U.S. patent application number 11/934147 was filed with the patent office on 2008-05-15 for sterol derivatives, liposomes comprising sterol derivatives and method of loading liposomes with active substances.
This patent application is currently assigned to NOVOSOM AG. Invention is credited to Anja Behrens, Gerold Endert, Stefan Fankhanel, STEFFEN PANZNER.
Application Number | 20080113017 11/934147 |
Document ID | / |
Family ID | 7675951 |
Filed Date | 2008-05-15 |
United States Patent
Application |
20080113017 |
Kind Code |
A1 |
PANZNER; STEFFEN ; et
al. |
May 15, 2008 |
STEROL DERIVATIVES, LIPOSOMES COMPRISING STEROL DERIVATIVES AND
METHOD OF LOADING LIPOSOMES WITH ACTIVE SUBSTANCES
Abstract
A sterol derivative with a pKa value of between 3.5 and 8,
according to the general formula cation-spacer 2-Y-spacer
1-X-sterol, wherein Y and X represent linking groups, is suggested,
as well as liposomes comprising such sterol derivatives.
Inventors: |
PANZNER; STEFFEN; (Halle,
DE) ; Endert; Gerold; (Halle, DE) ; Fankhanel;
Stefan; (Halle, DE) ; Behrens; Anja; (Koln,
DE) |
Correspondence
Address: |
AKERMAN SENTERFITT
P.O. BOX 3188
WEST PALM BEACH
FL
33402-3188
US
|
Assignee: |
NOVOSOM AG
Halle
DE
|
Family ID: |
7675951 |
Appl. No.: |
11/934147 |
Filed: |
November 2, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10468652 |
Feb 11, 2004 |
7312206 |
|
|
PCT/EP02/01879 |
Feb 21, 2002 |
|
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11934147 |
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Current U.S.
Class: |
424/450 ;
435/458; 514/44A; 540/107; 977/907 |
Current CPC
Class: |
C07J 41/0055 20130101;
C07J 43/00 20130101; A61K 31/58 20130101; C07J 43/003 20130101;
A61K 9/1272 20130101 |
Class at
Publication: |
424/450 ;
540/107; 514/44; 977/907; 435/458 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C07J 43/00 20060101 C07J043/00; A61K 9/127 20060101
A61K009/127; C12N 15/88 20060101 C12N015/88 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 21, 2001 |
DE |
10109898.7 |
Claims
1. A sterol derivative according to general formula (I):
cation-spacer 2-Y-spacer 1-X-sterol (1), wherein: said cation is a
nitrogen base selected from the group consisting of piperazines,
imidazoles, morpholines, purines, pyrimidines, and pyridines; said
spacers 1 and 2 are independently linear, branched or cyclic
C.sub.1-8 alkyl, and comprise 0-2 ethylenically unsaturated bonds,
wherein at least one of said spacers 1 and 2 is cyclic C.sub.1-8
alkyl; said linking group X is selected from the group consisting
of --(C=0)-O-- and --(C=0)-NH--; said linking group Y is selected
from the group consisting of --O-(0=C)--, --NH-(0=C)-1-(C=0)-O--,
and --(C=0)-NH--; said sterol is selected from the group consisting
of cholesterol, sitosterol, campesterol, desmosterol, fucosterol,
22-ketosterol, 20-hydroxysterol, stigmasterol,
22-hydroxycholesterol, 25-hydroxycholesterol, lanosterol,
7-dehydrocholesterol, dihydrocholesterol, 19-hydroxycholesterol,
5.alpha.-cholest-7-en-3.beta.-ol, 7-hydroxycholesterol,
epicholesterol, ergosterol, and dehydroergosterol; and said sterol
derivative has a pKa value of between about 3.5 and about 8.
2. The sterol derivative according to claim 1, wherein at least on
of said spacers 1 and 2 have hydroxyl groups.
3. The sterol derivative according to claim 1, wherein the sterol
derivative has a pKa value of between about 4 and about 7.
4. A liposome comprising the sterol derivative of claim 1.
5. The liposome of claim 4, wherein said liposome comprises between
about 5 mole-% and about 50 mole-% of sterol derivatives.
6. The liposome of claim 5, wherein said liposome comprises between
about 5 mole-% and about 40 mole-% of sterol derivatives.
7. The liposome of claim 6, wherein said liposome comprises between
about 10 mole-% and about 30 mole-% of sterol derivatives.
8. The liposome of claim 4, wherein the liposome comprises one or
more lipids selected from the group consisting of phosphatidyl
choline, phosphatidyl ethanolamine, and diacylglycerol.
9. The liposome of claim 8, wherein said liposome is neutral or
negatively charged at a pH of from about 7.0 to about 7.8.
10. The liposome of claim 4, wherein said liposome has an average
size of between about 50 and 1000 nm n.
11. The liposome of claim 10, wherein said liposome has an average
size of between about 50 and 300 nm.
12. The liposome of claim 11, wherein said liposome has an average
size of between about 60 and 130 nm.
13. The liposome of claim 4, wherein said liposome further
comprises an active substance.
14. The liposome claim 13, wherein said active substance is
selected from the group consisting of a protein, a peptide, a DNA,
an RNA, an antisense nucleotide, a decoy nucleotide, and a mixture
thereof.
15. The liposome of claim 13, wherein at least about 80% of said
active substance is situated inside the liposome.
16. A method of loading the liposome of claim 13 with an active
substance, said method comprising: encapsulating said active
substance in said liposome at a binding pH value; and removing
unbound active substances at a second pH value.
17. A method of loading the liposome of claim 13 with an active
substance, said method comprising: permeabilizing said liposome by
treatment at a pH value sufficient to enable loading of said active
substance, and sealing said liposome.
18. A method for the transport and release of an active substance
in a subject, said method comprising administering to said subject
the liposome of claim 13.
19. The method of claim 18, wherein said administration is
intravenous or peritoneal.
20. A transport and release system for the transport and release of
an active substance in a subject, said system comprising the
liposome of claim 13.
21. A depot formulation or circulative depot comprising the
liposome of claim 13.
22. A nanocapsule prepared from the liposome of claim 4.
23. A vector for transfecting cells in vivo, in vitro or ex vivo,
said vector comprising the liposome of claim 4 and a nucleic acid.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional application of U.S. patent application
Ser. No. 10/468,652, filed Feb. 11, 2004, which is a .sctn.371
national stage of PCT/EP02/01879, filed Feb. 21, 2002, which claims
priority to German Patent Application Number 101 09 898.7 filed
Feb. 21, 2001, all of which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The invention relates to polar compounds based on a sterol
skeleton, the 3-position of the ring system being substituted by an
organic cation having a pK value of between 3.5 and 8. The
invention also relates to liposomes containing such compounds.
BACKGROUND OF THE INVENTION
[0003] The term "lipids" summarizes three classes of natural
materials which can be isolated from biological membranes:
phospholipids, sphingolipids, and cholesterol, including its
derivatives.
[0004] These substances are of technical interest in the production
of liposomes. Inter alia, such liposomes can be used as containers
for active substances in pharmaceutical preparations. In such uses,
efficient and stable packaging of the cargo and controllable
release of the contents are desirable. Both of these requirements
are not easy to combine: the more stable and compact the packaging,
the more difficult the release of the entrapped active substance
therefrom. For this reason, liposomes changing their properties in
response to an external stimulus have been developed.
Thermosensitive and pH-sensitive liposomes are well-known. The
pH-sensitive liposomes are of special interest, because this
parameter undergoes changes even under physiological conditions,
e.g. during endocytotic reception of a liposome in a cell, or
during passage of the gastrointestinal tract.
[0005] The following abbreviations will be used hereinafter:
[0006] CHEMS Cholesterol hemisuccinate
[0007] PC Phosphatidyl choline
[0008] PE Phosphatidyl ethanolamine
[0009] PS Phosphatidyl serine
[0010] His-Chol Histaminylethaneamine-cholesterol hemisuccinate
[0011] Py-Chol Pyridylethaneamine-cholesterol hemisuccinate
[0012] Mo-Chol Morpholinoethaneamine-cholesterol hemisuccinate
[0013] PDEA-Chol Pyridyldithioethaneamino-cholesterol
hemisuccinate
[0014] According to the prior art, pH-sensitive liposomes
particularly comprise CHEMS. CHEMS, in mixture with phosphatidyl
ethanolamine, is used to produce pH-sensitive liposomes (Tachibana
et al. (1998); BBRC 251, 538-544, U.S. Pat. No. 4,891,208). Such
liposomes can enter cells by endocytosis and are capable of
transporting cargo molecules into the interior of cells on this
route, without doing damage to the integrity of the cellular
membrane.
[0015] One substantial drawback of CHEMS is its anionic character.
Liposomes produced using same have a negative overall charge and,
disadvantageously, are taken up by cells with low efficiency.
Despite the transfer mechanism described above, they are barely
suitable for the transport of macromolecules into cells.
[0016] For this purpose, the art uses cationic liposomes having a
preferably high and constant surface charge. The positive overall
charge of such particles leads to electrostatic adherence to cells
and subsequently to efficient transport into same. The use of these
compounds and of liposomes produced using same remains restricted
to in vitro or ex vivo applications, because such positively
charged liposomes disadvantageously result in uncontrolled
formation of aggregates with serum components.
SUMMARY OF THE INVENTION
[0017] The object was therefore to produce new compounds,
[0018] i) by means of which active substances can be entrapped in
liposomes and released therefrom when changing the pH value;
and
[0019] ii) the presence of which aids to achieve the production of
cationic liposomes which can be mixed with serum without formation
of larger aggregates.
[0020] Other objects of the invention involve finding ways allowing
easy and low-cost production of the desired compounds and
incorporation thereof in high amounts in liposomal membranes.
[0021] The object of the invention is accomplished by means of a
sterol derivative with a pKa value of between 3.5 and 8, according
to the general formula:
Cation-Spacer 2-Y-Spacer 1-X-Sterol,
wherein Y and X represent linking groups. Depending on the cation
used, compounds are obtained which undergo changes in their charge
at a specific pH value owing to the sterol component and allow
incorporation thereof in liposomal membranes in high amounts.
Ordinary and inexpensive sterols or derivatives thereof can be used
as starting compounds. Accordingly, the object of the invention can
be accomplished by conjugating pH-sensitive cations to the
3-position of a sterol skeleton.
[0022] Among the membrane-forming or membrane-bound groups of a
biological bilayer membrane, the sterols are of special interest
because these compounds are available at low cost, involve ordinary
chemistry, and allow incorporation in membranes in high amounts
without increasing the permeability thereof or even completely
destroying their membrane character. However, in order to retain
this latter feature, it is important that substitution with a polar
molecule be at the 3-position of the sterol.
[0023] The cation or cationic group can be e.g. a nitrogen base.
The sterol is cholesterol, for example. Situated between the
cationic group and the sterol skeleton are the molecule fragments
spacer 2-Y-spacer 1-X.
[0024] For example, spacer 1 is a lower alkyl residue of linear
structure, which has 8 C atoms and includes e.g. 2 ethylenically
unsaturated bonds. Spacer 2 is e.g. a lower alkyl residue of linear
structure, which may have 8 C atoms and includes 2 ethylenically
unsaturated bonds.
[0025] The overall molecule assumes its pH-dependent charge
characteristics by one or more organic cations with a pKa value
between 3.5 and 8. Typical molecules or molecule fragments with
this property are nitrogen bases. These nitrogen bases are linked
to the 3-position of the sterol skeleton via spacers and coupling
groups, thus forming a compound according to the formula of the
invention. In many cases, e.g. where the nitrogen bases are in the
form of a ring system, positional isomers are existing, wherein the
linking spacer is substituted to various positions of the organic
cation. Such positional isomers fall within the disclosure of this
invention. In many cases, the pKa values of the organic cation can
be influenced via said positional isomerism alone. The relevant
fundamental rules are well-known to those skilled in the art.
Alternatively, these effects can be estimated from tabular
compilations (Handbook of Chemistry and Physics, Vol. 73, pp.
8-37ff.).
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIG. 1: A. Structural formula of Histidineamido-cholesterol
hemisuccinate, m.w. 580 g/mol; B. structural formula of
Pyridyldithioethaneamido-cholesterol hemisuccinate, m.w. 655
g/mol.
[0027] FIG. 2: Shows transfection of HeLa cells according to
Example 12: A. Transfection by TRITC dextran with liposomes (DOPE
60/His-Chol 40); B. Transfection by TRITC dextran with liposomes
(POPC 60/His-Chol 40).
DETAILED DESCRIPTION
[0028] In a preferred embodiment of the invention, the sterol
derivative has a pKa value of between 4 and 6.5. Advantageously,
this pKa value falls in a range which is of crucial importance for
the physiology of numerous organisms.
[0029] In another preferred embodiment of the invention, the
cations are nitrogen bases. The cations preferably can be derived
from piperazines, imidazoles, morpholines, purines and/or
pyrimidines.
[0030] Coupling reactions result in amphiphilic organic cations,
e.g. those derived from the following classes of substances:
[0031] o-, m-, p-anilines; 2-, 3- or 4-substituted anisidines,
toluidines or phenetidines; 2-, 3-, 5-, 6-, 7- or 8-substituted
benzimidazoles, 2-, 3-, 4- or 5-substituted imidazoles, 1- or
5-substituted isoquinolines, 2-, 3- or 4-substituted morpholines,
2-, 3- or 4-substituted picolines, 1-, 2- or 3-substituted
piperazines, 2-, 5- or 6-modified pterines, 3-, 4-, 5-, 6- or
9-substituted purines, 2- or 3-substituted pyrazines, 3- or
4-substituted pyridazines, 2-, 3- or 4-modified pyridines, 2-, 4-,
5- or 6-substituted pyrimidines, 1-, 2-, 3-, 4-, 5-, 6- or
8-substituted quinolines, 2-, 4- or 5-substituted thiazoles, 2-, 4-
or 6-substituted triazines, or derivatives of tyrosine.
Particularly preferred are piperazines, imidazoles, morpholines,
purines and/or pyrimidines.
[0032] Highly preferred are molecule fragments such as occurring in
biological systems, i.e., for example: 4-imidazoles (histamines),
2-, 6- or 9-purines (adenines, guanines, adenosines, or
guanosines), 1-, 2- or 4-pyrimidines (uracils, thymines, cytosines,
uridines, thymidines, cytidines), or pyridine-3-carboxylic acids
(nicotinic esters or amides).
[0033] The above-mentioned structural fragments may also have
additional substituents. For example, these can be methyl, ethyl,
propyl, or isopropyl residues, more preferably in hydroxylated
form, including one or two hydroxyl groups. Also, these can be
hydroxyl or keto functions in the ring system. In addition, other
structural fragments are also possible unless anionically
dissociated molecule portions are formed in a pH range between 3.5
and 8.5, e.g. carboxylic acids, sulfonic acids, or some aromatic
hydroxyl groups or enols.
[0034] Nitrogen bases with preferred pKa values are also formed by
single or multiple substitution of the nitrogen atom with lower
alkanehydroxyls such as hydroxymethyl or hydroxyethyl groups.
Suitable organic bases from this group are e.g. aminopropanediols,
triethanolamines, tris(hydroxymethyl)methylamines,
bis(hydroxymethyl)methylamines, tris(hydroxyethyl)methylamines,
bis(hydroxyethyl)methylamines, or the corresponding substituted
ethylamines. Coupling of these fragments to the hydrophobic portion
of the molecule may proceed either via the nitrogen of the base or
via any of the hydroxyl functions.
[0035] In addition to sterol derivatives including a single organic
cation, those including two or three identical or different groups
are also preferred. All of these groups are required to have a pKa
value in the above-mentioned range. One suitable complex group is
the amide of histamine and histidine or of histamine and
histidylhistidine.
[0036] Anionic groups such as carboxylic acids, sulfonic acids,
enols, or aromatic hydroxyls are allowable as component of the
molecule only if undissociated in the claimed pH range between 3.5
and 8.5. In general, this is the case if the pKa value is above
9.5.
[0037] In another preferred embodiment of the invention, the
linking group X has the structure --(C.dbd.O)--O--;
--(C.dbd.O)--NH--; --(C.dbd.O)--S--; --O--; --NH--; --S--; or
--CH.dbd.N--, for example. In particular, the linking group Y
corresponds in its structure to the group X, and may additionally
assume the structure --O--(O.dbd.C)--; --S--(O.dbd.C)--;
--NH--(O.dbd.C)--; or --N.dbd.CH--. The Y group can be omitted in
those cases where the organic cation can be coupled directly to the
sterol skeleton, e.g. in the esterification of 4-imidazoleacetic
acid with cholesterol.
[0038] In another preferred embodiment of the invention, spacer 1
is a lower alkyl residue of linear, branched or cyclic structure,
which has from 1 to 8 C atoms and includes 0, 1 or 2 ethylenically
unsaturated bonds. Spacer 1 may have hydroxyl groups so as to
increase the polarity of the molecule. In particular, spacer 1 can
be a sugar. Spacer 2 is a lower alkyl residue of linear, branched
or cyclic structure, which has from 0 to 8 C atoms and includes 0,
1 or 2 ethylenically unsaturated bonds. Spacer 2 may have hydroxyl
groups so as to increase the polarity of the molecule. In
particular, spacer 2 can be a sugar.
[0039] Methods of performing such coupling reactions are well-known
to those skilled in the art and may vary depending on the starting
material and coupling component employed. Typical reactions are
esterification, amidation, addition of amines to double bonds,
etherification, or reductive amination.
[0040] A particularly preferred method of coupling is amidation of
sterol hemisuccinates. Inter alia, molecules satisfying the
requirements according to the object of the invention can be
produced by coupling of histamine, N-(2-aminoethyl)morpholine,
N-(2-aminoethyl)piperazine, N-(2-aminoethyl)pyridine, or
pyridyldithioethenylamine. The compounds thus obtained will be
referred to as His-Chol, Mo-Chol, Pip-Chol, Py-Chol, or PDEA-Chol
herein.
[0041] In another preferred embodiment of the invention, the
sterols are particularly cholesterol, sitosterol, campesterol,
desmosterol, fucosterol, 22-ketosterol, 20-hydroxysterol,
stigmasterol, 22-hydroxycholesterol, 25-hydroxycholesterol,
lanosterol, 7-dehydrocholesterol, dihydrocholesterol,
19-hydroxycholesterol, 5.alpha.-cholest-7-en-3.beta.-ol,
7-hydroxycholesterol, epicholesterol, ergosterol, and/or
dehydroergosterol, as well as other related compounds.
[0042] The sterols that are used may bear various groups in the
3-position thereof, which groups allow for ready and stable
coupling or optionally assume the function of a spacer.
Particularly suitable for direct coupling are the hydroxyl group
which is naturally present, but also, the chlorine of sterol
chlorides, or e.g. the amino group of sterolamines, or the thiol
group of thiocholesterol.
[0043] The invention also relates to liposomes comprising the
substances according to the invention. All of the substances or
compounds of the invention can be incorporated in high amounts in
liposomal membranes, resulting in a positive charge of the overall
particle only if the pH value of the medium is smaller than (pKa+1)
of the compounds according to the invention.
[0044] In a special embodiment of the invention, the amount of
sterol derivative is 50 mole-% at maximum. Compositions including
at least 5 mole-% of compound, but 40 mole-% at maximum, are
particularly preferred. Compositions including at least 10 mole-%
of sterol derivative and 30 mole-% at maximum are highly
preferred.
[0045] Another embodiment wherein the liposomes specifically
comprise phosphatidyl choline, phosphatidyl ethanolamine and/or
diacylglycerol is also convenient. Cholesterols themselves are
incapable of forming liposomes, and therefore, addition of further
lipid is necessary. In particular, this lipid can be a
phospholipid. Obviously, further modifications of the liposome are
possible. Thus, the use of polyethylene glycol-modified
phospholipids or analogous products is particularly
advantageous.
[0046] In another embodiment of the invention, the liposomes have
an average size of between 50 and 1000 nm, preferably between 50
and 300 nm, and more preferably between 60 and 130 nm.
[0047] In another preferred embodiment, the liposomes comprise
active substances. For example, the liposomes according to the
invention are suitable for parenteral application. They can be used
e.g. in cancer therapy and in the therapy of severe infections. To
this end, liposome dispersions can be injected, infused or
implanted. Thereafter, they are distributed in the blood or lymph
or release their active substance in a controlled fashion as a
depot. The latter can be achieved by highly concentrated
dispersions in the form of gels. The liposomes can also be used for
topical application on the skin. In particular, they may contribute
to improved penetration of various active substances into the skin
or even passage through the skin and into the body. Furthermore,
the liposomes can also be used in gene transfer. Due to its size
and charge, genetic material is usually incapable of entering cells
without an aid. For this purpose, suitable carriers such as
liposomes or lipid complexes are required which, together with the
DNA, are to be taken up by the respective cells in an efficient and
well-directed fashion. To this end, cell-inherent transport
mechanisms such as endocytosis are used. Obviously, the liposomes
of the invention can also be used as model membranes. In their
principal structure, liposomes are highly similar to cell
membranes. Therefore, they can be used as membrane models to
quantify the permeation rate of active substances through membranes
or the membrane binding of active substances.
[0048] Advantageously, liposomes produced using the substances of
the invention show low non-specific binding to cell surfaces. It is
this low non-specific binding which is an essential pre-condition
for achieving specific binding to target cells. Target control of
the vehicles is obtained when providing the above-described
liposomes with additional ligands. As a result, the active
substance can be accumulated specifically in such cells or tissues
which exhibit a pathological condition.
[0049] One important use of the substances according to the
invention is therefore in the construction of vectors for transfer
of active substances in living organisms. The vectors are
particularly suited for the transport of therapeutic macromolecules
such as proteins or DNA which themselves are incapable of
penetrating the cell membrane or undergo rapid degradation in the
bloodstream.
[0050] Advantageously, antibodies, lectins, hormones or other
active substances can be coupled to the surface of liposomes under
mild conditions in high yields. In one variant of the teaching
according to the invention, the liposomes for such a use comprise a
sufficient amount of PDEA-Chol in addition to other lipids,
including those mentioned in the present specification. The amount
of PDEA-Chol employed will depend on the desired use. Thus,
liposomes loaded with a marker require a high ratio of this signal
generator to the component determining the specificity. In this
case, only a low number of antibodies per liposome have to be
coupled.
[0051] In a preferred embodiment of the invention, the liposomes
comprise a protein, a peptide, a DNA, an RNA, an antisense
nucleotide, and/or a decoy nucleotide as active substance.
[0052] In a particularly preferred embodiment of the invention, at
least 80% of the active substance is inside the liposome.
[0053] The invention also relates to a method of loading liposomes
with active substances, wherein one defined pH value is used for
encapsulation, and a second pH value is adjusted to remove unbound
active substance.
[0054] The invention also relates to a method of loading liposomes
with active substances, wherein the liposomes are made permeable at
a well-defined pH value and sealed.
[0055] The invention also relates to the use of the liposomes in
the production of nanocapsules.
[0056] The invention also relates to the use of the liposomes in
the production of release systems in diagnostics.
[0057] Advantageously, the liposomes are used for the transport
and/or release of active substances.
[0058] In another embodiment, the liposomes conveniently are used
as depot formulation and/or as circulative depot.
[0059] Advantageously, the liposomes can be used in intravenous or
peritoneal application.
[0060] In another embodiment of the invention, the liposomes are
used with advantage as vector to transfect cells in vivo, in vitro
and ex vivo.
[0061] Surprisingly, it has been determined that the permeability
of the lipid layer of the inventive liposomes particularly depends
on the pH value and thus, on the state of charge of the sterol
derivative. In addition, when using the well-known CHEMS, an
increase in permeability occurs only in the simultaneous presence
of high amounts of phosphatidyl ethanolamine (PE) in the membrane.
This phospholipid does not form membranes by itself, being
stabilized artificially by CHEMS. However, one drawback of such
liposomes is their low stability which can be seen in the fact that
smaller molecules of active substance slowly diffuse out even
without a change in pH.
[0062] When using His-Chol, in particular, the membranes comprised
of phosphatidyl choline (PC) are made permeable in such a way that
entrapped active substances or markers will diffuse out within
minutes to hours. However, these membranes themselves are stable,
showing low initial permeability. Liposomes using the structures
according to the invention are therefore suited to construct
release systems wherein release of active substances is to proceed
in dependence on the pH value of the medium.
[0063] Surprisingly, it has also been found that amounts of
proteins or DNA above average can be enclosed in liposomes
including the compounds described herein in the membranes thereof.
The efficiency of such incorporation depends on the pH value of the
solution employed. Therefore, a process for efficient encapsulation
of proteins or DNA in liposomes can be performed by initially
adjusting a pH value that would result in good binding of the cargo
molecules to the liposomes. With DNA as polyanion, low pH values of
about 4 to 5 are used. With proteins, a useful pH value will depend
on the isoelectric point of the protein, which should be below the
pKa value of the substance according to the invention.
Encapsulation is particularly effective when the pH value of the
medium is selected so as to range between the isoelectric point of
the protein and the pKa value of the sterol derivative. The
proteins then will have a negative charge, while the lipid layer
already has a positive net charge. If necessary, non-incorporated
cargo molecules adhering on the outside can be removed by simply
increasing the pH value. This step is necessary in all those cases
where non-incorporated cargo molecules would give rise to
aggregation of the liposomes. One advantageous fact when using the
components of the invention is that the entrapped active substances
must be maintained under conditions allowing interaction with the
lipid layer only during the period of actual enclosure. Once the
lipid layer remains closed in itself, it is possible to change to
other conditions. Thereby, possible inactivation of active
substances, particularly of proteins, can be minimized.
[0064] Liposomes comprising the components of the invention can be
coated with polymers under conditions well-known to those skilled
in the art, where single or multiple deposition of such substances
on the surface is possible, in particular. In multiple deposition,
optionally in the presence of crosslinkers, liposomal nanocapsules
are formed as described in WO 00/28972 or WO 01/64330.
[0065] One advantageous fact when using the substances described
herein is that the electrostatic interaction with the
polyelectrolyte can be interrupted. As is well-known, the
interaction of a polyelectrolyte with charge carriers of the
liposomal membrane may give rise to demixing of membrane components
and formation of lipid clusters. In many cases, such demixing is
accompanied by a permeabilization of the liposomes. The substances
of the invention allow for elimination of this interaction
following the coating process. When increasing the pH value at this
point, the liposomes will be entrapped in the nanocapsules merely
in a steric fashion, and interaction between the membrane and
polyelectrolytes does no longer exist. In this way, cluster
formation of lipids and associated permeabilization of the membrane
can be circumvented.
[0066] In one variant of the teaching according to the invention,
these changes in permeability are used in a well-directed fashion
in loading liposomes. To this end, an active substance to be
enclosed can be added to a medium under conditions of high
permeability, followed by adjusting conditions of low permeability.
In this way, the active substance will remain inside the liposomes.
Thereafter, non-entrapped active substance can be removed, if
necessary. Such changes in permeability can be induced on liposomes
or on liposomal nanocapsules.
[0067] Surprisingly, it has also been found that liposomes
including e.g. His-Chol or Pip-Chol in the membranes thereof are
capable of chelating metal ions. This property results in an
increase of the positive charge of the liposome. This effect is
observed to be particularly strong at neutral pH values, because
the inherent charge of the compound is low in this case. Owing to
their chelating properties, such liposomes can be used in
biochemical diagnostics and in pharmaceutical therapy.
[0068] In a detection system, such liposomes can be loaded with
metal ions whose fluorescence is enhanced by chelate formation,
i.e., terbium or europium ions, for example. Liposomes for such
uses additionally include components determining the specificity,
i.e., antibodies, lectins, selectins, receptors, or hormones, or
RNA aptamers. In a particularly preferred embodiment of the use
according to the invention, the presence of these metal ions is
restricted to the volume of the liposomes so as to avoid
non-specific signals from slowly released metal ions adhering on
the outside. Coupling of pyridyldithioethenylamine to CHEMS
provides a compound with a special combination of desirable
properties. The terminal pyridyl group results in significant
positive charging of the membrane even under mild conditions (pH 6
to 7). Liposomes produced using this compound are capable of
binding proteins or nucleic acids in large amounts above average.
By reducing the disulfide bond, e.g. using dithiothreitol or
tris(2-carboxyethyl)phosphine, a free thiol function is generated,
resulting in neutralization of the surface. Under these conditions,
such enhanced binding of proteins or nucleic acids is decreased.
Ultimately, it is completely lost when increasing the pH value,
because the formation of thiolate ions results in a negatively
charged surface.
[0069] In those cases where biological macromolecules possess thiol
functions of their own, binding thereof can be retained. This is
the case with numerous proteins. Where other materials are
concerned, a person skilled in the art will be familiar with
procedures of introducing free thiol functions in such molecules,
while retaining the biological activity thereof (G. Hermanson,
Bioconjugate Techniques). Substances including a free thiol
function can be fixed covalently to the surface of such lipid
layers by means of a disulfide exchange reaction.
[0070] Owing to their particularly favorable properties in binding
and coupling of proteins, liposomes including PDEA-Chol are
especially suitable in the production of nanocapsules on liposomal
templates such as described in WO 00/28972.
[0071] Surprisingly, it has also been found that liposomes
including PDEA-Chol are capable of changing their permeability in
accordance with the redox state. Reductive removal of the pyridyl
group results in permeabilization of the membrane.
[0072] Surprisingly, it has also been found that the liposomes
according to the invention readily undergo fusion with other
membranes at low pH values. In general, this step requires the
presence of a larger amount of PE in the membrane. As a result of
its tendency of forming hexagonal phases, said PE assumes the
function of a helper lipid. However, the inferior stability of such
membranes is disadvantageous, and gradual release of entrapped
active substances is frequently observed.
[0073] However, liposomes produced using the substances according
to the invention undergo effective fusion even in the absence of
such a helper lipid. Thus, when using the substances of the
invention, it is possible to produce liposomes which are capable of
stably encapsulating an active substance, but undergo fusion with
cell membranes under the conditions of low pH values to release the
active substance there.
[0074] This combination of two properties is an important
precondition for the incorporation of cargo molecules in cells. In
fusion of liposomes with cell envelopes or components, the aqueous
volumes of both partners combine, with no opening of the membrane
structures to the medium taking place. As a result, uncontrolled
influx or efflux of other substances is avoided.
[0075] One essential precondition for the use of liposomes for
experimental or therapeutic purposes is their compatibility with
cells and tissues. A number of well-known compounds used to
incorporate DNA or proteins in cells (for example, the cationic
lipid DOTAP) are cytotoxic.
[0076] Surprisingly, it has also been found that the compounds of
the invention exhibit reduced cytotoxicity. These measurements will
be illustrated in the experimental section. Thus, compared to the
commonly used DOTAP, His-Chol shows lesser toxic effects in the MTT
test.
[0077] Another precondition for the construction of vectors to be
used in gene or protein transport into cells is their compatibility
with serum or blood. Due to their strong cationic charge, vectors
known at present form uncontrollable large aggregates, resulting in
formation of thrombi in the organism. Their use in vivo is
therefore practically impossible and is restricted to in vitro or
ex vivo applications.
[0078] Surprisingly, it has been found that liposomes constructed
using the components of the invention do not form any aggregates in
serum or blood.
[0079] Another precondition for the construction of vectors to be
used in protein or gene transfer is their stability under
physiological conditions. Upon application into the blood
circulation, liposomes are attacked by components of the complement
system and undergo rapid lysis. This reaction proceeds within
minutes. As a result, pores are formed in the membrane, which allow
even large molecules such as proteins to diffuse out therethrough.
At present, stabilization of liposomes with respect to this
mechanism is only possible by incorporating cholesterol in the
lipid layer. While such liposomes are highly stable, they are no
longer able to interact with cells or readily release their active
substance. Surprisingly, it has been found that liposomes
constructed using the components of the invention are stable in
serum or blood for several hours. Even under such conditions, the
release of active substance is low.
[0080] A liposomal vector for the transport of active substances
must satisfy at least three preconditions: it must have low
toxicity, entrap the active substance firmly and stably, and be
compatible with serum or blood.
[0081] All of these three preconditions are satisfied by liposomes
produced using the sub-stances according to the invention. The
liposomes disclosed herein are therefore well suited for
therapeutic uses. Other properties supporting such uses are good
loadability with active sub-stances and well-directed release of
these substances by permeabilization of the membrane at suitable pH
values or redox states.
[0082] Without intending to be limiting, the invention will be
explained in more detail with reference to the following
examples.
EXAMPLE 1
Synthesis of His-Chol
[0083] 1.31 g of cholesterol hemisuccinate is dissolved in 20 ml of
DMF at room temperature. The solution is added with 438 mg of
carbonyldiimidazole dissolved in 20 ml of DMF. The mixture is
allowed to stir for 1 hour and subsequently added with 300 mg of
histamine. The mixture is stirred overnight and concentrated
thoroughly in vacuum. The residue is purified by column
chromatography on silica (Kieselgel 60), with chloroform/methanol
10:1 being used as eluant. (Yield 54%; 1.45 mmol), pure in HPLC,
identity determined using MS and .sup.13C-NMR.
EXAMPLE 2
Synthesis of PDEA-Chol
[0084] The procedure for the synthesis of PDEA-Chol is as above.
Instead of histamine, 600 mg of pyridyldithioethaneamine
hydrochloride is used.
EXAMPLE 3
Synthesis of Mo-Chol
[0085] The procedure for the synthesis of Mo-Chol is as above.
Instead of histamine, 350 mg of 4-(2-aminoethyl)morpholine is
used.
EXAMPLE 4
Preparation of Cationic pH-Sensitive Liposomes
[0086] 5 mg of His-Chol and 9.8 mg of POPC are dissolved in 4 ml of
chloroform/methanol (1:1 v/v) and dried completely in a rotary
evaporator. The lipid film is hydrated with 4.3 ml of a
corresponding buffer (10 mM Kac, 10 mM HEPES, 150 mM NaCl, pH 7.5)
at a lipid concentration of 5 mM using brief ultrasonic treatment
(5 minutes). Finally, the suspension is frozen and, following
thawing, subjected to multiple extrusions (Avestine LiposoFast,
polycarbonate filter, pore width 200 nm).
[0087] The profile of the zeta potential at various pH values is
illustrated in the Table below.
TABLE-US-00001 pH value Zeta potential in mV 4.4 +52 6.2 -3 7.5
-13
EXAMPLE 5
Permeability
[0088] Liposomes are produced generally as in Example 4. The
following lipid mixtures are used (figures in mole-%)
TABLE-US-00002 A: DPPC 60 His-Chol 40 B: DPPC 60 CHEMS 40 C: POPC
60 His-Chol 40 D: POPC 60 CHEMS 40
[0089] The lipids are dissolved in the solvent mixture as indicated
and dried under vacuum. The lipid films are hydrated with 100 mM
carboxyfluoresceine, 50 mM NaCl, pH 7.5, at a lipid concentration
of 15 mM and frozen, thawed and extruded as above. Non-entrapped
carboxyfluoresceine is removed by gel filtration.
[0090] 20 .mu.l of the liposomes thus obtained are incubated with 2
ml of buffer (10 mM potassium acetate, 10 mM HEPES). After 90 min,
the amount of discharged carboxyfluoresceine is determined by
measuring the fluorescence intensity of the sample. A comparative
sample with complete release of the entrapped marker is obtained by
addition of 0.2% Triton X-100 to the batch.
TABLE-US-00003 A B C D pH 4.4 8 38 80 39 pH 5.4 7 7 35 18 pH 6.4 6
9 18 16 pH 7.4 7 7 17 15 pH 8.4 5 11 15 15
EXAMPLE 6
Chelating of Metal Ions
[0091] Liposomes are produced as in Example 4. 40 .mu.l of these
liposomes are suspended in 7 ml of buffer (10 mM potassium acetate,
10 mM HEPES, pH 4.2 or pH 7.5). Subsequently, the metal ions are
added with the concentrations as indicated, and the zeta potential
of the liposomes is measured.
TABLE-US-00004 Ions pH 4.2 pH 7.5 Ni.sup.2+ 10 mM +16.8 11.6
Ca.sup.2+ 10 mM +40.5 +4.6 Zn.sup.2+ 10 mM +65.4 not measurable No
addition +47.6 +2.4
[0092] At neutral pH, chelating of nickel ions is clearly
detectable. At slightly acidic pH, nickel ions are not bound
anymore, but zinc ions are. The non-transition metal calcium
behaves indifferently, forming no chelate complexes.
EXAMPLE 7
Binding of DNA
[0093] 1 mg of DNA (herring sperm, SIGMA D3159) is dissolved in 1
ml of water. Using the liposomes from Example 4, a 0.2 mM
suspension in buffer (10 mM potassium acetate, 10 mM HEPES, pH 4.2
or pH 7.5) is produced. 45 .mu.l of DNA solution each time is added
to 1 ml of these different liposome samples and mixed rapidly.
After 15 minutes of incubation, the sample is filled up with 6 ml
of the corresponding buffer, and the zeta potential of the
liposomes is measured.
TABLE-US-00005 Zeta potential in mV No DNA DNA added pH = 4.2 +27.1
-45.7 pH = 7.5 -6.3 -39.6
EXAMPLE 8
Fusion Properties
[0094] Liposomes having the following compositions are produced as
in Example 1 (all figures in mole-%)
[0095] A) POPC 60 His-Chol 40
[0096] X) POPC 100
[0097] Y) POPC 60 DPPG 40
[0098] The optionally cationic liposomes A are incubated with the
neutral liposomes X or with the anionic liposomes Y in buffer (10
mM HEPES, 10 mM potassium acetate, pH 4.2 or 7.5). Possible fusion
of liposomes is analyzed using size measurement by means of dynamic
light scattering.
TABLE-US-00006 Liposome 1 X Y Liposome 2 A A pH 4.2 181.6 nm 1689.3
nm pH 7.5 191.8 nm 250.0 nm
[0099] The initial values of the liposomes were 161.8 nm at pH 4.2
and 165.9 nm at pH 7.5
[0100] X) 199.2 nm
[0101] Y) 183.2 nm
[0102] The size of the YA pair of complementary charge is clearly
different from the size of the mixed suspensions including the
neutral liposome XA. The degree of interaction is determined by the
charge level of the optionally cationic liposomes. Fusion to form
larger units does not depend on the fusogenic PE lipid.
EXAMPLE 9
Permeability to Macromolecules
[0103] 15 .mu.mol of DOPE and 10 .mu.mol of His-Chol are dissolved
in isopropanol, and the solvent is removed under vacuum. The dried
lipid film is added with 2.5 ml of a solution of proteinase K in
buffer (1 mg/ml proteinase K, 10 mM potassium acetate, 10 mM HEPES,
150 mM NaCl, pH 4.2). Following hydration of the film, the
liposomes having formed are extruded through a 400 nm membrane.
Non-entrapped proteinase is removed by flotation of the liposomes
in a sucrose gradient.
[0104] The liposomes thus produced are incubated with 7.5 ml of
buffer at pH 4.2 and pH 7.2 (buffer as above, initial pH 4.2 and
8.0). Following incubation, the liberated proteinase K is separated
by ultrafiltration using a 0.1 .mu.m membrane. The liposomes
remaining in the filter are then treated with 7.5 ml of a solution
of Triton X-100 in buffer (as above, pH 8.0).
[0105] All of the filtrates are tested for presence of proteinase
K. To this end, a solution of azocasein (6 mg/ml azocasein in 1 M
urea, 200 mM Tris sulfate, pH 8.5) is used. 500 .mu.l of this
solution is mixed with 100 .mu.l of filtrate or buffer and
incubated for 30 minutes at 37.degree. C. The reaction is
terminated by addition of 10% trichloroacetic acid. Precipitated
proteins are removed by centrifugation. The coloration in the
supernatant is measured at 390 nm.
TABLE-US-00007 Absorption at 390 nm - pH Incubation Triton X-100
blank 4.2 - 0.0165 4.2 + 0.1731 7.2 - 0.1354 7.2 + 0.0260
[0106] When incubating the liposomes at a pH value of 4.2, no or
only a small amount of proteinase K is liberated. The enzyme is
liberated only after dissolving the liposomes with Triton
X-100.
[0107] When incubating the liposomes at a pH value of 7.2, a major
amount of the enzyme is liberated even without addition of Triton
and will be found in the first filtrate. Addition of Triton then is
barely capable of leaching further enzyme from the liposomes.
EXAMPLE 10
Cytotoxicity
[0108] The toxic effect of the substances was investigated using
the MTT test. To this end, HeLa cells were seeded at a density of
2.times.10.sup.4 cells per cavity of a 96-well titer plate and
cultured for two days. Liposomes of varying composition (see Table)
were added to the cells at a concentration of 0.5 mM and incubated
with these cells for 24 hours. Subsequently, the MTT test was
performed.
TABLE-US-00008 Liposome Viability (MTT) Notes -- 100% DOPE 60/DOTAP
40 82% DOPE 60/DC-Chol 40 57% Cells detaching DOPE 60/His-Chol 40
92% DOPE 60/Mo-Chol 40 103% POPC 60/His-Chol 40 90%
[0109] DOPE: Dioleoylphosphatidyl ethanolamine, 60 mole-% each time
POPC: Palmitoyloleoylphosphatidyl choline, 60 mole-% each time
[0110] DC-Chol: N,N-Dimethyl(2-aminoethyl)carbamoylcholesterol
[0111] Even at high concentrations, the liposomes comprising the
substances of the invention are well-tolerated by cells. Toxic
effects are barely detectable. In contrast, well-known cationic
lipids such as DC-Chol or DOTAP have a significant cytotoxic
effect.
EXAMPLE 11
Stability in Serum
[0112] Carboxyfluoresceine-loaded liposomes having the compositions
POPC/His-Chol 60:40, POPC/Mo-Chol 60:40, and POPC/DPPG 60:40 (all
figures in mole-%) were produced in analogy to Example 5. For
measurement, the liposomes were diluted to 0.1 mM in human serum
and incubated at 37.degree. C. Fluorescence was measured at
specific intervals. Complete liberation was achieved by addition of
Triton X-100 to the measuring buffer. The CF liberation data are
summarized in the Table below. For comparison, the negatively
charged POPC/DPPG liposomes virtually losing no CF during 4 hours
are illustrated. POPC/His-Chol liposomes show high serum stability
up to 2 hours, but lose some CF after 4 hours. POPC/Mo-Chol
liposomes exhibit a somewhat higher permeability than
POPC/His-Chol.
TABLE-US-00009 POPC/His-Chol POPC/Mo-Chol POPC/DPPG 0 min 0% 0% 0%
30 min 0% 4% 0% 60 min 2% 5% 1% 120 min 4% 9% 2% 240 min 19% 16%
2%
EXAMPLE 12
Transfection into Cells
[0113] HeLa cells (3.times.10.sup.5) were placed in each cavity of
a 6-well titer plate and cultured for three days. Liposomes having
the same compositions as in Example 10 were produced in the
presence of fluorescence-labelled dextran (TRITC dextran, 10 mg/ml
in hydration buffer). Non-incorporated TRITC dextran was removed by
gel filtration. The liposomes thus produced were added to the cells
and incubated for 6 hours at 37.degree. C. Subsequently, the cells
were washed twice with buffer. Dextran uptake was monitored by
microscopic imaging and quantified using fluorescence
spectroscopy.
[0114] The results are summarized in the following Table and in
FIG. 2.
TABLE-US-00010 TRITC dextran uptake Liposome in .mu.g - (TRITC
dextran in buffer) 0.1 DOPE 60/DOTAP 40 1.5 DOPE 60/DC-Chol 40 1.1
DOPE 60/His-Chol 40 0.3 DOPE 60/Mo-Chol 40 0.7 POPC 60/His-Chol 40
0.4
[0115] The new compounds do not quite achieve the efficiency of the
well-known cationic lipids DOTAP or DC-Chol. However, they are also
capable of mediating the transfection of macromolecules into cells.
It is to be expected that the efficiency can be increased
substantially when using ligands for cell adhesion.
* * * * *