloroform, dichloromethane, diethylether, cyclohexane,
cyclopentane, benzene, toluene, methanol, and other aliphatic
alcohols (e.g. C.sub.1 to C.sub.8) such as ethanol, propanol,
isopropanol, butanol, tert-butanol, iso-butanol, pentanol and
hexanol.
[0846] The mixing step can take place by any number of methods,
e.g., by mechanical means such as a vortex mixer.
[0847] The methods used to remove the organic solvent will
typically involve diafilitration or evaporation at reduced
pressures or blowing a stream of inert gas (e.g. nitrogen or argon)
across the mixture.
[0848] In other embodiments, the method further comprises adding
nonlipid polycations which are useful to effect the transformation
of cells using the present compositions. Examples of suitable
nonlipid polycations include, but are limited to, hexadimethrine
bromide (sold under the brandname POLYBRENE.RTM., from Aldrich
Chemical Co., Milwaukee, Wis., USA) or other salts of
hexadimethrine. Other suitable polycations include, e.g., salts of
poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine,
polyallylamine and polyethyleneimine.
[0849] In certain embodiments, the formation of the lipid
nanoparticles can be carried out either in a mono-phase system
(e.g. a Bligh and Dyer monophase or similar mixture of aqueous and
organic solvents) or in a two-phase system with suitable
mixing.
[0850] The lipid nanoparticle may be formed in a mono- or a
bi-phase system. In a mono-phase system, the cationic lipid(s) and
biologically active agent are each dissolved in a volume of the
mono-phase mixture. Combining the two solutions provides a single
mixture in which the complexes form. In a bi-phase system, the
cationic lipids bind to the biologically active agent (which is
present in the aqueous phase), and "pull" it into the organic
phase.
[0851] In one embodiment, the lipid nanoparticles are prepared by a
method which comprises: (a) contacting the biologically active
agent with a solution comprising noncationic lipids and a detergent
to form a compound-lipid mixture; (b) contacting cationic lipids
with the compound-lipid mixture to neutralize a portion of the
negative charge of the biologically active agent and form a
charge-neutralized mixture of biologically active agent and lipids;
and (c) removing the detergent from the charge-neutralized
mixture.
[0852] In one group of embodiments, the solution of neutral lipids
and detergent is an aqueous solution. Contacting the biologically
active agent with the solution of neutral lipids and detergent is
typically accomplished by mixing together a first solution of the
biologically active agent and a second solution of the lipids and
detergent. Preferably, the biologically active agent solution is
also a detergent solution. The amount of neutral lipid which is
used in the present method is typically determined based on the
amount of cationic lipid used, and is typically of from about 0.2
to 5 times the amount of cationic lipid, preferably from about 0.5
to about 2 times the amount of cationic lipid used.
[0853] The biologically active agent-lipid mixture thus formed is
contacted with cationic lipids to neutralize a portion of the
negative charge which is associated with the molecule of interest
(or other polyanionic materials) present. The amount of cationic
lipids used is typically the amount sufficient to neutralize at
least 50% of the negative charge of the biologically active agent.
Preferably, the negative charge will be at least 70% neutralized,
more preferably at least 90% neutralized.
[0854] The methods used to remove the detergent typically involve
dialysis. When organic solvents are present, removal is typically
accomplished by diafilitration or evaporation at reduced pressures
or by blowing a stream of inert gas (e.g. nitrogen or argon) across
the mixture.
[0855] There is herein disclosed an apparatus for making a
composition of the present invention. The apparatus typically
includes a first reservoir for holding an aqueous solution
comprising a biologically active agent and a second reservoir for
holding an organic lipid solution. The apparatus also typically
includes a pump mechanism configured to pump the aqueous and the
organic lipid solutions into a mixing region or mixing chamber at
substantially equal flow rates. In one embodiment, the mixing
region or mixing chamber comprises a T coupling or equivalent
thereof, which allows the aqueous and organic fluid streams to
combine as input into the T connector and the resulting combined
aqueous and organic solutions to exit out of the T connector into a
collection reservoir or equivalent thereof.
Example 69
Example Method of Making Compositions
[0856] The lipid nanoparticles (LNPs) are formed by mixing equal
volumes of lipids dissolved in alcohol with siRNA dissolved in a
citrate buffer by an impinging jet process. The lipid solution
contains a cationic lipid compound of the invention or a
comparative lipid, a helper lipid (cholesterol), an optional
neutral lipid (DSPC) and a PEG (PEG) lipid at a concentration of
8-16 mg/mL with a target of 12 mg/mL in an alcohol. The relative
molar ratios of each lipid component in the formulations of this
invention are reported in Tables 8 through 11. Where a LNP
formulation contains four lipid components, the molar ratios
correspond to the type of lipid as it appears in the first four
columns of the table, in the order that they appear. Where a LNP
formulation contains three lipid components, there is no neutral
lipid.
[0857] The ratio of the lipids ranges from 20 to 70 mole percent
for the cationic lipid with a target of 40-60, the mole percent of
helper lipid ranges from 20 to 70 with a target of 30 to 50, the
mole percent of neutral lipid ranges from 0-30, the mole percent of
PEG lipid has a range from 1 to 6 with a target of 2 to 5. The
concentration of siRNA solution ranges from 0.7 to 1.0 mg/mL with a
target of 0.8 to 0.9 mg/mL in a sodium citrate:sodium chloride
buffer pH 4. The LNPs are formed by mixing equal volumes of lipid
solution in ethanol with siRNA dissolved in a citrate buffer by an
impinging jet process through tubings with ID ranging from 0.25 to
2.0 mm at a total flow rate from 10 to 120 mL/min. The mixed LNP
solution is held at rt for 0-48 hrs prior to a dilution step. The
solution is then concentrated and diafiltered with suitable buffer
by ultrafiltration process using membranes with a MW cutoff from 30
to 100 KD. The final product is sterile filtered and stored at
4.degree. C.
Example 70
Transfection In Vivo in a Mouse Model
[0858] Female CD-1 mice are received from Charles River Labs and
maintained on standard lab chow and water ad libitum. The animals
weigh approximately 25 grams at time of dosing. Formulated siRNA is
administered as a single dose at various dose levels intravenously
via the lateral tail vein calculated on a (mg siRNAs/kg) basis
according to individual animal weights (10 ml/kg injection
volume).
[0859] Formulated siRNAs are made up of double stranded siRNA
sequences specific to a target mRNA sequence, and are in the form
of lipid nucleotid particles (LNPs) containing cationic lipids,
stealth lipids and neutral lipids, as provided in the Examples
below. The siRNA construct for use in targeting the liver is
specific to Factor VII. The siRNA construct for use in targeting
tumors is specific to PLK1-424, which is published by Judge et al.,
See, J Clin Invest. 2009 March; 119(3):661-73; doi:
10.1172/JCI37515).
TABLE-US-00016 1. FVII siRNA duplex sequence 5' UUu AAU UGA AAC cAA
GAc Auu 3' (SEQ ID NO: 1) 5' uGu cuu GGu uuc AAu uAA Auu 3' (SEQ ID
NO: 2) 2. PLK1-424 siRNA duplex sequence 5' UAU UUA AgG AGG GUG AuC
Uuu 3' (SEQ ID NO: 3) 5' AGA Uca cCC Ucc uuA AAU auu 3' (SEQ ID NO:
4)
[0860] The following abbreviations are used in these sequences:
[0861] A=adenosine [0862] U=uridine [0863] G=guanosine [0864]
C=cytosine [0865] a=2'-O-methyl-adenosine [0866]
u=2'-O-methyl-uridine [0867] g=2'-O-methyl-guanosine [0868]
c=2'-O-methyl-cytosine
Example 71
Factor VII Activity Assay
[0869] Formulated Factor VII siRNA is administered as a single dose
at various dose levels intravenously via the lateral tail vein
calculated on a mg siRNAs/kg basis according to individual animal
weights (10 ml/kg injection volume). Approximately 48 h after
injection, the mice are euthanized by CO.sub.2 inhalation followed
by exsanguinations through the vena cava. The blood is collected in
tubes containing 0.105M sodium citrate anticoagulant for plasma
Factor VII activity analysis. In some cases, small pieces
(.about.50 mg) of liver are collected and snap frozen in liquid
nitrogen for follow up mRNA quantitation.
[0870] Plasma collected from injected mice is assayed for Factor
VII activity using the Biophen FVII kit from Hyphen Biomedical
(catalog number 221304). An assay standard curve is prepared using
pooled plasma aliquots from the vehicle control animals. All
samples are diluted to fall within the linear range of the standard
curve and a relative Factor VII activity is reported.
[0871] In some cases, total liver RNA is prepared using Qiagen's
RNeasy isolation kit (catalog number 74106) according to the
manufacture's protocol. Factor VII mRNA is analyzed by quantitative
PCR and normalized to GAPDH. Applied Biosystems Factor VII gene
expression assay Mm00487333_m1 and mouse GAPDH endogenous control
cat number 4352339E are used for mRNA detection.
[0872] The results for the FVII assay are set out in the following
Table 8.
TABLE-US-00017 TABLE 8 Factor VII assay - In vivo liver results
Lipid ratio Final Neutral Helper Stealth (molar N/P size dose FVII
Lipid lipid.sup.1 lipid lipid.sup.2,3 ratio).sup.4 ratio (nm) PDI
pKa (mg/kg) inhib % E0001 DSPC CHOL AVANTI 50/28/18/4 3.43 71 0.101
4.84 3 27.6 E0001 CHOL AVANTI 50/0/46/4 3.43 77 0.16 4.84 3 20.1
E0002 DSPC CHOL AVANTI 50/28/18/4 3.43 109 0.221 3 10.4 E0002 DSPC
CHOL AVANTI 50/28/18/4 3.43 109 0.221 1 -0.3 E0003 DSPC CHOL AVANTI
50/28/18/4 3.43 106 0.224 3 2.9 E0004 CHOL AVANTI 50/0/46/4 3.43
186 0.201 7.48 3 41 E0004 DSPC CHOL AVANTI 50/28/18/4 3.43 142
0.113 7.48 3 23.4 E0005 CHOL AVANTI 50/0/46/4 3.43 146 0.07 1.5
59.9 E0005 DSPC CHOL AVANTI 50/28/18/4 3.43 168 0.069 3 12.2 E0006
CHOL AVANTI 50/0/46/4 3.43 95 0.142 4.88 3 21.2 E0006 DSPC CHOL
AVANTI 50/28/18/4 3.43 146 0.04 4.88 3 15.7 E0007 CHOL AVANTI
50/0/46/4 3.43 138 0.148 5.84 3 76.8 E0007 DSPC CHOL AVANTI
50/28/18/4 3.43 92 0.178 5.84 3 72.2 E0008 DSPC CHOL AVANTI
50/28/18/4 3.43 85 0.168 5.81 3 73 E0008 CHOL AVANTI 50/0/46/4 3.43
84 0.214 5.81 3 84.5 E0009 CHOL AVANTI 50/0/46/4 3.43 82 0.16 4.89
3 6.9 E0009 DSPC CHOL AVANTI 50/28/18/4 3.43 83 0.2 4.89 3 38.4
E0010 CHOL AVANTI 50/0/46/4 3.43 80 0.201 5.85 3 65.1 E0010 DSPC
CHOL AVANTI 50/28/18/4 3.43 90 0.184 5.85 3 46.8 E0011 DSPC CHOL
AVANTI 50/28/18/4 3.43 111 0.221 5.32 3 86.5 E0011 CHOL AVANTI
50/0/46/4 3.43 108 0.23 5.32 3 92.3 E0012 DSPC CHOL AVANTI
30/30/36/4 2.3 220 0.105 3 9.3 E0014 CHOL GM-020 60/0/36/4 3 101.5
0.182 6.4 1 68.5 E0014 CHOL AVANTI 50/0/46/4 3.43 148 0.05 6.4 3
97.4 E0014 DSPC CHOL AVANTI 50/28/18/4 3.43 104 0.151 6.4 3 98.6
E0014 DSPC CHOL AVANTI 50/28/18/4 3.43 104 0.151 6.4 1 72.1 E0014
DSPC CHOL AVANTI 50/28/18/4 3.43 95 0.148 6.4 3 95.8 E0014 CHOL
GM-020 60/0/36/4 3 94.6 0.092 6.4 3 94 E0014 CHOL GM-020 60/0/36/4
3 94.6 0.092 6.4 1 37.6 E0014 CHOL GM-020 60/0/34/6 3 85.8 0.106
6.4 3 96.6 E0014 CHOL GM-020 60/0/34/6 3 85.8 0.106 6.4 1 25 E0014
CHOL GM-020 60/0/38/2 3 141 0.122 6.4 3 96.2 E0014 CHOL GM-020
60/0/38/2 3 141 0.122 6.4 1 71.5 E0015 CHOL AVANTI 50/0/46/4 3.43
130 0.05 7.39 3 77.1 E0015 DSPC CHOL AVANTI 50/28/18/4 3.43 164
0.06 7.39 3 30.7 E0016 CHOL AVANTI 50/0/46/4 3.43 178 0.206 5.85 3
59.9 E0016 DSPC CHOL AVANTI 50/28/18/4 3.43 97 0.175 5.85 3 88.8
E0017 CHOL AVANTI 50/0/46/4 3.43 97 0.19 6.46 3 96.5 E0017 DSPC
CHOL AVANTI 50/28/18/4 3.43 106 0.17 6.46 3 98.3 E0017 DSPC CHOL
AVANTI 50/28/18/4 3.43 106 0.17 6.46 1 53.8 E0018 DSPC CHOL AVANTI
50/28/18/4 3.43 119 0.095 5.9 3 91.8 E0018 CHOL AVANTI 50/0/46/4
3.43 103 0.113 5.9 3 98 E0018 CHOL AVANTI 50/0/46/4 3.43 140 0.13
5.9 1 40.5 E0019 DSPC CHOL AVANTI 50/28/18/4 3.43 143 0.065 6.75 3
76.7 E0019 CHOL AVANTI 50/0/46/4 3.43 90.5 0.164 6.75 3 85.2 E0019
CHOL AVANTI 50/0/46/4 3.43 180 0.19 6.75 1 33.7 E0020 CHOL AVANTI
50/0/46/4 3.43 81 0.084 6.08 3 51.4 E0020 DSPC CHOL AVANTI
50/28/18/4 3.43 83 0.138 6.08 3 62.4 E0021 CHOL AVANTI 50/0/46/4
3.43 72 0.184 5.13 3 52.8 E0021 DSPC CHOL AVANTI 50/28/18/4 3.43 85
0.22 5.13 3 24.5 E0022 CHOL AVANTI 50/0/46/4 3.43 84 0.192 3 87.8
E0022 DSPC CHOL AVANTI 50/28/18/4 3.43 93 0.143 3 49.4 E0023 CHOL
AVANTI 50/0/46/4 3.43 99 0.122 5.85 3 68 E0023 DSPC CHOL AVANTI
50/28/18/4 3.43 95 0.221 5.85 3 59.1 E0024 CHOL AVANTI 50/0/46/4
3.43 149 0.092 6.62 3 99.5 E0024 DSPC CHOL AVANTI 50/28/18/4 3.43
100 0.148 6.62 3 99.9 E0024 DSPC CHOL AVANTI 50/28/18/4 3.43 100
0.148 6.62 1 85.3 E0024 CHOL AVANTI 50/0/46/4 3.43 125 0.08 6.62 1
95.7 E0024 CHOL S010 60/0/37/3 3 96.09 0.137 6.62 1 72.2 E0024 CHOL
S006 60/0/37/3 3 98.53 0.12 6.62 1 83.8 E0025 CHOL AVANTI 50/0/46/4
3.43 73 0.12 5.45 3 94.2 E0025 DSPC CHOL AVANTI 50/28/18/4 3.43 97
0.22 5.45 3 63 E0026 CHOL AVANTI 50/0/46/4 3.43 87 0.23 5.85 3 95.1
E0027 CHOL AVANTI 50/0/46/4 3.43 65 0.15 5.71 3 30.3 E0027 DSPC
CHOL AVANTI 50/28/18/4 3.43 146 0.311 5.71 3 36.3 E0028 CHOL AVANTI
50/0/46/4 3.43 68 0.125 4.8 3 38.7 E0028 DSPC CHOL AVANTI
50/28/18/4 3.43 129 0.216 4.8 3 12.5 E0029 CHOL AVANTI 50/0/46/4
3.43 113 0.08 4.95 3 -19.1 E0029 DSPC CHOL AVANTI 50/28/18/4 3.43
137 0.04 4.95 3 12.4 E0030 CHOL AVANTI 50/0/46/4 3.43 155 0.08 5.38
3 12.4 E0031 CHOL AVANTI 50/0/46/4 3.43 65 0.11 4.8 3 27.1 E0031
DSPC CHOL AVANTI 50/28/18/4 3.43 108 0.23 4.8 3 36.7 E0032 CHOL
AVANTI 50/0/46/4 3.43 63 0.21 6.8 3 85.5 E0032 DSPC CHOL AVANTI
50/28/18/4 3.43 87 0.18 6.8 3 64.5 E0033 CHOL AVANTI 50/0/46/4 3.43
105 0.05 3 15.3 E0033 DSPC CHOL AVANTI 25/28/18/4 1.9 161 0.16 3
-36.4 E0034 CHOL AVANTI 50/0/46/4 3.43 99 0.16 3 88.1 E0034 DSPC
CHOL AVANTI 50/28/18/4 3.43 99 0.16 3 81.3 E0035 CHOL AVANTI
50/0/46/4 3.43 79 0.21 5.93 3 66.8 E0035 DSPC CHOL AVANTI
50/28/18/4 3.43 103 0.2 5.93 3 22 E0036 CHOL AVANTI 50/0/46/4 3.43
76 0.24 5.74 3 -23.1 E0036 DSPC CHOL AVANTI 50/28/18/4 3.43 122
0.12 5.74 3 -47 E0037 CHOL AVANTI 50/0/46/4 3.43 117 0.1 3 66.2
E0037 DSPC CHOL AVANTI 50/28/18/4 3.43 96 0.24 3 69.3 E0038 CHOL
AVANTI 50/0/46/4 3.43 115 0.08 3 -6.2 E0038 DSPC CHOL AVANTI
50/28/18/4 3.43 116 0.25 3 -2.4 E0039 CHOL AVANTI 50/0/46/4 3.43 94
0.06 5.88 3 -5.4 E0039 DSPC CHOL AVANTI 50/28/18/4 3.43 113 0.15
5.88 3 -6.7 E0040 DSPC CHOL AVANTI 50/28/18/4 3.43 87 0.21 6.13 3
91.8 E0040 CHOL AVANTI 50/0/46/4 3.43 103 0.15 6.13 3 97.8 E0040
CHOL AVANTI 50/0/46/4 3.43 101 0.17 6.13 1 49.2 E0041 CHOL AVANTI
50/0/46/4 3.43 93 0.21 4.8 3 17.6 E0041 DSPC CHOL AVANTI 50/28/18/4
3.43 95 0.37 4.8 3 -7.4 E0042 CHOL AVANTI 50/0/46/4 3.43 73 0.19
6.7 3 84.5 E0042 DSPC CHOL AVANTI 50/28/18/4 3.43 83 0.11 6.7 3
49.1 E0043 DSPC CHOL AVANTI 50/28/18/4 3.43 110 0.24 6.33 3 95.7
E0043 CHOL AVANTI 50/0/46/4 3.43 76 0.15 6.33 3 -11.7 E0045 DSPC
CHOL AVANTI 50/28/18/4 3.43 100 0.18 4.73 3 10.4 E0045 CHOL AVANTI
50/0/46/4 3.43 103 0.25 4.73 3 90.7 E0046 CHOL AVANTI 50/0/46/4
3.43 194 0.116 5.75 3 12.7 E0047 CHOL AVANTI 50/0/46/4 3.43 112
0.061 5.55 3 -3.4 E0048 CHOL AVANTI 50/0/46/4 3.43 225 0.115 6.56 3
88.5 E0049 CHOL AVANTI 50/0/46/4 3.43 237 0.09 6.85 3 78.8 E0050
DSPC CHOL AVANTI 50/28/18/4 3.43 100 0.21 4.9 3 -7.2 E0050 CHOL
AVANTI 50/0/46/4 3.43 103 0.097 4.9 3 -10.4 E0051 CHOL AVANTI
50/0/46/4 3.43 142.9 0.057 5.73 3 77.5 E0051 DSPC CHOL AVANTI
50/28/18/4 3.43 157.7 0.131 5.73 3 17 E0052 DSPC CHOL AVANTI
50/28/18/4 3.43 100.4 0.185 6.95 3 98.1 E0052 DSPC CHOL AVANTI
50/28/18/4 3.43 100.4 0.185 6.95 1 69.8 E0053 DSPC CHOL AVANTI
50/28/18/4 3.43 103.3 0.221 6.95 3 90.1 E0053 DSPC CHOL AVANTI
50/28/18/4 3.43 103.3 0.221 6.95 1 -9.4 E0054 DSPC CHOL AVANTI
50/28/18/4 3.43 103 0.2 6.58 3 97.8 E0054 CHOL AVANTI 50/0/46/4
3.43 96 0.13 6.58 3 98.9 E0054 DSPC CHOL AVANTI 50/28/18/4 3.43 103
0.2 6.58 1 57.5 E0054 CHOL AVANTI 50/0/46/4 3.43 96 0.13 6.58 1 63
E0055 DSPC CHOL AVANTI 50/28/18/4 3.43 113 0.24 6.78 3 96.4 E0055
CHOL AVANTI 50/0/46/4 3.43 380 0.22 6.78 3 69.8 E0055 DSPC CHOL
AVANTI 50/28/18/4 3.43 113 0.24 6.78 1 28.2 E0055 CHOL AVANTI
50/0/46/4 3.43 380 0.22 6.78 1 14.8 E0060 CHOL GM-020 60/0/36/4 3
90.25 0.171 6.43 1 0.0 E0061 CHOL GM-020 60/0/36/4 3 139.4 0.073
6.28 1 52.6 E0062 CHOL GM-020 60/0/36/4 3 145.1 0.125 6.45 1 64.8
E0063 CHOL GM-020 60/0/36/4 3 155.5 0.09 6.48 1 33.3 E0064 CHOL
GM-020 60/0/36/4 3 169.2 0.144 6.83 1 37.2 E0065 CHOL GM-020
60/0/36/4 3 153.01 0.148 6.8 1 41.1 E0066 CHOL GM-020 60/0/36/4 3
140.7 0.071 6.5 1 44.2 E0068 CHOL GM-020 60/0/36/4 3 174.0 0.041
4.82 1 28.6 E0069 CHOL GM-020 60/0/36/4 3 113.4 0.199 6.07 1 29
E0070 CHOL GM-020 60/0/36/4 3 150.9 0.045 6.40 1 50.3 E0071 CHOL
GM-020 60/0/36/4 3 188.2 0.151 6.47 1 30.8 E0073 CHOL GM-020
60/0/36/4 3 130.1 0.118 5.66 1 37.4 E0075 CHOL GM-020 60/0/36/4 3
108.3 0.098 5.79 1 31.5 E0076 CHOL GM-020 60/0/36/4 3 89.47 0.143
5.85 1 29.3 E0077 CHOL GM-020 60/0/36/4 3 109.8 0.127 5.79 1 26.8
E0078 CHOL GM-020 60/0/36/4 3 122.6 0.098 5.72 1 51.9 E0079 CHOL
GM-020 60/0/36/4 3 106.2 0.052 6.79 1 33.5 E0082 CHOL GM-020
60/0/36/4 3 105.1 0.303 6.81 1 49.7 E0083 CHOL GM-020 60/0/36/4 3
113.4 0.199 6.79 1 55.6 E0084 CHOL GM-020 60/0/36/4 3 112.1 0.1
7.27 1 60.7 E0087 CHOL GM-020 60/0/36/4 3 119.2 0.148 6.38 1 69
E0088 CHOL GM-020 60/0/36/4 3 110.7 0.080 5.79 1 50.8 E0094 CHOL
GM-020 60/0/36/4 3 115.5 0.076 5.91 1 36.3 E0095 CHOL GM-020
60/0/36/4 3 125.1 0.091 5.84 1 13 E0096 CHOL GM-020 60/0/36/4 3
128.8 0.162 5.77 1 14.4 E0115 CHOL GM-020 60/0/36/4 3 162.5 0.098
6.41 1 68.8 E0118 DSPC CHOL AVANTI 50/28/18/4 3.43 88 0.21 6.88 3
98.1 E0118 CHOL AVANTI 50/0/46/4 3.43 93 0.23 6.88 3 99.2 E0118
CHOL AVANTI 50/0/46/4 3.43 98 0.22 6.88 1 76.1 E0170 CHOL AVANTI
50/0/46/4 3.43 170 0.05 3 14.3 E0170 DSPC CHOL AVANTI 50/28/18/4
3.43 133 0.095 3 9.3 E0171 CHOL AVANTI 50/0/46/4 3.43 90 0.18 5.85
3 54.1 E0172 CHOL AVANTI 50/0/46/4 3.43 129 0.184 8.5 3 40.8 E0172
DSPC CHOL AVANTI 50/28/18/4 3.43 170 0.181 8.5 3 21 E0173 CHOL
AVANTI 50/0/46/4 3.43 76 0.16 6.73 3 98.5 E0173 DSPC CHOL AVANTI
50/28/18/4 3.43 98 0.17 6.73 3 90.9 E0173 CHOL AVANTI 50/0/46/4
3.43 76 0.16 6.73 1 87.4 E0174 DSPC CHOL AVANTI 50/28/18/4 3.43 117
0.23 5.15 3 63.2 E0174 CHOL AVANTI 50/0/46/4 3.43 90 0.21 5.15 3
3.5 .sup.1wherein a blank cell indicates that no neutral lipid is
present .sup.2wherein AVANTI represents AVANTI 880150P
.sup.3wherein GM-020 represents GM-020 NOF .sup.4wherein the order
of the lipids types as they appear in the molar ratio corresponds
to the order in which the lipids appear in the first four columns
of the table. Where only three lipids are listed in the molar
ratio, the neutral lipid is absent.
Example 72
Hep3B Tumor Studies
[0873] Hep3B tumors are established in female nude mice by sc
injection of 7.times.10.sup.6 cells in 100 ul PBS into the left
flank. Mice are randomized into treatment groups 10-14 days after
seeding as tumors reached an average size of 100 mm.sup.3. siRNA
formulated LNP formulations are administered at various dose levels
intravenously via the lateral tail vein calculated on a mg
siRNAs/kg basis according to individual animal weights (10 ml/kg
injection volume). Mice are euthanized by CO.sub.2 inhalation at
various time points and the tumors are harvested for mRNA
quantitation. Lipids with a wide range of pKa (5.3-6.6) are used in
the LNP formulations. Tumors are measured in 2 dimensions
(width.times.length) to assess tumor growth using digital calipers.
Tumor volume is calculated using the equation
[a.times.b.times.b/2], where a equals the largest diameter and b
equals the smallest diameter.
Example 73
HepG2 Liver Tumor Studies
[0874] HepG2 tumors are established in female nude mice by sc
injection of 5.times.10.sup.6 cells in 100 ul PBS into the left
flank. Mice are randomized into treatment groups 10-14 days after
seeding as tumors reached an average size of 150 mm.sup.3. siRNA
formulated LNP formulations are administered at 3.times.3 mg/kg
dose levels intravenously via the lateral tail vein calculated on a
mg siRNAs/kg basis according to individual animal weights (10 ml/kg
injection volume). Mice are euthanized by CO.sub.2 inhalation at
various time points and the tumors are harvested 24 hrs after the
last dose for mRNA quantitation. Lipids with a wide range of pKa
(5.3-6.6) are used in the LNP formulations. Tumors are measured in
2 dimensions (width.times.length) to assess tumor growth using
digital calipers. Tumor volume is calculated using the equation
[a.times.b.times.b/2], where a=largest diameter and b=smallest
diameter.
Example 74
786-0 Renal Tumor Studies
[0875] 786-0 tumors are established as described before by sc
injection of 10.times.10.sup.6 cells in 200 ul PBS. 4 weeks post
implantation, the mice with the tumor size ranging from 200-250
mm.sup.3 are randomized into treatment groups. siRNA formulated
LNPs are then intravenously administered at either 5 or 10 mg/kg
dose levels. 48 hrs after the single dose, tumors are collected for
mRNA quantitation.
Example 75
Measurement of PLK-1 and GAPDH mRNA KD in Tumor Tissues
[0876] About 30-50 mg tumor tissue is homogenized in tissue lysis
buffer in a Qiagen homogenizer followed by centrifugation to
clarify lysates. Total RNA is isolated using RNeasy isolation kit
(catalog number 74106) according to the manufacture's protocol.
PLK-1 mRNA is analyzed by quantitative PCR and normalized to human
GAPDH. Applied Biosystems human PLK-1 gene expression assay
Hs00983229_ml and human GAPDH endogenous control cat number
4326317E are used for mRNA detection.
[0877] The results for knock down ("KD") of PLK1 mRNA levels in the
tumor experiments of Examples 72, 73, 74 and 75 are set out in the
following Tables and calculated as percent inhibition (PLK1%
inhibition). Results for targetting Hep3B liver tumors are reported
in Table 9. Results for targetting HepG2 liver tumors are reported
in Table 10. Results for targetting 786-0 renal tumors are reported
in Table 11. The pKa in all tables refers to the pKa of the
cationic lipid. Where multiple doses are indicated (e.g.,
3.times.5), the first number indicates the number of doses given
and the second number indicates the amount per dose in mg siRNA
(biologically active agent) per kg mouse. In general the multiple
doses are administered at 24 hour intervals, and tissues for mRNA
quantitation are harvested 24 hours after the last dose.
TABLE-US-00018 TABLE 9 Hep3B Tumor In vivo Assay results Lipid
Ratio Lipid Helper Neutral Stealth (molar N/P Final size # doses
.times. PLK1 ref. lipid Lipid.sup.1 lipid ratio).sup.2 (ratio) (nm)
pKa (mg/kg) inhibition % E0008 Chol S011 60/38/2 4 93.11 5.81 1
.times. 5 85 E0008 Chol S004 60/38/2 4 123.1 5.81 1 .times. 1 38
E0011 Chol DSPC S011 40/48/10/2 4 83.45 5.32 1 .times. 10 56 E0011
Chol S019 60/38/2 4 110 5.32 1 .times. 1 36 E0011 Chol S020 60/38/2
4 74.42 5.32 1 .times. 1 32 E0011 Chol S011 60/38/2 4 84.58 5.32 1
.times. 10 67 E0011 Chol S011 60/38/2 4 84.58 5.32 3 .times. 10 71
E0011 Chol S011 60/38/2 4 84.58 5.32 3 .times. 5 68 E0011 Chol DSPC
S004 40/48/10/2 4 85.09 5.32 1 .times. 10 65 E0011 Chol DSPC S012
40/48/10/2 4 91.46 5.32 1 .times. 10 18 E0011 Chol DSPC S002
40/48/10/2 4 89.73 5.32 1 .times. 10 37 E0011 Chol DSPC S003
40/48/10/2 4 100.5 5.32 1 .times. 10 42 E0011 Chol DSPC S011
40/53/5/2 4 100 5.32 1 .times. 10 60 E0011 Chol DSPC S011
40/43/15/2 4 94.21 5.32 1 .times. 10 44 E0011 Chol DSPC S011
40/38/20/2 4 95.4 5.32 1 .times. 10 38 E0011 Chol S011 60/38/2 4
83.01 5.32 1 .times. 5 69 E0011 Chol S011 60/38/2 4 83.02 5.32 1
.times. 2 46 E0011 Chol S004 60/38/2 4 95.47 5.32 1 .times. 10 70
E0011 Chol S004 60/38/2 4 95.47 5.32 3 .times. 5 67 E0011 Chol S004
60/38/2 4 96.31 5.32 1 .times. 2 56 E0011 Chol S004 60/38/2 4 96.31
5.32 1 .times. 5 66 E0011 Chol S007 60/38/2 4 94.2 5.32 1 .times. 2
65 E0011 Chol S009 60/38/2 4 98.41 5.32 1 .times. 2 64 E0011 Chol
S008 60/38/2 4 101.5 5.32 1 .times. 2 60 E0011 Chol S005 60/38/2 4
95.26 5.32 1 .times. 2 52 E0011 Chol S011 55/43/2 4 84.58 5.32 1
.times. 2 59 E0011 Chol S011 55/43/2 4 84.58 5.32 1 .times. 5 66
E0011 Chol S011 50/48/2 4 84.76 5.32 1 .times. 2 58 E0011 Chol S011
50/48/2 4 84.76 5.32 1 .times. 5 67 E0014 Chol DSPC S011 40/48/10/2
4 82.21 6.4 1 .times. 10 22 E0061 Chol S004 60/38/2 4 90.09 6.28 1
.times. 1 23 E0024 Chol DSPC S011 40/48/10/2 3 88.46 6.62 1 .times.
10 15 E0024 Chol S004 60/38/0/2 3 120.6 6.62 4 .times. 5 23 E0025
Chol S011 60/38/2 4 95.73 5.45 1 .times. 5 84 E0025 Chol DSPC S011
40/48/10/2 4 85.63 5.45 1 .times. 5 77 E0025 Chol DSPC S004
45/43/10/2 4 118.8 5.45 1 .times. 1 27 E0026 Chol S011 60/38/2 4
105.8 5.85 1 .times. 5 75 E0051 Chol DSPC S011 40/48/10/2 4 85.77
5.73 1 .times. 10 17 E0095 Chol S004 50/48/2 4 96.09 5.84 1 .times.
1 58 E0095 Chol S004 50/48/2 4 96.09 5.84 1 .times. 0.1 23 E0095
Chol S004 50/48/2 4 96.09 5.84 1 .times. 1 56 E0075 Chol S011
60/38/2 4 124.5 5.79 1 .times. 5 75 E0076 Chol S011 60/38/2 4 89.89
5.85 1 .times. 5 85 E0076 Chol S004 50/48/2 4 86.03 5.85 1 .times.
1 54 E0076 Chol S004 50/48/2 4 86.03 5.85 1 .times. 1 42 E0076 Chol
DSPC S004 45/43/10/2 4 80.98 5.85 1 .times. 1 19 E0076 Chol DSPC
S004 45/43/10/2 4 80.98 5.85 1 .times. 0.1 0 E0077 Chol S011
60/38/2 4 120.8 5.79 1 .times. 5 77 E0085 Chol DSPC S004 45/43/10/2
4 76.8 5.32 1 .times. 1 22 E0085 Chol DSPC S004 50/43/5/2 4 87.74
5.32 1 .times. 1 30 E0085 Chol DSPC S004 50/43/5/2 4 87.74 5.32 1
.times. 1 34 E0085 Chol DSPC S004 50/43/5/2 4 87.74 5.32 1 .times.
0.1 53 E0085 Chol S004 50/48/0/2 4 96.42 5.32 1 .times. 1 42 E0085
Chol DSPC S004 50/38/10/2 4 88.33 5.32 1 .times. 1 20 E0085 Chol
DSPC S011 40/48/10/2 4 81.83 5.32 1 .times. 10 55 E0085 Chol S004
60/38/2 4 90.06 5.32 1 .times. 2 62 E0085 Chol S004 60/38/2 4 90.06
5.32 1 .times. 5 69 E0088 Chol S011 60/38/2 4 146.2 5.79 1 .times.
5 71 E0093 Chol DSPC S011 40/48/10/2 4 82.42 4.85 1 .times. 10 2
E0104 Chol DSPC S004 45/43/10/2 4 87.59 6.1 1 .times. 1 48 E0104
Chol DSPC S004 45/43/10/2 4 87.59 6.1 1 .times. 0.1 34 E0104 Chol
DSPC S004 50/43/5/2 4 92.19 6.1 1 .times. 1 34 E0104 Chol DSPC S004
50/43/5/2 4 92.19 6.1 1 .times. 0.1 22 E0104 Chol S004 60/38/2 4
106.2 6.1 1 .times. 1 47 E0104 Chol S004 50/48/2 3 96.19 6.1 1
.times. 1 48 E0104 Chol S020 50/48/2 4 75.56 6.1 1 .times. 1 45
E0104 Chol S004 50/48/2 4 90.77 6.1 1 .times. 0.1 29 E0104 Chol
S004 50/48/2 4 90.77 6.1 1 .times. 1 55 E0104 Chol S004 50/48/2 4
95.05 6.1 1 .times. 1 50 E0104 Chol S004 50/48/2 4 95.05 6.1 1
.times. 0.1 15 E0104 Chol S004 50/48/2 4 95.05 6.1 1 .times. 1 50
E0104 Chol S004 50/48/2 4 95.05 6.1 1 .times. 1 40 E0104 Chol S004
50/48/2 4 90.87 6.1 1 .times. 1 56 E0102 Chol S004 60/38/2 4 94.69
6.31 1 .times. 1 37 E0102 Chol S004 60/38/2 4 94.69 6.31 1 .times.
1 47 E0045 Chol S004 60/38/2 4 103.2 4.73 1 .times. 1 36 E0119 Chol
S004 50/48/2 4 99.37 5.82 1 .times. 1 40 E0120 Chol S004 50/48/2 4
87 5.48 1 .times. 1 28 E0125 Chol S004 50/48/2 4 94.13 5.53 1
.times. 1 36 E0125 Chol DSPC S004 50/43/5/2 4 92.84 5.53 1 .times.
1 20 E0125 Chol DSPC S004 50/38/10/2 4 101.5 5.53 1 .times. 1 34
E0128 Chol S004 50/48/2 4 83.47 5.01 1 .times. 1 23 E0151 Chol S004
50/48/2 4 103.8 6.06 1 .times. 1 25 E0151 Chol S004 50/48/2 4 103.8
6.06 1 .times. 0.1 7 E0152 Chol S004 50/48/2 4 121.3 5.38 1 .times.
1 18 E0160 Chol S004 50/48/2 4 100.8 6.05 1 .times. 1 38 E0161 Chol
S004 50/48/2 4 101.4 6.3 1 .times. 1 45 E0167 Chol S004 50/48/2 4
86.57 5.21 1 .times. 1 23 E0175 Chol S004 60/38/2 4 78.74 5.67 1
.times. 1 42 E0175 Chol S020 60/38/2 4 69.33 5.67 1 .times. 1 44
E0175 Chol S004 50/48/2 4 67.98 5.67 1 .times. 1 41 E0175 Chol DSPC
S004 50/43/5/2 3.8 93.51 5.67 1 .times. 1 46 E0175 Chol
resorcinol.sup.3 S004 50/43/5/2 3.8 104.2 5.67 1 .times. 1 34 E0176
Chol S004 60/38/2 4 99.14 5.56 1 .times. 1 36 E0177 Chol S004
50/48/2 4 91.91 6.1 1 .times. 1 41 E0177 Chol S004 50/48/2 4 91.91
6.1 1 .times. 1 30 E0177 Chol S004 50/48/2 4 91.91 6.1 1 .times.
0.1 9 E0177 Chol DSPC S004 50/43/5/2 4 95.92 6.1 1 .times. 1 45
E0177 Chol DOPC S004 50/43/5/2 4 89.78 6.1 1 .times. 1 0 E0177 Chol
DSPC S004 50/38/10/2 4 88.15 6.1 1 .times. 1 36 E0178 Chol S004
50/48/2 4 88.39 5.92 1 .times. 1 35 E0178 Chol S004 50/48/2 4 88.39
5.92 1 .times. 0.1 0 E0178 Chol S004 50/48/2 4 88.39 5.92 1 .times.
1 56 E0178 Chol DSPC S004 50/43/5/2 4 94.67 5.92 1 .times. 1 58
E0178 Chol DSPC S004 50/43/5/2 4 94.67 5.92 1 .times. 1 32 E0179
Chol S004 50/48/2 4 114.6 5.95 1 .times. 1 53 E0180 Chol S004
50/48/2 4 77.35 1 .times. 1 16 .sup.1A blank cell indicates that
the neutral lipid is omitted .sup.2wherein the order of the lipids
types as they appear in the molar ratio corresponds to the order in
which the lipids appear in the first four columns of the table.
Where only three lipids are listed in the molar ratio, the neutral
lipid is absent. .sup.3wherein "resorcinol" represents
5-heptadecylbenzene-1,3-diol.
TABLE-US-00019 TABLE 10 HepG2 Tumor In vivo Assay results Lipid
Helper Neutral Stealth Molar Lipid N/P Final # doses .times. PLK1 %
ref. lipid lipid.sup.1 lipid Ratio.sup.2 ratio size pKa (mg/kg)
inhibition E0011 Chol S018 60/38/2 4 113.2 5.32 3 .times. 3 16
E0011 Chol S019 60/38/2 4 110 5.32 3 .times. 3 13 E0011 Chol S020
60/38/2 4 74.42 5.32 3 .times. 3 29 E0056 Chol S004 50/48/2 4 77
6.33 3 .times. 3 0 E0076 Chol S004 50/48/2 4 86.03 5.85 3 .times. 3
35 E0085 Chol DSPC S004 45/43/10/2 4 76.8 5.32 3 .times. 3 40 E0056
Chol S004 50/48/2 4 96.09 5.84 3 .times. 3 16 E0056 Chol S004
50/48/2 4 96.09 5.84 3 .times. 3 30 E0096 Chol S004 50/48/2 4 94.91
5.77 3 .times. 3 17 E0104 Chol DSPC S004 45/43/10/2 4 87.59 6.1 3
.times. 3 32 E0104 Chol DSPC S004 50/43/5/2 4 92.19 6.1 3 .times. 3
36 E0104 Chol DOPC S004 50/43/5/2 4 125.1 6.1 3 .times. 3 19 E0104
Chol S004 50/48/2 4 95.05 6.1 3 .times. 3 34 E0104 Chol S004
50/48/2 4 90.87 6.1 3 .times. 3 30 E0119 Chol S004 50/48/2 4 99.37
5.82 3 .times. 3 22 E0175 Chol S004 60/38/2 4 78.74 5.67 3 .times.
3 49 E0175 Chol DSPC S004 50/43/5/2 3.8 93.51 5.67 3 .times. 3 30
E0176 Chol S004 60/38/2 4 99.14 5.56 3 .times. 3 37 E0177 Chol S004
50/48/2 4 91.91 6.1 3 .times. 3 30 E0177 Chol DSPC S004 50/43/5/2 4
95.92 6.1 3 .times. 3 44 E0177 Chol DOPC S004 50/43/5/2 4 89.78 6.1
3 .times. 3 24 E0178 Chol DSPC S004 50/43/5/2 4 94.67 5.92 3
.times. 3 0 E0161 Chol S004 50/48/2 4 101.4 6.3 3 .times. 3 0 E0162
Chol S004 50/48/2 4 114.4 6.07 3 .times. 3 0 E0180 Chol S004
50/48/2 4 77.35 3 .times. 3 0 .sup.1A blank cell indicates that the
neutral lipid is omitted .sup.2wherein the order of the lipids
types as they appear in the molar ratio corresponds to the order in
which the lipids appear in the first four columns of the table.
Where only three lipids are listed in the molar ratio, the neutral
lipid is absent.
TABLE-US-00020 TABLE 11 786-0 Renal Tumor Assay results Lipid
Helper Neutral Stealth Lipid # doses .times. PLK1 % ref. lipid
lipid.sup.1 lipid Ratio.sup.2 N/P ratio Final size pKa (mg/kg)
Inhibition E0104 Chol S004 50/48/2 4 95.05 6.1 1 .times. 5 55 E0104
Chol DSPC S004 45/43/10/2 4 87.59 6.1 1 .times. 10 50 E0104 Chol
S004 50/48/2 4 95.05 6.1 1 .times. 10 50 .sup.1A blank cell
indicates that the neutral lipid is omitted .sup.2wherein the order
of the lipids types as they appear in the molar ratio corresponds
to the order in which the lipids appear in the first four columns
of the table. Where only three lipids are listed in the molar
ratio, the neutral lipid is absent.
[0878] In the above tables, the N/P ratio is equal to the
following: (number of moles of cationic lipid initially
formulated)/(number of moles of siRNA initially formulated*total
number of anionic charges per siRNA).
Example 76
Optimization of Lipid Formulations
[0879] Further optimization of the formulations utilizing cationic
lipids and stealth lipids, e.g., as shown in the above tables, is
considered within the knowledge of a skilled practitioner and may
be done without undue experimentation. For example, formulations
may be optimized for at least one parameter including but not
limited to individual selection of, e.g., the pKa of the cationic
lipid optimized for the type of cell or organ being targeted, the
cationic lipid used, the stealth lipid used, the helper lipid, the
neutral lipid used, whether the neutral lipid is present or absent,
the ratio of the selected helper lipid, optional neutral lipid,
stealth lipid and cationic lipid, the N/P ratio, the particle size,
the dosage regimen, the dose given, the formulation method, and the
like.
[0880] In one embodiment, when choosing the more optimal neutral
lipid, a skilled practitioner would more often opt for DSPC than
DOPC. In some embodiments, e.g., for E0177 in Table 9 and E0104 in
Table 10, compositions that differed only by the choice of these
two neutral lipids exhibit a lower KD or even zero KD when DOPC is
used compared to when DSPC is used, with all other aspects
considered equal. In certain compositions, omitting the neutral
lipid altogether, e.g., for at least one formulation of E0085 in
Table 9, results in a higher percent inhibition of PLK1 compared to
having a neutral lipid present, or for E0011 where progressively
decreasing the amount of DSPC in the formulations leads to
progressively increasing percent knockdown.
[0881] Dose in mg/kg and dosage regimen, e.g., number of doses
given and timing of said doses, may also be optimized. For example,
in one experiment shown shown in Table 9, administering 0.1 mg/kg
of a formulation with E0178 results in zero (0) % KD but
administering 1.0 mg/kg provides 35% KD. If larger doses are not
tolerated by a subject, a full treatment regimen may be
administered as multiple smaller doses provided over several days,
such in Table 9, wherein delivery of 1.times.10 mg/kg siRNA versus
3.times.5 mg/kg siRNA in formulations containing E0011 and S004
resulted in 70% KD and 67% KD, respectively.
Example 77
Liposomes for Delivery of Nucleic Acid Replicons
[0882] Various nucleic acid replicons, each about 10,000
nucleotides long, are delivered in liposomes as described below.
Nucleic acid is encapsulated in liposomes made essentially by the
methods of Jeffs et al. (2005) Pharmaceutical Research 22
(3):362-372 and Maurer et al. (2001) Biophysical Journal, 80:
2310-2326.
[0883] Reference liposomes are made of 10% DSPC (zwitterionic,
i.e., neutral lipid), 40% DlinDMA (cationic lipid), 48% cholesterol
(helper lipid) and 2% PEG-conjugated DMG (stealth lipid). The
DlinDMA lipid (1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane) is
synthesized using the procedure of Heyes et al. (2005) J Controlled
Release 107:276-87. DSPC
(1,2-Diastearoyl-sn-glycero-3-phosphocholine) is purchased from
Genzyme. Cholesterol is obtained from Sigma-Aldrich. PEG-conjugated
DMG
(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol), ammonium salt), DOTAP
(1,2-dioleoyl-3-trimethylammonium-propane, chloride salt) and
DC-chol(3.beta.-[N-(N',N'dimethyl
aminoethane)-carbamoyl]cholesterol hydrochloride) are available
from Avanti Polar Lipids.
[0884] Briefly, lipids are dissolved in ethanol (2 ml), replicon is
dissolved in buffer (2 ml, 100 mM sodium citrate, pH 6) and these
are mixed with 2 ml of buffer followed by 1 hour of equilibration.
The mixture is diluted with 6 ml buffer then filtered. The
resulting product contained liposomes, with .about.95%
encapsulation efficiency.
[0885] The percentage of encapsulated nucleic acid and the nucleic
acid concentration are determined with a commercial kit. Liposomes
are diluted 10.times. or 100.times. in 1.times.TE buffer (from kit)
before addition of the dye. Separately, liposomes are diluted
10.times. or 100.times. in 1.times.TE buffer containing 0.5% Triton
X before addition of the dye (to disrupt the liposomes and thus to
assay total nucleic acid). Thereafter an equal amount of dye is
added to each solution and then approximately 180 .mu.L of each
solution after dye addition is loaded in duplicate into a 96 well
culture plate. The fluorescence (Ex 485 nm, Em 528 nm) is read on a
microplate reader.
[0886] To assess in vivo expression of the nucleic acid a reporter
enzyme (SEAP; secreted alkaline phosphatase) is encoded in the
replicon. Expression levels are measured in sera diluted 1:4 in
1.times. Phospha-Light dilution buffer using a chemiluminescent
alkaline phosphate substrate. 8-10 week old BALB/c mice (5/group)
are injected intramuscularly on day 0, 50 .mu.l per leg with 0.1
.mu.g or 1 .mu.g nucleic acid dose. The same vector is also
administered without the liposomes (in PBS) at 1 .mu.g.
[0887] Encapsulation increases SEAP levels by about 1/2 log at the
1 .mu.g dose, and at day 6 expression from a 0.1 .mu.g encapsulated
dose matches levels seen with 1 .mu.g unencapsulated dose. Thus
expression increases when the nucleic acid is formulated in the
liposomes relative to the naked nucleic acid control, even at a
10.times. lower dose.
[0888] Further SEAP experiments show a clear dose response in vivo,
wherein expression is seen after delivery of as little as 1 ng
nucleic acid. Experiments comparing expression from encapsulated
and naked replicons indicate that 0.01 .mu.g encapsulated nucleic
acid is equivalent to 1 .mu.g of naked nucleic acid. At a 0.5 .mu.g
dose of nucleic acid the encapsulated material can give a 12-fold
higher expression at day 6; at a 0.1 .mu.g dose levels can be
24-fold higher at day 6.
[0889] As an alternative to using the reference lipid (DlinDMA),
cationic lipids of the invention are used. The reference liposomes
are formed using DlinDMA (see above) as the cationic lipid. DlinDMA
is replaced with various cationic lipids in series as described
below and shown in Table. Two different types of each liposome are
formed, using 2% PEG2000-DMG with either (01) 40% of the cationic
lipid, 10% DSPC, and 48% cholesterol, or (02) 60% of the cationic
lipid and 38% cholesterol. Thus a comparison of the (01) and (02)
liposomes shows the effect of the neutral zwitterionic lipid.
[0890] These liposomes are tested with the SEAP reporter described
above. The following Table 12 shows the size of the liposomes (Z
average and polydispersity index), the % of nucleic acid
encapsulation in each liposome, together with the SEAP activity
detected at days 1 and 6 after injection. SEAP activity is relative
to "DlinDMA (02)" liposomes made from DlinDMA, cholesterol and
PEG-DMG.
TABLE-US-00021 TABLE 12 Liposome delivery of nucleic acid replicons
% SEAP SEAP E no. Zav (pdI) encapsulation day 1 day 6 DlinDMA (01)
154.6 (0.131) 95.5 80.9 71.1 DlinDMA (02) 162.0 (0.134) 85.3 100
100 Comparative (01) 133.9 (0.185) 96.5 57 45.7 Comparative (02)
134.6 (0.082) 97.6 54.2 4.3 E0014 (01) 158.3 (0.212) 62.0 65.7 44.9
E0014 (02) 164.2 (0.145) 86 62.2 39.7 E0024 (01) 131.0 (0.145) 74.0
91 154.8 E0024 (02) 134.6 (0.117) 81.5 90.4 142.6 E0026 (01) 164.0
(0.162) 76.0 76.9 329.8 E0026 (02) 177.8 (0.117) 72.8 67.1 227.9
E0084 (01) 116.0 (0.180) 79.8 25.5 12.4 E0084 (02) 136.3 (0.164)
74.9 24.8 23.1 E0065 (01) 140.6 (0.184) 77 26.5 163.3 E0065 (02)
138.6 (0.122) 87 29.7 74.8 E0078 (01) 176.7 (0.185) 50 76.5 187
E0078 (02) 199.5 (0.191) 46.3 82.4 329.8 E0069 (01) 165.3 (0.169)
72.2 65.1 453.9 E0069 (02) 179.5 (0.157) 65 68.5 658.2 E0108 (01)
129.7 (0.184) 78.4 113.4 47.8 E0108 (02) 147.6 (0.131) 80.9 78.2
10.4 E0115 (01) 129.2 (0.186) 71 113.6 242.2 E0115 (02) .sup. 139
(0198) 75.2 71.8 187.2 E0099 (01) 135.7 (0.161) 78.8 65 10 E0099
(02) 158.3 (0.287) 69.4 78.8 8.2
[0891] Additional Aspects of the Invention
[0892] The invention comprises a composition comprising at least
one cationic lipid, at least one helper lipid and at least one
stealth lipid for delivery of a biologically active agent, wherein
the biologically active agent is for delivery to a tissue or cell
selected from: (a) the liver or liver cells, wherein the
composition has a cationic lipid with a pKa of from about 6.2 or
above; and (b) a tumor or tumor cell, wherein the composition has a
cationic lipid with a pKa of from about 6.2 or below.
[0893] The invention comprises a composition comprising at least
one cationic lipid, at least one helper lipid and at least one
stealth lipid for delivery of a biologically active agent, wherein
the biologically active agent is for delivery to a tissue or cell
selected from: (a) the liver or liver cells, wherein the
composition has a cationic lipid with a pKa of from about 5.1 to
about 7.4; and (b) a tumor or tumor cell, wherein the composition
has a cationic lipid with a pKa of from about 5.0 to about 6.7.
[0894] The invention comprises a composition of paragraphs [00706]
or [00707], wherein the composition further comprises an optional
neutral lipid.
[0895] The invention comprises the composition of any one of
paragraphs [00706], or [00708], wherein the biologically active
agent is in an amount effective for therapeutic treatment of a
disease or disorder.
[0896] The invention comprises the composition of paragraphs
[00709], wherein the biologically active agent is for delivery to
the liver or a liver cell and the cationic lipid 1 has a pKa at
least about 5.1 to about 7.4.
[0897] The invention comprises the composition of paragraph
[00709], wherein the biologically active agent is for delivery to a
tumor or tumor cells and the compound of paragraph 1 has a pKa of
from about 5.0 to about 6.7.
[0898] The invention comprises the composition of paragraph
[00711], wherein the cationic lipid has a pKa of from about 5.2 to
about 6.3.
[0899] The invention comprises the composition of paragraph
[00711], wherein the cationic lipid has a pKa of from about 5.4 to
about 6.2.
[0900] The invention comprises the composition of paragraph
[00711], wherein the cationic lipid has a pKa of from about 5.8 to
about 6.1.
[0901] The invention comprises the composition of any one or more
of paragraph [00711] through paragraph [00714], wherein the
composition is optimized by selection of at least one of a stealth
lipid, formulation method, N/P ratio, particle size, and molar
ratio of the cationic lipid, an optional neutral lipid, helper
lipid, stealth lipid and an optional alkyl resorcinol based
lipid.
[0902] The invention comprises the composition of paragraph
[00709], wherein the biologically active agent is for delivery to
the liver or a liver cell, the composition comprising a formulation
comprising at least one cationic lipid with a pKa of from about 5.1
to about 7.4.
[0903] The invention comprises the composition of paragraph
[00716], wherein the cationic lipid has a pKa of from about 5.3 to
about 7.3.
[0904] The invention comprises the composition of paragraph
[00716], wherein the Cationic lipid has a pKa of from about 5.9 to
about 7.0.
[0905] The invention comprises the composition of paragraph
[00716], wherein the cationic lipid has a pKa of from about 6.2 to
about 6.8.
[0906] The invention comprises the composition of any one or more
of paragraph [00716] through paragraph [00719], wherein the
composition is optimized by selection of at least one of a stealth
lipid, formulation method, N/P ratio, particle size, and molar
ratio of the cationic lipid, an optional neutral lipid, helper
lipid, stealth lipid and an optional alkyl resorcinol based
lipid.
[0907] The invention comprises the composition of paragraph
[00715], wherein the pKa of the cationic lipid is from about 5.4 to
about 5.9 for delivery to Hep3B-like tumors.
[0908] The invention comprises the composition of paragraph
[00715], wherein the pKa of the cationic lipid is from about 5.6 to
about 6.1 for delivery to HepG2-like and 786-O-like tumors.
[0909] The invention comprises a method for the treatment of a
disease or condition comprising the step of administering a
therapeutically effective amount of a composition of any of
paragraphs [00706] to [00722] to a patient in need thereof.
[0910] The invention comprises the method of paragraph [00723]
wherein the disease or condition is a tumor, a disease of the
liver, or a disease that is responsive to treatment with an RNAi
construct.
[0911] The invention comprises the method of paragraph [00723]
wherein the disease or condition is a tumor and the cationic lipid
in the composition has a pKa of from about 5.0 to about 6.7.
[0912] The invention comprises the method of paragraph [00723]
wherein the disease or condition is in the liver, and the cationic
lipid in the composition has a pKa of from about 5.1 to about
7.4.
[0913] The invention comprises a compound of formula (I):
##STR00329##
or a salt or pharmaceutically acceptable derivative thereof,
wherein:
[0914] R.sup.1 and R.sup.2 together with the nitrogen atom to which
they are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group;
[0915] a is absent or optionally substituted C.sub.1-4
alkylene;
[0916] b is absent or optionally substituted C.sub.1-4
alkylene;
[0917] c is absent or optionally substituted C.sub.1-4
alkylene;
[0918] X.sup.1 is O or S;
[0919] X.sup.2 is O or S;
[0920] Y.sup.1 is optionally substituted C.sub.10-30alkenyl,
C.sub.10-30alkynyl, C.sub.10-30heteroalkenyl or
C.sub.10-30heteroalkynyl;
[0921] L is absent or
-(L.sup.a).sub.d-(L.sup.b).sub.e-(L.sup.c).sub.f-, wherein [0922]
L.sup.a is optionally substituted C.sub.1-15alkylene,
C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [0923] L.sup.b is optionally
substituted C.sub.6-14arylene or C.sub.5-13heteroarylene; [0924]
L.sup.c is optionally substituted C.sub.1-15alkylene,
C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [0925] d is 0 or 1; [0926] e is 0 or 1;
and [0927] f is 0 or 1; and
[0928] Y.sup.2 is an optionally substituted steroid.
[0929] The invention comprises the compound of paragraph [00727],
wherein a is selected from optionally substituted C.sub.1-2
alkylene and optionally substituted C.sub.1 alkylene.
[0930] The invention comprises the compound of paragraphs [00727]
or [00728], wherein b is selected from optionally substituted
C.sub.0-2 alkylene and optionally substituted C.sub.1 alkylene.
[0931] The invention comprises the compound of any one of
paragraphs [00727] to [00729], wherein c is absent or is optionally
substituted C.sub.1 alkylene.
[0932] The invention comprises the compound of any one of
paragraphs [00727] to [00730], wherein a, b and c are
unsubstituted.
[0933] The invention comprises the compound of any one of
paragraphs [00727] to [00731], wherein R.sup.1 and R.sup.2 together
with the nitrogen atom to which they are attached form an
optionally substituted C.sub.3-20-heterocycloalkyl,
C.sub.3-20-heterocycloalkenyl or C.sub.3-20-heterocycloalkynyl
group.
[0934] The invention comprises the compound of any one of
paragraphs [00727] to [00732], wherein R.sup.1 and R.sup.2 together
with the nitrogen atom to which they are attached form a compound
selected from a cyclic optionally substituted C.sub.5-16 group and
a cyclic optionally substituted C.sub.5-12 group.
[0935] The invention comprises the compound of paragraph [00733],
wherein R.sup.1 and R.sup.2 together with the nitrogen atom to
which they are attached form a cyclic optionally substituted
C.sub.5 group, C.sub.6 group or C.sub.7 group.
[0936] The invention comprises the compound of any one of
paragraphs [00727] to [00734], wherein R.sup.1 and R.sup.2 together
with the nitrogen atom to which they are attached are selected from
at least one of the head groups H.sup.1 to H.sup.52.
[0937] The invention comprises the compound of any one of
paragraphs [00727] to [00735], wherein X.sup.1 is O.
[0938] The invention comprises the compound of any one of
paragraphs [00727] to [00736], wherein X.sup.2 is O.
[0939] The invention comprises the compound of any one of
paragraphs [00727] to [00737], wherein L comprises at least one
heteroatom.
[0940] The invention comprises the compound of paragraph [00738],
wherein L comprises at least one O atom.
[0941] The invention comprises the compound of any one of
paragraphs [00727] to [00739], wherein L' is selected from one of
formulae L.sup.c-i to L.sup.c-xxxxiii: [0942] L.sup.c-i
--(CH.sub.2).sub.2O(CH.sub.2).sub.2-- [0943] L.sup.c-ii
--(CH.sub.2).sub.4-- [0944] L.sup.c-iii --CO(CH.sub.2).sub.2CO--
[0945] L.sup.c-iv --CO-- [0946] L.sup.c-v --COCH.sub.2OCH.sub.2CO--
[0947] L.sup.c-vi --(CH.sub.2).sub.2O(CH.sub.2).sub.2NHCO-- [0948]
L.sup.c-vii --(CH.sub.2).sub.3O(CH.sub.2).sub.3-- [0949]
L.sup.c-viii --(CH.sub.2).sub.2-- [0950] L.sup.c-ix
--(CH.sub.2).sub.2O(CH.sub.2).sub.2O(CH.sub.2).sub.2O(CH.sub.2).sub.2--
[0951] L.sup.c-x
--(CH.sub.2).sub.2O(CH.sub.2).sub.2O(CH.sub.2).sub.2--
[0951] ##STR00330## [0952] L.sup.c-xv
--(CH.sub.2).sub.2O(CH.sub.2).sub.2OCH(CH.sub.3)-- [0953]
L.sup.c-xvi --(CH.sub.2).sub.2O(CH.sub.2).sub.2OC
(.dbd.O)(CH.sub.2).sub.2CO-- [0954] L.sup.c-xvii
--(CH.sub.2).sub.2OC(.dbd.O)(CH.sub.2).sub.2CO-- [0955]
L.sup.c-xviii --(CH.sub.2).sub.2O(CH.sub.2).sub.2OCO-- [0956]
L.sup.c-xix
--(CH.sub.2).sub.2NHC(.dbd.O)CH.sub.2OCH.sub.2C(.dbd.O)-- [0957]
L.sup.c-xx --(CH.sub.2).sub.2NHC(.dbd.O)(CH.sub.2).sub.2C(.dbd.O)--
[0958] L.sup.c-xxi --(CH.sub.2).sub.2NHC(.dbd.O)-- [0959]
L.sup.c-xxii --(CH.sub.2).sub.2NHC(.dbd.O)CH.sub.2NHC(.dbd.O)--
[0960] L.sup.c-xxiii
--(CH.sub.2).sub.2NHC(.dbd.O)CH(side-chain-1)NHC(.dbd.O)--, wherein
side-chain-1 represents the group
[0960] ##STR00331## the dashed line representing the bond to the
rest of the molecule; [0961] L.sup.c-xxiv
--(CH.sub.2).sub.2OC(.dbd.O)-- [0962] L.sup.c-xxv
--(CH.sub.2).sub.2O(CH.sub.2).sub.2OC(.dbd.O)CH.sub.2-- [0963]
L.sup.c-xxvi --(CH.sub.2).sub.2OC(.dbd.O)CH.sub.2-- [0964]
L.sup.c-xxvii --(CH.sub.2).sub.2OC(.dbd.O)CH.sub.2NHC(.dbd.O)--
[0965] L.sup.c-xxviii
--(CH.sub.2).sub.2OC(.dbd.O)(CH.sub.2).sub.2NHC(.dbd.O)--
[0965] ##STR00332## [0966] L.sup.c-xxxvi
--(CH.sub.2).sub.2OCO.sub.2(CH.sub.2).sub.2-- [0967] L.sup.c-xxxvii
--(CH.sub.2).sub.2OC(.dbd.O)CH.sub.2OCH.sub.2C(.dbd.O)-- [0968]
L.sup.c-xxxviii
--(CH.sub.2).sub.2OC(.dbd.O)(CH.sub.2).sub.3C(.dbd.O)-- [0969]
L.sup.c-xxxix
--(CH.sub.2).sub.3OC(.dbd.O)(CH.sub.2).sub.2C(.dbd.O)--
[0969] ##STR00333## [0970] L.sup.c-xxxxii
--(CH.sub.2).sub.2OCH.sub.2C(.dbd.O)--;
##STR00334##
[0971] The invention comprises the compound of any one of
paragraphs [00727] to [00740], wherein d is 0; e is 0, and f is
1.
[0972] The invention comprises the compound of any one of
paragraphs [00727] to [00741], wherein Y.sup.1 is a C.sub.12-28
group.
[0973] The invention comprises the compound of any one of
paragraphs [00727] to [00742], wherein Y.sup.1 has at least one
alkene group.
[0974] The invention comprises the compound of paragraph [00743],
wherein Y.sup.1 has at least one cis unsaturated alkene group.
[0975] The invention comprises the compound of any one of
paragraphs [00727] to [00741], wherein Y.sup.1 is selected from
Y.sup.1-i to Y.sup.1-vii.
[0976] The invention comprises the compound of any one of
paragraphs [00727] to [00745], wherein Y.sup.2 is linked to L via
an oxygen atom on the optionally substituted steroid.
[0977] The invention comprises the compound of paragraph [00746],
wherein Y.sup.2 is a sterol in which the hydrogen atom of the
hydroxy group at the 3-position of the A steroid ring has been
removed.
[0978] The invention comprises the compound of paragraph [00746],
wherein the sterol is selected from the group consisting of:
annasterol; avenasterol; beta-sitosterol; brassicasterol;
calciferol; campesterol; chalinosterol; chinasterol; cholestanol;
cholesterol; coprostanol; cycloartenol; dehydrocholesterol;
desmosterol; dihydrocalciferol; dihydrocholesterol;
dihydroergosterol; dinosterol; epicholesterol; ergosterol;
fucosterol; hexahydrolumisterol; hexaol; hydroxycholesterol;
lanosterol; lumisterol; parkeol; poriferasterol; saringosterol;
sitostanol; sitosterol; stigmastanol; stigmasterol; weinbersterol;
zymosterol; sterol bile acids (including one or more selected from
cholic acid; chenodeoxycholic acid; glycocholic acid; taurocholic
acid; deoxycholic acid, and lithocholic acid); and/or a salt or a
pharmaceutically acceptable derivative thereof.
[0979] The invention comprises the compound of paragraph [00748],
wherein the sterol is cholesterol.
[0980] The invention comprises the compound of paragraph [00727]
comprising formula (II):
##STR00335##
or a salt or pharmaceutically acceptable derivative thereof,
wherein:
[0981] R.sup.1 and R.sup.2 together with the nitrogen atom to which
they are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group;
[0982] a is absent or optionally substituted C.sub.1-4
alkylene;
[0983] b is absent or optionally substituted C.sub.1-4
alkylene;
[0984] c is absent or optionally substituted C.sub.1-4
alkylene;
[0985] X.sup.1 is O or S;
[0986] X.sup.2 is O or S;
[0987] Y.sup.1 is optionally substituted C.sub.10-30alkenyl,
C.sub.10-30alkynyl, C.sub.10-30heteroalkenyl or
C.sub.10-30heteroalkynyl;
[0988] L is -(L.sup.a).sub.d-(L.sup.b).sub.e-(L.sup.b).sub.f-,
wherein [0989] L.sup.a is optionally substituted
C.sub.1-15alkylene, C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [0990] L.sup.b is optionally
substituted C.sub.6-14arylene or C.sub.5-13heteroarylene; [0991]
L.sup.c is optionally substituted C.sub.1-15alkylene,
C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [0992] d is 0 or 1; [0993] e is 0 or 1;
and [0994] f is 0 or 1;
[0995] provided that L comprises one or more heteroatoms, and
[0996] Y.sup.2 is an optionally substituted steroid.
[0997] The invention comprises the compound of paragraph [00727]
comprising formula (III):
##STR00336##
or a salt or pharmaceutically acceptable derivative thereof,
wherein:
[0998] R.sup.1 and R.sup.2 together with the nitrogen atom to which
they are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group;
[0999] a is methylene;
[1000] b is methylene;
[1001] c is absent;
[1002] X.sup.1 is O or S;
[1003] X.sup.2 is O or S;
[1004] Y.sup.1 is optionally substituted C.sub.10-30alkenyl,
C.sub.10-30alkynyl, C.sub.10-30heteroalkenyl or
C.sub.10-30heteroalkynyl;
[1005] L is -(L.sup.a).sub.d-(L.sup.a).sub.e-(L.sup.c).sub.f-,
wherein [1006] L.sup.a is optionally substituted
C.sub.1-15alkylene, C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [1007] L.sup.b is optionally
substituted C.sub.6-14arylene or C.sub.5-13heteroarylene; [1008]
L.sup.c is optionally substituted C.sub.1-15alkylene,
C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [1009] d is 0 or 1; [1010] e is 0 or 1;
and [1011] f is 0 or 1; and
[1012] Y.sup.2 is an optionally substituted steroid.
[1013] The invention comprises the compound of paragraph [00727]
comprising formula (IV):
##STR00337##
or a salt or pharmaceutically acceptable derivative thereof,
wherein:
[1014] R.sup.1 and R.sup.2 together with the nitrogen atom to which
they are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group;
[1015] a is methylene;
[1016] b is methylene;
[1017] c is absent;
[1018] X.sup.1 is O or S;
[1019] X.sup.2 is O or S;
[1020] Y.sup.1 is optionally substituted C.sub.10-30alkenyl,
C.sub.10-30alkynyl, C.sub.10-30heteroalkenyl or
C.sub.10-30heteroalkynyl;
[1021] L is -(L.sup.a).sub.d-(L.sup.b).sub.e-(L.sup.c).sub.f-,
wherein [1022] L.sup.a is optionally substituted
C.sub.1-15alkylene, C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [1023] L.sup.b is optionally
substituted C.sub.6-14arylene or C.sub.5-13heteroarylene; [1024]
L.sup.c is optionally substituted C.sub.1-15alkylene,
C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [1025] d is 0 or 1; [1026] e is 0 or 1;
and [1027] f is 0 or 1; provided that L comprises one or more
heteroatoms, and [1028] Y.sup.2 is an optionally substituted
steroid.
[1029] The invention comprises the compound of paragraph [00727]
comprising formula (V):
##STR00338##
or a salt or pharmaceutically acceptable derivative thereof,
wherein: [1030] R.sup.1 and R.sup.2 together with the nitrogen atom
to which they are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group;
[1031] a is methylene;
[1032] b is methylene;
[1033] c is absent;
[1034] X.sup.1 is O;
[1035] X.sup.2 is O;
[1036] Y.sup.1 is optionally substituted C.sub.10-30alkenyl,
C.sub.10-30alkynyl, C.sub.10-30heteroalkenyl or
C.sub.10-30heteroalkynyl;
[1037] L is -(L.sup.a).sub.d-(L.sup.b).sub.e-(L.sup.b).sub.f-,
wherein [1038] L.sup.a is optionally substituted
C.sub.1-15alkylene, C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [1039] L.sup.b is optionally
substituted C.sub.6-14arylene or C.sub.5-13heteroarylene; [1040]
L.sup.c is optionally substituted C.sub.1-15alkylene,
C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [1041] d is 0 or 1; [1042] e is 0 or 1;
and [1043] f is 0 or 1;
[1044] provided that L comprises one or more heteroatoms, and
[1045] Y.sup.2 is an optionally substituted steroid.
[1046] The invention comprises the compound of paragraph [00727]
comprising formula (VI):
##STR00339##
or a salt or pharmaceutically acceptable derivative thereof,
wherein:
[1047] R.sup.1 and R.sup.2 together with the nitrogen atom to which
they are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group;
[1048] a is methylene;
[1049] b is methylene;
[1050] c is absent;
[1051] X.sup.1 is O;
[1052] X.sup.2 is O;
[1053] Y.sup.1 is optionally substituted C.sub.10-30alkenyl,
C.sub.10-30alkynyl, C.sub.10-30heteroalkenyl or
C.sub.10-30heteroalkynyl;
[1054] L is -L.sup.c-, wherein [1055] L.sup.c is optionally
substituted C1-15heteroalkylene, C1-15heteroalkenylene or
C1-15heteroalkynylene; and
[1056] Y.sup.2 is an optionally substituted steroid.
[1057] The invention comprises the compound of paragraph [00727]
comprising formula (VII):
##STR00340##
or a salt or pharmaceutically acceptable derivative thereof,
wherein:
[1058] R.sup.1 and R.sup.2 together with the nitrogen atom to which
they are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group;
[1059] a is methylene;
[1060] b is methylene;
[1061] c is absent;
[1062] X.sup.1 is O;
[1063] X.sup.2 is O;
[1064] Y.sup.1 is an optionally substituted C.sub.16-22 alkenyl
group;
[1065] L is -L.sup.c-, wherein [1066] L.sup.c is optionally
substituted C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; and
[1067] Y.sup.2 is an optionally substituted steroid.
[1068] The invention comprises the compound of paragraph [00727]
comprising formula (VIII):
##STR00341##
or a salt or pharmaceutically acceptable derivative thereof,
wherein:
[1069] R.sup.1 and R.sup.2 together with the nitrogen atom to which
they are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group;
[1070] a is methylene;
[1071] b is methylene;
[1072] c is absent;
[1073] X.sup.1 is O;
[1074] X.sup.2 is O;
[1075] Y.sup.1 is an optionally substituted C.sub.16-22 alkenyl
group;
[1076] L is -L.sup.c-, wherein [1077] L.sup.c is optionally
substituted C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; and
[1078] Y.sup.2 is cholesterol connected through the hydroxy group
at the 3-position of the A steroid ring, the hydrogen atom of said
hydroxy group being absent.
[1079] The invention comprises the compound of paragraph [00727]
comprising formula (IX):
##STR00342##
or a salt or pharmaceutically acceptable derivative thereof,
wherein:
[1080] R.sup.1 and R.sup.2 together with the nitrogen atom to which
they are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group;
[1081] a is methylene;
[1082] b is methylene;
[1083] c is absent;
[1084] X.sup.1 is O or S;
[1085] X.sup.2 is O or S;
[1086] Y.sup.1 is optionally substituted C.sub.10-30alkenyl,
C.sub.10-30alkynyl, C.sub.10-30heteroalkenyl or
C.sub.10-30heteroalkynyl;
[1087] L is -(L.sup.a).sub.d-(L.sup.b).sub.e-(L.sup.b).sub.f-,
wherein [1088] L.sup.a is optionally substituted
C.sub.1-15alkylene, C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [1089] L.sup.b is optionally
substituted C.sub.6-14arylene or C.sub.5-13heteroarylene; [1090]
L.sup.c is optionally substituted C.sub.1-15alkylene,
C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [1091] d is 0 or 1; [1092] e is 0 or 1;
and [1093] f is 0 or 1;
[1094] provided that L comprises one or more heteroatoms, and
[1095] Y.sup.2 is an optionally substituted steroid; and. wherein
the pKa of the compound is from about 5.1 to about 7.4.
[1096] The invention comprises the compound of paragraph [00727]
comprising formula (X):
##STR00343##
or a salt or pharmaceutically acceptable derivative thereof,
wherein:
[1097] R.sup.1 and R.sup.2 together with the nitrogen atom to which
they are attached form an optionally substituted
C.sub.3-20-heterocycloalkyl, C.sub.3-20-heterocycloalkenyl,
C.sub.3-20-heterocycloalkynyl or C.sub.5-20-heteroaryl group;
[1098] a is methylene;
[1099] b is methylene;
[1100] c is absent;
[1101] X.sup.1 is O or S;
[1102] X.sup.2 is O or S;
[1103] Y.sup.1 is optionally substituted C.sub.10-30alkenyl,
C.sub.10-30alkynyl, C.sub.10-30heteroalkenyl or
C.sub.10-30heteroalkynyl;
[1104] L is -(L.sup.a).sub.d-(L.sup.b).sub.e-(L.sup.c).sub.f-,
wherein [1105] L.sup.a is optionally substituted
C.sub.1-15alkylene, C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [1106] L.sup.b is optionally
substituted C.sub.6-14arylene or C.sub.5-13heteroarylene; [1107]
L.sup.c is optionally substituted C.sub.1-15alkylene,
C.sub.1-15alkenylene, C.sub.1-15alkynylene,
C.sub.1-15heteroalkylene, C.sub.1-15heteroalkenylene or
C.sub.1-15heteroalkynylene; [1108] d is 0 or 1; [1109] e is 0 or 1;
and [1110] f is 0 or 1;
[1111] provided that L comprises one or more heteroatoms, and
[1112] Y.sup.2 is an optionally substituted steroid; and wherein
the pKa of the compound is from about 5.0 to about 6.7.
[1113] The invention comprises a compound of any one of formulas I
through X in paragraphs [00727] through [00758], wherein the
compound is selected from any one or more of E0001-E0171 and
E0175-E0180.
[1114] The invention comprises a stealth lipid of formula (XI)
##STR00344##
or a salt or pharmaceutically acceptable derivative thereof,
wherein
[1115] Z is a hydrophilic head group component selected from PEG
and polymers based on poly(oxazoline), poly(ethylene oxide),
poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone),
poly[N-(2-hydroxypropyl)methacrylamide] and poly(amino acid)s,
wherein the polymer may be linear or branched, and wherein the
polymer may be optionally substituted;
[1116] wherein Z is polymerized by n subunits;
[1117] n is a number-averaged degree of polymerization between 10
and 200 units of Z, wherein n is optimized for different polymer
types;
[1118] L.sub.1 is an optionally substituted C.sub.1-10 alkylene or
C.sub.1-10 heteroalkylene linker including zero, one, two or more
of an ether (e.g., --O--), ester (e.g., --C(O)O--), succinate
(e.g., --O(O)C--CH.sub.2--CH.sub.2--C(O)O--)), carbamate (e.g.,
--OC(O)--NR'--), carbonate (e.g., --OC(O)O--), ketone (e.g.,
--C--C(O)--C--), carbonyl (e.g., --C(O)--), urea (e.g.,
--NRC(O)NR'--), amine (e.g., --NR'--), amide (e.g., --C(O)NR'--),
imine (e.g., --C(NR')--), thioether (e.g., --S--), xanthate (e.g.,
--OC(S)S--), and phosphodiester (e.g., --OP(O).sub.2O--); any of
which may be substituted by zero, one or more Z groups;
[1119] wherein R' is independently selected from H, --NH--, --O--,
--S--, a phosphate or an optionally substituted C.sub.1-10
alkylene;
[1120] X.sub.1 and X.sub.2 are independently selected from a carbon
or a heteroatom selected from --NH--, --O--, --S-- or a
phosphate;
[1121] A.sub.1 and A.sub.2 are independently selected from a
C.sub.6-30 alkyl, C.sub.6-30 alkenyl, and C.sub.6-30 alkynyl,
wherein A.sub.1 and A.sub.2 may be the same or different,
[1122] or wherein A.sub.1 and A.sub.2 together with the carbon atom
to which they are attached form an optionally substituted
steroid.
[1123] The invention comprises the stealth lipid of paragraph
[00760] comprising formula (XII)
##STR00345##
or a salt or pharmaceutically acceptable derivative thereof,
wherein
[1124] PEG is a poly(ethylene glycol) subunit, wherein the PEG may
be linear or branched;
[1125] n is a number-averaged degree of polymerization between 10
and 200 units of PEG, preferably about 23 units, about 45 units or
about 68 units;
[1126] L.sub.1 is an optionally substituted C.sub.1-10
heteroalkylene linker containing one, two or more of an ether,
ester, succinate, carbamate, carbonate, ketone, carbonyl, urea,
amine, amide, imine, thioether, xanthate, and phosphodiester; any
of which may be substituted by zero, one or more PEG groups;
[1127] X.sub.1 and X.sub.2 are independently selected from carbon
or oxygen;
[1128] A.sub.1 and A.sub.2 are independently selected from a
C.sub.6-30 alkyl, C.sub.6-30 alkenyl, and C.sub.6-30 alkynyl,
wherein A.sub.1 and A.sub.2 may be the same or different,
[1129] or wherein A.sub.1 and A.sub.2 together with the carbon atom
to which they are attached form an optionally substituted
steroid.
[1130] The invention comprises a compound of any one of paragraphs
[00760] and [00761], wherein the compound is selected from any one
or more of S001 though S009 and S012 through S026.
[1131] The invention comprises the compound of any one of
paragraphs [00727] to [00759], wherein the pKa of the compound is
from about 5.1 to about 7.4, for use in a formulation for delivery
of a biologically active agent to the liver or a liver cell.
[1132] The invention comprises the compound of any one of
paragraphs [00727] to [00759], wherein the pKa of the compound is
about 6.2 or above, for use in a formulation for delivery of a
biologically active agent to the liver or a liver cell.
[1133] The invention comprises the compound of paragraph [00764],
wherein the pKa of the compound is from about 5.9 to about 7.0, for
use in a formulation for delivery of a biologically active agent to
the liver or a liver cell.
[1134] The invention comprises the compound of any one of
paragraphs [00727] to [00759], wherein the pKa of the compound is
from about 5.0 to about 6.7, for use in a formulation for delivery
of a biologically active agent to a tumor or a tumor cell.
[1135] The invention comprises the compound of any one of
paragraphs [00727] to [00759], wherein the pKa of the compound is
from about 5.4 to about 6.2, for use in a formulation for delivery
of a biologically active agent to a tumor or a tumor cell.
[1136] The invention comprises the compound of any one of
paragraphs [00727] to [00759], wherein the pKa of the compound is
about 6.2 or below, for use in a formulation for delivery of a
biologically active agent to a tumor or a tumor cell.
[1137] The invention comprises the compound of any of paragraphs
[00727] to [00768] for use in a formulation for delivery of a
biologically active agent for therapy.
[1138] The invention comprises a composition comprising one or more
compounds of any one of paragraphs [00727] to [00769] for use in a
formulation for delivery of a biologically active agent for
therapy.
[1139] The invention comprises the composition of paragraph [00770]
further comprising at least one additional lipid component in
addition to the compound of any one of paragraphs to [00769].
[1140] The invention comprises the composition of any one of
paragraphs [00706] to and paragraph [00771] comprising a lipid
formulation comprising one or more lipid components, wherein the
lipid component is selected from the group consisting of one or
more of a cationic lipid, an optional neutral lipid, a helper
lipid, a stealth lipid and an optional alkyl resorcinol based
lipid.
[1141] The invention comprises the composition of paragraph
[00772], wherein the stealth lipid is selected from a stealth lipid
of formula XI or formula XII.
[1142] The invention comprises the composition of paragraph
[00772], wherein the composition is optimized for at least one
parameter including but not limited to individual selection of the
pKa of the cationic lipid optimized for the type of cell or organ
being targeted, the cationic lipid used, the stealth lipid used,
the helper lipid, the neutral lipid used, whether the neutral lipid
is present or absent, the ratio of the selected helper lipid,
optional neutral lipid, stealth lipid and cationic lipid, the N/P
ratio, the particle size, the dosage regiment, the dose given, the
formulation method, and the like.
[1143] The invention comprises the composition of any one of
paragraphs [00706] to [00722] and paragraphs [00770] to [00774],
further comprising a biologically active agent.
[1144] The invention comprises the composition of paragraph [00775]
wherein said biologically active agent is selected from the group
consisting of antibodies, cholesterol, hormones, antivirals,
peptides, polypeptides, proteins, nucleoproteins,
chemotherapeutics, low molecular weight drugs, vitamins,
co-factors, nucleosides, nucleoside derivatives, nucleotides,
oligonucleotides, enzymatic nucleic acids, antisense nucleic acids,
triplex forming oligonucleotides, 2,5-A antisense chimeras,
allozymes, aptamers, decoy RNA molecules and analogs thereof, and
small nucleic acid molecules, such as an RNA interfering agent
(RNAi), short interfering nucleic acid (siNA), short interfering
RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and
short hairpin RNA (shRNA).
[1145] The invention comprises the composition of paragraph [00776]
wherein said biologically active agent is a nucleoside or
nucleoside derivative.
[1146] The invention comprises the composition of paragraph [00777]
wherein said biologically active agent is selected from an RNAi, an
siNA, an RNAi inhibitor, a miRNA, an siRNA and a shRNA.
[1147] The invention comprises the composition of paragraph
[00772], wherein the cationic lipid is selected from one or more of
the cationic lipids of E0007, E0008, E0011, E0014, E0015, E0016,
E0017, E0018, E0019, E0022, E0024, E0025, E0026, E0032, E0034,
E0040, E0042, E0043, E0045, E0048, E0049, E0051, E0052, E0053,
E0054, E0055 and E0118.
[1148] The invention comprises the composition of paragraph
[00772], wherein the cationic lipid is selected from the cationic
lipids of E0008, E0011, E0025, E0026, E0075, E0076, E0077, E0085,
E0088, E0095, E0104, E0178 and E0179.
[1149] The invention comprises the composition of paragraph
[00772], the composition comprising at least one cationic lipid
selected from E0008, E0011, E0025, E0026, E0075, E0076, E0077,
E0085, E0088, E0095, E0104, E0178 and E0179.
[1150] The invention comprises the composition of paragraph
[00772], the composition comprising at least one cationic lipid
selected from E0011, E0025, E0026, E0075, E0076, E0077 and
E0088.
[1151] The invention comprises the composition of paragraph
[00772], wherein the stealth lipid is selected from the stealth
lipids of S001, S002, S003, S004, S005, S006, S007, S008, S009,
S010, S011, S012, S013, S014, S015, S016, S017, S018, S019, S020,
S021, S022, S023, S024, S025 and S026.
[1152] The invention comprises any one or more of the above
compounds, formulations and/or compositions, which further
comprises a pharmaceutically acceptable carrier.
[1153] The invention comprises a kit comprising any one or more of
the above compounds, formulations and compositions, and
instructions for use.
[1154] The invention comprises a method for the treatment of a
disease or condition in a subject in need thereof, the method
comprising the step of administering a therapeutically effective
amount of a biologically active agent in a formulation comprising
one or more compositions of any of paragraphs [00706] to [00722]
and paragraphs [00770] to [00783].
[1155] The invention comprises a method for delivering a
biologically active agent to a cell or tissue, which method
comprises administering the composition of any one of paragraphs
[00706] to [00722] and paragraphs [00770] to [00783] to the cell or
tissue.
[1156] The invention comprises the method of paragraph [00787]
wherein the disease or condition is a tumor, a disease of the
liver, or a disease that is responsive to treatment with an RNAi
construct.
[1157] The invention comprises the method of paragraph [00787]
wherein the disease or condition is a tumor and the cationic lipid
in the composition has a pKa of from about 5.0 to about 6.7.
[1158] The invention comprises the method of paragraph [00787]
wherein the disease or condition is a tumor and the cationic lipid
in the composition has a pKa of from about 6.2 or below.
[1159] The invention comprises the method of paragraph [00787]
wherein the disease or condition is a tumor and the cationic lipid
in the composition has a pKa of from about 5.0 to about 6.7.
[1160] The invention comprises the method of paragraph [00787]
wherein the disease or condition is in the liver, and the cationic
lipid in the composition has a pKa of from about 5.1 to about
7.4.
[1161] The invention comprises the method of paragraphs [00786] to
[00792] wherein said biologically active agent is selected from the
group consisting of antibodies, cholesterol, hormones, antivirals,
peptides, polypeptides, proteins, nucleoproteins,
chemotherapeutics, low molecular weight drugs, vitamins,
co-factors, nucleosides, nucleoside derivatives, nucleotides,
oligonucleotides, enzymatic nucleic acids, antisense nucleic acids,
triplex forming oligonucleotides, 2,5-A antisense chimeras,
allozymes, aptamers, decoy RNA molecules and analogs thereof, and
small nucleic acid molecules, such as an RNA interfering agent
(RNAi), short interfering nucleic acid (siNA), short interfering
RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and
short hairpin RNA (shRNA).
[1162] The invention comprises the method of paragraph [00793]
wherein said biologically active agent is a nucleoside or
nucleoside derivative.
[1163] The invention comprises the method of paragraph [00794]
wherein said biologically active agent is selected from an RNAi, an
siNA, an RNAi inhibitor, a miRNA, an siRNA and a shRNA.
[1164] Unless defined otherwise, the technical and scientific terms
used herein have the same meaning as that usually understood by a
specialist familiar with the field to which the invention
belongs.
[1165] Unless indicated otherwise, it is assumed that all other
methods, steps, techniques and manipulations that are not
specifically described in detail can be performed and have been
performed in a manner known to the skilled person. Reference is
e.g., made to the standard handbooks and the general background art
and to the further references cited therein.
[1166] Claims to the invention are non-limiting and are provided
below.
[1167] Although particular embodiments and claims have been
disclosed herein in detail, this has been done by way of example
for purposes of illustration only, and is not intended to be
limiting with respect to the scope of the appended claims, or the
scope of subject matter of claims of any corresponding future
application. In particular, it is contemplated by the inventors
that various substitutions, alterations, and modifications may be
made to the invention without departing from the spirit and scope
of the invention as defined by the claims. The choice of starting
material, biological material of interest, or liposome assembly
method is believed to be a matter of routine for a person of
ordinary skill in the art with knowledge of the embodiments
described herein. Other aspects, advantages, and modifications
considered to be within the scope of the following claims.
Redrafting of claim scope in later filed corresponding applications
may be due to limitations by the patent laws of various countries
and should not be interpreted as giving up subject matter of the
claims.
TABLE-US-00022 Table of Sequences SEQ ID NO: Sequence (5' to 3')
Type 1 UUu AAU UGA AAC cAA GAc Auu Artificial 2 uGu cuu GGu uuc AAu
uAA Auu Artificial 3 UAU UUA AgG AGG GUG AuC Uuu Artificial 4 AGA
Uca cCC Ucc uuA AAU auu Artificial
Sequence CWU 1
1
4121RNAartificialsiRNA construct 1uuuaauugaa accaagacau u
21221RNAartificialsiRNA construct 2ugucuugguu ucaauuaaau u
21321RNAArtificialsiRNA construct 3uauuuaagga gggugaucuu u
21421RNAArtificialsiRNA construct 4agaucacccu ccuuaaauau u 21
* * * * *