U.S. patent application number 09/779791 was filed with the patent office on 2005-01-27 for compound containing a labile disulfide bond.
Invention is credited to Budker, Vladimir G., Monahan, Sean D., Rozema, David B., Slattum, Paul M., Wolff, Jon A..
Application Number | 20050020518 09/779791 |
Document ID | / |
Family ID | 46257498 |
Filed Date | 2005-01-27 |
United States Patent
Application |
20050020518 |
Kind Code |
A9 |
Wolff, Jon A. ; et
al. |
January 27, 2005 |
Compound containing a labile disulfide bond
Abstract
A labile disulfide-containing compound under physiological
conditions containing a labile disulfide bond and a transduction
signal.
Inventors: |
Wolff, Jon A.; (Madison,
WI) ; Monahan, Sean D.; (Madison, WI) ;
Budker, Vladimir G.; (Middleton, WI) ; Slattum, Paul
M.; (Madison, WI) ; Rozema, David B.;
(Madison, WI) |
Correspondence
Address: |
Mark K. Johnson
P.O. Box 510644
New Berlin
WI
53151-0644
US
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 0044417 A1 |
November 22, 2001 |
|
|
Family ID: |
46257498 |
Appl. No.: |
09/779791 |
Filed: |
February 8, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09779791 |
Feb 8, 2001 |
|
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09312351 |
May 14, 1999 |
|
|
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60085764 |
May 16, 1998 |
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Current U.S.
Class: |
554/85 ; 514/1.3;
530/350; 536/23.1 |
Current CPC
Class: |
A61K 31/155 20130101;
A61K 38/08 20130101; C07C 323/62 20130101; A61K 31/198 20130101;
C07C 323/60 20130101; C07D 207/46 20130101; C07H 21/04 20130101;
A61K 48/00 20130101 |
Class at
Publication: |
514/044 ;
514/002; 530/350; 536/023.1 |
International
Class: |
A61K 048/00; A61K
038/00; C07H 021/04; C07K 014/00 |
Claims
We claim:
1. A compound for inserting into an organism, comprising: the
compound having a disulfide bond that is labile under physiologic
conditions selected from the group consisting of (a) a disulfide
bond that is cleaved more rapidly than oxidized glutathione and (b)
a disulfide bond constructed from thiols in which one of the
constituent thiols has a lower pKa than glutathione and (c) a
disulfide bond that is activated by intramolecular attack from a
free thiol wherein the compound contains a transduction signal.
2. The compound of claim 1 wherein the transduction signal consists
of Tat.
3. The compound of claim 1 wherein the transduction signal consists
of VP22.
4. The compound of claim 1 wherein the transduction signal consists
of ANTP.
5. The compound of claim 1 wherein the transduction signal consists
of a polymer containing a cationic charge.
6. The compound of claim 5 claim 1 wherein the transduction signal
consists of a peptide containing cationic residues.
7. A process for delivering a compound having a labile disulfide
bond into a mammal, comprising: a) forming the compound having a
disulfide bond selected from the group consisting of (i) a
disulfide bond that is cleaved more rapidly than oxidized
glutathione, and (ii) a disulfide bond constructed from thiols in
which one of the constituent thiols has a lower pKa than
glutathione, and (iii) a disulfide bond that is activated by
intramolecular attack from a free thiol; b) attaching a
transduction signal to the compound; c) inserting the compound into
the mammal; and, d) releasing the bond between the sulfur atoms in
the disulfide.
8. The process of claim 7 wherein the transduction signal consists
of Tat.
9. The process of claim 7 wherein the transduction signal consists
of VP22.
10. The process of claim 7 wherein the transduction signal consists
of ANTP.
11. The process of claim 7 wherein the transduction signal consists
of a peptide containing a cationic charge.
12. The process of claim 11 wherein the transduction signal
consists of a peptide containing cationic residues.
13. The compound of claim 1 wherein the compound consists of
nucleic acids.
Description
[0001] This application is a continuation-in-part of Ser. No.
09/312,351 filed on May 14, 1999.
BACKGROUND
[0002] Bifunctional molecules, commonly referred to as
crosslinkers, are used to connect two molecules together.
Bifunctional molecules can contain homo or heterobifunctionality.
The disulfide linkage (RSSR') may be used within bifunctional
molecules. The reversibility of disulfide bond formation makes them
useful tools for the transient attachment of two molecules.
Disulfides have been used to attach a bioactive compound and
another compound (Thorpe, P. E. J. Natl. Cancer Inst. 1987, 79,
1101). The disulfide bond is reduced thereby releasing the
bioactive compound. Disulfide bonds may also be used in the
formation of polymers (Kishore, K., Ganesh, K. in Advances in
Polymer Science, Vol. 21, Saegusa, T. Ed., 1993).
[0003] There are many commercially available reagents for the
linkage of two molecules by a disulfide bond. Additionally there
are bifunctional reagents that have a disulfide bond present.
Typically, these reagents are based on 3-mercaptopropionic acid,
i.e. dithiobispropionate. However, the rate at which these bonds
are broken under physiological conditions is slow. For example, the
half life of a disulfide derived from dithiobispropionimidate, an
analog of 3-mercaptopropionic acid, is 27 hours in vivo (Arpicco,
S., Dosio, F., Brusa, P., Crosasso, P., Cattel, L. Bioconjugate
Chem. 1997, 8, 327.). A stable disulfide bond is often desirable,
for example when purification of linked molecules or long
circulation in vivo is needed. For this reason, attempts have been
made to make the disulfide less susceptible to cleavage.
[0004] It has been demonstrated that both stability, measured as
reduction potential, and rate, measured as rate constants, of
disulfide reduction are both related to the acidity of the thiols
which constitute the disulfide. Additional factors that may affect
the rate of reduction are steric interactions, and intramolecular
disulfide cleavage. Looking at the difference in the rates for the
reactions RSH+R'SSR'.fwdarw.RSSR'+R'S- H and
RSH+R"SSR".fwdarw.RSSR"+R"SH, it has been demonstrated that log
k"/k'=.beta.(pK.sub.a.sup.R'-pK.sub.a.sup.R"), where k' and k" are
the rate constant for the reactions with R'SSR' and R"SSR"
respectively, pK.sub.a.sup.R' and pK.sub.a.sup.R" are the acidities
of the thiol groups R'SH and R"SH, and .beta. is a constant
determined empirically to be 0.72. From this equation, one would
predict that the reduction of a disulfide composed from relatively
acidic thiols would be reduced more quickly than one composed of
less acidic thiols. In support of this observation, it has been
demonstrated that the disulfides cystine (pK.sub.a 8.3) and
cystamine (pK.sub.a 8.2) are reduced 3-15 times faster than
oxidized glutathione (pK.sub.a 8.9) (Bulaj, G., Kortemme, T.,
Goldenberg, D. P. Biochemistry 1998, 37, 8965).
[0005] It has been demonstrated that both stability
(thermodynamics), measured as reduction potential (Keire D. A. J.
Org. Chem. 1992, 57, 123), and rate (kinetics), measured as rate
constants, of disulfide reduction are both related to the acidity
of the thiols which constitute the disulfide (Szajewski, R. P.,
Whitesides, G. M. J. Am. Chem. Soc. 1980, 102, 2011). The increase
in acidity of a thiol is dependent upon one or more of the
following structural factors: the presence of electron withdrawing
groups which stabilize the thiolate through sigma and pi bonds
(inductive effect), the presence of electron withdrawing groups
that stabilize the thiolate through space or solvent (field
effects), pi bonds which allow the negative charge to be placed on
other atoms (resonance stabilization), and hydrogen bond donating
groups within the molecule that can interact internally with the
thiolate. For example, cysteine has an amino group two atoms from
the thiol, which is more electron withdrawing than the amide
nitrogen that is two atoms from the thiol in glutathione. As a
consequence of this difference in electron withdrawing groups, the
thiol of cysteine is 0.6 pK units more acidic than glutathione, and
as mentioned previously, cystine is reduced 3-15 times faster than
oxidized glutathione. Another example of a relatively acidic thiol
is 5-thio-2-nitrobenzoic acid, pK.sub.a 5. Its acidity is due to
resonance stabilization and inductive effects. Its disulfide is
rapidly reduced by all standard alkyl thiols and its colored
thiolate makes it a convenient assay for thiol concentration.
SUMMARY
[0006] Described in a preferred embodiment is a process for the
delivery of a compound to a cell, comprising associating a
compound, containing a disulfide bond that can be cleaved under
physiological conditions, with a polymer, then delivering the
polymer to the cell. The polymer may comprise a first polymer and a
second polymer. The first polymer and the second polymer may
comprise nucleic acids, proteins, genes, antisense polymers,
DNA/RNA hybrids, or synthetic polymers.
[0007] In another preferred embodiment, a biologically active
compound is associated with a disulfide-containing compound,
comprising: the disulfide-containing compound having a labile
disulfide bond that is selected from the group consisting of (a) a
disulfide bond that is cleaved more rapidly than oxidized
glutathione and (b) a disulfide bond constructed from thiols in
which one of the constituent thiols has a lower pKa than
glutathione and (c) a di sulfide bond that is activated by
intramolecular attack from a free thiol.
[0008] In another preferred embodiment, a compound is provided for
inserting into an organism, comprising: the compound having a
disulfide bond that is labile under physiologic conditions selected
from the group consisting of (a) a disulfide bond that is cleaved
more rapidly than oxidized glutathione and (b) a disulfide bond
constructed from thiols in which one of the constituent thiols has
a lower pKa than glutathione and (c) a disulfide bond that is
activated by intramolecular attack from a free thiol.
[0009] In another preferred embodiment, a process is provided for
forming a compound having a labile disulfide bond for use with an
organism, comprising: forming the compound having a disulfide bond
selected from the group consisting of (i) a disulfide bond that is
cleaved more rapidly than oxidized glutathione, and (ii) a
disulfide bond constructed from thiols in which one of the
constituent thiols has a lower pKa than glutathione, and (iii) a
disulfide bond that is activated by intramolecular attack from a
free thiol; inserting the compound into the organism.
[0010] In another preferred embodiment, a process is described for
compacting a nucleic acid for delivery to a cell, comprising
associating a polymer containing a disulfide bond with a nucleic
acid and delivering the nucleic acid to the cell.
[0011] In another preferred embodiment, a process is described for
compacting a nucleic acid for delivery to a cell comprising
associating a polymer with the nucleic acid, then associating a
compound containing a disulfide bond that can be cleaved under
physiological conditions with the nucleic acid polymer complex,
then delivering the complex to a cell.
[0012] In another preferred embodiment, a process is described for
compacting a nucleic acid for delivery to a cell, comprising
associating a polymer containing a disulfide bond with a nucleic
acid, then associating another polymer with the disulfide
containing polymer--nucleic acid complex, then delivering the
complex to the cell.
[0013] In another preferred embodiment, a process is described for
compacting a nucleic acid for delivery to a cell comprising
associating a polymer with the nucleic acid, then associating a
compound containing a disulfide bond that can be cleaved under
physiological conditions with the nucleic acid polymer complex,
then associating another polymer with the complex, then delivering
the complex to a cell.
[0014] In another preferred embodiment, a compound is described
which contains a disulfide bond that can be cleaved under
physiological conditions and possesses heterobifunctional or
homobifunctional groups. Such a compound can be described as a
disulfide containing bifunctional molecule.
A.sub.1--S--S--A.sub.2
[0015] More particularly, a compound that contains an aliphatic
disulfide bond with one or more electronegative (electron
withdrawing groups) substituted alpha or beta to one or both of the
sulfur atoms. These groups serve to lower the pK.sub.a of the
constituent thiols. 1
[0016] Where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
R.sub.7, R.sub.8--at least one of which is an electronegative atom
or functionality such as OH, OR (an ether), NH.sub.2,(also
secondary, tertiary, and quaternary amines), SO.sub.3.sup.-, COOH,
COOR (an ester), CONH.sub.2, CONR.sub.2 (substituted amide), a
halogen (F, Cl, Br, O), NO.sub.2. L is defined as a linker or
spacer group that provides a connection between the disulfide and
the reactive heterobifunctional or homobifunctional groups, A.sub.1
and A.sub.2. L may or may not be present and may be chosen from a
group that includes alkanes, alkenes, alkynes, esters, ethers,
glycerol, amide, urea, saccharides, polysaccharides, heteroatoms
such as oxygen, sulfur, or nitrogen. The spacer may be charge
positive, charge negative, charge neutral, or zwitterionic. A.sub.1
and A.sub.2 are reactive groups they may be identical as in a
homobifunctional bifunctional molecule, or different as in a
heterobifunctional bifunctional molecule. In a preferred
embodiment, the disulfide compounds contain reactive groups that
can undergo acylation or alkylation reactions. Such reactive groups
include (but not limited to) isothiocyanate, isocyanate, acyl
azide, acid halide, O-acyl urea, N-hydroxysuccinimide esters,
succinimide esters, amide, urea, sulfonyl chloride, aldehyde,
ketone, ether, epoxide, carbonate, alkyl halide, imidoester,
carboxylate, alkylphosphate, arylhalides (e.g.
difluoro-dinitrobenzene) or anhydrides.
[0017] If functional group A1, A2 is an amine then A1, A2 can react
with (but not restricted to) an activated carboxylic acid,
isothiocyanate, isocyanate, acyl azide, alkyl halide, acid halide,
N-hydroxysuccinimide ester, sulfonyl chloride, aldehyde, ketone,
epoxide, carbonate, imidoester, amide, carboxylate, or
alkylphosphate, arylhalides (difluoro-dinitrobenzene) or
anhydrides. In other terms when function A1, A2 is an amine, then
an acylating or alkylating agent can react with the amine.
[0018] If functional group A1, A2 is a sulflhydryl then A1, A2 can
react with (but not restricted to) a haloacetyl derivative,
activated carboxylic acid, maleimide, aziridine derivative,
acryloyl derivative, fluorobenzene derivatives, or disulfide
derivative (such as a pyridyl disulfide or 5-thio-2-nitrobenzoic
acid{TNB} derivatives).
[0019] If functional group A1, A2 is carboxylate then A1, A2 can
react with (but not restricted to) a diazoacetate, alcohol, thiol
or an amine once the acid has been activated.
[0020] If functional group A1, A2 is an hydroxyl then A1, A2 can
react with (but not restricted to) an activated carboxylic acid,
epoxide, oxirane, or an amine in which carbonyldiimidazole is
used.
[0021] If functional group A1, A2 is an aldehyde or ketone then A1,
A2 can react with (but not restricted to) an hydrazine, hydrazide
derivative, amine (to form a Schiff Base that may or may not be
subsequently reduced by reducing agents such as NaCNBH.sub.3), or a
diol to form an acetal or ketal.
[0022] If functional group A1, A2 is activated carboxylic acid,
isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide ester,
sulfonyl chloride, aldehyde, ketone, epoxide, carbonate,
imidoester, alkylphosphate, arylhalides (difluoro-dinitrobenzene),
anhydride, alkyl halide, or acid halide, p-nitrophenyl ester,
o-nitrophenyl ester, pentachlorophenyl ester, pentafluorophenyl
ester, carbonyl imidazole, carbonyl pyridinium, or carbonyl
dimethylaminopyridinium, then A1, A2 can react with (but not
restricted to) an amine, a hydroxyl, hydrazine, hydrazide, or
sulfhydryl group.
[0023] If functional group A1, A2 an activated carboxylic acid,
haloacetyl derivative, maleimide, aziridine derivative, acryloyl
derivative, fluorobenzene derivatives, or disulfide derivative
(such as a pyridyl disulfide or 5-thio-2-nitrobenzoic acid{TNB}
derivatives) then A1, A2 can react with (but not restricted to) a
sulfhydryl.
[0024] If functional group A1, A2 is an aldehyde, ketone, epoxide,
oxirane, or an amine in which carbonyldiimidazole or
N,N'-disuccinimidyl carbonate is used, then A1, A2 can react with
(but not restricted to) a hydroxyl.
[0025] If functional group A1, A2 is a hydrazine, hydrazide
derivative, or amine (primary or secondary) then A1, A2 can react
with (but not restricted to) an aldehyde or ketone (to form a
Schiff Base that may or may not be reduced by reducing agents such
as NaCNBH.sub.3).
[0026] Additionally, a compound which contains an aromatic
disulfide bond in which the sulfur atom is bonded directly to the
aromatic ring. The ring may contain 5 or more atoms. 2
[0027] R.sub.1-R.sub.4, R.sub.6-R.sub.9--The substitution pattern
on the ring may be varied to alter the reduction potential of the
disulfide bond. The substiuents may be selected from the group that
includes but is not limited to OH, OR (an ether), NH.sub.2,(also
secondary, tertiary, and quaternary amines), SO.sub.3.sup.-, COOH,
COOR (an ester), CONH.sub.2, CONR.sub.2 (substituted amide), a
halogen (F, Cl, Br, I), NO.sub.2, CH.sub.3 (or longer branched or
straight chain, saturated, or unsaturated aliphatic group). L is
defined as a linker or spacer group that provides a connection
between the disulfide and the reactive heterobifinctional or
homobifunctional groups. L may or may not be present and may be
chosen from a group that includes alkanes, alkenes, esters, ethers,
glycerol, amide, saccharides, polysaccharides, heteroatoms such as
oxygen, sulfur, or nitrogen. The spacer may be charge positive,
charge negative, charge neutral, or zwitterionic. R.sub.5,
R.sub.10--are reactive groups they may be identical as in a
homobifunctional bifunctional molecule, or different as in a
heterobifunctional bifunctional molecule. In a preferred
embodiment, the disulfide compounds contain reactive groups that
can undergo acylation or alkylation reactions. Such reactive groups
include isothiocynanate, isocynanate, acyl azide,
N-hydroxysuccinimide esters, succinimide esters, sulfonyl chloride,
aldehyde, epoxide, carbonate, imidoester, carboxylate,
alkylphosphate, arylhalides (e.g. difluoro-dinitrobenzene) or
succinic anhydride.
[0028] If functional group R.sub.5, R.sub.10 is an amine then
R.sub.5, R.sub.10 can react with (but not restricted to) an
activated carboxylic acid, isothiocyanate, isocyanate, acyl azide,
alkyl halide, acid halide, N-hydroxysuccinimide ester, sulfonyl
chloride, aldehyde, ketone, epoxide, carbonate, imidoester, amide,
carboxylate, or alkylphosphate, arylhalides
(difluoro-dinitrobenzene) or anhydrides. In other terms when
function R5, R10 is an amine, then an acylating or alkylating agent
can react with the amine.
[0029] If functional group R5, R10 is a sulfhydryl then R5, R10 can
react with (but not restricted to) a haloacetyl derivative,
activated carboxylic acid, maleimide, aziridine derivative,
acryloyl derivative, fluorobenzene derivatives, or disulfide
derivative (such as a pyridyl disulfide or 5-thio-2-nitrobenzoic
acid{TNB} derivatives).
[0030] If functional group R5, R10 is carboxylate then R5, R10 can
react with (but not restricted to) a diazoacetate, alcohol, thiol
or an amine once the acid has been activated.
[0031] If functional group R5, R10 is an hydroxyl then R5, R10 can
react with (but not restricted to) an activated carboxylic acid,
epoxide, oxirane, or an amine in which carbonyldiimidazole is
used.
[0032] If functional group R5, R10 is an aldehyde or ketone then
R5, R10 can react with (but not restricted to) an hydrazine,
hydrazide derivative, amine (to form a Schiff Base that may or may
not be subsequently reduced by reducing agents such as
NaCNBH.sub.3), or a diol to form an acetal or ketal.
[0033] If functional group R5, R10 is activated carboxylic acid,
isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide ester,
sulfonyl chloride, aldehyde, ketone, epoxide, carbonate,
imidoester, alkylphosphate, arylhalides (difluoro-dinitrobenzene),
anhydride, alkyl halide, or acid halide, p-nitrophenyl ester,
o-nitrophenyl ester, pentachlorophenyl ester, pentafluorophenyl
ester, carbonyl imidazole, carbonyl pyridinium, or carbonyl
dimethylaminopyridinium, then R5, R10 can react with (but not
restricted to) an amine, a hydroxyl, hydrazine, hydrazide, or
sulfhydryl group.
[0034] If functional group R5, R10 an activated carboxylic acid,
haloacetyl derivative, maleimide, aziridine derivative, acryloyl
derivative, fluorobenzene derivatives, or disulfide derivative
(such as a pyridyl disulfide or 5-thio-2-nitrobenzoic acid{TNB}
derivatives) then R5, R10 can react with (but not restricted to) a
sulfhydryl. If functional group R5, R10 is an aldehyde, ketone,
epoxide, oxirane, or an amine in which carbonyldiimidazole or
N,N'-disuccinimidyl carbonate is used, then R5, R10 can react with
(but not restricted to) a hydroxyl.
[0035] If functional group R5, R10 is a hydrazine, hydrazide
derivative, or amine (primary or secondary) then R5, R10 can react
with (but not restricted to) an aldehyde or ketone (to form a
Schiff Base that may or may not be reduced by reducing agents such
as NaCNBH.sub.3).
[0036] Additionally, a compound which contains a disulfide bond
that is connected directly to a heterocyclic ring. The heterocyclic
ring may be aromatic or aliphatic. The heterocyclic ring may
contain 5 or more atoms of which 1 or more is a heteroatom (O, N,
S, P), and the rest being carbon atoms 3
[0037] H is a heteroatom selected from the group including sulfur,
oxygen, nitrogen, or phosphorus. R.sub.1-R.sub.3, R.sub.5-R.sub.7
are substiuents that may be selected from the group that includes
but is not limited to OH, OR (an ether), NH.sub.2,(also secondary,
tertiary, and quaternary amines), SO.sub.3.sup.-, COOH, COOR (an
ester), CONH.sub.2, CONR.sub.2 (substituted amide), a halogen (F,
Cl, Br, I), NO.sub.2, CH.sub.3 (or longer branched or straight
chain, saturated, or unsaturated aliphatic group). The substitution
pattern on the aromatic ring may be varied to alter the reduction
potential of the disulfide bond. L is defined as a linker or spacer
group that provides a connection between the disulfide and the
reactive heterobifunctional or homobifunctional groups. L may or
may not be present and may be chosen from a group that includes
alkanes, alkenes, esters, ethers, glycerol, amide, saccharides,
polysaccharides, heteroatoms such as oxygen, sulfur, or nitrogen.
The spacer may be charge positive, charge negative, charge neutral,
or zwitterionic. R.sub.4, R.sub.8 are reactive groups they may be
identical as in a homobifunctional bifunctional molecule, or
different as in a heterobifunctional bifunctional molecule. In a
preferred embodiment, the disulfide compounds contain reactive
groups that can undergo acylation or alkylation reactions. Such
reactive groups include isothiocynanate, isocynanate, acyl azide,
N-hydroxysuccinimide esters, succinimide esters, sulfonyl chloride,
aldehyde, epoxide, carbonate, imidoester, carboxylate,
alkylphosphate, arylhalides (e.g. difluoro- dinitrobenzene) or
succinic anhydride.
[0038] If functional group R4, R8 is an amine then R4, R8 can react
with (but not restricted to) an activated carboxylic acid,
isothiocyanate, isocyanate, acyl azide, alkyl halide, acid halide,
N-hydroxysuccinimide ester, sulfonyl chloride, aldehyde, ketone,
epoxide, carbonate, imidoester, amide, carboxylate, or
alkylphosphate, arylhalides (difluoro- dinitrobenzene) or
anhydrides. In other terms when function R4, R8 is an amine, then
an acylating or alkylating agent can react with the amine.
[0039] If functional group R4, R8 is a sulfhydryl then R4, R8 can
react with (but not restricted to) a haloacetyl derivative,
activated carboxylic acid, maleimide, aziridine derivative,
acryloyl derivative, fluorobenzene derivatives, or disulfide
derivative (such as a pyridyl disulfide or 5-thio-2-nitrobenzoic
acid{TNB} derivatives).
[0040] If functional group R4, R8 is carboxylate then R4, R8 can
react with (but not restricted to) a diazoacetate, alcohol, thiol
or an amine once the acid has been activated.
[0041] If functional group R4, R8 is an hydroxyl then R4, R8 can
react with (but not restricted to) an activated carboxylic acid,
epoxide, oxirane, or an amine in which carbonyldiimidazole is
used.
[0042] If functional group R4, R8 is an aldehyde or ketone then R4,
R8 can react with (but not restricted to) an hydrazine, hydrazide
derivative, amine (to form a Schiff Base that may or may not be
subsequently reduced by reducing agents such as NaCNBH.sub.3), or a
diol to form an acetal or ketal.
[0043] If functional group R4, R8 is activated carboxylic acid,
isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide ester,
sulfonyl chloride, aldehyde, ketone, epoxide, carbonate,
imidoester, alkylphosphate, arylhalides (difluoro-dinitrobenzene),
anhydride, alkyl halide, or acid halide, p-nitrophenyl ester,
o-nitrophenyl ester, pentachlorophenyl ester, pentafluorophenyl
ester, carbonyl imidazole, carbonyl pyridinium, or carbonyl
dimethylaminopyridinium, then R4, R8 can react with (but not
restricted to) an amine, a hydroxyl, hydrazine, hydrazide, or
sulfhydryl group.
[0044] If functional group R4, R8 an activated carboxylic acid,
haloacetyl derivative, maleimide, aziridine derivative, acryloyl
derivative, fluorobenzene derivatives, or disulfide derivative
(such as a pyridyl disulfide or 5-thio-2-nitrobenzoic acid{TNB}
derivatives) then R4, R8 can react with (but not restricted to) a
sulfhydryl.
[0045] If functional group R4, R8 is an aldehyde, ketone, epoxide,
oxirane, or an amine in which carbonyldiimidazole or N,
N'-disuccinimidyl carbonate is used, then R4, R8 can react with
(but not restricted to) a hydroxyl.
[0046] If functional group R4, R8 is a hydrazine, hydrazide
derivative, or amine (primary or secondary) then R4, R8 can react
with (but not restricted to) an aldehyde or ketone (to form a
Schiff Base that may or may not be reduced by reducing agents such
as NaCNBH.sub.3).
[0047] Additionally, a compound which contains a disulfide bond
that is connected directly to a ring system(aromatic or
non-aromatic) through one of the sulfur atoms and to a aliphatic
carbon through the other sulfur atom. The cyclic ring may contain 5
or more atoms. 4
[0048] R.sub.1-R.sub.4 are substiuents selected from the group that
includes but is not limited to H, OH, OR (an ether), NH.sub.2,(also
secondary, tertiary, and quaternary amines), SO.sub.3.sup.-, COOH,
COOR (an ester), CONH.sub.2, CONR.sub.2 (substituted amide), a
halogen (F, Cl, Br, I), NO.sub.2, CH.sub.3 (or longer branched or
straight chain, saturated, or unsaturated aliphatic group). The
substitution pattern on the aromatic ring may be varied to alter
the reduction potential of the disulfide bond. R.sub.6-R.sub.9 are
substiuents selected from the group that includes but is not
limited to H, OH, OR (an ether), NH.sub.2,(also secondary,
tertiary, and quaternary amines), SO.sub.3.sup.b-, COOH, COOR (an
ester), CONH.sub.2, CONR.sub.2 (substituted amide), a halogen (F,
Cl, Br, I), NO.sub.2, CH.sub.3 (or longer branched or straight
chain, saturated, or unsaturated aliphatic group). L is defined as
a linker or spacer group that provides a connection between the
disulfide and the reactive heterobifunctional or homobifunctional
groups. L may or may not be present and may be chosen from a group
that includes alkanes, alkenes, esters, ethers, glycerol, amide,
saccharides, polysaccharides, heteroatoms such as oxygen, sulfur,
or nitrogen. The spacer may be charge positive, charge negative,
charge neutral, or zwitterionic. R.sub.5, and R.sub.10 are reactive
groups that may be identical as in a homobifunctional bifunctional
molecule, or different as in a heterobifunctional bifunctional
molecule. In a preferred embodiment, the disulfide compounds
contain reactive groups that can undergo acylation or alkylation
reactions. Such reactive groups include isothiocynanate,
isocynanate, acyl azide, N-hydroxysuccinimide esters, succinimide
esters, sulfonyl chloride, aldehyde, epoxide, carbonate,
imidoester, carboxylate, alkylphosphate, arylhalides (e.g.
difluoro-dinitrobenzene) or succinic anhydride.
[0049] If functional group R5, R10 is an amine then R5, R10 can
react with (but not restricted to) an activated carboxylic acid,
isothiocyanate, isocyanate, acyl azide, alkyl halide, acid halide,
N-hydroxysuccinimide ester, sulfonyl chloride, aldehyde, ketone,
epoxide, carbonate, imidoester, amide, carboxylate, or
alkylphosphate, arylhalides (difluoro-dinitrobenzene) or
anhydrides. In other terms when function R5, R10 is an amine, then
an acylating or alkylating agent can react with the amine.
[0050] If functional group R5, R10 is a sulfhydryl then R5, R10 can
react with (but not restricted to) a haloacetyl derivative,
activated carboxylic acid, maleimide, aziridine derivative,
acryloyl derivative, fluorobenzene derivatives, or disulfide
derivative (such as a pyridyl disulfide or 5-thio-2-nitrobenzoic
acid{TNB} derivatives).
[0051] If functional group R5, R10 is carboxylate then R5, R10 can
react with (but not restricted to) a diazoacetate, alcohol, thiol
or an amine once the acid has been activated.
[0052] If functional group R5, R10 is an hydroxyl then R5, R10 can
react with (but not restricted to) an activated carboxylic acid,
epoxide, oxirane, or an amine in which carbonyldiimidazole is
used.
[0053] If functional group R5, R10 is an aldehyde or ketone then
R5, R10 can react with (but not restricted to) an hydrazine,
hydrazide derivative, amine (to form a Schiff Base that may or may
not be subsequently reduced by reducing agents such as
NaCNBH.sub.3), or a diol to form an acetal or ketal.
[0054] If functional group R5, R10 is activated carboxylic acid,
isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide ester,
sulfonyl chloride, aldehyde, ketone, epoxide, carbonate,
imidoester, alkylphosphate, arylhalides (difluoro-dinitrobenzene),
anhydride, alkyl halide, or acid halide, p-nitrophenyl ester,
o-nitrophenyl ester, pentachlorophenyl ester, pentafluorophenyl
ester, carbonyl imidazole, carbonyl pyridinium, or carbonyl
dimethylaminopyridinium, then R5, R10 can react with (but not
restricted to) an amine, a hydroxyl, hydrazine, hydrazide, or
sulfhydryl group.
[0055] If functional group R5, R10 an activated carboxylic acid,
haloacetyl derivative, maleimide, aziridine derivative, acryloyl
derivative, fluorobenzene derivatives, or disulfide derivative
(such as a pyridyl disulfide or 5-thio-2-nitrobenzoic acid{TNB}
derivatives) then R5, R10 can react with (but not restricted to) a
sulfhydryl.
[0056] If functional group R5, R10 is an aldehyde, ketone, epoxide,
oxirane, or an amine in which carbonyldiimidazole or
N,N'-disuccinimidyl carbonate is used, then R5, R10 can react with
(but not restricted to) a hydroxyl.
[0057] If functional group R5, R10 is a hydrazine, hydrazide
derivative, or amine (primary or secondary) then R5, R10 can react
with (but not restricted to) an aldehyde or ketone (to form a
Schiff Base that may or may not be reduced by reducing agents such
as NaCNBH.sub.3).
[0058] Additionally, a compound which contains a disulfide bond
that is connected directly to a heterocyclic ring system through
one of the sulfur atoms and to a aliphatic carbon through the other
sulfur atom. The heterocyclic ring may contain 5 or more atoms of
which 1 or more is a heteroatom (O, N, S, P) or combinations of
heteroatoms, and the rest 5
[0059] being carbon atoms.
[0060] H is a heteroatom selected from the group including sulfur,
oxygen, nitrogen, or phosphorus. Where R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8,R.sub.9, R.sub.10,
R.sub.11, R.sub.12, R.sub.14--at least one of which is an
electronegative atom or functionality such as OH, OR (an ether),
NH.sub.2, (also secondary, tertiary, and quaternary amines),
SO.sub.3.sup.-, COOH, COOR (an ester), CONH.sub.2, CONR.sub.2
(substituted amide), a halogen (F, Cl, Br, I), NO.sub.2. L is
defined as a linker or spacer group that provides a connection
between the disulfide and the reactive heterobifunctional or
homobifunctional groups, A.sub.1 and R.sub.9. L may or may not be
present and may be chosen from a group that includes alkanes,
alkenes, alkynes, esters, ethers, glycerol, amide, urea,
saccharides, polysaccharides, heteroatoms such as oxygen, sulfur,
or nitrogen. The spacer may be charge positive, charge negative,
charge neutral, or zwitterionic. A.sub.1 and R9 are reactive groups
they may be identical as in a homobifunctional bifunctional
molecule, or different as in a heterobifunctional bifunctional
molecule. In a preferred embodiment, the disulfide compounds
contain reactive groups that can undergo acylation or alkylation
reactions. Such reactive groups include (but not limited to)
isothiocynanate, isocynanate, acyl azide, acid halide, 0-acyl urea,
N-hydroxysuccinimide esters, succinimide esters, amide, urea,
sulfonyl chloride, aldehyde, ketone, ether, epoxide, carbonate,
alkyl halide, imidoester, carboxylate, alkylphosphate, arylhalides
(e.g. difluoro-dinitrobenzene) or anhydrides.
[0061] If functional group A1, R9 is an amine then A1, R9 can react
with (but not restricted to) an activated carboxylic acid,
isothiocyanate, isocyanate, acyl azide, alkyl halide, acid halide,
N-hydroxysuccinimide ester, sulfonyl chloride, aldehyde, ketone,
epoxide, carbonate, imidoester, amide, carboxylate, or
alkylphosphate, arylhalides (difluoro-dinitrobenzene) or
anhydrides. In other terms when function A1, R9 is an amine, then
an acylating or alkylating agent can react with the amine.
[0062] If functional group A1, R9 is a sulfhydryl then A1, R9 can
react with (but not restricted to) a haloacetyl derivative,
activated carboxylic acid, maleimide, aziridine derivative,
acryloyl derivative, fluorobenzene derivatives, or disulfide
derivative (such as a pyridyl disulfide or 5-thio-2-nitrobenzoic
acid{TNB} derivatives).
[0063] If functional group A1, R9 is carboxylate then A1, R9 can
react with (but not restricted to) a diazoacetate, alcohol, thiol
or an amine once the acid has been activated.
[0064] If functional group A1, R9 is an hydroxyl then A1, R9 can
react with (but not restricted to) an activated carboxylic acid,
epoxide, oxirane, or an amine in which carbonyldiimidazole is
used.
[0065] If functional group A1, R9 is an aldehyde or ketone then A1,
R9 can react with (but not restricted to) an hydrazine, hydrazide
derivative, amine (to form a Schiff Base that may or may not be
subsequently reduced by reducing agents such as NaCNBH.sub.3), or a
diol to form an acetal or ketal.
[0066] If functional group A1, R9 is activated carboxylic acid,
isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide ester,
sulfonyl chloride, aldehyde, ketone, epoxide, carbonate,
imidoester, alkylphosphate, arylhalides (difluoro-dinitrobenzene),
anhydride, alkyl halide, or acid halide, p-nitrophenyl ester,
o-nitrophenyl ester, pentachlorophenyl ester, pentafluorophenyl
ester, carbonyl imidazole, carbonyl pyridinium, or carbonyl
dimethylaminopyridinium, then A1, R9 can react with (but not
restricted to) an amine, a hydroxyl, hydrazine, hydrazide, or
sulfhydryl group.
[0067] If functional group A1, R9 an activated carboxylic acid,
haloacetyl derivative, maleimide, aziridine derivative, acryloyl
derivative, fluorobenzene derivatives, or disulfide derivative
(such as a pyridyl disulfide or 5-thio-2-nitrobenzoic acid{TNB}
derivatives) then A1, R9 can react with (but not restricted to) a
sulfhydryl.
[0068] If functional group A1, R9 is an aldehyde, ketone, epoxide,
oxirane, or an amine in which carbonyldiimidazole or
N,N'-disuccinimidyl carbonate is used, then A1, R9 can react with
(but not restricted to) a hydroxyl.
[0069] If functional group A1, R9 is a hydrazine, hydrazide
derivative, or amine (primary or secondary) then A1, R9 can react
with (but not restricted to) an aldehyde or ketone (to form a
Schiff Base that may or may not be reduced by reducing agents such
as NaCNBH.sub.3).
[0070] Additionally, a compound which contains a disulfide bond
that is connected directly to a heterocyclic ring system (aromatic
or non-aromatic) through one of the sulfur atoms and to an aromatic
ring system through the other sulfur atom. The heterocyclic ring
may contain 5 or more atoms of which 1 or more is a heteroatom (O,
N, S, P) or combinations of heteroatoms, and the rest being carbon
atoms. 6
[0071] H is a heteroatom selected from the group including sulfur,
oxygen, nitrogen, or phosphorus. Where R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.10, R.sub.11,
R.sub.12, R.sub.13--at least one of which is an electronegative
atom or functionality such as OH, OR (an ether), NH.sub.2, (also
secondary, tertiary, and quaternary amines), SO.sub.3.sup.-, COOH,
COOR (an ester), CONH.sub.2, CONR.sub.2 (substituted amide), a
halogen (F, Cl, Br, I), NO.sub.2. L is defined as a linker or
spacer group that provides a connection between the disulfide and
the reactive heterobifunctional or homobifunctional groups, R.sub.9
and R.sub.14. L may or may not be present and may be chosen from a
group that includes alkanes, alkenes, alkynes, esters, ethers,
glycerol, amide, urea, saccharides, polysaccharides, heteroatoms
such as oxygen, sulfur, or nitrogen. The spacer may be charge
positive, charge negative, charge neutral, or zwitterionic. R.sub.9
and R.sub.14 are reactive groups they may be identical as in a
homobifunctional bifunctional molecule, or different as in a
heterobifunctional bifunctional molecule. In a preferred
embodiment, the disulfide compounds contain reactive groups that
can undergo acylation or alkylation reactions. Such reactive groups
include (but not limited to) isothiocynanate, isocynanate, acyl
azide, acid halide, O-acyl urea, N-hydroxysuccinimide esters,
succinimide esters, amide, urea, sulfonyl chloride, aldehyde,
ketone, ether, epoxide, carbonate, alkyl halide, imidoester,
carboxylate, alkylphosphate, arylhalides (e.g.
difluoro-dinitrobenzene) or anhydrides.
[0072] If functional group R9, R14 is an amine then R9, R14 can
react with (but not restricted to) an activated carboxylic acid,
isothiocyanate, isocyanate, acyl azide, alkyl halide, acid halide,
N-hydroxysuccinimide ester, sulfonyl chloride, aldehyde, ketone,
epoxide, carbonate, imidoester, amide, carboxylate, or
alkylphosphate, arylhalides (difluoro-dinitrobenzene) or
anhydrides. In other terms when function R9, R14 is an amine, then
an acylating or alkylating agent can react with the amine.
[0073] If functional group R9, R14 is a sulfhydryl then R9, R14 can
react with (but not restricted to) a haloacetyl derivative,
activated carboxylic acid, maleimide, aziridine derivative,
acryloyl derivative, fluorobenzene derivatives, or disulfide
derivative (such as a pyridyl disulfide or 5-thio-2-nitrobenzoic
acid{TNB} derivatives).
[0074] If functional group R9, R14 is carboxylate then R9, R1 4 can
react with (but not restricted to) a diazoacetate, alcohol, thiol
or an amine once the acid has been activated.
[0075] If functional group R9, R14 is an hydroxyl then R9, R14 can
react with (but not restricted to) an activated carboxylic acid,
epoxide, oxirane, or an amine in which carbonyldiimidazole is
used.
[0076] If functional group R9, R14 is an aldehyde or ketone then
R9, R14 can react with (but not restricted to) an hydrazine,
hydrazide derivative, amine (to form a Schiff Base that may or may
not be subsequently reduced by reducing agents such as
NaCNBH.sub.3), or a diol to form an acetal or ketal.
[0077] If functional group R9, R14 is activated carboxylic acid,
isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide ester,
sulfonyl chloride, aldehyde, ketone, epoxide, carbonate,
imidoester, alkylphosphate, arylhalides (difluoro-dinitrobenzene),
anhydride, alkyl halide, or acid halide, p-nitrophenyl ester,
o-nitrophenyl ester, pentachlorophenyl ester, pentafluorophenyl
ester, carbonyl imidazole, carbonyl pyridinium, or carbonyl
dimethylaminopyridinium, then R9, R14 can react with (but not
restricted to) an amine, a hydroxyl, hydrazine, hydrazide, or
sulfhydryl group.
[0078] If functional group R9, R14 an activated carboxylic acid,
haloacetyl derivative, maleimide, aziridine derivative, acryloyl
derivative, fluorobenzene derivatives, or disulfide derivative
(such as a pyridyl disulfide or 5-thio-2-nitrobenzoic acid {TNB}
derivatives) then R9, R14 can react with (but not restricted to) a
sulfhydryl.
[0079] If functional group R9, R14 is an aldehyde, ketone, epoxide,
oxirane, or an amine in which carbonyldiimidazole or
N,N'-disuccinimidyl carbonate is used, then R9, R14 can react with
(but not restricted to) a hydroxyl.
[0080] If functional group R9, R14 is a hydrazine, hydrazide
derivative, or amine (primary or secondary) then R9, R14 can react
with (but not restricted to) an aldehyde or ketone (to form a
Schiff Base that may or may not be reduced by reducing agents such
as NaCNBH.sub.3).
DETAILED DESCRIPTION
[0081] Counterintuitive to previous efforts to synthesize
bifunctional molecules with stabile disulfides, the object of the
current invention is to synthesize labile disulfide molecules. In
vivo, disulfides are primarily reduced by the cysteine-based thiol
glutathione (.gamma.-glutamylcystylglycine), which is present in
millimolar concentrations in the cell. To increase the lability of
the disulfide bond in a bifunctional molecule and its construct, we
have synthesized several disulfide bond-containing bifunctional
molecules that are more rapidly reduced than oxidized
glutathione.
[0082] Disulfide Bond Containing Bifunctional Molecules
[0083] Bifunctional molecules, possessing either homo or
heterobifunctionality (commonly referred to as crosslinkers), are
used to connect two molecules together. The disulfide linkage
(RSSR') may be used within bifunctional molecules. The
reversibility of disulfide bond formation makes them useful tools
for the transient attachment of two molecules. Physiologically,
disulfides are reduced by glutathione.
[0084] A disulfide bond that is labile under physiological
conditions means: the disulfide bond is cleaved more rapidly than
oxidized glutathione or any disulfide constructed from thiols in
which one of the constituent thiols is more acidic, lower pKa, than
glutathione or is activated by intramolecular attack by a free
thiol. Constituent in this case means the thiols that are bonded
together in the disulfide bond. Cleavable means that a chemical
bond between atoms is broken.
[0085] The present invention describes physiologically labile
disulfide bond containing bifunctional molecules. The present
invention is also meant to include constructs prepared from the
bifunctional molecules, including polymers, peptides, proteins,
nucleic acids, polymer nucleic acid complexes. Construct means any
compound resulting from the chemical reaction of at least one of
the reactive centers of the bifunctional molecule resulting in new
chemical bond other that that resulting from hydrolysis of both
reactive centers of the bifunctional molecule. Further chemical
modification may occur after the formation of the construct.
Crosslinking refers to the chemical attachment of two or more
molecules with a bifunctional reagent. A bifunctional reagent is a
molecule with two reactive ends. The reactive ends can be identical
as in a homobifunctional molecule, or different as in a
heterobifunctional molecule.
[0086] Polymers
[0087] A polymer is a molecule built up by repetitive bonding
together of smaller units called monomers. In this application the
term polymer includes both oligomers which have two to about 80
monomers and polymers having more than 80 monomers. The polymer can
be linear, branched network, star, comb, or ladder types of
polymer. The polymer can be a homopolymer in which a single monomer
is used or can be copolymer in which two or more monomers are used.
Types of copolymers include alternating, random, block and
graft.
[0088] To those skilled in the art of polymerization, there are
several categories of polymerization processes that can be utilized
in the described process. The polymerization can be chain or step.
This classification description is more often used that the
previous terminology of addition and condensation polymer. "Most
step-reaction polymerizations are condensation processes and most
chain-reaction polymerizations are addition processes" (M. P.
Stevens Polymer Chemistry: An Introduction New York Oxford
University Press 1990). Template polymerization can be used to form
polymers from daughter polymers.
[0089] Step Polymerization: In step polymerization, the
polymerization occurs in a stepwise fashion. Polymer growth occurs
by reaction between monomers, oligomers and polymers. No initiator
is needed since there is the same reaction throughout and there is
no termination step so that the end groups are still reactive. The
polymerization rate decreases as the functional groups are
consumed.
[0090] Typically, step polymerization is done either of two
different ways. One way, the monomer has both reactive functional
groups (A and B) in the same molecule so that A--B yields
--[A--B]--Or the other approach is to have two bifunctional
monomers. A--A+B--B yields --[A--A--B--B]--Generally, these
reactions can involve acylation or alkylation. Acylation is defined
as the introduction of an acyl group (--COR) onto a molecule.
Alkylation is defined as the introduction of an alkyl group onto a
molecule. If functional group A is an amine then B can be (but not
restricted to) an isothiocyanate, isocyanate, acyl azide,
N-hydroxysuccinimide, sulfonyl chloride, aldehyde (including
formaldehyde and glutaraldehyde), ketone, epoxide, carbonate,
imidoester, carboxylate activated with a carbodiimide,
alkylphosphate, arylhalides (difluoro-dinitrobenzene), anhydride,
or acid halide, p-nitrophenyl ester, o-nitrophenyl ester,
pentachlorophenyl ester, pentafluorophenyl ester, carbonyl
imidazole, carbonyl pyridinium, or carbonyl
dimethylaminopyridinium. In other terms when function A is an amine
then function B can be acylating or alkylating agent or amination
agent.
[0091] If functional group A is a sulfhydryl then function B can be
(but not restricted to) an iodoacetyl derivative, maleimide,
aziridine derivative, acryloyl derivative, fluorobenzene
derivatives, or disulfide derivative (such as a pyridyl disulfide
or 5-thio-2-nitrobenzoic acid{TNB} derivatives).
[0092] If functional group A is carboxylate then function B can be
(but not restricted to) a diazoacetate or an amine in which a
carbodiimide is used. Other additives may be utilized such as
carbonyldiimidazole, dimethylamino pyridine (DMAP),
N-hydroxysuccinimide or alcohol using carbodiimide and DMAP.
[0093] If functional group A is an hydroxyl then function B can be
(but not restricted to) an epoxide, oxirane, or an amine in which
carbonyldiimidazole or N,N'-disuccinimidyl carbonate, or
N-hydroxysuccinimidyl chloroformate or other chloroformates are
used. If functional group A is an aldehyde or ketone then function
B can be (but not restricted to) an hydrazine, hydrazide
derivative, amine (to form a Schiff Base that may or may not be
reduced by reducing agents such as NaCNBH3) or hydroxyl compound to
form a ketal or acetal.
[0094] Yet another approach is to have one bifunctional monomer so
that A--A plus another agent yields --[A--A]--.
[0095] If function A is a sulfhydryl group then it can be converted
to disulfide bonds by oxidizing agents such as iodine (I2) or NaIO4
(sodium periodate), or oxygen (O2). Function A can also be an amine
that is converted to a sulfhydryl group by reaction with
2-iminothiolate (Traut's reagent) which then undergoes oxidation
and disulfide formation. Disulfide derivatives (such as a pyridyl
disulfide or 5-thio-2-nitrobenzoic acid {TNB} derivatives) can also
be used to catalyze disulfide bond formation. Functional group A or
B in any of the above examples could also be a photoreactive group
such as aryl azide (including halogenated aryl azide), diazo ,
benzophenone, alkyne or diazirine derivative.
[0096] Reactions of the amine, hydroxyl, sulfhydryl, carboxylate
groups yield chemical bonds that are described as amide, amidine,
disulfide, ethers, esters, enamine, imine, urea, isothiourea,
isourea, sulfonamide, carbamate, alkylamine bond (secondary amine),
carbon-nitrogen single bonds in which the carbon contains a
hydroxyl group, thioether, diol, hydrazone, diazo, or sulfone.
[0097] Chain Polymerization: In chain-reaction polymerization
growth of the polymer occurs by successive addition of monomer
units to limited number of growing chains. The initiation and
propagation mechanisms are different and there is usually a
chain-terminating step. The polymerization rate remains constant
until the monomer is depleted. Monomers containing (but not limited
to) vinyl, acrylate, methacrylate, acrylamide, methacrylamide
groups can undergo chain reaction which can be radical, anionic, or
cationic. Chain polymerization can also be accomplished by cycle or
ring opening polymerization. Several different types of free
radical initiators could be used that include peroxides, hydroxy
peroxides, and azo compounds such as 2,2'-Azobis(-amidinopropane)
dihydrochloride (AAP).
[0098] Types of Monomers
[0099] A wide variety of monomers can be used in the polymerization
processes. These include positive charged organic monomers such as
amine salts, imidine, guanidine, imine, hydroxylamine, hydrozyine,
heterocycle (salts) like imidazole, pyridine, morpholine,
pyrimidine, or pyrene. The amines could be pH-sensitive in that the
pKa of the amine is within the physiologic range of 4 to 8.
Specific amines include spermine, spermidine,
N,N'-bis(2-aminoethyl)-1,3-propanediamine (AEPD), and
3,3'-Diamino-N,N-dimethyldipropylammonium bromide.
[0100] Monomers can also be hydrophobic, hydrophilic or
amphipathic. Amphipathic compounds have both hydrophilic
(water-soluble) and hydrophobic (water-insoluble) parts.
Hydrophilic groups indicate in qualitative terms that the chemical
moiety is water-preferring. Typically, such chemical groups are
water soluble, and are hydrogen bond donors or acceptors with
water. Examples of hydrophilic groups include compounds with the
following chemical moieties; carbohydrates, polyoxyethylene,
peptides, oligonucleotides and groups containing amines, amides,
alkoxy amides, carboxylic acids, sulfurs, or hydroxyls. Hydrophobic
groups indicate in qualitative terms that the chemical moiety is
water-avoiding. Typically, such chemical groups are not water
soluble, and tend not to hydrogen bonds. Hydrocarbons are
hydrophobic groups.
[0101] Monomers can also be intercalating agents such as acridine,
thiazole organge, or ethidium bromide. Monomers can also contain
chemical moieties that can be modified before or after the
polymerization including (but not limited to) amines (primary,
secondary, and tertiary), amides, carboxylic acid, ester, hydroxyl,
hydrazine, alkyl halide, aldehyde, and ketone.
[0102] Other Components of the Monomers and Polymers
[0103] The polymers have other groups that increase their utility.
These groups can be incorporated into monomers prior to polymer
formation or attached to the polymer after its formation. These
groups include: targeting groups, signals, reporter or marker
molecules, spacers, steric stabilizers, chelators, polycations,
polyanions, and polymers.
[0104] Targeting groups are used for targeting the polymer-nucleic
acid complexes to specific cells or tissues. Examples of targeting
agents include agents that target to the asialoglycoprotein
receptor by using asiologlycoproteins or galactose residues.
Proteins such as insulin, EGF, or transferrin can be used for
targeting. Protein refers to a molecule made up of 2 or more amino
acid residues connected one to another by peptide bonds between the
alpha-amino group and carboxyl group of contiguous amino acid
residues as in a polypeptide. The amino acids may be naturally
occurring or synthetic. Peptides that include the RGD sequence can
be used to target many cells. Peptide refers to a linear series of
amino acid residues connected to one another by peptide bonds
between the alpha-amino group and carboxyl group of contiguous
amino acid residues. Polypeptide includes proteins and peptides,
modified proteins and peptides, and non-natural proteins and
peptides.
[0105] Chemical groups that react with sulfhydryl or disulfide
groups on cells can also be used to target many types of cells.
Folate and other vitamins can also be used for targeting. Other
targeting groups include molecules that interact with membranes
such as fatty acids, cholesterol, dansyl compounds, and
amphotericin derivatives.
[0106] Other targeting groups can be used to increase the delivery
of the drug or nucleic acid to certain parts of the cell. For
example, agents can be used to disrupt endosomes and a nuclear
localizing signal (NLS) can be used to target the nucleus. A
variety of ligands have been used to target drugs and genes to
cells and to specific cellular receptors. The ligand may seek a
target within the cell membrane, on the cell membrane or near a
cell. Binding of ligands to receptors typically initiates
endocytosis. Ligands could also be used for DNA delivery that bind
to receptors that are not endocytosed. For example peptides
containing RGD peptide sequence that bind integrin receptor could
be used. In addition viral proteins could be used to bind the
complex to cells. Lipids and steroids could be used to directly
insert a complex into cellular membranes. The polymers can also
contain cleavable groups within themselves. When attached to the
targeting group, cleavage leads to reduce interaction between the
complex and the receptor for the targeting group. Cleavable groups
include but are not restricted to disulfide bonds, diols, diazo
bonds, ester bonds, sulfone bonds, acetals, ketals, enol ethers,
enol esters, enamines and imines, acyl hydrazones, and Schiff
bases.
[0107] In a preferred embodiment, a chemical reaction can be used
to attach a signal to a nucleic acid complex. The signal is defined
in this specification as a molecule that modifies the nucleic acid
complex, or biologically active molecule, and can direct it to a
cell location (such as tissue cells) or location in a cell (such as
the nucleus, or cytoplasm) either in culture or in a whole
organism. By modifying the cellular or tissue location of the
foreign gene, the expression of the foreign gene can be
enhanced.
[0108] The signal can be a protein, peptide, lipid, steroid, sugar,
carbohydrate, nucleic acid or synthetic compound. The signals
enhance cellular binding to receptors, cytoplasmic transport to the
nucleus and nuclear entry or release from endosomes or other
intracellular vesicles.
[0109] A certain subset of signals, termed transduction signals in
this application, transport themselves and attached molecules
across membranes (Schwarze and Dowdy Trends Pharm. Sci. 2000, 21,
45). Examples of these transduction signals are derived from viral
coat proteins such as Tat from HIV and VP22 from herpes simplex
virus, and a transcriptional factor from Drosophila, ANTP. The
peptides Tat (Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-- Arg-Arg-Arg), VP22
(Asp-Ala-Ala-Thr-Ala-Thr-Arg-Gly-Arg-Ser-Ala-Ala-Ser-Ar-
g-Pro-Thr-Glu-Arg-Pro-Arg-Ala-Pro-Ala-Arg-Ser-Ala-Ser-Arg-Pro-Arg-Arg-Pro--
Val-Glu), and ANTP
(Arg-Gln-Iso-Lys-Iso-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Tr- p-Lys-Lys)
share no sequence motif other than number of cationic (lysine and
arginine) residues. In addition, reports of synthetic peptides
possessing no homology other than a propensity of cationic charge
(net overall cationic charge) have also been shown to posses
transduction activity (Service, R. F. Science 2000, 288, 28.)
[0110] Nuclear localizing signals enhance the targeting of the gene
into proximity of the nucleus and/or its entry into the nucleus.
Such nuclear transport signals can be a protein or a peptide such
as the SV40 large T ag NLS or the nucleoplasmin NLS. These nuclear
localizing signals interact with a variety of nuclear transport
factors such as the NLS receptor (karyopherin alpha) which then
interacts with karyopherin beta. The nuclear transport proteins
themselves could also function as NLS's since they are targeted to
the nuclear pore and nucleus.
[0111] Signals that enhance release from intracellular compartments
(releasing signals) can cause DNA release from intracellular
compartments such as endosomes (early and late), lysosomes,
phagosomes, vesicle, endoplasmic reticulum, golgi apparatus, trans
golgi network (TGN), and sarcoplasmic reticulum. Release includes
movement out of an intracellular compartment into cytoplasm or into
an organelle such as the nucleus. Releasing signals include
chemicals such as chloroquine, bafilomycin or Brefeldin A1 and the
ER-retaining signal (KDEL sequence), viral components such as
influenza virus hemagglutinin subunit HA-2 peptides and other types
of amphipathic peptides. Cellular receptor signals are any signal
that enhances the association of the gene or particle with a cell.
This can be accomplished by either increasing the binding of the
gene to the cell surface and/or its association with an
intracellular compartment, for example: ligands that enhance
endocytosis by enhancing binding the cell surface. This includes
agents that target to the asialoglycoprotein receptor by using
asiologlycoproteins or galactose residues. Other proteins such as
insulin, EGF, or transferrin can be used for targeting. Peptides
that include the RGD sequence can be used to target many cells.
Chemical groups that react with sulfhydryl or disulfide groups on
cells can also be used to target many types of cells. Folate and
other vitamins can also be used for targeting. Other targeting
groups include molecules that interact with membranes such as
lipids fatty acids, cholesterol, dansyl compounds, and amphotericin
derivatives. In addition viral proteins could be used to bind
cells.
[0112] Reporter or marker molecules are compounds that can be
easily detected. Typically they are fluorescent compounds such as
fluorescein, rhodamine, Texas red, cy 5, cy 3 or dansyl compounds.
They can be molecules that can be detected by UV or visible
spectroscopy or by antibody interactions or by electron spin
resonance. Biotin is another reporter molecule that can be detected
by labeled avidin. Biotin could also be used to attach targeting
groups.
[0113] A spacer is any linker known to those skilled in the art to
enable one to join one moiety to another moiety. The moieties can
be hydrophilic or hydrophobic. Preferred spacer groups include, but
are not limited to C1-C12 alkyl, C1-C12 alkenyl, C1-C12 alkynyl,
C6-C18 aralkyl, C6-C 18 aralkenyl, C6-C 18 aralkynyl, ester, ether,
ketone, alcohol, polyol, amide, amine, polyglycol, polyamine,
thiol, thio ether, thioester, phosphorous containing, and
heterocyclic.
[0114] A Steric stabilizer is a long chain hydrophilic group that
prevents aggregation of final polymer by sterically hindering
particle to particle electrostatic interactions. Examples include:
alkyl groups, PEG chains, polysaccharides, hydrogen molecules,
alkyl amines. Electrostatic interactions are the non-covalent
association of two or more substances due to attractive forces
between positive and negative charges.
[0115] A polycation is a polymer containing a net positive charge,
for example poly-L-lysine hydrobromide. The polycation can contain
monomer units that are charge positive, charge neutral, or charge
negative, however, the net charge of the polymer must be positive.
A polycation also can mean a non-polymeric molecule that contains
two or more positive charges. A polyanion is a polymer containing a
net negative charge, for example polyglutamic acid. The polyanion
can contain monomer units that are charge negative, charge neutral,
or charge positive, however, the net charge on the polymer must be
negative. A polyanion can also mean a non-polymeric molecule that
contains two or more negative charges. The term polyion includes
polycation, polyanion, zwitterionic polymers, and neutral polymers.
The term zwitterionic refers to the product (salt) of the reaction
between an acidic group and a basic group that are part of the same
molecule. Salts are ionic compounds that dissociate into cations
and anions when dissolved in solution. Salts increase the ionic
strength of a solution, and consequently decrease interactions
between nucleic acids with other cations.
[0116] A chelator is a polydentate ligand, a molecule that can
occupy more than one site in the coordination sphere of an ion,
particularly a metal ion, primary amine, or single proton. Examples
of chelators include crown ethers, cryptates, and non-cyclic
polydentate molecules. A crown ether is a cyclic polyether
containing (--X--(CR1-2)n)m units, where n=1-3 and m=3-8. The X and
CR1-2 moieties can be substituted, or at a different oxidation
states. X can be oxygen, nitrogen, or sulfur, carbon, phosphorous
or any combination thereof. R can be H, C, O, S, N, P. A subset of
crown ethers described as a cryptate contain a second
(--X--(CR1-2)n)z strand where z=3-8. The beginning X atom of the
strand is an X atom in the (--X--(CR1-2)n)m unit, and the terminal
CH2 of the new strand is bonded to a second X atom in the
(--X--(CR1-2)n)m unit. Non-cyclic polydentate molecules containing
(--X--(CR1-2)n)m unit(s), where n=1-4 and m=1-8. The X and CR1-2
moieties can be substituted, or at a different oxidation states. X
can be oxygen, nitrogen, or sulfur, carbon, phosphorous or any
combination thereof. A polychelator is a polymer associated with a
plurality of chelators by an ionic or covalent bond and can include
a spacer. The polymer can be cationic, anionic, zwitterionic,
neutral, or contain any combination of cationic, anionic,
zwitterionic, or neutral groups with a net charge being cationic,
anionic or neutral, and may contain steric stabilizers, peptides,
proteins, signals, or amphipathic compound for the formation of
micellar, reverse micellar, or unilamellar structures. Preferably
the amphipathic compound can have a hydrophilic segment that is
cationic, anionic, or zwitterionic, and can contain polymerizable
groups, and a hydrophobic segment that can contain a polymerizable
group.
[0117] The present invention provides for the transfer of
polynucleotides, and biologically active compounds into parenchymal
cells within tissues in situ and in vivo, utilizing disulfide bonds
that can be cleaved under physialogicval condidtions, and delivered
intravasculary (U.S. patent application Ser. No. 08/571,536),
intrarterially, intravenous, orally, intraduodenaly, via the
jejunum (or ileum or colon), rectally, transdermally,
subcutaneously, intramuscularly, intraperitoneally,
intraparenterally, via direct injections into tissues such as the
liver, lung, heart, muscle, spleen, pancreas, brain (including
intraventricular), spinal cord, ganglion, lymph nodes, lymphatic
system, adipose tissues, thryoid tissue, adrenal glands, kidneys,
prostate, blood cells, bone marrow cells, cancer cells, tumors, eye
retina, via the bile duct, or via mucosal membranes such as in the
mouth, nose, throat, vagina or rectum or into ducts of the salivary
or other exocrine glands.
[0118] "Delivered" means that the polynucleotide becomes associated
with the cell. The polynucleotide can be on the membrane of the
cell or inside the cytoplasm, nucleus, or other organelle of the
cell. The process of delivering a polynucleotide to a cell has been
commonly termed "transfection" or the process of "transfecting" and
also it has been termed "transformation". The polynucleotide could
be used to produce a change in a cell that can be therapeutic. The
delivery of polynucleotides or genetic material for therapeutic and
research purposes is commonly called "gene therapy". The
polynucleotides or genetic material being delivered are generally
mixed with transfection reagents prior to delivery.
[0119] A biologically active compound is a compound having the
potential to react with biological components. More particularly,
biologically active compounds utilized in this specification are
designed to change the natural processes associated with a living
cell. For purposes of this specification, a cellular natural
process is a process that is associated with a cell before delivery
of a biologically active compound. In this specification, the
cellular production of, or inhibition of a material, such as a
protein, caused by a human assisting a molecule to an in vivo cell
is an example of a delivered biologically active compound.
Pharmaceuticals, proteins, peptides, polypeptides, hormones,
cytokines, antigens, viruses, oligonucleotides, and nucleic acids
are examples of biologically active compounds. Bioactive compounds
may be used interchangeably with biologically active compound for
purposes of this application.
[0120] The term "nucleic acid" is a term of art that refers to a
polymer containing at least two nucleotides. "Nucleotides" contain
a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate
group. Nucleotides are linked together through the phosphate
groups. "Bases" include purines and pyrimidines, which further
include natural compounds adenine, thymine, guanine, cytosine,
uracil, inosine, and synthetic derivatives of purines and
pyrimidines, or natural analogs. Nucleotides are the monomeric
units of nucleic acid polymers. A "polynucleotide" is distinguished
here from an "oligonucleotide" by containing more than 80 monomeric
units; oligonucleotides contain from 2 to 80 nucleotides. The term
nuclei acid includes deoxyribonucleic acid (DNA) and ribonucleic
acid (RNA). DNA may be in the form of anti-sense, plasmid DNA,
parts of a plasmid DNA, vectors (P1, PAC, BAC, YAC, artificial
chromosomes), expression cassettes, chimeric sequences, chromosomal
DNA, or derivatives of these groups. RNA may be in the form of
oligonucleotide RNA, tRNA (transfer RNA), snRNA (small nuclear
RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA,
ribozymes, chimeric sequences, or derivatives of these groups.
"Anti-sense" is a polynucleotide that interferes with the function
of DNA and/or RNA. This may result in suppression of expression.
Natural nucleic acids have a phosphate backbone, artificial nucleic
acids may contain other types of backbones and bases. These include
PNAs (peptide nucleic acids), phosphothionates, and other variants
of the phosphate backbone of native nucleic acids.
[0121] In addition, DNA and RNA may be single, double, triple, or
quadruple stranded. "Expression cassette" refers to a natural or
recombinantly produced polynucleotide molecule which is capable of
expressing protein(s). A DNA expression cassette typically includes
a promoter (allowing transcription initiation), and a sequence
encoding one or more proteins. Optionally, the expression cassette
may include trancriptional enhancers, non-coding sequences,
splicing signals, transcription termination signals, and
polyadenylation signals. An RNA expression cassette typically
includes a translation initiation codon (allowing translation
initiation), and a sequence encoding one or more proteins.
Optionally, the expression cassette may include translation
termination signals, a polyadenosine sequence, internal ribosome
entry sites (IRES), and non-coding sequences.
[0122] The term "naked polynucleotides" indicates that the
polynucleotides are not associated with a transfection reagent or
other delivery vehicle that is required for the polynucleotide to
be delivered to the cardiac muscle cell. A "transfection reagent"
or "delivery vehicle" is a compound or compounds used in the prior
art that bind(s) to or complex(es) with oligonucleotides or
polynucleotides, and mediates their entry into cells. The
transfection reagent also mediates the binding and internalization
of polynucleotides into cells. Examples of transfection reagents
include cationic liposomes and lipids, polyamines, calcium
phosphate precipitates, histone proteins, polyethylenimine, and
polylysine complexes (polyethylenimine and polylysine are both
toxic). Typically, the transfection reagent has a net positive
charge that binds to the polynucleotide's negative charge. The
transfection reagent mediates binding of polynucleotides to cell
via its positive charge (that binds to the cell membrane's negative
charge) or via ligands that bind to receptors in the cell. For
example, cationic liposomes or polylysine complexes have net
positive charges that enable them to bind to DNA or RNA. Other
delivery vehicles are also used, in the prior art, to transfer
genes into cells. These include complexing the polynucleotides on
particles that are then accelerated into the cell. This is termed
"biolistic" or "gun" techniques.
[0123] Ionic (electrostatic) interactions are the non-covalent
association of two or more substances due to attractive forces
between positive and negative charges, or partial positive and
partial negative charges.
[0124] Condensed Nucleic Acids: A method of condensing a polymer is
defined as decreasing its linear length, also called compacting.
Condensing a polymer also means decreasing the volume that the
polymer occupies. An example of condensing nucleic acid is the
condensation of DNA that occurs in cells. The DNA from a human cell
is approximately one meter in length but is condensed to fit in a
cell nucleus that has a diameter of approximately 10 microns. The
cells condense (or compacts) DNA by a series of packaging
mechanisms involving the histones and other chromosomal proteins to
form nucleosomes and chromatin. The DNA within these structures is
rendered partially resistant to nuclease DNase) action. The process
of condensing polymers can be used for delivering them into cells
of an organism. A delivered polymer can stay within the cytoplasm
or nucleus apart from the endogenous genetic material.
Alternatively, the polymer could recombine (become a part of) the
endogenous genetic material. For example, DNA can insert into
chromosomal DNA by either homologous or non-homologous
recombination.
[0125] Intravascular: An intravascular route of administration
enables a polymer or polynucleotide to be delivered to cells more
evenly distributed and more efficiently expressed than direct
injections. Intravascular herein means within a tubular structure
called a vessel that is connected to a tissue or organ within the
body. Within the cavity of the tubular structure, a bodily fluid
flows to or from the body part. Examples of bodily fluid include
blood, lymphatic fluid, or bile. Examples of vessels include
arteries, arterioles, capillaries, venules, sinusoids, veins,
lymphatics, and bile ducts. The intravascular route includes
delivery through the blood vessels such as an artery or a vein. An
administration route involving the mucosal membranes is meant to
include nasal, bronchial, inhalation into the lungs, or via the
eyes.
[0126] Buffers are made from a weak acid or weak base and their
salts. Buffer solutions resist changes in pH when additional acid
or base is added to the solution. Biological, chemical, or
biochemical reactions involve the formation or cleavage of ionic
and/or covalent bonds. Biomolecule refers to peptides,
polypeptides, proteins, enzymes, polynucleotides, oligonucleotides,
viruses, antigens, carbohydrates (and conjugates), lipids, and
saccharides. Enzymes are proteins evolved by the cells of living
organisms for the specific function of catalyzing chemical
reactions. A chemical reaction is defined as the formation or
cleavage of covalent or ionic bonds. As a result of the chemical
reaction a polymer can be formed. A polymer is defined as a
compound containing more than two monomers. A monomer is a compound
that can be attached to itself or another monomer and thus a form a
polymer.
[0127] Transdermal refers to application to mammal skin in which
drug delivery occurs by crossing the dermal layer.
[0128] Hydrocarbon means containing carbon and hydrogen atoms; and
halohydrocarbon means containing carbon, halogen (F, Cl, Br, I),
and hydrogen atoms.
[0129] Alkyl means containing sp.sup.3 hybridized carbon atoms;
alkenyl means containing two or more sp.sup.2 hybridized carbon
atoms; aklkynyl means containing two or more sp hybridized carbon
atoms; aralkyl means containing one or more aromatic ring(s) in
addition containing sp.sup.3 hybridized carbon atoms; aralkenyl
means containing one or more aromatic ring(s) in addition to
containing two or more sp.sup.2 hybridized carbon atoms; aralkynyl
means containing one or more aromatic ring(s) in addition to
containing two or more sp hybridized carbon atoms; steroid includes
natural and unnatural steroids and steroid derivatives.
[0130] A steroid derivative means a sterol, a sterol in which the
hydroxyl moity has been modified (for example, acylated), or a
steroid hormone, or an analog thereof.
[0131] Carbohydrates include natural and unnatural sugars (for
example glucose), and sugar derivatives (a sugar derivative means a
system in which one or more of the hydroxyl groups on the sugar
moiety has been modified (for example acylated), or a system in
which one or more of the hydroxyl groups is not present).
[0132] Polyoxyethylene means a polymer having two to six (n=2-3000)
ethylene oxide units (--(CH.sub.2CH.sub.2O).sub.n--) or a
derivative thereof.
[0133] R is meant to be any compatible group, for example hydrogen,
alkyl, alkenyl, alkynyl, aralkyl, aralkenyl, or aralkynyl, and can
include heteroatoms (N, O, S), and carbonyl groups.
[0134] A compound is a material made up of two or more
elements.
[0135] Electron withdrawing group is any chemical group or atom
composed of electronegative atom(s), that is atoms that tend to
attract electrons. Resonance stabilization is the ability to
distribute charge on multiple atoms through pi bonds. The inductive
effective, in a molecule, is a shift of electron density due to the
polarization of a bond by a nearby electronegative or
electropositive atom.
[0136] Steric hindrance, or sterics, is the prevention or
retardation of a chemical reaction because of neighboring groups on
the same molecule.
[0137] An activated carboxylate is a carboxylic acid derivative
that reacts with nucleophiles to form a new covalent bond.
Nucleophiles include nitrogen, oxygen and sulfur-containing
compounds to produce ureas, amides, carbonates, esters, and
thioesters. The carboxylic acid may be activated by various agents
including carbodiimides, carbonates, phosphoniums, uroniums to
produce activated carboxylates acyl ureas, acylphosphonates, and
carbonates. Activation of carboxylic acid may be used in
conjunction with hydroxy and amine-containing compounds to produce
activated carboxylates N-hydroxysuccinimide esters,
hydroxybenzotriazole esters,
N-hydroxy-5-norbomene-endo-2,3-dicarboximide esters, p-nitrophenyl
esters, pentafluorophenyl esters, 4-dimethylaminopyridinium amides,
and acyl imidazoles.
[0138] A nucleophile is a species possessing one or more
electron-rich sites, such as an unshared pair of electrons, the
negative end of a polar bond, or pi electrons.
EXAMPLES
Example 1
Synthesis of Cysteine-terminal Tat Peptide (Tat-Cys).
[0139] Peptide syntheses were performed using standard solid phase
peptide techniques using FMOC chemistry. A cysteine was added to
the amino terminus of Tat to allow for conjugation through the
thiol group to make the peptide
Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys (Tat-Cys).
Example 2
Synthesis of Noncleavably Linked (Irreversible Covalent) Tat-Cys
and Fluorescein through a Thioether Bond.
[0140] To a solution of succinimidyl-4-(N-maleimidomethyl)
cyclohexane-carboxylate (SMCC from Pierce) 1.0 mg in 0.1 mL
dimethylformamide was added 1.2 mg (1 eq) of
4'-(aminomethyl)fluorescein. After two hours, this solution was
added to a 1 mL aqueous solution of 8.4 mg Tat-Cys (1 eq). The
solution was buffered to pH 8 by the addition of potassium
carbonate. This solution was used for transport studies without
further purification.
Example 3
Synthesis of Unactivated (Non-labile) Disulfide Linked Lissamine
Dimer (Dilissamine Cystamine)
[0141] To a solution of cystamine dihydrochloride (10 mg) in water
(1 mL) was added diisopropylethylamine (15 .mu.L, 2 eq). To this
was added lissamine chloride (Rhodamine B sulfonyl chloride,
Molecular Probes) 77 mg (3 eq) in 5 mL of methanol. The solution
was stirred for 1 hour and then chromatographed by reverse-phase
HPLC using an Aquasil C-18 column using a gradient from 100% 0.1%
trifluoroacetic acid in water to 100% 0.1% triflouroacetic acid in
acetonitrile. The fraction containing the product was determined by
mass spectroscopy. The molecular weight of compound is 1234, which
was detected in positive ion mode. The concentration of the
product-containing fraction was determined by the absorbance of the
solution at 588 nm (.epsilon.=88,000 M.sup.-1 cm.sup.-1).
Example 4
Attachment of Lissamine to Tat-Cys by an Unactivated Disulfide
[0142] To a solution of Tat-Cys (100 .mu.g) in 100 .mu.L water was
added dilissamine cystamine (41 .mu.g, 1 eq). The pH of the
solution was adjusted to 7-8 by the addition of potassium
carbonate.
Example 5
Synthesis of Activated (Labile) Disulfide-containing Lissamine
Adduct (Lissamine 4-aminophenyl Disulfide)
[0143] To a solution of 10 mg of lissamine chloride (Rhodamine B
sulfonyl chloride, Molecular Probes) in 0.2 mL dimethylformamide
was added over five minutes ten 10 .mu.L aliquots of 4-aminophenyl
disulfide (2 mg, 0.5 eq) and diisopropylethylamine (3 .mu.L, 1 eq).
Two hours after final addition of disulfide the solution was
diluted into 2 mL of acetonitrile and chromatographed by
reverse-phase HPLC using an Aquasil C-18 column applying a gradient
from 20% acetonitrile and 80% water containing 0.1% trifluoroacetic
acid to 100% 0.1% triflouroacetic acid in acetonitrile. We were
unable to isolate the lissamine dimer, but were able to isolate the
product of monoaddition. The fraction containing the monoaddition
product was determined by mass spectroscopy. The molecular weight
of compound is 789, which was detected in positive ion mode. The
concentration of the product-containing fraction was determined by
the absorbance of the solution at 588 nm (.epsilon.=88,000 M.sup.-1
cm.sup.-1).
Example 6
Attachment of Lissamine to Tat-Cys by an Activated Disulfide
[0144] To a solution of Tat-Cys (100 .mu.g) in 100 .mu.L water was
added Lissamine 4-aminophenyl disulfide (26 .mu.g, 1 eq). The pH of
the solution was adjusted to 7-8 by the addition of potassium
carbonate.
Example 7
Synthesis of Activated (Labile) Disulfide Fluorescein Dimer
(Difluorescein 4-aminophenyl Disulfide)
[0145] To a solution of 20 mg of fluorescein isothiocyanate in 0.5
mL dimethylformamide was added 4-aminophenyl disulfide (4 mg, 0.33
eq) in 100 .mu.L dimethylformamide and diisopropylethylamine (3
.mu.L, 0.33 eq). After two hours, the solution was diluted into 2
mL of water that was brought to pH 8 with potassium carbonate. This
aqueous solution was filtered and chromatographed by reverse-phase
HPLC using an Aquasil C-18 column applying a gradient from 100%
water containing 0.1% trifluoroacetic acid to 100% 0.1%
triflouroacetic acid in acetonitrile. The fraction containing the
product was determined by mass spectroscopy. The molecular weight
of compound is 1025, which was detected in negative ion mode. The
concentration of the product-containing fraction was determined by
the absorbance of the solution at 494 nm (.epsilon.=75,000 M.sup.-1
cm.sup.-1).
Example 8
Measurement of the Reduction of Unactivated (Non-labile) Disulfide
(Dilissamine Cystamine).
[0146] To a solution containing 0.44 .mu.M dilissamine cystamine
and 100 mM sodium phosphate pH 7.5 was added glutathione to a
concentration of 250 .mu.M. The solution was irradiated with 555 nm
light and the fluorescence of the solution was measured at 585 nm.
The amount of time required to reach half maximum fluorescence was
2000-2400 sec.
Example 9
Measurement of the Reduction of Activated (Labile) Disulfide
(Difluorescein 4-aminophenyl Disulfide).
[0147] To a solution containing 0.44 .mu.M fluorescein
4-aminophenyl disulfide and 100 mM sodium phosphate pH 7.5 was
added glutathione to a concentration of 250 .mu.M. The solution was
irradiated with 495 nm light and the fluorescence of the solution
was measured at 520 nm. The amount of time required to reach half
maximum fluorescence was 30-50 sec.
Example 10
Analysis of Delivery to Cells by TAT Peptide.
[0148] Grow HeLa cells on glass coverslips by incubating at
4.degree. C. in Delbecco's Modified Eagle's Media (DMEM)
supplemented with 50 .mu.g TAT peptide-fluorophore chimera (pulse).
At this temperature, endocytosis is believed to be completely
inhibited. Incubate the cells for two hours at 4.degree. C. and
then wash with DMEM to remove external TAT-fluorophore. Remove the
media and then either process cells for fluorescence microscopy or
incubate three more hours at 4.degree. C. with DMEM with media
changes every hour (chase). The cells that are chased are then
processed for fluorescence microscopy. Cells processed for
fluorescence microscopy are washed 3.times. in phosphate-buffered
saline (PBS), fixed in PBS+4% formaldehyde for 20 min, washed
3.times. in PBS, and coverslips are mounted on slides.
[0149] The presence of fluorophore was detected by confocal
microscopy (Zeiss LSM 510). In the case of irreversible covalent
thioether linkage between TAT and fluorophore, fluorescence was
detected inside of the cell after the initial two hour incubation.
Subsequent incubation of the cells with fluorophore-free media
(chase) resulted in cells with no internalized fluorophore.
Similarly, TAT-fluorophore adducts linked through an unactivated
disulfide cystamine bond also had initial internalization that
disappeared upon incubation with chase solutions. For the activated
disulfide 4-aminophenyl disulfide, fluorescence was detected inside
of the cell after the initial two hour incubation. In contrast to
the other attachments between flourophore and TAT, a chase of the
fluorophore with fluorophore-free media did not show a reduction in
the amount of internalized fluorophore.
Example 11
Synthesis of 5,5'-Dithiobis(2-nitrobenzoate)propionitrile
[0150] 5,5'-dithiobis(2-nitrobenzoic acid) (500 mg, 1.26 mmol,
Aldrich Chemical Company) was taken up in 4.0 mL dioxane.
Dicylohexylcarbodiimide (540 mg, 2.6 mmol, Aldrich Chemical
Company) and 3-hydroxypropionitrile (240 .mu.L, 188 mg, 2.60 mmol,
Aldrich Chemical Company) were added. The reaction mixture was
stirred overnight at room temperature. The precipitate was removed
by centrifugation, and the solvent concentrated under reduced
pressure. The residue was washed with saturated sodium bicarbonate,
water, and brine; and dried over magnesium sulfate. Solvent removal
(aspirator) yielded 696 mg yellow/orange foam. The residue was
purified using normal phase HPLC (Alltech econosil, 250.times.22
nm), flow rate=9.0 mL/min, mobile phase=1% ethanol in chloroform,
retention time=13 min. Removal of solvent (aspirator) afforded 233
mg (36.8%) of 5,5'-dithiobis(2-nitrobenzoate)propionitrile as a
yellow oil. TLC (silica: 5% methanol in chloroform; Rf=0.51).
H.sup.1NMR .differential. 8.05 (d, 4 H), 7.75 (m, 4H), 4.55 (t,
4H), 2.85 (t, 4H).
Example 12
Synthesis of Dimethyl
5,5'-dithiobis(2-nitrobenzoate)propionimidate-2 HCl
[0151] 5,5'-Dithiobis(2-nitrobenzoate)propionitrile (113 mg, 0.226
mmol) was taken up in 500. .mu.L anhydrous chloroform. Anhydrous
methanol (20.0 .mu.L, 0.494 mmol, Aldrich Chemical Company) was
added. The resulting solution was cooled to 0.degree. C. on an ice
bath, and HCl gas was bubbled through the solution for a period of
10 minutes. The resulting solution was placed in a -20.degree. C.
freezer for a period of 48 hours. During this time a yellow oil
formed. The oil was washed thoroughly with chloroform and dried
under vacuum to afford 137 mg (95.8%) of dimethyl
5,5'-dithiobis(2-nitrobenzoate)propionimidate-2 HCl as a yellow
foam.
Example 13
Polymerization of N-(2-Aminoethyl)-1,3-propanediamine and Dimethyl
5,5'-dithiobis(2-nitrobenzoate)propionimidate-2 HCl on a DNA
Template.
[0152] Procedure:
[0153] Template polymerization was carried out in 25 mM HEPES
buffer, pH 8.0. N-(2-Aminoethyl)-1,3-propanediamine (48 .mu.g, 0.3
mM, Aldrich Chemical Company) was added to a 0.5 mL solution of
pCIluc DNA (25 mg, 0.075 mM in phosphate, 2.6 .mu.g/.mu.L pCIluc;
prepared according to Danko, I., Williams, P., Herweijer, H. et al.
Hum. Mol. Genetics (1997) in press). Dimethyl
5,5'-dithiobis(2-nitrobenzoate)propionimidate-2 HCl (500 .mu.g,
0.78 mM) was added, and the solution was vortexed. The reaction was
incubated at room temperature for one hour. A fine yellow
precipitate was observed to form during the incubation period. The
reaction was centrifuged to remove the precipitate. A portion of
the reaction (10 .mu.L) was reduced with 10 mM dithiothreitol (10
.mu.L) to break the disulfide bonds forming the polymer. Portions
(0.5 .mu.g) of the intact polymer and the reduced polymer were
analyzed on a 1% agarose gel.
Example 14
Formation of DNA/Poly-L-Lysine/Dimethyl
5,5'-Dithiobis(2-nitrobenzoate) propionimidate -2 HCl Complexes
[0154] pDNA/Poly-L-lysine hydrobromide complexes were prepared by
combining plasmid DNA (25 .mu.g) with Poly-L-lysine hydrobromide
(95 .mu.g, MW 35 kDa, Aldrich Chemical Company) in 0.5 mL 25 mM
Hepes buffer pH 8.0, and the solution was vortexed to mix. The
resulting solution was divided into 3 portions. One portion was
incubated at room temperature for 2 hrs. To the second portion was
added dimethyl 5,5'-dithiobis(2-nitrobenzoate)propionimidate-2 HCl
(472 mg, 1.5 mmol), the solution was mixed, and incubated at room
temperature for 2 hrs. To the third sample was added dimethyl
3,3'-dithiobispropionimidate (1.1 mg, 1.5 mmol), the solution was
mixed, and incubated at room temperature for 2 hrs. After 2 hrs.
the samples were then centrifuged at 12000 rpm for five
minutes.
[0155] Ninety degree light scattering measurements were performed
(Shimadzo RF-1501 Fluorescence Spectrophotometer). The wavelength
setting was 700 nm for both the incident beam and detection of
scattering light. The slits for both beams were fixed at 10 nm. The
particle size of the resulting complex was determined by light
scattering (Brookhaven ZetaPlus Particle Sizer). After determining
the initial intensity of scattered light, 15 .mu.L 5 M NaCl
solution was added to the complexes while the intensity of
scattered light was monitored.
[0156] The addition of salt to the non-caged particles led to an
immediate increase in the turbidity of the solution indicating
aggregation. The non-caged sample also became visibly cloudy. The
addition of salt to the particles caged using dimethyl
3,3'-dithiobispropionimidate led to an increase in turbidity of
approximately 33%. The addition of salt to the dimethyl
5,5'-dithiobis(2-nitrobenzoate)propionimidate-2 HCl caged complexes
lead to no visible rise in turbidity. The particle size of the
dimethyl 5,5'-dithiobis(2-nitrobenzoate) propionimidate-2 HCl caged
particles was determined (Brookhaven Zeta Plus Particle Sizer) in
150 mM NaCl (physiological concentration). The mean particle
diameter was found to be 89.7 nm, 67% of the total number of
particles were under 100 nm in size.
[0157] The example indicates that dimethyl
5,5'-dithiobis(2-nitrobenzoate)- propionimidate-2 HCl caged DNA.
The particles formed are stable in physiological salt, and are
under 100 nm in size.
Example 15
Demonstration of Reducibility of Disulfide Bond in vitro.
[0158] pDNA (pCI Luc)/polyethyleneimine (25 kDa, Aldrich Chemical
Company)/dimethyl 3,3'-dithiobispropionimidate and
pDNA/polyethyleneimine/dimethyl
5,5'-dithiobis(2-nitrobenzoate)propionimi- date-2 HCl complexes
were prepared in 25 mM HEPES buffer pH 8.0. All complexes were
prepared at pDNA/polyethyleneimine ratios of 1/3. Dimethyl
3,3'-dithiobispropionimidate and dimethyl
5,5'-dithiobis(2-nitrobenzoate) propionimidate-2 HCl were added at
the following ratios: 0, 3, 6, 12, and 25. Complexes were incubated
0.5 hour at room temperature, and centrifuged 5 minutes at 12,000
rpm prior to transfection. Transfections were carried out in 35 mm
wells. At the time of transfection, HepG2 monolayers, at
approximately 50% confluency, were washed once with PBS (phosphate
buffered saline), and subsequently stored in serum-free media
(Opti-MEM, Gibco BRL). The complexes were diluted in Opti-MEM and
added by drops, 5.0 .mu.g DNA/well, to the cells. After a 4 hour
incubation period at 37.degree. C., the media containing the
complexes was aspirated from the cells, and replaced with complete
growth media, DMEM with 10% fetal bovine serum (Sigma). After an
additional incubation of 42 hours, the cells were harvested and the
lysate was assayed for luciferase expression (Wolff, J. A., Malone,
R. W., Williams, P., Chong, W., Acsadi, G., Jani, A. and Feigner,
P. L. Direct gene transfer into mouse muscle in vivo. Science,
1465-1468,1990.). A Lumat LB 9507 (EG&G Berthold, Bad-Wildbad,
Germany) luminometer was used.
[0159] pDNA/polyethyleneimine/methyl 3,3'-dithiobispropionimidate
and pDNA/polyethyleneimine/dimethyl
5,5'-dithiobis(2-nitrobenzoate)propionimi- date-2 HCl particles
were transfected into Hep G2 cells. pDNA/polyethyleneimine
complexes were also transfected as a control. The cell lysates were
then analyzed for the expression of luciferin. The results show
that while the dimethyl 3,3'-dithiobispropionimidate complexes gave
expression results below baseline (<200 RLU), the dimethyl
5,5'-dithiobis(2-nitrobenzoate)propionimidate-2
HCl/pDNA/polyethyleneimine complexes gave levels of expression that
were as high as 120,000 RLU.
[0160] The physiologically labile disulfide bonds present in the
dimethyl 5,5'-dithiobis(2-nitrobenzoate)propionimidate-2 HCl
complexes can be reduced by cultured cells, while the disulfide
bonds present in the dimethyl 3,3'-dithiobispropionimidate
complexes cannot.
Example 16
Synthesis of 5,5'-dithiobis[(3"-bromopropyl)-2-nitrobenzoate]
[0161] 5,5'-dithiobis-(2-nitrobenzoic acid) (500 mg, 1.26 mmol,
Aldrich Chemical Company) and 3-bromopropanol (368 mg, 2.65 mmol,
Aldrich Chemical Company) were taken up in 7.0 mL THF.
Dicyclohexylcarbodiimide (545 mg, 2.65 mmol, Aldrich Chemical
Company) was added, and the reaction mixture was stirred overnight
at ambient temperature. The precipitate was removed by filtration,
and the solution was concentrated under reduced pressure to afford
430 mg (54%) of 5,5'-dithiobis[(3"-bromopropyl)-2-nitr- obenzoate]
as a yellow oil.
Example 17
Synthesis of
5,5'-dithiobis[(3"-ammonio-{N,N-dimethyl-N-propionitrile}prop- yl
bromide)2-nitrobenzoate]
[0162] 5,5'-dithiobis[(3"-bromopropyl)-2-nitrobenzoate] was taken
up in 2.0 mL THF, and 3-dimethylaminopropionitrile (193 mg, 1.96
mmol, Aldrich Chemical Company) was added. After 3 days at ambient
temperature, the salt was precipitated from solution with
Et.sub.2O, and purified by reverse phase HPLC (C-18 Aquasil
200.times.20 mm) using a gradient from 20 to 80% methanol over 20
minutes (elution at 15 minutes). The solvent was removed under
reduced pressure to afford 15.2 mg (3%)
5,5'-dithiobis[(3"-ammonio-{N,N-dimethyl-N-propionitrile}propyl
bromide)2-nitrobenzoate]. H.sup.1-NMR (CD.sub.3OD) .differential.
8.4-8.6 (m, 6H), 5.0 (t, 4 H), 4.35 (t, 4H), 4.1 (m, 4H), 2.85 (m,
4H), 3.75 (m, 16H). Synthesis of Dimethyl
5,5'-dithiobis[(3"-ammonio-(N,N-dimethyl-N-pr- opioimidate)propyl
chloride) 2-nitrobenzoate]-hydrochloride
Example 18
Synthesis of
5,5'-dithiobis[(3"-ammonio-(N,N-dimethyl-N-propioimidate)prop- yl
chloride) 2-nitrobenzoate]
[0163]
5,5'-dithiobis[(3"-ammonio-{N,N-dimethyl-N-propionitrile}propyl
bromide)2-nitrobenzoate] (15.2 mg, 0.018 mmol) was taken up in 1 mL
of methanol. The solution was saturated with HCl at 0.degree. C.
The resulting solution was held at -20.degree. C. for 1 week.
Et.sub.2O was added and the precipitate collected by filtration to
afford 8.3 mg (47%) of dimethyl
5,5'-dithiobis[(3"-ammonio-(N,N-dimethyl-N-propioimidate)prop- yl
chloride) 2-nitrobenzoate]-hydrochloride.
Example 19
Synthesis of N,N'-Bis(t-BOC)-L-cystine
[0164] To a solution of L-cystine (1 gm, 4.2 mmol, Aldrich Chemical
Company) in acetone (10 mL) and water (10 mL) was added
2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile (2.5 gm, 10
mmol, Aldrich Chemical Company) and triethylamine (1.4 mL, 10 mmol,
Aldrich Chemical Company). The reaction was allowed to stir
overnight at room temperature. The water and acetone was then by
rotary evaporation resulting in a yellow solid. The diBOC compound
was then isolated by flash chromatography on silica gel eluting
with ethyl acetate 0.1% acetic acid.
Example 20
Synthesis of L-cystine-1,4-bis(3-aminopropyl)piperazine
Copolymer
[0165] To a solution of N,N'-Bis(t-BOC)-L-cystine (85 mg, 0.15
mmol) in ethyl acetate (20 mL) was added
N,N'-dicyclohexylcarbodiimide (108 mg, 0.5 mmol) and
N-hydroxysuccinimide (60 mg, 0.5 mmol). After 2 hr, the solution
was filtered through a cotton plug and 1,4-bis(3-aminopropyl)pip-
erazine (54 .mu.L, 0.25 mmol) was added. The reaction was allowed
to stir at room temperature for 16 h. The ethyl acetate was then
removed by rotary evaporation and the resulting solid was dissolved
in trifluoroacetic acid (9.5 mL), water (0.5 mL) and
triisopropylsilane (0.5 mL). After 2 h, the trifluoroacetic acid
was removed by rotary evaporation and the aqueous solution was
dialyzed in a 15,000 MW cutoff tubing against water (2.times.2 l)
for 24 h. The solution was then removed from dialysis tubing,
filtered through 5 .mu.M nylon syringe filter and then dried by
lyophilization to yield 30 mg of polymer.
Example 21
Synthesis of Guanidino-L-cystine
[0166] To a solution of cystine (1 gm, 4.2 mmol) in ammonium
hydroxide (10 mL) in a screw-capped vial was added O-methylisourea
hydrogen sulfate (1.8 gm, 10 mmol). The vial was sealed and heated
to 60.degree. C. for 16 h. The solution was then cooled and the
ammonium hydroxide was removed by rotary evaporation. The solid was
then dissolved in water (20 mL), filtered through a cotton plug.
The product was then isolated by ion exchange chromatography using
Bio-Rex 70 resin and eluting with hydrochloric acid (100 mM).
Example 22
Synthesis of Guanidino-L-cystine-L-1,4-bis(3-aminopropyl)piperazine
Copolymer
[0167] To a solution of guanidino-L-cystine (64 mg, 0.2 mmol) in
water (10 mL) was slowly added N,N'-dicyclohexylcarbodiimide (82
mg, 0.4 mmol) and N-hyroxysuccinimide (46 mg, 0.4 mmol) in dioxane
(5 mL). After 16 hr, the solution was filtered through a cotton
plug and 1,4-bis(3-aminopropyl)pip- erazine (40 .mu.L, 0.2 mmol)
was added. The reaction was allowed to stir at room temperature for
16 h and then the aqueous solution was dialyzed in a 15,000 MW
cutoff tubing against water (2.times.2 l) for 24 h. The solution
was then removed from dialysis tubing, filtered through 5 .mu.M
nylon syringe filter and then dried by lyophilization to yield 5 mg
of polymer.
Example 23
The Particle Size of
pDNA-L-cystine-1,4-bis(3-aminopropyl)piperazine Copolymer and
DNA-guanidino-L-cystine1,4-bis(3-aminopropyl)piperazine Copolymer
Complexes.
[0168] To a solution of pDNA (10 .mu.g/mL) in 0.5 mL 25 mM HEPES
buffer pH 7.5 was added 10 .mu.g/mL
L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer or
guanidino-L-cystine1,4-bis(3-aminopropyl)piperazine copolymer. The
size of the complexes between DNA and the polymers were measured.
For both polymers, the size of the particles were approximately 60
nm.
Example 24
Condensation of DNA with L-cystine-1,4-bis(3-aminopropyl)piperazine
Copolymer and Decondensation of DNA upon Addition of
Glutathione
[0169] Fluorescein labeled DNA was used for the determination of
DNA condensation in complexes with
L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer. pDNA was
modified to a level of 1 fluorescein per 100 bases using Mirus'
LabelI.TM. Fluorescein kit. The fluorescence was determined using a
fluorescence spectrophotometer (Shimadzu RF-1501
spectrofluorometer) at an excitation wavelength of 495 nm and an
emission wavelength of 530 nm. (Trubetskoy, V. S., Slattum, P. M.,
Hagstrom, J. E., Wolff, J. A., Budker, V. G., "Quantitative
Assessment of DNA Condensation," Anal. Biochem (1999) incorporated
by reference).
[0170] The intensity of the fluorescence of the fluorescein-labeled
DNA (10 .mu.g/mL) in 0.5 mL of 25 mM HEPES buffer pH 7.5 was 300
units. Upon addition of 10 .mu.g/mL of
L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer, the intensity
decreased to 100 units. To this DNA-polycation sample was added 1
mM glutathione and the intensity of the fluorescence was measured.
An increase in intensity was measured to the level observed for the
DNA sample alone. The half life of this increase in fluorescence
was 8 minutes.
[0171] The experiment indicates that DNA complexes with
physiologically-labile disulfide-containing polymers are cleavable
in the presence of the biological reductant glutathione.
Example 25
Mouse Tail Vein Injection of
DNA-L-cystine-1,4-bis(3-aminopropyl)piperazin- e Copolymer and
DNA-guanidino-L-cystinel,4-bis(3-aminopropyl)piperazine Copolymer
Complexes
[0172] Plasmid delivery in the tail vein of ICR mice was performed
as described. To PCILuc DNA (50 .mu.g) in 2.5 mL H.sub.2O was added
either L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer,
guanidino-L-cystine1,4-bis(3-aminopropyl)piperazine copolymer, or
poly-L-lysine (34,000 MW, Sigma Chemical Company) (50 .mu.g). The
samples were then injected into the tail vein of mice using a 30
gauge, 0.5 inch needle. One day after injection, the animal was
sacrificed, and a luciferase assay was conducted.
1 Polycation ng/liver poly-L-lysine 6.2
L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer 439
guanidino-L-cystine1,4-bis(3-aminopropyl)piperazine copolymer
487
[0173] The experiment indicates that DNA complexes with the
physiologically-labile disulfide-containing polymers are capable of
being broken, thereby allowing the luciferase gene to be
expressed.
Example 26
Rat Intramuscle injection of
DNA-L-cystine-1,4-bis(3-aminopropyl)piperazin- e copolymer and
DNA-guanidino-L-cystine1,4-bis(3-aminopropyl)piperazine copolymer
complexes.
[0174] Plasmid delivery intro rat leg was performed as described
(Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi, G.,
Jani, A. and Felgner, P. L. Direct gene transfer into mouse muscle
in vivo. Science, 1465-1468,1990.). To pCILuc DNA (100 .mu.g/mL,
2.5 mL) was added L-cystine-1,4-bis(3-aminopropyl)piperazine
copolymer or guanidino-L-cystine1,4-bis(3-aminopropyl)piperazine
copolymer (100 .mu.g/mL) and then injected into the leg muscles of
a rat. After 7 days, the animal was sacrificed and a luciferase
assay was conducted.
2 amount luciferase DNA complex (ng) per leg no polycation 3.3
L-cystine-1,4-bis(3-aminopropyl)piperazi- ne copolymer 4.5
guanidino-L-cystine1,4-bis(3-aminopropyl)piperazin- e 6.5
copolymer
[0175] The experiment indicates that DNA complexes with the
physiologically-labile disulfide-containing polymers are capable of
being broken, thereby allowing the luciferase gene to be
expressed.
Example 27
Injection of DNA-L-cystine-1,4-bis(3-aminopropyl)piperazine
Copolymer Complex and pDNA (pCI Luc)/5,5'-Dithiobis(2-nitrobenzoic
acid)-1,4-Bis(3-aminopropyl)piperazine Copolymer Complex and pDNA
(pCI Luc)/5,5'-dithiobis(2-nitrobenzoic
Acid)-1,4-bis(3-aminopropyl)piperazine- --Folate Copolymer
Complexes into the Intestinal Lumen of Mice.
[0176] Intestinal cells were transfected by injecting pDNA
solutions into the mesenteric vasculature. A 3-cm section of the
small intestines was clamped, blocking both vascular inflow and
outflow. A volume of 250 .mu.l containing 50 .mu.g pCILuc and 50
.mu.g poly(ethylenimine) (Aldrich Chemical Co. MW 25,000 MW),
L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer, pDNA (pCI
Luc)/5,5'-dithiobis(2-nitrobenzoic
acid)-1,4-bis(3-aminopropyl)piperazine copolymer, and pDNA (pCI
Luc)/5,5'-dithiobis(2-nitrobenzoic
acid)-1,4-bis(3-aminopropyl)piperazine- -folate copolymer complexes
were injected into the intestinal lumen of mice. After 3 minutes,
the clamps were removed. One day after DNA delivery, the mice were
sacrificed, the injected section of the intestines was excised, cut
in 3 cm sections and assayed for luciferase expression. Different
areas of the intestines were targeted (duodenum, jejunum,
ileum).
3 Amount luciferase (pg) Complex Duodenum jejunum ileum
DNA-poly(ethylenimine) 0.5 3.0 1.7 DNA-L-cystine-1,4-bis 6.2 3.7
2.8 (3-aminopropyl)piperazine copolymer pDNA (pCI
Luc)/5,5'-dithiobis(2-nitro- 42 20 226 benzoic
acid)-1,4-bis(3-amino- propyl)piperazine copolymer pDNA (pCI
Luc)/5,5'-dithiobis(2-nitro- 36 1.9 51 benzoic
acid)-1,4-bis(3-amino- propyl)piperazine-folate copolymer
[0177] The experiment indicates that DNA complexes with labile
disulfide-containing polymers are capable of being broken, thereby
allowing the luciferase gene to be expressed.
Example 28
Synthesis of 5,5'-Dithiobis[succinimidyl(2-nitrobenzoate)]
[0178] 5,5'-dithiobis(2-nitrobenzoic acid) (50.0 mg, 0.126 mmol,
Aldrich Chemical Company) and N-hyroxysuccinimide (29.0 mg, 0.252
mmol, Aldrich Chemical Company) were taken up in 1.0 mL
dichloromethane. Dicylohexylcarbodiimide (52.0 mg, 0.252 mmol) was
added and the reaction mixture was stirred overnight at room
temperature. After 16 hr, the reaction mixture was partitioned in
EtOAc/H.sub.2O. The organic layer was washed 2.times.H.sub.2O,
1.times.brine, dried (MgSO.sub.4) and concentrated under reduced
pressure. The residue was taken up in CH.sub.2Cl.sub.2, filtered,
and purified by flash column chromatography on silica gel
(130.times.30 mm, EtOAc:CH.sub.2Cl.sub.2 1:9 eluent) to afford 42
mg (56%) 5,5'-dithiobis[succinimidyl(2-nitrobenzoate)] as a white
solid. H.sup.1NMR (DMSO) .differential. 7.81-7.77 (d, 2H),
7.57-7.26 (m, 4H), 3.69 (s, 8 H).
Example 29
Synthesis of 5,5'-Dithiobis(2-nitrobenzoic
Acid)-1,4-Bis(3-aminopropyl)pip- erazine Copolymer
[0179] 1,4-Bis(3-aminopropyl)piperazine (10 .mu.L, 0.050 mmol,
Aldrich Chemical Company) was taken up in 1.0 mL methanol and HCl
(2 mL, 1 M in Et.sub.2O, Aldrich Chemical Company) was added.
Et.sub.2O was added and the resulting HCl salt was collected by
filtration. The salt was taken up in 1 mL DMF and
5,5'-dithiobis[succinimidyl(2-nitrobenzoate)] (30 mg, 0.050 mmol)
was added. The resulting solution was heated to 80.degree. C. and
diisopropylethylamine (35 .mu.L, 0.20 mmol, Aldrich Chemical
Company) was added by drops. After 16 hr, the solution was cooled,
diluted with 3 mL H.sub.2O, and dialyzed in 12,000-14,000 MW cutoff
tubing against water (2.times.2 L) for 24 h. The solution was then
removed from dialysis tubing and dried by lyophilization to yield
23 mg (82%) of 5,5'-dithiobis(2-nitrobenzoic
acid)-1,4-bis(3-aminopropyl)piperazine copolymer.
Example 30
Particle Sizing of pDNA (pCI Luc)/5,5'-Dithiobis(2-nitrobenzoic
acid)-1,4-Bis(3-aminopropyl)piperazine Copolymer Complexes
[0180] To 50 .mu.g pDNA in 3 mL Ringers (0.85% sodium chloride,
0.03% potassium chloride, 0.03% calcium chloride) was added 170
.mu.g 5,5'-Dithiobis(2-nitrobenzoic
acid)-1,4-Bis(3-aminopropyl)piperazine Copolymer. Particle sizing
(Brookhaven Instruments Coporation, ZetaPlus Particle Sizer, 190,
532 nm) indicated an effective diameter of 92 nm for the complex. A
50 .mu.g pDNA in 3 mL Ringers sample indicated no particle
formation.
[0181] 5,5'-Dithiobis(2-nitrobenzoic
acid)-1,4-Bis(3-arninopropyl)piperazi- ne Copolymer condenses pDNA,
forming small particles.
Example 31
Mouse Tail Vein Injections of pDNA (pCI
Luc)/5,5'-Dithiobis(2-nitrobenzoic
Acid)-1,4-Bis(3-aminopropyl)piperazine Copolymer Complexes
[0182] Four complexes were prepared as follows:
[0183] Complex I: pDNA (pCI Luc, 200 .mu.g) in 1 mL H.sub.2O and
diluted with 9 mL Ringers prior to injection.
[0184] Complex II: pDNA (pCI Luc, 200 .mu.g) was mixed with
poly-L-lysine (378 .mu.g, MW 3400, Sigma Chemical Company) in 1 mL
H.sub.2O and diluted with 9 mL Ringers prior to injection.
[0185] Complex III: pDNA (pCI Luc, 200 .mu.g) was mixed with
5,5'-Dithiobis(2-nitrobenzoic
acid)-1,4-Bis(3-aminopropyl)piperazine Copolymer (400 .mu.g) in 1
mL H.sub.2O and diluted with 9 mL Ringers prior to injection.
[0186] Complex IV: pDNA (pCI Luc, 200 .mu.g) was mixed with Histone
H1 (1.2 mg, Sigma Chemical Company) in 1 mL H.sub.2O and diluted
with 9 mL Ringers prior to injection.
[0187] 2.5 mL and 250 .mu.L tail vein injections of the complex
were performed (Zhang, G., Budker, V., Wolff, J, High Levels of
Foreign Gene Expression in Hepatocytes from Tail Vein Injections of
Naked Plasmid DNA. Human Gene Therapy, July, 1999, incorporated by
reference). Results reported are for liver expression. Luciferase
expression was determined as previously reported (Wolff, J. A.,
Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A. and
Felgner, P.L. Direct gene transfer into mouse muscle in vivo.
Science, 1465-1468,1990.). A Lumat LB 9507 (EG&G Berthold,
Bad-Wildbad, Germany) luminometer was used.
[0188] Results from 2.5 mL Injections
[0189] Complex I: 1,976,000
[0190] Complex II: 128,000
[0191] Complex III: 5,025,000
[0192] Complex IV: 1,960
[0193] Results from 250 .mu.L Injections
[0194] Complex I: 985
[0195] Complex III: 1,140
[0196] Results indicate an increased level of luciferase expression
in pDNA/5,5'-Dithiobis(2-nitrobenzoic
acid)-1,4-Bis(3-aminopropyl)piperazine Copolymer complexes over pCI
Luc DNA itself, pCI Luc DNA/poly-L-lysine complexes, and pCI Luc
DNA/Histone H1 complexes. These results also indicate that the pDNA
is being released from the pDNA/5,5'-Dithiobis(2-n- itrobenzoic
acid)-1,4-Bis(3-aminopropyl)piperazine Copolymer complexes, and is
accessible for transcription.
[0197] 250 .mu.L injection results were similar for both
pDNA/5,5'-Dithiobis(2-nitrobenzoic acid)
1,4-Bis(3-aminopropyl)piperazine Copolymer complexes and pCI Luc
DNA.
Example 32
Synthesis of 5,5'-Dithiobis(2-nitrobenzoic
acid)-1,4-Bis(3-aminopropyl)pip- erazine-Tris(2-aminoethyl)amine
Copolymer
[0198] 1,4-Bis(3-aminopropyl)piperazine (2.4 .mu.L, 0.012 mmol,
Aldrich Chemical Company) and tris(2-aminoethyl)amine (0.51 .mu.L,
0.0034 mmol, Aldrich Chemical Company) were taken up in 0.5 mL
methanol and HCl (1 mL, 1 M in Et.sub.2O, Aldrich Chemical Company)
was added Et.sub.2O was added and the resulting HCl salt was
collected by filtration. 5,5'-dithiobis[succinimidyl
(2-nitrobenzoate)] (10 mg, 0.016 mmol) was added and the mixture
was taken up in 0.4 mL DMSO and 0.4 mL THF . The resulting solution
was stirred at room temperature and diisopropylethylamine (5.9
.mu.L, 0.042 mmol, Aldrich Chemical Company) was added by drops.
After 16 hr, the solution was diluted with 3 mL H.sub.2O, and
dialyzed in 12,000-14,000 MW cutoff tubing against water (2.times.2
L) for 48 h. The solution was then removed from dialysis tubing and
dried by lyophilization to yield 2.7 mg (30%) of
5,5'-dithiobis(2-nitrobenzoic
acid)-1,4-bis(3-aminopropyl)piperazine-tris- (2-aminoethyl)amine
copolymer.
Example 33
Synthesis of 5,5'-Dithiobis(2-nitrobenzoic
acid)-Tetraethylenepentamine Copolymer
[0199] Tetraethylenepentamine (3.2 .mu.L, 0.017 mmol, Aldrich
Chemical Company) was taken up in 1.0 mL dichloromethane and HCl (1
mL, 1 M in Et.sub.2O, Aldrich Chemical Company) was added Et.sub.2O
was added and the resulting HCl salt was collected by filtration.
The salt was taken up in 1 mL DMF and 5,5'-dithiobis[succinimidyl
(2-nitrobenzoate)] (10 mg, 0.017 mmol) was added. The resulting
solution was heated to 80.degree. C. and diisopropylethylamine (15
.mu.L, 0.085 mmol, Aldrich Chemical Company) was added by drops.
After 16 hr, the solution was cooled, diluted with 3 mL H.sub.2O,
and dialyzed in 12,000-14,000 MW cutoff tubing against water
(2.times.2 L) for 24 h. The solution was then removed from dialysis
tubing and dried by lyophilization to yield 5.8 mg (62%) of
5,5'-dithiobis(2-nitrobenzoic acid)-tetraethylenepentamine
copolymer.
Example 34
Mouse Tail Vein Injections of pDNA (pCI
Luc)/5,5'-Dithiobis(2-nitrobenzoic Acid)-Tetraethylenepentamine
Copolymer Complexes
[0200] Complexes were prepared as follows:
[0201] Complex I: pDNA (pCI Luc, 200 .mu.g) was added to 300.mu.L
DMSO then 2.5 mL Ringers was added.
[0202] Complex II: pDNA (pCI Luc, 200 .mu.g) was added to 300.mu.L
DMSO then 5,5'-Dithiobis(2-nitrobenzoic
acid)-Tetraethylenepentamine Copolymer (336 .mu.g) was added
followed by 2.5 mL Ringers.
[0203] 2.5 mL tail vain injections of the complex were performed as
previously described. Results reported are for liver expression,
and are the average of two mice. Luciferase expression was
determined as previously reported (Wolff, J. A., Malone, R. W.,
Williams, P., Chong, W., Acsadi, G., Jani, A. and Felgner, P. L.
Direct gene transfer into mouse muscle in vivo. Science, 1465-1468,
1990.). A Lumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany)
luminometer was used.
[0204] 250 .mu.L Injections
[0205] Complex I: 25,200,000
[0206] Complex II: 21,000,000
[0207] Results indicate that pDNA (pCI
Luc)/5,5'-Dithiobis(2-nitrobenzoic acid)-tetraethylenepentamine
copolymer complexes are nearly equivalent to pCI Luc DNA itself in
2.5 mL injections. This indicates that the pDNA is being released
from the complex and is accessible for transcription.
Example 35
Synthesis of 5,5'-Dithiobis(2-nitrobenzoic
Acid)-Tetraethylenepentamine-Tr- is(2-aminoethyl)amine
Copolymer
[0208] Tetraethylenepentamine (2.3 .mu.L, 0.012 mmol, Aldrich
Chemical Company) and tris(2-aminoethyl)amine (0.51 .mu.L, 0.0034
mmol, Aldrich Chemical Company) were taken up in 0.5 mL methanol
and HCl (1 mL, 1 M in Et.sub.2O, Aldrich Chemical Company) was
added. Et.sub.2O was added and the resulting HCl salt was collected
by filtration. The salt was taken up in 1 mL DMF and
5,5'-dithiobis[succinimidyl (2-nitrobenzoate)] (10 mg, 0.017 mmol)
was added. The resulting solution was heated to 80.degree. C. and
diisopropylethylamine (15 .mu.L, 0.085 mmol, Aldrich Chemical
Company) was added by drops. After 16 hr, the solution was cooled,
diluted with 3 mL H.sub.2O, and dialyzed in 12,000-14,000 MW cutoff
tubing against water (2.times.2 L) for 24 h. The solution was then
removed from dialysis tubing and dried by lyophilization to yield
6.9 mg (77%) of 5,5'-dithiobis(2-nitrobenzoic
acid)-tetraethylenepentamine-tris(- 2-aminoethyl)amine
copolymer.
Example 36
Mouse Tail Vein Injections of pDNA (pCI
Luc)/5,5'-Dithiobis(2-nitrobenzoic
Acid)-Tetraethylenepentamine-Tris(2-aminoethyl)amine Copolymer
Complexes
[0209] Complexes were prepared as follows:
[0210] Complex I: pDNA (pCI Luc, 200 .mu.g) was added to 300 .mu.L
DMSO then 2.5 mL Ringers was added.
[0211] Complex II: pDNA (pCI Luc, 200 .mu.g) was added to 300 .mu.L
DMSO then 5,5'-Dithiobis(2-nitrobenzoic
acid)-Tetraethylenepentamine-Tris(2-am- inoethyl)amine Copolymer
(324 .mu.g) was added followed by 2.5 mL Ringers.
[0212] 2.5 mL tail vain injections of the complex were preformed as
previously described. Results reported are for liver expression,
and are the average of two mice. Luciferase expression was
determined as previously reported (Wolff, J. A., Malone, R. W.,
Williams, P., Chong, W., Acsadi, G., Jani, A. and Felgner, P. L.
Direct gene transfer into mouse muscle in vivo. Science,
1465-1468,1990.). A Lumat LB 9507 (EG&G Berthold, Bad-Wildbad,
Germany) luminometer was used.
[0213] 250 .mu.L Injections
[0214] Complex 1: 25,200,000
[0215] Complex II: 37,200,000
[0216] pDNA (pCI Luc)/5,5'-Dithiobis(2-nitrobenzoic
acid)-tetraethylenepentamine-Tris(2-aminoethyl)amine Copolymer
Complexes are more effective than pCI Luc DNA in 2.5 mL injections.
Indicating that the pDNA is released from the complex and is
accessible for transcription.
Example 37
Synthesis of 5,5'-Dithiobis(2-nitrobenzoic
Acid)-N,N'-Bis(2-aminoethyl)-1,- 3-propanediamine Copolymer
[0217] N,N'-Bis(2-aminoethyl)-1,3-propanediamine (2.8 .mu.L, 0.017
mmol, Aldrich Chemical Company) was taken up in 1.0 mL
dichloromethane and HCl (1 mL, 1 M in Et.sub.2O, Aldrich Chemical
Company) was added. Et.sub.2O was added and the resulting HCl salt
was collected by filtration. The salt was taken up in 1 mL DMF and
5,5'-dithiobis[succinimidyl(2-nitrobenz- oate)] (10 mg, 0.017 mmol)
was added. The resulting solution was heated to 80.degree. C. and
diisopropylethylamine (12 .mu.L, 0.068 mmol, Aldrich Chemical
Company) was added by drops. After 16 hr, the solution was cooled,
diluted with 3 mL H.sub.2O, and dialyzed in 12,000-14,000 MW cutoff
tubing against water (2.times.2 L) for 24 hr. The solution was then
removed from dialysis tubing and dried by lyophilization to yield
5.9 mg (66%) of 5,5'-dithiobis(2-nitrobenzoic
acid)-N,N'-bis(2-aminoethyl- )-1,3-propanediamine Copolymer.
Example 38
Mouse Tail Vein Injections of pDNA (pCI
Luc)/5,5'-Dithiobis(2-nitrobenzoic
Acid)-N,N'-Bis(2-aminoethyl)-1,3-propanediamine Copolymer
Complexes
[0218] Complexes were prepared as follows:
[0219] Complex I: pDNA (pCI Luc, 200 .mu.g) was added to 300 .mu.L
DMSO then 2.5 mL Ringers was added.
[0220] Complex II: pDNA (pCI Luc, 200 .mu.g) was added to 300 .mu.L
DMSO then 5,5'-Dithiobis(2-nitrobenzoic
acid)-N,N'-Bis(2-aminoethyl)-1,3-propa- nediamine Copolymer (474
.mu.g) was added followed by 2.5 mL Ringers.
[0221] Tail vain injections of 2.5 mL of the complex were preformed
as previously described. Results reported are for liver expression,
and are the average of two mice. Luciferase expression was
determined as previously reported.
[0222] Results: 2.5 mL Injections
[0223] Complex 1: 25,200,000
[0224] Complex II: 341,000
[0225] pDNA (pCI Luc)/5,5'-Dithiobis(2-nitrobenzoic
acid)-tetraethylenepentamine Copolymer Complexes provides
luciferase expression indicating that the pDNA is being released
from the complex and is accessible for transcription.
Example 39
Synthesis of 5,5'-Dithiobis(2-nitrobenzoic
acid)-N,N'-Bis(2-aminoethyl)-1,-
3-propanediamine-Tris(2-aminoethyl)amine Copolymer
[0226] N,N'-Bis(2-aminoethyl)-1,3-propanediamine (2.0 .mu.L, 0.012
mmol, Aldrich Chemical Company) and tris(2-aminoethyl)amine (0.51
.mu..mu.L, 0.0034 mmol, Aldrich Chemical Company) were taken up in
0.5 mL methanol and HCl (1 mL, 1 M in Et.sub.2O, Aldrich Chemical
Company) was added. Et.sub.2O was added and the resulting HCl salt
was collected by filtration. The salt was taken up in 1 mL DMF and
5,5'-dithiobis[succinim- idyl(2-nitrobenzoate)] (10 mg, 0.017 mmol)
was added. The resulting solution was heated to 80.degree. C. and
diisopropylethylamine (12 .mu.L, 0.068 mmol, Aldrich Chemical
Company) was added by drops. After 16 hr, the solution was cooled,
diluted with 3 mL H.sub.2O, and dialyzed in 12,000-14,000 MW cutoff
tubing against water (2.times.2 L) for 24 hr. The solution was then
removed from dialysis tubing and dried by lyophilization to yield
6.0 mg (70%) of 5,5'-dithiobis(2-nitrobenzoic
acid)-N,N'-bis(2-aminoethyl)-1,3-propanediamine-tris(2-aminoethyl)amine
copolymer.
Example 40
Mouse Tail Vein Injections of pDNA (pCI
Luc)/5,5'-Dithiobis(2-nitrobenzoic
acid)-N,N'-Bis(2-aminoethyl)-1,3-propanediamine-Tris(2-
aminoethyl)amine Copolymer Complexes
[0227] Complexes were prepared as follows:
[0228] Complex I: pDNA (pCI Luc, 200 .mu.g) was added to 300 .mu.L
DMSO then 2.5 mL Ringers was added.
[0229] Complex II: pDNA (pCI Luc, 200 .mu.g) was added to 300 .mu.L
DMSO then 5,5'-Dithiobis(2-nitrobenzoic
acid)-N,N'-Bis(2-aminoethyl)-1,3-propa-
nediamine-Tris(2-aminoethyl)amine Copolymer (474 .mu.g) was added
followed by 2.5 mL Ringers.
[0230] Tail vain injections of 2.5 mL of the complex were preformed
as previously described. Results reported are for liver expression,
and are the average of two mice. Luciferase expression was
determined as previously reported.
[0231] Results: 2.5 mL Injections
[0232] Complex I: 25,200,000
[0233] Complex II: 1,440,000
[0234] Results indicate that pDNA (pCI
Luc)/5,5'-Dithiobis(2-nitrobenzoic
acid)-N,N'-Bis(2-aminoethyl)-1,3-propanediamine-Tris(2-aminoethyl)amine
Copolymer Complexes are less effective than pCI Luc DNA in 2.5 mL
injections. Although the complex was less effective, the luciferase
expression indicates that the pDNA is being released from the
complex and is accessible for transcription.
Example 41
Intramuscular Injections of Complexes from pDNA (pCI
Luc)/Physiologically Labile Disulfide Bond Containing Polymers on
Mouse.
[0235] Seven complexes were prepared as follows:
[0236] Complex I: pDNA (pCI Luc, 40 .mu.g) was added to 586 .mu.L
glucose (290 mM)-HEPES (5 mM, pH 8).
[0237] Complex II: pDNA (pCI Luc, 40 .mu.g) was added to 577 .mu.L
glucose (290 mM)-HEPES (5 mM, pH 8). To this solution was added
5,5'-Dithiobis(2-nitrobenzoic
acid)-1,4-Bis(3-aminopropyl)piperazine Copolymer (9 .mu.L, 200
.mu.g).
[0238] Complex III: pDNA (pCI Luc, 40 .mu.g) was added to 573 .mu.L
glucose (290 mM)-HEPES (5 mM, pH 8). To this solution was added
5,5'-Dithiobis(2-nitrobenzoic
acid)-1,4-Bis(3-aminopropyl)piperazine Copolymer (13 .mu.L, 200
.mu.g).
[0239] Complex IV: pDNA (pCI Luc, 40 .mu.g) was added to 574 pL
glucose (290 mM)-HEPES (5 mM, pH 8). To this solution was added
5,5'-Dithiobis(2-nitrobenzoic acid)-Tetraethylenepentamine
Copolymer (12 .mu.L, 70 .mu.g).
[0240] Complex V: pDNA (pCI Luc, 40 .mu.g) was added to 576 .mu.L
glucose (290 mM)-HEPES (5 mM, pH 8). To this solution was added
5,5'-Dithiobis(2-nitrobenzoic
acid)-Tetraethylenepentamine-Tris(2-aminoet- hyl)amine Copolymer
(10 .mu.L, 65 .mu.g).
[0241] Complex VI: pDNA (pCI Luc, 40 .mu.g) was added to 581 .mu.L
glucose (290 mM)-HEPES (5 mM, pH 8). To this solution was added
5,5'-Dithiobis(2-nitrobenzoic
acid)-N,N'-Bis(2-aminoethyl)-1,3-propanedia- mine Copolymer (5
.mu.L, 94 .mu.g).
[0242] Complex VII: pDNA (pCI Luc, 40 .mu.g) was added to 570 .mu.L
glucose (290 mM)-HEPES (5 mM, pH 8). To this solution was added
5,5'-Dithiobis(2-nitrobenzoic
acid)-N,N'-Bis(2-aminoethyl)-1,3-propanedia-
mine-Tris(2-aminoethyl)amine Copolymer (16 .mu.L, 94 .mu.g).
[0243] Direct muscle injections of 150 .mu.L of the complex were
preformed as previously described (See Budker, V., Zhang, G.,
Danko, I., Williams, P., and Wolff, J., "The Efficient Expression
Of Intravascularly Delivered DNA In Rat Muscle," Gene Therapy 5,
272-6(1998); Wolff, J. A., Malone, R. W., Williams, P., Chong, W.,
Acsadi, G., Jani, A. and Felgner, P. L. Direct gene transfer into
mouse muscle in vivo. Science, 1465-1468, 1990. which are
incorporated herein by reference.). Seven days post injection, the
animals were sacrificed, and the muscle harvested. Samples were
homogenized in lux buffer (I mL), and centrifuged for 15 minutes at
4000 RPM. Luciferase expression was determined as previously
reported. Results reported for left quadracep: right quadracep
(Complex IV-only injected into left quadracep).
[0244] Results:
[0245] Complex I: RLU 1,900: 4,316
[0246] Complex II: RLU 13,433: 20,640
[0247] Complex III: RLU 10,156 : 39,491
[0248] Complex IV: RLU 9,888:
[0249] Complex V: RLU=19,565: 5,806
[0250] Complex VI: RLU =270: 427
[0251] Complex VII: RLU =973: 6,000
[0252] The complexes prepared from pCI Luc DNA/Physiologically
Labile Disulfide Bond Containing Polymers are effective in direct
muscle injections. The luciferase expression indicates that the
pDNA is being released from the complex and is accessible for
transcription. Complexes prepared with
5,5'-Dithiobis(2-nitrobenzoic acid)-1,4-Bis(3-aminopropyl)p-
iperazine Copolymer were the most effective, giving luciferase
expression levels 2 to 10 times as high as pDNA.
Example 42
Synthesis of 5,5'-Dithiobis(2-nitrobenzoic
Acid)-Pentaethylenehexamine Copolymer
[0253] Pentaethylenehexamine (4.2 .mu.L, 0.017 mmol, Aldrich
Chemical Company) was taken up in 1.0 mL dichloromethane and HCl (1
mL, 1 M in Et.sub.2O, Aldrich Chemical Company) was added Et.sub.2O
was added and the resulting HCl salt was collected by filtration.
The salt was taken up in 1 mL DMF and
5,5'-dithiobis[succinimidyl(2-nitrobenzoate)] (10 mg, 0.017 mmol)
was added. The resulting solution was heated to 80.degree. C. and
diisopropylethylamine (12 .mu.L, 0.068 mmol, Aldrich Chemical
Company) was added by drops. After 16 hr, the solution was cooled,
diluted with 3 mL H.sub.2O, and dialyzed in 12,000-14,000 MW cutoff
tubing against water (2.times.2 L) for 24 hr. The solution was then
removed from dialysis tubing and dried by lyophilization to yield
5.9 mg (58%) of 5,5'-dithiobis(2-nitrobenzoic
acid)-pentaethylenehexamine Copolymer.
Example 43
Synthesis of 5,5'-Dithiobis(2-nitrobenzoic
Acid)-Pentaethylenehexamine-Tri- s(2-aminoethyl)amine Copolymer
[0254] Pentaethylenehexamine (2.9 .mu.L, 0.012 mmol, Aldrich
Chemical Company) and tris(2-aminoethyl)amine (0.51 .mu.L, 0.0034
mmol, Aldrich Chemical Company) were taken up in 0.5 mL methanol
and HCl (1 mL, 1 M in Et.sub.2O, Aldrich Chemical Company) was
added. Et.sub.2O was added and the resulting HCl salt was collected
by filtration. The salt was taken up in 1 mL DMF and
5,5'-dithiobis[succinimidyl(2-nitrobenzoate)] (10 mg, 0.017mmol)
was added. The resulting solution was heated to 80.degree. C. and
disopropylethylamine (12 .mu.L, 0.068 mmol, Aldrich Chemical
Company) was added by drops. After 16 hr, the solution was cooled,
diluted with 3 mL H.sub.2O, and dialyzed in 12,000-14,000 MW cutoff
tubing against water (2.times.2 L) for 24 h. The solution was then
removed from dialysis tubing and dried by lyophilization to yield
6.0 mg (64%) of 5,5'-dithiobis(2-nitrobenzoic
acid)-pentaethylenehexamine-tris(2-aminoeth- yl)amine
copolymer.
Example 44
Synthesis of 5,5'-Dithiobis(2-nitrobenzoic
Acid)-N-(3-Aminopropyl)-1,3-pro- panediamine Copolymer
[0255] 5,5'-Dithiobis[succinimidyl(2-nitrobenzoate)] (2.5 mg,
0.0042 mmol) was taken up in 10 .mu.L of DMF.
N-(3-aminopropyl)-1,3-propanediamine (0.6 .mu.L, 0.004 mmol,
Aldrich Chemical Company) was added with 10 .mu.L HEPES 250 mM, pH
7.5. After 1 hr the solution was concentrated under reduced
pressure. The resulting residue was dissolved in 0.42 mL DMSO.
Analysis of the solution on SDS-PAGE versus poly-L-lysisne
hydrobromide (MW of 1000, 7500, 15000) indicated an approximate
molecular weight range of 3500-8000 for the polymer.
Example 45
Synthesis of 5,5'-dithiobis(2-nitrobenzoic
Acid)-1,4-bis(3-aminopropyl)pip- erazine-Folate Copolymer
[0256] Folate-PEG(3400 MW)-NH2 was prepared according to the known
procedure (Lee, R. J., Low, P. S. Biochimica et Biophysica Acta
1233, 1995, 134-144). Folate-PEG-NH2 was acylated with succinylated
N-(3-(BOC)aminopropyl)-1,3-propaneamine(BOC)amine. Removal of the
BOC protecting groups afforded the Folate monomer.
1,4-bis(3-aminopropyl)pipe- razine (5.0 .mu.L, 0.023 mmol, Aldrich
Chemical Company) and folate monomer (5.0 mg, 0.0012 mmol) were
taken up in 0.4 mL methanol and HCl (1 mL, 1 M in Et.sub.2O,
Aldrich Chemical Company) was added. The resulting suspension was
concentrated under reduced pressure to afford a white solid. The
salt was taken up in 0.5 mL DMF and 5,5'-dithiobis[succinimidy-
l(2-nitrobenzoate)] (14 mg, 0.025 mmol) was added. The resulting
solution was heated to 80.degree. C. and diisopropylethylamine (18
.mu.L, 0.10 mmol, Aldrich Chemical Company) was added by drops.
After 16 hr, the solution was cooled, diluted with 3 mL H.sub.2O,
and dialyzed in 12,000-14,000 MW cutoff tubing against water
(2.times.2 L) for 24 h. The solution was then removed from dialysis
tubing and dried by lyophilization to yield 13 mg (68%) of
5,5'-dithiobis(2-nitrobenzoic acid)-1,4-bis(3-aminopropyl)
piperazine-folate copolymer.
Example 46
Synthesis of 5,5'-Dithiobis(2-nitrobenzoic acid)-Poly-Glutamicacid
(8mer) Copolymer
[0257] H.sub.2N-EEEEEEEE-NHCH.sub.2CH.sub.2NH.sub.2 (5.0 mg, 0.0052
mmol, Genosis) was taken up in 0.1 mL HEPES (250 mM, pH 7.5).
5,5'-dithiobis[succinimidyl(2-nitrobenzoate)] (3.1 mg, 0.0052) was
added with 0.2 mL DMSO and the mixture was stirred overnight at
room temperature. After 16 hr the solution was heated to 70.degree.
C. for 10 min, cooled to room temperature and diluted to 1.10 mL
with DMSO.
Example 47
Complex Formation with 5,5'-Dithiobis(2-nitrobenzoic
acid)-PolyGlutamicacid (8mer) Copolymer
[0258] Fluorescein labeled DNA was used for the determination of
DNA condensation in complexes with 5,5'-Dithiobis(2-nitrobenzoic
acid)-Poly-Glutamicacid (8mer) Copolymer. pDNA was modified to a
level of 1 fluorescein per 20 bases using Mirus' LabellT.TM.
Fluorescein kit. The fluorescence was determined using a
fluorescence spectrophotometer (Shimadzo RF-1501 Fluorescence
Spectrophotometer), at an excitation wavelength of 497 nm, and an
emission wavelength of 520 nm.
[0259] To fluorescein labeled DNA (10 .mu.g) in 1 mL HEPES (25 mM,
pH 7.5) was added polyornithine (18 .mu.g, Sigma Chemical Company).
The mixtures were held at room temperature for 5 minutes and the
fluorescence was determined. (see: Trubetskoy, V. S., Slattum, P.
M., Hagstrom, J. E., Wolff, J. A., Budker, V. G., "Quantitative
Assessment of DNA Condensation," Anal. Biochem (1999) incorporated
by reference) Since fluorescence intensity is decreased by DNA
condensation, results indicate that polyornithine compacts DNA. To
the resulting complex was added 5,5'-Dithiobis(2-nitrobenzoic
acid)-Poly-Glutamicacid (8mer) Copolymer (60 .mu.g), and the
fluorescence was again determined. The fluorescence of the sample
decreased further.
[0260] Upon the addition of 5,5'-Dithiobis(2-nitrobenzoic
acid)-Poly-Glutamicacid (8mer) Copolymer to the sample, the
fluorescence decreased, indicating the formation a triple complex.
No competition of the 5,5'-Dithiobis(2-nitrobenzoic
acid)-Poly-Glutamicacid (8mer) Copolymer for the polyornithine was
observed (increase in fluorescence).
Example 48
Transfection of 3T3 Cells with 5,5'-Dithiobis(2-nitrobenzoic
acid)-Poly-Glutamicacid (8mer) Copolymer
[0261] Three complexes were formed:
[0262] Complex I) To 300 .mu.L Opti-MEM was added LT-1TM (12 .mu.g,
Mirus Corporation) followed by pDNA (pCI Luc, 4 .mu.g).
[0263] Complex II) To 300 .mu.L Opti-MEM was added LT-1.TM. (12
.mu.g, Mirus Corporation) followed by pDNA (pCI Luc, 4 .mu.g), and
5,5'-Dithiobis(2-nitrobenzoic acid)-Poly-Glutamicacid (8mer)
Copolymer (4 .mu.g).
[0264] Complex III) To 300 .mu.L Opti-MEM was added LT-1.TM. (12
.mu.g, Mirus Corporation) followed by pDNA (pCI Luc, 4 .mu.g), and
Poly-Glutamicacid (4 .mu.g, MW 49000, Sigma Chemical Company).
[0265] Transfections were carried out in 35 mm wells. At the time
of transfection, 3T3 cells, at approximately 50% confluency, were
washed once with PBS (phosphate buffered saline), and subsequently
stored in serum-free media (2.0 mL, Opti-MEM, Gibco BRL). 150 .mu.L
of complex was added to each well. After a 3.25 h incubation period
at 37.degree. C., the media containing the complexes was aspirated
from the cells, and replaced with complete growth media, DMEM with
10% fetal bovine serum (Sigma). After an additional incubation of
48 hours, the cells were harvested and the lysate was assayed for
luciferase expression as previously reported (Wolff, J. A., Malone,
R. W., Williams, P., Chong, W., Acsadi, G., Jani, A. and Felgner,
P. L. Direct gene transfer into mouse muscle in vivo. Science,
1465-1468,1990.). A Lumat LB 9507 (EG&G Berthold, Bad-Wildbad,
Germany) luminometer was used.
[0266] Results:
[0267] Complex I: RLU =17,000,000
[0268] Complex II: RLU =14,000,000
[0269] Complex III: RLU =26,000,000
[0270] The addition of Poly-Glutamicacid (4 .mu.g, MW 49000, Sigma
Chemical Company) in the transfection experiment improved the pDNA
expression. The addition of 5,5'-Dithiobis(2-nitrobenzoic
acid)-Poly-Glutamicacid (8mer) Copolymer (4 .mu.g) while not
improving the pDNA expression was not detrimental to the
expression.
Example 49
Demonstration of Reduction of by 5,5'-dithiobis(2-nitrobenzoic
acid)-Containing Copolymers by Glutathione
[0271] To a solution of 5,5'-Dithiobis(2-nitrobenzoic
acid)-1,4-Bis(3-aminopropyl)piperazine Copolymer (100 .mu.g) in 0.5
mL HEPES (25 mM, pH 8) was added glutathione (final concentration
of 2 mM). The absorbance of the sample was measured at .lambda. 412
(The cleaved disulfide has an absorbance maximum at .lambda. 412.
See Hermanson, G. T. Bioconjugate Techniques, Academic Press, New
York, New York, 1996, pp 88) versus time (Beckman DU-7
Spectrophotometer).
[0272] To a solution of 5,5'-dithiobis(2-nitrobenzoic
acid)-tetraethylenepentamine copolymer (50 .mu.g) in 0.5 mL HEPES
(25 mM, pH 8) was added glutathione (final concentration of 2 mM).
The absorbance of the sample was measured at .lambda. 412 (The
cleaved disulfide has an absorbance maximum at .lambda. 412. See
Hermanson, G. T. Bioconjugate Techniques, Academic Press, New York,
New York, 1996, pp 88) versus time (Beckman DU-7
Spectrophotometer).
[0273] To a solution of 5,5'-Dithiobis(2-nitrobenzoic
acid)-Poly-Glutamicacid (8mer) Copolymer (50 .mu.g) in 0.5 mL HEPES
(25 mM, pH 8) was added glutathione (final concentration of 2 mM).
The absorbance of the sample was measured at .lambda. 412 (The
cleaved disulfide has an absorbance maximum at .lambda. 412. See
Hermanson, G. T. Bioconjugate Techniques, Academic Press, New York,
New York, 1996, pp 88) versus time (Beckman DU-7
Spectrophotometer).
[0274] Each sample showed a rapid increase in the absorbance at
.lambda. 412 upon the addition of glutathione, indicating cleavage
of the disulfide bond. Half life values were estimated as:
[0275] 5,5'-Dithiobis(2-nitrobenzoic
acid)-1,4-Bis(3-aminopropyl)piperazin- e Copolymer t.sub.1/2=42
sec.
[0276] 5,5'-dithiobis(2-nitrobenzoic acid)-tetraethylenepentamine
copolymer t.sub.1/2=75 sec.
[0277] 5,5'-Dithiobis(2-nitrobenzoic acid)-Poly-Glutamicacid (8mer)
Copolymer t.sub.1/2=24 sec.
[0278] The experiment demonstrates rapid cleavage of the disulfide
bond of 5,5'-dithiobis(2-nitrobenzoic acid)-containing copolymers
with the physiological reducing agent glutathione.
Example 50
Analysis of Delivery to Cells by VP22 Peptide:
[0279] Grow HeLa cells on glass coverslips by incubating at
4.degree. C. in Delbecco's Modified Eagle's Media (DMEM)
supplemented with 50 .mu.g VP22 peptide-fluorophore chimera
(pulse). At this temperature, endocytosis is believed to be
completely inhibited. Incubate the cells for two hours at 4.degree.
C. and then wash with DMEM to remove external VP22-fluorophore.
Remove the media and then either process cells for fluorescence
microscopy or incubate three more hours at 4.degree. C. with DMEM
with media changes every hour (chase). The cells that are chased
are then processed for fluorescence microscopy. Cells processed for
fluorescence microscopy are washed 3.times. in phosphate-buffered
saline (PBS), fixed in PBS+4% formaldehyde for 20 min, washed
3.times. in PBS, and coverslips are mounted on slides. The presence
of fluorophore is detected by confocal microscopy (Zeiss LSM
510).
Example 51
Analysis of Delivery to Cells by ANTP Peptide:
[0280] Grow HeLa cells on glass coverslips by incubating at
4.degree. C. in Delbecco's Modified Eagle's Media (DMEM)
supplemented with 50 .mu.g ANTP peptide-fluorophore chimera
(pulse). At this temperature, endocytosis is believed to be
completely inhibited. Incubate the cells for two hours at 4.degree.
C. and then wash with DMEM to remove external ANTP-fluorophore.
Remove the media and then either process cells for fluorescence
microscopy or incubate three more hours at 4.degree. C. with DMEM
with media changes every hour (chase). The cells that are chased
are then processed for fluorescence microscopy. Cells processed for
fluorescence microscopy are washed 3.times. in phosphate-buffered
saline (PBS), fixed in PBS+4% formaldehyde for 20 min, washed
3.times. in PBS, and coverslips are mounted on slides. The presence
of fluorophore is detected by confocal microscopy (Zeiss LSM
510).
[0281] The foregoing is considered as illustrative only of the
principles of the invention. Furthermore, since numerous
modifications and changes will readily occur to those skilled in
the art, it is not desired to limit the invention to the exact
construction and operation shown and described. Therefore, all
suitable modifications and equivalents fall within the scope of the
invention.
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