Note: Descriptions are shown in the official language in which they were submitted.
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1
CATIONIC LIPIDS FOR TRANSFECTION OF NEGATIVELY
CHARGED OR NEUTRAL MOLECULES INTO LIVING CELLS
Field of the Invention
This invention relates to polycationic lipids useful for the delivery
(transfection) of
nucleic acids (DNA,RNA) and other negatively charged or neutral molecules into
living
cells, either in vivo or in vitro.
Background of the Invention
Liposomes aggregates made with polycationic lipids are useful structures
capable
of complexing with negatively charged macromolecules such as DNA or RNA. These
complexes can be taken up by living cells and then , once inside the cytosol,
through an
uriknown mechanism, they are presumed to migrate into the cell nucleus. In the
nucleus,
there are enzymes capable of "reading" and "expressing" the message coded by
the nucleic
acids so delivered and produce new proteins, which were not being produced by
the cell
before the transfection of the foreign nucleic acid. When cells so transfected
divide and
their daughter cells still have the capability to produce the proteins encoded
by the initially
transfected DNA , the transfection is said to be stable. That is, the new DNA
has stably
integrated into the cell nucleus changing the cell's genetic make-up. If , on
the other
hand, the parent cells can produce the protein encoded by the transfected DNA,
but their
daughter cells are not capable of expressing the such DNA , the transfection
is said to be
transient. RNA transfection is always transient. Stable transfection of human
or animal
cells is the basis of the so called gene therapy, since cells which are
deficient on a crucial
protein for the organism's survival could be in principle repaired by stably
transfecting the
DNA needed to produce the absent protein. Another type of potential use of
DNAIRNA
transfection for therapy is the antisense therapy . In this approach, a short
piece of nucleic
acid (oligonucleotide) capable of adhering (hybridizing) to defective DNA (or
RNA)
which is being expressed by the cells to produce an undesired protein, such as
an
' oncoprotein (cancer causing protein), is transfected into the cells in order
to stop the
expression of the undesired protein by virtue of its adherence to the
defective nucleic
acid. This method of therapy does not change the genetic make-up of the cell,
but blocks
the effect of the genetic disorder already present in the cell's genome.
Besides this
potential applications of polycationic lipids for use in human therapy, there
is already a
SUBSTITUTE SHEET (RULE 26)
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well established market for these types of chemicals in the research products
field. They
are currently being used by researchers to deliver nucleic acids and proteins
into cells in
order to study how the expression of different genes affect cell growth and
function.
There are two possible ways to deliver DNA into cells for gene therapy : ex
vivo
and in vivo transfection. In the ex vivo modality , cells from a patient are
removed from
the body, cultured and transfected in vitro. Then, the cells are returned into
the patient
where the beneficial DNA message is hopefully expressed. In the in vivo mode,
the
DNA is delivered directly into the patient, which makes this procedure simpler
and less
expensive. To date the only effective way to deliver DNA in vivo is by using a
virus
which naturally infects cells of an specific organ (targets that organ) within
the body, and
whose genetic make up has been modified by adding the DNA beneficial to the
patient.
Once inside the cells of the patient, the virus can incorporate the new DNA in
the genome
of the cell (stable transfection) and the parent cell and its daughters can
express the
beneficial protein. The pathological component of the virus has been deleted
before the
patient is exposed to such a virus and only the targeting component left
intact. Virus can
do this process sometimes with nearly 100% efficiency. However, there are
risks
associated with their use, they can produce immunological reactions which may
be fatal
to the patient; the DNA incorporation in the cell's genome is random,
therefore it might
disrupt needed genes or activate oncogenes; they are also difficult to mass
produce, etc.
Liposomes or lipid aggregates do not have the side effects of viruses , but
are not
as efficient as viruses are. There is a constant need to develop newer lipids
that can
approach the efficiency of viruses without their undesirable side effects (E.
Marshall,
Sciefrce 269,1050 (1995)) . There are several lipids for nucleic acids
transfection already
in the market. The most relevant of these lipids are: DOTMA (N-[1-(2,3-
dioleoyloxy)propyl]- N,N,N-trimethylamonium chloride, U.S. Pat. No. 4,897,355
to D.
Eppstein et al.), DMRIE (D,L-1,2-O-dimyristyl-3-dimethylaminopropyl-b-
hydroxyethylammoniumchloride, U.S. pat. No 5,264,618 to Felgner, P.L. et al.),
DOTAP
(I,2-bis(oleoyloxy)-3-3(trimethylammonia)propane) Boehringer-Mannheim Catalog
No.l
202 375) , DOGS (5-carboxysperminyglycine dioctadecylamide, U.S. pat. No
5,171,678
to Behr, J-P. et al. DOGS is sold under the trade name TransfectamTM by the
Promega
... ~....~ ,~ ....
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Corp. Madison, WI ), DOSPA (2,3-dioleyloxy-N-[2(sperminecarboxyamido)ethyl]-
N,N-
dimethyl-propanaminium trifluoroacetate, U.S. Pat. No. 5,334,761 to Gebeyehu,
G. et
al.), DDAB (Dimethyloctadecylammoniumbromide, U.S. Pat. No. 5,279,833 to Rose,
- J.K.), TMTPS (N,N,N,N-Tetramethyltetrapalmylspermine, PCT Int.Pub.No. WO
95/17373. Haces, A. et al.). DOTMA, DOSPA, DDAB and TMTPS are sold by Life
Technologies, Inc., Gaithersburg, MD under the trade names of Lipofectin,
LipofectAMINE, LipofectACE and CelIFECTIN, respectively. A recent relevant
publication which deals with art related to the present invention has been
reported by
Ruysschaert et al.((1994)Biochem. Biophys. Res. Commun.203,1622-1228). All
these
lipids, except DOGS, are formulated with dioleolylphosphatidylethanolamine
(DOPE),
which is a neutral lipid devoid of transfection activity, in order to make the
active
. liposomes. These lipids posses some desirable characteristics, however they
are far from
the ideal vehicle to deliver DNA. Their main drawbacks are low efficiency, non-
specificity
of targeting, considerably toxicity, low water solubility, and serum
inhibition of their
action.
Although progress has been made in overcoming some of these obstacles, there
is
considerable room for improvement an experimentation. The design of these
lipids is still
a semi-empirical endeavor , since very little is known about the mechanism by
which they
act.
Therefore, it is the object of this invention to improve the desired
characteristics of
these lipids by exploring and incorporating new chemical functionalities as
well as
spatial or topological arrangements which improve the transfection efficiency
and lower
the toxicity.
It is also an object of this invention to synthesize polycationic lipids which
incorporate a small , non lipid-bilayer-disturbing moiety that mimics a
natural molecule,
.. which cells can recognize as their natural e~'ector or ligand, thus
facilitating the
transfection as well as the specificity of targeting of the macromolecule.
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Summary of the Invention
In this invention a series of new polycationic lipids and their method of
preparation
is described Such lipids are useful as transfection reagents for: nucleic
acids,
oligonucleotides,mononucleotides, polypeptides and proteins. In addition, some
of these
lipids are also useful as more effective detergents for cleaning and as
vehicles in the
cosmetic field.
The present invention describes novel oxo and sulfinyl backbone substituted
polycationic lipids with ammonium, guanidinium and imidinium positively
charges
moieties as anchoring groups having the formula:
Formula I
1 Rt
2 Y- I
R2 N+ (CH~)m (X)p (CH2)n N+ R2
3)i
~ R3)i
Y= Cl-, Br-, I-, Ac0-, or any pharmaceutically acceptable anion
X = O,S(O),CH2 RI,Rl' = independently: C1-Clg linear hydrocarbon.
m,n = 1,2,3 R2,R2' = independently : H ; Cl-Clg linear alkyl ; cyanoethyl;
aminopropyl ; aminobutyl; C2 -C4 alkyl guanidinium or
amidinium;N,N,N independently(C ~ C 1 g)aminopropyl
or butyl; C or N substituted spermine or spermidine;
N,N-(C4-C 1 g )alkyl-4- aminonobutyrylaminopropyl
p = 0, 1 R3,R3' = independently: CI-C6 linear alkyl, acetoxyethyl,
~ = 0, 1 CH2C02CH2CH3
X ~ CH2 when R2,R2' = Cl-C6 linear alkyl
When p = I, m and n $ 1
It is also disclosed in this invention a series of novel phosphatidyl and
glyceryl
guanidiniurn cationic lipids having the formula II.
_..... ~ . ~ . 1..
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Formula II
CH2 O R4
CH O Ra
CH2 o L A
R4 = Independently: linear (CO)C~-C2o , C6-C2o
L = 2-10 atoms linker
A = Guanidinium; imidinium; guanidylated polyamines.
These compounds can be used alone or in mixtures with other liposome forming
compounds (co-lipids) to prepare lipid aggregates which are useful to deliver
macromolecules, specifically negatively charged macromolecules to living cells
either in
culture or in viva.
Compounds of Formula I:
The lipids depicted in Formula I have a hydrocarbon backbone substituted with
heteroatoms which are sterically smaller, but equally or more flexible as the
methylene
group that they replace. This feature makes these new lipids fit more closely
to the
macromolecule to be delivered to the cells. This closer fit combined with the
polycationic
nature of the backbone produces a tighter binding. In addition, these
heteroatoms are
hydrophilic; thus, they not only confer an increased amphiphilic character to
the lipids but
also make the backbone more linear or "stretched" as compared to the all-
methylene
groups backbone. The latter being hydrophobic tends to wrap around itself in
an aqueous
environment, therefore pulling the positively charged moieties away from the
negatively
charged phosphates on the DNA/RNA backbone, this results on a weaker binding
between the polycationic lipid backbone and the polyanionic DNA backbone,
since the
opposite charges can not align properly in this arrangement. The hydrophilic
backbone
being linear allows for proper alignment of the opposite charges, also leading
to a tighter
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binding. In addition, the higher hydrophilicity conferred by the heteroatoms
on the
polycationic backbone, make possible the addition of more hydrophobic tails
without loss
of water solubility, thus making these compounds more densely packed than
compounds
of the prior art. This is an advantage since the same molar amount of lipid
will produce a
higher hydrophobic coating of the nucleic acid to be delivered. In fact, one
of the
preferred embodiments is compound {Sa) which has four hydrophobic tails and
two
positive charges (two tails per charge).
(CHz)i7CH3 (CHzOCH3
~+ O ~ +
CH3(CHZ)i7 N ~ ~ N (CH2)17CH3
2I_
CH3 CH3
Sa
Some of the compounds herein described, such as (l3a,b) have fewer hydrophobic
tails and a more hydrophobic backbone ( no heteroatom substitution, but less
hydrophobic
overall). However, these compounds have moieties which mimic small natural
biological
effectors such as the neurotransmitters gamma amino butyric acid (GABA),
acetylcholine
etc. These moieties bind to their corresponding cell receptors targeting the
delivery of
nucleic acids to those cells rich in these type of receptors such as muscle
and neural cells.
The latter type of cells are among the most difficult to transfect since they
are postmitotic
cells (non-dividing). These small chemical moieties do not perturb the ability
of the lipids
to form Iiposomes aggregates and at the same time confer more amphiphilic
character to
said lipids, since they are polar entities. A particular preferred embodiment
of the latter
compounds are compounds of formulae (13a) and more specifically compound
(13d).
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(CHZ)»CH3 (CHz)»CH3 (~ H2}»CH3 (~ H2)«CHa
O
+ N
N ~ N+ N ~/~/ ~OCH2CH3
CHaCHzO
2 I-
CH2COOCHzCH3 C~OOCHzCHa
(13a)
(CI-)r)nCH3 (C~)nCH~ (~ I~)nCH~ (~ ~)nCI-b
~ + NON
N N NHz
HzN
2 I- O
O CH, CH3
(13d)
Another novel feature of the compounds disclosed here is the fact that, in
addition
to the traditional quaternary ammonium salts, guanidino and amidino moieties
are used as
permanent positively charged centers. These functional groups are strongly
basic an
have the same charge as their ammonium counterparts, but have the advantage of
being
sterically smaller , since they are planar. Thus, they can get closer to the
negatively
charged phosphates of the DNA/ RNA backbone producing a stronger binding
interaction
than that of ammonium salts. Furthermore, these guanidinium and amidinium
moieties
have the ability to form hydrogen bridges with the nucleic acids bases
(guanidinium salts
are used as chaotropic agents to precipitate DNA) therefore they have an
additional
binding mode not available to ammonium salts. Moreover, the guanidino moiety
can also
be used to target neural cells, since compounds such as Guanethidine , which
possess such
a functional group, are internalized by neurons (Wiener, N. In, The
Pharmacological
Basis of Therapeutics, (Gilman, A.G.; Goodman,L.S.; Rall, T.W.; and Murad, F.;
Eds.)
Macmillan Pubs. Co. New York, 1985,p~ 181-214.) . Thus , by including the
lipidic
content as well as the amine and guanidino moieties of Guanethidine in our
novel
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liposome reagents we can target this difficult to transfect cell type. A
particularly useful
and preferred embodiment of these compounds is compound (6d), which is the
most
active of compounds tested.
NH
N ~ (CH2 )i7CH3
NH3
2I- O
NH
NH N
~(CHZ ),7CH3
~+
3
(6d)
Additionally, reduced or no toxicity was observed for these lipids at the
concentrations
tested.
Compounds of Formula II:
Despite all the reasons given above in order to "rationally design" these
lipids, it is
still impossible to predict their DNA transfection activity at this time. In
fact, cationic
lipids for DNA transfection already in the market such DOTAP, DOTMA, DMRIE and
DORI whose chemical structures are almost identical to that of the cationic
lipid known
as the Rosenthal Inhibitor. (Rosenthal, A.F. and Geyer, R.P., J. Biol. Chem.
235(8):2202
(1960)) have significant transfection activity, unlike the Rosenthal Inhibitor
which is
reported to be inactive as a DNA transfection reagent (see U.S. pat No
5,264,618 to
Felgner, P.L. et al,). The following formulae illustrate this point more
clearly
~ . ~ .r
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CH2 O R CH2 O R
CH O R CH O R
,
CH2 N (CH3)~ CI - CH2 N (CH3~ CI
CH OH
CH2 2 CH3
R = Stearoyl, Rosenthal inhibitor (inactive} R = Oleyl, DOTMA (active)
R = Myristyl, DMRIE (active} R = Oleoyl, DOTAP (active)
R = Oleoyl, DORI diester (active)
Compounds of formula II described herein have also a similar structure to that
of
the Rosenthal Inhibitor. However, these compounds differ from the Rosenthal
Inhibitor on
that a guanidinium or amidinium functionality is used as the positively
charged anchoring
group, and they also lack a quaternary ammonium group at the C-1 position of
the
glycerol backbone. A preferred embodiment of these latter type of transfection
reagents is
compound (15).
(IS)
O
CH2 O O (CH2)7 ~~(CH2)7CH3
CHZ ~ CH CH
CH O O ( )7 ( 2)7 3
~2
CHI O P O ~' NH
NH
OH
An interesting feature of this compound is that it has the ability to form
liposomes without
the need of co-adjuvants such as DOPE or DOPC. Thus, it can be used to form
liposomes with other cationic lipid compounds.
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Specific Examples of the Invention.
Scheme I
This reactions scheme shows the general synthetic route to prepare
poiycationic
lipids having a heteroatom substituted anchoring backbone. Thus, diglycolyl
chloride (1)
is treated with a suitable primary or secondary amine (2a-d) in methylene
chloride in the
presence of a base such as triethyl amine under an inert gas such as argon at
room
temperature to obtain the corresponding diglycolamides (3a-d). These amides
can then be
reduced with lithium aluminum hydride or borane in refluxing anhydrous
tetrahydrofurane
(THF) to afford the corresponding amines (4a-d). Secondary amine (4c) was
easily
converted to the corresponding tertiary amine (4e) upon treatment with
acrylonitrile.
Compound (4e) can be treated with ammonium chloride at high temperature to
produce
the corresponding amidine(6e). Alternatively, the latter amidine derivatives
can be
obtained by reacting the dinitrile (4e) with anhydrous hydrogen chloride in
ethanol,
followed of treatment of the imidoester so obtained with ammonium hydroxide.
Primary
amine (4d) can be converted to the target compound (6) by treatment with S-
methyl
isothiouronium hydroiodide (S-methyl thiourea) in tetrahydrofurane in the
presence of
triethylamine. Additionally , this guanidinium derivative can be alkylated
with for
example iodo methane to produce the corresponding quaternary ammonium salt.
Tertiary
amines (4a,b,e) are treated with an alkylating agent such as iodo methane ,
iodoethyl
acetate or 2- bromo ethyl acetate to afford the quaternary ammonium salts
(Sa,b,f). The
latter compounds were also synthesized by treating the corresponding tertiary
amines
with the commercially available 2-bromoethyl ether (lower panel, scheme I).
This route
has only two steps , but is not as flexible or prolific as the route depicted
on scheme I.
Scheme II
Compound (7) is easily synthesized by treating commercially available 1,4-
diaminobutane with acrylonitrile. Diamide-dinitrile (8) is then easily
obtained by treatment
of compound (7) with an acyl halide such as palmitoyl chloride in methylene
chloride in
the presence of triethyl amine. The diamide-dinitrile (8) can be reduced with
lithium
aluminum hydride or borane in THF to the corresponding tertiary and primary
amines
... , , t
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functionalities to afford compound (9). Guanidinium compound { 10) can be
obtained in a
similar fashion as shown in scheme I for compound (6d) by reacting the primary
amines of
compound (9) with S-methyl thiourea in THF and triethylamine.
Scheme III
Tetrapalmyl spermine (11) ( Haces, A. et al. PCT Int. Pub. No WO 95/17373) was
treated with ethyl iodoacetate at room temperature to afford the
tetraalkylated derivative
( 13 a). Similarly, compound ( 11 ) can be treated with 2-bromoethyl acetate
at high
temperature to afford ( 13b). Reaction of ( 11 ) with 4-bromo or 4- chloro
butyryl chloride
in methylene chloride in the presence of triethyl amine at low temperature
gives the
corresponding 4-bromobutyramide derivative (12). Intermediate (12) is
immediately
treated with iodo methane to produce the N,'N"- dimethylated intermediate
(13c) which in
turn is treated with an excess of ammonium hydroxide in tetrahydrofurane at
elevated
temperature to convert the bromide (or chloride) into the corresponding
primary amine
(13d). All these compounds have moieties which resemble or mimic the
neurotransmitters
gamma aminobutyric acid (GABA, compound (13d)) and acetyl choline (compounds
{l3a,b)). These small groups do not change substantially the liposomal forming
ability of
the lipid molecule and at the same time are capable of being recognized by
neural or
muscles cells. This preferential recognition by these type of cells makes
these lipids target
specific DNA/RNA delivery agents.
Scheme IV
Commercially available dioleoylphosphatidylethanolamine ( 14) was treated with
an
excess of S-methyl isothiouronium hydroiodide in tetrahydrofurane and in the
presence of
triethylamine to afford the corresponding guanidinium compound (15).
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EXAMPLES
Example 1: Synthesis of octadecylc3ranoethylamine (2d).
Octadecylamine (2g, 7.4 mmol) and acrylonitrile (15m1) were heated for 3h at
70oC in a thick wall test tube capped with a teflon lined cap. TLC (silica
gel; ethyl
acetate) shows a new spot at rf = 0.65 . The excess acrylonitrile was removed
in vacuo to
afford pure product (2.40g, 100% yield). H-NMR (CDCl3) 8: 0.88 (t, 3H), 2.25
(br.s.,32H), 2.53 (t, 2H), 2.62 (t,2H), 2.93 (t,2H). FT-IR {cm-1 ) 2250 (CN).
Example 2: Synthesis of bis !mono and dialk~rl)di~lycolamides(3a-d) general
procedure.
To a solution of diaIkylamine ( 2mmo1) and triethylamine {2 mmol) in methylene
chloride (250m1 ) under argon was added diglycolyl chloride ( lmmol) and the
resulting
solution was stirred for 18h at room temperature. TLC (silica gel; MeOH or
CH2CL2/THF,3:1) shows absence of starting material and a new spot. The
methylene
chloride solution was washed with sodium bicarbonate { 10% in water), dried
{Na2S04)
and the solvent removed, to afford the desired diamide.
Proceeding as described before and using the appropriate mono or dialkylmine
the
following compounds were prepared:
3a. Bis {dioctadecyl) diglycolamide (81% yield), H-NMR (CDCL3) 8: 0.88
(t,l2H), 1.25
{s, 124H), 1.5 (br.s, 8H), 3.15(t,4H), 2.9 (t,4H), 3.2 (s, 4H). R (cm-1): 1651
(C=O);
3b. Bis (didecyl)diglycolamide (73% yield), H-NMR{CDCL3)d X: 0.85 (t,12H),
1.25 (s,
64H), 1.5 (br.s,BH), 3.18 (t,4H), 3.3 (t,4H), 4.4 (s, 4H). FT-IR(cm-1):
1657(C=O);
3c. Bis (octadecyl)diglycolamide (100% yield),H- NMR(CDCL3) b: 0.85 (t, 6H),
1.25
(br.s, 60H), 1.5 (br.s,4H), 3.3 (q,4H), 4.05(s,4H), 6.4 (br.s,2H);
3d. Bis (octadecylcyanoethyl) diglycolamide (98%yield), H-NMR (CDC13) 8: 0.85
(br.t,
6H), 1.1-1.6 (br.s, 64H), 2.65 {t, 4H), 3.25 (br.t, 4H), 3.55 (br.t, 4H), 4.3
(s, 4H);
...~.. ~_....w.._ . . _.._...__. ~. . r i . i
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Example 3: Synthesis of N.N.N'.N' -Mono and dialkyl 2 2'-oxybis ethylamines 4a-
d)
e~ neral procedure
To a solution of lithium aluminum hydride {6 to 64 molar excess) in dry
tetrahydrofurane (THF) was added the corresponding diamide in small portions
under a
blanket of argon. The resulting mixture was refluxed for two to three days
under argon.
The progress of the reaction was followed by TLC (silica gel; CH2CI2/TFH, 3:1
for
dialkylamides; 10% triethylamine in CH2C12 for monoalkylamides and
triethylamine for
cyanoethylamides). The reactions were quenched with sodium hydroxide (10% in
water).
The mixture was filtered, the filtrate dried (Na2S04), and the solvent removed
in vacuo to
afford the desired products.
Proceeding as described above and using the appropriate diamide the following
compounds were obtained:
4a. N,N,N',N'-dioctadecyl-2,2'-oxobis ethylamine (77% yield), H-NMR(CDCI3) 8:
0.88
(t, 12H), 1.25 and 1.43 (br.s., 128H), 2.42 (t, 4H), 2.62 (t, 4H), 3.48 (t,
4H), 3.7, (t,4H);
4b. N,N,N',N'-didecyl-2,2'-oxobis ethylamine (93% yield ), H-NMR {CDCL3) 8:
0.88
(t,12H), 1.25 and 1.42 (br.s, 64H), 2.42 (t, 8H), 2.63 (t, 4H), 3.5 (t, 4H);
4c. N,N',-octadecyl-2,2'-oxobis ethylamine (71% yield), H-NMR (CDC13) 8: 0.87
(t, 6H),
1.25 and 1.45 (br.s., 64H), 2.6 (t,4H), 2.78 (t, 4H), 3.65 (t,4H);
4d. N,N,N',N'-octadecylaminopropyl-2,2'-oxobis ethylamine (80% yield), H-NMR
(CDCL3) 5:0.88 (t,6H), 1.25 and 1.45 (br.s,70H), 2.4-2.8 (br.m,16H), 3.4-3.7
(br.m, 4H).
Example 4: Synthesis of N.N.N,N' N' N' - dioctadecylmethyl-2 2'-
oxybisethylammonium
iodide.(Sa~
N,N,N',N'-dioctadecyl-2,2'-oxy bis ethylamine (l9mg, 0.017mmo1) was dissolved
in iodo methane (lml) inside a capped thick-wall test tube, and the resulting
solution
heated for 20h at 75oC. TLC (silicagel; chloroform:acetone:methanol:water;
50:15:5:5:1)
shows only one spot at Rf = 0.8 ,which gives a negative ninhydrin test and no
starting
material. The excess iodo methane was removed in vacuo to afford desired
product (23
mg, 96%). H-NMR(CDC13) b: 0.88 (t,l2H), 1.15 - 1.5 andl.7 (br.s.,128H), 3.35
(s, 6H),
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3.46 (br.m.,BH), 3.88 (br.s.,4H), 4.28 (br.s., 4H). Proceeding in a similar
fashion as per
compound (Sa) , compound (Sb) was obtained in 100% yield.
Example S: S~rnthesis ofN_N.N'.N'-cvanoethvloctadecyl-2.2'-oxybis
ethylamine~4e1
A suspension of N,N'-octadecyI-2,2'-oxybis ethylamine (100mg,0.16mmo1) in
acrylonitrile (4m1) was heated for 18h at 80oC in a capped, thick-wail test
tube (the
initially liquid two phase system became a clear homogenous solution after
2h). TLC
(silicagel ; dichloromethane / THF, 3:1) shows absence of starting material
and a spot
corresponding to desired material at rf = 0.95. The excess acrylonitrile was
removed in
vacuo to afford desired material. H-NMR(CDCl3) 8: 0.88 (t, 6H), 1.25 {br.s.,
64H), 2.45
(2t,8H), 2.68 {t, 4H), 2.87 (t,4H), 3.5 (t, 4H).
Example 6: Synthesis of N.N,N,N',N'.N'-cyanoethyloctadecMethyl-2 2'-oxybis
ethyl
ammonium iodide (S~.
A solution of N,N,N',N'-cyanoethyloctadecyl-2,2'-oxybis ethylamine (60 mg,0.08
mmol) in iodo methane (l.Sm1) was heated for 3h at 80oC in a capped, thick
wall tested
tube. The excess iodo methane was removed in vacuo to afford desired product.
H-NMR
(CDC13) 8: 0.88 (t, 6H), 1.25 (br.S., 60H), 1.8 (br.s., 4H), 3.45 (br.s., 6H),
3.65 (br.s.,
4H), 3.95-4.4 (br.m., 16H).
Example 7: Synthesis of N.N,N,N'.N',N'- acetoxyethyldioctadecvl-2,2'-ox,~rbis
ethXl
ammonium iodid~5f~
A solution of N,N,N',N'-dioctadecyl-2,2'- oxybis ethylamine {l6mg, 14.3 mmol)
and ethyl iodoacetate (O.SmI) in chloroform (lml) was heated for 18h at 75 oC
in a
capped thick wall test tube. The chloroform was removed in vacuo and the
residue
dissolved in tetrahydrofurane (lOml}. To this solution was added thiourea and
the mixture
stirred at room temperature until no more thiourea went into solution
{stoichiometric
excess after all iodide is converted to the isothiouronium salt). The mixture
was heated for
2h at 70oC and the excess solvent removed in vacuo. The residue was then
redissolved in
~ ~.r
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dichloromethane (lOml) and the solution washed with water (4x 5 ml), dried
(Na2S04) ,
filtered and the solvent removed in vacuo to afford l Omg of desired product.
Example 8: Synthesis of S-methylisothiouronium hydroiodide.
A solution of thiourea (0.8g,10.5mmo1) and iodo methane (8.8g,62mmo1) in
methanol (25m1) was heated in a capped thick-wall test tube for 4.5h at 50oC.
The
reaction mixture was rotaevaporated to afford pure desired material in 100%
yield.H-
NMR (CD30D) 8: 2.62(s, 3H), 4.8 (br.s., 4H).
Example 9: Synthesis of N N N' N'=guanidinopro~yloctade~rl oxv bis-2 2'-
ethvlamine
h~rdroiodide (,6d).
A solution of N,N,N',N'-aminopropyloctadecyl-2,2'- oxybis ethylamine {100mg,
0.14 mmol) , S- methyl isothiouronium hydroiodide (300mg, 1.3 mmol) and
triethylamine
(300mg, 3 mmol) in tetrahydrofurane (lOml) were heated in an argon flushed,
capped
thick-wall test tube for 20h at 95oC. The solvent and methyl mercaptan by
product were
removed in vacuo in a chemical fumes hood and the residue dissolved in
methylene
chloride (30m1), the organic phase was washed with brine ( 3x, 20 ml), water
(2x IOmI),
dried (Na2S04}, filtered and the solvent removed to obtain a reddish solid
(100mg,80%).
H-NMR(CDC13) 8: 0.86 (t,6H), I.1-1.6 (br.s., 64H), 2.4-2.8 (br.m., 16H), 3.15-
3.7
(br.m., I2H}. FTIR (cm-1): 1653 (C=NH).
Example 10: Synthesis ofN,N'-cyanoethyl-1 4-diaminobutane (7)
1,4-diaminobutane (2g, 22 mmol) was cooled to OCo (ice bath) and to this solid
was added acrylonitrile (4ml) . The mixture was let reach room temperature
slowly (ca
30min) and then let react for additional 18h at room temperature with stirnng.
The excess
acrylonitrile was removed in vacuo to afford the desired product. H-NMR
(CDC13) b: 1.3
(br.s.,2H), 1.5 (br.s, 4H), 2.5 (t, 4H), 2.65 (br.m.,4H), 3.9 (t,4H). FTIR (cm-
I) : 2247
(C=N).
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16
Example 11: Synthesis of N.N.N'.N'-cyanoethyl~amitoyl-1.4-diaminobutane(8)
To a solution of N,N'-cyanoethyl-1,4-diaminobutane (0.714g , 3.68mmo1) and
triethylamine (0.744g ,7.36mmol) in dichloromethane (1 SOmI) was slowly added
palmitoyl
chloride (2.028, 7.36 mmol) and the resulting mixture let react at room
temperature
overnight. The reaction was washed with sodium bicarbonate ( 10%,2x SOmI),
water (2x
SOmI), dried (Na2S04) , filtered and the solvent removed in vacuo to afford
the desired
product (2.3g, 93 %). H-NMR (CDCL3) S: 1.9 (t,6H), 1.25 and 1.6 (br.s., 56H),
2.32
(m,4H), 2.72 (t, 4H), 3.42 (m, 4H), 3.55 {t,4H). FTIR (cm-1) 1643 (C=O).
Example 12: Synthesis of N.N,N',N'-aminopr~vl~almyl-1 4-diaminobutane (9)
To a solution of lithium aluminum hydride (600mg,15.9 mmol) in
tetrahydrofurane
(SOmI) was added N,N,N',N'-cyanoethylpalmitoyl-1,4-diaminobutane (300mg, 0.45
mmol) and the reaction mixture was refluxed for 72 h. Then, a procedure
essentially the
same as per example 3 (supra) was followed to afford desired diamine
(200mg,70%). H-
NMR(CDCl3) b: 0.88 (t,6H), 1.25 and 1.5 (br.s, 64H), 2.3-2.7 (br.m, overlap.
t, 16H).
Example 13: Synthesis of N.N,N'.N'-guanidinopro~~palmyl-1,4-diaminobutane
(10~,
A procedure identical as per example 6 (supra) was followed. H-NMR (CDCl3) 8:
0.9 (t, 6H), 1.2-1.35 (br.s., 68H), 1.5-2.0 (br.m., 4H), 2.65-3.5 (m, 24H).
FTIR (ctri')
1650 (C=N).
Example 14: Synthesis of N,N"'-4-bromabutyryl-N,N'.N"N"'-tetrapamyl~ermine
12).
To a cooled (0 °C) solution of N,N',N",N"'-tetrapalmylspermine
(400mg, 0.36
mmol) and triethylamine (80mg, 0.8 mmol) in dichloromethane ( 14m1) was added
4-
bromobutyryl chloride (156mg, 0.8 mmol) and the resulting mixture let react
for lh at 0
oC. The reaction was quenched and washed with cold sodium bicarbonate solution
(10
in water,3x Sml), dried {sodium sulfate), filtered and the solvent removed at
room
temperature in vacuo to afford a white foam.. H-NMR (CDC13) 8: 0.88 (t, 12H),
1.15-
1.5 (br.s., 136H), 2.12 (t, 4H), 2.3-2.55 (br.m.,l2H), 3.2-3.36 (br.m., 8H),
3.62 (t, 4H).
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Example 15: Synthesis of N.N' N" N"'-tetrapalmyltetraacetoxyethylspermine
iodide
salt 13a
A solution of tetrapalmylspermine (Races et aI.,PCT Int. Pub. No WO/95/17373),
130mg, mmol) in neat ethyl iodoacetate (l.Sm1) was heated to 75 oC for 18h.
The
reaction was worked up following essentially the same procedure as per example
13
(supra) to afford the desired product.
Example 16: Synthesis of N.N"'-4-bromobutyrvl-N N' N" N"'-tetrapalmvl-N~N"
dimeth ~~lspermine(13c).
N,N"'-4-bromobutyryl-N,N',N",N"'-tetrapalmylspermine (350mg,0.25 mmol) was
dissolved in iodo methane (3ml) and the resulting solution iet react for 2
days at room
temperature . Excess iodo methane was evaporated to afford desired product,
which is
negative for ninhydrin test. H-NMR (CDC13) b: 0.88 (t, 12H), 3.41 (br.s., 6H).
Example 17: Synthesis of N N"'-4-aminobut~ryl-N,N' N" N"'-tetrapalm r~N' N"
dimeth~s ermine(13d~.
To a solution of N,N"'-4-bromobutyryl-N,N',N",N"'-tetrapalmyl-N',N"-
dimethylspermine (100mg,0.05 mmol) in tetrahydrofurane (lOml) was added
ammonium
hydroxide (20 mI, 28% by weight) and the resulting mixture heated to 70 oC for
2 days in
a capped, thick wall reaction tube. The solvent was azeotropically evaporated
(ethanol)
to afford a brown solid which is strongly positive for ninhydrin test.H-NMR
(CDC13) b:
0.88 (t,12H), 1.1-1.4 (br.s,130H),1.5-2.2 (br.m,16H), 3.0-3.8 (br.m, lOH), 3.4
(br.s, 6H).
Example 18: Synthesis of dioleo ly~hosphatidvl ethanol~uanidine (15).
To a solution of dioleolylphosphatidyl ethanoamine (70mg,0.094 mmol) and
triethylamine (lml) in tetrahydrofurane (10 ml) was added S-
methyiisothiouronium
hydroiodide (70mg, 0.32 mmol) and the resolting solution heated for 18h at 70
oC. The
solvent was removed in vacuo and the residue redissolved in dichloromethane
(25m1).
This solution was washed with water (2 x 10 ml), dried ( sodium sulfate),
filtered and the
solvent evaporated to afford the desired product (40 mg, 47%). H-NMR (CDC13)
b: 0.88
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18
(t,6H), 1.2-1.44 (br.s, 40H), 2.00 {br.d,lOH), 2.29 (t, 4H), 3.19 (q,lH), 3.42
(br.s,lH),
3.85-4.20 {br.m,2H), 4.38 (br.m,lH), 5.3 (br.d, 4H). FTIR (cni') : 2361, 1741.
Example 19: Liposomes formulation:
Lipids were formulated by mixing the appropriated molar amounts of the active
lipid with dioleoylphophatidyl ethanolamine (DOPE) in dichloromethane and
dispersing
this mixture in the final amount of water using the solvent vaporization
method. (David
W. Deamer, in Liposome Technology, vol.I, p-29, CRC press Boca Raton, Fl,
1984).
Cell culture and plasmids
Cell lines were from the American Type Culture Collection (Rockville,
Maryland)
and were cultured in RPMI11649, 10% FCS, pen/strep . Plasmid pCMV (3-gal,
which
contains the E. Coli (3-galactosidase {gene) under the control of the powerful
cytomegalovirus promoter (McGregor et al. (1989) Nucleic Acids Res., 7 :2365)
was
purchased from Clontech, Inc. Primary cells were from human tracheal isolates
and
neonatal foreskin.
Example 20: Transfection of H~G2 and HeLa cells.
Cells were plated in 48-well plates (lcm2 ) at a concentration of lx 105
ceils/well
in 0.5 ml of RPMI-1640, 10% FCS, Pen/step. The next day, lipids aliquats ( 1,3
and 5~1
of lmg/ml liposome in water) were diluted in polystyrene tubes containing
100u1 of
serum-free, antibiotic-free RPMI-1640 and to these tubes were added 150 ng of
plasmid
in 100ly1 of the same medium (suboptimal amount in polypropylene tubes) and
incubate
for 1 S min. The cells were washed twice with Dulbecco's PBS, the
lipid:plasmid
complexes added to them and then incubated for 7h at 37 Co in 5% C02
atmosphere.
Growth medium was added to the cells for a final volume of 1 ml and a final
concentration
of 10 % FCS, pen/strep, SOug/ml gentamicin in RPMI-1640 and were incubated
overnight.
r ., i . r
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Example 21: Transfection of Primary Human Tracheobronchial and Epidermal
Keratinocvtes.
Cells were grown in serum free medium (SFM) and plated on 35 mm plates
(6wells) such that the confluence after 24h was above 50%. Plasmid reporter
(2~tg and
S~tg, respectively) was mixed with variable amounts of liposomes (see tables
IV and V)
and the complex formed added to the cells. The cells were transfected during
4h and 6h,
respectively. The DNA/liposome complex was removed by rinsing with SFM and the
cells
incubated for 48h under normal growth conditions and then assayed for the
appropiate
marker.
Example 22: Transfection and CAT assay of Jurkat Cells (suspension cells
The cell suspension culture was transferred to a 50 ml conical tube and
centrifuged at 4008 for 10 min. The cells were washed twice by aspirating off
the
supernatant and gently resuspending the cell pellet in 25 ml of sterile PBS
and centrifuging
again at 4008 for l Omin. The pellet was resuspended in a volume of serum-free
growth
medium such that a final concentration of 6.25 x 106 cells /ml is obtained
(about 10 ml).
35 mm cell culture plates were inoculated with 0.8 ml of the cell suspension.
For each
well, 10 yg of CAT plasmid were dissolved in 110 pl of serum-free medium and a
separately in another tube were diluted 30 yl of the lipid solution in 70 ~ul
of serum-free
medium. The plasmid and lipid solution were mixed and gently swirled and let
stand at
room temperature for 10 min. The complex DNA/lipid solution was then randomly
dropped over the culture well. The wells were gently swirled and then
incubated at 37 ° C
under a 7 % COZ atmosfere for 5 hours. After Sh incubation, 4m1 of 12.5 % FBS
growth
medium were added to the wells and the incubation continued for additional 72
h under
the above conditions. The cells were then transferred to 10 ml Falcon tubes
and the
wells rinsed with Sml of sterile PBS . The cell suspension was washed twice
with Sml of
sterile PBS as previously. The final pellet was resuspended in 400 pl of lysis
buffer and
transferred to 1.5 mi centrifugation tubes. The tubes were capped and placed
horizontally
on a rocker and the cells lysated for 30 min.
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100 pl aliquats were then assayed for CAT activity following the procedure of
Neumann et al. (1987) Biotechniques 5: 444
Example 23: Assav for transient transfection~adherent cellsl.
The cells were washed twice with Dulbecco's PBS and stained with freshly
prepared fixative (2%formaldehyde/ 0.2% glutaraldehyde in PBS) for 5 min,
washed
twice with Dulbecco PBS.). Then, stained with 0.5 ml of (3-galactosidase
histochemical
stain (0.1% x-gal, SmM potassium ferrocyanide, SmM potassium ferrocyanide, 2mM
MgCl2 in PBS) for 24h at 37 Co in a S% C02 atmosphere. Blue cells (~i-gal
positive)
were counted.
Example 24: Results and Discussion.
Results are summarized on tables I , II ,III, IV and V. Tables I and II show
the
relative transfection efficiency of compounds Sa, 6d and 13d versus control
compound
TMTPS (Compound 3 in PCT Int.Pub.No. W095/17373. Haces, A. et al.) in HepG2
(human hepatocarcinoma) and HeLaS3 (human cervical carcinoma) cells,
respectively.
And under suboptimal conditions for activity. In these cell lines, compounds
6d and 13d
show a 2-2.4 fold higher efficiency than the TMTPS control , and compound Sa
is half as
active as control in HepG2 cells and showed negligible activity in HeLaS3
cells. Table III
shows an analogous comparison using Jurkat cells (T-cell leukemia). In this
experiment,
compounds Sa and 13d show similar efficiency as TMTPS, but compound 6d shows
almost 38% more activity than that of the control. Tables IV and V shows the
relative
efficiency of compounds 6d and 13d in the primary human tracheobronchial
epithelial and
human keratinocytes cells. Primary cells are cells that are freshly isolated
from humans or
animals and which , unlike the cultured cell lines, reflect the potential
behavior of a
compound in vivo more closely. Thus, for genetic therapy to work, it is
necessary to be
able to transfect these types of cell lines before any in vivo experiments are
tried. These
types of cells are alsa the most difficult to transfect and their transfection
effciencies are
usually below I%. Table IV shows the relative efficiency of compounds 6d and
13d
..._....~_._ _ . . .._. . . ~ . . ~ , r
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versus DOTMA (LipofectinTM Reagent, Life Technologies, Inc.,Su ra in primary
human
Tracheo brochial cells. Both of these compounds show a relative range of
activities of
5.3 to 6.0 times higher than that of the Lipofectin control. At the same time
their cell
toxicity was below 5%, unlike the control which showed toxicity in the 10-20%
range.
Thus, these lipid reagents are superior to the commercial standards in both
respects. In
addition, this is a very significant result since tracheobronchial cells are
involved in the
genetic disease cystic fibrosis. There are several genetic therapy clinical
trials being
conducted at the present time targeting these cells using either viral or
liposomal vectors
(see 10th Annual North America Cystic Fibrosis Conference, Orlando, Fl., Oct
24-27
( 1996), Abstracts or Pediatr Pz~lmofrol Suppl, 13: 74-365, Sept, 1996 ) .
Table V depicts the the percentages of [3-gal positive cells (absolute number)
which were obtained in primary human epidermal keratinocytes with compounds Sa
and
6d versus that obtained with DOSPA control (LipofectamineTM Reagent, Life
Technologies, Inc. Supra) . Compounds Sa and 6d gave, respectively, 35% and
50%
positive cells as compared with 2% positives for the control. This represents
a 1 S-25 fold
better efficiency for these novel liposome reagents when compared with this
well known
standard. Moreover, primary human keratinocytes are also a potential target
cells for
genetic therapy ( Fenjves, E.S. et al., Hrmr Gene Ther 5: 10,1241-8, Oct.
1994.), but its
use has been restricted due to the lack of highly efficient transfection
vectors.
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TABLE I
TRANSFECTION OF HEP G2 CELLS
Lipid/DOPE (molar ratio) Optimal Liposome Amount(pg) J3- Gal Positive Cells
(%)
Compound Sa ( 1: 1.8) 3 pg 0.8
Compound 6d ( 1: 1.5) 3 pg 4.3
Compound 13d (1:1.5) ~ 3 beg 2.9
Control TMTPS/DOPE(1:1.5) 5 ug 1.5
Cells were plated in 48 well plates at a density of 1 x 105 per well in 0.5 ml
of growth
medium. After 24h, the cells were washed with serum free medium and
transfected with
a suboptimal amount( 150 ng ) of plasmid pCMV-J3gal. using 1,3 and Sul (i,3
and S~tg)
of lipid "formulation. The amount giving the highest level of transfection
efficiency is
shown. The experiment was run in tr~phcate.
TABLE II
TRANSFECTION OF He La S3 CELLS
Lipid/DOPE {molar Optimal Liposome Amount ~i-Gal Positive Cells (%)
ratio) (ug)
Compound Sa (1:1.8) 5ltg 0.001
Compound 6d ( 1:1.5) 3 l.tg 2.4
Compound 13d (1:1.5) 1 ~g 2.1
Control 5 pg 0.9
TMTPS/DOPE(1:1.5)
Cells were plated in 48 well plates at a density of 1 x 105 per well in 0.5 ml
of growth
medium. After 24h, the cells were washed with serum free medium and
transfected with
a suboptimal amount 150 ng ) of plasmid pCMV-(3gal. using 1,3 and Sul (1,3 and
S~tg)
of lipid formulation. 'l,he amount giviWtha highest level of transfection
efficiency is
shown. The experiment was run in tr~phca~e.
..~.,~~~. ...... t . ~ . T
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TABLE III
TRANSFECTION OF JURKAT CELLS
LipidIDOPE (molar ratio) Optimal Liposome Amount CAT Activity (mUlwell)
Compound Sa (1:1.8) 30 pg 196.00
Compound 6d (1:1.5) 30 ug 298.20
Compound 13d(1:1.5) 30 pg 220.00
Control TMTPS/DOPE(1:1.5) 30 pg 216.44
Wells were inoculated with 6.25 x 106 cells . 10 yg of CAT plasmid were mixed
with
30yg (optimal amount known for the control) of the lipids and then added to
the cells.
Atter Sh the transfection was quenched with FBS containing medium and the
cells
incubated for 72h. Cells were lysated in 400 yl of buffer. 100 yl aliquats
were assayed
for CAT activity.
TABLE IV
TRANSFECTION OF PRIMARY HUMAN TRACHEOBRONCHIAL
EPITHELIAL CELLS
LipidIDOPE (molar ratio) Optimal Liposome Amount Luciferase Activity
(pg) (counts)
Compound 6d (1:1.5) 12 ug 7297022
Compound 13d (1:1.5) i2 pg 8343975
Control , LipofectinTM* 6 ug 1379341
35 mm plates were inoculated with cell isolates and transfected at 90%
confluence. tug
of f refly luciferase plasmid were mixed with 12 yg of the Lipids and then
added to the
cells. After Sh, the transfection was quenched by removal of the DNA/Lipid
complex and
the cells incubated for 72h. Cells were lysated and aliquats assayed for
luciferase activity.
Cell toxicity , determined by the trypan blue method, was below 5% for lipids
6d and 13d
and between 10-20% for Lipofectin. * Lipofectamine was also run as a control,
but its
efficiency was negligible.
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24
TABLE V
TRANSFECTION OF PRIMARY HUMAN EPIDERMAL KERATINOCYTES
LipidIDOPE (molar ratio) Optimal Liposome Amount j3-Gal Positive Cells (%)
...............................................................................
........~la.~~.................................................................
.............................................................
Compound Sa ( 1:1.5) 40 lxg 35
Compound 6d ( 1:1.5) 20 ug 50
Control , LipofectamineTM 2~ pg 2
Cells were seeded at 2x105 /well in 35 mm wells and transfected the next day.
Sp,g of
J3ga1 DNA were mixed with the appropiate amount of lipids and added to the
cells. After
4h, the medium was replaced and the cells incubated for additional 48h and
then assayed.
Blue cells were observed under the microscope and counted.
