Note: Descriptions are shown in the official language in which they were submitted.
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Cationic Amphiphiles and Pl~smi~c for
Intracellular Delivery of Therapeutic Molecules
Background of the Invention
The ~res~llt ~, Iv~l~lion relates to novel cationic amphiphilic
compounds that fa~ ilit~te the intrAcPlllllar delivery of biologically active
(therapeutic) molecules. The present invention relates also to
pharmaceutical compositions that comprise such cationic amphiphiles, and
10 that are useful to deliver into the cells of patients therapeutically effective
amounts of biologically active molecules. The novel cationic amphiphilic
compounds of the invention are particularly useful in relation to gene
therapy.
Effective therapeutic use of many type.s of biolot, cally active
15 molecules has not been achieved simply because methods are not available
to cause delivery of therapeutically effective amounts of such substances
into the particular cells of a patient for which treatment therewith would
provide therapeutic benefit. ~ffi~ i~nt delivery of therapeutically sufficient
amounts of such molecules into cells has often proved difficult, if not
20 impossible, since, for example, the cell membrane presents a selectively-
permeable barrier. Additionally, even when biologically active molecules
successfully enter targeted cells, ~hey r.ay be degraded directly in rhe ceii
cytoplasm or even transported to structures in the the cell, such as
lysosomal compartments, spe( iali7er7 for degradative processes. Thus both
25 the nature of substances that are allowed to enter cells, and the amounts
thereof that ultimately arrive at targeted locations within cells, at which theycan provide therapeutic benefit, are strictly limited.
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Although such selectivity is generally necf~s~ry in order that proper
cell function can be m~int~ined, it comes with the disadvantage that many
therapeutically valuable substances (or therapeutically effective amounts
thereof) are excluded. Additionally, the complex structure, behavior, and
~LIvirol~ment presented by an intact tissue that is targeted for intrac~ r ,,
delivery of biologically active molecules often iLIL~ e substantially with
such delivery, in comparison with the case presented by populations of cells
cultured in vitro
Examples of biologically active molecules for which effective
targeting to a patients' tissues is often not achieved: (1) numerous proteins
including immunoglobin ploL~iLLs, (2) polynucleotides such as genomic
DNA, cDNA, or mRNA (3) antisense polynucleotides; and (4) many low
molecular weight compounds, whether synthetic or naturally occurring,
such as the peptide hormones and antibiotics.
One of the fundamental challenges now facing medical practicioners
is that although the defective genes that are associated with numerous
inherited diseases (or that represent disease risk factors including for
various cancers) have been isolated and characterized, methods to correct
the disease states themselves by providing patients with normal copies of
such genes (the technique of gene therapy) are substantially lacking.
Accordingly, the development of i,l.~roved methods of intrac~ r
delivery therefor is of great m~1ic~1 importance. Examples of diseases
that it is hoped can be treated by gene therapy include inherited disorders
such as cystic fibrosis, Gaucher's disease, Fabry's disease, and muscular
dystrophy. Representative of acquired disorders that can be treated are~
for cancers--multiple myeloma, leukemias, melanomas, ovarian carcinoma
and small cell lung cancer; (2) for cardiovascular conc1itions--progressive
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heart failure, restenosis, and hemophilias; and (3) for neurological
conditions--traumatic brain injury.
Gene therapy requires successful transfection of target cells in a
patient. Transfection may generally be defined as the process of introducing
5 an expressible polynucleotide (for example a gene, a cDNA, or an mRNA
patterned thereon) into a cell. Sllcc~sful expression of the encoding
polynucleotide leads to production in the cells of a normal protein and leads
to correction of the disease state associated with the abnormal gene.
Therapies based on providing such ploLe"-s directly to target cells (protein
10 replacement therapy) are often ineffective for the reasons mentioned above.
Cystic fibrosis, a common lethal genetic disorder, is a particular
example of a disease that is a target for gene therapy. The disease is caused
by the presence of one or more mutations in the gene that encodes a protein
known as cystic fibrosis trarl~m~mhrane conductance regulator ("CFTR"),
15 and which regulates the movement of ions (and therefore fluid) across the
cell membrane of epithelial cells, including lung epithelial cells. Abnormnal
ion transport in airway cells leads to abnormal mucous secretion,
inflammmation and infection, tisssue damage, and eventually death.
It is widely hoped that gene therapy will provide a long lasting and
20 predictable form of therapy for certain disease states, and it is likely the only
form of therapy suitable for many inhereted diseases. There remains
however a critical need to develop compounds that faciliate entry of
functional genes into cells, and whose activity in this regard is sufficient to
provide for in vivo delivery of genes or other such biologically active
25 therapeutic molecules in concentrations thereof that are sufficient for
intracellular therapeutic effect.
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Reported Developments
In as much as compounds designed to facilitate intracellular delivery
of biologically active molecules must interact with both non-polar and polar
el,vilolu.lents ( in or on, for example, the plasma membrane, tissue fluids,
compartments within the cell, and the biologically achve molecule itself ),
such compounds are designed typically to contain both polar and non-polar
domains. Compounds having both such ~70mains may be f~rme~
amphiphiles, and many lipids and synthetic lipids that have been disclosed
for use in facilitating such intracellular delivery (whether for in vitro or in
0 vivo application) meet this definition. One particularly important class of such amphiphiles is the cationic amphiphiles. In general, cationic
amphiphiles have polar groups that are capable of being positively charged
at or around physiological pH, and this property is understood in the art to
be important in defining how the amphiphiles interact with the many types
of biologically active (therapeutic) molecules including, for example,
negatively charged polynucleotides such as DNA.
Examples of cationic amphiphilic compounds that have both polar
and non-polar domains and that are stated to be useful in relation to
intrac~ 7lar delivery of biologically active molecules are found, for
example, in the following references, which contain also useful discussion of
(1) the properties of such compounds that are understood in the art as
making them su*able for such applications, and (2) the nature of structures,
as understood in the art, that are formed by complexing of such
amphiphiles with therapeutic molecules intended for intracellular delivery.
(1) Felgner, et al., Proc. Natl. Acad. Sci. USA, 84, 7413-7417 (1987)
disclose use of positively-charged synthetic cationic lipids including N-
[1(2,3-dioleyloxy)~,~yl]-N,N,N-trimethylammonium chloride ("DOTMA"),
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to form lipid/DNA complexes suitable for transfections. See also Felgner et
al.,
The Tournal of Biological Chemistry, 269(4), 2550-2561 (1994).
(2) Behr et al., Proc. Natl. Acad. Sci. USA 86, 6982-6986 (1989) disclose
numerous amphiphiles including dioctadecylamidologlycylspermine
("DOGS").
(3) U.S. Patent 5, 283,185 to Epand et al. describes additional classes and
species of amphiphiles including 3~ [N-(Nl,Nl - dimethylaminoethane)-
carbamoyl] cholesterol, termed "DC-chol".
(4) Additional compounds that facilitate transport of biologically active
molecules into cells are disclosed in U.S. Patent No. 5,264,618 to Felgner et
al. See also Felgner et al., The Journal Of Biological Chemistry 269(4), pp.
2550- 2561 (1994) for disclosure therein of further compounds including
"DMRIE" 1,2- dimyristyloxypr~yl-3-dimethyl-hydroxyethyl ammonium
bromide, which is discussed below.
(5) Reference to amphiphiles suitable for intracellular delivery of
biologically active molecules is also found in U.S. Patent No. 5,334,761 to
Gebeyehu et al., and in Felgner et al., Methods(Methods in Enzymology), 5,
67- 75 (1993).
Although the compounds mentioned in the above-identified
Lefelel-ces have been demonstrated to facilitate (although in many such
cases only in vitro ) the entry of biologically active molecules into cells, it is
believed that the uptake efficiencies provided thereby are insufficient to
support numerous therapeutic applications, particulary gene therapy.
" 25 Additionally, since the above-identified compounds are understood to have
only modest activity, substantial quantities thereof must be used leading to
concerns about the toxicity of such compounds or of the metabolites thereof.
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Accordingly there is a need to develop a "second generation" of cationic
amphiphiles whose activity is so sufficient that successful theraples can be
achieved therewith.
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Sl~mn-~v of the Invention
This invention provides for cationic amphiphiles that are parhcl1larly
effective to facilitate transport of biologically active molecules into cells.
The cationic amphiphiles of the invention are divided into four (4) groups,
5 although it will be seen that there are certain structural and functional
features that many of the amphiphiles share.
Accordingly, there are provided cationic amphiphiles of Group I (see
Figure 1, panels A, B, and C) capable of facilitating transport of biologically
active molecules into cells, said amphiphiles having the structure (I),
(R3)--(Rl)
(~ (Y) (~
(R4) (R2) (I)
wherein:
Z is a steroid;
15 X is a carbon atom or a nitrogen atom;
Y is a short linking group, or Y is absent;
R3 is X or a saturated or unsaturated aliphatic group;
R1 is --NH--, an alkylamine, or a polyalkylamine;
R4 is H, or a saturated or unsaturated aliphatic group;
20 R2 is--NH--, an alkylamine, or a polyalkylamine;
and wherein R1 is the same or is different from R2, except that both R1 and
R2 cannot be --NH--.
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In one ~re~l red embodiment, the steroid component "Z" is selected
from the group consisting of 3-sterols, wherein said sterol molecule is linked
by the 3-0- group thereof, or by N- in replacement thereof, to Y (or directly
to X, if Y is absent ). According to this aspect of the invention, particularly
5 effective amphiphiles include, for example, spermidine cholesterol
carbamate ( N4 -spermidine cholesteryl carbamate, amphiphile No. 53), and
spermine cholesterol carbamate ( N4-spermine cholesteryl carbamate,
amphiphile No. 67), and amphiphiles patterned thereon.
In a further ~re~el~ed embodiment, the steroid group is linked to Y
10 (or directly to X, if Y is absent) from ring position 17 of the steroid nucleus
(see Figures 1 and 22), or from the arm that normally extends from position
17 in many steroids( see the structure of cholesterol in Figure 1), or from any
shortened form of said arm.
In other ~re~rled embodiments, within linking group Y are
15 contained no more than about three or four atoms that themselves form a
bridge of covalent bonds between X and Z. In a specific ~reL~lled
embodiment of the invention, Y is a linking group wherein no more than
one atom of said group forms a bond with both X and Z, or Y is absent.
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Repres~l~ldLiv~ amphiphiles provided according to Group I include:
H2N /'N~O~
,I No. 53 NH2
N4-sp~rmi~linf~ cholesteryl
carbamate
O ~
H2N~'N~
H~
No. 67 NH2
N4-spermine cholesteryl
~rb~m~t~
~",
H N~~'N
'~ N H
No. 75 ~NH
Nl,N8-Bis (3-aminopropyl)- N4 -
spermidine cholesteryl carbamate
~ 0~
H2N~N No. 78
NH2
N(N4-3-amin~ opyl~rmi~in~)
cholesteryl carbamate
-
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Additionally there are provided cationic amphiphiles of Group :~ (see
Figure 5) capable of facilitating transport of biologically active molecules
into cells said amphiphiles having the structure (II),
(R3) (R1)
(~ (Y) (~
(R4) (R2) (II)
wherein:
Z is a steroid;
10 X is a carbon atom or a nitrogen atom;
Y is a linking group or Y is absent;
R3 is an amino acid, a derivatized amino acid, H or alkyl;
Rl is --NH--, an alkylamine, or a polyalkylamine;
R4 is an amino acid, a derivatized amino acid, H or alkyl;
15 R2 is --NH--, an alkylamine, or a polyallcylamine;
and wherein Rl is the same or is different from R2, except that both Rl and
R2 cannot be --NH~.
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Represel~LaLive amphiphiles provided according to Group II include:
,~<
, HN ~~/
~0
H2N ~ N~ H NH2
No. 91
H2N~ NH
HN~
H2N~O
NH
o~ NH2
No.95
NH
HN NH2
Nl,N8-Bis(argiI~ine carboxarnide)-
N4-spermidine rhoh~t~ry carbamate
Additionally there are provided cationic amphiphiles of Group m
(see Figure 6) capable of facilitating transport of biologically active
molecules into cells said amphiphiles having the structure (III),
(R3) (R )~
(X) (~') (Z)
(R4)--(R2)/ (m)
wherein:
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Z is an alkylamine or a dialkylamine, linked by the N-atom thereof, to Y ( or
directly to X, if Y is absent ), wherein if Z is a dialkylamine, the alkyl groups
thereof can be the same or dif~l'~-l,
X is a carbon atom or a nitrogen atom;
5 Y is a short linking group, or Y is absent;
R3 is H, or a saturated or unsaturated aliphatic group;
Rl is--N~I--, an alkylamine, or a polyalkylamine;
R4 is H, or a saturated or unsaturated aliphatic group;
R2 is --NH--, an alkylamine, or a polyalkylamine;
10 and wherein R1 is the same or is different from R2, except that both R1 and
R2 cannot be NH--.
With respect to amphiphiles provided according to Structure (III), it
is again preferred that within linking group Y there are contained no more
than about three or four atoms that themselves ~orm a bridge of covalent
15 bonds between X and Z. In a specific prer~lred embodiment of the
invention, Y is a linking group, such as > C=O, wherein no more than one
atom of said group forms a bond with both X and Z, or Y is absent.
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Representative amphiphiles provided according to Group III include: .
o
H2N--~N~ [CH2]17cH3
2 HCl NH2 [CH2]l7(~H3
No. 43
N,N-dioctadecylly.~in~mi~le diHCl salt
[CH2]l7CH3
NH2 ~N~
3HCl NH2 [CH2]l 7CH3
No. 47
Nl,Nl-dioctadecyl-1,2,~
hi~min-~h~xane tri HCl salt
Additionally there are provided cationic amphiphiles of Group IV
(see Figure 7) capable of facilitating transport of biologically active
molecules into cells said amphiphiles having the structure (IV),
I~(C)~ ~(R ) (R )
(R ) ¦ (R2)l (R4)
(R6) (IV)
wherein:
A and B are independently 0, N or S;
15 R5 and R6 are independently alkyl or acyl groups and may be saturated or
contain sites of unsaturation;
C is selected from the group consisting of --CH2--, >C=O, and >C=S;
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E is a carbon atom or a nitrogen atom;
D is a linking group such as -NH(C=O)- or -O(C=O)-, or D is absent;
R3 is H, or a saturated or unsaturated aliphatic group;
Rl is NH--, an alkylamine, or a polyalkylamine;
R4 is H, or a saturated or unsaturated aliphatic group;
R2 is --NH , an alkylamine, or a polyalkylamine;
and wherein Rl is the same or is different from R2, except that both Rl and
R2 cannot be ~
14
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Representative amphiphiles of Group IV include:
~----~'~--~~ N
No. 89 ~,NH
NH2
1-(N4-sperrnine)-2,3-dilauryl-
glycerol carbarnate
~H2
~0--~NJ
O J
No. 102 ~, N H
NH2
N4-spermine-2,3-dilauryl-
uky~ ylamine O
The invention provides also for pharmaceutical compositions that
comprise one or more cationic amphiphiles, and one or more biologically
active molecules, wherein said compositions facilitate intracellular delivery
in the tissues of patients of therapeutically effective amounts of the
biologically active molecules. The pharmaceutical compositions of the
invention may be formulated to contain one or more additional
physiologically acceptable substances that stabilize the compositions for
storage and/or contribute to the successful intracellular delivery of the
biologically active molecules.
In a further aspect, the invention provides a method for facilitating
,~ 15 the transfer of biologically active molecules into cells co~ ising the steps
of. preparing a dispersion of a cationic amphiphile of the invention;
contacting said dispersion with a biologically active molecule to form a
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complex between said amphiphile and said molecule, and contacting cells
with said complex thereby facilitating transfer of said biologically-active
molecule into the cells.
For pharmaceutical use, the cationic amphiphile(s) of the invention
5 may be formulated with one or more additional cationic amphiphiles
including those known in the art, or with neutral co-lipids such as
dioleoylphosphatidyl-ethanolamine, (" DOPE"), to facilitate delivery to cells
of the biologically active molecules. Additiona~ly, compositions that
comprise one or more cationic amphiphiles of the invention can be used to
10 introduce biologically active molecules into plant cells, such as plant cells in
tissue culture.
Additionally, the present application provides for novel plasmids
suitable for complexing with the amphiphiles of the invention in order to
treat patients by gene therapy, so that a high level of expression of the
15 appropriate therapeutic transgene can be achieved. Representative
examples thereof include the plasmid pCMVHI and pCFI. pCF1 plasmid
contains the enhancer/promoter region from the immediate early gene of
cytomegalovirus. The plamid also contains a hybrid intron located between
the promoter and the transgene cDNA. The polyaden!rlation signal of the
20 bovine growth hormone gene was selected for placement downstream from
the transgene. These and other features contribute substantially to the
improved transgene expression possible with this plasmid.
Further enhancements in plasmid performance are made possible by
the provision of replicating episomal plasmids. Additional therapeutic
25 enhancements are made possible by providing plasmids in which
expression of the therapeutic transgene is placed under the control of a
transcriptional promoter that is sensitive to the concentration of
16
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i~flammation-related substances in the target tissue. Such plasmids are of
particular use for the treatment of clinical cases in which inflammation is a
major complication.
In a still further embodiment of the invention, particular organs or
5 tissues may be targeted for gene therapy, by intravenous administration of
amphiphile/transgene complexes, by adjusting the ratio of amphiphile to
DNA in such complexes, and by adjusting the apparent charge or zeta
potential thereof.
Further additional and representative aspects of the invention are
10 described according to the Detailed Description of the Invention which
follows directly.
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Brief Description of the Drawings
FIGURE1 depicts representative Group I cationic amphophiles.
FIGUl~E 2 depicts repres~nldliv~ steroid lipophilic groups.
FIGURE 3 depicts represelllaliv~ steroid lipophilic groups.
5 FIGURE 4 depicts a transacylation reaction.
FIGURE 5 depicts represenative Group II cationic amphiphiles.
FIGURE 6 depicts represenative Group m cationic amphiphiles.
FIGURE 7 depicts representative Group IV cationic amphiphiles.
FIGURE 8 provides a route of synthesis for spermidine cholesterol
10 carbamate.
FIGU3~E 9 provides a route of synthesis for spermine cholesterol carbamate
FIGURE 10 provides a comparison of in vivo transfection efficiency for
certain cationic amphiphiles under particular conditions.
FIGURE 11 is a depiction of in vivo transfection effeciency as a function of
15 DNA concentration for a particular cationic amphiphile.
FIGURE 12 is a depiction of in vivo transfection effeciency as a function of
concentration of a particular cationic amphiphile.
FIGURE 13 provides relative transfection efficiencies for Group I
amphiphiles.
20 FIGURE 14 provides relative transfection efficiencies for Group II
amphiphiles.
FIGURE 15 provides relative transfection efficiencies for Group IV
amphiphiles.
FIGURE 16 provides a map of pCMVHI-CAT plasmid.
25 FIGURE 17 shows the hybrid intron of pCMVHI-CAT.
FIGURE 18 (panel A) provides a map of pCF1/CAT pl~mi~l
FIGURE 18 (panel B) provides a map of pCF2/CAT pl~mill
18
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FIGURE 19 shows a plot of corrected chloride ion transport in nasal polyp
epithelial cells from a cystic fibrosis patient.
FIGURE 20 provides a map of pMyc4-CFTR plasmid.
FIGURE 21 demonstrates intravenous targeting of the heart and lung.
19
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Detailed Description of the Invention
Information Concerning the Structure of Cationic Amphiphiles of the
Invention
This invention provides for cationic amphiphile compounds, and
5 compositions containing them, that are useful to facilitate transport of
biologically active molecules into cells. The amphiphiles are particularly
useful in facilitating the transport of biologically active polynucleotides intocells, and in particular to the cells of patients for t-he purpose of gene
therapy.
Cationic amphiphiles according to the practice of the invention
possess several novel features. These features may be seen in comparison
with, for example, cationic amphiphile structures such as those disclosed in
U.S. Patent No. 5, 283,185 to Epand et al., a representative structure of which
is is 3~ [N-(N 1,N 1 dimethylaminoethane)-carbamoyl] cholesterol,
15 commonly known as "DC-chol", and to those disclosed by Behr et al. Proc.
Natl. Acad. Sci. USA, 86, 6982- 6986 (1989), a representative structure of
which is dioctadecylamidolo-glycylspermine ("DOGS").
Cationic amphiphiles of the present invention contain distinctive
structural features: (1) the presence of a lipophilic group which is connected
20 directly, or through a linking group, to two cationic groups (see below) that themselves comprise amino, alkylamine or polyalkylamine groups, there
resulting an overall and novel "T-shaped" structure; and (2) in many cases,
and in comparison with numerous art-recognized amphiphiles, the use of a
relatively short linking group to bring into close proximity the lipophilic
25 and cationic regions of the amphiphile. Without being limited as to theory,
it is believed that these features contribute substantially to the transfection-enhancing capability of these compounds. As an example of this, Figure 10
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below demonstrates the very substantial in vivo transfection-enhancing
capability of sp~ 1ine cholesterol carbamate (a novel amphiphile of the
invention) in comparision to DC- chol and DMRIE--two well recognized
transfectants.
In connection with the practice of the present invention, it is noted
that "cationic" means that the R groups, as defined herein, tend to have one
or more positive charges in a solution that is at or near physiological pH.
Such cationic character may enhance interaction of the amphiphile with
therapeutic molecules (such as nucleic acids) or with cell structures (such as
plasma membrane glyco~role" ls) thereby contributing to successful entry of
the therapeutic molecules into cells, or processing within subcompartments
(such as the nucleus) thereof. In this regard, the reader is referred to the
numerous theories in the art concerning transfection-enhancing function of
cationic amphiphiles, none of which is to be taken as limiting on the practice
of the present invention.
Biological molecules for which transport into cells can be facilitated
according to the practice of the invention include, for example, genomic
DNA, cDNA, mRNA, antisense RNA or DNA, polypeptides and small
molecular weight drugs or hormones. Representative examples thereof are
mentioned below in connection with the description of therapeutic
(pharmaceutical) compositions of the invention.
In an imporant embodiment of the invention the biologically active
molecule is an encoding polynucleotide that is expressed when placed in the
cells of a patient leading to the correction of a metabolic defect. In a
particularly important example, the polynucleotide encodes for a
polypeptide having an amino acid sequence sufficiently duplicative of that
of human cystic fibrosis transmembrane regulator ("CFTR") to allow
-
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possession of the biological property of epithelial cell anion channel
regulation.
As aforementioned, characteristic and novel features of the
amphiphiles of the invention include first, that the linking group that
5 connects the two cationic amine groups to the lipophilic group is very short,
or absent entirely, and second, that the resultant linking of the the two
cationic R groups to the lipophilic group forms a T-shaped structure when
viewed from the position of atom "X" (a carbon or nitrogen atom) as
depicted, for example, in Structures (I), (II), (m) and (IV, see atom "E").
As examples of the cationic amphiphiles of the invention, both
sp~ ine cholesterol carbamate ( N4 -spermidine cholesteryl carbamate)
and spermine cholesterol carbamate ( N4 -spermine cholesteryl carbamate)
have been determinedto be superior transfectants in vivo in comparison
with non "T-shaped" amphiphiles having otherwise equivalent amounts of
15 cationic alkylamine structure. Superior performance (see also Example 3)
has been determined for:
~'~
~ ,C~
H2N /'N,~_
NH2 ( spermidine cholesterol carbamate )
in comparison with, for example,
H2N '--N----NJIo~
H (N1-spermidine cholesteryl
carbamate).
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Additionally, superior performance has been determined for
H N~--N ~
H
NH2 ( spermine cholesterol carbamate )
5 in comparison with, for example,
~ ~ 4
H2N ~ N----N----N O
H H- H (Nl-Thermospermine
cholesteryl
carbamate), and
H O ~<
H2N N ~ N----N~O
H (Nl-Spermine
cholesteryl carbamate).
Applicants have also noted that numerous of the cationic
15 amphiphiles of the invention have structural features in common with
naturally occurring polyamines such as spermine and spermidine (including
~ N-atom spacing). In this regard, the structures of amphiphiles 53, 67, 78, 90,
and 91 are representative. As can be seen by examination of the data in
Figures 13, 14 and 15, the placement of the nitrogen atoms in the polar head
20 groups of the amphiphiles such that they are separated by one or more
combinations of 3 and 4 carbon atoms leads to high in vivo transfection
efRciency for plasmid transgenes complexed therewith. Applicants have
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also noted that these in-common structural features may have a useful effect
upon the binding of the amphiphiles to DNA, and on interaction with cell
surface polyamine rece~lol~. Interaction with cell polyamine receptors may
be particularly important with respect to the treatment of cancer cells by
5 gene therapy, since the DNA replication requirements of such cells may lead
to high level expression of such receptors.
Group I Amphiphiles
In connection with the design of the Group I amphiphiles of the
invention, the following considerations are of note. Many of these design
10 features are then discussed in connection with the other amphiphiles of the
invention, those ~l~ssifie~l under Groups II, II and IV.
Accordingly, there are provided cationic amphiphiles of Group I (see
Figure 1, panels A, B, and C) capable of facilitating transport of biologically
active molecules into cells, said amphiphiles having the structure (I),
(R3) (R1)
(~ (Y) (~
(R4) (R2) (I)
wherein:
Z is a steroid;
20 X is a carbon atom or a nitrogen atom;
Y is a short linking group, or Y is absent;
R3 is H, or a saturated or unsaturated aliphatic group;
R1 is --NH--, an alkylamine, or a polyalkylamine;
24
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R4 is H, or a saturated or unsaturated aliphatic group;
R2 is NH--, an aLkylamine, or a polyalkylamine;
and wherein R1 is the same or is dif~r~lt from R2, except that both R1 and
R2 cannot be --NH--.
5 The Linking Group
Preferably the linking group that connects the lipophilic group to the
two cationic R groups is relatively short. It is ~refeired that within linking
group Y are contained no more than about three or four atoms that
themselves form a bridge of covalent bonds between X and Z. Examples of
10 Y groups include--
-(CH2)2-; -(cH2)3-; -(cH2)-(c=o)-; -(cH2)n-NH-(c=o)- where n is
plereLdbly 4 or less. Additional linking groups useful in the practice of the
invention are those patterned on small amino acids such as glycinyl, alanyl,
beta-alanyl, serinyl, and the like.
With respect to the above representations, the left hand side thereof-
as depicted- is intended to bond to atom "X", and the right hand side
thereof to group "Z"( see structure I).
In certain preferred embodiments of the invention, Y is a linking
group wherein no more than one atom of this group forms a bond with both
20 "X"and "Z". Examples of ~le~erred linking groups include --CH2--, >C=S,
and >C=0. Alternatively, the linking group "Y"may be absent entirely.
As aforementioned (see Structure I, directly above), "X" forms a
connecting point in the amphiphiles to which is also attached the two
cationic R groups. As can be seen therein (see also Figure 1), the placement
25 of the nitrogen atom that represents "X" clearly causes the molecule to
assume a T-shape.
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Steroid Lipophilic Groups
Cationic amphiphiles according to the practice of the invention may
include a variety of structures as lipophilic group. Steroids represent a
p~efelred group of such structures.
With respect to the design and orientation of steroids as lipophilic
groups according to the practice of the invention, the following
considerations are of note. Steroids are widely distributed in the animal,
microbial and plant kingdoms. They may be defined as solid alcohols that
typically contain, as their basic skeleton, 17 carbon atoms arranged in the
form of a perhydrocyclopenteno-phenanthrene ring system. Accordingly,
such compounds include bile acids, cholesterol and related substances,
vitamin D, certain insect molting hormones, certain sex hormones, corticoid
hormones, certain antibiotics, and derivatives of all of the above wherein
additional rings are added or are deleted from the basic structure. [see
Natural Products Chemistry, K. Nalcanashi et al. eds., Academic Press, Inc.,
New York (1974), volume 1, at Chapter 6 for a further discussion of the
broad classes of molecules that are understood in the art to be steroids].
Additionally, for the purposes of the invention, the term steroid is used
broadly to include related molecules derived from multiple isoprenoid
units, such as vitamin E. Steroids representative of those useful in the
practice of the invention are shown in Figures 1, 2, 3 and 5.
As elaborated below, certain ~Lefelled amphiphiles of the invention
include a steroid component "Z" that is selected from the group consisting
of 3-sterols, wherein said sterol molecule is linked by the 3-O- group thereof
, or by N- in replacement thereof, to Y (see Figure 1). Such structures
include, for example, spPrmi~1ine cholesterol carbamate, spermine
cholesterol carbamate, spermidine 7-dehydrocholesteryl carbamate, lysine
26
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3-N-dihydrocholesteryl carbamate, spermi(line cholestamine urea, and N-
3-amino~ro~yl-N-~aminobutylcholestamine.
In a further ~refelled embodiment, the steroid group is linked to Y
(or directly to X if Y is absent) from ring position 17 of the steroid nucleus
5 (see Figures 1 and 3), or from the arm that normally extends from position
17 in many steroids (see Figures 1 and 3), or from any shortened form of
said arm.
In connection with the selection of steroids for inclusion in the
amphiphiles of the invention, it is preferred that the molecules have
10 structures which can be metabolized by the body and are nontoxic at the
doses thereof that are used. Prerelred are steroids such as cholesterol and
ergosterol that are substantially non toxic and which possess biologically
normal stereospecificity in order to facilitate their safe metabolism in
patients. Additional steroids useful in the practice of the invention include,
15 for example, ergosterol B1, ergosterol B2, ergosterol B3, androsterone, cholic
acid, desoxycholic acid, chenodesoxycholic acid, lithocholic acid and, for
example, various derivatives thereof as are shown in the panels of Figures 2
and 3.
With respect to the orientation of the steroid lipophilic group, that is,
20 how the group is attached(with or without a linker) to the cationic (alkyl)
amine groups of an amphiphile, the following further information is of note.
Any ring position or substituent on the steroid can in general be used as
point of attachment. It is plererred, however, to use a point of attachment
that (1) rnimimi7~ the complexity of chemical syntheses, and (2) is
25 positioned near either "end" of the steroid molecule, for example, a
position near ring position 3, or near ring position 17( or the arm that
typically extends thererloln). Such positions provide an orientation of the
27
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W O96118372 PCTnUS95/16174
steroid with respect to the rest of the amphiphile structure that faciliates
bilayer formation, and~or micelle formation, and/or stabilizes interaction
with the biologically active molecules to be carried into the target cells.
Representative structures showing attachment of the cationic (alkyl) amine
groups to the steroid lipophilic group through the arm extending from ring
position 17 therof are shown in Figure 3 (panels A, B). With respect to this
type of structure, it is further ple~Lr~d that any polar groups on the steroid,
such as may be attached to ring position 3, be either removed or capped (for
example, hydroxy as methoxy) to avoid potentially destabilizing bilayer or
micelle struc~ures.
The representation in Figure 3 of cationic amphiphiles in which the
steroid lipophilic group thereof is linked to the cationic alkylamine groups
through steroid ring position 17 is but an example of the invention.
Similarly, the representation in Figures 1 to 4 of cationic amphiphiles in
which the steroid lipophilic group thereof is linked to the cationic
alkylamine groups through steroid ring position 3 is an example of the
invention. As aforementioned, use of any steroid ring position (or moiety or
branch extending therefrom) as point of attachment is within the practice of
the invention.
Preferred steroids for use as group "Z" according to the practice of
the invention include:
3- sterols (derived from cholesterol)
~Y
~ ~ 15
- 0~
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3-N steryl groups (patterned on cholesterol)
~ 15
_ NJ~
5 ergosterol and derivatives
HOJ~
Representative species of steroid that are patterened on ergosterol
and that may be used to define the structure of cationic amphiphiles of the
invention include: ergosterol (double bonds as shown); ergosterol Bl (~ 8, 9;
~ 14, 15; ~ 22, 23); ergosterol Bl (~ 6, 7; ~ 8, 14; ~ 22, 23); ergosterol Bl (~ 7, 8;
~14, 15; ~ 22, 23); and lumisterol ( the 9b-H isomer of ergosterol).
cholic acid and derivatives
COOH
r2~
-
,L3 4 6 7-~
HO ~ r
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Representative species of steroid that are patterened on cholic acid
and that may be used to define the structure of cationic amphiphiles of the
,.,velLlion include: cholic acid wherein r1 and r2 = OH; desoxycholic acid
wherein r1 - H and r2 = OH; chenodesoxycholic acid wherein rl = OH and
5 r2 = H; and lithocholic acid wherein r 1 and r2 = H.
androsterone and derivatives thereof
,~,
r~
HO~J
Selection of Groups R1, _2, _3, and R4
For _3and R4:
According to the practice of the invention R3 and R4 are,
15 independently, H, or saturated or unsaturated aliphatic groups. The
aliphatic groups can be branched or unbranched. Representative groups
include alkyl, alkenyl, and cycloalkyl.
For Rl and R2:
R1 and R2 represent structures recognized in the art as being amine;
20 alkylamines (including primary, secondary, and tertiary amines), or
extended versions thereof-herein tPrme~1 "polyalkylamines". It is
understood that both alkylamine and polyalkylamine groups as defined
herein may include one or more carbon-carbon double bonds and the use of
such alkenylamines is therefore within the practice of the invention.
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RepresenLalive alkylamines include: (a) NH-(CH2)z--where z is
other than 0; (b) -- [[CH3(CH2)y]N] -(CH2)z--where z is other than 0; and
(c)--[[CH3(CH2)X][CH3(cH2)y]]N-(cH2)z -- where z is other than 0.
With respect to the circumstance where one or both of Rl and R2 are
5 tertiary amines, such as is represented in Structure (c) above, it is
understood that a hydrogen atom corresponding to either R3 or R4, as
a~pro~riate, may or may not be present since such hydrogen atoms
correspond to the N:H(+) structure whose level of protonation will vary
according to pH.
The term "polyalkylamine" as referred to herein defines a polymeric
structure in which at least two alkylamines are joined. The alkylamine units
that are so joined may be primary or secondary, and the polyalkylamines
that result may contain primary, secondary, or tertiary N-atoms. The
alkylamine (sub)units may be saturated or unsaturated, and therefore the
term "alkylamine" encompasses alkenylamines in the description of the
venLion.
Representative resultant polyalkylamines include: (d) -- [NH-
(CH2)(z)]q --, where z is other than 0, and q is 2 or higher; (e) -- ~NH-
(CH2)(y)]p--[NH~(CH2)(z)]q--, where y and z are each other than 0, and p
and q are each other than 0; (f)--[NH-(CH2) (x)] n --[NH-(CH2) (y) ~p--[NH-
(CH2) (z) ~q --, where x, y, and z are each other than 0, and n, p and q are
each other than 0; (g) -- [NH-(cH2)(w)]m -- [NH-(CH2)(x)]n--[NH-
(CH2)(y)]p--[NH-(CH2)(z)]q--, where w, x, y, and z are each other than 0,
and m, n, p, and q are each other than 0; (h) -- [NH-(CH2) (w) ]m--[NH-
(CH2)(x)]n--[[cH3(cH2)y]N] -(cH2)z --, where x, n and z are each other
than 0; (i) --[NH-(CH2)(w)]p--[[CH3(CH2)x]N]-(CH2)y--[NH-(CH2)(z)]q
r ~~ ~ where w, p, y, z, and p are each other than 0; and G)
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-- [NH-(CH2) (v) ]l--[NH-(CH2) (w) ]m [NH-(CH2) (x)]n--[NH-(CH2) (y) ]p
-- INH-(CH2) (z)]q --, where v, w, x, y, and z are each other than 0, and l, m,
n, p, and q are each other than 0.
As mentioned above R1 and R2, independently, can be NH--, an
5 alkylamine, or a polyalkylamine, and can be the same or different from each
other, except that both R1 and R2 cannot be --NH-- in order to (1) preserve
the "T- shape" of the resultant compound, and (2) to provide for the
stability thereof. It is ~refelled that - in combination- the combined
backbone length of R3R1 (or of R4R2) be less than about 40 atoms of
10 nitrogen and carbon, more
~re~elldbly less than about 30 atoms of nitrogen and carbon.
In the case where the R1 group adjacent to R3 (or R2 adjacent to R4)
includes a terrninal nitrogen atom that defines a tertiary center, then a
quaternary amine is formed (at that nitrogen atom of R1) if R3 is an
15 aliphatic group, and a tertiary amine remains (at that nitrogen atom of R1) if
R3 is H. Accordingly, with respect to such resultant R3R1 or R4R2
structures, repres~llLalive respective formulas are:
(k) H-(CH2)(w)--~cH3(cH2)x]~cH3(cH2)y]N] -(cH2)z--, where w and
z are each other than zero; and (l) H--[[CH3(CH2)x][CH3(CH2)y]Nl-
20 (CH2)z--, where z is other than zero.
In connection with inlelpreLi~lg the structural diagrams describedherein, it is intended that the attachment of R3R1--(or R4R2_ ) structures
to atom "X" is through the right hand side (as depicted) of the R3R1--, that
is, through a CH2--moiety. The coefficents (i.e. v, w, x, y, and z and l, m, n,
25 p, and q) as depicted herein represent whole numbers. For the purposes of
the invention, "whole number" means 0 and the natural numbers
1,2,3,4,5,6.. and up, unless spe~ifi~lly restricted.
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With respect to the amphiphiles of the invention including those
represented by formulas (a) to (l), it is noted that there are certain
~ ~re~eLellces concerning the design of such groups depending on whether
atom 'X" as it is shown according to structure (I) above, is a nitrogen atom
5 or a carbon atom. If "X" is nitrogen, then amphiphiles containing R3-R1 (
or R4-R2 ) groups that end in an N atom [ i.e formula (e) where z equals 0
and q=1; formula (h) where z equals 0] are not preferred, since the resultant
N-N linkage involving position X results in an amphiphile that may be
unstable and/or difficult to prepare. An additional group of structures that
10 are difficult to prepare and/or are unstable is represented, for example, by
the R sequence (whether in Rl, or bridging Rl and R3)--NH- CH2-NH-
CH2-- . Accordingly, use of such structures [ i.e. formula (a) where Z equals
1, formula (e) where one or both of y and z equals 1]in the practice of the
invention is not ple~lled.
With respect to the design of structures (such as those depicted
above) for inclusion in cationic amphiphiles, the following further
considerations are of note. Any combination of alternating amine and alkyl
moieties creates an R structure within the scope of the invention. A
polyalkylamine may be represented, for example, by the formulas above,
although many more structures (such structures being within the scope of
the invention) can be depicted by extending the number of, or types or
combinations of, alkylamine subunits within the amphiphile structure.
That further such variations can be made is apparent to those skilled in the
art.
It is noted that a polyaLkylamine group (or resultant R3R1 group) that
is very long may il~LeL~ele, for example, with the solubility of the resultant
amphiphile, or interfere with its ability to stably interact with the
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biologically active molecule selected for intracellular delivery. In this
regard, polyalkyl~mine~ (or resultant R3Rl groups) having a backbone
length of about 40 nitrogen and carbon atoms, or more, may not be suitable
for inclusion in amphiphiles. However, for each such proposed structure,
5 its properties may be determined by experimentation, and its use is
nonetheless within the practice of the invention.
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Accordingly, specific alkylamine and polyalkylamine structures
result as follows:
Table 1
ForRl and/or
R2
.,
(1) -NH-
(2) -NH-(CH2) (2)-
(3) -NH-(cH2) (3) ~
(4) -NH-(CH2) (4)~
(5) -NH-(CH2) (6) ~
(6) -NH-(CH2) (3) - NH-(cH2) (4)-
(7) -NH-(CH2) (2)- NH-(CH2) (2)-
(8) -NH-(CH2) (4)- NH-(CH2) (3)-
(9) -NH-(CH2) (y) - NH-(CH2) (z) -
(10) -NH-(CH2) (X)-NH-(CH2) (y) -NH-(cH2) (z) -
(11) -NH-(CH2) (W) -NH-(cH2) (x)-NH-(cH2) (y) -NH-(CH2) (Z) -
(12) -NH-(CH2) (v) -NH-(CH2) (w) -NH-(CH2) (x)-NH-(CH2) (y) -NH-
(CH2) (Z)-
(13) -[NH-(CH2)(w)]m -- [NH-(CH2)(x)]n--[[CH3(CH2)y]N] -(CH2)z-
(14)-[NH-(CH2)(x)]n--[[CH3(CH2)y]N]-(CH2)z~
(15) -[NH-(CH2) (w)]m -- [NH-(CH2) (x)]n--[[CH3(CH2)y]N] -(CH2)z -
(16) - [[CH3(CH2)X][CH3(cH2)y]N]-(cH2)z--
(17) -NH-(cH2) (z)- NH--
(18) -NH-(CH2) (y)-NH-(cH2) (z)-NH--
25 (19) -NH-(CH2) (y)-CH=CH-(CH2)z--
(20) --[NH-(CH2)(w) ]p--[[CH3(CH2)x]N] -(CH2)y - [NH-(cH2) (z) ]q -
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FQrR3 and/or
R4
(1) H--
(2) CH3--
(3) CH3-(CH2)2-
(4) CH3-(CH2)4--
(5) CH3-(CH2)z-
(6) CH3-[CH3-(CH2)Z]CH--
(7) CH3-~CH3-(CH2)2]CH--
(8) CH3-[1CH3-(CH2)y][CH3~(CH2)z]]C~~
(9) CH3-(CH2)z-CH=CH~CH2--
(10) CH3-1CH3-(CH2) y-CH=CH-(CH2)z ]CH--
(11) CH3-[[cH3-(cH2)w-cH=cH-(cH2)x][cH3-(cH2)y-cH=
(CH2)z]]cH--
(12) CH3-[CH3-(CH2)y]CH-(CH2)z-
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Group II Amphiphiles
Additionally there are provided cationic amphiphiles of Group II (see
Figure 5) capable of facilitating transport of biologically active molecules
into cells said amphiphiles having the structure (II),
R3) - (R
(~ (Y) (~
(R4) (R2) (II)
wherem:
Z is a steroid;
10 X is a carbon atom or a nitrogen atom;
Y is a linking group or Y is absent;
R3 is an amino acid, a derivatized amino acid, H or alkyl;
R1 is --NH--, an alkylamine, or a polyalkylamine;
R4 is an amino acid, a derivatized amino acid, H or alkyl;
15 R2 is --NH--, an alkylamine, or a polyalkylamine;
and wherein R1 is the same or is different from R2, except that both R1 and
R2 cannot be --NH--.
Representative amphiphiles provided according to Group II include
amphiphiles 87, 91, 93, 95, 97, 99, 100, and 103. With respect to the structural20 features of these amphiphiles, and the other amphiphiles of Group II, the
following should be considered.
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The steroid group may be selected according to the criteria defined
above for the Group I amphiphiles. Accordingly, pre~lred amphiphiles
include those sf:~lectf~ from 3- sterols, wherein the sterol molecule is linked
by the 3-O- group thereof, or by N in replac~ nt thereof, to "Y".
The linking group Y of the Group II amphiphiles consists of an N-
acylamino acid (or a derivative thereofl, or consists of a group (such as >
C=O or > C=S) wherein no more than one atom of said group forms a bond
with both "X" and "Z". Optionally, group Y may be absent. Representative
N-acylamino groups include an N-Acyl serine ( No. 87), an N-Acyl glycine
(No. 91), and an N-Acyl aspartic acid ( No. 103). With respect to the use of
N-Acyl aspartic acid in amphiphile No. 103, it is noted that, as provided, the
gamma carboxyl thereof is further derivatized to an additional alkylamine
moiety.
The crtiteria for selection of Rl and R2 are as set forth for the Group I
amphiphiles. R3 and R4 represent H or alkyl, or may be natural or artificial
amino acids ~ncluding derivatives of either. Representative examples of R3
or R4 amino acid groups include those derived from l,yplo~han ( No. 97)
and from arginine ( No. 95).
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Group III Amphiphiles
Additionally there are provided cationic amphiphiles of Group III
(see Figure 6) capable of facilitating transport of biologically active
molecules into cells said amphiphiles having the structure (m),
(R3) (Rl)
(~ (Y) (~
(R4) (R2) (m)
wherein:
Z is an alkylamine or a dialkylamine, linked by the N-atom thereof, to Y, or
directly to X if Y is absent, wherein if Z is a dialkylamine, the alkyl groups
thereof can be the same or diL~lellL,
X is a carbon atom or a nitrogen atom;
Y is a short linking group, or Y is absent;
R3 is H, or a saturated or unsaturated aliphatic group;
Rl is ~NH--, an aL~cylamine, or a polyalkylamine;
R4 is H, or a saturated or unsaturated aliphatic group;
R2 is --NH--, an alkylamine, or a polyalkylamine;
and wherein Rl is the same or is different ~rom R2, except that both Rl and
R2 cannot be --NH
Representative cationic amphiphiles according to the practice of the
invention that contain an alkyl amine or dialkylamine as lipophilic group
r include, for example, N,N-dioctadecyllysine~mi~1e; 1~l, Nl-dioctadecyl-
1,2,6- triaminohexane; N,N-didodecyllysineamide; N,N-
didecyllysineamide; spermidine- N,N- dioctadecyl urea; N-
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myristyllysin~micle; and N-(dioctyldecylaminoethyl)-lysi~e~mit1e .
Representative amphiphiles are depicted (Figure 6) as amphiphiles 43, 47,
56, 60, and 73. With respect to the structural features of these amphiphiles,
and the other amphiphiles of Group III, the following should be considered.
With respect to the selection of the lipophilic alkylamine or
dialkylamine group "Z", Table 2 below provides representative structures.
Table 2
For "Z"
(1) CH3-(CH2)13-NH--
(2) CH3-(CH2)z-NH--
(3) 1ICH3(CH2) 17][CH3(CH2) 17]]N
(4) 1[CH3(CH2) 11][CH3(CH2) 11]]N--
(5) [[CH3(CH2) 9] [CH3(CH2) g]]N
(6) [[CH3(CH2)x][CH3(CH2)y]]N--
(7) [[CH3(CH2) x] [CH3(CH2) yCH=CH(CH 2) z]]N--
(8) [lcH3(cH2)w][cH3(cH2)xcH=cH(cH2)ycH=cH(cH2)z]]N--
In connection with the selection of suitable alkylamine or
dialkylamine groups for inclusion at position Z in the amphiphiles of the
invention, an alkyl chain(s) of the group should not be so large in molecular
weight that it interferes with the solubility of the amphiphile, or interferes
with its ability to interact with plasmid DNA. Additionally, an alkyl chain
of an alkylamine or dialkylamine may include one or more points of
unsaturation. The selection of R groups R1, R2, R3, and R4 follows that
disclosed for the Group I amphiphiles, and these R groups may be selected,
for example, from Table I. Linking group Y may be seleected as for the
Group I amphiphiles, and plef~lled examples thereof include--CH2--,
and > C=O.
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Group IV Amphiphiles
Additionally there are provided cationic amphiphiles of Group IV
(see Figure 7) capable of facilitating transport of biologically active
molecules into cells said amphiphiles having the structure (IV),
(j)~ ~ <(R ) (R3)
(B) ~R2~--(R4)
(R5)
(R6) (IV)
wherem:
A and B are independently O, N or S;
10 R5 and R6 are independently alkyl or acyl groups and may be saturated or
col,laill sites of unsaturation;
C is selected from the group consisting of -CH2-, >C=O, and >C=S;
E (analogous to "X" in structures I, II, m) is a carbon atom or a nitrogen
atom;
15 D is a linking group such as -NH(C=O)- or -O(C=O)-, or D is absent;
R3 is H, or a saturated or unsaturated aliphatic group;
R1 is --NH--, an alkylamine, or a polyalkylamine;
R4 is H, or a saturated or unsaturated aliphatic group;
R2 is NH--, an alkylamine, or a polyalkylamine;
20 and wherein R1 is the same or is different from R2, except that both Rl and
R2 cannot be NH--.
Representative amphiphiles of Group IV include Nos. 64, 76, 85, 89,
94, 98, 102, 105, 110, and 111. With respect to the structural features of these
41
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amphiphiles, and the other amphiphiles of Group IV, the following should
be considered.
With respect to the s~lechon of R1, R2, R3, and R4, the teachings
provided for Group I, II, and m amphiphiles are applicable. As
5 aforemenh-)ne~l, group l'E" represents a carbon atom or a nitrogen atom.
R5 and R6 are independently alkyl or acyl groups, ~re~lldbly
containing about 8 to about 30 carbon atoms, and such groups may contain
one or more points of unsaturation.
With respect to the selection of Group D, linkers such as -NH(C=O)-
10 or -O(C=O)- are ple~lled, and are depicted such that the left side thereof
in intended to bond to "C" and the right side thereof is intended to bond to
"E". Optionally, group D may be absent (amphiphile No.94). Additional
linkers may be selected based on the teachings provided with respect to
Groups I, II, and m above, and based upon the in vivo test date derived (
15 Figure 15), it is yre~lred that the linker D be short or absent.
Co-lipids
Representative co-lipids that are useful according to the practice of
the invention for mixing with one or more cationic amphiphiles include
dioleoylphosphatidylethanolamine ("DOPE"), diphytanoylphosphatidyl-
20 ethanolamine, lyso-phosphatidylethanolamines other phosphatidyl-
ethanolamines, phosphatidylcholines, lyso-phosphatidylcholines and
cholesterol. Typically, a ~re~lled molar ratio of cationic amphiphile to
colipid is about 1:1. However, it is within the practice of the invention to
vary this ratio (see Example 3 below), including also over a considerable
25 range.
It is generally believed in the art that preparing cationic amphiphiles
as complexes with co-lipids (particularly neutral co-lipids) enhances the
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capability of the amphiphile to facilitate transfections. Although colipid-
enhanced performance has been observed for numerous of the amphiphiles
of the invention, the amphiphiles of the invention are active as transfectants
without co-lipid. Accordingly, the practice of the present invention is
5 neither to be considered limite(l by theories as to co-lipid participation in
intracellular delivery mechanisms, nor to require the involvement of co-
lipids.
Transacylation Reactions
Although heretofore unrecognized in the art, it has been determined
10 also that certain co-lipids may react chemically with certain types of
cationic amphiphiles under conditions of co-storage, there resulting new
molecular species. Generation of such new species is believed to occur via
mechanisms such as transacylation. In this regard, see Figure 4 which
depicts a transacylation reaction involving spermine cholesterol
15 carbamate(No.67) and DOPE, there resulting lyso PE species and multiple
forms of particular acyl- cationic amphiphile ( designated No. 80).
With respect to such reactions, the following remarks are of interest.
With respect to use of amphiphile No.67, it has been observed that a mixture
of amphiphile and DOPE, in chloroform solvent, does not appear to
20 participate in such reactions. However, preparing the amphiphile and co-
lipid in an aqueous solution where bilayer-containing structures such as
liposomes can form will permit transacylation. Additionally, if amphiphile
and co-lipid are dried down to a thin film, such as from chloroform (thereby
placing the 2 species in intimate contact), then transacylation also occurs,
25 possibly as a result of ellLl'~ic effects. It is expected that these phenomena
would also apply to lyophilized amphiphile/DOPE preparations.
43
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Accordingly, it is highly ~reLell~d to maintain such amphiphile
/DOPE preparations at very cold temperatures, such as -70 degrees C.
Preparation of amphiphile No. 67 as a mono, di, or tri acetate salt has also
been determined to slow transacylations.
It is to be understood that therapeutically-effective pharmaceutical
compositions of the present invention may or may not contain such
transacylation byproducts, or other byproducts, and that the presence of
such byproducts does not prevent the therapeutic use of the compositions
containing them. Rather use of such compositions is within the practice of
the invention, and such compositions and the novel molecular species
thereof are therefore spe~ifi~lly claimed.
Preparation of Pharmaceutical Compositions and Admimstration Thereof
The present invention provides for pharmaceutical compositions that
facilitate intracPlllllar delivery of therapeutically effective amounts of
biologically active molecules. Ph~ ceutical compositions of the invention
facilitate entry of biologically active molecules into tissues and organs such
as the gastric mucosa, heart, lung, and solid tumors. Additionally,
compositions of the invention facilitate entry of biologically active molecules
into cells that are maintained in vifro , such as in tissue culture. The
amphiphilic nature of the compounds of the invention enables them to
associate with the lipids of cell membranes, other cell surface molecules, and
tissue surfaces, and to fuse or to attach thereto. One type of structure that
can be formed by amphiphiles is the liposome, a vesicle formed into a more
or less spherical bilayer, that is stable in biological fluids and can entrap
biological molecules targeted for intracellular delivery. By fusing with cell
membranes, such liposomal compositions permit biologically active
molecules carried therewith to gain access to the interior of a cell through
44
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one or more cell processes including endocytosis and pinocytosis.
However, unlike the case for many classes of amphiphiles or other lipid-like
molecules that have been proposed for use in therapeutic compositions, the
cationic amphiphiles of the invention need not form highly organized
5 vesicles in order to be effective, and in fact can assume (with the biologically
active molecules to which they bind) a wide variety of loosely organized
structures. Any of such structures can be present in pharmaceutical
preparations of the invention and can contribute to the effectivenesss
thereof.
Biologically active nlolec~ s that can be provided intracellularly in
therapeutic amounts using the amphiphiles of the invention include:
(a) polynucleotides such as genomic DNA, cDNA, and mRNA that encode
for therapeutically useful proleills as are known in the art,
(b) ribosomal RNA;
15 (c) antisense polynucleotides, whether RNA or DNA, that are useful to
inactivate transcription products of genes and which are useful, for
example, as therapies to regulate the growth of malignant cells; and
(d) ribozymes.
In general, and owing to the potential for leakage of contents
20 therefrom, vesicles or other structures formed from numerous of the cationic
amphiphiles are not ~rerelled by those skilled in the art in order to deliver
low molecular weight biologically active molecules. Although not a
preferred embodiment of the present invention, it is nonetheless within the
practice of the invention to deliver such low molecular weight molecules
25 intracellularly. Representative of the types of low molecular weight
biologically active molecules that can be delivered include hormones and
antibiotics.
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Cationic amphiphile species of the invention may be blended so that
two or more species thereof are used, in combination, to facilitate entry of
- biologically active molecules into target cells and/or into subcellularcompartments thereof. Cationic amphiphiles of the invention can also be
5 blended for such use with amphiphiles that are known in the art.
Dosages of the ph~ ceutical compositions of the invention will
vary, depending on factors such as half-life of the biologically-active
molecule, potency of the biologically-active molecule, half-life of the
amphiphile(s), any potential adverse effects of the amphiphile(s) or of
10 degradation products thereof, the route of administration, the condition of
the patient, and the like. Such factors are capable of determination by those
skilled in the art.
A variety of methods of administration may be used to provide
highly accurate dosages of the pharmaceutical compositions of the
15 invention. Such preparations can be administered orally, parenterally,
topically, transmucosally, or by injection of a preparation into a body cavity
of the patient, or by using a sustained-release formulation containing a
biodegradable material, or by onsite delivery using additional micelles, gels
and liposomes. Nebulizing devices, powder inhalers, and aerosolized
20 solutions are representative of methods that may be used to administer such
preparations to the respiratory tract.
Additionally, the therapeutic compositions of the invention can in
general be formulated with excipients (such as the carbohydrates lactose,
trehalose, sucrose, mannitol, maltose or galactose) and may also be
2~ lyophilized (and then rehydrated) in the presence of such excipients prior to use. Conditions of optimized formulation for each amphiphile of the
invention are capable of determination by those skilled in the
46
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pharmaceutical art. By way of example, for spPrmi~ine cholesterol
carbamate (amphiphile No. 53), it has been determined that use of sucrose is
ereLled over mannitol in order to pr~velll formation of amphiphile/DNA
aggregates, particularly as the concentration of DNA is increased therein.
Addition of such excipients maintains the consistency of lyophilized
pharmaceutical compositions during storage, and prevent difficulties such
as aggregation, or insolubity, that may likely occur upon rehydration from
the lyophili7e~ state.
Accordingly, a principal aspect of the invention involves providing a
composition that comprises a biologically active molecule (for example, a
polynucleotide) and one or more cationic amphiphiles (including optionally
one or more co-lipids), and then maintaining said composition in the
presence of one ore more excipients as aforementioned, said resultant
composition being in liquid or solid (plereldbly lyophilized) form, so that:
(1) the therapeutic activity of the biologically active molecules is
substantially preserved; (2) the transfection-enhancing nature of the
amphiphile( or of amphiphile/ DNA complex) is maintained. Without
being limited as to theory, it is believed that the excipients stabilize the
interaction (complexes)of the amphiphile and biologically active molecule
through one or more effects including:
(1) minimizing interactions with container surfaces,
(2) preventing irreversible aggregation of the complexes, and
(3) maintaining amphiphile/DNA complexes in a chPmil ~lly-stable state,
i.e., preventing oxidation and/or hydrolysis.
Although the presence of excipients in the pharmaceutical
compositions of the invention stabilizes the compositions and fa~ tP~
storage and manipulation thereof, it has also been determined that moderate
47
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concentrations of numerous excipients may, ' ,lel~r~ with the transfection-
enhancing capability of p~rm~c~utical formulations containing them. In
this regard, an additional and valuable characteristic of the amphiphiles of
the invention is that any such potentially adverse effect can be minimi7e(1
5 owing to the ~,leaLly enhanced in vivo activity of the amphiphiles of the
invention in comparison with amphiphilic compounds known in the art.
Without being 1imite-1 as to theory, it is believed that osmotic stress ( at lowtotal solute concentration) may contribute positively to the successful
transfection of polynucleotides into cells in vivo . Such a stress may occur
10 when the pharmaceutical composition, provided in unbuffered water,
contacts the target cells. Use of such otherwise pre~. led compositions may
therefore be incompatible with treating target tissues that already are
stressed, such as has damaged lung tissue of a cystic fibrosis patient.
Accordingly, and using sucrose as an example, selection of concentrations of
this excipient that range from about 15 mM to about 200 mM provide a
compromise betweeen the goals of (1) stabilizing the pharmaceutical
composition to storage and (2) mimizing any effects that high concentrations
of solutes in the composition may have on transfection performance.
Selection of optimum concentrations of particular excipients for
20 particular formulations is subject to experimentation, but can be determined
by those skilled in the art for each such formulation.
An additional aspect of the invention concerns the protonation state
of the cationic amphiphiles of the invention prior to their contacting plasmid
DNA in order to form a therapeutic compositiol . It is within the practice of
25 the invention to utilize fully protonated, partially protonated, or free baseforms of the amphiphiles in order to form such therapeutic compositions.
With respect to amphiphile No. 67 (spermine cholesterol carbamate), it has
48
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been observed that when providing this amphiphile for a transfecting
composition with DOPE (itself provided as a zwitterion), transgene
expression was best for the free base, but decreased if the amphiphile was
prepared as an acetate salt. Activity decreased step-wise through the mono
5 and di acetate salts and was minimal for the tri-acetate salt. Under the
circumstances described, the plasmid DNA provided for contacting with the
amphiphile was prepared (without buffer) as a sodium salt in water.
49
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Methods of Syntheses
The following methods illustrate production of certain of the cationic
amphiphiles of the invention. Those skilled in the art will recognize other
methods to produce these compounds, and to produce also the other
5 compounds of the invention.
Group I amphiphiles
(A! N4-Spermidine cholesteryl carbamate
Spermidine cholesterol carbamate (Figure 1, No. 53) was synthesized
according to the following procedure which is outlined in Figùre 8.
10 Synthesis of N1 N8-DiCBZ-N4-Spermidine Cholesterol Carbamate
Nl, N8 dicarbobenzoxyspermidine (61% yield, m.p. 104 - 105~ C)
was prepared according to the procedure of S. K. Sharma, M. J. Miller, and
S. M. Payne, J. Med. Chem., 1989, 32, 357-367. The Nl, N8
dicarbobenzoxyspPrmit1ine (25 g, 60.5 mmol) and triethylamine (25 ml, 178
15 mmol) were dissolved in 625 ml of anhydrous methylene chloride, cooled to
0 - 4~C and stirred under N2. Cholesteryl chloroformate (27.2 g, 60.6 mmol)
was dissolved in 250 ml of methylene chloride and added to the reaction
over a 20 minute period. A white precipitate formed upon addition. After
the addition was complete, the reaction was stirred at 0 - 4~C for 10 minutes
20 and then at room temperature for 1.5 hr. At this point, the white precipitatecompletely dissolved. The reaction was followed by TLC with hexane /
ethyl acetate 6 i 4 as eluent (product Rf = 0.25). To this reaction mixture
was added 625 ml of methylene chloride and 625 ml of water. The layers
were then allowed to separate. The organic layer was dried over MgSO4
25 and filtered. The filtrate was concenLlaLed in v~cuo to give an oil. Vacuum
drying was then carried out overnight. This crude product had a glue-like
consistency. The crude product was purified by column chromatography (2
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kg silica gel, eluent- hexane / ethyl acetate 6 / 4) to give 46.8 g of the 3-~-
IN4-(Nl,N8-dicarbobenzoxyspPrmitline)carbamoyl] cholesterol (also
~ described herein as N1, N8- diCBZ-N4- spermidine cholesterol carbamate)
in 93% yield.
Final Synthesis of Spermidine Cholesterol Carbamate
To 6.0 grams of 10% palladium on activated carbon under N2 was
added a solution of 30 grams of 3-i3-[N~(Nl,N8-
dicarbobenzox ,yspermidine)carbamoyl] cholesterol in 1 liter of ethanol, see
Figure 13. The reaction mixture was purged with N2 and stirred under H2
(atmospheric pressure) for 18 hr. The mixture was again purged with N2
and filtered through a 10 g bed of celite. The filter cake was washed with 2
liters of 10% triethylamine in ethanol and the combined filtrates were
concentrated in vacuo to a gel. The product was then dried under vacuum
overnight to a sticky solid. This crude product was purified by column
chromatography (2 kg of silica gel, eluent - 4 L of chloroform / methanol 95
/ 5 followed by 30 L of chloroform / methanol / iso-propylamine 95 / 5 / 5,
Rf = 0.24) to obtain 13.1 g of the desired spermidine cholesterol carbamate in
64% yield. HPLC (C-18 reversed phase column, linear gradient elution
profile - methanol / iso-propanol / water / trifluoroacetic acid 60 / 20 / 20
/ 0.1 to methanol / iso-propanol / trifluoroacetic acid 70 / 30 / 0.1 to
methanol / iso-propanol / chloroform / trifluoroacetic acid 60 / 20 / 20 /
0.1) analysis of this material showed it to be 99.2% pure with the 7-
dehydrocholesterol analog present at a level of 0.8%.
In connection with this example and those that follow, it is noted that
all TLC plates were visualized with phosphomolybdic acid.
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(B) N4 Spermine cholesteryl carbamate
Sperrnine cholesterol carbamate (Figure 1, No. 67) was prepared
according to the following procedure which is outlined in Figure 9.
N1,N12 -diCBZ-spermine
Benzylchloroformate (1.76g, 1.5 ml, 10.36 mmol) was dissolved in
methylene chloride (5 ml) and placed in a three neck flask under a nitrogen
atmosphere. Imidazole (1.4 g, 20.6 mmol) was dissolved in methylene
chloride (20 ml) and placed in an addition funnel. The three neck flask was
cooled to 0~C and the imirl~7.ole solution was added gradually over 20 min.
The mixture was stirred at room temperature for 1 hour and then methylene
chloride ( 25 mL) and citric acid (10%, 25 ml) were added. The layers were
separated and the organic fraction was washed with citric acid (10%, 25 ml).
The organic component was dried over magnesium sulfate and
concentrated in vacuo. The residue was dried under high vacuum for 1 hour
at ambient temperature.
To the residue was added dimethylaminopyridine (35 mg),
methylene chloride (25 ml) and the mixture was cooled to 0~C, under a
nitrogen atmosphere. To an addition funnel was added a solution of
spermine (lg, 4.94 mmol) in methylene chloride (25 ml). The spermine
solution was added gradually over 15 min. The reaction mixture was stirred
overnight at ambient temperature and then concentrated in vacuo. The
- residue was dissolved in ethyl acetate (80 ml) and washed three times with
water (15 ml). The organics were dried over magnesium sulfate, filtered and
concentrated in vacuo to give a crude white solid. The material was purified
by flash chromatography (65g silica gel, 100:100:10 CHCl3: MeOH: NH4OH,
product l~f.=0.33), to give after drying under high vacuum 1;01g (2.146
mmol, 43 % yield) of product.
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N1,N12-diCBZ- N4- spermine cholestryl carbamate
Cholesteryl chloroformate (964 mg, 2.15 mmol) was dissolved in
chloro~olm (10 ml) and added dropwise to a cooled (0~C) solution of
Nl,N12-diCBZ spermine (1.Olg, 2.15 mmol), triethylamine (1 ml) in
5 chloroform (10 ml). The reaction was allowed to warm to room temperature
and stirred for 2 hours. To the reaction solution was added water (25 ml)
and chloroform (25 ml). The layers were separated and the organic fraction
dried over magnesium sulfate. The solution was concentrated in vacuo to
give a crude material that was purified by flash chromatography (68g silica
gel, MeOH / CHCl3 1/4, product Rf. =0.36) to give 1.23 g (1.39 mmol, 65%
yield) of product.
final s~nthesis of N4-Spermine Cholester~l Carbamate
N1,N1~diCBZ-N4-spermine cholesteryl carbamate (262 mg, 0.300
mmol) was dissolved in 5 ml of acetic acid and 45 mg of 10% Pd on C was
15 added. The solution was purged with nitrogen and stirred under hydrogen
at atmospheric pressure. The hydrogenolysis was allowed to proceed for 7
hours. The reaction mixture was filtered and the cata~yst was washed with
40 ml of ethyl acetate / acetic acid 9 / 1 and the filtrate will be concentratedin vacuo to give a residue. The crude product ~ as dissol~ed in 35 mL of lN
20 NaOH and extracted three times with 40 ml of chloroform / methanol 9 / 1.
The combined organic fractions were washed with 20 mL of water and dried
over Na2SO4. The solution was filtered, concentrated in vacuo and dried
under vacuum to give 125 mg of the desired product in 67% yield.
In connection with the above procedure, it is noted that the
25 hydrogenolysis should be carried out under acidic conditions, in order to
minimize the poisoning of the catalyst.
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Urea analogs - such as spermine or spermidine cholestamine urea -
can be prepared by a sequence of reactions well known to those versed in
the art of organic synthesis. For example an amine can be treated with an
equal molar amount of carbonyldiimidazole followed by the addition of a
5 second amine to give the desired urea.
(C! N,N Bis (3-amino~ yl)-O-cholesteryl -3-carbamate
N,N Bis (3-aminopropyl)-O-cholesteryl-3-carbamate (Figure 1, No.
69) was prepared according to the following procedure.
Bis (3-CBZ amin~pro~yl) amine was prepared using the method described
above for Nl,;N12 -diCBZ-spermine, except that N-(3-aminopropyl)1,3-
propan~ mine was substituted for Termine as reactant. The pure product
was isolated in 34 % yield by silica gel flash chromatography using as
solvent CHC13/ MeOH/ NH40H 80/20/0.5.
The Bis (3-CBZ aminopropyl) amine so prepared was then reacted
with cholesteryl chlorofolll.ate according to the method described above for
the synthesis of Nl, N8-DiCBZ -N4-spermidine cholesteryl carbamate. The
pure product (N,N Bis ( 3-CBZ aminopropyl)-O-cholesteryl-3-carbamate)
was obtained in 73% yield.
Synthesis of N,N Bis(3-aminopropyl)-O-cholesteryl-3-carbamate was
completed by hydrogenolysis of the CBZ groups from N,N Bis(3-CBZ
aminopropyl)-O-cholesteryl-3-carbamate following the procedure described
above in relation to the synthesis of N4-spermidine cholesteryl carbamate.
The product was obtained in 23% yield without silica gel chromatography
purification.
(D! N,N Bis (6-aminohexyl)-O-cholesteryl -3- carbamate.
N,N Bis (6-aminohexyl)-O-cholesteryl-3-carbamate (Figure 1, No. 70)
was prepared according to the following procedure.
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First, Bis (6-CBZ aminohexyl) amine was prepared using the method
described above for N1,N 12 -diCBZ-spermine, except that
Bis(hexamethylene)triamine was substituted for spermine as reactant. Pure
product was isolated in 24% yield by recrystallization from toluene.
Bis (6-CBZ aminohexyl) amine was then reacted with cholesteryl
chloroformate according to the method described above for the synthesis of
N1, N8-DiCBZ -N4-spermidine cholesteryl carbamate. Product N,N Bis(6-
CBZ aminohexyl)-O-cholesteryl-3-carbamate was isolated in 40% yield by
silica gel flash chromatography using hexanes/ethyl acetate 7/3 .
(E) Lysine 3-N- dihydrocholesteryl carbamate
Lysine 3-N- dihydrocholesteryl carbamate (Figure 1, panel C) was
prepared according to the following procedure.
To a solution of dihydrocholesterol (5.0 g, 12.9 mmol, Aldrich),
phth~limi-le (2.0 g, 13.6 mmol, Aldrich), and triphenylphosphine (3.8 g, 13.6
mmol, Aldrich) in THF (20 ml, Aldrich) stirred at 0~ C under a nitrogen
atmosphere was added dropwise diethylazodicarboxylate (2.3 ml, 14.5
mmol, Aldrich). Upon the completion of addition the reaction mixture was
allowed to warm to ambient temperature and stirred overnight. The
reaction mixture was concentrated in vacuo to a residue. This residue was
dissolved in 50 ml hexane / ethyl acetate 95 / 5 and a precipitate formed.
The mixture was filtered. The filtrate was concentrated to dryness in vacuo,
dissolved in 25 ml of hexane / ethyl acetate 95 / 5 and chromatographed on
200 g silica gel (eluent 2 L hexane / ethyl acetate 95 / 5 then 1 L hexane /
ethyl acetate 90 / 10). A 76% yield of the desired 3-phthalimidocholestane
(5.43 g) was obtained.
The 3-phthalimidocholestane (5.40 g, 9.75 mmol) was dissolved in 60
mL of methanol and anhydrous hydrazine (3.1 ml, 99 mmol) was added.
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The reaction mixture was stirred and heated at reflux under a nitrogen
atmosphere for 4 hr. This mixture was then cooled to room temperature, 3.1
mL of concentrated HCl was added and the resulting mixture was heated at
reflux overnight. Upon cooling to ambient temperature, 100 ml of die~yl
ether and 50 ml of 1 N NaOH were added (final pH of 10.1) and the layers
were separated. The aqueous layer was extr~cte~ with 50 ml of diethyl
ether and the combined organic fractions were filtered. The filtrate was
concentrated in vacuo and the residue was purified by siIica gel
chromatography (chloroform / methanol 90 / 10) to give 2.24 g of 3-
aminocholestane in 59 % yield.
L-Noc,N~-diBOClysine N-hydroxysuccinimide ester (286 mg, 0.644
mmol, Sigma) and 3-aminocholestane (250 mg, 0.644 mmol) were dissolved
in 5 mL of methylene chloride, 0.1 mL of triethylamine was added and the
resulting solution was stirred under a nitrogen atmosphere at ambient
temperature overnight. To the reaction mixture was added 10 mL of water
and 25 mL of methylene chloride and the layers were separated. The
aqueous layer was extracted with 25 mL of methylene chloride and the
combined organic fractions were dried over MgSO4 and filtered. The
filtrate was concentrated in vacuo and the residue was purified by
chromatography on 25 g of silica gel (eluent - hexane / ethyl acetate 6 / 4,
sample applied in hexane / ethyl acetate 9 / 1). The purified material was
dissolved in 25 mL of chloroform and HCl gas was bubbled through the
solution for 2 hr. followed by nitrogen for 10 min. The solution was
concentrated in vacuo to give 299 mg of the desired product in 79% yield as
the dihydrochloride salt.
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N_N8-Bis(3-aminopropyl)-N4-spermidine cholesteryl carbamate
Nl,N8-Bis(3-amino~r~yl)-N4-spermidine cholesteryl carbamate
(Figure 1, No. 75) was prepared according to the following procedure.
N4 SpPrmi~line cholesteryl carbamate (1.14g, 2.04 mmol) was dissolved
in MeOH (5 mL). Freshly distilled acrylonitrile (0.28 mL, 4.29 mmol) was
added and the solution was stirred at room temperature for 18 h. The
solvent was COIlCellLLd~ed in vacuo to give an oil. Vacuum drying was then
carried out overnight. The crude product was purified by column
chromatography (125 g silica gel, eluent - CHC13 MeOH 1/9) to give 1.15 g
(85 %) of the Nl,N8-Bis (cyanoethyl) N4-Spermidine cholesteryl carbamate.
Raney Nickel 50% slurry (1.2 g, Aldrich) was placed in a Parr Bomb with
lM NaOH in 95% EtOH (50 mL). The Nl,N8-Bis (cyanoethyl) N4-
Spermidine cholesteryl carbamate. was dissolved in EtOH (35 mL) and
added to the bomb. The vesicle was evacuated and placed under Argon
pressure (80-100 psi), three times and then evacuated and placed under
Hydrogen pressure (100 psi), three times. The reaction was stirred under
hydrogen pressure (100 psi) at room temperature for 72h. The vesicle was
evacuated and placed under argon pressure. The catalyst was removed by
filtration. The filtrate was concentrated in vacuo . The resulting oil was
dissokTed in 2:1 CH2C12: MeOH (100 mL) and washed with H2O (35 and 25
mL). The organic layer was dried over Na2SO4 and filtered. The filtrate was
concentrated in vacuo and the residue was purified by chromatography on
100 g of silica gel (eluent - CHC13/MeOH/conc. NH40H 40/25/10, sample
applied in CHC13/MeOH 40/25). The purified material was concentrated in
vacuo with iPrOH (3 X 50 mL) and CH2C12(3X50 mL) and then vacuum
dried to give 986 mg (85%) of Nl,N8-Bis(3-aminopLo~yl)-N4-sp~ line
cholesteryl carbamate.
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(G) N(N--3-amin~ru~yl-spermidine) cholesteryl carbamate
N~4-3-aminupro~yl-spermi~line) cholesteryl carbamate (Figure 1, No.
78) was prepared as follows:
Nl, N8-dicarbobenzoxyspermidine (1.0 g, 2.4 mmol) was dissolved in
MeOH (10 mL). Freshly distilled acrylonitrile (0.3 mL, 4.5 mmol) was added
and the reaction was stirred at room temperature for 18 h. The solvent was
concentrated in vacuo to give an oil. The crude product was purified by
column chromatography (100 g silica gel, eluent - CHC13/MeOH 1/19) to
give 1.10 g (97 %) of N4-2-Cyanoethyl-Nl, N8 dicarbobenzoxyspermidine.
The N4-2-Cyanoethyl-Nl, N8-dicarbobenzoxysp~rmic~ine (0.5 g, 1.07
mmol) was dissolved in MeOH (5 mL) and CoCk (280 mg, 2.15 mmol,
Aldrich) was added. The blue solution was cooled in an ice bath and NaBH4
(405 mg, 10.7 mmol, Aldrich) was added in portions over 15 min. The
resulting black solution was stirred at room temperature for 1 h. The black
solution turned blue over this period. To the reaction was added
CH2C12/MeOH 2/1 (30 mL). A black ppt formed. To this was added H20
(20mL) and the mixture was filtered. The resulting layers were separated
and the organic layer dried with MgS04. The drying agent was filtered and
the filtrate concentrated in vacuo to give an oil. The crude product was
purified by column chromatography (50 g sillca gel, eluent -
CHC13/MeOH/conc NH40H 100/100/5) to give 309 mg (62 %) of the N4
3-amino~ru~yl-Nl, N8 dicarbobenzoxyspermidine.
To the N4-3-aminopropyl-Nl, N8 - dicarbobenzoxyspermidine (300 mg,
0.66 mmol) dissolved in CH2Cl2 was added Et3N under N2. Cholesteryl
chloro formate (326 mg, 0.726 mmol, Aldrich) was dissolved in CH2C12 and
added to the reaction dropwise. The mixture was stirred for 2h at room
temperature. After adding CH2a2 (25 mL) an-d H20 (10 mL), the layers
58
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were separated. The organic layer was dried with MgSO4 and filtered. The
filtrate was conc~nLL~Led in vacuo to give 640 mg of crude product. The
residue was purified by chromatography on 80 g of silica gel (eluent -
CHC13 / MeOH 90 /10, sample applied in CHC13 / MeOH 90/10). The
5 purified material was concentrated in vacuo and then vacuum dried to give
329 mg (57%) of N-(N4-3-amino~r~yl-Nl, N8 dicarbobenzoxyspermi~ine)
cholesteryl carbamate.
To 10% Pd on carbon (65 mg, Aldrich) was added a solution of N-(N4-3-
aminopr~yl-Nl, N8 dicarbobenzoxyspermidine) cholesteryl carbamate
(300 mg) in acetic acid (25 mL). The reaction was placed under H2 and
stirred at room temperature overnight. After being placed under N2, the
reaction was filtered. The catalyst was washed with 10 % acetic acid in
EtOAc (50 mL). The filtrate was concentrated in vacuo to give an oil. The oil
was dissolved in 2/1 CH2Cl2/MeOH (35 mL) and washed with 1 M NaOH
(15 mL). The organic layer was dried with MgSO4 and filtered. The filtrate
was concentrated in vacuo and vacuum dried to give 196 mg (93%) of N-(N4-
3-amino~r~ylspPrmi~ine) cholesteryl carbamate.
(H) N-~N-,N4,N~Tris (3-aminopropyl) spermidine~ cholestervl
carbamate
N-[Nl,N4,N3~Tris (3-aminopropyl) spermidine] cholesteryl carbamate
(Figure 1, No. 96) was prepared by reacting N-(N~3-,
amin~.~L~ylspermidine) cholesteryl carbamate with acrylonitrile (90%
yield) and subsequent reduction of the di adduct with Raney nickel (75 %
yield) as described for the preparation of Nl,N3Bis(3-aminopropyl)-N4-
sp~rmitline cholesteryl carbamate.
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~ N N-Bis(4-aminobutyl) cholesteryl carbamate
N,N-Bis(4-aminobutyl) cholesteryl carbamate (Figure 1, No. 82) was
prepared as follows.
To a mixture of Benzylamine (2.0 g, 18.6 mmol, Aldrich), Na2C03
(4.4g, 42 mmol) and Kl (1.4 g, 9.5 mmol) in n-butanol (50 mL) was added 4-
Chlorobutyronitrile (4.0 mL, 95 mmol) under nitrogen. The reaction was
stirred at reflux of 48 h under nitrogen. After cooling to room temperature,
diethyl ether (50 mL) was added and the precipitate filtered off. The filtrate
was concentrated in vacuo to an oil. Toluene (100 mL) was added and the
solution was conc~nlldled in vacuo . Chloroform (100 mL) was added and
again the solution was concentrated in vacuo and then vacuum dried for 18
h. The resulting oil was dissolved in Chloroform (100 mL) filtered and
concentrated in vaczlo . The crude product was purified by column
chromatography (250 g silica gel, eluent - hexanes/EtOAc 60/40) to give
3.75g (97 %) of N,N-Bis (3-cyano~ro~yl) benzylamine.
The N,N-Bis (3-cyanopropyl) benzylamine (3.7 g, 17.8 mmol) was
dissolved in EtOH (150 mL) and Acetic acid (4 mL) was added. This solution
was added to 10% Pd on carbon (400 mg) under N2. The mixture was placed
under H2 and the reaction stirred for 18 h at room temperature. The reaction
was placed under N2. The catalyst was filtered off and washed with EtOH
(150 mL). The filtrate was concel~l. dL~d in vacuo, chloroform (50 mL) was
added and again concel.lLdled in vacuo . The resulting oil was vacuum dried
for 0.5 h and used directly in the next reaction. To this oil dissolved in
CH2C12 (lOOmL) was added Et3N (5 mL, 35 mn~ol) under N2 and the
solution cooled in an ice bath. Cholesteryl chloro formate (6.2 g, 13.87 mmol)
was dissolved in CH2C12 (100 mL) and this solution was added to t~e
reaction dropwise over 10 min. The cooling bath was removed and the
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reaction stirred at room temperature for 18 h under N2. CH2C12 (100 mL)
and H2O (100 mL) was added and the resulting layers were separated. The
organic layer was dried with MgSO4 and filtered. The filtrate was
concentrated in vacuo and vacuum dried for 1 h. The crude product was
5 purified by column chromatography (600 g silica gel, eluent -
hexanes/EtOAc 60/40) to give 1.05g (10 %) of N,N-Bis (3-cyanu~ro~yl)
cholesteryl carbamate.
Raney Nickel 50% slurry (1.2 g) was placed in a Parr Bomb with lM
NaOH in 95% EtOH (50 mL). The N,N-Bis (3-cyano~ro~yl) cholesteryl
carbamate (1.0 g, 1.77 mmol was dissolved in EtOH (100 mL) and added to
~he bomb. The vesicle was evacuated and placed under Argon pressure (80-
100 psi), three times and then evacuated and placed under Hydrogen
pressure (100 psi), three times. The reaction was stirred under hydrogen
pressure (100 psi) at room temperature for four days. The vesicle was
evacuated and placed under argon pressure The catalyst was removed by
filtration. The filtrate was concentrated in vacllo . The resulting oil was
dissolved in 2:1 CH2C12: MeOH (250 mL) and washed twice with H2O (75
and 50 mL). The organic layer was dried over Na2SO4 and filtered. The
filtrate was concentrated in vacuo and the residue was purified by
chromatography on 110 g of silica gel (eluent - CHCI3/MeOH/iPrNH2
95/5/5, sample applied in CHCl3/MeOH 95/5). The purified material was
concentrated in vacuo and then vacuum dried to give 900 mg (85%) of N,N-
Bis(4-aminobutyl) cholesteryl carbamate.
m N,N-Bis(N'-3-amino~ropyl-4-aminobutyl) cholesteryl carbamate
N,N-Bis(N'-3-aminopropyl-4-aminobutyl) cholesteryl carbamate
(Figure 1, No. 83) was prepared by reacting N,N~Bis(4-aminobutyl)
cholesteryl carbamate with acrylonitrile (82% yield) and subsequent
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reduction of the di acrylonitrile adduct with Raney nickel (81 % yield) as
described for the preparation of Nl~N8-Bis(3-aminoplo~yl)-N4-sp~ ne
cholesteryl carbamate.
(K! N4 Spermidine cholesteryl carboxamide
N4 SpPrmi~line cholesteryl carboxamide ( Figure 1, No. 90) was
prepared as follows.
A solution of cholesteryl chloride (5.0 g, 12.3 mmol) in THP (50 mL)
was added dropwise over 0.5 h under reflux to Magnesium turnings (390
mg) in THF (25 mL). Initially a pinch of Iodine and three drops of
Iodomethane were added to inihate the reaction. After refluxing for 3 h. the
reachon was cooled to room temperature. This mixture was poured onto
Dry ice (10 g) and then shrred for lh. This solution was cooled in an ice bath
and added to ice cold 1 M H2SO4 (100 mL). After shrring for 5 min., sodium
chloride (1 g) and diethyl ether (100 mL) was added. The layers were
separated and the aqueous layer was extracted with diethyl ether (100 mL).
The combined organic layers were washed with a solution of Sodium
thiosulfate pentahydrate(120 mg) in H2O (30 mL). The organic layer was
concentrated in vacuo and vacuum dried for 18 h. The crude solid was
titrated with hexanes (25 mL). After filtration the solid was washed with ice
cold hexanes (10 mL). The solid was vacuum dried for lh. The cholesteryl
carboxylic acid obtained (3.0 g, 59 %) was ca. 90 % pure and used without
further purificahon.
Cholesteryl carboxylic acid (500 mg, 1.2 mmol) and N-
hydroxysuccinimide (140 mg, 1.2 mmol) was dissolved in CH2C12 . To this
solution was added Dicyclohexylcarbodiimide (275 mg, 1.32 mmol) was
added and t~e reaction was shrred under N2 for 2h. Nl, N8-
dicarbobenzoxyspermidine (474 mg, 1.2 mmol) and Et3N (1.0 mL, 7.1 mmol)
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was added and the reaction was stirred under N2 for 72 h. The reaction was
filtered and the precipitate was washed with CH2Cl2 (50 mL). The filtrate
was washed with H20 (25mL). The separated organic layer was dried over
MgSO4 and filtered. The filtrate was concentrated in vacuo and the residue
was purified by chromatography on 150 g of silica gel (eluent - hexanes /
EtOAc 1/ 1). The purified material was concentrated in vacuo and then
vacuum dried to give 680 mg (70%) of N1,N8-dicarbobenzoxy-N4-
spermidine cholesteryl carbox~mi~le.
The carbobenzoxy group were removed from Nl,N8-
dicarbobenzoxy-N4-spermidine cholesteryl carboxamide as described in the
preparation of N4-sp~rmi~line cholesteryl carbamate. The purified product,
N4 Spermidine cholesteryl carboxamide, was obtained in 53 % yield.
Group II Amphiphiles
(A) __ N8-Bis(Arginine carboxamide)-N--spermidine cholesteryl
carbamate
N1, N8-Bis(Arginine carboxamide)-N4-spermidine cholesteryl
carbamate (Pigure 5, No. 95) was prepared as follows.
To N(a) ,N(e) ,N(e) (alpha, epsilon, epsilon) -tricarbobenzoxyArginine
in CH2Cl2 (25 mL) was added N-hydroxysuccinimide (100 mg, 0.89 mmol)
and dicyclohexylcarbodiimide (240 mg, 0.89 mmol). The mixture was stirred
under N2 at room temperature for 2.5 hours. N4- Spermidine Cholesteryl
Carbamate (250 mg, 0.448 mmol) and Et3N ( 0.25 mL, 1.8 mmol) was added
and the reaction stirred at room temperature under N2 for 72 h. The
reaction was filtered and the precipitate was washed with CH2Cl2 (20 mL).
The filtrate was washed with H20 (20 mL). The separated organic layer was
dried over MgSO4 and filtered. The filtrate was concentrated in vacuo and
the residue was purified by chromatography on 70 g of silica gel (eluent -
63
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CHCl3 / MeOH 95/ 5). The purified material was concentrated in vacuo
and then vacuum dried to give 533 mg (71%) of N1, N8-Bis (N(a),N(e),N(e)-
tricarboberlzoxyArginine carboxamide)-N4-spermidine cholesteryl
carbamate.
The carbobenzoxy group were removed from N1, N8-Bis (N(a),N(e),N(e)-
tricarbobenzoxyArginine carboxamide)-N4-spermidine cholesteryl
carbamate as described in the preparation of N-(N4-3-
amino~ro~ylspermidine) cholesteryl carbamate. The product, N 1, N8
Bis(Arginine carboxamide)-N4-spermidine cholesteryl carbamate was
obtained in 27 % yield.
Group III Amphiphiles
(A) N,N-Dioctadecyllysineamide
N,N- dioctadecyllysineamide( Figure 6, No.73) was prepared
according to the following procedure. N,N-dioctadecylamine (1.35 g, 2.58
mmol, Fluka) and L-Noc,N~-diBOClysine N-hydroxysuccinimide ester ( 1.00
g, 2.58 mmol, Sigma) were combined in 15 ml of methylene chloride and 2
ml triethylamine was added. The reaction mixture was heated briefly to
effect complete dissolution and then stirred at ambient temperature
overnight. Water (20 ml) and methylene chloride (50 ml) were added to the
reaction mixture and the layers were separated. The aqueous fraction was
extracted a second time with 50 ml methylene chloride. The combined
organic fractions were dried over MgSO4, filtered and concentrated in vacuo.
The residue was purified by column chromatography (150 g silica gel,
eluent - hexane/ethyl acetate 8/2). The purified material, N,N-dioctadecyl-
N(x,N~-diBOC lysineamide(1.59 g) was dissolved in 25 ml of chloroform
and stirred for 2 hr. while HCl gas was bubbled through the solution. This
solution was purged with N2 gas and concentrated in vacuo. N,N
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-dioctadecyllysine~mi~e (1.34 g) was obtained in 68% yield as the di HCl
salt.
(B! Nl,Nl-Dioctadecyl-1,2,6-triaminohexane
Nl,Nl-Dioctadecyl-1,2,6-triaminohexane (Figure 6, No. 47) was
5 prepared as follows. To N,N-Dioctadecyl-Noc,N~-diBOClysineamide (760
mg, 0.823 mmol) in 30 ml anhydrous THF stirred at ambient temperature
was added LiAlH4 (185 mg, 4.87 mmol) in portions. The reaction mixture
was stirred at ambient temperature overnight under a nitrogen atmosphere.
The reaction was quenched by the dropwise addition of 2 ml water and the
10 resulting solution was concentrated in vacuo. To this residue was added in
order 10 mL of 1 M HCl, 50 ml of methylene chloride, and 10 ml of lM
NaOH (final pH 10). The layers were separated and the aqueous fraction
was extracted a second time with 50 ml of methylene chloride. The
combined organic layers were dried over MgSO4 and filtered. The filter
15 cake was washed with 50 ml of methylene chloride. The combined filtrates
were conC~llLLdLed in vacuo to give 700 mg of crude product. The crude
product was purified by column chromatography (80 g silica gel, eluent -
hexane/ethyl acetate 7/3). The fractions containing the purified product
were combined and concellLldLed in vacuo to obtain 490 mg of the product
20 protected as the diBOC derivative. To 200 mg of this diBOC derivative was
added 4 ml of chloroform and 1 ml of TFA. The resulting reaction mixture
was stirred at ambient temperature for 2 hr and concentrated in vacuo. The
residue was dissolved in 25 ml of water and 25 mL of methylene chloride
and adjusted to pH 10 with approximately 2 ml of concentrated ammonium
25 hydroxide. The layers were separated and the aqueous layer was extracted
a second time with 25 ml of methylene chloride. The organic fractions were
combined, dried over Na2SO4 and concentrated in vacuo. The resulting
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residue was dissolved in 10 ml of diethyl ether, HCI gas was bubbled
through the solution for 2 minutes and the solution was cooled at 4~ C
overnight. The precipitated product was collected by filtration, washed
with cold (4~ C) diethyl ether, and dried under vacuum to obtain 160 mg of
5 the desired product in 67% yield.
Group IV Amphiphiles
(A! 1-(N4-spermine)-2,3-dilaurylglycerol carbamate
1-(N4-spermine)-2,3-dilaurylglycerol carbamate (Figure 7, No. 89)
was prepared as follows. A solution of 3-benzyloxy-1,2-propanediol (1.00 g,
5.49 mmol) in THF (20 mL) was added to a suspension of sodium hydride
(60% w/w in oil, 550 mg, 13.725 mmol) in THF (30 mL) and allowed to
reflux overnight under dry nitrogen. A solution of dodecyl methane
sulfonate (3.39 g, 12.078 mmol) in THF (20 mL) was added and the reaction
was refluxed for another two days. After cooling to room temperature the
15 reaction was filtered through a bed of Celite, rinsing with T~. The filtrate
was reduced in vacuo to a yellow oil which was redissolved in diethyl e'cher
(100 mL). The ether solution was washed with 0.1 N NaOH (30 mL) and
dH20 (2 x 30 mL). The organic layer was dried over magnesium sulfate,
filtered and reduced in vacuo to a red-brown oil. The crude material was
20 purified by flash column chromatography (300 g silica gel) eluting with 3%
ethyl acetate/ hexanes. The desired product was isolated as a pale yellow
oil and characterized by lH NMR as 3-OBn-1,2-dilaurylglycerol (1.70 g,
60%). 3-OBn-1,2-dilaurylglycerol (1.70 g, 3.28 mmol) in ethanol (100 mL)
was stirred with 10% Pd/C (250 mg, 15 wt%) under a hydrogen atmosphere
25 for 24 hours. The reaction was flushed with nitrogen and filtered through
Celite, rinsing with ethanol, to remove the catalyst. The filtrate was reduced
in vacuo to a solid. The crude material was purified by flash columrL
66
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chromatography (140 g silica gel) eluting with 10% ethyl acetate/ hexanes.
The desired product was isolated as a white solid and characterized by 1H
NMR as 1,2-dilaurylglycerol (1.23g, 88%).
A 1.93 M solution of phosgene in toluene (0.77 mL, 1.49 mmol) was
added to a solution of 1,2-dilaurylglycerol (580 mg, 1.35 mmol) and N,N-
diisopropylethylamine (0.26 mL, 1.49 mmol) in methylene chloride (10 mL)
and stirred overnight. A solution of N1,N12-di-CBz-spermine-2HCl (734
mg, 1.35 mmol) in 60: 25: 4 chloroform/ methanol/ water (80 mL) was
added. After 3 hours another equivalent of N,N-diisopropylethylamine
(0.26 mL, 1.49 mmol) was added. An additional 0.5 equivalents of N,N-
diisopropylethylamine (0.13 mL, 0.75 mmol) was added three hours later
and the reaction was allowed to stir overnight under nitrogen at ambient
temperature. The reaction was washed with lM NaOH (20 mL) and dH20
(15 mL). The organic layer was separated, dried over magnesium sulfate,
filtered and reduced in vacuo to a white solid The crude material was
purified by flash column chromatography (125 g silica gel) eluting with 90:
10: 0.5 chloLorolm/ methanol/ ammonium hydroxide. The desired product
was isolated as an oil and characterized by lH NMR as l-(N4-(N1,N12-di-
CBz-spermine))-2,3-dilaurylglycerol carbamate (188 mg, 15%).
The 1-(N4-(N1,N12-di-CBz-spermine))-2,3-dilaurylglycerol carbamate
(188 mg, 0.203 mmol) was dissolved in glacial acetic acid (10 mL) and stirred
with 10% Pd/C (45 mg, 24 wt %) under a hydrogen atmosphere for 5 hours.
The catalyst was removed by vacuum filtration rinsing with 10% acetic
acid/ ethyl acetate (10 mL) The filtrate was reduced to an oil by rotary
evaporation. The resulting oil was dissolved in 10% methanol/ chloroform
(85 mL) and was washed with lM NaOH (15 mL) and dH20 (10 mL). The
organic layer was separated, dried over magnesium sulfate, filtered and
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reduced in vacuo to an oil. The product was characterized by lH NMR as 1-
(N4-spermine)-2,3-dilaurylglycerol carbamate (125 mg, 94%).
Other amphiphiles of the invention may be prepared according to
procedures that are within the knowledge of those skilled in art.
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Examples
The following Examples are representative of the practice of the
il~v~llion.
Example 1- Cell Transfection Assay
Separate 3.35 ,umole samples of spermidine cholesterol carbamate
(amphiphile No. 53) and the neutral lipid
dioleoylphosphatidylethanolamine ("DOPE") were each dissolved in
chloroform as stock preparations. Following combination of the solutions, a
thin film was produced by removing chloroform from the mixture by
10 evaporation under reduced pressure (20 mm Hg). The film was further
dried under vacuum (1 mm Hg) for 24 hours. As aforementioned, some of
the amphiphiles of the invention participate in transacylation reactions with
co-lipids such as DOPE, or are subject to other reactions which may cause
decomposition thereof. Accordingly, it is ~re~lr~d that amphiphile/co-
15 lipid compositions be stored at low temperature, such as -70 degrees C, until use.
To produce a dispersed suspension, the lipid film was then hydrated
with sterile deionized water (1 ml) for 10 minutes, and then vortexed for 1
minute ( sonication for 10 to 20 seconds in a bath sonicator may also be
20 used, and sonication has proved useful for other amphiphiles such as DC-
chol). The resulting suspension was then diluted with 4 ml of water to yield
a solution that is 670~1M in cationic amphiphile and 670,uM in neutral
colipid.
Experiments were also performed using spermine cholesterol
25 carbamate (amphiphile No. 67) and other amphiphiles of the invention.
With respect to spermine cholesterol carbamate, the optimum molar ratio of
amphiphile to DOPE under the conditions tested was determined to be 1:2,
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not 1:1. Optimi7e~1 ratios for many of the amphiphiles of the invention are
reported in Figures 13, 14 and 15, and are readily determined by those
skilled in the art.
For preparation of the transfecting solution, DNA encoding for ~-
galactosidase (pCMV~, ClonTech., Palo Alto, CA) was dissolved in
OptiMEM culture m~ m~ (Gibco/ BRL No. 31885-013). The resulting
solution had a DNA conce~ dLion of 960 ,uM (assuming an average
molecular weight of 330 daltons for nucleotides in the encoding DNA).
The following procedure was used to test a 1:1 molar mixture of the
cationic amphiphile spPrmitline cholesterol carbamate in combination with
DOPE. A 165 ~Ll aliquot of spPrmic~ine cholesterol carbamate (670 ~LM)
containing also the colipid ( at 670 ,uM ) was pipetted into 8 separate wells ina 96-well plate containing OptiMEM (165,~L1) in each well. The resulting 335
,~LM solutions were then serially f~illlte~l 7 times to generate 8 separate
amphiphile-containing solutions having concentrations ranging from 335
,uM to 2.63 ~M, with each resultant solution having a volume of 165 ,ul.
Thus, 64 solutions were prepared in all, there being 8 wells each of 8
different concentrations of amphiphile/DOPE.
Independently, DNA solutions (165~11, 960,uM) were pipetted into 8
wells conLaining OptiMEM (165,ul), and the resulting 480~M solutions were
then serially diluted 7 times to generate 8 separate 165 ,ul solutions from
each well, with the concentrations of DNA in the wells ranging from 480,~LM
to 3.75 ,uM.
The 64 test solutions (cationic amphiphile: neutral lipid) were then
combined with the 64 DNA solutions to give separate mixtures in 64 wells,
each having a volume of 330,u1, with DNA concentrations ranging from 240
,uM to 1.875 ,uM along one axis, and lipid concentrations ranging from 167
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IlM to 1.32 ,uM aiong the other axis. Thus 64 solutions were prepared in all,
each having a different amphiphile: DNA ratio and/or concentration. The
solutions of DNA and amphiphile were allowed to stand for 15 to 30
minutes in order to allow complex formation.
A CFT-1 cell line (human cystic fibrosis bronchial epithelial cells
irnmortalized with papillomavirus) provided by Dr. James Yankaskas,
University of North Carolina, Chapel Hill, was used for the in vitro assay.
The cells are homozygous for a mutant allele (deletion of phenylalanine at
position 508, hereinafter A F508 ) of the gene encoding for cystic fibrosis
transmembrane conductance regulator ("CFTR") protein. CFTR is a cAMP-
regulated chloride (Cl-) channel ploLeill. Mutation of the CFTR gene results
typically in complete loss ( or at least substantial impairment) of Cl- channel
activity across, for example, cell membranes of affected epithelial tissues.
The ~ F508 mutation is the most common mutation associated with
cystic fibrosis disease. For a discussion of the properties of the ~ F508
mutation and the genetics of cystic fibrosis disease see, in particular, Cheng
et al., Cell, 63, 827-834 (1990). See also Riordan et al., Science 245~ 1066-1073
(1989); published European Patent Application No. 91301819.8 of Gregory et
al., bearing publication number 0 446 017 A1; and Gregon~ et al., Nature,
347, 382-385 (1990).
The cells were cultured in Hams F12 nutrient media (Gibco/ BRL No.
31765-027) supplemented with 2% fetal bovine serum ("FBS", Irvine
Scientific, No. 3000) and 7 additional supplements. Cells were then plated
into 96-well tissue culture plates at a density of approximately 7,500
cells/well. Before being used in the assay, cells were allowed to grow for
periods of 5-7 days until a confluent pattern had been achieved.
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Following the allotted time period, three 96-well plates with CFT-l
cells were aspirated in order to remove the growth medium. The various
concentrations of DNA-lipid complex (in 100 ~Ll aliquots) were transferred to
each of three 96-well plates bringing the DNA-lipid complexes in contact
5 with the cells. DNA-only/cell and lipid-only/cell control wells were also
prepared on one of the three plates.
The 100 ~11 solutions of DNA-lipid complex were maintained over the
cells for 6 hours, after which 50 ,ul of 30% FBS (in OptiMEM) was added to
each well. After a further 20-hour incubation period, an additional 100 ,ul of
10 10% FBS in OptiMEM was also added. Following a further 24-hour
incubation period, cells were assayed for expression of protein and ~-
galactosidase.
~ or the assays, the resultant medium was removed from the plates
and the cells washed with phosphate buffered saline. Lysis buffer (50 ,ul,
250 mM Tris-HCl, pH 8.0, 0.15% Triton X-100) was then added, and the cells
were lysed for 30 minutes. The 96-well plates were carefully vortexed for 10
seconds to dislodge the cells and cell debris, and 5 ~ll volumes of lysate from
each well were transferred to a plate containing 100~ olumes of Coomassie
Plus(~ protein assay reagent (Pierce Compan~, No. 23236). The protein
assay plates were read by a Bio-Rad Model 450 plate-reader containing a
595nm filter, with a protein standard curve included in every assay.
The level of ~-galactosidase activity in each well was measured by
adding phosphate buffered saline (50 ~11) to the remaining lysates, followed
by addition of a buffered solution consisting of chlorophenol red
galaclo~yldnoside (100 ,ul, 1 mg per ml, Calbiochem No. 220588), 60 mM
disodium hydrogen phosphate pH 8.0, 1 mM magnesium sulfate, 10 mM
potassium chloride, and 50 mM 2-meLca~loethanol. The chlorophenol red
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gala~ L~yldlloside, following enzymatic ( ~-galactosidase) hydrolysis, gave
a red color which was detected by a plate-reader containing a 570 nm filter.
A ~-galactosidase (Sigma No. G6512) standard curve was included to
calibrate every assay.
Following subtraction of background readings, optical data
determined by the plate-reader allowed determination of ~-galactosidase
activity and yroLe~l~ content. In comparison to the amount of ~-
galactosidase expressed by known transfectants, for example, DMRIE (1,2-
dimyristyloxy~ro~yl-3-dimethyl-hydroxyethyl ammonium bromide),
compounds of the invention are particularly effective in transfecting airway
epithelial cells and inducing therein ~-galactosidase expression. Relative to
DMRIE:DOPE (1:1), the spermidine cholesterol carbamate: DOPE mixture
~also 1:1) dPmonc~af.ed transl~ction efficiency iInproved by a factor of about
5 (see, for example, Figures 13,14 and 15).
Example 2 - Transfection of the Gene Encoding for Human Cystic Fibrosis
Transmembrane Conductance Regulator Protein
The ability of the cationic amphiphiles of the invention to transfect
cells and to induce therein biochemical corrections was demonstrated with a
separate i~ vitro assay. Irnmortalized human cystic fibrosis airway cells
(CFT-1, as above) were used.
In preparation for the assay, the cells were grown on glass coverslips
until approximately 60% confluent. The cells were then transfected with a
complex of spermidine cholesterol carbamate:DOPE (1:1) and a
plasmid(pCMV- C~Tl~) col~Lail ing a cDNA that encodes wild type human
CFTR. pCMV-CFTR plasmid is a construct containing the encoding
sequence for CFTR and the following regulatory elements, a CMV promoter
and enhancer, and an SV40 polyadenylation signal. Additional constructs
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suitable for the practice of this example include pMT-CFTR, Cheng et al.,
Cell, 63, 827-834 (1990). The complex used was 10.5 ,~Lmolar of sp~rmi~line
cholesterol carbamate (also of DOPE) and 30,umolar of pCMV-CFTR based
on nucleotide.
48 hours after amphiphile-mediated transfection, cells were tested for
cAMP-stimulated Cl- channel activity using the ~-methoxy-N-(3-
sulfu~rûpyl)quinolinium ("SPQ") assay. See S. Cheng et al., Cell, 66, 1027-
1036 (1991) for further information concerning assay methodology. In the
assay, cAMP-dependent Cl- channel activity was assessed using "SPQ"
(from Molecular Probes, Eugene, Oregon), a halide-sensitive fluorophore.
Increases in halide permeability results in a more rapid increase in SPQ
fluorescence, and the rate of change (rather than the absolute change in
fluorescence) is the important variable in assessing Cl- permeability. See
also Rich et al., Nature, 347, 358-363 (1990) for background information.
Fluorescence of the SPQ molecule in individual cells was measured
using an inverted microscope, Nikon,, a digital imaging system from
Universal Imaging, and an ICCD camera, Hamamatsu, Inc.. Cells were
selected for analysis without prior knowledge of their expected rate-of-
change- in-fluorescence characteristics.
In each experiment, up to five microscope fields of between 90 and
100 cells were examined on a given day, and studies under each condition
were repeated on at least 3 different days. Since expression of CFTR is
heterogenous (i.e. cells do not produce identical amounts of CFTR), the data
presented were for the 20% of cells in each field exhibiting the greatest
response.
As expected, cells that were mock transfected failed to exhibit any
measurable increase in cAMP-stimulated halide fluorescence. In contrast,
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cells that had been transfected with the wild type CFTR cDNA displayed a
rapid increase in SPQ fluorescence upon stimulation with cAMP agonist,
indicating increased p~orme~hility to anions. Approximately 60% of the cells
assayed exhibited measurable cAMP-stimulated Cl- channel activity.
5 Accordingly, spermidine cholesterol carbamate, and other cationic
amphiphiles of the invention similarly tested, are effective in transferring
CFTR-encoding plasmid into immortalized CF airway cells.
Example 3 - CAT Assay
part A
This assay was used to assess the ability of the cationic amphiphiles
of the invention to transfect cells in vivo from live specimens. In the assay,
the lungs of balb/c mice were instilled intra-nasally (the procedure can also
be performed trans-tracheally) with 100 ,ul of cationic amphiphile:DNA
complex, which was allowed to form during a 15-minute period prior to
administration according to the following procedure. The amphiphile
(premixed with co-lipid, see below) was hydrated in water for 10 minutes, a
period sufficient to yield a suspension at twice the final concentration
required. This was vortexed for two minutes and aliquoted to provide 55
microliter quantities for each mouse to be instilled. Similarly, DNA
encoding the reporter (CAT) gene was diluted with water to a concentration
twice the required final concentration, and then aliquoted at 55 microliters
for each mouse to be instilled. The lipid was gently combined with the
DNA (in a poly~Lyr~l~e tube), and the complex allowed to form for 15
minutes before the mice were instilled therewith.
The plasmid used (pCMVHI-CAT, see Example 4) provides an
encoding DNA for chloramphenicol transferase enzyme. Specifics on the
amphiphile:DNA complexes are provided below.
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Two days following transfection, mice were sacrificed, and the lungs
and trachea removed, weighed, and homogenized in a buffer solution (250
mM Tris, pH 7.8, 5mM EDTA). The homogenate was t larifie~ by
centrifugation, and the deacetylases therein were inactivated by heat
5 treatment at 70 ~C for 10 minutes. Lysate was incubated overnight with
acetyl coen_yme A and C 14 --chloramphenicol. CAT enzyme activity was
then visll~li7e~ by thin layer chromatography ("TLC") following an ethyl
acetate extraction. Enzyme activity was quantitated by comparison with a
CAT standard curve.
The presence of the en_yme CAT will cause an acetyl group to be
transferred from acetylcoenzyme A to C 14 --chloramphenicol. The
acetylated/radiolabeled chloramphenicol migrates faster on a TLC plate and
thus its presence can be detected. The amount of CAT that had been
necessary to generate the determined amount of acetylated chloramphenicol
can then be calculated from standards.
The activity of spermidine cholesterol carbamate (amphiphile No.53)
was determined in the CAT assay in relation to the recognized transfection
reagents DMRIE and DC-Chol. Figure 10 demonstrates dramatically (as ng
CAT activity per 100 mg tissue) the enhanced ability of spermidine
cholesterol carbamate (amphiphile No. 53) to transfect cells in vivo, which
enhancement is about 20-fold, or greater, in this assay. In the assay, activity
was measured as ng CAT enzyme per 100 mg lung tissue. As a comparison,
it is generally observed that DMRIE, a well known transfectant, when
prepared as a 1:1 molar mixture with DOPE and then complexed with
plasmid DNA (1.7 mM DMRIE, 1.7 mM DOPE, 1.2 mM plasmid DNA
measured as nucleotide) gives about 1 to 2 ng activity per 100 mg lung
tissue in this assay.
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With respect to the comparison provided by Figure 10, the following
conditions are of note. The transfection solution for sp~rmi-line cholesterol
carbamate contained 6mM DNA measured as concentration of nucleotide,
and 1.5 mM of cationic amphiphile. Following generally the procedure of
5 Example 1, each amphiphile had also been premixed with DOPE, in this
case at 1:1 molar ratio. For transfection with DC-chol, the molar ratio of DC-
chol to DOPE was 3:2, and the concentrations of cationic amphiphile and of
DNA (as nucleotide) were 1.3 mM and 0.9 mM, respectively. For
transfection with DMPIE, the molar ratio of DMRIE to DOPE was 1:1 and
10 the concentrations of cationic amphiphile and of DNA were 1.7 mM and 1.2
mM, respectively. These concentrations (and concentration ratios) for each
amphiphile, and colipid and DNA, had been determined to be optimal for
transfection for that respective amphiphile, and accordingly were used as
the basis for the comparison presented herein.
For spermidine cholesterol carbamate (amphiphile No. 53),
op7imi7~t70n experiments were also performed to determine ~le~.l~d
concentrations of plasmid for a particular amphiphile concentration (see
Figure 11), and also to determine ~rerelled concentrations of the same
amphiphile in relation to a particular plasmid concentration (see Figure 12).
Transfection efficiency was optimal at an amphiphile concentration of 1.5
mM (DOPE also being present at 1.5 mM), and about 6 mM (by nucleotide)
of plasmid, or about at a ratio of 1:4. It was noted, however, that
concentrations of about 0.75 mM of amphiphile, and 3.0 mM of plasmid
were less toxic to the target cells.
Intra-nasal transfection with pCMVHI-CAT vector was also
performed in mice using spermidine cholesterol carbamate as cationic
amphiphile but with cholesterol as co-lipid. In this experiment, the
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concentrations of spermidine cholesterol carbamate tested were between 1.0
and 1.5 mM (cholesterol being present at a 1:1 molar ratio in each case, with
the mixing of amphiphile and co-lipid being performed as above). The
DNA concentration ( measured as nucleotide concentration) was between
4.0 and 6.0 mM. Transfection efficiency (again measured as ng CAT/100 mg
tissue) was less effective than with DOPE as co-lipid; however, the
transfections were substantially more effective than those achieved using
DC-Chol/DOPE.
part B
Additional experiments were performed to compare in vivo the
transfection efficiency of cationic amphiphiles depicted in Figures 1, 5 and 7.
Results therefor are reported in Figures 13,14 and 15 respectively. The
compounds were administered intra-nasally using between 12 and 15 mice
per compound. As in part A above, ng CAT activity was measured per 100
mg of tissue. However, improved vectors (pCF1/CAT and its near
equivalent pCF2/CAT) were used. In part resulting from improved vector
performance, incubations of lysate with acetyl coenzyme A and C14-
chloramphenicol were conducted for only 30 minutes. Construction of
pCF1/CAT and pCF2/CAT is described below in Example 4.
The in vivo data reported in Figures 13, 14 and 15 were compiled
generally as follows. As aforementioned, Figures 10 and 11 report data
from the complete in vivo optimization of amphiphile No. 53. Amphiphile
No. 67 was subjected to a similar partial optimization. With respect to all of
the other cationic amphiphiles reported on, and taking advantage of
numerous structural ~imil~rities, optimi7e-1 compositions for in vivo testing
were extrapolated from in vitro results. This facilitated the screening of
large numbers of amphiphiles and produced broadly, if not precisely,
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comparable data. For all amphiphiles other than Nos. 53 and 67, the ratio,
for in vivo testing, of amphiphile concentration to DOPE conc~lLralion was
~ taken from the in vitro experiments, as was the optimi~e~ ratio of
amphiphile concentration to DNA concentration (see Example 1).
5 Accordingly, for such amphiphiles the in vivo test concentration was fixed
at lmM, thereby fixing also the co-lipid concentration. [Broadly, the molar
ratio of the amphiphile to co-lipid DOPE ranged from 1:2 (for example,
spermine cholesterol carbamate, No. 67) through 1:1 (for example,
spermidine cholesterol carbamate, No. 53) to about 2:1 (for example,
10 amphiphile No. 75)]. The concentration of plasmid DNA varied for each
amphiphile species tested in order to duplicate the optimized
amphiphile/DNA ratio that had been determined in vitro.
part C
That the novel amphiphiles of the invention are an important
15 contribution to the art is immediately seen by comparing their performance
- as in vivo transfection enhancers - to that of closely related cationic
amphiphiles that lack the novel T-shape. It has been determined that
spermidine cholesterol carbamate provides a much greater level of
enhancement than N1-spermidine cholesteryl carbamate which contains the
20 same number of carbon and nitrogen atoms in its cationic alkylamine
component but which is lirlear and not "T-shaped". Following generally the
procedures of Example 3, part B, and using respectively 6mM (as
nucleotide), 1.5 mM, and 1.5 mM concentrations of DNA, amphiphile and of
co-lipid, the transfection enhancement provided by spermidine cholesterol
25 carbamate (amphiphile No.53), in relation to Nl-spermidine cholesteryl
carbamate, was determined to be about 30 fold.
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Also following the procedures of Example 3, part B, and using
respectively 4mM (as nucleotide), lmM, and 2 mM concentrations of DNA,
amphiphile and co-lipid, the transfection enhancement provided by
sperrnine cholesterol carbamate (amphiphile No. 67)--in relation to NL
5 thermospermine cholesteryl carbamate and Nl-spermine cholesteryl
carbamate to which spermine cholesterol carbamate is similarly related--is
at least about 30 fold.
Example ~ Construction of vectors
As aforementioned, numerous types of biologically active molecules
10 can be transported into cells in therapeutic compositions that comprise one
or more of the cationic amphiphiles of the invention. In an important
embodiment of the invention, the biologically active macromolecule is an
encoding DNA. There follows a description of novel vectors (plasmids) that
are ~re~lled in order to facilitate expression of such encoding DNAs in
15 target cells.
part A--pCMVHI-CAT
pCMVHI-CAT is representative of plasmid constructs useful in the
practice of the invention. Although the plasmid is provided in a form
carrying a reporter gene (see Example 3), transgenes having therapeutic
20 utility may also be included therein.
The pCMVHI-CAT vector is based on the commercially available
vector pCMV~ (Clontech). The pCMV~ construct has a pUC19 backbone a
Vieira, et al., Gene ,19, 259-268,1982) that includes a procaryotic origin of
replication derived originally from pBR322. Basic features of the pCMVHI-
25 CAT plasmid (as constructed to include a nucleotide sequence coding forCAT) are as follows. Proceeding clockwise--the human cytomegalovirus
immediate early gene promoter and enhancer, a fused tripartite leader from
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adenovirus and a hybrid intron, a linker sequence, the CAT cDNA, an
additional linker sequence, the late SV40 polyadenylation signal, and the
pUC origin of replication and backbone that includes the gene for ampicillin
resistance.
The human cytomeg~l~virus immediate early gene promoter and
enhancer spans the region from nucleotides 1-639. This corresponds to the
region from -522 to +72 relative to the transcriptional start site (+1) and
includes almost the entire enhancer region from -524 to -118 as originally
defined by Boshart et al, Cell 41:521-530, 1985. The CAAT box is located at
nucleotides 487-491 and the TATA box is at nucleotides 522-526 in
pCMVHI-CAT. The CAT transcript is predicted to initiate at nucleotide 549,
which is the transcriptional start site of the CMV promoter. The tripartite
leader-hybrid intron is composed of a fused tri-partite leader from
adenovirus containing a 5' splice donor signal, and a 3' splice acceptor signal
derived from an IgG gene. The elements in the intron are as follows: the
first leader, the second leader, part of the third leader, the splice donor
sequence and intron region from the first leader, and the mouse
immunoglobulin gene splice donor sequence. The length of the intron is 230
nucleotides. The CAT coding region comprises nucleotides 1257-1913. The
SV40 poly A signal extends from nucleotide 2020 to 2249.
Accordingly, construction of the pCMVHI-CAT plasmid proceeded
as follows. The vector pCMVI~ (Clontech, Palo Alto, CA) was digested with
Not I to excise the ~-galactosidase gene. The vector fragment lacking the ~-
galactosidase gene was isolated and ligated to form pCMV.
The hybrid intron (Figure 17) was obtained from the plasmid pAD~3
(Clontech). The hybrid intron had been isolated from a 695 base pair XhoI-
EcoRI fragment of p91023(B), see Wong et al., Science, 228, 810-815 (1985).
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The hybrid intron contains the fused tripartite leader from adenovirus, the
donor site from the first segment of the tripartite leader, and the acceptor
site from an IgG gene, and has a length of 230 bp.
pAD~ was digested with Pml I and Not I, and the ~500 base-pair (bp)
5 fragment was isolated, and then ligated into the Not I site of pBluescriptII
KS(-) (Stratagene, La Jolla, CA) to form pBlueII-HI.
pBlueII-HI was digested with XhoI and NotI to excise the hybrid
intron fragment. This fragment was ligated into the XhoI and NotI sites of
pCMV, replacing the SV40 intron to form pCMVHI.
The CAT gene was obtained from the Chloramphenicol
Acetyltransferase GenBlock (Pharmacia, Piscataway, NJ). This 792 bp Hind
m fragment was blunted with the Klenow fragment of DNA Polymerase I,
then Not I linkers (New England Biolabs) were ligated to each end. After
digestion with Not I to expose the Not I sticky ends, the fragment was
subcloned into the Not I site of pCMV to form pCMV-CAT. pCMV-CAT
was digested with Not I to excise the CAT fragment. The CAT fragment
was ligated into pCMVHI to form pCMVHI-CAT which is depicted in
Figure 16.
part B--pCF1 and pCF2
Although pCMV~ is suitable for therapeutic transfections, further
performance enhancements (including increased expression of transgenes)
are provided by the pCF1 and pCF2 plasmids. A map of pCFl/CAT is
shown in Figure 18, panel A, and a map of pCF2/CAT is shown in panel B.
Briefly, pCF1 contains the enhancer/promoter region from the
imme~ te early gene of cytomegalovirus (CM~I) . A hybrid intron is
located between the promoter and the transgene cDNA. The
polyadenylation signal of the bovine growth hormone gene was selected for
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placement downstream from the transgene. The vector also contains a
drug-resistance marker that encodes the aminoglycosidase 3'-
phosphotransferase genè (derived from the transposon Tn903, A. Oka et al.,
Journal of Molecular Blology, 147, 217-226, 1981) thereby conferring
resistance to kanamycin. Further details of pCFl structure are provided
directly below, including description of placement therein of a cDNA
sequence encoding for cystic fibrosis transmembrane conductance regulator
(CFTR) ~roLei~L.
The pCFl vector is based on the commercially available vector
pCMV~ (Clontech). The pCMVI~ construct has a pUCl9 backbone U Vieira,
et al., Gene, 19, 259-268, 1982) that includes a procaryotic origin of
replication derived originally from pBR322.
Basic features of the pCPl-plasmid (as constructed to inciude a
nucleotide sequence coding for CFTR) are as follows. Proceeding clockwise
--the human cytomegalovirus imme~7iate early gene promoter and
enhancer, a fused tripartite leader from adenovirus and a hybrid intron, a
linker sequence, the CFTR cDNA, an additional linker sequence, the bovine
growth hormone polyadenylation signal, pUC origin of replication and
backbone, and the kanamycin resistance gene. The pCFl-CFTR plasmid has
been completely sequenced on both strands.
The human cytomegalovirus immediate early gene promoter and
enhancer spans the region from nucleotides 1-639. This corresponds to the
region from -522 to ~72 relative to the transcriptional start site (+1) and
includes almost the entire enhancer region from -524 to -118 as originally
defined by Boshart et al., Cell 41, 521-530 (1985). The CAAT box is located
at nucleotides 486-490 and the TATA box is at nucleotides 521-525 in pCFl-
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C~Tl~. The CFTR transcript is pre~1icted to initiate at nucleotide 548, which
is the transcriptional start site of the CMV promoter.
The hybrid intron is composed of a fused tri-partite leader from
adenovirus containing a 5' splice donor signal, and a 3' splice acceptor signal
derived from an IgG gene. The elements in the intron are as follows: the
first leader (nucleotides 705-745), the second leader (nucleotides 746-816),
the third leader (partial, nucleotides 817-877), the splice donor sequence and
intron region from the first leader (nucleotides 878-1042), and the mouse
immunoglobulin gene splice donor sequence (nucleotides 1043-1138). The
donor site (G I S~E) is at nucleotides 887-888, the acceptor site (AG I G) is atnucleotides 1128-1129, and the length of the intron is 230 nucleotides. The
CFTR coding region comprises nucleotides 1183-5622.
Within the CFTR-encoding cDNA of pCF1-CFTR, there are two
differences from the originally-published predicted cDNA sequence a.
Riordan et al., Science, 245, 1066-1073, 1989); (1) an A to C change at
position 1990 of the CFTR cDNA which corrects an error in the original
published sequence, and (2) a T to C change introduced at position 936. The
change at position 936 was introduced by site-directed mutagenesis and is
silent but greatly increases the stability of the cDNA when propagated in
bacterial plasmids (R. J. Gregory et al. et al., Nature, 347, 382-386, 1990).
The 3' untranslated region of the predicted CFTR transcript comprises 51
nucleotides of the 3' untranslated region of the CFTR cDNA, Z1 nucleotides
of linker sequence and 114 nucleotides of the BGH poly A signal.
The BGH poly A signal contains 90 nucleotides of flanking sequence
5' to the conserved AAUAAA and 129 nucleotides of flanking sequence 3' to
the AAUAAA motif. The primary CFTR transcript is predicted to be
cleaved downstream of the BGH polyadenylation signal at nucleotide 5808.
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There is a deletion in pCF1-CFTR at position +46 relative to the cleavage
site, but the deletion is not predicted to effect either polyadenylation
efficiency or cleavage site accuracy, based on the studies of E.C. Goodwin et
al., J. Biol. Chem., 267, 16330-16334 (1992). After the addition of a poly A
5 tail, the size of the resulting transcript is approximately 5.1 kb.
pCF2 plasmid, Figure 18 (E~), contains a second CMV enhancer, in
tandem with the first. Enhanced expression of transgenes from pCF1 or
pCF2 results from the combination of a strong promoter, the presence of a
highly efficient polyadenylation signal, a leader sequence that enhances
10 translation, and an intron to increase m~c~ge stability.
Example 5- Correction of Chloride Ion Transport Defect in Nasal Polyp
Epithelial Cells of a Cystic Fibrosis Patient by Cationic Amphiphile-
Mediated Gene Transfer
Primary (non-immortalized) nasal polyp cells from an adult male
15 cystic fibrosis patient (homozygous for the ~ F508mutation) were grown on
collagen- coated permeable filter supports (~illic~ ) to form a polarized
and confluent epithelial monolayer. Once the monolayer was electrically
tight (about 5 to 7 days post seeding, and as indicated by the development
of resistance across the cell sheet), the apical surface can be exposed to
20 formulations of cationic amphiphile: DNA complex.
In this case, the amphiphile (spermidine cholesterol carbamate ) was
provided as a 1:1 (by mole) mixture with DOPE, and this mixture was then
complexed with pCMV-C~ plasmid vector (a construct encoding wild
type human cystic fibrosis trar ~m~mhrane conductance regulator protein,
25 see above). Conc~nLldLions in the final mixture were 42 ,umolar of
spermidine cholesterol carbamate(and also of DOPE) and 60 ~lmolar (based
on molarity in nucleotides) of the plasmid expression vector.
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Expression of CFTR was r~et~rrnined by measuring cAMP-stimulated
transepithelial chloride secretion in a modified Ussing chamber, Zabner et
al., Nature Genetics ,6, 75-83 (1984). The mucosal side of the epithelium was
bathed in Ringer's bicarbonate solution bubbled with 95% ~2 and 5% CO2.
The composition of the submucosal solution was similar to the mucosal
solution with the exception that sodium gluconate replaced sodium
chloride. Transepithelial voltage was clamped to 0 mV and short circuit
current was recorded. Amiloride (10 ,uM) was applied into the apical bath,
followed by the mucosal addition of forskolin and IBMX (at 100 ~lM each).
5-nitro-2-(3-phenylpropylamino) benzoic acid ("NPPB"), an inhibitor of
CFTR chloride channels, was then added to the mucosal solution at 10 to 30
~M ==
Chloride secretion (i.e. movement of chloride from the epithelial cells
to the mucosal solution) is shown as an upward deflection (see Figure 19).
The same plasmid vector, but containing a reporter gene, was used as a
negative control. A cAMP-stim~ current (0.5 to 2.5 11ampere/cm2) was
observed in monolayers transfected with wild type CFTR gene. Current
was not detected with the pCMV-~-galactosidase control.
Example 6- Correction of Chloride Ion Transport Defect in Airwav
Epithelial Cells of a Cystic Fibrosis Patient by Cationic Amphiphile-
Mediated Gene Transfer
A recommended procedure for formulating and using the
pharmaceutical compositions of the invention to treat cystic fibrosis in
human patients is as follows.
Following generally the procedures described in Example 1, a thin
film (evaporated from chloroform) is produced wherein spermine
cholesterol carbamate (amphiphile No. 67) and DOPE are present in the
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molar ratio of 1: 2. The amphiphile-containing film is rehydated in water-
for -injection with gentle voLL~ g to a resultant amphiphile concentration
of about 3mM. However, in order to increase the amount of
amphiphile/DNA complex that may be stably delivered by aerosol as a
5 homogeneous phase (for example, using a Puritan Bennett Raindrop
nebulizer from Lenexa Medical Division, Lenexa, KS, or the PARI LC JetTM
nebulizer from PARI Respiratory Equipment, Inc., Richmond, VA), it may
be advantageous to prepare the amphiphile-containing film to include also
one or more further ingredients that act to stablize the final
10 amphiphile/DNA composition. Accordingly, it is presently ~reL~ d to
prepare the amphiphile-containing film as a 1: 2: 0.05 molar mixture of
amphiphile No. 67, DOPE, and PEG(sooo)-DMpE. ~A suitable source of
PEG-DMPE, polyethylene glycol 5000 - dimyristoylphoshatidyl
ethanolamine, is Catalog No. 880210 from Avanti Polar Lipids, Alabaster,
15 AL]. Additional fatty acid species of PEG-PE may be used in replacement
therefor.
Without being limited as to theory, PEG (5000) -DMPE is believed to
stablize the therapeutric compositions by preventing further agrregation of
formed amphiphile/DNA complexes. Additionally it is noted that
20 PEG (2000) -DMPE was found to be less effective in the practice of the
invention.
pCF1-CFTR plasmid (containing an encoding sequence for human
cystic fibrosis transmembrane conductance regulator, see Example 4) is
provided in water-for-injection at a concentration, measured as nucleotide,
25 of 4 mM. Complexing of the plasmid and amphiphile is then allowed to
proceed by gentle contacting of the two solutions for a period of 10 minutes.
CA 0220~968 1997-0~-23
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It is presently ~re~lled to deliver aerosolized DNA to the lung at a
concentration thereof of between about 2 and about 12 mM (as nucleotide).
A sample of about 10 to about 40 ml is generally sufficient for one aerosol
administration to the lung of an adult patient who is homozygous for the A
5 F508 mlltatic)n in the CFTR-encoding gene.
It is expected that this procedure (using a freshly prepared sample of
amphiphile/DNA) will need to be repeated at time intervals of about two
weeks, but depending considerably upon the response of the patient,
duration of expression from the transfected DNA, and the appearance of
10 any potential adverse effects such as inflammation, all of which can be
determined for each individual patient and taken into account by the
patient's physicians.
One important advantage of the cationic amphiphiles of the present
invention is that they are substantially more effective--in vivo--than
15 other presently available amphiphiles, and thus may be used at
substantially lower concentrations than known cationic amphiphiles. There
results the opportunity to substantially minimize side effects (such as
amphiphile toxicity, i~flAmmatory response) that would otherwise affect
adversely the success of the gene therapy. A further particular
20 advantage associated with use of many of the amphiphiles of the invention
should again be mentioned. Many of the amphiphiles of the invention were
designed so that the metabolism thereof would rapidly proceed toward
relatively ~rml~s biologically-compatible components. In this regard,
highly active amphiphiles 53, 67, and 75 are of particular note.
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Example 7- Further Enhancements in Plasmid Design for Gene Therapy:
Replicating Episomal Plasmids
Although the above design features substantially enhance the
performance of available pl~smi~.s, further modifications are desirable in
5 order that therapeutic compositions comprising such plasmids and cationic
amphiphiles have optimal performance for gene therapy.
It is desirable that plasmids for gene therapy also be able to replicate
in the cells of patients, since continued presence of the plasmid will provide
correction of the genetic defect (in the case of cystic fibrosis, lack of
10 functioning CFTR protein in the cell membrane of lung epithelial cells or
other cells) over an extended period of time. There is concern that plasmids
representative of the current art (that is, those that cannot replicate in the
targeted cells of a patient) may be degraded after only a relatively short
period of maintenance in the patient, thus requiring excessive repeat
15 administrations.
Long term correction could perhaps be achieved using a vector
designed to integrate into chromosomes in the patient's targeted cells (for
example, vectors patterned on retrovirus). Such a strategy, however,
involves risks including (1) that the vector will integrate into an essential
20 region of a chromosome, or (2) that the vector will integrate adjacent to an
oncogene and activate it.
Accordingly, it would be desirable to provide for continued
maintenance of gene therapy vectors (pl~.smit1.s) in target cells by other
means. One such strategy is to construct a plasmid capable of being
25 maintained separately in the nucleus of a target cell, and that is also able to
replicate there (i.e. an episome).
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Pl~mi~1s provided according to this aspect of the invention can be
constructed as follows. It has been deLe~ ed (C. McWhinney et al.,
~ Nucleic Acids Research, 18, 1233-1242, 1990) that the 2.4 kb Hindm-XhoI
fragment that is present imrne~ ly 5' to exon 1 of the human c-myc gene
5 contains an origin of replication. The fragment was then cloned into a
plasmid that if transfected into HeLa cells was shown to persist therein for
more than 300 generations under drug selection. Replication was shown to
be semiconservative (C. McWhirmey et al.). Although a~ploxi Inately 5% of
the plasmid population was lost per cell generation without drug selection
10 in those experiments, this result nonetheless demonstrates substantial
stabilization would be of benefit with respect to the design of therapeutic
pl~smi~ls for gene therapy.
Accordingly, in one example of a replicating episomal vector, a
variant of pCF1-CFTR (or pCP1-CAT) can be constructed in which a copy of
15 the 2.4 kb Hindm-XhoI fragment is placed just 5' to the CMV
enhancer/promoter region of the pCF1 backbone. Alternatively, between 2
and about 4 - in tandem - copies of the 2.4 kb fragment may be simil~rly
positioned. The increase in plasmid size that results from insertion of the
2.4 kb fragment (or multiple copies thereofl is predicted to provide an
20 additional benefit, that is, to facilitate plasmid unwinding, thus facilitating
the activity of DNA polymerase.
- Use of this origin of replication, or multiple copies thereof, allows the
resultant plasmid to replicate efficiently in human cells. Other DNA
sequences containing other origins of replication may also be used (for
25 exa~nple, as found in the human ~-globin gene, or the mouse DHFR gene.
A plasmid that can be constructed according to this aspect of the
invention and containing the cytomeg~lovirus promoter and enhancer, an
CA 0220~968 1997-0~-23
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intron, the CFTR cDNA, the bovine growth hormone polyadenylation
signal, the kanamycin resistance transposon Tn903, and 4 copies of the 2.4
kb 5' flanking region of the human c-myc gene is shown in Figure 20.
Example 8- Further Enhancements in Plasmid Design for ~ene Therapy:
Use of Cytokine Promoters to Modulate Expression
of Transgenes in Gene Therapy
Chronic inflammation is associated with numerous of the disease
10 states that can be treated by gene therapy. Representative of such disease
states are cystic fibrosis (using CFTR), bronchitis, adult respiratory distress
syndrome (using alpha-1 antitrypsin), and metastatic cancers (through
upregulation of p53, TIMP-1, and TIMP-2). Inflammatory conditions
typically involve many interrelated processes (for example, involvement by
15 many types of immune system cells and liver proteins), whereby the body
attempts to heal a damaged or infected tissue. However, chronic
infla~m~tion which persists as a result of an unresolved condition may lead
to permanent tissue damage, as is the case with respect to lung tissue
affected by cystic fibrosis and associated and unresolved lung infections. In
20 fact, permanent damage to the lung tissue of cystic fibrosis patients is a
leading cause of their mortality. It would be desirable to provide gene
therapy in such a manner as to treat inflammatory conditions associated
with the targeted disease state.
Accordingly, a further aspect of the present invention involves
25 construction of gene therapy vectors in which the therapeutic transgene is
placed under control of an RNA polymerase promoter from a cytokine gene
(or a gene that encodes another similar regulatory protein) such as, for
example, the promoter for any of interleukin 2, interleukin 8, interleukin 1,
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interleukin 11, interleukin 6, endothelin -1, monocyte chemoattractant
~roLe"l -1, IL-lra (receptor agonist), or for GM-CSF.
Cytokines may be defined as hormone-like intercellular signal
pr~L~I.ls that are involved in regulation of cell proliferation, differentiation,
5 and function, such as conceming haematopoiesis and lymphopoiesis. The
interleukins are a particular group of cytokines having promoters that are
useful in the practice of the invention. The interleukins are ~roLe~, Is,
typically of unrelated origin, which act as intercellular signals mediating
reactions between immunoreactive cells. However, it is understood that
10 many "interleukins" have effects upon additional cell types including
endothelial cells, epithelial cells, and fibroblasts.
Since the concentration of many cytokines is upregulated at an
affected site in response to the level of inflammation that is present, gene
therapy vectors can be designed wherein the level of therapeutic transgene
15 expressed thelerloln is rlet~rmined~ in part, by the level of inflammation
present. There follows hereafter description of how such vectors are
designed using primarily properties of the interleukin 8 gene as an example.
It has been determined that numerous biologically active molecules are
present in tissues at concentrations thereof that increase with the severity of an
20 inflammatory condition (for example, tumor necrosis factor "TNF" and
potentially transcription factors such as N~-kB, .9P-1, NF-IL6 and octamer
binding ~roL~" 1).
It has also been determined that interleukin 8, a polypeptide of 8,500 MW,
is upregulated by inflammation and acts as a potent chemoattractant for T
25 lymphocytes and neutrophil cells that are themselves involved in the
inflammation response. The interleukin 8 gene is regulated primarily at the
transcriptional level, and it has also been determined (H. Nakamura et al.,
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Joumal of Biological Chemistry, 266, 19611-19617, 1991) that TNF can
increase interleukin 8 transcription by more than 30 -fold in vitro in
bronchial epithelial cells. Accordingly, there follows description of gene
therapy vectors which take advantage of the above.
A plasmid can be constructed that is substantially similar to pCF1,
that is, derived from a pUC plasmid containing a bacterial-derived origin of
replication and a gene conferring resistance to kanamycin. The resultant
plasmid contains also, in sequence, a CMV enhancer, a promoter, a hybrid
intron, a cDNA sequence encoding CFTR, and the bovine growth hormone
polyadenylation signal. As l~NA polymerase promoter there is selected the
-335 to +54 region of the interleukin 8 promoter. This region gave the
highest ratio in terms of promoter activity plus TNF over minus TNF
(Nakamura, 1991)
Such a plasmid has particularly valuable performance attributes. As
inflammation increases in a cystic fibrosis-affected lung (and therefore the
need to treat the lung with gene therapy also increases), the concentration of
various inflAmm~tion-related molecules ( such as TNF) will increase. By
placing the CFTR-encoding cDNA of the therapeutic plasmid under the
control of a transcriptional promoter (that of interleukin 8, for example) that
is itself sensitive to the concentration of inflammation-related substances in
contact with the cell, the promoter will function as a natural gene switch
such that the amount of beneficial CFTR transcription will be tailored to the
amount of infl~mm~tion. As aforementioned, RNA polymerase promoter
sequences derived from the other aforementioned genes are also useful in
the practice of the invention.
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Example 9 Intravenous Delivery of Transgenes
For some ~ P~e states, such as cystic fibrosis, it is desirable to
deliver transgenes to the lung. Delivery by aerosol is the most direct
approach to achieve this goal. However, given the difficulties inherent
with the delivery of an aerosol together with the potential need to
target organs other than the lung (for example, the pancreas for cystic
fibrosis), it is important to evaluate the feasibility of lung delivery
using non-aerosol delivery formats. Accordingly, intravenous
delivery of a reporter transgene was performed using a mouse model
and the feasibility of intravenous organ targeting was assessed. A
comparison was made of feasibility of delivery to the lung and the
heart.
The reporter plasmid pCF-l CAT (Example 4) was used and was
purified to minimize endotoxin (<1 EU/mg pDNA), and also
chromosomal DNA colllalnination (< 2%). Amphiphile No. 53 (1:1
with DOPE) / DNA complex was prepared according to the
procedures of Example 3. The amphiphile was provided as the free
base, the plasmid was prepared as a sodium salt in water, and the
DOPE was provided in zwitterionic form.
The animal model was the BALBIc mouse. Females 6-8 weeks
old weighing 16-18 g were injected intravenously using the tail vein,
using 5 animals per group. The volume of lipid:pDNA complex used
was 100 ,ul in all experiments. Unless noted otherwise, mice were
sacrificed 48 h following adminstration of the complex. Organs were
frozen immediately on dry ice to store for subsequent analysis.
Expression of chloramphenicol acetyl transferase (CAT) was
quantitated using a radiochPmic~l assay for CAT enzymatic activity.
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Organs were weighed and homogenized on ice in a lysis buffer
containing protease inhibitors. The lysate was freeze-thawed 3X,
centrifuged, and heated to 65~C to inactivate deacetylases before
adding it to a reaction mixture containing 14C-chloramphenicol. After
5 an incubation at 37~C, the mixture was extracted with ethyl acetate,
concentrated, spotted onto TLC plates and eluted with CHCl3/MeOH.
Spots corresponding to the acylated reaction products were
quantitated (Betagen) and converted to ng CAT activity using
authentic CAT standards.
It was surprisingly determined that targeting to the heart could
be substantially improved by altering the molar ratio (at a constant
DNA concentration of 0.9 mM, measured as nucleotide) of
amphiphile/DNA in the therapeutic composition. This information is
of value in connection with gene therapy for the heart, such as for
15 coronary disease. However, targeting to the lung remained relatively
constant over a range of amphiphile/DNA ratios, all at constant DNA
concentration (Figure 21).
At molar ratios of less than about 0.5, the organ distribution was
found to be strongly weighted toward the lung. At this molar ratio, the
20 zeta potential of the complex is negative (about -30 mV) due, in part, to
excess negative charge from the DNA relative to the amphiphile. At an
amphiphile/DNA ratio of 1.25, however, where the complex has a
positive zeta potential (about +30mV), organ distribution was
rem~rk~hly altered and substantial expression was found in the heart
25 (Figure 21).
Zeta potentials of the samples can be measured (using typically
5 measurements per sample) employing a Malvern Zetasizer 4
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(Malvern Instruments, Southborough, MA.) and a zeta cell (AZ-104
cell, Malvern Instruments Co.). Dried lipid films containing the
cationic lipid and DOPE are hydrated in distilled water (dH2O). DNA
typically should be diluted to a concentration of about 300 ~lM in
dH2O. The DNA solution (1.5 mL) can then be added to an equal
volume of cationic lipid vesicles and incubated at room temperature
for 10 min. Enough NaCl (for example, 4 mM stock) may be added to
result in a final conc~nl~alion of 1 mM NaCl. If necessary, the sample
can be diluted further with 1 mM NaCl (to maintain a photomultiplier
signal below 4000 counts per second), and distilled water can be used
in place of the NaCl solutions.
According to this aspect of the invention, amphiphiles No. 53
and No. 67 are among those pLer~lled for use in intravenous targeting
of the heart, as are many other amphiphiles selected from Groups I and
II.
Example 10- Additional Experimental Procedures
(A! Additional synthesis procedure for N--spermine cholesteryl carbamate,
amphiphile No. 67
(Synthesis of Nl,N12 -diCbz-spermine di-HCl salt)
Benzylchloroformate (15 mL, 105 mmol) was dissolved in methylene
chloride (335 mL) and placed in a three neck flask under a nitrogen
atmosphere. In~idazole (14 g, 206 mmol) was dissolved in methylene
chloride (200 mL). The three neck flask was cooled to 0-2 ~C using an ice-
water bath and the imidazole solution was added gradually over 30 min.
The cooling bath was removed and the mixture stirred at room temperature
for 1 hour. Methylene chloride ( 250 mL) and aqueous citric acid (10%, 250
mL) were added to the mixture. The layers were separated and the organic
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layer was washed with aqueous citric acid (10%, 250 mL). The organic
fraction was dried over magnesium sulfate and concentrated in vacuo. The
resulting oil was vacuum dried for 2 hours at ambient temperature. To the
oil was added dimethylaminopyridine (530 mg, 4.3 mmol) and methylene
5 chloride (250 mL). The mixture was cooled to 0-2 ~C and kept under a
nitrogen atmosphere. A solution of spermine (lOg, 49 mmol) in methylene
chloride (250 mL) was added gradually over 15 minutes, maintaining a
reaction temperature of 0-2 ~C. The reaction mixture was stirred overnight at
ambient temperature and then concentrated in vacuo. To the resulting
material was added lM hydrochloric acid (67 mL) and methanol (400 mL).
The solution was cooled overnight at 4 ~C yielding a white precipitate. The
precipitate was isolated by vacuum filtration using Whatman #l filter paper.
The Nl,N12-diCbz-spermine di HCl salt (13.38g, 24.7 mmol, 50% yield) thus
obtained was dried under vacuum at ambient temperature for 17 hours.
(Synthesis of Nl,N12-diCbz-N4-spermine cholesteryl
carbamate)
N~ 2-diCbz-spermine di HCl salt (13.38g, 24.7 mmol) was
dissolved in a chloroform, methanol and water mixture in the ratio 65:25:4
(940 mL). The solution was stirred at room temperature and cholesteryl
20 chloroformate (llg, 24.5 mmol) was added. The solution was stirred at
ambient temperature for 1.5 hours and then diluted with lM sodium
hydroxide solution (165 mL). The organic and aqueous layers were
separated and the organic layer containing the product was washed with
water (110 mL). The organic fraction was dried over sodium sulfate,
25 concentrated in vacuo and vacuum dried. The crude oil was purified by
chromatography using silica gel (60A, 1 Kg) . The silica was packed in 10%
- MeOH / CHC13 and the column was eluted with 25% MeOH / CHC13.
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Fractions of 900 mL were collected and analyzed by thin layer
chromatography. Fractions containing the product (Rf. = 0.5 in 20% MeOH /
CHC13) were combined and conC~nLLdLed in vacuo. The resulting oil was
dried under vacuum for 17 hours to give 8.5g (9.67 mmol, 39% yield) of
5 product.
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(Synthesis of N4-spermine cholesteryl carbamate)
N1,N1~diCbz-N4-spermine cholesteryl carbamate (8.5g, 9.67 mmol)
was dissolved in 200 mL of acetic acid and 1.66 g of 10% Pd on carbon was
added. The solution was purged with nitrogen and stirred under hydrogen
5 at atmospheric pressure. The hydrogen was supplied to the reaction flask
using a balloon filled with hydrogen gas. The hydrogenolysis was allowed
to proceed for 3 hours. The reaction mixture was filtered through Whatman
#1 filter paper and the catalyst was washed with 250 mL of 10% acetic acid in
ethyl acetate. The filtrate was concentrated in vacuo to give a residue,
10 coevaporation with chloroform aids removal of the acetic acid. To the crude
product was added lM sodium hydroxide solution (400 mL) and the solution
was extracted three times with 10% MeOH / CHCl3 (700 mL). The
combined organic fractions were washed with water (600 mL) and dried over
sodium sulfate. The solution was filtered, concentrated in vacuo and
15 vacuum dried at ambient temperature for 48 hours. The crude material was
purified by chromatography on silica gel (500 g). The column was packed in
40:25 MeOH: CHC13 and eluted with 40:25 MeOH: CHCl3 and then 40:25:10
MeOH: CHCl3: NH40H. The fractions collected were analyzed by thin
layer chromatography and the product containing fractions ~ere combined
20 and concentrated in vacuo (the evaporation was assisted by the addition of
iso-propanol in order to azeotrope the residual water). The material was
vacuum dried at ambient temperature for 48 hours to give N4-spermine
cholesteryl carbamate (4g, 6.5 mmol, 67% yield).
(B! N~(N'-cholesteryl carbamate glvcineamide)-spermine (amphiphile
25 No. 91)
N-t-BOC-glycine-N-hydroxysuccinimide ester (0.5 g, 1.83 mmol) was
added to a solution of diCbz-spermine.2HCl (1.0 g, 1.94 mmol) and N,N-
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diiso~Lopylethylamirle (0.3 mL, 1.72 mmol) in 65/ 25/ 4 chloroform/
methanol/ water (50 mL). The solution was stirred overnight at room
temperature. Analysis of the reaction by TLC (20% methanol/ chloroform)
indicated the presence of a new spot. The reaction was washed first with
5 lM NaOH (10 mL) then with H2O (10 mL). The organic layer was
separated, dried over sodium sulfate, vacuum filtered, and reduced in vacuo
to an oil. The crude material was purified by flash column chromatography
(85 g silica gel) eluting with 20% methanol/ chloroform. The desired
product was isolated and characterized by lH NMR as Nl,Nl~diCbz-N4-
(N'-t-BOC-glycineamide)-spermine (402 mg, 0.65 rnmol, 35%).
Benzyl chloroformate (100 mg, 0.58 mmol) was added to a solution of
Nl,Nl~diCbz-N4-(N'-t-BOC-glycineamide)-spermine (220 mg, 0.354 mmol)
and triethylamine (4 drops) in methylene chloride (20 mL). The reaction
was stirred overnight at room temperature. Analysis of the reaction by TLC
(20% methanol/ chloroform) indicated the presence of a new, higher
running spot. The reaction was quenched by the addition of lM HCl (5
mL). The organic layer was isolated, washed with H2O (5 mL), dried over
sodium sulfate, filtered, and reduced in vacuo .
The resulting crude material was dissolved in chloroform (30 mL)
and anhydrous HCl gas was bubbled through the solution for 2 hours.
Analysis of the reaction by TLC (10% methanol/ chloroform) indicated the
complete disappearance of the starting material. The reaction was purged
with dry nitrogen, and washed with lM NaOH (2 x 10 mL) and dH20 (10
mL). The organic layer was isolated, dried over sodium sulfate, filtered, and
reduced in vacuo to give Nl,N9,Nl~triCbz-N4-glycineamide-spermine (219
mg, 0.33 mmol, 93% yield for two steps).
100
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Cholesteryl chloroformate (148 mg, 0.33 mmol) was added to a
solution of Nl,N9,N12-triCbz-N4-glycin~mi-l~-spermine (219 mg, 0.33
mmol) and triethylamine (0.3 mL, 2.15 mmol) in methylene chloride (30
mL). The reaction was stirred at room temperature for 3 hours. The
5 reaction was washed with H2O (10 mL). The organic layer was separated,
dried over sodium sulfate, filtered, and reduced in vacuo . The crude
material was purified by flash column chromatography (30 g silica gel)
eluting with 65% ethyl acetate/ hexanes. The desired product was isolated
and characterized by 1H NMR as N1,N9,N12-triCbz-N4-(N'-cholesteryl
carbamate glycineamide)-spermine (221 mg, 0.2 mmol, 62% yield).
N1,N9,N12-tri-Cbz-N4-(N'-cholesteryl carbamate glycineamide)-
spermine (221 mg, 0.2 mmol) was stirred with 10% Pd/C (50 mg) in glacial
acetic acid (10 mL) under a hydrogen atmosphere for 2.5 hours. Analysis of
the reaction by TLC (65% ethyl acetate/ hexanes) indicated the complete
15 disappearance of the starting m~ri~l. The flask was purged with nitrogen
and the catalyst was removed by vacuum filtration through filter paper
rinsing with 10% acetic acid/ ethyl acetate (20 mL). The filtrate was reduced
in vacuo to an oil which was dissolved in 10% methanol/ chloroform (100
mL) and washed with lM NaOH (20 mL) and H2O (15 mL). The organic
20 layer was separated, dried over sodium sulfate, filtered, and reduced in
vacuo. The isolated product was characterized by 1H NMR as N~(N'-
cholesteryl carbamate glycin~mitle)-spermine (128 mg, 0.19 mmol, 95%
yield).
(C) Synthesis of N4-spermidine-2 3-dilauryloxypropylamine amphiphile No.
25 ~
2,3 Dimyristoylglycerol (600 mg, 1.4 mmol) was dissolved in pyridine
and the solution cooled to 0~C. The solution was stirred under a nitrogen
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atmosphere and p-toluenesulfonyl chloride (300 mg, 1.57 mmol) was added.
The solution was allowed to warm to room temperature and was then
stirred overnight at ambient temperature. To the solution was added
hydrochloric acid (2.5M, 20 mL) and the solution was extracted three times
5 with methylene chloride (25 mL). The combined organic extracts were dried
over sodium sulfate, filtered and conc~nLLd~ed in vacuo to give a crude oil.
The oil was purified by flash chromatography (50g of silica gel, 60A) eluting
with 5% ethyl acetate / hexane. The oil obtained by flash chromatography
was dried under high vacuum at ambient temperature to give 2,3-
Dimyristoylglycerol-tosylate(630 mg, 77% yield).
2,3-Dimyristoylglycerol-tosylate (300 mg, 0.51 mmol) and N1,N8-
diCbz-sp~ line (1.5g, 3.6 mmol) were dissolved in toluene (15 mL). The
solution was stirred under a nitrogen atmosphere and heated at reflux
(110 ~C). The reaction was heated for 5 days at reflux temperature. The
15 reaction was cooled to room temperature and then filtered through
Whatman #1 filter paper. The filtrate was concentrated in vacuo . The
residue was dissolved in chloroform (50 mL) and washed with sodium
hydroxide solution (1 M, 10 mL) and water (10 mL). The organic fraction
was dried over sodium sulfate, filtered and concentrated in vacuo . The
20 crude material was purified by flash chromatography (30g silica gel, 60A)
eluting with 5% methanol / chloroform. The product containing fractions
were concentrated in vacuo. The material was purified by a second flash
chromatography column (20 g silica, 60A) eluting with 50% ethyl acetate /
hexane. Chromatography gave, after drying the product under high
25 vacuum at ambient temperature, N~(N1,N8)-diCbz-spermidine-2,3-
dilauryloxypropylamine, as an oil (142 mg, 35~/O yield).
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N4-(N1,N8)-diCbz-spermidine-2,3-dilauryloxypropylamine (142 mg,
0.18 mmol) in glacial acetic acid (5 mL) was stirred with 10% Pd/C (50 mg)
under a hydrogen atmosphere, for 2 hours. The catalyst was removed by
vacuum filtration through Whatman #1 filter paper. The catalyst was
washed with ethyl / acetate hexane (10%, 10 mL). The filtrate was
concentrated in vacuo and dried for 2 hours under high vacuum. To the
residue was added sodium hydroxide solution (1 M, 8 mL) and the solution
was extracted three times with methanol / chloroform (10%, 20 mL). The
combined organic extracts were dried over sodium sulfate, filtered and
concentrated in vacuo to give after drying under high vacuum N4-
sp~rmi~line-2,3-dilauryloxy~r~ylamine (52 mg, 52% yield).
(D! Synthesis of N--spermine-2 3-dilauryloxypropylamine amphiphile
No. 102
Nl,N12-diCbz-spermine (0.87g, 1.85 mmol) and 2,3-
dimyristoylglycerol-tosylate (280mg, 0.48 mmol) were dissolved in toluene
(25 mL) and heated at reflux temperature (110~C) for 3 days. The solution
was concentrated in vacuo and the resulting material was purified by flash
chromatography (30g silica gel, 60~) eluting with 10% methanol
/chloroform. The material isolated was dissolved in methanol / chloroform
(10%, 85 mL) and washed twice with sodium hydroxide solution (1 M, 15
mL) and water (10 mL). The organic fraction was dried over sodium sulfate,
filtered and concentrated in vacuo . The material was dried under high
vacuum overnight, at ambient temperature, to yield N4-(N1,N 12-diCbz-
spermine)-2,3-dilauryloxypropylamine (180 mg, 43 % yield).
N4-(N1,N12-diCbz-spermine)-2,3-dilauryloxypropylmine (180 mg,
0.2 mrnol) in glacial acetic acid (10 mL) was stirred with 10% Pd/C (50 mg)
under a hydrogen atmosphere, for 3 hours. The catalyst was removed by
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vacuum filtration through Whatman #l filter paper. The catalyst was
washed with ethyl / acetate hexane (10%, 30 mL). The filtrate was
conc~nlldled in vacuo and dried for 2 hours under high vacuum. To the
residue was added methanol / chloroform (10%, 85 mL) and the organic
5 layer was washed twice with sodium hydroxide solution (1 M, 15 mL) and
water (10 mL). The organic fraction was dried over sodium sulfate, filtered
and concentrated in vacuo to give after drying under high vacuum N4-
spermine-2,3-dilauryloxypropylamine (50 mg, 40% yield).
104
