U.S. patent application number 10/021421 was filed with the patent office on 2002-10-17 for lipid compounds and compositions containing them which can be used for the transfer of at least one active substance, in particular a polynucleotide, into a target cell and use in gene therapy.
Invention is credited to Bischoff, Rainer, Cordier, Yves, Nazih, Abdesslame.
Application Number | 20020151070 10/021421 |
Document ID | / |
Family ID | 9504295 |
Filed Date | 2002-10-17 |
United States Patent
Application |
20020151070 |
Kind Code |
A1 |
Bischoff, Rainer ; et
al. |
October 17, 2002 |
Lipid compounds and compositions containing them which can be used
for the transfer of at least one active substance, in particular a
polynucleotide, into a target cell and use in gene therapy
Abstract
The invention relates to new lipid compounds of formula:
R--HN--[--(CH.sub.2).sub.m--NR--].sub.n-1--(CH.sub.2).sub.m--NH--R
I in which: the R residues are, independently of each other, a
hydrogen atom or a group of formula II: 1 for which: R.sub.1 and
R.sub.2 are, independently of each other, C.sub.6-C.sub.23 alkyl or
alkenyl radicals, which are linear or branched, or radicals
--C(.dbd.O)--(C.sub.6-C.sub.23) alkyl or
--C(.dbd.O)--(C.sub.6-C.sub.23) alkenyl, which are linear or
branched, aryl radicals, cycloalkyl radicals, fluoroalkyl radicals,
polyethylene glycol groups, oxyethylene or oxymethylene groups
which are optionally repeated, linear or branched, optionally
substituted, p is a positive integer from 1 to 4, n is a positive
integer from 1 to 6, m is a positive integer from 1 to 6 which may
be different for each motif --(CH.sub.2).sub.m, and more
particularly for each motif --(CH.sub.2).sub.m--NR-- when n>1,
the number of R groups of formula II being between 1 and 4 said
compounds being optionally in a cationic form and being combined
with one or more biologically acceptable anions. It also relates to
new complexes comprising at least one said cationic compound and an
active substance comprising negative charges allowing the
introduction of said active substances into cells. It relates in
particular to new complexes, in which the active substance consists
of one or more nucleic acids, useful for transfecting cells.
Inventors: |
Bischoff, Rainer;
(Barsebacksby, SE) ; Nazih, Abdesslame;
(Strasbourg, FR) ; Cordier, Yves; (Strasbourg,
FR) |
Correspondence
Address: |
Norman H. Stepno
BURNS, DOANE, SWECKER & MATHIS, L.L.P.
P.O. Box 1404
Alexandra
VA
22313-1404
US
|
Family ID: |
9504295 |
Appl. No.: |
10/021421 |
Filed: |
December 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10021421 |
Dec 19, 2001 |
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09171845 |
Oct 28, 1998 |
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6335199 |
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09171845 |
Oct 28, 1998 |
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PCT/FR98/00389 |
Feb 27, 1998 |
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Current U.S.
Class: |
435/458 ;
514/44R; 554/56 |
Current CPC
Class: |
A61K 9/1272 20130101;
A61K 47/54 20170801; C12N 15/88 20130101; C12N 15/87 20130101; A61K
47/543 20170801 |
Class at
Publication: |
435/458 ; 514/44;
554/56 |
International
Class: |
A61K 048/00; C12N
015/88; C11C 003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 28, 1997 |
FR |
97 02420 |
Claims
1. Lipid compound of
formula:R--HN--[--(CH.sub.2).sub.0--NR--].sub.n-1--(C-
H.sub.2).sub.m--NH--R Iin which: the R residues are, independently
of each other, a hydrogen atom or a group of formula II: 7 for
which: R.sub.1 and R.sub.2 are, independently of each other,
C.sub.6-C.sub.23 alkyl or alkenyl radicals, which are linear or
branched, or radicals --C(.dbd.O)--(C.sub.6-C.sub.23) alkyl or
--C(.dbd.O)--(C.sub.6-C.sub.23) alkenyl, which are linear or
branched, aryl radicals, cycloalkyl radicals, fluoroalkyl radicals,
polyethylene glycol groups, oxyethylene or oxymethylene groups
which are optionally repeated, linear or branched, optionally
substituted, p is a positive integer from 1 to 4, n is a positive
integer from 1 to 6, m is a positive integer from 1 to 6 which may
be different for each motif --(CH.sub.2).sub.m, and more
particularly for each motif --(CH.sub.2).sub.m--NR-- when n>1,
the number of R groups of formula II being between 1 and 4.
2. Compound according to claim 1, chosen from the compounds of
formulae:H.sub.2N--[--(CH.sub.2).sub.m--NH--].sub.nR
IIIRNH--[--(CH.sub.2).sub.m--NH].sub.nH IIIain which: R is a group
of formula II as defined in claim 1, n is a positive integer from 1
to 6, m is a positive integer from 1 to 6 which may be different
for each motif --(CH.sub.2).sub.m.
3. Compound according to claim 1 of
formula:H.sub.2N--[--(CH.sub.2).sub.m--
-NR].sub.n-1--(CH.sub.2).sub.m--NH.sub.2 IIIbin which: R has one of
the meanings indicated for the formula I of claim 1 provided that
at least one R group is of formula II, n is a positive integer from
1 to 6, m is a positive integer from 1 to 6 which may be different
for each motif --(CH.sub.2).sub.m, and more particularly for each
motif --(CH.sub.2).sub.m--NR-- when n>1.
4. Compound according to claim 3, characterized in that it contains
one or two R groups of formula II.
5. Compound according to claims 1 to 4, characterized in that
R.sub.1 and R.sub.2 are, independently of each other, linear
--C(.dbd.O)-alkyl or linear --C(.dbd.O)-alkenyl radicals.
6. Compound according to claim 5, characterized in that said alkyl
or alkenyl comprises from 12 to 20 carbon atoms and in that said
compound comprises 1 or 2 R groups of formula II.
7. Compound according to claim 6, characterized in that said alkyl
or alkenyl comprises 12, 16 or 18 carbon atoms.
8. Compound according to claims 1 to 7, characterized in that n is
an integer chosen from the numbers 2, 3 or 4.
9. Compound according to claims 1 to 8, characterized in that m is
an integer chosen from the numbers 2, 3 or 4.
10. Compound according to claim 1, characterized in that it is
chosen from the group consisting of the compounds of the following
formulae:
4 IV 8 IVa 9
in which R.sub.1 and R.sub.2 are identical and are chosen from the
stearoyl and oleoyl radicals.
11. Compound according to one of claims 1 to 10, characterized in
that it is conjugated with one or more ligands of interest via one
of the secondary or primary nitrogen atoms of the polyamine chain
or of the diaminocarboxylic acid.
12. Compound according to claim 11, characterized in that said
targeting component is chosen from the group consisting of all or
part of sugars, peptides, oligonucleotides, lipids, hormones,
vitamins, antigens, antibodies, ligands specific for membrane
receptors, ligands capable of reacting with an anti-ligand,
fusogenic peptides, nuclear localization peptides, or a combination
of such compounds.
13. Compound according to one of claims 1 to 12, characterized in
that it is in a cationic form.
14. Composition characterized in that it comprises at least one
compound according to one of the preceding claims and optionally at
least one adjuvant capable in particular of enhancing the formation
of the complex between said compound and an active substance.
15. Composition according to claim 14, characterized in that said
adjuvant is a neutral or zwitterionic lipid.
16. Composition according to claim 15, characterized in that said
neutral or zwitterionic lipid is or is derived from a triglyceride,
a diglyceride, cholesterol, a phosphatidylethanolamine (PE),
phosphatidylcholine, phosphocholine, sphyngo-myelin, ceramide or
cerebroside.
17. Composition according to claim 16, characterized, in that said
neutral or zwitterionic lipid is dioleylphosphatidylethanolamine
(DOPE).
18. Composition according to claims 14 to 17, characterized in that
the compound/adjuvant weight ratio is between 0.1 and 10.
19. Complex comprising at least one compound according to claim 13
or at least one composition according to one of claims 14 to 18 and
at least one active substance, in particular a therapeutically
active substance, comprising at least one negative charge.
20. Complex according to claim 19, characterized in that said
active substance is chosen from nucleic acids and proteins.
21. Complex according to claim 20, characterized in that said
active substance is a nucleic acid chosen from the group consisting
of a cDNA, a genomic DNA, a plasmid DNA, an antisense
polynucleotide, a messenger RNA, a ribosomal RNA, a ribozyme, a
transfer RNA, or a DNA encoding such RNAs.
22. Complex according to claim 21, characterized in that said
nucleic acid comprises a gene of interest and components allowing
the expression of said gene of interest.
23. Complex according to one of claims 19 to 22, characterized in
that it has a size of less than 500 nm, advantageously less than
200 nm.
24. Complex according to one of claims 23, characterized in that it
has a size of less than 100 nm.
25. Complex according to one of claims 19 to 24, characterized in
that the ratio between the number of positive charges of the
cationic compound(s) and/or composition(s) and the number of
negative charges of said active substance varies from 0.05 to 20,
more particularly from 0.1 to 15, and preferably from 5 to 10.
26. Process for preparing a complex according to claims 19 to 25,
characterized in that one or more compounds according to claim 13
and/or at least one composition according to one of claims 14 to 18
are brought into contact with one or more active substances
comprising at least one negative charge and in that said complex is
recovered, optionally after a purification step.
27. Process of preparation according to claim 26, characterized in
that said compounds and/or compositions are dissolved beforehand in
a solvent which is miscible with water, in particular ethanol or
dimethyl sulfoxide or a mixture of both.
28. Process of preparation according to claim 26, characterized in
that said compounds and/or compositions are suspended beforehand in
a detergent solution.
29. Process of preparation according to claim 28, characterized in
that, in addition, a step of purification of said complex by
dialysis is carried out.
30. Use of a compound according to any one of claims 1 to 13, of a
composition according to any one of claims 14 to 18, of a complex
according to any one of claims 19 to 25 to transfer at least one
active substance, especially a therapeutically active substance, in
particular a nucleic acid, into target cells in vitro, ex vivo or
in vivo, more particularly in vivo.
31. Use according to claim 30, characterized in that said target
cell is a mammalian cell.
32. Use according to claim 31, characterized in that said target
cell is selected from a muscle cell, a hematopoietic stem cell, a
cell of the airways, more particularly a tracheal or pulmonary
cell.
33. Process for transferring an active substance comprising at
least one negative charge, into a cell, characterized in that said
cell is brought into contact with a complex according to any one of
claims 19 to 25.
34. Complex according to one of claims 19 to 25, as a medicament
for curative, preventive or vaccinal purposes.
35. Complex according to one of claims 19 to 25 for carrying out a
method of therapeutic treatment which consists in transferring at
least one therapeutically active substance, in particular a nucleic
acid, into target cells.
36. Complex according to claim 35, characterized in that said
target cell is a mammalian cell.
37. Complex according to claim 36, characterized in that said
target cell is selected from a muscle cell, a hematopoietic stem
cell, a cell of the airways, more particularly a tracheal or
pulmonary cell, a cell of the respiratory epithelium.
38. Use of a compound according to any one of claims 1 to 13, of a
composition according to any one of claims 14 to 18, of a complex
according to any one of claims 19 to 25 for the preparation of a
medicament for curative, preventive or vaccinal purposes, intended
for the treatment of the human or animal body, in particular by
gene therapy.
39. Use according to claim 38, characterized in that the medicament
is intended to be administered by intramuscular injection, by
inhalation, by intratracheal injection, by instillation, by
aerosolization, by the topical route or by the oral route.
40. Pharmaceutical preparation characterized in that it comprises
at least one complex according to any one of claims 19 to 25.
41. Preparation according to claim 40, characterized in that it
comprises, in addition, at least one adjuvant capable of enhancing
the transfecting power of said complex.
42. Preparation according to claim 41, characterized in that said
adjuvant is chosen from the group consisting of chloroquine, a
protic polar compound chosen in particular from propylene glycol,
polyethylene glycol, glycerol, ethanol, 1-methyl-L-2-pyrrolidone or
derivatives thereof, or an aprotic polar compound chosen in
particular from dimethyl sulfoxide (DMSO), diethyl sulfoxide,
di-n-propyl sulfoxide, dimethyl sulfone, sulfolane,
dimethylformamide, dimethylacetamide, tetramethylurea, acetonitrile
or derivatives thereof.
43. Preparation according to one of claims 40 to 42, characterized
in that it comprises, in addition, a pharmaceutically acceptable
carrier allowing its administration to humans or animals.
44. Cell transfected with a complex according to any one of claims
19 to 25.
Description
[0001] The present invention relates to new lipid compounds and new
compositions containing them. More particularly, the present
invention relates to the use of said compounds or of said
compositions to prepare a vector for transferring an active
substance, in particular a therapeutically active substance
comprising negative charges, in particular a polynucleotide, into a
target cell, particularly a vertebrate cell, and more particularly
a mammalian cell.
[0002] The transfer of a gene into a given cell is the very basis
of gene therapy. This new technology, whose field of application is
vast, makes it possible to envisage the treatment of serious
diseases for which the conventional therapeutic alternatives are
not very effective, or are even inexistent, and applies to diseases
which are either of genetic origin (hemophilia, cystic fibrosis,
myopathy and the like) or acquired (cancer, AIDS and the like).
[0003] During the past 30 years, numerous tools have been developed
which allow the introduction of various heterologous genes into
cells, in particular mammalian cells. These different techniques
may be divided into two categories. The first category relates to
physical techniques such as microinjection, electroporation or
particle bombardment which, although effective, are greatly limited
to applications in vitro and whose implementation is cumbersome and
delicate. The second category involves techniques relating to
molecular and cell biology in which the gene to be transferred is
combined with a vector of a biological or synthetic nature which
promotes the introduction of said material.
[0004] Currently, the most effective vectors are viral, in
particular adenoviral or retroviral, vectors. The techniques
developed are based on the natural properties which these viruses
have to cross the cell membranes, to escape degradation of their
genetic material and to cause their genome to penetrate into the
nucleus. These viruses have already been the subject of numerous
studies and some of them are already used experimentally as vectors
for genes in humans for the purpose, for example, of a vaccination,
an immunotherapy or a therapy intended to make up for a genetic
deficiency. However, this viral approach has many limitations, in
particular because of the limited capacity for cloning into the
viral genome, the risks of spreading in the host organism and in
the environment the infectious viral particles produced, the risk
of artefactual mutagenesis by insertion into the host cell in the
case of retroviral vectors, and the high induction of immune and
inflammatory responses in vivo during the therapeutic treatment,
considerably limiting the number of administrations which can be
envisaged (McCoy et al., 1995, Human Gene Therapy, 6, 1553-1560;
Yang et al., 1996, Immunity, 1, 433-442)1 These numerous
disadvantages, in particular in the context of a use in humans,
have led several teams to develop alternative systems of
transferring poly-nucleotides.
[0005] Several nonviral methods are currently available. By way of
example, there may be mentioned coprecipitation with calcium
phosphate, the use of receptors mimicking viral systems (for a
review see Cotten and Wagner, 1993, Current Opinion in
Bio-technology, 4, 705-710), or the use of polymers such as
polyamidoamine (Haensler and Szoka, 1993, Bioconjugate Chem., 4,
372-379) or of polymer such as those presented in WO 95/24221
describing the use of dendritic polymers, the document WO 96/02655
describing the use of polyethyleneimine, or of polypropyleneimine
and the documents U.S. Pat. No. 5,595,897 and FR 2,719,316
describing the use of conjugates of polylysine. Other non-viral
techniques are based on the use of liposomes whose value as agent
allowing the introduction, into cells, of certain biological
macromolecules, such as for example DNA, RNA, proteins or certain
pharmaceutically active substances, has been widely described in
the literature. To this end, several teams have already proposed
the use of cationic lipids which have a high affinity for the cell
membranes and/or the nucleic acids. Indeed, although it has been
shown, in the case of nucleic acids, that this type of
macromolecule is capable of crossing the plasma membrane of some
cells in vivo (WO 90/11092), it is nevertheless the case that the
observed transfection efficiency is still highly limited, because
of in particular the polyanionic nature of the nucleic acids which
prevent their passage across the cell membrane, which itself has a
negative net apparent charge. Since 1989 (Felgner et al., Nature,
337, 387-388), cationic lipids have been presented as molecules
which are advantageous for promoting the introduction of large
anionic molecules, such as nucleic acids, into certain cells. These
cationic lipids are capable of complexing anionic molecules, thus
tending to neutralize the negative charges on said molecules and to
promote their coming close to the cells. Many teams have already
developed various cationic lipids. By way of example, there may be
mentioned DOTMA (Felgner et al., 1987, PNAS, 84, 7413-7417), DOGS
or Transfectam.TM. (Behr et al., 1989, PNAS, 86, 6982-6986), DMRIE
and DORIE (Felgner et al., 1993, Methods 5, 67-75), DC-CHOL (Gao
and Huang, 1991, BBRC, 179, 280-285), DOTAP.quadrature. (McLachlan
et al., 1995, Gene Therapy, 2,674-622) or
Lipofectamine.quadrature., as well as those described in Patent
Applications WO9116024 or WO9514651
[0006] More particularly, Patent Application WO-A-9116024 describes
cationic lipids of formula: 2
[0007] in which:
[0008] R.sub.1 and R.sub.2 are in particular alkyl or alkenyl
radicals;
[0009] Y.sub.1 and Y.sub.2 are radicals --OCH.sub.2--,
--OC(.dbd.O)-- or --O--;
[0010] R.sub.3 and R.sub.4 are alkyl or alkenyl radicals;
[0011] R.sub.5 is an alkylene chain;
[0012] R.sub.6 is C(.dbd.O)--(CH.sub.2).sub.m--NH--, a
diaminocarboxylic acid or --C(.dbd.O)--(CH.sub.2).sub.m--NH-- bound
to said diaminocarboxylic acid;
[0013] R.sub.7 is H, spermine, spermidine, histone, a protein, an
amino acid or a polypeptide.
[0014] However, several studies (by way of examples, see Mahato et
al., J. Pharm. Sci., 1995, 84, 1267-1271, Thierry et al., 1995,
P.N.A.S., 92, 9742-9746) have demonstrated that the efficiency of
transferring the anionic macromolecule into cells could vary
depending in particular on the interaction between the complexes
and the cell membranes, the cell considered, the lipid composition
of the cationic compounds, the size of the complexes formed with
the anionic molecules and more particularly the ratio between the
positive and negative charges on the different components of said
complex. The mechanisms which allow in particular the interaction
of the complexes with the cell membranes and the transfer of the
complexes into the cell are still to a large extent poorly
understood and researchers proceed in their studies based on a
highly empirical approach. Other factors such as, for example, the
formation of the complexes, the stability, the behavior in vivo, or
possibly their toxicity make, in addition, the choice of the lipids
to priori non-obvious. It is consequently desirable to provide
other cationic lipids possibly having improved properties or
properties which are different from the cationic lipids already
described.
[0015] The Applicant has now identified new lipid compounds, which
can be provided in cationic form, useful in particular for
transferring an active substance, in particular a therapeutically
active substance, comprising negative charges, in particular a
polynucleotide, into a target cell, whose use may be envisaged in
particular in vivo in the context of a gene therapy.
[0016] Accordingly, the subject of the present invention is first
of all a lipid compound of formula:
R--HN--[--(CH.sub.2).sub.m--NR--].sub.n-1--(CH.sub.2).sub.m--NH--R
I
[0017] in which:
[0018] the R residues are, independently of each other, a hydrogen
atom or a group of formula II: 3
[0019] in which:
[0020] R.sub.1 and R.sub.2 are, independently of each other,
C.sub.6-C.sub.23 alkyl or alkenyl radicals, which are linear or
branched, or radicals --C(.dbd.O)--(C.sub.6-C.sub.23) alkyl or
--C(.dbd.O)--(C.sub.6-C.sub.23) alkenyl, which are linear or
branched, aryl radicals, cycloalkyl radicals, fluoroalkyl radicals,
polyethylene glycol groups, oxyethylene or oxymethylene groups
which are optionally repeated, linear or branched, optionally
substituted,
[0021] is a positive integer from 1 to 4,
[0022] n is a positive integer from 1 to 6,
[0023] m is a positive integer from 1 to 6 which may
[0024] be different for each motif --(CH.sub.2).sub.m, and more
[0025] particularly for each motif --(CH.sub.2).sub.m--NR-- when
n>1,
[0026] the number of R groups of formula II being between 1 and
4.
[0027] The expression "alkenyl" is understood to mean that the
carbon chain may comprise one or more double bond(s) along said
chain.
[0028] According to a specific case, the invention relates to a
compound chosen from the compounds of formulae:
H.sub.2N--[--(CH.sub.2).sub.m--NH--].sub.nR III
RNH--[--(CH.sub.2).sub.m--NH].sub.nH IIIa
[0029] in which:
[0030] R is a group of formula II as defined above and
H.sub.2N--[--(CH.sub.2).sub.m--NR].sub.n-1--(CH.sub.2).sub.m--NH.sub.2
IIIb
[0031] in which:
[0032] R has one of the meanings indicated for the formula I
provided that at least one R is a group of formula II and for each
of the formulae:
[0033] n is a positive integer from 1 to 6,
[0034] m is a positive integer from 1 to 6 which may be different
for each motif --(CH.sub.2).sub.m, and more particularly for each
motif --(CH.sub.2).sub.m--NR-- when n>1.
[0035] According to a preferred case, the compounds of formula IIIb
contain one or two R groups of formula II.
[0036] According to preferred embodiments of the invention, the
variations presented below, taken in combination with each other or
otherwise, will be chosen:
[0037] R.sub.1 and R.sub.2 are, independently of each other, linear
--C(.dbd.O)-alkyl or linear --C(.dbd.O)-alkenyl radicals,
[0038] R.sub.1 and R.sub.2 are, independently o each other,
--C(.dbd.O)-alkyl or --C(.dbd.O)-alkenyl radicals comprising from
12 to 20 carbon atoms, preferably 12, 16 or 18 carbon atoms, when
the lipid comprises 1 or 2 R groups of formula II,
[0039] n is an integer chosen from the numbers 2, 3 or 4,
[0040] m is an integer chosen from the numbers 2, 3 or 4.
[0041] According to a specific case, it is possible to reduce the
length of the alkyl or alkenyl chain so that R.sub.1 and R.sub.2
are radicals having 6 to 10 carbon atoms, but in this case
compounds for which the number of R groups of formula II is equal
to 2, 3 or 4 will be preferably chosen.
[0042] Preferably, the lipids according to the invention are chosen
from the group consisting of the compounds of the following
formulae:
1 IV 4 IVa 5
[0043] for which R.sub.1 and R.sub.2 are identical and are chosen
from the stearoyl and oleoyl radicals.
[0044] The compounds according to the invention are prepared by
reacting a compound or formula: 6
[0045] in which:
[0046] R.sub.3 and R.sub.4 are protecting groups, in particular
Fmoc (Grandas et al., 1989, Int. Journal pept. prot. Res. vol 33,
386-390), with an amine of formula:
R.sub.5NH[(--CH.sub.2).sub.m--NR.sub.5].sub.n-1--(CH.sub.2).sub.mNHR.sub.5
VI
[0047] m and n having the same meaning as for the formula I,
[0048] R.sub.5 being a protecting group, in particular
t-butoxycarbonyl (BOC) or a hydrogen atom, at least one of the
R.sub.5 radicals and at most four of the R.sub.5 radicals
corresponding to the hydrogen atom.
[0049] The functional groups N--R.sub.3 and --N--R.sub.4 are then
deprotected so as to bind, by amidation or alkylation, the radicals
R.sub.1 and R.sub.2 in a known manner, in particular by the action
of the corresponding --N-hydroxy-succinimide ester.
[0050] The compound obtained is deprotected in the presence of
trifluoroacetic acid.
[0051] The amines of formula VI are prepared in a known manner.
[0052] In the case where the compound of formula VI is
1-4-di-boc-spermidine, reference will be made to the examples
indicated below in order to know the practical modalities for the
synthesis. The processes described are applicable in general to the
syntheses of the compounds according to the invention subject to
adaptations within the capability of persons skilled in the
art.
[0053] However, the compounds of the invention cannot be limited to
those obtained by the modes of preparation described above.
[0054] The compounds according to the invention may, in addition,
be substituted. Such substitutions may in particular consist-of a
labeling molecule (see labeling molecules in U.S. Pat. No.
4,711,955) which makes it possible, for example, to visualize the
distribution of the compounds or of the complexes containing them
after administration in vitro or in vivo, a cell targeting molecule
or an anchoring molecule. The invention consequently also relates
to a compound as presented above, conjugated with one or more
targeting components, also called ligands of interest, via the
intermediacy of at least a) one of the carbon atoms, in particular
chosen from those present on the groups R.sub.1 and/or R.sub.2, or
b) one of the secondary or primary nitrogen atoms of the polyamine
chain or of the diaminocarboxylic acid. Such components may allow
targeting to a specific cell type, facilitate penetration into the
cell, lysis of the endosomes or alternatively intracellular
transport and are widely described in the literature. They may be,
for example, all or part of sugars, peptides (GRP peptide, Gastrin
Releasing Peptide, for example), oligonucleotides, lipids,
hormones, vitamins, antigens, antibodies, ligands specific for
membrane receptors, ligands capable of reacting with an
anti-ligand, fusogenic peptides, nuclear localization peptides, or
a combination of such compounds. There may be mentioned more
particularly the galactosyl residues which make it possible to
target the asyaloglycoprotein receptor at the surface of hepatic
cells, the fusogenic peptide INF-7 derived from the influenza virus
hemagglutinin subunit HA-2 (Plank et al., 1994, J. Biol. Chem. 269,
12918-12924) or a nuclear localization signal derived from the SV40
virus T antigen (Lanford and Butel, 1984, Cell 37, 801-813) or the
Epstein Barr virus EBNA-1 protein (Ambinder et al., 1991, J. Virol.
65, 1466-1478).
[0055] Such conjugates can be easily obtained by techniques widely
described in the literature, and more particularly by chemical
coupling, in particular using protecting groups such as
trifluoroacetyl or Fmoc or Boc; onto the polyamine and more
particularly using one or more orthogonal protecting groups such as
those described in Protective Groups in Organic Synthesis (p.
309-406, 1991, eds. T. W. Greene, P. G. M. Wuts, Wiley) onto the
polyamine or the diaminocarboxylic acid. The selective deprotection
of a protecting group then makes it possible to couple the
targeting component, and the lipid is then deprotected. It should
be stated, however, that the substitution of the nonreactive groups
such as the carbon atoms in the CH or CH2 groups will be carried
out during synthesis of the compounds of the invention by methods
known to a person skilled in the art, whereas the reactive groups,
such as the primary or secondary amines, may be the subject of
substitutions on the neosynthesized lipids of the invention.
[0056] According to an advantageous case of the invention, said
compound is in a cationic form, that is to say that it is in a form
which is protonated by binding of a proton onto one or more
nitrogen atoms present on the polyamine chain. In this case, said
cationic lipid is combined with one or more biologically acceptable
anions, such as for example the trifluoroacetate, halide,
monomethylsulfate, acetate or phosphate, iodide, chloride, or
bromide anion and the like. It is also possible to obtain compounds
in cationic form by substitution of the amines, for example, with a
methyl or ethyl radical, and the like.
[0057] According to another aspect, the invention also relates to a
composition comprising at least one compound as described above and
optionally at least one adjuvant capable of enhancing the formation
of the complex between a said compound and an active substance, or
of enhancing the function of these complexes toward the cell.
[0058] Preferably, such an adjuvant will be a neutral or
zwitterionic lipid, such as for example a lipid which is or is
derived from a triglyceride, a diglyceride, cholesterol (see for
example U.S. Pat. No. 5,438,044), in particular, a neutral or
zwitterionic lipid which is or is derived from a
phosphatidylethanolamine (PE), phosphatidylcholine, phosphocholine,
sphyngomyelin, ceramide or cerebroside. Advantageously,
dioleoylphosphatidylethanolamine (DOPE) will be chosen.
[0059] The weight ratio between the compound of the invention and
the neutral or zwitterionic lipid is generally between 0.1 and 10,
it being understood that this ratio may vary depending on the
nature of the components considered. Persons skilled in the art
have sufficient knowledge to allow these minor adaptations. It is
also possible to use a mixture of neutral and/or zwitterionic
lipids or alternatively a mixture of cationic lipids and neutral
and/or zwitterionic lipids.
[0060] The invention relates, in addition, to a complex comprising
at least one compound or at least one composition as described
above and at least one active substance, in particular a
therapeutically active substance, comprising at least one negative
charge. According to a variant of the invention, said complex may,
in addition, contain one or more cationic amphiphilic agents such
as those described in the literature of which examples were
provided above.
[0061] According to a specific embodiment, said active substance is
chosen from nucleic acids and proteins. Preferably, the active
substance of the complex according to the invention is a
polynucleotide, said compound or said composition then making it
possible to enhance the transfecting power of the polynucleotide in
a cell.
[0062] "Polynucleotide" is understood to designate a DNA and/or RNA
fragment which is double-stranded or single-stranded, linear or
circular, natural, isolated or synthetic, designating a precise
succession of nucleotides, which are modified or otherwise (see by
way of example U.S. Pat. No. 5,525,711), labeled or otherwise (see
for example U.S. Pat. No. 4,711,955 or EP 302175), making it
possible to define a fragment or a region of a nucleic acid without
size limitation. Polynucleotide is understood to designate in
particular a cDNA, a genomic DNA, a plasmid DNA, a messenger RNA,
an antisense RNA, a ribozyme, a transfer RNA, a ribosomal RNA or a
DNA encoding such RNAs. "Polynucleotide" or "nucleic acid" are
synonymous terms in the context of the present application.
"Antisense" is understood to designate a nucleic acid having a
sequence complementary to a target sequence, for example an mRNA
sequence for which it is sought to block the expression by
hybridization with the target sequence; "sense" is understood to
designate a nucleic acid having a sequence homologous or identical
to a target sequence, for example a sequence which binds to a
proteinaceous transcription factor and which is involved in the
expression of a given gene.
[0063] According to a specific embodiment of the invention, said
polynucleotide comprises a gene of interest and components allowing
the expression of said gene of interest. In this embodiment, said
poly-nucleotide is advantageously in the form of a plasmid. The
components allowing expression are all the components allowing the
transcription of said DNA fragment into RNA (antisense RNA or mRNA)
and the translation of the mRNA into a polypeptide. They are in
particular promoter sequences and/or regulatory sequences which are
effective in said cell, and optionally the sequences required to
allow excretion or expression of said polypeptide at the surface of
the target cells. By way of example, there may be mentioned
promoters such as the promoters of the viruses RSV, MPSV, SV40, CMV
or 7.5 k, of the vaccinia virus, the promoters of the gene encoding
muscle creatine kinase, actin, or pulmonary surfactant. It is, in
addition, possible to choose a promoter sequence specific for a
given cell type or which can be activated under defined conditions.
The literature provides a large amount of information relating to
such promoter sequences. Moreover, said polynucleotide may comprise
at least two sequences, which are identical or different,
exhibiting a transcriptional promoter activity and/or at least two
coding DNA sequences, which are identical or different, situated,
relative to each other, contiguously, far apart, in the same
direction or in the opposite direction, as long as the
transcriptional promoter function or the transcription of said
sequences is not affected. Likewise, it is possible to introduce
into this type of nucleic acid construct "neutral" nucleic
sequences or introns which do not affect transcription and are
spliced before the translation step. Such sequences and their uses
are described in the literature. Said polynucleotide may also
contain sequences required for intracellular transport, for
replication and/or for integration. Such sequences are well known
to persons skilled in the art. Moreover, the polynucleotides
according to the present invention may also be polynucleotides
which are modified such that it is not possible for them to become
integrated into the genome of the target cell or polynucleotides
which are stabilized with the aid of agents such as, for example,
spermine.
[0064] In the context of the present invention, the polynucleotide
may be homologous or heterologous to the target cell. It may be
advantageous to use a polynucleotide which encodes all or part of a
polypeptide, in particular a polypeptide having a therapeutic or
prophylactic activity, and more particularly an immunogenic
activity of the cellular or humoral type. The term polypeptide is
understood without restriction as to its size or its degree of
modification (for example glycosylation). There may be mentioned,
by way of examples, the genes encoding an enzyme, a hormone, a
cytokine, a membrane receptor, a structural polypeptide, a
polypeptide forming a membrane channel, a transport polypeptide, an
adhesion molecule, a ligand, a factor for regulation of
transcription, of translation, of replication, or of the
stabilization of the transcripts, or an antibody, such as for
example the gene encoding the CFTR protein, dystrophin, factor VIII
or IX, E6/E7 of HPV, MUC1, BRAC1, .beta.-interferon,
.gamma.-interferon, interleukin (IL)2, IL-4, IL-6, IL-7, IL-12,
tumor necrosis factor (TNF) type alpha, GM-CSF (Granulocyte
Macrophage Colony Stimulating Factor), the Herpes Simplex virus
type 1 (HSV-1) tk gene, the gene associated with retinoblastoma or
p53 or all or part of immunoglobulins, such as the fragments
F(ab).sub.2, Fab', Fab or the anti-idiotypes (U.S. Pat. No.
4,699,880). This list is of course not limiting and other genes may
be used.
[0065] According to a preferred embodiment, the complexes according
to the invention are small in size (less than 500 nm,
advantageously less than 200 nm and preferably less than 100
nm).
[0066] Moreover, the transfection experiments carried out show that
advantageously the weight ratio of the lipid compound according to
the invention to said polynucleotide is 0.01 to 100. The optimum
ratio is between 0.05 and 10.
[0067] The invention also relates to a process for preparing the
complexes cationic compounds/anionic active substances, said
process being characterized in that one or more lipids in cationic
form or a composition according to the invention whose lipid is in
cationic form are brought into contact with one or more active
substances comprising at least one negative charge and in that said
complex is recovered, optionally after a purification step. It also
relates to the kits for preparing such complexes comprising one or
more lipids or one or more compositions according to the
invention.
[0068] In a first instance, according to a first variant, one or
more cationic compounds are dissolved with an appropriate quantity
of solvent or mixture of solvents which are miscible in water, in
particular ethanol, dimethylsulfoxide (DMSO), or preferably a 1:1
(v:v) ethanol/DMSO mixture, so as to form lipid aggregates
according to a known method described, for example, in Patent
Application WO-A-9603977, or according to a second variant, are
suspended with an appropriate quantity of a solution of detergent
such as an octylglucoside such as n-octyl-.beta.-D-glucopyranoside,
or 6-O-(N-heptylcarbomoyl)-methyl-.alph- a.-D-glucopyranoside.
[0069] The suspension may then be placed in a buffer medium and
mixed with a solution of active substance comprising negative
charges.
[0070] In the case where it is desirable that a neutral or
zwitterionic lipid is present in the final complex, a film is
formed, in the known manner, prior to the dissolution in the
solvent which is miscible with water or in the solution of
detergent, with a mixture containing a said cationic compound and a
said neutral or zwitterionic lipid, such as for example DOPE.
[0071] One of the important characteristics of the process consists
in the choice of the ratio between the positive charges of the
cationic lipid and the negative charges of the active
substance.
[0072] Without wishing to be limited by a specific ratio,
quantities of the different charges will be chosen so that the
ratio between the number of positive charges of the compound or of
the cationic composition and the number of negative charges of the
active substance is between 0.05 and 20, in particular between 0.1
and 15, and preferably between 5 and 10.
[0073] This ratio between the number of positive charges of the
cationic compound(s) and/or composition(s) and the number of
negative charges of said active substance also constitutes an
advantageous characteristic of the complex according to the
invention.
[0074] The calculation to arrive at such a ratio will take into
consideration the negative charges carried by the active substance
and the quantity of compound necessary to satisfy the ratio
indicated above will be adjusted. The quantities and the
concentrations for the other components are adjusted according to
their respective molar masses and the number of their positive
and/or negative charges.
[0075] In the case of the second variant and optionally, subsequent
dialysis may be carried out in order to reduce the detergent and to
recover the complexes. The principle of such a method is for
example described by Hofland et al. (1996, PNAS 93, p 7305-7309)
and in chapter II of the Philippot et al. document (G. Gregoriadis,
81-89, CRC Press 1993).
[0076] It has been shown that the first variant leads to excellent
results in terms of the size of the complexes obtained.
[0077] According to a third variant, one or more cationic
compositions or compounds are suspended in a buffer and then the
suspension is subjected to sonication until visual homogeneity is
obtained. The lipid suspension is then extruded through two
microporous membranes under appropriate pressure. The lipid
suspension is then mixed with a solution of active substance
comprising negative charges. In the case where a neutral lipid is
present in the complex, a film of the mixture of cationic lipid and
neutral lipid such as DOPE is formed in a known manner prior to the
preparation as a suspension. The same remarks relating to the ratio
between the positive charges of the cationic lipid and the negative
charges of the active substance as those indicated in the first
variant are applicable to this third variant. This so-called
sonication-extrusion technique is well known in the art.
[0078] The characteristics of the complexes formed may be evaluated
by several means which make it possible to determine, for
example:
[0079] the state of complex formation with the active substance, in
particular by identification of the free nucleic acids by agarose
gel electrophoresis in the case where the substances are nucleic
acids,
[0080] the size of the particles by a quasi-elastic scattering of
light,
[0081] the absence of precipitation over the long term.
[0082] The object of the present invention is also the complexes
obtained using the processes listed above.
[0083] The invention also relates to the use of a compound, of a
composition or of a complex according to the invention to transfer
at least one active substance, especially a therapeutically active
substance, more particularly a nucleic acid, into target cells, in
vitro, ex vivo or in vivo, more particularly in vivo.
[0084] "Target cells" according to the invention is understood to
mean prokaryotic cells, yeast cells and eukaryotic cells, plant
cells, human or animal cells, and in particular mammalian cells.
Cancer cells should, moreover, be mentioned. In vivo, the invention
may be applied at the level of the interstitial or luminal space of
tissues such as the lungs, trachea, skin, muscle, brain, liver,
heart, spleen, bone marrow, thymus, bladder, lymph, blood,
pancreas, stomach, kidney, ovaries, testicles, rectum, peripheral
or central nervous system, eyes, lymphoid organs, cartilages and
endothelium. According to an advantageous choice of the invention,
the target cell will be a muscle cell, a hematopoietic stem cell or
alternatively a cell of the airways, more particularly a tracheal
or pulmonary cell, and advantageously a cell of the respiratory
epithelium.
[0085] The complexes according to the invention can be used as a
medicament for curative, preventive or vaccinal purposes.
Accordingly, the subject of the invention is also the complexes of
the invention as a medicament for curative, preventive or vaccinal
purposes. Such complexes may be used in a method of therapeutic
treatment which consists in transferring at least one
therapeutically active substance, in particular a polynucleotide,
into target cells, in particular a mammalian cell, and more
precisely a muscle cell, a hematopoietic stem cell, a cell of the
airways, more particularly a tracheal or pulmonary cell, a cell of
the respiratory epithelium.
[0086] A compound according to the invention is most particularly
advantageous for transferring a nucleic acid into a muscle cell or
a pulmonary cell. It is the compound noted pcTG37 (see
example).
[0087] More widely, the present invention also relates to a process
for introducing an active substance comprising negative charges
into a cell in particular in vitro, characterized in that cells, in
particular cultured on an appropriate medium are brought into
contact with a complex cationic compound/active substance
comprising at least one negative charge according to the invention,
in particular in the form of a suspension of complexes. After a
certain incubation time, the cells are washed and recovered. The
introduction of the active substance may be checked (optionally
after lysis of the cell) by any appropriate means.
[0088] The process of introduction is well known per se. The term
"introduction" is understood to mean that the active substance
comprising negative charges is transferred into the cell and is
located, at the end of the process, inside said cell or at the
level of the membrane thereof. In the case where the active
substance is a nucleic acid, reference will be made more
particularly to "transfection". in this case, the verification of
the transfection of the nucleic acid can be carried out by any
appropriate means, for example by measuring the expression of the
gene considered or the concentration of the expressed protein.
[0089] The invention relates more particularly to the use of a
compound, of a composition or of a complex according to the
invention for the preparation of a medicament for curative,
preventive or vaccinal purposes, intended for the treatment of the
human or animal body, in particular by gene therapy.
[0090] According to a first possibility, the medicament may be
administered directly in vivo (for example into a muscle, into the
lungs by aerosol and the like) . It is also possible to adopt the
ex vivo approach which consists in collecting cells from the
patient (bone marrow stem cells, peripheral blood lymphocytes,
muscle cells and the like), transfecting them in vitro according to
the present invention and readministering them to the patient.
[0091] The complexes according to the invention may be administered
by the intramuscular, intratracheal, intranasal, intracerebral,
intrapleural, intratumoral, intracardiac, intragastric,
intraperitoneal, epidermal, intravenous or intraarterial route by a
syringe or by any other equivalent means, systems suitable for the
treatment of the airways or of the mucous membranes such as
inhalation, instillation or aerosolization. There may also be
mentioned the modes of administration by application of a cream, by
oral administration or any other means perfectly known to the
person skilled in the art and applicable to the present
invention.
[0092] It is also within the scope of the invention to target
specific organs or tissues by administration, in particular by the
intravenous route, of a complex according to the invention prepared
so as to adjust the ratio compound or composition/therapeutically
active substance in said complex, the apparent charge of the
complex (see in particular Liu et al., 1997, Gene Therapy, 4,
517-523; Thierry et al., 1995, P.N.A.S., 92, 9742-9746).
[0093] The invention also relates to a method of gene therapy
consisting in administering to a patient an appropriate quantity of
a composition according to the invention. According to the present
invention and in the context of gene therapy in vivo, it is
possible to repeat several times, in a given patient, the method as
proposed without any major immune reaction being elicited against
one of the compounds administered. The administration may take
place in a single dose or repeated once or several times after a
certain time interval. The repeated administration would make it
possible to reduce the quantity of therapeutically active
substance, more particularly of DNA, to be administered for a given
dose. The appropriate route of administration and dosage vary
according to various parameters, for example the individual or
disease to be treated or alternatively the polynucleotide to be
transferred.
[0094] The invention relates more particularly to a pharmaceutical
preparation comprising at least one complex as described above,
optionally containing, in addition, at least one adjuvant capable
of stabilizing said pharmaceutical preparation for the purpose of
its storage for example and/or of enhancing the transfecting power
of said complex. Such an adjuvant could, for example, be chosen
from the group consisting of chloroquine, a protic polar compound
chosen in particular from propylene glycol, polyethylene glycol,
glycerol, ethanol, 1-methyl-L-2-pyrrolidone or derivatives thereof,
or an aprotic polar compound chosen in particular from dimethyl
sulfoxide (DMSO), diethyl sulfoxide, di-n-propyl sulfoxide,
dimethyl sulfone, sulfolane, dimethylformamide, dimethylacetamide,
tetramethylurea, acetonitrile or derivatives thereof. Likewise,
said preparation may contain a pharmaceutically acceptable carrier
allowing its administration to humans or animals.
[0095] In the context of the use of a method of treatment in vivo
according to the present invention, it is, in addition, possible to
carry out, before the administration of a pharmaceutical
preparation as described above, a treatment of the patient designed
to observe a temporary depletion of the macrophages making it
possible to enhance the transfection rate. Such a technique is
described in the literature; see in particular Van Rooijen et al.,
1997, TibTech, 15, 178-184.
[0096] Finally, the invention relates to a cell transfected with a
complex as defined above, particularly a prokaryotic cell, a yeast
cell or eukaryotic cell, especially an animal cell, in particular a
mammalian cell, and more particularly a cancer cell. According to a
preferred case of the invention, said cell is a cell of the
airways, more particularly a tracheal or pulmonary cell, and
advantageously a cell of the respiratory epithelium.
[0097] The examples below illustrate the invention without limiting
it in any manner. An explanatory diagram of synthesis which forms
an integral part of the description is appended (FIG. 1). FIG. 2
indicates the results observed after intravenous injection of
complexes according to the invention, the luciferase activity is
indicated in RLU/mg of protein).
EXAMPLES
[0098] The preparation of the cationic lipid of formula I in which
R.sub.1 and R.sub.2, which are identical, are oleoyl or stearoyl
radicals, n=2; m.alpha.=3, m.beta.=4 is described below with
reference to the diagram of synthesis represented in FIG. 1.
.alpha. and .beta. are the positions of the
--(CH.sub.2).sub.m--NH-- motifs from the carbonyl.
Synthesis of the Cationic Lipid of Formula I in which n=2,
m.alpha.=3, m.beta.=4, R=oleoyl (pcTG37) (see FIG. 1)
Synthesis of the Intermediate 1-4-di-boc-spermidine
[0099] The protocol applied in order to obtain
1,4-di-boc-spermidine is published by Goodnow et al. (1990,
Tetrahedron 46, 3267-3286). In practice, the intermediate
N-propionitrile-1,4-diaminobutane is obtained by reacting 37.6 mmol
of diaminobutane (Fluka; reference 32790) diluted in 3.8 ml of
dichloromethane +2 ml of methanol at 0.degree. C. with 18.8 mmol of
acrylonitrile (Fluka; reference 01710) for 1 hour at 20.degree. C.
The amino groups of the product of the reaction are protected with
99 mmol of BOC-ON [(2-boc-oxyimino)-2-phenylacetonitrile] (Fluka;
reference 15475) in 50 ml of 2/1 CH.sub.2Cl.sub.2/CH.sub.3OH
solvent. The protecting reaction is left overnight at room
temperature. The solvents are evaporated off and the product is
taken up in 75 ml of ethyl acetate for 3 extractions with 75 ml of
1 M NaOH. The organic phase is again extracted with 5% citric acid
and then dried and filtered over Na.sub.2SO.sub.4 before
evaporation to dryness. The step of reducing the nitriles is then
carried out in ether at 0.degree. C. in the presence of 20 mmol of
LiAlH4 (Sigma; L0260). After 1 hour 30 min, the reaction is stopped
by addition of 20 ml of 1 M NaOH at 0.degree. C., with evolution of
hydrogen. The mixture is filtered and extracted 3 times with a 20%
NaCl solution in water. After drying the ethereal fraction, the
product is deposited on a silica column in a 2/1
CH.sub.2Cl.sub.2/CH.sub.3OH solvent and then eluted in a 15/5/1
CH.sub.2Cl.sub.2/CH.sub.3OH/(C.sub.2H- .sub.5).sub.3N solvent. The
fractions containing the di-boc-spermidine are determined by
thin-layer chromatography.
[0100] NMR data: 1H NMR (200 MHz, CDC13): 1.43-1.48 ppm (s, 18 H,
Boc), 1.48-1.72 ppm (m, 6H,
NH(Boc)--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--N(-
Boc)--CH.sub.2--CH.sub.2--CH.sub.2--NH.sub.2), 2.69 ppm (t, 2H,
J=6.7 Hz, --CH.sub.2--NH.sub.2), 3.07-3.25 ppm (m, 6H,
NH(Boc)--CH.sub.2--CH.sub.2--
-CH.sub.2--CH.sub.2--N(Boc)--CH.sub.2--CH.sub.2--CH.sub.2--NH.sub.2),
4.59 ppm (b, 1H, NH(Boc)). Quantity synthesized (1.45 g; 5.1
mmol)--27% yield relative to the quantity of acrylonitrile.
Synthesis of pcTG37
[0101] 140 .mu.mol of N-hydroxybenzotriazole (Sigma; H2006), then
140 .mu.mol of di-boc-spermidine in 1 ml of anhydrous
tetrahydrofuran, and finally 180 .mu.mol of
dicyclohexylcarbodiimide (Sigma; D3128) diluted in 2 ml
CH.sub.2Cl.sub.2 are added to 140 .mu.mol of N-.alpha. N-.beta.,
di-Fmoc-diaminopropionic (Bachem B2265). After 2 hours of reaction,
the dicyclohexylurea formed is filtered and the product is purified
on a silica column in a 99/1 CH.sub.2Cl.sub.2/CH.sub.3OH solvent.
After evaporation, the Fmoc groups are removed from the product in
a solution of 20% piperidine, 20% tetrahydrofuran and 60%
CH.sub.2Cl.sub.2 for 1 hour. After evaporation, another
purification is carried out on a silica column in a 1/1
CH.sub.2Cl.sub.2/CH.sub.3OH solvent. The fractions containing the
product are determined by thin-layer chromatography. The product is
dried before the last coupling. 510 .mu.mol of the
N-hydroxysuccinimide ester of oleic acid (Sigma 0 9506) diluted in
5 ml of anhydrous CH.sub.2Cl.sub.2 are added to
di-boc-spermidine-diaminopropi- onamide. After evaporation, the
product is purified on a silica column (ether/ethyl acetate; 1/1).
The final product, taken up in 1 ml of CH.sub.2Cl.sub.2, is Boc
deprotected in 10 ml of trifluoroacetic acid (Fluka 91699). To
remove the trifluoroacetic acid, evaporation is carried out under
reduced pressure after dilution in 50 ml of n-hexane.
[0102] Overall yield of synthesis: 64% (89 mg; 90 .mu.mol). .sub.1H
NMR (200 MHz, CDCl.sub.3/CD.sub.3OD: 0.81 ppm (t, j=6.4 Hz, 6H
--CH.sub.3), 1.22-1.3 ppm (m, 44 H, --CH.sub.2--), 1.51 ppm (m, 4
H, --CH.sub.2--CH.sub.2--CO--NH--), 1.72-1.90 ppm (m, 6 H,
NH.sub.3.sup.+--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--NH.sub.2.sup.+--C-
H.sub.2--CH.sub.2--CH.sub.2--NH--), 1.92 ppm (m, 8 H,
CH.sub.2--CH.dbd.), 2.07-2.2 ppm (m, 4 H, CH.sub.2--CO--NH--), 2.89
ppm (m: 6 H,
NH.sub.3.sup.+--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--NH.sub.2.sup.+--C-
H.sub.2--CH.sub.2--CH.sub.2--NH--), 4.22 ppm (t, 1 H,
NH--CO--CHR--NH--), 5.27 ppm (m, 4H, --CH.dbd.). Mass spectrometry:
measured 759.7 Da (calculated 760.2 Da).
Synthesis of the Cationic Lipid of Formula I in which n=2,
m.alpha.3, mp=4; R=stearoyl (lipid pcTG39)
[0103] The compound pcTG39 is prepared in an amount of 140 .mu.mol
in the same manner using stearic acid and dicyclohexylcarbodiimide
or (benzotriazol-1-yloxy) tri-pyrrolidinophosphonium
hexafluorophosphate (PyBOP) as coupling agent.
[0104] Overall yield of synthesis: 22% (30 mg; 30 .mu.mol). .sup.1H
NMR (200 MHz, CDCl.sub.3/CD.sub.3OD/D.sub.2O): 0.68 ppm (t, j=6.8
Hz, 6H, --CH.sub.3), 1.06 ppm (m, 48 H, --CH.sub.2--), 1.37 ppm (m,
4 H, --CH.sub.2--CH.sub.2--CO--NH--), 1.53-1.75 ppm (m, 6 H,
NH.sub.3.sup.+--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--NH.sub.2.sup.+--C-
H.sub.2--CH.sub.2--CH.sub.2--NH--), 1.90-2.30 ppm (m, 4 H,
CH.sub.2--CO--NH.dbd.), 2.74 ppm (m, 6H,
NH.sub.3.sup.+--CH.sub.2--CH.sub-
.2--CH.sub.2--CH.sub.2--NH.sub.2.sup.+--CH.sub.2--CH.sub.2--CH.sub.2--NH---
), 3.25-3.55 ppm (m, 4 H, --CH.sub.2--NH--CO--). Mass spectrometry:
measured 764.5 Da (calculated 764.3 Da).
Preparation of the Lipid-DNA Complexes by Suspension in Ethanol
[0105] 1. Preparation of the Complexes Lipid pcTG37, Optionally
DOPE,-DNA
[0106] The quantities of lipids are calculated based on the
concentration of final DNA (0.1 mg/ml for the tests in vitro), the
desired charge ratio, the molar mass and the number of positive
charges of the chosen cationic lipid. To obtain a complex between
pcTG37/DOPE and the plasmid DNA in a ratio of 10 between positive
charges provided by the cationic lipid and negative charges
provided by the DNA at a final DNA concentration of 0.1 mg/ml, the
different ingredients are mixed according to the following
calculation:
[0107] 0.1 mg of DNA/ml, that is to say (0.1/330) mmol of negative
charges (330 Da is the average molecular weight of a nucleotide)
per ml correspond to 0.30 .mu.mol/ml of negative charges. To obtain
10 times more positive charges, a concentration of 3.0 .mu.mol/ml
of positive charges provided by the cationic lipid is required. The
molar mass of pcTG37 in trifluoroacetate form is 988 g/mol and the
molecule contains 2 positive charges. Therefore, 1.5 .mu.mol/ml of
pcTG37 is required, which corresponds to 1.49 mg/ml.
[0108] To obtain an equimolar concentration of
L-.alpha.-dioleoyl-phosphat- idylethanolamine (DOPE, 744 g/mol,
Sigma; P0510), 1.12 mg/ml is required in the lipid preparation. The
quantities and the concentrations for the other compounds are
adjusted according to their respective molar masses and the number
of their positive charges.
[0109] The lipids are taken up in chloroform, dried by evaporation
and then solubilized in chloroform/methanol (v:v) and again dried.
The cationic lipids are weighed and the quantity of DOPE is added
from a stock solution of 10 or 20 mg/ml in chloroform to a glass
tube sterilized with alcohol and with UV in order to obtain a
cationic lipid concentration of 2 mM. The solvents are evaporated
under vacuum (0.2.times.105 Pa (200 mbar)) for 45 min at 45.degree.
C. using a vortex of 40 revolutions per minute (Labconco, Rapidvap,
Uniequip, Martinsried, Germany). The lipid film is taken up in
ethanol so as to be at the cationic lipid concentration of 50
mg/ml.
[0110] pcTG37/DOPE 1.49 mg+1.12 mg=2.61 mg in 30 .mu.l of ethanol.
This solution is adjusted to 270 .mu.l with 20 mM HEPES pH 7.5
(adjusted with NaOH) in order to prepare a solution at 5 mg/ml
final.
[0111] The plasmid DNA is prepared from a stock solution at 1 mg/ml
(10 mM Tris, 1 mM EDTA, pH 7.5).
[0112] For a solution of 0.5 ml final, 50 .mu.l of the stock
solution (50 .mu.g DNA) are collected to which 300 .mu.l of 20 mM
HEPES pH 7.5 are added.
[0113] To complex the DNA with the lipid preparations, the lipids
are added to the DNA. The suspension is mixed by
aspiration/discharge using a pipette (10 times). The complexes are
stored at +4.degree. C.
[0114] 150 .mu.l of pcTG37/DOPE are added to 350 .mu.l of the DNA
solution in order to obtain 0.5 ml of complex at 0.1 mg/ml DNA and
a charge ratio of 10.
[0115] The preparation of the complexes is carried out under a
laminar flow cabinet.
[0116] The complexes are obtained whose characteristics are
indicated in Table 1 below.
[0117] 2. Preparation of the Complexes Lipids pcTG39, Optionally
DOPE,-DNA
[0118] The complexes are obtained according to the same protocol as
above.
Preparation of the Lipid-DNA Complexes by Suspension in a Detergent
Solution
[0119] 1. Preparation of the Complexes Lipids pcTG37, Optionally
DOPE, DNA
[0120] The quantities of lipids are calculated as described above
based on the concentration of final DNA (0.1 mg/ml for the tests in
vitro), the desired charge ratio, the molar mass and the number of
positive charges of the cationic lipid chosen. The lipids are mixed
in a glass tube, sterilized with alcohol and with UV, in order to
obtain a 2 mM cationic lipid solution (see above). The solvents are
evaporated and the lipid film is taken up in a solution of n-octyl,
.beta.-D-glucopyranoside (octylglucoside, Sigma, O 9882) according
to a cationic lipid/detergent ratio of 1:5 (mol:mol).
[0121] 375 .mu.l of a 20 mM octylglucoside solution in 20 mM HEPES
pH 7.5 are collected and used to take up the film of pcTG37/DOPE
lipid mixture. The plasmid DNA is prepared from a stock solution of
plasmid DNA at 1 mg/ml of which 50 .mu.l are removed for a final
volume of 0.5 ml (0.1 mg/ml final) to which 262.5 .mu.l of 20 mM
HEPES pH 7.5 are added. 187.5 .mu.l of the lipid suspension are
added to the DNA by aspirating and discharging 10 times using a
pipette in order to obtain the final suspension at 0.1 mg/ml of DNA
and a +/- charge ratio of 10. To remove the detergent, a dialysis
of 3 times 4 hours at room temperature against 20 mM HEPES pH 7.5
is carried out in dialysis microbags (cut-off of 13.2 kD;
Sartorius, Gottingen, Germany). The dialyzed DNA/lipid complexes
are stored at +4.degree. C. The preparation is carried out in a
laminar flow cabinet.
[0122] The complexes are obtained whose characteristics are
indicated in Table 1 below.
[0123] 2. Preparation of the Complexes Lipids pcTG39, Optionally
DOPE,-DNA
[0124] Based on the same protocol, the complexes whose
characteristics are indicated in Table 1 below are obtained.
Preparation of the Lipid-DNA Complexes by Sonication Extrusion
[0125] The quantities of lipids are calculated as described above
based on the concentration of final DNA (0.1 mg/ml for the tests in
vitro), the desired charge ratio, the molar mass and the number of
positive charges of the cationic lipid chosen. The lipids are mixed
in a glass tube, sterilized with alcohol and with UV, in order to
obtain a 2 mM cationic lipid solution, as indicated above. The
solvents are evaporated and the lipid film is taken up in 900 .mu.l
of 20 mM HEPES pH 7.5 at 4.degree. C. for about 16 h. The
suspension is sonicated in a sonication bath (Bransonic 221) to
visual homogeneity. The lipid suspension is extruded through two
membranes with a pore diameter of 0.2 .mu.m (Nucleopore, Costar,
Cambridge, Mass., USA) and rinsed with 20 mM HEPES pH 7.5 (extruder
from Lipex Biomembranes, Vancouver, Canada) at a maximum pressure
of 50 bars. The lipid suspension is kept at room temperature for 1
hour. 450 .mu.l of the lipid suspension are added to 50 .mu.l of a
stock solution of plasmid DNA (1 mg/ml) and mixed by
aspirating/discharging 10 times using a pipette. The lipid/DNA
complexes are stored at +4.degree. C. The preparations are carried
out under a laminar flow cabinet.
Protocol for Evaluation of the Complexing of the DNA by the
Lipids
[0126] A 1% (w:v) agarose gel is prepared in a TAE buffer (TAE:
Tris 4.86 g/l+sodium acetate 0.68 g/l+EDTA 0.336 g/l pH 7.8. If
necessary, the sample is diluted in TAE and then the sample buffer
0.083% bromophenol blue, 0.083% cyanol xylene FF, 10% glycerol in
water) is added so as to have 50 ng DNA/.mu.l. The sample is
briefly subjected to a vortex and left for 30 min at room
temperature. As a control, the non-complexed plasmid prepared at
the same concentration is used. 10 .mu.l (500 ng of DNA) are
deposited on the gel and the migration is carried out at 60 mV for
3 hours. The gel is developed in TAE containing 0.006% (v:v) of
ethidium bromide at 10 mg/ml for at least 30 min. Next, the gel is
rinsed in TAE and analyzed under UV.
Protocol for Measuring the Size of the Particles by Quasi-elastic
Scattering of Light
[0127] The analyses are carried out on a Coulter N4Plus
(Coultronics France S.A., Margency, France) at 25.degree. C. after
equilibration of the sample for 20 min. An aliquot of the sample is
aspirated and discharged several times before being pipetted. The
sample is diluted in the measuring tank and homogenized. The
measurement of the light diffracted at 90.degree. is carried out
for 180 sec after a 180 sec wait. The range used goes from 3 nm to
10 000 nm using 31 bins. To be valid, the sample should give
between 50 000 and 1 000 000 counts/sec.
Physicochemical Characteristics
[0128] The three methods of formulation "injection of ethanol",
"dialysis of detergent" and "sonication/extrusion" are applied to
the cationic lipids according to the invention with or without
equimolar quantities of DOPE at charge ratios of about 10, or 5.
The formulations are considered to be appropriate when the DNA is
completely complexed (no migration in the agarose gel) and when the
complexes have diameters, determined by quasi-elastic scattering of
light, less than 500 nm. The table 1 summarizes the results of
these analyses. All the DNA/lipid complexes indicated in the table
complex the DNA completely.
2 TABLE 1 Lipid Ratio.sup.1 Size (nm).sup.2 Formulation pcTG37 10
77 ethanol pcTG37 5 80 ethanol pcTG37/DOPE 10 79 ethanol
pcTG37/DOPE 5 64 ethanol pcTG37 10 240 detergent pcTG37 5 59
detergent pcTG37/DOPE 10 312 detergent pcTG39/DOPE 10 312 detergent
pcTG37/DOPE 5 280 sonication .sup.1Ratio between the positive
charges of the cationic lipid and the negative charges of the DNA.
.sup.2The populations which represent less than 10% of the
intensity are not indicated. The size is determined 24-48 hours
after the preparation.
[0129] These analyses show that the formulations meet the necessary
requirements. The "injection of ethanol" method gives the best
results, the different preparations tested being less than 100 nm
in size. The method by dialysis of detergent or
sonication/extrusion also makes it possible to obtain complexes
meeting the criteria of the present invention.
In vitro Transfection of Satellite Cells
[0130] Cultures of dog muscle and human muscle [lacuna] are carried
out in an HamF 14 medium (Life Technologies) supplemented with 10%
fetal calf serum (FCS, Hyclone, Logan, Utah), 10 .mu.g/ml of
insulin (Sigma), 10 ng/ml of EGF (Sigma) and of FGF (Pepro Tech
Inc., Rocky Hill, N.J.) 2 mM of glutamine (bioMrieux), and 40
.mu.g/ml of gentamycin (Schering Plough).
[0131] The cells are inoculated 24 h to 48 h before the
transfection into a 96-well culture plate with about
5.times.10.sup.3 to 10.sup.4 cells per well, at about 30%
confluence, and kept at 37.degree. C. under a 5% CO.sub.2 and 95%
air atmosphere.
[0132] The transfections are carried out with mixtures of variable
quantities of lipids and plasmid DNA in order to determine the
charge ratios and the optimum DNA concentrations per well.
[0133] The complexes used are prepared 24 h to 48 h before the
transfection and diluted in HamF 14 medium containing 40 .mu.g/ml
of gentamycin and 2 mM glutamine.
[0134] After removing the culture medium, 100 .mu.l of transfection
mixtures with or without 10% FCS are transferred into each of the
wells and the plates are incubated for 4 h at 37.degree. C.
[0135] All the transfection media are then adjusted to 10% FCS, 10
.mu.g/ml of insulin (Sigma), 10 ng/ml of EGF (Sigma) and of FGF
(Pepro Tech Inc., Rocky Hill, N.J.), 2 mM glutamine (bioMrieux),
and 40 .mu.g/ml of gentamycin (Schering Plough) for a final volume
of 250 .mu.l. The cultures are incubated for 48 h and then the
cells are recovered and tested for their capacity to express the
luciferase gene. The protein concentrations are determined by the
system for testing quantity of protein (Promega).
Transfection of A549 Cells with Lipid Complexes
[0136] The A549 cells (epithelial cells derived from human
pulmonary carcinoma) are cultured in Dulbecco's modified Eagle's
medium (DMEM) containing 10% fetal calf serum (Gibco BRL) 24 hours
before the start of the transfection in 96-well plates
(2.times.10.sup.4 cells per well) in a humid atmosphere at
37.degree. C. and 5% CO.sub.2/95% air. For the transfection in the
absence of serum, the medium is removed and replaced with
serum-free medium. In another microplate, the following suspensions
of lipid/DNA complexes are prepared (lipid/DNA complexes at 0.1
mg/ml of DNA and at the indicated charge ratio):44 .mu.l (4.4 .mu.g
DNA), 22 .mu.l (2.2 .mu.g DNA), 5.5 .mu.l (0.55 .mu.g DNA) of stock
solution in the first 3 wells, and 11 .mu.l (0.11 .mu.g DNA) of the
stock solution diluted 10-fold in the next well. The volume is
adjusted to 110 .mu.l with DMEM and 100 .mu.l are transferred over
the A549 cells. The incubation is carried out with 4, 2, 0.5 and
0.1 .mu.g of DNA per well for 4 hours. 50 .mu.l of DMEM+30% fetal
calf serum are added 4 hours after the start of transfection and
then 100 .mu.l of DMEM+10% FCS 24 hours after the start of
transfection. The transfections in the presence of 10% fetal calf
serum are carried out in an identical manner except that the
transfection occurs in medium with serum.
Analysis of the Transfection
[0137] 48 h after the transfection, the medium is removed and the
cells are washed with 100 .mu.l of PBS phosphate solution and lyzed
with 50 .mu.l of lysis buffer (Promega). The lysates are frozen at
-80.degree. C. until the luciferase activity is measured. The
latter is carried out on 20 .mu.l of mixture for one minute using
the Luciferase" determination system (Promega) (LB96P Berthold
luminometer) in 96-well plates in kinetic mode.
Transfection in vitro
[0138] The preparations of cationic lipids pcTG37 were evaluated in
transfection in vitro using the A549 cells and the dog primary
myoblasts.
[0139] The results are summarized in Table 2 below and show the
relative light units (RLU) per well. The values given are obtained
with 0.5 .mu.g of DNA per well. All the complexes are prepared
using the injection of ethanol method. The total protein
concentration per well is determined by the conventional techniques
(BCA test, Pierce) . As a guide, a well contains about 20 to 30
.mu.g of protein.
3TABLE 2 Lipid Ratio.sup.1 A549.sup.2 A549 + serum myoblasts
myoblast + serum pcTG37 10 1.7 .times. 10.sup.6 3.2 .times.
10.sub.4 0 1.2 .times. 10.sup.5 (4.0 .times. 10.sub.8 RLU/mg/min)
pcTG37 5 9.9 .times. 10.sup.6 3.9 .times. 10.sup.3 8.5 .times.
10.sup.7 8.6 .times. 10.sup.3 (1.4 .times. 10.sub.9 RLU/mg/min)
pcTG37/DOPE 10 3.5 .times. 10.sup.6 1.2 .times. 10.sup.6 1.5
.times. 10.sup.4 4.9 .times. 10.sup.5 pcTG37/DOPE 5 5.0 .times.
10.sup.4 6.7 .times. 10.sup.6 1.9 .times. 10.sup.6 4.9 .times.
10.sup.4
Another Mode of Synthesis of the Compounds According to the
Invention has been Developed
[0140] 1. Synthesis of the Polyamine Part
[0141] 1.1. In order to be able to synthesize the "T-branched"
compounds for which the group R of formula II is bound to the
secondary amines, an N,N'-di-boc-spermine was synthesized. The
protocol applied in order to obtain the 1,4-diboc-spermidine is
published by Goodnow et al. (1990, Tetrahedron 46, 3267-3286).
[0142] The intermediate
N-N'-(di-boc)-N,N'-(dipropio-nitrile)-1,4-diaminob- utane is
obtained by reacting 37.6 mmol of diaminobutane (Fluka; reference
32790) diluted in 3.8 ml of dichloromethane+2 ml of methanol at
0.degree. C. with 75.2.mmol of acrylonitrile (Fluka; reference
01710), for 1 hour at 0.degree. C. The product of the reaction is
blocked with 100 mmol of BOC-ON
[(2-boc-oxyimino)-2-phenylacetonitrile] (Fluka; reference 15475) in
50 ml of 2/1 CH.sub.2Cl.sub.2/CH.sub.3OH solvent. The blocking
reaction is placed overnight at room temperature. The solvents are
evaporated and the product is taken up in 200 ml of ethyl acetate
and then subjected to 3 extractions with 200 ml of 1 M NaOH. The
organic phase is again extracted with 5% citric acid, dried and
filtered over Na.sub.2SO.sub.4 before evaporation to dryness. A
step of reducing the nitrites is then carried out in ether at
0.degree. C. in the presence of 260 mmol of LiAlH4 (Sigma; L0260).
After 15 hours, the reaction is stopped by addition of 100 ml of 1
M sodium hydroxide at 0.degree. C., with evolution of hydrogen. The
mixture is filtered and extracted three times with a 20% NaCl
solution. After drying the ethereal fraction, the product is
deposited on a silica column in a 1/1 CH.sub.2Cl.sub.2/CH.sub.- 3OH
solvent and then eluted in a 15/5/1
CH.sub.2Cl.sub.2/CH.sub.3OH/(C.sub- .2H.sub.5).sub.3N solvent. The
fractions containing the di-boc-spermine are determined by
thin-layer chromatography. Weighing of the final product: 2.45 g
(5.8 mmol). Overall yield: 15.5%.
[0143] 1.2. Synthesis of N,N'-di-boc-spermidine from spermidine. 20
mmol of spermidine (Sigma S2626) diluted in 20 ml of
dichloromethane added to 20 ml of methanol, 11 mmol (2 ml) of
diisopropylethylamine at 0.degree. C. and 40 mmol of BOC-O-BOC
[di-(tert-butyl)-dicarbonate] (Fluka; reference 34660) in 80 ml of
1/1 CH.sub.2Cl.sub.2/CH.sub.3OH solvent. The blocking reaction is
placed overnight at room temperature. The solvents are evaporated
and then the product is deposited on a silica column and eluted in
steps of 9/1 to 8/2 CH.sub.2Cl.sub.2/CH.sub.3OH solvents. The
fractions containing the N,N'-di-boc-spermidine are determined by
RP-HPLC chromatography. A pure portion of N,N'-di-boc-spermidine is
thus isolated from the other position isomers of di-boc-spermidine.
Weighing of the final product: 0.58 g (1.68 mmol) Overall yield:
8.5%.
[0144] 1.3. New process for the synthesis of 1-4-di-boc-spermidine:
50 mmol of diaminobutane (Fluka; reference 32790) diluted in 50 ml
of dichloromethane are added to 20 ml of methanol at 0.degree. C.
with 58 mmol of acrylonitrile (Fluka; reference 01710) and placed
for 1 hour at 0.degree. C. The product of the reaction is blocked
with 109 mmol of BOC-ON [(2-boc-oxyimino)-2-phenylacetonitrile]
(Fluka; reference 15475) in 50 ml of 2/1
CH.sub.2Cl.sub.2/CH.sub.3OH solvent. The blocking reaction is
placed overnight at room temperature. The solvents are evaporated
and the product is taken up in 300 ml of ethyl acetate for 3
extractions with 300 ml of 1 M NaOH. The organic phase is again
extracted with 5% citric acid, dried and filtered over
Na.sub.2SO.sub.4 before evaporation to dryness. The step of
reduction of the nitrites is then carried out in ether at 0.degree.
C. in the presence of 140 mmol of LiAlH4 (Sigma; L0260). After 15
hours, the reaction is stopped by addition of 100 ml of 1 M sodium
hydroxide at 0.degree. C., with evolution of hydrogen. The mixture
is filtered and extracted three times with a 20% NaCl solution.
After drying the ethereal fraction, the product is ready to be
deposited on a silica column for the final purification.
[0145] 2. Synthesis of the Hydrophobic Part
[0146] Synthesis of N-.alpha.-N-.beta.-di[oleylamido]propionic
acid. 9.04 mmol (3.43 g) of the N-hydroxysuccinimide ester of oleic
acid (Sigma O 9506) diluted in 10 ml of dioxane are added to 4.1
mmol (0.58 g) of N-.alpha.-N-.beta.-diaminopropionic acid,HCl
(Bachem F3040) in a mixture of 22 ml of water, 44 ml of dioxane and
20.5 mmol (2.65 ml) of diisopropylethylamine. After 2 hours of
reaction, 2 extractions with ether/HCl 5% are carried out, followed
by saturated NaCl washing and drying over Na.sub.2SO.sub.4 before
evaporating the ethereal fraction to dryness. The product is
deposited on a silica column in a 95/5 CH.sub.2Cl.sub.2/CH.sub.3OH
solvent, and then eluted in steps of 95/5, then 92.5/7.5, and
finally 90/10 CH.sub.2Cl.sub.2/CH.sub.3OH solvents. The fractions
containing n-.alpha.-N-.beta.-di[oleyl-amido]propionic acid are
determined by thin-layer chromatography. Control on TLC
(CH2Cl2/CH3OH 90/10) and NMR. Drying. Weighing: 0.916 g (1.45
mmol), yield: 35.4%.
[0147] Synthesis of N-.alpha.-N-.beta.-di-[laurylamido]propionic
acid. 2.2 mmol (0.65 g) of the N-hydroxysuccinimide ester of lauric
acid (Sigma L3900) diluted in 10 ml of dioxane are added to 1 mmol
(0.14 g of N-.alpha.-N-.beta.-diaminopropionic acid,HCl (Bachem
F3040) in a mixture of 10 ml of water, 20 ml of dioxane and 5 mmol
(0.65 ml) of diisopropylethylamine. The same purification protocol
(see N-.alpha.-N-.beta.-di[oleylamido]-propionic acid) is applied.
Weighing: 0.4 g (0.5 mmol), yield: 50%.
[0148] Synthesis of N-.alpha.-N-.beta.-di[palmitylamido]propionic
acid. 2.2 mmol (0.79 g) of the N-hydroxysuccinimide ester of
palmitic acid (Sigma P1162) diluted in 10 ml of dioxane are added
to 1 mmol (0.14 g) of N-.alpha.-N-.beta.-diaminopropionic acid,HCl
(Bachem F3040) in a mixture of 10 ml of water, 20 ml of dioxane and
5 mmol (0.65 ml) of diisopropylethylamine. The activated ester
precipitates upon the addition and the reaction takes place in a
heterogeneous phase for 15 hours, with stirring, at room
temperature. And the same purification protocol (see
N-.alpha.-N-.beta.-di[oleylamido]-propionic acid) is applied.
Weighing: 0.14 g (0.15 mmol), yield: 15%.
[0149] 3. New synthesis of pcTG37
[0150] 1.45 mmol 916 mg of
N-.alpha.-N-.beta.-di[oleylamido]-propionic acid, 1.74 mmol (235
mg) of N-hydroxybenzo-triazole (Sigma; H2006), 2 mmol (265 .mu.l)
of diisopropylethylamine, and finally 2.175 mmol (449 mg) of
dicyclohexylcarbodiimide (Sigma; D3128) diluted in 10 ml of
CHCl.sub.3 are added to 1.45 mmol (500 mg) of 1,4-di-boc-spermidine
diluted in 30 ml of anhydrous CHCl.sub.3. After 2 hours of
reaction, the medium is filtered and after evaporation, rediluted
in 50/50 ether/hexane, and another filtration-evaporation is
carried out. This product is purified on a silica column deposited
in ether and eluted in steps of ether/ethyl acetate solvent: 75/25,
50/50, 25/75 and 100% ethyl acetate. Control on TLC (ethyl acetate)
and NMR. The final product, taken up in 10 ml of CH.sub.2Cl.sub.2,
is Boc deprotected by adding dropwise at 0.degree. C. 50 ml of
trifluoroacetic acid (Fluka 91699) redistilled at 72.degree. C. and
diluted in 50 ml of CH.sub.2Cl.sub.2. After 4 hours, the medium is
evaporated twice at reduced pressure after dilution in 50 ml of
n-hexane. The product is taken up in 10 ml of ether, precipitated
in hexane and filtered on filter paper. The product is taken up by
washing the paper in a 1/1 CH.sub.2Cl.sub.2/CH.sub.3OH solvent.
Controls on TLC (ethyl acetate), HPLC and NMR. Evaporation and
weighing: 670 mg (0.68 mmol) yield: 47%.
[0151] 4. Synthesis of pcTG89 (equivalent of pcTG37 but in a
T-branched form)
[0152] The same protocol is applied as for pcTG37 but by replacing
1,4-di-boc-spermidine with N,N'-di-boc-spermidine. Weighing of the
protected product: 170 mg (0.18 mmol), yield: 12.2%.
Intravenous Injection of Complexes According to the Invention
[0153] The results are summarized in FIG. 2. Complexes according to
the invention were synthesized according to the methods described
above from the compound pcTG337, in the presence or in the absence
of DOPE (2:1, mol:mol), at a fixed charge ratio of 5, using a
plasmid containing the gene for luciferase pTG11033 (French Patent
Application No. 97/08267).
[0154] The mice used are 9- to 11-week-old C57BL/6 female mice. One
day before the injection with the complex according to the
invention, the mice are pretreated by injecting 50 .mu.l of a
preparation of chlodronate encapsulated in a liposome (see Van
Rooijen and Sanders, 1994, J. Immunol. Methods, 174, 83-93)
incorporated in a total volume of 200 .mu.l (+150 .mu.l of PBS
buffer). The intravenous injections are carried out into the tail
after disinfecting the skin with 70% ethanol. The volume injected
is 250 .mu.l and the quantity of DNA is 60 .mu.g, the quantity of
lipid is 345 .mu.g.
[0155] Two days after the injections, the mice are sacrificed.
After extraction, the tissues are frozen in liquid nitrogen and
stored at -80.degree. C. To measure the luciferase activity, the
tissues are mechanically ground with the aid of a pestle in a
mortar placed on dry ice. 500 .mu.l of a lysis buffer (Promega) are
added to the tissue debris obtained from the lungs and subjected to
three freeze/thaw steps. The cellular debris is removed by
centrifugation and the luciferase activity (in RLU/min, relative
light unit per minute) is measured on 20 .mu.l of supernatant in
accordance with the manufacturer's instructions (Promega) by adding
100 .mu.l of reagent and by measuring the activity by luminescence.
The luciferase activity measured is standardized relative to the
protein quantity with the aid of a calibration series prepared by
commercially available luciferase (Promega). The total protein
quantity is, moreover, determined by the bicinchoninic acid BCA
colorimetric method (Smith et al., 1985, Anal. Biochem., 150, 76-85
Pierce) using an aliquot of supernatant. This makes it possible to
express the luciferase activity in RLU per milligram of protein
extracted from the tissues. Controls are carried out for which the
mice were not injected or were injected with DNA alone (60 .mu.g)
in a 20 mM HEPES buffer pH 7.5. The results (FIG. 2) show that the
expression of the luciferase reporter gene in the lungs after
intravenous injection of complexes containing the compound
according to the invention at a charge ratio of 5, in the presence
of DOPE is markedly enhanced relative to the injection of DNA
alone. The values indicated are the mean values obtained from 3
mice injected.
* * * * *