U.S. patent application number 13/378484 was filed with the patent office on 2012-09-20 for amphoteric liposomes comprising imino lipids.
This patent application is currently assigned to MARINA BIOTECH, INC.. Invention is credited to Steffen Panzner, Evgenios Siepi.
Application Number | 20120237589 13/378484 |
Document ID | / |
Family ID | 42555953 |
Filed Date | 2012-09-20 |
United States Patent
Application |
20120237589 |
Kind Code |
A1 |
Panzner; Steffen ; et
al. |
September 20, 2012 |
AMPHOTERIC LIPOSOMES COMPRISING IMINO LIPIDS
Abstract
The invention concerns lipid assemblies, liposomes having an
outer surface comprising a mixture of anionic and cationic
moieties; wherein at least a portion of the cationic moieties are
imino moieties that are essentially charged under physiological
conditions, and their use for serum resistant transfection of
cells.
Inventors: |
Panzner; Steffen; (Halle,
DE) ; Siepi; Evgenios; (Frenaros, CY) |
Assignee: |
MARINA BIOTECH, INC.
Bothell
WA
|
Family ID: |
42555953 |
Appl. No.: |
13/378484 |
Filed: |
July 2, 2010 |
PCT Filed: |
July 2, 2010 |
PCT NO: |
PCT/EP2010/059487 |
371 Date: |
June 8, 2012 |
Current U.S.
Class: |
424/450 ;
435/458; 514/20.9; 514/44A; 514/44R; 546/304; 554/104; 554/78 |
Current CPC
Class: |
A61K 9/1272 20130101;
C07C 279/14 20130101; C07C 233/43 20130101; C07J 41/0055 20130101;
C12N 2310/14 20130101; C07D 233/61 20130101; C12N 15/88 20130101;
C07C 233/36 20130101; C07C 237/22 20130101; C07C 279/12 20130101;
C12N 2320/32 20130101; Y02P 20/582 20151101; C07D 213/75 20130101;
C12N 15/1137 20130101 |
Class at
Publication: |
424/450 ;
514/44.R; 514/44.A; 514/20.9; 435/458; 554/78; 554/104;
546/304 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 31/713 20060101 A61K031/713; C07D 213/73 20060101
C07D213/73; C12N 15/85 20060101 C12N015/85; C07F 9/06 20060101
C07F009/06; C07C 279/14 20060101 C07C279/14; A61K 31/7088 20060101
A61K031/7088; A61K 38/14 20060101 A61K038/14 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 9, 2009 |
EP |
09165106.7 |
Sep 23, 2009 |
EP |
09171102.8 |
Claims
1. Lipid assemblies comprising anionic and cationic amphiphiles;
wherein at least a portion of the cationic amphiphiles are imino
lipids that are substantially charged under physiological
conditions, and wherein the anionic amphiphiles are carboxyl or
phosphate lipids and wherein further the charge ratio between the
cationic and anionic amphiphiles is 1.5 or less.
2. Lipid assemblies as in claim 1 comprising anionic and cationic
amphiphiles; wherein at least a portion of the cationic amphiphiles
are imino lipids that are substantially charged under physiological
conditions, and wherein further at least a portion of the anionic
amphiphiles are carboxyl lipids, and wherein the ratio between the
cationic and anionic amphiphiles is lower or equal to 1.5.
3. Lipid assemblies as in claim 1 comprising a combination of
lipids wherein the cationic lipids of said combination comprise a
guanido moiety and the anionic lipids of said combination comprise
a carboxyl group, further characterized in that the ratio between
the guanido moieties and the carboxyl groups is lower or equal to
1.5.
4. Lipid assemblies as in claim 1 comprising anionic and cationic
amphiphiles wherein at least a portion of the cationic amphiphiles
are imino lipids that are substantially charged under physiological
conditions, and wherein further at least a portion of the anionic
amphiphiles are phosphate lipids, and wherein the charge ratio
between the cationic and anionic amphiphiles is lower or equal to
1.5.
5. Lipid assemblies as in claim 1, further characterized in that
the charged imino groups of the cationic amphiphiles have a pK of
greater than 7.5 and are selected from imines, amidines, pyridines,
2-aminopyridines, heterocyclic nitrogen bases, guanido moieties,
isoureas and thioisoureas.
6. Lipid assemblies as in claim 1, wherein in cationic amphiphiles
are selected from the group comprising structures of I1 to I113,
structures A1 to A21 or structures L1 to L17, wherein members of
said group are further selected according to a pK greater 7.5.
7. Lipid assemblies as in claim 1, wherein the imino lipids are
selected from the group of PONA, CHOLGUA, GUADACA, MPDACA and
SAINT-18.
8. Lipid assemblies as in claim 1, wherein the anionic amphiphiles
are selected from the group of CHEMS, DMGS, DOGS, DOPA and
POPA.
9. Lipid assemblies as in claim 1, wherein said assemblies have a
charge ratio of the cationic and anionic lipids of between 0.5 and
1.5.
10. Lipid assemblies as in claim 1, wherein said assemblies are
liposomes.
11. Liposomes as in claim 10, further comprising a neutral or
zwitterionic lipid selected from cholesterol, phosphatidylcholine,
phosphatidylethanolamine, sphingomyeline and mixtures thereof.
12. Liposomes as in claim 11, wherein the neutral lipid is
cholesterol and the molar fraction of cholesterol in the lipid
mixture is between 10 and 50 mol %.
13. Liposomes as in claim 10, further comprising PEG lipids.
14. Liposomes as in claim 13, wherein the PEG lipids are situated
in the outermost membrane leaflet.
15. Liposomes as in claim 10, further comprising an active
pharmaceutical ingredient.
16. Liposomes as in claim 15, wherein said pharmaceutical
ingredient is an oligonucleotide.
17. Liposomes as in claim 16, wherein said oligonucleotide is a
decoy oligonucleotide, and antisense oligonucleotide, a siRNA, an
agent influencing transcription, a ribozyme, DNAzyme or an
aptamer.
18. Liposomes as in claim 16, wherein said oligonucleotides
comprise modified nucleosides such as DNA, RNA, LNA, PNA, 2'OMe
RNA, 2' MOE RNA, 2'F RNA in their phosphodiester or phosphothioate
forms.
19. The liposomes of claim 14, produced by a process comprising the
steps of (i) formation and sealing of the liposomes in the presence
of an active ingredient and (ii) a separate addition of PEG-lipids
after said step (i).
20. (canceled)
21. (canceled)
22. A method for transfecting cells comprising preparing liposomes
as in claim 10 and contacting the cells with the liposomes.
23. An aerosol for the transfection of lung cells comprising
liposomes as in claim 10.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to lipid assemblies or
liposomes that are capable of overcoming a lipoprotein mediated
uptake blockade. More specifically, this invention relates to
improvements in liposomes comprising both negatively charged lipids
having a carboxylic or phosphate head group and positively charged
lipids having imino or guanido moieties or derivatives thereof in
the respective polar regions.
BACKGROUND TO THE INVENTION
[0002] Liposomes have widespread use as carriers for active
ingredients. Neutral or negatively charged liposomes are often used
for the delivery of small molecule drugs, whereas positively
charged (cationic) or the recently introduced class of amphoteric
liposomes are mainly used for the delivery of nucleic acids such as
plasmids or oligonucleotides. Important examples for cationic
liposomes used for the delivery of nucleic acid cargoes include,
but are not limited to Semple et al., Nat. Biotech. (2010)
28:172-176; Akinc et al., Nat. Biotech. (2008) 26:561-569; Chien et
al., Cancer Gene Ther. (2005) 12:321-328; de Fougerolles, Nat. Rev.
Drug Discov. (2007) 6:443-453; Kim et al., Mol. Ther. (2006)
14:343-350; Morrissey, Nat. Biotech. (2005) 23: 1002-1007; Peer,
Science (2008) 319: 627-630 and Santel, Gene Ther. (2006) 13:
1222-1234. Application of amphoteric liposomes for the delivery of
nucleic acids has been demonstrated in Andreakos et al., Arthritis
Rheum. (2009) 60:994-1005.
[0003] Amphoteric liposomes belong to the larger family of
pH-sensitive liposomes, which further comprise pH-sensitive anionic
or cationic liposomes, prototypes of which have been presented in
Lai et al., Biochemistry (1985) 24:1654-1661 and Budker et al.,
Nat. Biotech. (1996) 14:760-764. Unlike the pH-sensitive anionic or
cationic liposomes, amphoteric liposomes are complex structures and
comprise at least a pair of lipids having complementary charge. WO
02/066012 discloses a key feature of amphoteric liposomes in that
these have a stable phase at both low and neutral pH. WO 02/066012
and WO07/107,304 describe a method of loading such particles with
nucleic acids starting from a low pH.
[0004] Hafez, et al. (Biophys. J. 2000, 79(3), 1438-1446) and WO
02/066012 provide some guidance as to how to select lipid mixtures
with truly amphoteric properties and more specifically how to
determine their isoelectric point and onset of fusion. Neutral
lipids can be additional constituents of amphoteric liposomes. The
inclusion of one or more such neutral lipids significantly adds to
the complexity of the mixture, especially since the individual
amounts of all the components may vary. The very high number of
possible combinations of lipids represents a practical hurdle
towards a more rapid optimisation of amphoteric liposomes. In this
regard, WO08/043,575 reveals strategies for the optimization of
stability, fusogenicity and cellular transfection of amphoteric
liposomes, particularly a method of predicting which mixtures of
lipids form satisfactorily stable lamellar phases at high and low
pH, whilst forming a fusogenic, hexagonal phase at an intermediate
pH.
[0005] The amphoteric liposomes according to the abovementioned
references are potent transfectants of cells. However, it was
observed that the function of some of these liposomes could be
blocked by the addition of certain sera, thereby potentially
limiting the activity of these liposomes for the targeting of
certain cells in vivo. This is further illustrated in the Examples
presented herein, e.g., Example 3.
[0006] The inhibition of the uptake of amphoteric liposomes
observed in different sera is apparently opposite to the recently
published activation of cationic carrier through complex formation
with lipoproteins, in this case ApoE, as demonstrated in Akinc at
al., Mol. Ther. (2010) electronic publication on May 11th, ahead of
print. DOI: 10.1038/mt.2010.85
[0007] A more detailed investigation revealed lipoproteins as
mediators of this inhibitory effect. As shown in Example 4 herein,
human serum deficient of lipoproteins is no longer able to inhibit
the uptake of liposomes as indicated by the functional delivery of
siRNA to the challenged cells. The inventors have now surprisingly
and unexpectedly found that certain species of cationic imino
lipids in combination with anionic lipids having a carboxyl or
phosphate moiety in their polar head groups are particularly
advantageous in maintaining transfection activity in the presence
of serum. Frequently, a particular advantage was observed when the
lipid assemblies or liposomes created from said lipid mixtures were
formulated according to the method described herein and in
WO08/043,575.
OBJECT OF THE INVENTION
[0008] It was therefore an object of the invention to provide lipid
assemblies or liposomes that can transfect cells in the presence of
various sera.
[0009] Another object of the invention is to provide pharmaceutical
compositions comprising such liposomes as a carrier for the
delivery of active agents or ingredients, including drugs such as
nucleic acid drugs, e.g., oligonucleotides and plasmids into cells
or tissues.
SUMMARY OF THE INVENTION
[0010] The present invention provides lipid assemblies, liposomes
and their use for transfection of cells wherein said lipid
assemblies comprise anionic and cationic amphiphiles and wherein at
least a portion of the cationic amphiphiles are imino lipids that
are substantially charged at pH7.5, and wherein the anionic
amphiphiles are carboxyl or phosphate lipids and wherein further
the charge ratio between the cationic and anionic amphiphiles is
1.5 or less.
[0011] In various embodiments of the invention, lipid assemblies
comprising anionic and cationic amphiphiles are provided wherein at
least a portion of the cationic amphiphiles are imino lipids that
are substantially charged under physiological conditions, and
wherein further at least a portion of the anionic amphiphiles are
carboxyl lipids, and wherein the ratio between the cationic and
anionic amphiphiles is lower or equal to 1.5.
[0012] In more specific aspects of the invention, lipid assemblies
comprising a combination of lipids are provided wherein the
cationic lipids of said combination comprise a guanido moiety and
the anionic lipids of said combination comprise a carboxyl group,
further characterized in that the ratio between the guanido
moieties and the carboxyl groups is lower or equal to 1.5.
[0013] In other embodiments of the invention, lipid assemblies
comprising anionic and cationic amphiphiles are provided wherein at
least a portion of the cationic amphiphiles are imino lipids that
are substantially charged under physiological conditions, and
wherein further at least a portion of the anionic amphiphiles are
phosphate lipids, and wherein the ratio between the cationic and
anionic amphiphiles is lower or equal to 1.5. In further preferred
aspects of such embodiments, the imino lipids are guanido
lipids.
[0014] The charged imino groups of the cationic amphiphiles of the
inventions have a pK of greater than 7.5 and are selected from
imines, amidines, pyridines, 2-aminopyridines, heterocyclic
nitrogen bases, guanido moieties, isoureas or thioisoureas. In
preferred embodiments, the cationic lipids are selected from the
group of PONA, CHOLGUA, GUADACA, MPDACA or SAINT-18.
[0015] In preferred embodiments, the anionic lipids are selected
from the group of CHEMS, DMGS, DOGS, DOPA or POPA.
[0016] In many embodiments, the lipid assemblies of the invention
are liposomes.
[0017] In further embodiments, the lipid assemblies also comprise
neutral lipids such as cholesterol, phosphatidylcholine,
phosphatidylethanolamine or sphingomyelin or mixtures thereof.
[0018] In preferred embodiments the neutral lipid is cholesterol
and the molar fraction of cholesterol in the lipid mixture is
between 10 and 50 mol %.
[0019] In some embodiments, the lipid assemblies also comprise
PEGylated lipids and in preferred aspects of such embodiments the
liposomes are produced by a process comprising the steps of (i)
formation and sealing of the liposomes in the presence of an active
ingredient and (ii) a separate addition of PEG-lipids after said
step (i).
[0020] It was unexpectedly found that serum resistant transfection
can be achieved with lipid assemblies or liposomes having an outer
surface comprising a mixture of anionic and cationic moieties;
wherein at least a portion of the cationic moieties are imino
moieties that are essentially charged under physiological
conditions. In numerous embodiments, the lipid assemblies and
liposomes of the present invention are formulated using a method
described in WO08/043,575 and also described in more detail
herein.
DETAILED DESCRIPTION OF THE INVENTION
Lipid Chemistry
[0021] By "chargeable" is meant that the amphiphile has a pK in the
range between 4 to pH 8. A chargeable amphiphile may therefore be a
weak acid or base. "Stable" in connection with charged amphiphiles
means a strong acid or base with a pK outside this range, which
results in substantially stable charge on the range pH 4 to pH
8.
[0022] By "amphoteric" herein is meant a substance, a mixture of
substances or a supra-molecular complex (e.g., a liposome)
comprising charged groups of both anionic and cationic character
wherein: [0023] 1) at least one, and optionally both, of the cation
and anionic amphiphiles is chargeable, having at least one charged
group with a pK between 4 and 8, [0024] 2) the cationic charge
prevails at pH 4, and [0025] 3) the anionic charge prevails at pH
8.
[0026] As a result the substance or mixture of substances has an
isoelectric point of neutral net charge between pH 4 and pH 8.
Amphoteric character is by this definition different from
zwitterionic character, as zwitterions do not have a pK in the
range mentioned above. In consequence, zwitterions are essentially
neutrally charged over a range of pH values; phosphatidylcholines
and phosphatidylethanolamines are neutral lipids with zwitterionic
character.
[0027] By "charge ratio" or "C/A" herein is meant the absolute
value or modulus of the ratio between the nominal charges usually
assigned to the cationic and anionic amphiphiles, respectively. The
nominal charge of a carboxyl group is "-1", that of a phosphate
moiety is "-2" and the nominal charge of an imino compound is "+1".
The "charge ratio" in a given mixture of amphiphiles or in a lipid
assembly is then calculated from the product of these nominal
charges and the respective molar fractions of the compounds
considered, neutral compounds such as cholesterol or zwitterionic
amphiphiles such as POPC or DOPE are not taken into account.
C/A=(x.sub.c1*z.sub.c1+x.sub.c2*z.sub.c2+ . . .
x.sub.cn*z.sub.cn)/(x.sub.a1*z.sub.a1+x.sub.a2*z.sub.a2+ . . .
x.sub.an*z.sub.an)
Wherein x.sub.c1 . . . n represents the molar fraction of a given
cationic compound, x.sub.a1 . . . n represents the molar fractions
of anionic compounds, z.sub.c1 . . . n stands for the nominal
charge of a given cationic compound and z.sub.a1 . . . n represents
the nominal charge of the anionic compound.
[0028] As an example, a mixture comprising 42 mol % of a carboxyl
lipid, 38% of an imino lipid and 20 mol % of a neutral lipid has a
charge ratio or CIA of 38/42=0.91. Another mixture comprising 27%
of a phosphate lipid, 43 mol % of an imino lipid and 30 mol % of a
neutral lipid has a charge ratio or C/A of 43/54=0.8 due to the
double nominal charge of the phosphate group.
[0029] It becomes apparent from the definition and examples, that
molar ratios or--for the sake of brevity--ratios between lipids and
charge ratios have the same meaning for single-charged species and
that these terms can be mutually exchanged within that group. This
is for example the case for combinations of imino and carboxy
lipids. In contrast to that, the molar ratio is different from the
charge ratio for phosphate lipids, since these compounds may bear a
double charge, e.g. in cases where the phosphate group is present
as a primary phosphate ester as in DOPA. As shown in the
calculation example above, the molar ratio or lipid ratio is then
double the charge ratio. For the sake of clarity only, the term
"charge ratio" is used with preference throughout this
disclosure.
[0030] By "physiological pH" or "physiological conditions" herein
is meant a pH of about 7.5.
[0031] Anionic lipids comprising carboxyl moieties in their polar
head groups are well known to the skilled artisan. Examples of
anionic lipids comprising carboxyl moieties in the polar head
groups can be selected from the structures (1)-(4) below,
##STR00001##
wherein n or m is an integer between 0 and 29, R.sub.1 and R.sub.2
are independently from each other an alkyl, alkenyl or alkinyl
moieties having between 8 and 24 carbon atoms and 0, 1 or 2
unsaturated bonds, A, B or D are independently from each other
absent, --CH2-, --CH.dbd., .dbd.CH--, --O--, --NH--, --C(O)--O--,
--O--C(O)--, --C(O)--NH--, --NH--C(O)--, --O--C(O)--NH--,
--NH--C(O)--O--, a phosphoric or phosphorous acid diester, and
"sterol" can be a cholesterol attached via its C3 atom.
[0032] The list below provides further specific examples of lipids
carrying a carboxyl group.
CHEMS Cholesterolhemisuccinate
[0033] Chol-COOH or Chol-C1Cholesteryl-3-carboxylic acid
Chol-C2 Cholesterolhemioxalate
Chol-C3 Cholesteroihemimalonate
Chol-C3N N-(Cholesteryl-oxycarbonyl)glycine
Chol-05 Cholesterolhemiglutarate
Chol-C6 Cholesterolhemiadipate
Chol-C7 Cholesterolhemipimelate
Chol-C8 Cholesterolhemisuberate
[0034] Chol-C12 Cholesterolhemidodecane dicarboxylic acid Chol-C13N
12-Cholesteryloxycarbonylaminododecanoic acid Chol-C16
Cholesterolhemihexadecane dicarboxylic acid
[0035] Cholesterolhemidicarboxylic acids and
Cholesteryloxycarbonylaminocarboxylic acids of following general
formula:
##STR00002##
wherein Z is C or --NH-- and n is any number between 0 and 29. DGS
or DG-SuccDiacylglycerolhemisuccinate (unspecified membrane
anchor)
DOGS or DOG-Succ Dioleoylglycerolhemisuccinate
DMGS or DMG-Succ Dimyristoylglycerolhemisuccinate
DPGS or DPG-Succ Dipalmitoylglycerolhemisuccinate
DSGS or DSG-Succ Distearoylglycerolhemisuccinate
[0036] POGS or POG-Succ
1-Palmitoyl-2-oleoylglycerol-hemisuccinate
DOGM Dioleoylglycerolhemimalonate
DOGG Dioleoylglycerolhemiglutarate
DOGA Dioleoylglycerolhemiadipate
DMGM Dimyristoylglycerolhemimalonate
DMGG Dimyristoylglycerolhemiglutarate
DMGA Dimyristoylglycerolhemiadipate
[0037] DOAS 4-{(2,3-Dioleoyl-propyl)amino}-4-oxobutanoic acid DOAM
3-{(2,3-Dioleoyl-propyl)amino}-3-oxopropanoic acid DOAG
5-{(2,3-Dioleoyl-propyl)amino}-5-oxopentanoic acid DOAA
6-{(2,3-Dioleoyl-propyl)amino}-6-oxohexanoic acid DMAS
4-{(2,3-Dimyristoyl-propyl)amino}-4-oxobutanoic acid DMAM
3-{(2,3-Dimyristoyl-propyl)amino}-3-oxopropanoic acid DMAG
5-{(2,3-Dimyristoyl-propyl)amino}-5-oxopentanoic acid DMAA
6-{(2,3-Dimyristoyl-propyl)amino}-6-oxohexanoic acid DOP
2,3-Dioleoyl-propanoic acid DOB 3,4-Dioleoyl-butanoic acid DOS
5,6-Dioleoyl-hexanoic acid DOM 4,5-Dioleoyl-pentanoic acid DOG
6,7-Dioleoyl-heptanoic acid DOA 7,8-Dioleoyl-octanoic acid DMP
2,3-Dimyristoyl-propanoic acid DMB 3,4-Dimyristoyl-butanoic acid
DMS 5,6-Dimyristoyl-hexanoic acid DMM 4,5-Dimyristoyl-pentanoic
acid DMG 6,7-Dimyristoyl-heptanoic acid DMA
7,8-Dimyristoyl-octanoic acid DOG-GluA Dioleoylglycerol-glucoronic
acid (1- or 4-linked) DMG-GluA Dimyristoylglycerol-glucoronic acid
(1- or 4-linked) DO-cHA
Dioleoylglycerolhemicyclohexane-1,4-dicarboxylic acid DM-cHA
Dimyristoylglycerolhemicyclohexane-1,4-dicarboxylic acid PS
Phosphatidylserine (unspecified membrane anchor)
DOPS Dioleoylphosphatidyiserine
DPPS Dipalmitoylphosphatidylserine
MA Myristic Acid
PA Palmitic Acid
OA Oleic Acid
LA Linoleic Acid
SA Stearic Acid
NA Nervonic Acid
BA Behenic Acid
[0038] POGA Palmitoyl-oleoyl-glutamic acid DPAA Dipalmitoylaspartic
acid
[0039] Any dialkyl derivatives of the anionic lipids comprising
diacyl groups listed above are also within the scope of the present
invention.
[0040] Preferred anionic lipids having a carboxyl group can be
selected from the group of Chol-C1 to Chol-C16 including all its
homologues, in particular CHEMS. Also preferred are the anionic
lipids DMGS, DPGS, DSGS, DOGS, POGS.
[0041] Anionic lipids comprising phosphate moieties in their polar
head groups are well known to the skilled artisan. Examples for
phosphate lipids can be selected from structures (P1)-(P4)
below:
##STR00003##
wherein n or m is an integer between 0 and 29, R.sub.1 and R.sub.2
are independently from each other an alkyl, alkenyl or alkinyl
moieties having between 8 and 24 carbon atoms and 0, 1 or 2
unsaturated bonds, A, B or D are independently from each other
absent, --CH2-, --CH.dbd., .dbd.CH--, --O--, --NH--, --C(O)--O--,
--O--C(O)--, --C(O)--NH--, --NH--C(O)--, --O--C(O)--NH-- or
--NH--C(O)--O-- and "sterol" can be a cholesterol attached via its
C3 atom.
[0042] The list below provides further specific examples of lipids
carrying a phosphatidic acid group.
Chol-P Cholesterol-3-phosphate DOPA Dioleoyl-phosphatidic acid POPA
Palmitoyl-oleoyl-phosphatidic acid DPPA Dipalmitoyl-phosphatidic
acid DMPA Dimyristoylphosphatidic acid. Cetylphosphate or
phosphoric acid ester homologues with R1 having between 16 and 24
carbon atoms.
[0043] The cationic lipids that can be used with this invention are
amphipathic molecules comprising an imino moiety in their polar
head group, wherein such imino moiety is substantially charged
under physiological conditions. Therefore, in preferred embodiments
the pK value of this functional group is 7.5 or greater, in further
preferred forms the pK value of the imino group is 8.5 of higher.
Imino moieties having such characteristics can be imines itself or
be part of larger functional groups, such as amidines, pyridines,
2-aminopyridines, heterocyclic nitrogen bases, guanido functions,
isoureas, isothioureas and the like.
[0044] The following structures (I1) . . . (I113) represent some
specific examples of such imino moieties,
##STR00004## ##STR00005## ##STR00006## ##STR00007## ##STR00008##
##STR00009##
wherein L represents the apolar region and optionally linker or
spacer moieties of the amphipathic lipid molecule. Examples of L
can further be selected from the following general structures (11)
to (15),
##STR00010##
wherein n or m represent an integer between 0 and 29, R.sub.1 and
R.sub.2 are independently from each other an alkyl, alkenyl or
alkinyl moieties having between 8 and 24 carbon atoms and 0, 1 or 2
unsaturated bonds, A, B or D are independently from each other
absent, --CH2-, --CH.dbd., .dbd.CH--, --O--, --NH--, --C(O)--O--,
--O--C(O)--, --C(O)--NH--, --NH--C(O)--, --O--C(O)--NH-- or
--NH--C(O)--O-- and "sterol" can be a cholesterol attached via its
C3 atom.
[0045] The following Table 1 provides calculated or database values
for the pK of the imino containing moieties (I1) through to (I113).
For quarternized imino moieties, a hypothetical value of 99 was
introduced to merely highlight this fact.
TABLE-US-00001 TABLE 1 pK values for the moieties I1-I113 pK moiety
imino amino ring N guanido N I1 10.49 I2 7.23 I3 7.23 I4 7.08 I5
8.41 I6 8.06 I7 7.87 I8 7.52 I9 11.58 I10 6.18 I11 6.61 I12 7.01
I13 n.d. I14 n.d. I15 5.62 I16 5.89 I17 0.63 I18 4.53 I19 6.22 I20
6.99 I21 5.36 I22 5.11 I23 5.85 I24 6.03 I25 12.06 -5 I26 12.37
-4.91 I27 12.37 -4.91 I28 12.37 -4.91 I29 12.37 -3.58 I30 12.37
-3.68 I31 12.37 -3.58 I32 12.06 -5 I33 12.68 -3.49 I34 12.66 -3.58
I35 10.98 -5.43 I36 12.98 -4.25 I37 12.52 -3.12 I38 12.82 -4.01 I39
13.13 -3.93 I40 13.12 -3.68 I41 12.37 -3.25 I42 12.68 -4.04 I43
12.99 -3.96 I44 12.98 -3.71 I45 9.1 -4.89 I46 9.37 -3.47 I47 10.66
-3.56 I48 99 -3.47 I49 8.47 -4.89 I50 9.02 -3.47 I51 10.31 -3.56
I52 99 -3.47 I53 7.73 I54 10.62 -6.91 I55 1.92 -5.58 I56 10.63
-6.87 I57 8.62 -7.89 I58 11.03 -5.39 I59 9.31 -4.75 I60 8.67 -6.83
I61 9.37 -3.47 I62 10.66 -3.56 I63 99 -3.47 I64 7.19 -7.59 I65 7.41
-2.85 I66 8.37 -2.58 I67 99 -2.7 I68 13.72 -1.04 I69 14.03 2.05 I70
14.14 1.71 I71 11.11 0.94 I72 14.33 1.68 I73 14.25 -0.71 I74 14.73
-0.4 I75 13.9 -0.09 I76 14.04 -0.1 I77 14.18 -0.72 I78 14.67 -0.41
I79 14.18 -0.2 I80 14.33 -0.2 I81 9.85 -1.92 I82 10.17 -0.57 I83
11.41 -0.65 I84 99 -0.57 -13.15 I85 14.33 -0.98 I86 14.33 -0.57 I87
14.47 -0.68 I88 99 -0.57 -11.28 I89 10 -8.4 I90 8.69 -9.2 I91 10.93
-7.8 I92 10.08 -6.76 I93 10.32 -6.88 I94 3.51 I95 3.51 I100 8.98
-8.16 I101 8.85 -8.94 I102 9.9 -7.55 I103 9.69 -6.76 I104 9.29
-6.88 I105 8.82 -9.73 I106 10.58 -8.09 I107 12.49 -3.67 I108 12.49
-3.67 I109 12.36 -3.67 I110 12.8 -3.58 I111 12.78 -3.58 I112 10.62
-3.58 I113 10.27 -3.58
[0046] It becomes apparent from the data presented here, that most
of the structures I1-I113 comprise preferred imino moieties having
a pK greater 7.5 or even greater than 8.5.
[0047] The pK values can be taken from public databases.
Alternatively, there is expert software in the public domain that
can calculate, predict or extrapolate such values, e.g., ACD/Labs
v7 (by Advanced Chemistry Development, Ontario, Canada) or the
like.
[0048] The imino moieties analyzed above are illustrating the
teachings of this invention, without limiting it to the specific
examples. It is of course possible to change the position of
substituents, in particular when ring systems such as pyrrols or
pyridins are used for practicing this invention. It is also
possible to replace the aliphatic radicals used throughout I1-I13
with aromatic residues or aryl moieties. The following list of
compounds (A1) through to (A21) provides a few examples that should
further illustrate such modifications, wherein L is defined as
above.
##STR00011## ##STR00012##
[0049] The following Table 2 provides calculated or database values
for the pK of the imino containing moieties (A1) through to (A21)).
For quarternized imino moieties, a hypothetical value of 99 was
introduced to merely highlight this fact.
TABLE-US-00002 TABLE 2 pK values for structures (A1) to (A21).
structure atom pK atom pK atom pK A1 ring 7.29 out -7.16 A2 ring 99
A3 ring 99 out -6.76 A4 ring 7.06 out -6.91 A5 ring 4.74 A6 imino
12.15 amidin -4.95 A7 imino 3.07 amidin -12.14 ring 99 A8 imino
14.24 ring -1.31 A9 imino 14.18 amidin -0.72 A10 imino 12.52 amidin
-3.12 A11 imino 14.18 ring -1.27 A12 imino 14.25 amidin -0.71 A13
imino 12.31 amidin -5 A14 imino 13.75 amidin -0.76 A15 imino 10.98
amidin -5.43 A16 imino 7.96 A17 imino 9.44 amidin -8.39 A18 imino
9.78 amidin 0.95 A19 imino 8.52 out -1.86 A20 imino 11.97 amidin
-6.3 A21 imino 12.5 amidin -3.6
[0050] Again, many of the structures presented in the above Table 2
comprise preferred imino moieties having a pK greater 7.5 or even
greater than 8.5.
[0051] As mentioned above, the charged imino moieties can be
combined with lipid anchors or hydrophobic portions to yield lipids
or amphiphiles that are capable of forming lipid bilayers by
themselves or can be integrated into lipid membranes formed from
other lipids or amphiphiles. In some embodiments, specific lipids
or amphiphiles are selected from the examples L1 to L17 presented
below,
##STR00013## ##STR00014## ##STR00015##
wherein R.sub.1 and R.sub.2 are independently from each other an
alkyl, alkenyl or alkinyl moieties having between 8 and 24 carbon
atoms and 0, 1 or 2 unsaturated bonds.
[0052] Some of these lipids have been presented earlier in the
literature, for example the guanido lipids in WO91/16024,
WO97/43363, WO98/05678, WO01/55098, WO2008/137758 (amino acid
lipids), in EP 0685234 (based on diacylglycerols), U.S. Pat. No.
5,965,434 (also based on diacylglycerols) or the pyridinium
compounds in U.S. Pat. No. 6,726,894. Furthermore, as demonstrated
in WO29086558 or illustrated in structure (15), it is also possible
to use alternative lipid backbones, e.g. those comprising a
dioxolane linker segment while maintaining the functionality of the
respective head groups.
Lipid Mixtures and Optional Other Lipids
[0053] The present invention discloses lipid mixtures comprising
anionic and cationic amphiphiles; wherein at least a portion of the
cationic amphiphiles are imino lipids that are substantially
charged under physiological conditions, and wherein further at
least a portion of the anionic amphiphiles are carboxyl lipids or
phosphate lipids.
[0054] A co-presence of both cationic lipids comprising a charged
imino moiety in their polar head group and anionic lipids
comprising a carboxyl or phosphate function in their polar head
group is a central feature of this invention. That is, liposomes or
lipid assemblies that substantially lack one of these elements are
not contemplated in the practice of the present invention. The
cationic imino lipids and the anionic lipids can be present in
different ratios; said ratios are characterized herein as "charge
ratios" (cation:anion ratios, C/A, see definitions) throughout this
disclosure. In many embodiments the C/A ratio is above 0.33, in
preferred embodiments this ratio is above 0.5 and in some
embodiments the ratio is equal or above 0.66. In preferred aspects
of said embodiments the C/A is equal or below 3, in further
preferred aspects the ratio is equal or below 2 and in particularly
preferred aspects the ratio is equal or below 1.5.
[0055] In many aspects of said embodiments, the resulting lipid
mixture has amphoteric character. Imino lipids having a pK of more
than 7.5, and even more so the preferred imino lipids having a pK
of 8.5 or higher are essentially charged under physiological
conditions, their actual charge becomes close and eventually
identical with their nominal charge. The typical pK of carboxyl
lipids is between 4.5 and 6 and these lipids are therefore also
charged at physiological pH. Mixtures of both the imino and the
carboxyl lipid therefore have net negative charge at physiological
pH whenever C/A is smaller than 1, the net charge become 0 at C/A=1
and positive for C/A>1.
[0056] At low pH, the anionic charge disappears around the pK of
the carboxyl lipid, which renders lipid mixtures having a C/A<1
first neutral and then positively charged. The charge reversal is
characteristic for C/A<1 and defines the amphoteric character.
Lipid mixtures having C/A=1 or C/A>1 also undergo a reduction of
negative charges at low pH, but no charge reversal. It should
however be noted, that the relationship between C/A and amphoteric
character of the resulting lipid assemblies implies a statistic,
essentially equal distribution of the charged moieties across a
given bilayer. That means that the inner and outer leaflet of a
membrane must have the same composition of charged lipids to
maintain the full validity of these calculations. This may not
always be the case as demonstrated in example 9 and liposomes of
amphoteric character can be formed even with lipid mixtures having
C/A>1. Still, the correlations between membrane composition and
amphoteric character disclosed here give good guidance for the
selection of lipid mixtures.
[0057] The lipid mixtures may further comprise additional cationic,
anionic, neutral/zwitterionic, or functionalized lipids. Additional
cationic lipids may be known components such as DOTAP, DODAP,
DC-Chol and the like. Additional anionic lipids may be selected
from negatively charged phospholipids, such as
phosphatidylglycerol, phosphatidic acid, dicetylphosphoric acid,
cardiolipin and the like. Neutral or zwitterionic lipids are
cholesterol, phosphatidylethanolamine, phosphatidylcholine,
sphingomyelin and the like.
[0058] In preferred embodiments the neutral lipid is cholesterol.
Further preferred are variants wherein the lipid mixtures comprise
between 10 mol % and 50 mol % of cholesterol, even more preferred
are variants with about 20 mol % and 40 mol % cholesterol.
[0059] An important group of functionalized lipids are those
comprising polymer extensions such polyethylenglycol (PEG-lipids).
Numerous PEGylated lipids are known in the state of the art and
essential differences can be found in (i) the size and degree of
branching of the PEG-chain, (ii) the type of the linker group
between PEG and the membrane-inserted portion of the molecule and
(iii) the size of the hydrophobic, membrane inserted domain of a
PEGylated lipid. Further aspects of PEGylation are (iv) the density
of the modification in the lipid assemblies and (v) their
orientation within such lipid assemblies.
[0060] In many embodiments of the aspect (i), the PEG fragment has
a molecular weight between 500 Da and 5,000 Da, in more preferred
embodiments, this fragment has a molecular weight of about 700 Da
to 2,500 Da and even more preferred are PEG fragments of about
2,000 Da. In many such embodiments, the PEG moiety is a single
chain, non-branched PEG.
[0061] Typical embodiments of aspect (ii) are phosphoethanolamine
moieties, diacylglycerols moieties or the polar head groups of
ceramides.
[0062] The size of the hydrophobic, membrane inserted domain
characterized in aspect (iii) is a further important feature of
such molecules as it determines the membrane residence time of the
PEG lipid within a bilayer. As an example, PEGylated lipids having
a short hydrophobic domain such as DMPE-PEG2000 (a
dimyristoylphosphatidylethanolamine-PEG conjugate, wherein the PEG
chain has a molecular weight of 2000 Da) diffuse from a given
membrane within seconds, whereas the DSPE-PEG2000 homologue resides
in a bilayer for many hours or days (see Silvius, J. R. and
Zuckermann, M. J. (1993) Biochemistry 32, 3153-3161 or Webb, M. S.
et al (1998) in Biochim Biophys Acta 1372: 272-282 or Wheeler et
al. (1999) in Gene Ther 6: 271-281.
[0063] PEGylation at the same time provides colloidal stability to
liposomes, in particular to combinations of cationic liposomes with
anionic nucleic acid cargoes as illustrated in U.S. Pat. No.
6,287,591 but also impairs the cellular uptake and/or endosomal of
liposomes (see Shi, F. et al. (2002) in Biochem. J. 366:333-341). A
transient PEGylation is now state of the art and satisfies the need
for both colloidal stability and activity of the particles.
[0064] A further aspect (iv) of PEGylation is the density of such
modification, which should be between 0.5 and 10 mol % of the lipid
mixture, in preferred embodiments the degree of PEGylation is about
1 to 4 mol %.
[0065] Since PEGylation of a given bilayer stabilizes the lamellar
phase of the lipid assembly and impairs lipid fusion associated
with the formation of a hexagonal phase, the amount of residual PEG
moieties in a bilayer must be minimal. This can be achieved by
titration of the required amounts of PEGlipids. In some embodiments
of aspect (v) the liposomes are thus PEGylated on both membrane
leaflets and the amount of PEG is minimized. In another variant,
PEG removal is as complete as possible. While this is easily
achieved for the PEG lipids associated with the outer bilayer,
diffusion is essentially not possible for PEG lipids attached to
the interior of the lipid structure. It is thus a preferred
embodiment of the aspect (v) of this invention to provide Liposomes
comprising charged imino and carboxyl or phosphate lipids further
comprising PEGylated lipids, wherein said PEGylated lipids are
essentially situated on the outer surface.
[0066] Such liposomes can be characterized by the process of their
production, wherein liposomes are formed in a first step and this
step also comprises encapsulation of cargo molecules. The
PEG-lipids are then inserted into the outer bilayer of the
pre-fabricated liposomes in a second step, e.g. by addition of a
micellar solution of PEGylated lipids to the liposome suspension.
In a specific embodiment of such process, the liposomes
sequestering nucleic acids are formed by mixing of a watery
solution of nucleic acids with an alcoholic solution of lipids.
Liposomes entrapping nucleic acids are formed spontaneously and the
PEGylated lipids are added in a subsequent step.
[0067] With particular advantage, such process can be practiced
with amphoteric liposomes, as these liposomes already provide
colloidal stability and the time element between liposome formation
and PEGylation is less critical. The preparation of amphoteric
liposomes encapsulating nucleic acids is disclosed in WO 02/066012,
its continuation US2007/0252295 or further in WO 07/107,304.
[0068] In a preferred embodiment, amphoteric liposomes comprising
imino and carboxyl or phosphate lipids are PEGylated on their outer
surface by providing the required amounts of PEG lipid together
with the neutralization buffer. For that, the PEG lipids can be
dissolved in the neutralization buffer. In another embodiment, said
liposomes are formed and neutralized and the PEG lipid is added
separately after a time interval of between 0.1 s and several days.
In yet another embodiment, the liposomes are formed and neutralized
and the liposome suspension is further concentrated and the PEG
lipids are added after the concentration of the materials. In yet
another embodiment, the liposomes are formed and neutralized and
concentrated and the non-encapsulated nucleic acid is removed and
optionally the buffer for the liposome suspension is exchanged and
the PEG lipids are added afterwards. In summary, the PEG lipids can
be added at any time after the formation and closure of the
liposomes.
[0069] In other embodiments the liposomes comprising imino and
carboxyl or phosphate lipids have pH-sensitive cationic character
and are PEGylated on their outer surface by providing the required
amounts of PEG lipid upon formation and closure of said liposomes,
following the steps outline above. Since pH-sensitive liposomes are
more prone to form aggregates in the presence of nucleic acids, a
rapid PEGylation is preferred and the PEG lipids are added
immediately upon closure of the liposomes, e.g. between 0.1 s and 1
min after their production.
[0070] In contrast to the above methods yielding product liposomes
that are essentially PEGylated on their outer surface, presence of
PEGylated lipids during the actual formation of liposomes; that is
before the nascent structures close, results in a different
product. Although structural data have not yet been obtained, the
skilled artisan would expect in such situation that a substantial
amount of PEG moieties also resides in the inner leaflet of the
membrane. This is similar to the situation of the nucleic acid
cargo which also has access to both leaflet of the nascent liposome
and of which a substantial portion can be detected inside the
liposomes, once these have closed.
Lipid Assemblies
[0071] The components mentioned herein can be assembled in various
structures known to the skilled artisan. These can be liposomes
comprising one or a number of individual bilayers, other
supramolecular lipid assemblies or vesicles having a sizeable
interior volume that provides an aqueous phase. It also can be
emulsion droplets or structures in the form of lipoplex assemblies,
the latter in many embodiments comprising electrostatic complexes
between the lipids and nucleic acids. In preferred embodiments,
these structures are liposomes or vesicles. In many embodiments,
the liposomes or vesicles have a sizeable aqueous interior. In many
aspects of this invention, an active pharmaceutical ingredient is
complexated, encapsulated, sequestered or otherwise associated with
the lipid assemblies.
[0072] Given the large number of useful imino and carboxyl or
phosphate and additional lipids, a very high number of potentially
useful combinations does exist, thereby creating a further need for
selection and optimization amongst the many variants. WO08/043,575
gives specific guidance and provides a method for the optimization
of complex lipid assemblies, specifically for lipid bilayers, as
discussed in further detail herein. In brief, the teachings in
WO08/043,575 demonstrate that amphoteric lipid mixtures form stable
bilayers both under acidic and neutral pH conditions, however, the
bilayers formed from these lipid mixtures can undergo phase
transition and fusion at their isoelectric point, which typically
is at slightly acidic conditions. WO08/043,575 discloses the use of
moderately sized or small lipid head groups for the charged lipid
components. WO 08/043,575 also teaches the use of large or bulky
buffer ions to stabilize the lamellar phases at low pH during the
loading procedure, as well as the use of large or bulky buffer ions
to stabilize the lamellar phases at neutral pH during storage. In
particular, reference is made to pages 44-57 of WO 08/043,575,
which feature the essential elements cited above. The reference
further discloses the use of neutral lipids bearing a small head
group such to maximize the fusion activity. Typical neutral lipids
for improved fusion are cholesterol or DOPE. Specific
considerations and optimization rules for the neutral lipids are
further presented in WO 09/047,006, in particular on pages 63
through to 70.
[0073] Altogether, WO 08/043,575 or WO 09/047,006, together
referred to as "the References" herein provide rational guidance
for the optimization of lipid assemblies. The References are not
restricted to amphoteric liposomes, but provide a comprehensive
model for the structure-activity relationship of lipid
assemblies.
[0074] The present invention represents an advance in the art, as
it provides optimized methods of formulating liposomes that are
capable of circumventing cellular binding, interaction or
competition with lipoproteins or other serum components. While the
methods taught by References provide the information for the
necessary fusogenicity of lipid assemblies, they are silent with
respect to a prediction of the cellular binding of the
liposomes.
[0075] Thus, it is an object of the present invention to provide
lipid assemblies, lipid mixtures, and liposomes formulated by the
method disclosed in the References in combination with the
unexpected properties observed when using an imino lipid that is
substantially charged under physiological conditions is used in
combination with an anionic lipid having a carboxyl or phosphate,
that is, negatively charged moiety. Without wishing to be bound by
theory, the novel compositions formulated herein can better
facilitate lipoprotein-like cellular binding and uptake--a feature
that is not known in the art.
[0076] The lipid mixtures described herein can have amphoteric or
pH-sensitive cationic properties, both of which are generally
conveyed towards the lipid assemblies or liposomes by the lipids
forming them. Charge properties can easily predicted as described
in WO 02/066012 for a symmetrical distribution of the lipids
towards both leaflets of a lipid membrane or bilayer. However, in
some cases the lipid distribution of the outermost leaflet may
differ from other parts of the assembly. Macroscopically, lipid
mixtures comprising charged imino lipids in combination with
carboxyl or phosphate lipids having C/A somewhat larger than 1 may
therefore still form liposomes having amphoteric character, as
demonstrated in example 9 and FIG. 1
[0077] For purposes of in silico optimization and prediction, lipid
mixtures of the present invention having a C/A<1 are considered
amphoteric and can form lipid assemblies categorized as "amphoter
I" mixtures according to the classification of the References. In
other embodiments, lipid mixtures are used that have C/A=1 or
C/A>1; these are pH-sensitive cationic lipid mixtures, that is
their charge is neutral or cationic at physiological pH and becomes
more cationic with descending pH. The pH-sensitive cationic
mixtures of said embodiments do no longer have an isoelectric point
as it is the case with their amphoteric counterparts. Still, the
structure-activity relationships provided in the References are
applicable as these provide a universal understanding of the phase
behaviour of lipid assemblies in combination with solute and ions
irrespective of their charge.
[0078] For the sake of clarity, lipid mixtures of the present
invention comprise one or more cationic lipids having an imino
group that is substantially charged at physiological pH, further
comprising one or more anionic lipids having a carboxyl or
phosphate group, optionally further comprising neutral lipids.
[0079] The amphoteric character of liposomes has further
advantages. The negative surface charge of such liposomes or lipid
assemblies improves greatly the colloidal stability of the
liposomes in suspension. This is of particular importance in
combinations with polyanionic cargoes such as nucleic acids, which
easily produce aggregates with cationic liposomes.
[0080] The negative to neutral surface charge of the amphoteric
lipid assemblies or liposomes is also advantageous when
administering the liposomes in vivo, where it prevents unspecific
adsorption on endothelia or the formation of aggregates with serum
components as observed with cationic liposomes (see Santel et al.,
(2006) in Gene Therapy 13: 1222-1234 for endothelial adsorption of
cationic liposomes or Andreakos et al., (2009) in Arthritis and
Rheumatism, 60:994-1005 for the prevention of aggregate formation
with amphoteric liposomes).
[0081] Thus, in preferred embodiments, the liposomes of this
invention have amphoteric character. Within this group, it is of
advantage to avoid very low percentages of the cationic component
to maintain effective loading of the particles with polyanionic
cargos, e.g. nucleic acids. In further preferred embodiments, the
C/A is greater 0.5.
[0082] When applied systemically, that is, into the bloodstream,
the liposomes undergo a certain distribution within the body.
Typical target sites are liver and spleen, but also include the
circulating phagocytic cells. The liposomes also contact the
endothelia surrounding the blood vessels and may transfect these
cells. The accumulation of liposomes in inflamed sites and tumors
is of particular therapeutic relevance.
[0083] The skilled artisan would be aware of methods to direct the
distribution of particles towards one or the other site. It is well
known that liposomes having a small diameter of about 150 nm or
less can penetrate the liver endothelium, thus gaining access to
the hepatocytes and other cells of the liver parenchyme. In aspects
where targeting of the liver hepatocytes is of therapeutic
interest, the liposomes of this inventions can be 150 nm or less in
diameter, in preferred embodiments, the liposomes can be less than
120 nm in diameter.
[0084] It is also well known that particles having a diameter of
100 nm or more are well recognized by phagocytic cells. Therefore,
in embodiments where macrophages or dendritic cells constitute the
target of interest, the liposomes of this invention are 120 nm or
larger. In some embodiments, these liposomes are 150 nm or larger.
In other embodiments these liposomes can be as a large as 250 nm,
or up to 400 nm in size.
[0085] It has also been described that surface charge may influence
the circulation time, hence the biodistribution of liposomes and it
is well established that PEGylation reduces the surface charge and
results in prolonged circulation of the liposomes. Prolonged
circulation is generally thought to maximize the distribution
towards tumors. Therefore, in aspects where tumors constitute the
target of interest, the liposomes of this invention have a small
net surface charge and are characterized by a C/A of between 0.67
and 1.5. In preferred embodiments for such applications the lipid
mixtures forming said liposomes have a C/A between 0.8 and 1.25.
Also, the liposomes targeting tumors are of small size. In
preferred embodiments such liposomes are smaller than 150 nm, in
further preferred embodiments the liposomes are smaller than 120
nm. In some embodiments, the liposomes further comprise PEG
lipids.
Cargoes for the Liposomes of this Invention
[0086] The liposomes or lipid assemblies of this invention can
sequester or encapsulate at least one active agent. Said active
agent may comprise a drug. In some embodiments, said active agent
may comprise one or more nucleic acids. In preferred embodiments,
the active ingredient consists of nucleic acids.
[0087] Without being limited to such use, the liposomes or lipid
assemblies described in the present invention are well suited for
use as carriers for nucleic acid-based drugs, such as for example,
oligonucleotides, polynucleotides and DNA plasmids. These drugs are
classified into nucleic acids that encode one or more specific
sequences for proteins, polypeptides or RNAs and into
oligonucleotides that can specifically regulate protein expression
levels or affect the protein structure through, inter alia,
interference with splicing and artificial truncation.
[0088] In some embodiments of the present invention, therefore, the
nucleic acid-based therapeutic may comprise a nucleic acid that is
capable of being transcribed in a vertebrate cell into one or more
RNAs, which RNAs may be mRNAs, shRNAs, miRNAs or ribozymes, wherein
such mRNAs code for one or more proteins or polypeptides. Such
nucleic acid therapeutics may be circular DNA plasmids, linear DNA
constructs, like MIDGE vectors (Minimalistic Immunogenically
Defined Gene Expression) as disclosed in WO 98/21322 or DE
19753182, or mRNAs ready for translation (e.g., EP 1392341).
[0089] In other embodiments of the invention, oligonucleotides may
be used that can target existing intracellular nucleic acids or
proteins. Said nucleic acids may code for a specific gene, such
that said oligonucleotide is adapted to attenuate or modulate
transcription, modify the processing of the transcript or otherwise
interfere with the expression of the protein. The term "target
nucleic acid" encompasses DNA encoding a specific gene, as well as
all RNAs derived from such DNA, being pre-mRNA or mRNA. A specific
hybridisation between the target nucleic acid and one or more
oligonucleotides directed against such sequences may result in an
inhibition or modulation of protein expression. To achieve such
specific targeting, the oligonucleotide should suitably comprise a
continuous stretch of nucleotides that is substantially
complementary to the sequence of the target nucleic acid.
[0090] Oligonucleotides fulfilling the abovementioned criteria may
be built with a number of different chemistries and topologies. The
oligonucleotides may comprise naturally occurring or modified
nucleosides comprising, but not limited to, DNA, RNA, locked
nucleic acids (LNA's), unlocked nucleic acids (UNA's), 2'O-methyl
RNA (2'Ome), 2' O-methoxyethyl RNA (2'MOE) in their phosphate or
phosphothioate forms or Morpholinos or peptide nucleic acids
(PNA's). Oligonucleotides may be single stranded or double
stranded.
[0091] Oligonucleotides are polyanionic structures having 8-60
charges. In most cases, these structures are polymers comprising
nucleotides. The present invention is not limited to a particular
mechanism of action of the oligonucleotides and an understanding of
the mechanism is not necessary to practice the present invention.
The mechanisms of action of oligonucleotides may vary and might
comprise inter alia effects on splicing, transcription,
nuclear-cytoplasmic transport and translation.
[0092] In a preferred embodiment of the invention, single stranded
oligonucleotides may be used, including, but not limited to
DNA-based oligonucleotides, locked nucleic acids, 2'-modified
oligonucleotides and others, commonly known as antisense
oligonucleotides. Backbone or base or sugar modifications may
include, but are not limited to, Phosphothioate DNA (PTO),
2'O-methyl RNA (2'Ome), 2'Fluoro RNA (2'F), 2' O-methoxyethyl-RNA
(2'MOE), peptide nucleic acids (PNA), N3'-P5' phosphoamidates (NP),
2' fluoroarabino nucleic acids (FANA), locked nucleic acids (LNA),
unlocked nucleic acids (UNA), Morpholine phosphoamidate
(Morpholino), Cyclohexene nucleic acid (CeNA), tricyclo-DNA (tcDNA)
and others. Moreover, mixed chemistries are known in the art, being
constructed from more than a single nucleotide species as
copolymers, block-copolymers or gapmers or in other
arrangements.
[0093] In addition to the aforementioned oligonucleotides, protein
expression can also be inhibited using double stranded RNA
molecules containing the complementary sequence motifs. Such RNA
molecules are known as siRNA molecules in the art (e.g., WO
99/32619 or WO 02/055693). Other siRNAs comprise single stranded
siRNAs or double stranded siRNAs having one non-continuous strand.
Again, various chemistries were adapted to this class of
oligonucleotides. Also, DNA/RNA hybrid systems are known in the
art. Other varieties of siRNA's comprise three-stranded constructs
wherein two smaller strand hydridize to one common longer strand,
the so-called meroduplex or sisiRNA's having nicks or gaps in their
architecture.
[0094] In another embodiment of the present invention, decoy
oligonucleotides can be used. These double stranded DNA molecules
and chemical modifications thereof do not target nucleic acids but
transcription factors. This means that decoy oligonucleotides bind
sequence-specific DNA-binding proteins and interfere with the
transcription (e.g., Cho-Chung, et al., in Curr. Opin. Mol. Ther.,
1999).
[0095] In a further embodiment of the invention, oligonucleotides
that may influence transcription by hybridizing under physiological
conditions to the promoter region of a gene may be used. Again,
various chemistries may adapt to this class of
oligonucleotides.
[0096] In a still further alternative of the invention, DNAzymes
may be used. DNAzymes are single-stranded oligonucleotides and
chemical modifications thereof with enzymatic activity. Typical
DNAzymes, known as the "10-23" model, are capable of cleaving
single-stranded RNA at specific sites under physiological
conditions. The 10-23 model of DNAzymes has a catalytic domain of
15 highly conserved deoxyribonucleotides, flanked by 2
substrate-recognition domains complementary to a target sequence on
the RNA. Cleavage of the target mRNAs may result in their
destruction and the DNAzymes recycle and cleave multiple
substrates.
[0097] In yet another embodiment of the invention, ribozymes can be
used. Ribozymes are single-stranded oligoribonucleotides and
chemical modifications thereof with enzymatic activity. They can be
operationally divided into two components, a conserved stem-loop
structure forming the catalytic core and flanking sequences which
are reverse complementary to sequences surrounding the target site
in a given RNA transcript. Flanking sequences may confer
specificity and may generally constitute 14-16 nt in total,
extending on both sides of the target site selected.
[0098] In other embodiments of the invention, aptamers may be used
to target proteins. Aptamers are macromolecules composed of nucleic
acids, such as RNA or DNA, and chemical modifications thereof that
bind tightly to a specific molecular target and are typically 15-60
nt long. The chain of nucleotides may form intramolecular
interactions that fold the molecule into a complex
three-dimensional shape. The shape of the aptamer allows it to bind
tightly against the surface of its target molecule including but
not limited to acidic proteins, basic proteins, membrane proteins,
transcription factors and enzymes. Binding of aptamer molecules may
influence the function of a target molecule.
[0099] All of the above-mentioned oligonucleotides may vary in
length between as little as 5 or 10, preferably 15 and even more
preferably 18, and as many as 50 or 60, preferably 30 and more
preferably 25, nucleotides per strand. More specifically, the
oligonucleotides may be antisense oligonucleotides of 8 to 50
nucleotides length that catalyze RNAseH mediated degradation of
their target sequence or block translation or re-direct splicing or
act as antagomirs; they may be siRNAs of 15 to 30 basepairs length;
or they may further represent decoy oligonucleotides of 15 to 30
basepairs length. Alternatively, they can be complementary
oligonucleotides influencing the transcription of genomic DNA of 15
to 30 nucleotides length; they might further represent DNAzymes of
25 to 50 nucleotides length or ribozymes of 25 to 50 nucleotides
length or aptamers of 15 to 60 nucleotides length. Such subclasses
of oligonucleotides are often functionally defined and can be
identical or different or share some, but not all, features of
their chemical nature or architecture without substantially
affecting the teachings of this invention. The fit between the
oligonucleotide and the target sequence is preferably perfect with
each base of the oligonucleotide forming a base pair with its
complementary base on the target nucleic acid over a continuous
stretch of the abovementioned number of oligonucleotides. The pair
of sequences may contain one or more mismatches within the said
continuous stretch of base pairs, although this is less preferred.
In general, the type and chemical composition of such nucleic acids
is of little impact for the performance of the inventive liposomes
as vehicles be it in vivo or in vitro, and the skilled artisan may
find other types of oligonucleotides or nucleic acids suitable for
combination with the amphoteric liposomes of the invention.
[0100] In certain aspects and as demonstrated herein, the liposomes
according to the present invention are useful to transfect cells in
vitro, in vivo or ex vivo.
Specific Embodiments
Cholesterol Based Lipids
[0101] To illustrate the teachings of this invention, cationic
derivatives of cholesterol comprising guanido moieties (charged
imino group, CHOL-GUA), imidazol moieties (non-charged imino group,
CHIM) or dimethylamino or trimethyl ammonium moieties (non-imino,
but charged groups, DC-CHOL or TC-CHOL) were systematically
combined with different anionic lipids.
##STR00016##
[0102] The anionic lipids used were CHEMS (cholesterol as
hydrophobic portion, carboxylic acid charge group), DMGS or DOGS
(diacylglycerols hydrophobic portion, carboxylic acid charge group)
or DOPA (diacyl glycerol as hydrophobic portion, phosphate ester
charge group). For most of the cation/anion combinations, a series
of 8 binary mixtures having C/A ratios between 0.33 and 2 was
prepared, combinations of the cationic lipids with DOPA were tested
at CIA 0.75 and 1. Cholesterol was added to all lipid mixtures to
constitute between 20 and 40 mol %, as indicated.
[0103] All liposomes were loaded with PLK-1 siRNA, an
oligonucleotide capable of inhibiting the production of the cell
cycle kinase PLK-1 and successful transfection was measured by
inhibition of cell viability of the test cells (see also Haupenthal
et al., Int. J. Cancer (2007), 121:206-210. Unspecific inhibition
of the cell viability, that is, cytotoxic effects, were monitored
by control preparations comprising a non-targeting siRNA of the
same general composition and in the same amounts.
[0104] The transfection of cells was followed in regular cell
culture medium or with the additional presence of 10% mouse serum,
a potent inhibitor of cellular uptake for many amphoteric
liposomes. The efficacy of transfection is expressed as IC50, the
concentration needed to achieve a 50% inhibition of the cell
viability.
[0105] The ratio between the IC50 in regular medium and the IC50
upon addition of mouse serum is used as a metric for the inhibition
of the cellular uptake by mouse serum. This ratio is 5 or higher
for liposomes without specific targeting properties. It is 5 or
lower for the liposomes of this invention; that is liposomes
comprising charged imino groups in combination with negatively
charged lipids.
[0106] As further demonstrated in examples 14, the best
serum-resistant transfection of HeLa cells can be achieved by
combinations of CHOLGUA with the carboxyl lipid DOGS. Particular
good results were obtained in the presence of less than 40%
cholesterol and for mixtures having a C/A of between 0.5 and 1.5.
If all other components such as DOGS or cholesterol were kept
constant and the GUA head group was exchanged against a
dimethylamine as in DC-CHOL, the liposomes are still active in the
absence, but no longer in the presence of mouse serum. The same can
be observed for combinations of CHIM and DMGS.
[0107] Combinations of cholesterol-based cationic lipids with the
phosphate lipid DOPA resemble the findings in that the best
activities was observed for the imino lipid CHOLGUA. Also,
serum-resistant transfection of CHOLGUA:DOPA liposomes could be
observed, although with substantial inhibition compared to the
absence of serum. Combinations for DOPA with CHIM or DC-CHOL did
not result in any transfection in the presence of serum.
DACA-Based Lipids
[0108] To further investigate the dependence of the serum resistant
transfection from head group chemistry, the following lipids were
synthesized using a common dialkyl-carboxylic acid (DACA) anchor as
their hydrophobic domain:
##STR00017##
[0109] Wherein the DACA moiety was obtained by addition of
oleyliodide to oleic acid as described in the example 10 and the
resulting compound is:
##STR00018##
[0110] Out of the cationic lipids, GUADACA, MPDACA or BADACA have a
charged imino moiety in their polar head groups. The head group of
PDACA is essentially uncharged due to the low pK of the pyridine
moiety (calculated pK is 5.9) while the methylated variant results
in the formation of the constantly charged pyridinium compound
MPDACA. ADACA has a high enough pK of about 9, but lacks the imino
component. However, small amounts of the respective enamine may
form from that component as the amino group is situated in
.quadrature.-position from the amide, allowing mesomeric
stabilization of the imine form.
[0111] Combinations with the anionic lipids CHEMS, DMGS, DOGS and
DOPA were prepared as described above for the cholesterol based
lipids and similar series of different liposomes having various C/A
ratios of between 0.33 and 2 (or 0.75 and 1 for the phosphate
lipid) were produced.
[0112] Also, the liposomes were loaded with siRNA targeting PLK-1
or an unrelated sequence and the transfection properties were
tested on HeLa cells in the presence or absence of mouse serum.
[0113] As further demonstrated in examples 14 and 15,
serum-resistant transfection of HeLa cells can be achieved by
combinations of GUADACA or MPDACA with carboxyl lipids or phosphate
lipids. In addition, these lipids yield very efficient transfection
of PLK-1 siRNA also in the absence of serum. This implies that
there is no activation of the liposomes with serum components as
recently described for liposomes having a dimethylamino head group
(Akinc et al., Mol. Ther. (2010) electronic publication on May
11th, ahead of print. DOI: 10.1038/mt.2010.85). Very high levels of
carrier activity are also observed for C/A ratios between 0.5 and
1.5 for the combinations with the carboxylic lipids and for C/A
0.75 or 1 for the phosphate lipids. In many of these cases,
formulations have amphoteric charge properties.
[0114] A lack of methylation of the pyridinium compound MPDACA
gives the related PDACA. While still bearing an imine function,
this function is no longer charged as in MPDACA; PDACA is also not
active as a cationic lipid for transfection purposes. In yet
another variant the aromatic ring of the head group was kept, but
the charged imine was then presented as part of an extra-annular
aminide group. This compound was found active as a lipid for
transfection, e.g. in combinations with CHEMS or DMGS where it also
resulted in serum-resistant transfection.
Additional Lipids Based on Dialkylcarboxylic Acids.
[0115] Similar findings have been made using the pyridinium lipid
SAINT-18 as described in U.S. Pat. No. 6,726,894 (structure
31).
##STR00019##
[0116] SAINT-18 was combined with various lipid anions, such as
CHEMS, DMGS or DOGS. The ratios of the cationic and anionic lipids
were varied in a systematic way and the resulting binary mixtures
optionally were further supplied with 20 or 40 mol % cholesterol.
The individual lipid mixtures were transformed into liposomes and
used for the encapsulation of an active and control siRNA. When
tested on HeLa cells in the presence of normal cell culture medium,
efficient and specific inhibition of the cell viability was
observed for numerous of the tested formulations, as demonstrated
in Example 8. However, none of the liposomes having a C/A>=1
yielded transfection of cells in the presence of mouse serum. In
stark contrast, a great many of the amphoteric formulations
resisted the serum challenge and did transfect the cells
effectively. Furthermore, the effect was specific to the PLK-1
siRNA and much higher concentrations of liposomes loaded with an
unrelated siRNA (SCR) were needed to unspecifically inhibit cell
proliferation. The best results were obtained by using SAINT18 in
combination with DMGS. Liposomes comprising SAINT-18 and DMGS,
further characterized by C/A<1 are therefore within the purview
of this invention.
Amino Acid Based Lipids
[0117] To further illustrate the teachings of this invention, the
cationic guanido lipid PONA (palmitoyl-oleoyl-nor-arginine,
structure 21) was combined with various lipid anions such as CHEMS
or DMGS. The ratios of the cationic and anionic lipids were varied
in a systematic way and the resulting binary mixtures optionally
were further supplied with 20 mol % cholesterol. The individual
lipid mixtures were transformed into liposomes and used for the
encapsulation of an active and control siRNA. When tested on HeLa
cells, efficient and specific inhibition of the cell viability was
observed for most of the tested formulations, as demonstrated in
Example 5. The activity was not or only marginally affected by the
presence of human or mouse serum.
[0118] In Example 6, the anionic lipid CHEMS was combined with
derivatives of PONA, wherein the guanido moiety was substituted by
an amino group (PONamine) or an quarternized ammonium group
(PONammonium) as shown in the structures (21) and (23).
##STR00020##
[0119] Again, the ratios between the anionic and cationic lipid
components were systematically varied and 20% cholesterol was
present in all lipid mixtures. The material was formulated into
liposomes and used for the encapsulation of active and control
siRNA. When tested on HeLa cells, efficient and specific inhibition
of the cell viability was observed for all formulations comprising
a molar excess of the cationic lipids. For mixtures comprising
higher molar amounts of the anionic lipid CHEMS, the best activity
was observed in combinations with PONA, while PONamine: CHEMS
combinations were only effective in some cases. The
PONammonium:CHEMS combinations were not effective when an excess of
the anionic lipid was used.
[0120] Moreover, out of the mixtures comprising an excess of the
anionic lipid CHEMS, the transfection activity of the PONA:CHEMS
combinations was only marginally affected by the presence of human
or mouse serum, while the activity of PONamine:CHEMS combinations
was completed suppressed in the presence of mouse serum. The
PONammonium formulations remained inactive in the presence of
sera.
[0121] Combinations of PONA, PONamine or PONammonium with the
phosphate lipids DOPA were also tested as further described in
example 15. Both PONA and PONamine, but not PONammonium resulted in
serum-resistant transfection of HeLa cells.
[0122] The combined data support a preferred uptake of lipid
combinations comprising guanido lipids in combination with
negatively charged, e.g. carboxyl or phosphate lipids. This may
relate to the mechanistic considerations made further below. The
constant and high activity of the formulations having an excess of
the cationic lipid component may be due to electrostatic
interaction between these particles and the cell surface, which
however is unspecific. In line with this view is the fact that the
activity of the cationic formulations did not depend on either the
nature of the anionic or the cationic lipid.
[0123] In further experiments, the guanido lipid PONA was combined
with CHEMS, DMGS or DOGS. Again, a systematic variation of the
ratios of both the anionic and cationic lipid compound in the
respective binary mixtures was performed and the formulations were
further supplied with 0, 20 or 40 mol % of cholesterol. When tested
as above, the great majority of the formulations were active in
inhibiting the cell proliferation of HeLa cells with an IC.sub.50
being lower than 6 nM (see Example 7). A comparison between the
concentrations needed for the efficacy of the active and inactive
siRNA, however, revealed substantial differences between the
formulations. A measure for such comparison is the ratio between
the IC.sub.50 values for both siRNA's, here expressed as SCR/PLK
ratio. Only selected formulations reach values significantly higher
than 5. Even more preferred formulations have SCR/PLK>=10. All
of these preferred formulations can be characterized by their ratio
between the cationic and anionic lipid component, which is lower
than 1.
[0124] This invention identifies specific lipid head group
chemistry as critical for the uptake into certain cells in the
presence of otherwise inhibitory sera. With preference, amphoteric
combinations of anionic lipids comprising carboxyl groups and
cationic lipids comprising charged imino moieties result in the
desired properties. In contrast, cationic formulations comprising
the same lipids do not depend on a specific head group chemistry
and are less tolerated by cells.
Lipoprotein Binding
[0125] The lipoproteins competing with the transfection of
liposomes comprise a variety of structures, according to their
density. These are known as chylomicrons, VLDL, LDL, IDL or HDL
particles. In the endogenous pathway, chylomicrons are synthesized
in the epithelial lining of the small intestine and are assembled
using ApoB-48, a shorter variant of the ApoB gene product. Further
exchange of lipoproteins with HDL particles leads to transfer of
ApoC-II and ApoE to the chylomicron particle, the first mediating
the activation of lipoprotein lipase, an enzyme needed for the
release of lipids from the particle. The hydrolyzed chylomicrons
form so called remnants which are taken up mainly in the liver via
recognition of their ApoE portion. The synthesis, maturation, use
and recycling of VLDL particles follows the very same pathway, but
starts in the liver and is using the ApoB-100 protein as its
structure forming unit. Again, ApoE mediates the eventual uptake
and recycling of the VLDL-remnants, the so-called IDL particles.
(see also http://en.wikipedia.org/wiki/Lipoprotein)
[0126] ApoE shares structural homology to the apolipoproteins A and
C in that they all comprise amphipathic tandem repeats of 11 amino
acids. Crystallographic data confirm the existence of extended
amphipathic helical structures for ApoA-I and ApoE fragment and
also reveal a mixed charge organization on the polar face of these
helices. These data are publicly available from the RCSB Protein
Data Bank (available at wwwscsb.org/pdb/home/home.do) and entry
1AV1.pdb gives the protein structure of ApoA-I. The amino acids 129
to 166 of 1lpe.pdb represent the LDL-receptor binding fragment of
ApoE. In contrast to their overall similarity, the three
apolipoproteins display specific deviations when their amino acid
composition is analyzed. In ApoE, arginine is the prevailing
cationic amino acid in the tandem repeats. In contrast, ApoA has
equal amounts of lysine and arginine, while ApoC has an excess of
lysine residues.
TABLE-US-00003 TABLE 3 Analysis of the amino acid composition in
tandem repeats of related apolipoproteins. Sequence data were
obtained from Swiss-Protavailable at
www.expasy.ch/sprot/sprot-top.html). Sequence ApoAI ApoE ApoC-II
SwissProt Entry P02647 P02649 P02655 Endpoints 68-267 80-255 23-101
lenght 199 175 78 IP 5.55 9.16 4.66 # of lysine 18 8 6 # of
arginine 14 25 1 # of histidine 5 1 0 # of aspartic acid 10 8 4 #
of glutamic acid 28 22 7 Lysine (%) 9% 5% 8% Arginine (%) 7% 14% 1%
Histidine (%) 3% 1% 0% Aspartic acid (%) 5% 5% 5% Glutamic acid (%)
14% 13% 9%
[0127] In summary, the polar surface of natural lipoproteins is
covered with apolipoproteins, of which ApoE is a common binding
motif for the cellular uptake of these particles. The water-exposed
portions of ApoE represent a mosaic of anionic and cationic
charges, wherein the anionic charges are created from the free
carboxyl termini of aspartic and glutamic acid residues. The
cationic charges comprise a mixture of amino and guanido groups
with a very few imidazols being present.
[0128] In order to emulate the recognition pattern of the ApoE
binding cassette on the surface of liposomes, different
alternatives can be followed. It is possible to synthesize ApoE
peptide fragment and graft such peptides on the surface of
liposomes. This has been demonstrated by Mims et al., J. Biol.
Chem. 269, 20539 (1994); Rensen at al., Mol. Pharmacol. 52, 445
(1997); Rensen et al., J. Lipid Res. 38, 1070 (1997); Sauer et al.,
Biochemistry 44, 2021 (2005) or Versluis et al., J. Pharmacol. Exp.
Ther 289, 1 (1999). However, the high cost associated with peptide
synthesis and derivatization call for alternative approaches.
[0129] A direct presentation of the required charged moieties using
mixtures of different charged lipids, potentially further
comprising neutral lipids would yield a much simpler structure and
eliminate the needs for costly peptide production and
derivatization. A considerable challenge of such an approach is the
planar diffusion of the charged groups within the lipid bilayer; it
was heretofore unclear whether the affinity of such a less
organized assembly would effectively compete with the affinities
provided by the authentic lipoproteins. Moreover, the oppositely
charged lipid headgroups may form salt bridges with each other,
while only few hydrogen bonds between functional groups are
detected in the binding cassette of lipoproteins, e.g. ApoE. This
may explain the activity of imino:phosphate lipid combinations such
as GUADACA:DOPA or MPDACA:DOPA. While DOPA provides two negative
charges under physiological conditions, steric hindrance disables
the formation of a salt from one DOPA and two GUADACA lipids. As
such, in these membranes the negatively charged salt between DOPA
and GUADACA must co-exist with free GUADACA molecules, thereby
facilitating the simultaneous presence of separated anionic and
cationic elements in a common lipid assembly.
[0130] The theory above is mentioned without limiting the findings
of this invention. Without wishing to be bound to this particular
theory, one can assume that the combinations of charged imino
lipids with negatively charged carboxyl or phosphate lipids emulate
the surface properties of lipoproteins covered with ApoE. The
particles can of course be used, developed and optimized without
such knowledge. The theoretical background may however be helpful
to understand guiding principles or applicability of the vectors
described in the various embodiments of this invention.
[0131] It is for example known, that lipoprotein receptors have
different expression profiles in various cell types and such
knowledge can be used to assess target cell populations for the
liposomes of this invention.
[0132] The LDL-receptor is highly expressed on tumors and on the
bronchoepithelial cells of the lung (see Su Al, Wiltshire T,
Batalov S, et al (2004). Proc. Natl. Acad. Sci. U.S.A. 101 (16):
6062-7, also published at
http://en.wikipedia.org/wiki/File:PBB_GE_LDLR.sub.--202068_s_at_tn.png)
[0133] The liposomes of this invention are thus specifically suited
for applications in the field of oncology, but also for
transfection of specific lung cells. While tumors are accessible
from systemic circulation through the EPR-effect (enhanced
permeability and retention), that is via leaky tumor vasculature,
the bronchoepithelial cells can be targeted also from the
airways.
[0134] In a specific embodiment of this invention, aerosols from
liposomes comprising charged imino and carboxyl or phosphate lipids
are thus used for inhaled dosage forms for the targeting of lung
cells, in particular bronchoepithelial cells.
FIGURE LEGENDS
[0135] FIGS. 1-6 display the results of the screening experiment
described in example 14. The nature of the cationic lipids is
indicated in the smaller figures and other legends and axis are
similar for all display items and are given in the separate smaller
figure below. The double bars denote liposomes with 20% cholesterol
(left bar) and 40% cholesterol (right bar), respectively.
[0136] Bars represent the IC50 values for the respective
liposome/siRNA combinations under the experimental conditions for
each figure, that is, either in the presence of absence of mouse
serum. These IC50 values denote the concentrations needed for a
half-maximal inhibition of the cell growth and are given in nM. The
maximum concentrations of the test items were 40 and 36 nM for the
absence or presence of mouse serum, respectively.
[0137] The order of the test items is as follows:
[0138] FIG. 1 the anionic lipid is CHEMS-no addition of mouse
serum
[0139] FIG. 2 the anionic lipid is CHEMS+addition of mouse
serum
[0140] FIG. 3 the anionic lipid is DMGS-no addition of mouse
serum
[0141] FIG. 4 the anionic lipid is DMGS+addition of mouse serum
[0142] FIG. 5 the anionic lipid is DOGS-no addition of mouse
serum
[0143] FIG. 6 the anionic lipid is DOGS+addition of mouse serum
EXAMPLES
[0144] The teachings of this invention may be better understood
with the consideration of the following examples. However, these
examples should by no means limit the teachings of this
invention.
Example 1
Liposome Production, Characterization and Encapsulation of
siRNA
[0145] Liposomes were prepared using methods as disclosed in
WO07/107,304. More specifically, lipids were dissolved in
isopropanol and liposomes were produced by adding siRNA solution in
NaAc 20 mM, Sucrose 300 mM, pH 4.0 (pH adjusted with HAc) to the
alcoholic lipid mix, resulting in a final alcohol concentration of
30%. The formed liposomal suspensions were shifted to pH 7.5 with
twice the volume of Na.sub.2HPO.sub.4 136 mM, NaCl 100 mM (pH 9),
resulting in a final lipid concentration of 3 mM and a final
isopropanol concentration of 10%.
[0146] Liposomes were characterized with respect to their particle
size using dynamic light scattering (MALVERN 3000HSA).
[0147] Active siRNA: 21mer blunt ended targeting mouse and human
PLK-1 mRNA as in Haupenthal et al., Int. J. Cancer (2007),
121:206-210.
[0148] Control siRNA (SCR): 21 mer from the same source.
Example 2
General Cell Culture and Proliferation Assay
[0149] HeLa cells were obtained from DSMZ (German Collection of
Micro Organism and Cell Cultures) and maintained in DMEM
(Gibco-Invitrogen) and supplemented with 10% FCS. The cells were
plated at a density of 2.5.times.10.sup.4 cells/ml and cultivated
in 100 .mu.l medium at 37.degree. C. under 5% CO.sub.2. After 16 h,
the liposomes containing siRNA were diluted and 10 .mu.l were added
to the cells to yield final concentrations between 0.4 to 100 nM
Plk1 or scrambled siRNA; 10 .mu.l dilution buffer were also added
to untreated cells and into wells without cells. Cell culture
dishes were incubated for 72 h at 37.degree. C. under 5%
CO.sub.2.
[0150] Cell proliferation/viability was determined by using the
CellTiter-Blue Cell Viability assay (Promega, US) according to the
instructions of the supplier.
Example 3
Inhibition of Transfection by Sera
[0151] Liposomes from DODAP:DMGS:Cholesterol (24:36:40 mol %) were
loaded with active and control siRNA as above and 25 .mu.l of the
liposomes were incubated with 75 .mu.l sera from different species
(SIGMA-Aldrich) for 30 min. Following that, liposomes were added to
the cells, incubation was continued for 72 h and cell viability was
determined as above.
[0152] When incubated without serum, administration of the active
siRNA results in a strong inhibition of cell proliferation. As
demonstrated in the Table 7 below, this process is inhibited by the
addition of sera.
TABLE-US-00004 TABLE 7 Inhibition of cellular transfection by sera
of different origin. siRNA type siRNA concentration Serum Cell
viability (%) PLK1 50 nM no 7 PLK1 50 nM Human 98 PLK1 50 nM
Hamster 80 PLK1 50 nM Rat 108 PLK1 50 nM Mouse 102 No No No 100
Example 4
Inhibition is Lipoprotein Dependent
[0153] Liposomes as in Example 3 were incubated with human serum
devoid of certain complement factors or lipoproteins
(SIGMA-Aldrich) as above and analyzed for their ability to mediate
the RNAi effect on HeLa cells.
[0154] As demonstrated in Table 8, the efficacy of transfection can
be restored by a depletion of lipoproteins. Removal of complement
factors was ineffective.
TABLE-US-00005 TABLE 8 Restoration of cellular transfection in sera
being deficient of various factors. Cell viability siRNA type siRNA
concentration Serum (%) PLK1 50 nM no 7 PLK1 50 nM Human, complete
98 PLK1 50 nM Human, no C3 91 complement factor PLK1 50 nM Human,
no C9 98 complement factor PLK1 50 nM Human, lipoprotein 18
deficient No No No 100
Example 5
Serum Resistant Transfection Using a Guanido Lipid
[0155] A series of liposomes was constructed from PONA:Anionic
Lipid:Cholesterol (x:y:20 mol %) and loaded with active and control
siRNA as in Example 1. Within that series, the ratio between the
cationic component PONA and the anionic lipids CHEMS or DMGS was
systematically varied between 0.33 and 2 as indicated in the table.
Liposomes having a ratio of the cationic:anionic lipid of 1 or
greater were further supplied with 2 mol % DMPE-PEG2000 (Nippon
Oils and Fats) to avoid aggregation of the particles. This
modification is indicated by a "+" in the table. Control reactions
with particles having C/A<1 did not reveal a change of
transfection properties in the presence or absence of PEG
lipids.
[0156] HeLa cells were grown and maintained as in Example 2 and
sera of human or mice origin (SIGMA-Aldrich) was added directly to
the cells for 120 min. Following that, the liposomes were added to
the cells in concentrations between 50 pM and 50 nM, incubation was
continued for 72 h and cell viability was determined as above. The
efficacy of transfection is expressed here as IC.sub.50, the
concentration needed to inhibited cell proliferation by 50%. Low
IC50 values therefore represent highly effective transfection.
[0157] It becomes apparent from the results in the Table 9, that
the addition of sera only marginally affects the transfection of
siRNA mediated by the liposomes of the example. Some inhibition is
still observed for liposomes from PONA:CHEMS comprising low amounts
of the anionic lipid (ratios 0.33 and 0.5, particular strong
inhibition with mouse serum).
TABLE-US-00006 TABLE 9 Efficacy of transfection of liposomes
comprising guanido moieties in the presence of sera. Ratio
cationic/anionic lipid 0.33 0.50 0.67 0.82 1+ 1.22+ 1.5+ 2+ CHEMS
No Serum 38.54 1.21 0.40 0.56 1.83 1.61 0.70 1.42 Human 199.00 2.10
0.62 1.13 2.16 1.92 1.70 1.83 Serum Mouse 199.00 50.00 1.56 1.94
2.47 1.90 0.76 1.44 Serum DMGS No Serum 0.23 0.54 0.01 0.01 Human
1.50 2.39 2.88 2.21 Serum Mouse 0.67 0.69 1.41 1.81 Serum
Example 6
Criticality of the Guanido Head Group
[0158] Series of liposomes having systematically varied ratios
between the cationic and anionic lipid components were produced and
loaded with siRNA as in Example 5. The cationic lipid components
were PONA, PONamine and PONammonium, the anionic lipid was CHEMS
and the cholesterol content was fixed to 20 mol %. Liposomes having
a ratio of the cationic anionic lipid of 1 or greater were further
supplied with 2 mol % DMPE-PEG2000 (Nippon Oils and Fats) to avoid
aggregation of the particles. This modification is indicated by a
"+" in the table.
[0159] HeLa cells were grown and maintained as in Example 2 and
sera of human or mice origin (SIGMA-Aldrich) was added directly to
the cells for 120 min. Following that, the liposomes were added to
the cells in concentrations between 50 pM and 50 nM, incubation was
continued for 72 h and cell viability was determined as above. The
efficacy of transfection is expressed here as IC.sub.50 as in
Example 5.
[0160] It becomes apparent from the data in Table 10, that only
PONA, but neither PONamine and even less so PONammonium mediates
the transfection of HeLa cells in the presence of serum. This is
most striking in the case of mouse serum, which inhibits the
transfection more aggressively. An excess of the cationic lipid
components to some extent compensate the serum mediated loss of
activity, but may be due to unspecific electrostatic adsorption of
these liposomes to the cells.
TABLE-US-00007 TABLE 10 Criticality of the guanido head group for
the serum resistant transfection of cells. C/A ratio 0.33 0.5 0.67
0.82 1+ 1.22+ 1.5+ 2+ PONA no serum 42.9 1.8 0.6 1.0 4.1 5.4 2.4
6.8 human serum 80.0 2.5 2.2 2.0 1.8 2.8 6.2 5.2 mouse serum 80.0
31.1 55.0 5.7 2.1 5.3 8.1 7.5 PONamine no serum 3.1 65.0 7.5 100.0
3.0 5.2 3.0 2.5 human serum 100.0 55.0 11.9 100.0 2.2 2.8 6.1 5.1
mouse serum 70.0 100.0 100.0 100.0 75.0 70.0 39.3 8.7 PONammonium
no serum 80.0 100.0 90.0 90.0 65.0 9.5 9.5 5.2 human serum 95.0
90.0 90.0 80.0 90.0 11.8 12.4 15.7 mouse serum 85.0 100.0 100.0
100.0 100.0 90.0 75.0 55.0
Example 7
Optimization of the Liposome Composition
[0161] Series of liposomes having systematically varied ratios
between the cationic and anionic lipid components were produced and
loaded with siRNA as in Example 5. The cationic lipid component was
PONA, the anionic lipids were CHEMS, DMGS or DOGS and the
cholesterol content was varied between 0 and 40 mol %. Liposomes
having a ratio of the cationic:anionic lipid of 1 or greater but
also some of the other liposomes were further supplied with 2 mol %
DMPE-PEG2000 (Nippon Oils and Fats) to avoid aggregation of the
particles. This modification is indicated by a "+" in the
table.
[0162] HeLa cells were grown and maintained as in Example 2 and
liposomes were added to the cells in concentrations between 6 nM
and 200 nM, incubation was continued for 72 h and cell viability
was determined as above. The efficacy of transfection is expressed
here as IC.sub.50 as in the examples above. In addition, the
IC.sub.50 was determined for the liposomes carrying the inactive
siRNA (SCR) and the ratio between IC.sub.50 (SCR) and IC.sub.50
(PLK1) was determined. A high value for this parameter indicates a
very specific inhibition of the cellular viability by the PLK1
siRNA, low unspecific effects contributed by the carrier and low
levels of cytotoxicity in general.
TABLE-US-00008 TABLE 11 Optimization results for CHEMS. Lowest and
highest detectable IC.sub.50 values are 6 and 200 nM, respectively.
C/A 0.33 0.33+ 0.5 0.5+ 0.67 0.67+ 0.82 0.82+ 1+ 1.22+ 1.5+ 2+ PLK
0% Chol 44 77 6 6 6 6 6 6 6 6 6 6 20% Chol 54 79 6 6 6 6 6 6 6 6 6
6 40% Chol 67 94 6 6 6 6 6 6 6 6 6 6 SCR 0% Chol 90 86 113 152 23
200 16 21 15 16 14 11 20% Chol 73 90 109 128 200 200 26 23 21 11 16
10 40% Chol 94 117 198 200 200 200 6 6 30 14 27 12 SCR/ 0% Chol
2.05 1.12 18.86 25.33 3.81 83.33 2.60 3.52 2.50 2.68 2.30 1.84 PLK
20% Chol 1.37 1.14 18.10 21.39 83.33 83.33 4.26 3.77 3.45 1.84 2.65
1.69 40% Chol 1.40 1.24 32.96 83.33 83.33 83.33 1.00 1.00 5.00 2.39
4.48 1.97
TABLE-US-00009 TABLE 12 Optimization results for DMGS. Lowest and
highest detectable IC.sub.50 values are 6 and 200 nM, respectively.
C/A 0.33 0.5 0.67 0.82 1+ 1.22+ 1.5+ 2+ PLK 0% Chol 98 200 200 188
6 6 6 6 20% Chol 6 6 6 6 6 6 6 6 40% Chol 6 6 6 6 6 6 6 6 SCR 0%
Chol 200 200 200 158 14 6 10 14 20% Chol 200 54 8 8 13 9 9 10 40%
Chol 155 23 11 6 6 14 9 12 SCR/ 0% Chol 5.11 no effect no effect
0.84 2.26 1.00 1.66 2.36 PLK 20% Chol 83.33 9.01 1.27 1.26 2.20
1.55 1.45 1.69 40% Chol 25.85 3.90 1.83 1.00 1.00 2.27 1.54
1.97
TABLE-US-00010 TABLE 13 Optimization results for DOGS. Lowest and
highest detectable IC.sub.50 values are 6 and 200 nM, respectively.
C/A 0.33 0.5 0.67 0.82 1+ 1.22+ 1.5+ 2+ PLK 0% Chol 200 200 200 200
6 6 6 6 20% Chol 22 200 200 200 6 6 6 6 40% Chol 6 170 200 200 6 6
6 6 SCR 0% Chol 200 200 200 200 14 10 16 10 20% Chol 200 200 200
200 21 10 12 8 40% Chol 15 197 200 200 12 7 9 9 SCR/ 0% Chol no
effect no effect no effect no effect 2.40 1.59 2.65 1.63 PLK 20%
Chol 22.42 no effect no effect no effect 3.45 1.65 2.07 1.29 40%
Chol 2.48 1.16 no effect no effect 1.93 1.09 1.48 1.55
Example 8
Liposomes Comprising a Pyridinium Lipid
[0163] SAINT-18 was used as the cationic lipid, its methylated
pyridinium structure provides a charged imino moiety. CHEM, DMGS
and DOGS were individually used as anionic lipids providing the
carboxyl functional group. Series of liposomes having
systematically varied ratios between the cationic and anionic lipid
components were produced and loaded with siRNA as in Example 5. The
lipid mixture was further supplied with 20 or 40 mol % cholesterol.
Liposomes having a ratio of the cationic:anionic lipid of 1 or
greater were further supplied with 2 mol % DMPE-PEG2000 (Nippon
Oils and Fats) to avoid aggregation of the particles. This
modification is indicated by a "+" in the table.
[0164] HeLa cells were grown and maintained as in Example 2 and
liposomes were added to the cells in concentrations between 50 pM
and 50 nM, incubation was continued for 72 h and cell viability was
determined as above. The efficacy of transfection is expressed here
as IC.sub.50 as in the examples above. In addition, the IC.sub.50
was determined for the liposomes carrying the inactive siRNA (SCR)
and the ratio between IC.sub.50 (SCR) and IC.sub.50 (PLK1) was
determined. A high value for this parameter indicates a very
specific inhibition of the cellular viability by the PLK1 siRNA,
low unspecific effects contributed by the carrier and low levels of
cytotoxicity in general.
TABLE-US-00011 TABLE 14 transfection results for liposomes from
SAINT-18, CHEMS and cholesterol C/A ratio 0.33 0.5 0.67 0.82 1+
1.22+ 1.5+ 2+ lipid anion CHEMS, no serum PLK1 20% Chol 2.2 no eff.
1.7 33.9 17.8 7.2 4.1 2.7 40% Chol 7.8 no eff. 1.5 32.0 7.2 4.4 2.1
6.4 SCR 20% Chol no eff. no eff. 11.9 no eff. 37.4 19.6 20.7 29.0
40% Chol no eff. no eff. no eff. no eff. no eff. 16.9 18.6 28.0
SCR/PLK-1 20% Chol >22.7 7.2 >1.5 2.1 2.7 5.0 10.6 40% Chol
>6.4 >32.5 >1.6 >7.0 3.9 8.7 4.4 lipid anion CHEMS,
plus mouse serum PLK1 20% Chol no eff. no eff. 14.4 no eff. no eff.
no eff. no eff. 31.7 40% Chol no eff. no eff. no eff. no eff. no
eff. no eff. 35.5 23.1 SCR 20% Chol no eff. no eff. no eff. no eff.
no eff. no eff. no eff. no eff. 40% Chol no eff. no eff. no eff. no
eff. no eff. no eff. 41.5 38.1 SCR/PLK-1 20% Chol >3.5 >1.6
40% Chol 1.2 1.6
TABLE-US-00012 TABLE 15 transfection results for liposomes from
SAINT-18, DMGS and cholesterol C/A ratio 0.33 0.5 0.67 0.82 1+
1.22+ 1.5+ 2+ lipid anion DMGS, no serum PLK1 20% Chol 0.8 2.3 1.7
43.6 24.3 7.5 5.2 3.8 40% Chol 1.6 2.3 1.8 2.2 11.4 8.9 3.8 5.8 SCR
20% Chol 7.7 8.2 5.3 36.0 28.1 27.6 10.5 10.3 40% Chol 4.7 no eff.
22.6 5.7 27.7 28.5 8.1 8.2 SCR/PLK-1 20% Chol 9.2 3.6 3.1 0.8 1.2
3.7 2.0 2.7 40% Chol 2.9 >22.1 12.6 2.5 2.4 3.2 2.1 1.4 lipid
anion DMGS; plus mouse serum PLK1 20% Chol 4.0 8.0 2.7 no eff. 26.5
28.8 no eff. no eff. 40% Chol 2.0 2.2 1.6 1.6 no eff. 21.0 no eff.
no eff. SCR 20% Chol 10.1 no eff. 23.4 no eff. 29.1 31.2 25.7 28.4
40% Chol 7.7 18.0 25.8 6.3 28.0 37.4 31.7 25.7 SCR/PLK-1 20% Chol
2.5 >6.2 8.6 1.1 1.1 40% Chol 3.9 8.0 16.5 3.9 1.8
TABLE-US-00013 TABLE 16 transfection results for liposomes from
SAINT-18, DOGS and cholesterol C/A ratio 0.33 0.5 0.67 0.82 1+
1.22+ 1.5+ 2+ lipid anion DOGS, no serum PLK1 20% Chol 36.9 38.0 no
eff. no eff. 9.2 8.1 7.0 6.1 40% Chol 6.9 19.4 no eff. no eff. 22.7
8.7 6.6 8.5 SCR 20% Chol no eff. no eff. no eff. no eff. 27.5 20.5
10.2 25.9 40% Chol no eff. no eff. no eff. no eff. no eff. no eff.
no eff. no eff. SCR/PLK-1 20% Chol >1.4 >1.3 3.0 2.5 1.5 4.3
40% Chol >7.3 >2.6 >2.2 >5.7 >7.6 >5.9 lipid
anion DOGS, plus serum PLK-1 20% Chol 2.2 18.4 no eff. no eff. 27.5
30.5 26.3 28.1 40% Chol 2.7 7.7 no eff. no eff. 27.4 29.2 30.4 30.8
SCR 20% Chol 2.8 no eff. no eff. no eff. 32.6 34.4 30.9 33.2 40%
Chol no eff. 8.2 no eff. no eff. 30.6 no eff. no eff. 42.8
SCR/PLK-1 20% Chol 1.3 >2.7 1.2 1.1 1.2 1.2 40% Chol >18.6
1.1 1.1 >1.7 >1.6 1.4
[0165] As it becomes clear from the data in tables 14 to 16, a
large number of amphoteric liposomes facilitate the transfection of
cells even in the presence of mouse serum. Particularly useful are
liposomes comprising SAINT-18 in combination with the
diacylglycerols DMGS and DOGS, while the combination with CHEMS was
only effective at C/A=0.67. As with the PONA combinations, the
amphoteric constructs transfect the cells with high specificity,
while the compositions having C/A>1 do not provide a highly
specific transfection as indicated by SCR/PLK1 being below 2.
[0166] Other embodiments and uses of the invention will be apparent
to those skilled in the art from consideration of the specification
and practice of the invention disclosed herein. The specification
and examples should be considered exemplary only with the true
scope and spirit of the invention indicated by the following
claims.
Example 9
Zeta Potential Measurements
[0167] 9.1 Analysis of the Zeta Potential for Liposomes Formed from
PONA:CHEMS:CHOL
[0168] 100 .mu.l of a lipidmix comprising x Mol % PONA, y mol %
CHEMS and 20 Mol % cholesterol (20 mM total lipid concentration,
solvent:isopropanol) was injected in 900 .mu.l of a buffer
comprising 10 mM acetic acid and 10 mM phosphoric acid pH4. X and
Y, the molar percentages for PONA and CHEMS were adjusted to yield
the C/A ratios in table 17.
[0169] The suspension was immediately vortexed and 3 mL of a pH
adjusting buffer was added. Buffers were selected from the group
of: 50 mM acetic acid and 50 mM phosphoric acid, adjusted to pH 4,
5, 6.5 or 7.5 using NaOH or 50 mM Na.sub.2HPO.sub.4/50 mM sodium
acetate pH9.4. The mixing pH is was recorded and is given in the
table 17 below together with the zeta potentials of the resulting
lipid particles that were monitored using a Zetasizer HSA3000.
TABLE-US-00014 TABLE 17 Zeta Potentials for lipid particles from
PONA:CHEMS:CHOL C/A ratio Final pH 0.5 0.67 0.82 1.00 1.22 1.5 2.00
7.56 -54.40 -58.90 -58.20 -61.80 -21.80 22.60 #NV 7.20 -48.47
-46.00 -44.90 -50.00 -21.10 14.97 #NV 6.32 -44.33 -37.07 -31.37
0.64 23.43 9.60 #NV 4.84 19.67 18.00 22.15 31.80 32.77 32.57 28.37
3.93 35.53 41.73 43.75 46.63 46.20 43.40 43.23
[0170] Clearly, the particles display amphoteric character even for
mixtures having a C/A of 1.22, that is, greater than 1. Particles
having a C/A of 0.67, 0.82 or 1 were also produced at pH7.4 and
subsequently exposed to lower pH. There were no apparent changes to
the zeta potentials shown in table 17.
9.2 Zeta Potential Measurements for Combinations Wherein DOPA is
the Anionic Lipid
[0171] Lipid particles were also prepared from binary mixtures of
GUADACA and DOPA, an imino/phosphate combination of lipid head
groups. The particles were prepared in the same fashion as
described in 9.1 and the zeta potentials of table 18 were recorded
for mixtures having different C/A ratios:
TABLE-US-00015 TABLE 18 Zeta Potentials for lipid particles from
GUADACA:DOPA C/A final pH 0.65 0.75 0.98 1.16 1.4 4.5 21 13 38 46
51 5.32 -24 22 20 33 35 6.25 -8 -45 -30 2 24 7.02 -61 -67 -8 -56 -6
7.81 -67 -78 -76 -65 -21
[0172] As with the particles obtained in 9.1, particles with
amphoteric character are also obtained with C/A>1. Still, the
drift in the isoelectric point follows the expectations.
9.3 Zeta Potential Measurements for DOTAP:CHEMS:CHOL
[0173] For comparison, the same measurements were performed with
lipid mixtures wherein PONA was substituted by DOTAP. The results
are shown in table 19. In contrast to PONA:CHEMS, amphoteric
particles from DOTAP:CHEMS are only found at C/A<1.
TABLE-US-00016 TABLE 19 Zeta potential for lipid particles from
DOTAP:CHEMS:CHOL Ratio C/A Final pH 0.67 0.82 1 1.22 7.56 -37.7
-21.63 4.9 13.25 7.20 -50.17 -24.1 #NV 12.55 6.32 #NV #NV 11.43
7.37 4.84 25.6 32.1 20.27 9.3 3.93 52.13 43.93 47.77 12.15
Example 10
Synthesis of CHOLGUA
[0174] 25 g cholesterolchloroformiate and 50 equivalents (eq.)
ethylendiamine were dissolved in dichloromethane and allowed to
react for 6 h at 20.degree. C. The
aminoethylcarbamoyl-cholestererol was isolated using chromatography
and crystallization. Yield was 28.7 g, purity 90%.
[0175] CHOLGUA was synthesized from the
aminoethylcarboamoyl-cholesterol isolated before. 30 g of the
substance were incubated with 1.5 eq. of
1H-pyrazole-1-carboxamidinium hydrochloride and 4 eq.
N,N-diisopropylethylamin in dichloromethane/ethanol for 16 h at
20.degree. C., after which the product was isolated by
chromatography. Purity was 95%, Yield 16.5 g.
Example 11
Synthesis of DACA, PDACA and MPDACA
[0176] 42.4 g of oleyl alcohol, 2.5 eq. of
diisoproylazodicarboxylate, 2.5 eq. triphenylphosphine and 5 eq.
Lil were reacted in tetrahydrofuran (THF) for 24 h at 20.degree. C.
Oleyliodid was isolated by chromatography with a purity of 90%,
yield was 13.4 g.
[0177] In a second step, 10 g oleic acid were mixed with 2.2 eq. of
lithiumdiisopropylamide in THF for 0.5 h at 20.degree. C., after
which 1 eq. oleyliodide was added. The mixture was incubated for 2
h at 20.degree. C. and DACA purified from the reaction mix using
chromatography. Purity was 95%, Yield 14.96 g.
[0178] For the synthesis of PDACA, 2 g of DACA, 1.2 eq. of
4-picolylamine, 1.4 eq. of
O-benzotriazole-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate
and 4 eq. of N-methylmorpholine were mixed in THF for 24 h at
20.degree. C. The reaction mixture was purified including
chromatography. Purity of PDACA was 95%, yield was 1.72 g.
[0179] For the synthesis of MPDACA, 2 g of PDACA was dissolved in
THF together with 2 eq. of dimethylsulphate and the mixture was
incubated for 16 h at 20.degree. C., after which MPDACA was
purified by chromatography. Purity of MPDACA: 95%, Yield: 1.71
g
Example 12
Synthesis of GUADACA
[0180] In a first step, 3.5 g DACA and 1.5 eq. of
1,1'-carbonyldiimidazol were dissolved in dichloromethane and
incubated for 16 h at 20.degree. C., after which 30 eq.
ethylenediamine were added. The reaction mixture was incubated for
4 h at 20.degree. C. after which aminoethyl-DACA was purified
including chromatography. Purity was 90%, Yield 3.2 g.
[0181] GUADACA was synthesized from aminoethyl-DACA and for that,
3.2 g of aminoethyl-DACA, 2.5 eq. 1H-pyrazole-1-carboxamidine
hydrochloride and 12 eq. N,N-diisopropylethylamine were incubated
for 3 h at 20.degree. C., after which GUADACA was isolated. Purity:
95%, Yield: 2.24 g.
Example 13
Synthesis of BADACA
[0182] BADACA was synthesized from DACA according to the following
procedure: 4.15 g DACA, 1.2 eq. p-aminobenzamidine, 1.2 eq.
N,N'-dicyclohexylcarbodiimid and 3 eq. of 4-Dimethylaminopyridine
were mixed in dry dimethylformamide and incubated for 16 h at
70.degree. C. BADACA was isolated from the reaction using
chromatography. Purity: 95%, Yield: 1.62 g
Example 14
Serum Resistant Transfection of DACA or Cholesterol Based Cationic
Lipids in Combination with Carboxyl Lipids
[0183] Series of liposomes having systematically varied ratios
between the cationic and anionic lipid components were produced and
loaded with siRNA as in Example 5. The cationic lipid components
were CHOLGUA, CHIM, DC-CHOL, TC-CHOL, GUADACA, MPDACA, BADACA and
PDACA. The anionic lipids were CHEMS, DMGS or DOGS and the
cholesterol content was either 20 or 40 mol %, all lipid mixtures
are identified in the data tables. Liposomes having a ratio of the
cationic:anionic lipid of 1 or greater (C/A>=1) were further
supplied with 1.5 mol % DMPE-PEG2000 (Nippon Oils and Fats).
[0184] HeLa cells were grown and maintained as in Example 2 and
mouse serum (SIGMA-Aldrich) was added directly to the cells for 120
min. Following that, the liposomes were added to the cells,
incubation was continued for 72 h and cell viability was determined
as above. The highest concentrations of liposomes were 40 nM and 36
nM for experiments in the absence or presence of mouse serum,
respectively. The efficacy of transfection is expressed here as
IC.sub.50 (in nM siRNA) as in Example 5. All results from this
screening experiment are shown in FIGS. 1-6.
[0185] Many of the transfecting mixtures resulted in very potent
transfection of HeLa cells with siRNA, as indicated by the very low
IC50 values. Combinations of lipids comprising imino lipids such as
CHOLGUA, but more so MPDACA, GUADACA or PONA remain potent
transfectants even in the presence of mouse serum.
Example 15
Serum Resistant Transfection of Several Cationic Lipids in
Combination with Phosphate Lipid
[0186] Series of liposomes having C/A ratios of either 0.75 or 1
were produced and loaded with siRNA as in Example 5. The cationic
lipid components were CHOLGUA, CHIM, DC-CHOL, GUADACA, MPDACA,
BADACA, PONA, DOTAP or DODAP. The anionic lipid was DOPA and the
cholesterol content was 40 mol %, all lipid mixtures are identified
in table 20. Liposomes were further supplied with 1.5 mol %
DMPE-PEG2000 (Nippon Oils and Fats).
[0187] HeLa cells were grown and maintained as in Example 2 and
mouse serum (SIGMA-Aldrich) was added directly to the cells for 120
min. Following that, the liposomes were added to the cells,
incubation was continued for 72 h and cell viability was determined
as above. The efficacy of transfection is expressed here as
IC.sub.50 (in nM of siRNA) as in Example 5.
[0188] Many of the transfecting mixtures resulted in very potent
transfection of HeLa cells with siRNA, as indicated by the very low
IC50 values. Combinations of lipids comprising imino lipids such as
CHOLGUA, but more so MPDACA, GUADACA or PONA remain potent
transfectants even in the presence of mouse serum.
TABLE-US-00017 TABLE 20 IC50 values (nM siRNA) for various
liposomes in the presence and absence of mouse serum. Serum
inhibition "not potent" refers to a lack of minimum potency in the
presence of mouse serum, in these cases the inhibition factor
cannot be defined. The highest concentration of siRNA in the test
was 146 nM. -mouse serum +mouse serum IC50 IC50 IC50 IC50 serum C/A
Cation PLK1 Scr. PLK1 Scr. inhibition 0.75 CholGUA 8 160 104 146 12
CHIM 26 160 146 146 not potent DC-Chol 28 160 146 146 not potent
MPDACA 5 67 10 146 2 GUADACA 6 39 26 146 4 BADACA 159 160 146 146
not potent PONA 6 24 146 146 not potent DOTAP 21 152 146 146 not
potent DODAP 160 160 146 146 not potent 1 CholGUA 9 141 128 146 14
CHIM 33 160 146 146 not potent DC-Chol 29 160 146 146 not potent
MPDACA 12 100 4 146 0.3 GUADACA 9 89 7 146 1 BADACA 38 160 146 146
not potent PONA 2 66 21 146 10 DOTAP 13 160 76 146 6 DODAP 94 160
146 146 not potent
Example 16
Serum Resistant Transfection is Poor in the Absence of Negatively
Charged Lipids
[0189] A series of liposomes was produced from cationic lipids and
cholesterol as a neutral lipid. No anionic lipids were used in
these preparations. The cationic lipid components were CHOLGUA,
CHIM, DC-CHOL, ADACA, GUADACA, MPDACA, BADACA, PONA, DOTAP and
DODAP and the liposomes were produced with the procedure described
in example 5.
[0190] The cholesterol content was 40 mol % and liposomes were
further supplied with 1.5 mol % DMPE-PEG2000 (Nippon Oils and Fats)
to avoid aggregate formation in the presence of siRNA.
[0191] HeLa cells were grown and maintained as in Example 2 and
mouse serum (SIGMA-Aldrich) was added directly to the cells for 120
min. Following that, the liposomes were added to the cells,
incubation was continued for 72 h and cell viability was determined
as above. The efficacy of transfection is expressed here as
IC.sub.50 (in nM of siRNA) as in Example 5.
[0192] The results obtained are shown in table 21 below. In all
cases, the transfection efficacy is substantially lower than that
of the mixtures further comprising an anionic lipid. With the
exception of GUADACA or PONA, there was no activity detectable in
the presence of mouse serum.
TABLE-US-00018 TABLE 21 IC50 values (nM siRNA) for various
liposomes in the presence and absence of mouse serum. Serum
inhibition "not potent" refers to a lack of minimum potency in the
presence of mouse serum, in these cases the inhibition factor
cannot be defined. The highest concentration of siRNA in the test
was 160 or 146 nM in the absence of presence of mouse serum,
respectively. no mouse serum with mouse serum IC50 IC50 serum
Cation IC50 PLK1 Scr. IC50 PLK1 Scr. inhibition CholGUA 93 160 146
146 not potent CHIM 160 160 146 146 not potent DC-Chol 101 109 146
146 not potent MPDACA 27 154 146 146 not potent GUADACA 22 69 95
146 4 BADACA 99 160 146 146 not potent PONA 30 100 70 99 2 DOTAP
160 160 146 146 not potent DODAP 160 160 146 146 not potent
* * * * *
References