U.S. patent application number 10/444662 was filed with the patent office on 2003-11-27 for reversible modification of membrane interaction.
Invention is credited to Ekena, Kirk, Hagstrom, James E., Rozema, David B., Wakefield, Darren, Wolff, Jon A..
Application Number | 20030220264 10/444662 |
Document ID | / |
Family ID | 46282375 |
Filed Date | 2003-11-27 |
United States Patent
Application |
20030220264 |
Kind Code |
A1 |
Rozema, David B. ; et
al. |
November 27, 2003 |
Reversible modification of membrane interaction
Abstract
An process for the reversible modification of membrane
interaction of a compound is described. Modification of membrane
interaction can be used to facilitate delivery of molecules to
cells in vitro and in vivo. The described modifiers, which are used
to reversibly inactivate the membrane active compounds, can also be
utilized as cross-linkers or to reverse the charge of a
molecule.
Inventors: |
Rozema, David B.; (Madison,
WI) ; Wakefield, Darren; (Madison, WI) ;
Wolff, Jon A.; (Madison, WI) ; Ekena, Kirk;
(Madison, WI) ; Hagstrom, James E.; (Middleton,
WI) |
Correspondence
Address: |
Mark K. Johnson
Mirus Corporation
505 S. Rosa Rd.
Madison
WI
53719
US
|
Family ID: |
46282375 |
Appl. No.: |
10/444662 |
Filed: |
May 23, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60383298 |
May 24, 2002 |
|
|
|
Current U.S.
Class: |
530/333 ;
530/350; 530/406 |
Current CPC
Class: |
A61K 38/00 20130101;
A61K 48/00 20130101; C07H 21/04 20130101; A61K 47/60 20170801; C12N
15/87 20130101; C07K 7/06 20130101; A61K 48/0041 20130101; C07H
21/00 20130101; A61K 48/0008 20130101 |
Class at
Publication: |
514/12 ; 530/350;
530/406 |
International
Class: |
A61K 038/16; C07K
014/16 |
Claims
We claim:
1. A process for reversibly modifying an amine-containing compound
to alter interaction of the compound with membrane lipids
comprising: covalently attaching a substituted maleic anhydride to
the amine on the compound.
2. The process of claim 1 wherein the substituted maleic anhydride
consists of a disubstituted maleic anhydride.
3. The process of claim 2 wherein the disubstituted maleic
anhydride consists of a negatively charged disubstituted maleic
anhydride.
4. The process of claim 3 wherein the disubstituted maleic
anhydride consists of 2-propionic-3-methylmaleic anhydride.
5. The process of claim 1 wherein the substituted maleic anhydride
consists of a bifuntional maleic anhydride.
6. The process of claim 5 wherein the substituted maleic anhydride
consists of a thioester substituted maleic anhydride.
7. The process of claim 6 wherein the substituted maleic anhydride
consists of CDM-thioester.
8. The process of claim 1 wherein the substituted maleic anhydride
consists of a PEG substituted maleic anhydride.
9. The process of claim 1 wherein the compound consists of a
membrane active compound.
10. The process of claim 9 wherein modification of the membrane
active compound inactivates the membrane activity of the
compound.
11. The process of claim 9 wherein the membrane active compound
consists of melittin.
12. The process of claim 1 wherein the compound consist of a cell
penetrating compound.
13. The process of claim 11 wherein the cell permeable compound
consists of an arginine-rich peptide.
14. The process of claim 11 wherein the cell penetrating compound
consists of a TAT peptide.
15. The process of claim 1 wherein the compound consists of a
membrane permeable drug.
16. The process of claim 1 wherein modification of the compound
consists of reducing positive charge on the compound.
17. The process of claim 1 wherein modification of the compound
consists of reversing the charge of the compound.
18. The process of claim 1 wherein modification reduces toxicity of
the compound.
19. A process for delivery of a molecule to cell comprising:
reversibly modifying an amine-containing membrane active compound
by covalently attaching a substituted maleic anhydride to the amine
on the compound to alter interaction of the compound with membrane
lipids, associating the molecule with the modified compound and
delivering the compound to the cell.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims priority status of U.S. Provisional
Application Serial No. 60/383,298 filed May 24, 2002.
STATEMENT REGARDING SEQUENCE LISTING
BACKGROUND
[0002] A variety of methods and routes of administration have been
developed to deliver pharmaceuticals, small molecular drugs and
biologically active compounds such as peptides, hormones, proteins,
and enzymes, to their site of action. Parenteral routes of
administration include intravascular (intravenous, intraarterial),
intramuscular, intraparenchymal, intradermal, subdermal,
subcutaneous, intratumor, intraperitoneal, and intralymphatic
injections that use a needle or catheter. The blood circulatory
system provides systemic spread of the pharmaceutical. Polyethylene
glycol and other hydrophilic polymers have been used to provided
protection and to increase the circulatory time of the
pharmaceutical in the blood stream by preventing interaction with
blood components and preventing opsonization, phagocytosis and
uptake by the reticuloendothelial system. For example, the enzyme
adenosine deaminase has been covalently modified with polyethylene
glycol to increase the circulatory time and persistence of this
enzyme in the treatment of patients with adenosine deaminase
deficiency. The controlled release of pharmaceuticals after their
administration is under intensive development.
[0003] In the rational design of synthetic delivery vehicles for
biologically-active compounds and macromolecules, the problem of
providing for endosomal escape can be a critical barrier to
efficient delivery to cytoplasmic or nuclear sites of action.
Various methods have been employed in attempts overcome this
barrier including: liposomes, which hypothetically fuse with cell
endosomal membranes; viruses; which may either fuse with or rupture
endosomal membranes; and polymer-based or other non-viral systems;
which must destabilize or rupture endosomal membranes. Currently,
none of these systems affects efficient escape of co-endocytosed
material from internal membrane enclosed compartments.
Understanding of endosomal release and ability to synthetically
enhance it remain a largely unsolved process.
[0004] Both viral and synthetic processes for accomplishing
endosomal release often rely upon acidification of the endosome
and/or lysosome to trigger either membrane fusion or disruption.
For viruses, the reduced pH of the endocytic compartment triggers a
conformational change that induces endosomal escape. The pH
gradient between cytoplasm and endosome also causes monoamines such
as chloroquinine to concentrate within endosomes thus destroying
the pH gradient. The pH-sensitive amines present on polyamines such
as PEI may also play a role in endosomal release although it is
unclear what role protonation plays. For some liposome-based
systems, pH-sensitive groups have been incorporated into lipids
that enable the lipid to undergo phase transformations upon
protonation. Finally, peptides and synthetic polymers containing
protonatable groups have been modeled after viral sequences to
become more amphipathic and membrane active in acidic environments.
By limiting membrane activity to acidic vesicles, effects on the
plasma membrane, and thereby cellular toxicity, are theoretically
attenuated.
[0005] Unfortunately, the use of protonation to effect membrane
disruption is beset with a theoretical conundrum: Endosome
disruption destroys the pH-gradient (i.e. membrane integrity is
essential to the maintenance of a pH gradient). With loss of a pH
gradient, activity of the pH-dependent endosomalytic agent is
reversed. Thus, delivery is potentially limited by the endosomal
membrane resealing before macromolecules are able to diffuse out.
The use of linkages that are labile within an endosomal or
lysosomal milieu has previously been used in liposomes or for
coupling drugs with carriers [Blattler et al. 1985,]. Specifically,
citraconic anhydride has been used to reversibly modify the primary
amine of DOPE (dioleoylphosphatidylethanolamine) [Reddy and Low
2000]. However, the kinetics of reversal of this reagent are too
slow to be effective in cells. Furthermore, this compound was found
to be ineffective in inhibiting membrane activity of endosomalytic
agents to which it was attached. In order to address the problems
associated with currently available endosomalytic agents, we have
developed a process of rapid irreversible activation that relies
upon chemical bond cleavage to unmask a biological agent's
activity.
[0006] In addition to endocytosis-dependent delivery systems, cell
permeable compounds that do not rely on endocytosis for delivery of
compounds to the interior of a cell have been recently described.
Delivery by these carriers involves attachment of the compound to a
highly cationic, arginine-rich peptide [Lindgren et al. 2000]. One
such import peptide, GRKKRRQRRR (SEQ ID 1), is derived from the TAT
protein of the HIV virus. This peptide, when attached to a variety
of molecules facilitates their intracellular delivery both in vitro
and in vivo [Schwarze et al. 1999; Lee and Pardridge 2001]. Other
import molecules include peptides from VP22, a herpes simplex virus
protein, and the ANT protein, (RQIKIWFQNRRMKWKK, SEQ ID 2) derived
from the homeo-domain of the Drosophila transcription factor
antennapedia. These peptides contain no obvious homology other than
the high content of cationic residues, especially arginine. In
fact, peptides and peptide analogs composed solely of arginine
residues have been shown to have import properties [Futaki 2002].
For all of these peptides, import does not appear to occur via
endocytosis since import occurs readily at the endocytosis
impermissible temperature of 4.degree. C. However, delivery of
molecules by conjugation to these cationic import peptides is not
selective. This lack of specificity results in organ distribution
of TAT-modified streptavidin that is similar to the distribution of
unconjugated streptavidin [Lee and Pardridge 2001]. The compounds
and processes we have developed for use with membrane active
compounds also functions in controlling the activity of the cell
permeable compounds.
SUMMARY
[0007] In a preferred embodiment, we describe a process to modify
an amine-containing molecule with a substituted maleic anhydride or
derivative thereof wherein cleavage of the modification is
accelerated upon exposure to pH less than 7. Upon cleavage of the
modification, the amine on the molecule is regenerated. The
molecule may be modified alter interaction of the molecule with
cell membranes, to attach a functional group, to link the molecule
to another compound or delivery agent, to alter the activity of the
molecule, or to inactivate the molecule. A preferred substituted
maleic anhydride is a disubstituted maleic anhydride.
[0008] In a preferred embodiment, compounds are described that
enable modification of an amine-containing molecule to reversibly
alter membrane interaction of the molecule comprising: substituted
maleic anhydrides having the general structure shown in FIG. 1A
wherein the R to anhydride bonds can be carbon-hydrogen or
carbon-carbon bonds and either R.sub.1 or R.sub.2, but not both,
can be a hydrogen atom. A preferred substituted maleic anhydride is
a disubstituted maleic anhydride wherein neither R.sub.1 or R.sub.2
is a hydrogen atom. We show that disubstitution increases the
pH-lability of the covalent bond formed between the anhydride and
an amine. A preferred disubstituted maleic anhydride is carboxy
dimethylmaleic anhydride (CDM), or 2-propionic-3-methylmaleic
anhydride, wherein R.sub.1 or R.sub.2 is --CH.sub.3 and R.sub.2 or
R.sub.2 is --(CH.sub.2).sub.2COOH. The addition of the carboxyl
group increases the charge and water solubility of the anhydride
and enhances inactivation of membrane active and cell permeable
compounds. Other functional groups may be added to either R.sub.1,
R.sub.2, or both.
[0009] In a preferred embodiment, we describe reversible covalent
crosslinkers comprising bifunctional substituted maleic anhydrides
having the general structure shown in FIG. 1A wherein R.sub.1 or
R.sub.2 contains a thioester. The thioester group enables
attachment to terminal cysteines or other molecules containing a
thiol separated from an amine by two to three bonds. A preferred
bifunctional disubstituted maleic anhydride is carboxy
dimethylmaleic anhydride thioester (CDM-thioester) wherein R.sub.1
or R.sub.2 is --CH.sub.3 and R.sub.2 or R.sub.1 is
--(CH.sub.2).sub.2COSCH.sub.2COOH. The thioester-containing
disubstituted maleic anhydride can be use to covalently link an
amine containing molecule to a molecule containing an appropriate
thiol. Attachment to the thiol occurs through native chemical
ligation. The thioester itself is relatively stable when compared
to activated esters such as N-hydroxysuccinimidyl esters, but will
couple readily and selectively with N-terminal cysteine groups. The
reaction mixture may contain other amines and other thiols, but
only an appropriate thiol couples to form a stable amide with the
thioester.
[0010] In a preferred embodiment, we describe a process to
reversibly inactivate an amine-containing biologically active
molecule wherein the molecule is modified with a substituted maleic
anhydride. Efficiency of inactivation of the molecule is affected
by the groups at positions R.sub.1 and R.sub.2 of the maleic
anhydride (see FIG. 1A). Reversal of the modification regenerates
the amine on the biologically active molecule, and thus activity of
the molecule, and is accelerated by exposure to pH<7.
[0011] In a preferred embodiment, we describe a process for
delivering a molecule to the interior of a cell comprising:
reversibly inactivating a membrane active compound and associating
the modified membrane active compound and the molecule with the
cell wherein the compound and the molecule are endocytosed and the
activity of the membrane active compound is restored causing
disruption of the endosomal/lysosomal membrane. Reversibly
inactivating the membrane active compound consists of modifying the
compound by reaction with a substituted maleic anhydride. Exposure
to the acidic pH environment of an endosome or lysosomes causes
reactivation of the membrane active compound by cleavage of the
anhydride. A preferred substituted maleic anhydride is a
disubstituted maleic anhydride. A preferred maleic anhydride is CDM
or CDM-thioester. The additional carboxylic acid present on CDM
enhances the inactivation of certain membrane active compounds. The
cell may be in vitro or in vivo.
[0012] In a preferred embodiment, we describe a process for the
reversible inactivation of a cationic import molecule comprising:
modifying the molecule by reaction with a substituted maleic
anhydride. The reversibly inactivated import molecule can then be
associated with a cell and the activity of the import molecule is
restored. The reactivation of the import molecule by cleavage of
the anhydride modification is enhanced in an acidic environment
such as in an endosome/lysosomes or tumor region. The import
molecule may be further modified by attaching other functional
groups or biologically active compounds. The other groups or
compounds may be attached to the import compound or to the
anhydride. The modified cationic import molecule may be delivered
to a cell that is in vitro or in vivo. In a preferred embodiment,
we describe a process for linking or coupling two molecules via a
reversible covalent bond wherein one of the molecules contains an
amine. The linkage comprises a bifunctional substituted maleic
anhydride (FIG. 1A) wherein R.sub.1 or R.sub.2 contains a reactive
group that can form a covalent or noncovalent interaction with a
molecule. An example of a reactive group that forms a noncovalent
interaction is biotin, which forms a stable interaction with
streptavidin. An example of a reactive group that forms a covalent
interaction is a thioester. The bifunctional substituted maleic
anhydride may provide reversible attachment of a molecule to a
biologically active compound containing an amine. Upon exposure to
a reduced pH, the molecule is cleaved, regenerating the
biologically active compound or pharmaceutical. The molecule may
facilitate targeting or delivery of the compound, reversibly
inactivate the compound, or serve another purpose. A preferred
thioester-containing linker is CDM-thioester. To link two molecules
with CDM-thioester, the anhydride of CDM-thioester is reacted with
an anime and then the thioester is reacted with an appropriate
thiol on another molecule. Alternatively, the bifunctional
anhydride can be used to crosslink an amine and a thiol that are
both present on the same molecule.
[0013] In a preferred embodiment, we describe a process to
reversibly modify an amine by reacting the amine with a substituted
maleic anhydride. Incubation at reduced pH, such as in endosome or
lysosomes or regions of tumors, accelerates cleavage of the
substituted maleic anhydride and regeneration of the amine.
Modification of an amine can alter the chemical and physical
properties of an amine-containing molecule. A preferred substituted
maleic anhydride is a disubstituted maleic anhydride.
[0014] In a preferred embodiment, we describe a process for
targeting the activity of a biologically active compound to the
region near a tumor in vivo comprising: reversibly inactivating the
compound by modification with a disubstituted maleic anhydride and
injecting the modified compound into an animal. In the region of
the acidic tumor, the reversal of the modification is accelerated,
regenerating the active compound. If the compound contains an
amine, the compound itself may be inactivated. Alternatively, the
compound can be attached to a reversibly inactivated targeting
molecule, such as TAT-CDM-thioester. TAT is a cell permeable
peptide. In another example, association with non-tumor cells is
inhibited by modification with a disubstituted maleic anhydride
such as CDM-PEG. In a preferred embodiment, we describe a process
for reversibly altering the charge of an amine comprising:
modifying the amine by reacting the it with a substituted maleic
anhydride. The substituted maleic anhydride may contain an acidic
functional group such as, but not limited to, CDM, enabling the
reversible conversion of the positively charged amine to a
negatively charged carboxyl. The amine can be on a polyamine. In
this way, a polyamine can be partially or completely modified,
allowing a wide range of charge neutralization or reversal.
Regeneration of the amine by cleavage of the negatively charged
anhydride modification is accelerated by exposure to pH<7.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIGS. 1A-1B. Illustrations for the structures of (A) maleic
anhydride derivatives and (B) succinic anhydride derivatives.
[0016] FIG. 2. Illustration of the reversible covalent bond formed
between a maleic anhydride derivative and a primary amine.
[0017] FIG. 3. Illustration of the steps in the synthesis of
carboxy dimethylmaleic anhydride.
[0018] FIG. 4. Delivery of a Streptavidin to hepatocytes in vivo.
Streptavidin was linked to a heptocyte-targeting peptide and to the
TAT cell permeable peptide. The cell permeable peptide was either
unmodified (A) and reversible inactivated by modification with CDM
(B).
[0019] When the highly cationic TAT peptide was reversibly
inactivated by CDM modification, a broader more even distribution
of streptavidin was observed.
DETAILED DESCRIPTION
[0020] We describe maleic anhydride derivatives that are usable for
the reversible modification of amine-containing compounds. We
further describe methods to reversibly inactivate, crosslink, or
otherwise modify biologically active compounds. The described
molecules and methods offer several significant advances over other
methods of reversible modification. First, unlike many reversible
reagents, such as disulfide containing crosslinkers, cleavage of
the maleic anhydride derivatives regenerates the starting
unmodified amine. Second, under mildly acidic physiological
conditions, the described anhydrides are cleaved very rapidly; in
only a few minutes compared to the hours or even days required for
other labile bonds to be cleaved. Nevertheless, using our
processes, modification of amine-containing compounds is
straightforward and facile. Furthermore, reversal of the
modification in vivo or in vitro is essentially unidirectional.
This characteristic is especially useful when the modification is
used to reversibly inhibit a membrane active compound. Once the
modification is cleaved, the membrane active compound retains
activity even if the local pH does not stay acidic. Conversely,
many compounds which rely on protonation to become active and
disrupt membranes, such as cationic lipids, are potentially
inactivated if the pH rises. Anhydrides form covalent bonds with
primary amines as shown in FIG. 2 for maleic anhydride. For
succinic anhydrides (FIG. 1 B), the resultant covalent bond is
stable and not reversible under physiological conditions. For
maleic anhydrides (FIG. 1A), the resultant covalent bond can be
cleaved under physiological conditions. The lability of the amide
bond is directly affected by the degree of substitution at the
unsaturated carbon-carbon bond of maleic anhydride [Kirby and
Lancaster 1972; Nieto and Palacian 1983]. The parent anhydride,
maleic anhydride, has no substitution and forms the most stable
maleamic acid. Citraconic and cis-aconitic-derived maleamic acids
each have one substitution and are more pH-labile. Maleamic acids
derived from disubstituted dimethyl maleic anhydride are the most
pH-labile.
[0021] We describe maleic anhydride derivatives that add additional
functionality to the anhydride reactive group. As an example, a
disubstituted maleaic anhydride was made in which R.sub.1 or 2
contains a thioester reactive group, thus forming a bifunctional
molecule (CDM-thioester) which can be used as a coupling or
crosslinking reagent. In this molecule, the R.sub.1 or 2 carboxyl
group is pre-activated as a thioester. The thioester can react with
an N-terminal cysteine or other thiol linked to an amine by two to
three bonds to form an amide bond in a process termed native
chemical ligation. The stability of the thioester allows one to
react the anhydride of CDM-thioester with an amine on a compound to
form maleamic acid groups without reaction between amines and the
thioester. The thioester is then able to react with an N-terminal
cysteine group or other appropriate thiol on a second compound.
[0022] We have developed coupling techniques using maleic anhydride
derivatives to link membrane active compounds with biologically
active compounds. Coupling of a membrane active compound to a
biologically active compound is beneficial for delivery of certain
biologically active compounds to cells, especially to cells in
vivo. The process, in addition to coupling the membrane active
compound to another molecule, may also reversibly inhibit the
activity of the membrane active compound, thus reducing toxicity.
In a similar way, it is possible to attach both a biologically
active compound and a membrane active compound to a third molecule
such as a polymer. Both compounds are attached through the
acid-labile maleamic group of CDM-thioester. The thioesters can be
coupled to a cysteine containing polymer via native chemical
ligation. The same technique can be applied to attaching a
biologically active compound to a targeting group or other desired
compound.
[0023] Other functional groups can be envisioned that can be
attached to the anhydride at either position R.sub.1 or R.sub.2.
Any group that allows covalent or noncovalent interaction with
another molecule without inhibiting coupling of the anhydride to an
amine can be used.
[0024] Definitions
[0025] Functional group. Functional groups include cell targeting
signals, nuclear localization signals, compounds that enhance
release of contents from endosomes or other intracellular vesicles
(releasing signals), cell penetrating compounds, and other
compounds that alter the behavior or interactions of the compound
or complex to which they are attached. Functional groups may also
include reactive groups, molecules that provide noncovalent
interaction with another molecule or any other R group.
[0026] Cell targeting signals are any signals that enhance the
association of the biologically active compound with a cell. These
signals can modify a biologically active compound such as drug or
nucleic acid and can direct it to a cell location (such as tissue)
or location in a cell (such as the nucleus) either in culture or in
a whole organism. The signal may increase binding of the compound
to the cell surface and/or its association with an intracellular
compartment. By modifying the cellular or tissue location of the
foreign gene, the function of the biologically active compound can
be enhanced. The cell targeting signal can be, but is not limited
to, a protein, peptide, lipid, steroid, sugar, carbohydrate,
(non-expressing) polynucleic acid or synthetic compound. Cell
targeting signals such as ligands enhance cellular binding to
receptors. 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 include 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 thiol, 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.
[0027] After interaction of a compound or complex with the cell,
other targeting groups can be used to increase the delivery of the
biologically active compound to certain parts of the cell.
[0028] Nuclear localizing signals enhance the targeting of the
pharmaceutical into proximity of the nucleus and/or its entry into
the nucleus during interphase of the cell cycle. Such nuclear
transport signals can be a protein or a peptide such as the SV40
large T antigen 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. For example, karyopherin beta itself
could target the DNA to the nuclear pore complex. Several peptides
have been derived from the SV40 T antigen. Other NLS peptides have
been derived from the hnRNP A1 protein, nucleoplasmin, c-myc,
etc.
[0029] Many biologically active compounds, in particular large
and/or charged compounds, are incapable of crossing biological
membranes. In order for these compounds to enter cells, the cells
must either take them up by endocytosis, i.e., into endosomes, or
there must be a disruption of the cellular membrane to allow the
compound to cross. In the case of endosomal entry, the endosomal
membrane must be disrupted to allow for movement out of the
endosome and into the cytoplasm. Either entry pathway into the cell
requires a disruption of the cellular membrane. Compounds that are
able to alter the structure of a membrane are called membrane
active compounds. This change in structure can be shown by the
compound inducing one or more effects on a membrane selected from
the group comprising: alterations that allow small molecule
permeability, pore formation, fusion and/or fission of membranes,
alterations that allows large molecule permeability, dissolving or
disrupting the membrane, or membrane phase transitions. This
alteration can be functionally defined by the compound's activity
in at least one assay selected from the group comprising: red blood
cell lysis (hemolysis), liposome leakage, liposome fusion, cell
fusion, cell lysis and endosomal release. An example of a membrane
active agent is the bee venom peptide melittin, whose membrane
activity is demonstrated by its ability to release heme from red
blood cells (hemolysis).
[0030] More specifically membrane active compounds allow for
transport of molecules with molecular weight greater than 50 atomic
mass units to cross a membrane. This transport may be accomplished
by either the total loss of membrane structure, the formation of
holes (or pores) in the membrane structure, the assisted transport
of the molecule through the membrane, or other rearrangement or
disorganization of the lipid bilayer structure. In addition,
transport between liposomes or cell membranes may be accomplished
by the fusion of the two membranes thus mixing of the contents of
the two membranes.
[0031] These membrane active compounds, or releasing signals,
enhance release of endocytosed material 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 the 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, SEQ ID 3), viral components
such as influenza virus hemagglutinin subunit HA-2 peptides and
other types of amphipathic peptides. The control of when and where
the membrane active compound is active is crucial to effective
transport. If the membrane active agent is operative in a certain
time and place it would facilitate the transport of the
biologically active compound across the biological membrane. If the
membrane active compound is too active or active at the wrong time,
then no transport occurs or transport is associated with cell
rupture and cell death. Nature has evolved various strategies to
allow for membrane transport of biologically active compounds
including membrane fusion and the use of membrane active compounds
whose activity is modulated such that activity assists transport
without toxicity. Many lipid-based transport formulations rely on
membrane fusion and some membrane active peptides' activities are
modulated by pH. In particular, viral coat proteins are often
pH-sensitive, inactive at neutral or basic pH and active under the
acidic conditions found in the endosome.
[0032] Membrane active peptides are membrane active compounds that
are peptides. There are many naturally occurring membrane active
peptides including, but not limited to: cecropin (insects),
magainin (wasp), CPF 1, PGLa, Bombinin BLP-1 (all three from
amphibians), seminalplasmin (bovine), indolicidin, bactenecin (both
from bovine neutrophils), tachyplesin 1 (crabs), protegrin (porcine
leukocytes), and defensins (from human, rabbit, bovine, fungi, and
plants), gramicidin A and gramicidin S (bacillus brevis), the nisin
(a lantibiotic from lactococcus lactis), androctonin (scorpion),
cardiotoxin I (cobra), caerin (frog litoria splendida), and
dernaseptin (frog) and the like. Viral peptides have also been
shown to have membrane activity and include, but are not limieted
to: hemagglutinin subunit HA-2 (influenza virus), E1 (Semliki
forest virus), F1 (Sendai and measles viruses), gp41 (HIV), gp32
(SIV), vp1 (Rhino, polio, and coxsackie viruses), and the like.
Melittin is a highly cytotoxic and hemolytic membrane active
26-residue peptide component of bee venom
(GIGAILKVLATGLPTLISWIKNKRKQ, little honey bee peptide sequence, SEQ
ID 4). Multiple variations of this sequence, both naturally
occurring and synthetic, also possess membrane activity.
Additionally, synthetic peptides have been synthesized that have
membrane activity. Synthetic peptides rich in leucines and lysines
(KL or KL.sub..eta.motif) have been shown to have membrane
activity. In particular, the peptide
H.sub.2N-KLLKLLLKLWLKLLKLLLKLL--CO.sub.2 (SEQ ID 5), termed KL3, is
membrane active. There exists little to no homology or structural
similarity between all the different membrane active peptides.
Therefore, they are defined by their membrane activity.
[0033] Membrane active polymers are polymers that have membrane
activity.
[0034] Membrane Active Amphiphiles are membrane active compounds
that are amphiphilic, containing both hydrophobic and hydrophilic
sections. Examples include such compounds as surfactants or
detergents such as dodecylamine and dodecylsulfate.
[0035] An amphipathic compound is a molecule that contains one end
that is hydrophilic while the other end is hydrophobic. The term
hydrophobic indicates in qualitative terms that the chemical moiety
is water-avoiding. Hydrocarbons are hydrophobic groups. The term
hydrophilic indicates 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,
oligonucleotides, and groups containing amines, amides, alkoxy
amides, carboxylic acids, sulfurs, or hydroxyls.
[0036] Cell penetrating compounds, which include cationic import
peptides (also called peptide translocation domains, membrane
translocation peptides, arginine-rich motifs, cell-penetrating
peptides, and peptoid molecular transporters), are capable of
crossing biological membranes. In addition, they are capable of
transporting molecules to which they are attached across membranes.
Cell penetrating compounds are another form of membrane active
compound. Examples of cationic import peptides, which are typically
rich in arginine and lysine, include TAT (SEQ ID 1), VP22 peptide,
and an ANTp peptide (SEQ ID 2). Cell penetrating compounds,
however, are not strictly peptides. Short, non-peptide polymers
that are rich in amines or guandinium groups are also capable of
carrying molecules crossing biological membranes. Like membrane
active peptides, cationic import peptides are defined by their
activity rather than by strict amino acid sequence
requirements.
[0037] Another functional group comprises compounds, such as
polyethylene glycol, that decrease interactions between molecules
and themselves and with other molecules. Such groups are useful in
limiting interactions such as between serum factors or cells and
the molecule or complex to be delivered.
[0038] Another functional group comprises alkyl chains and other
hydrophobic groups such as cholesterol and cholesterol derivatives.
These hydrophobic groups can be used to bind to membranes, disrupt
membranes, or provide hydrophobic interactions.
[0039] A membrane permeable molecule is a molecule than can pass
through a cell membrane bilayer. Movement of these molecules though
the bilayer does not require additional proteins, compounds or
chemicals. In fact, because of the impermeable nature of the cell
membrane to charged or highly polar molecules, most small molecule
drugs which need to reach the cell cytoplasm are membrane
permeable. Drub design must often balance water solubility with
membrane permeability. However, for certain drugs, such as the
anticancer drug doxorubicin, not-specific membrane permeability
leads to negative non-specific effects in non-target cells. The
ability of reversibly inactivate the membrane permeable
characteristics of such drugs could be used to improve efficacy of
the drug while decreasing toxic effects.
[0040] A reactive group is a group that is able to form a covalent
bond with another group. Reactive groups that form covalent bonds
may be selected from the group comprising: isothiocyanate,
isocyanate, acyl azide, acid halide, O-acyl urea,
N-hydroxysuccinimide esters, succinimide esters, thioesters, amide,
urea, sulfonyl chloride, aldehyde, ketone, ether, epoxide,
carbonate, alkyl halide, imidoester, carboxylate, alkylphosphate,
arylhalides (e.g. difluoro-dinitrobenzene), and anhydrides.
[0041] R groups comprise any group that one may desire to attach to
the maleic anhydride. R groups may be selected from the group that
comprises: peptides, proteins, antigens, haptens, biotin, nucleic
acids, alkyl groups, etc.
[0042] CDM (2-propionic-3-methylmaleic anhydride). CDM is a
carboxylic acid-containing derivative of the disubstituted
dimethylmaleic anhydride. CDM is synthesized via a Horner-Emmons
reaction between dimethyloxoglutarate and
triethyl-2-phosphonopropionate followed by saponification of the
ester groups according to the published procedure (FIG. 3)
[Naganawa et al. 1994]. The addition of a carboxylate-containing
group increases charge and water solubility of the compound. The
cis-aconitic anhydride has a similar carboxylic acid group but
lacks the disubstitution at second R position.
[0043] Phosphorodiamidate morpholino oligonucleotides (PMOs). PMOs
are assembled from morpholino subunits, each of which contains one
of the genetic bases linked to a 6-membered morpholine ring. The
subunits are joined by non-ionic phosphorodiamidate intersubunit
linkages. PMOs exert their effects by steric hindrance mechanisms
and can be used to block translation or splicing of a target RNA.
Like PEG and dextran, oligonucleotides are internalized by fluid
phase endocytosis and are unable to diffuse directly across cell
membranes.
[0044] Crosslinking refers to the attachment of two or more
molecules with a bifunctional reagent (crosslinker). 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. The attachment can be a covalent
bond or a stable non-covalent bond.
[0045] A labile bond is a covalent bond that is capable of being
selectively broken. That is, the labile bond may be broken in the
presence of other covalent bonds without the breakage of other
covalent bonds. For example, a disulfide bond is capable of being
broken in the presence of thiols without cleavage of any other
bonds, such as carbon-carbon, carbon-oxygen, carbon-sulfur,
carbon-nitrogen bonds, which may also be present in the molecule.
Labile also means cleavable.
[0046] A labile linkage is a chemical compound that contains a
labile bond and provides a link or spacer between two other groups.
The groups that are linked may be chosen from compounds such as
biologically active compounds, membrane active compounds, compounds
that inhibit membrane activity, functional reactive groups,
monomers, and cell targeting signals. The spacer group may contain
chemical moieties chosen from a group that includes alkanes,
alkenes, esters, ethers, glycerol, amide, saccharides,
polysaccharides, and heteroatoms such as oxygen, sulfur, or
nitrogen. The spacer may be electronically neutral, may bear a
positive or negative charge, or may bear both positive and negative
charges with an overall charge of neutral, positive or
negative.
[0047] pH-labile refers to the selective breakage of a covalent
bond under acidic conditions (pH<7). That is, the pH-labile bond
may be broken under acidic conditions without the breakage of other
covalent bonds. The term pH-labile includes both linkages and bonds
that are pH-labile, very pH-labile, and extremely pH-labile.
[0048] A subset of pH-labile bonds is very pH-labile. For the
purposes of the present invention, a bond is considered very
pH-labile if the half-life for cleavage at pH 5 is less than 45
minutes.
[0049] A subset of pH-labile bonds is extremely pH-labile. For the
purposes of the present invention, a bond is considered extremely
pH-labile if the half-life for cleavage at pH 5 is less than 15
minutes.
[0050] A molecule is modified, to form a modification through a
process called modification, by a second molecule if the two become
bonded through a covalent bond. That is, the two molecules form a
covalent bond between an atom from one molecule and an atom from
the second molecule resulting in the formation of a new single
molecule. A chemical covalent bond is an interaction, bond, between
two atoms in which there is a sharing of electron density.
[0051] Native chemical ligation is the formation of an amide bond
between the carboxylate of thioester and the amine of a compound
that also contains a thiol two or threee atoms away from the amine,
such as an terminal cysteine.
[0052] Charge, Polarity and Sign. The charge, polarity, or sign of
a compound refers to whether or not a compound has lost one or more
electrons (positive charge, polarity, or sign) or gained one or
more electrons (negative charge, polarity, or sign).
EXAMPLES
[0053] 1. Synthesis of 2-carboxyethyl-3-methyl-maleic anhydride
(CDM-thioester). CDM anhydride (100 mg) was dissolved in 10 mL
methylenechloride. To this solution as added 2 mL oxalyl chloride.
After stirring overnight, excess oxalyl chloride was removed by
rotary evaporation to yield a clear oil. The acid chloride of CDM
was then dissolved in 10 mL methylenechloride. To this solution was
added 200 mg mercaptoacetic acid and 100 .mu.L
diisopropylethylamine. The solution was stirred for 1 h and the
solvent was removed by rotary evaporation. The resulting oil was
placed under high vacuum (0.2 torr) for 3 h to remove excess
mercaptoacetic acid and diisopropylethylamine. The product was
dissolved in water and acetonitrile (9:1) and purified by HPLC
using a reverse-phase C18 column loading in water:acetonitrile (9:1
with 0.1% trifluoroacetic acid) and eluting with acetonitrile (0.1%
trifluoroacetic acid).
[0054] 2. Charge reversal of a polyamine by modification with
disubstituted maleic anhydrides. Cyclic anhydrides were reacted
with the polyamine poly-L-lysine (PLL). To a solution containing
200 .mu.g PLL, 2 mg HEPES, and 0.4 mg NaOH in 100 .mu.L water was
added 0.4 mg CDM or dimethylmaleic anhydride (Aldrich) in 50 .mu.L
ethanol with rapid vortexing. For cis-aconitic anhydride (Aldrich),
1 mg of anhydride, 5 mg of HEPES, and 1 mg of NaOH were used.
Similarly, PLL was reacted with two equivalents of succinic or
citraconic anhydride (Aldrich). Following reaction of PLL with the
anhydrides, trinitrobenzenesulfonic acid (TNBS) assay indicated
complete conversion of the F-amines of PLL to carboxylates. The
charge density of these polyanions was then determined by assessing
their ability to condense cationic, fluorescein-labeled PLL. At
neutral pH, one functional equivalent of succinylated PLL is
required to condense PLL. Similarly, one equivalent of
citraconylated PLL and one half equivalent of cis-aconitylated PLL
(which has two carboxylate groups per repeating unit) are required
to condense PLL. In contrast, PLL modified with dimethylmaleic
anhydride is unable to condense PLL, even when 10 equivalents are
added. Similarly, the charge density of CDM-modified PLL is one
charge per two carboxylates. The distal carboxylate of the
anhydride adds charge to the modified polymer while the carboxylate
of the anhydride appears to contribute no charge to the
polyanion.
[0055] 3. Acylation of the dipeptide glycinylalanine and the
kinetics of cleavage. The rates of acid-catalyzed cleavage of the
maleamates were evaluated using the dipeptide glycinylalanine. CDM
and dimethyl maleamic modified glycinylalanine (GA) were
synthesized by addition of 400 .mu.g CDM or dimethylmaleic
anhydride in 20 .mu.L ethanol to a solution of 200 .mu.g GA and 2.4
mg HEPES base in 44 .mu.L water. Cleavage was allowed to proceed by
addition of the modified peptides to a pH 5 solution. At various
times, 10 .mu.g aliquots were removed and added to 0.5 mL of a 100
mM NaHCO.sub.3 solution (pH 9) containing 0.4 mM TNBS (pH 9). The
disubstituted maleamic acids cleaved much more rapidly than
monosubstituted maleamic acids. A plot of In [l-(A.sub.t/A.sub.0)]
as a function of time was a straight line whose slope is -k, the
rate constant for the cleavage reaction, where A.sub.t is the
absorbance at time t and A.sub.0 is the absorbance of unmodified
GA. The rate constants for the cleavage of dimethylmaleamic acid
and CDM modified glycinylalanine were 0.4/min (t.sub.1/2=1.5 min)
and 0.3/min (t.sub.1/2=2 min), respectively. Similar analysis for
the reversal of cis-aconitic modification revealed much slower
cleavage kinetics with an approximate half-life of 300 min (5
h).
1 compound half-life (minutes) DM 1.5 CDM 2.0 cis-aconitic 300
[0056] 4. Reversible inactivation of a membrane active peptide
modification with a disubstituted maleic anhydride. Little honey
bee Melittin (SEQ ID 4) was acylated at pH 7.5 with two molar
equivalents, relative to the four lysine residues, with succinic,
cis-aconitic, dimethylmaleic, citraconic, and CDM anhydrides. To a
solution containing 200 .mu.g melittin, 500 .mu.g HEPES, and 100
.mu.g NaOH in 20 .mu.L water was added 0.1 .mu.g CDM or
dimethylmaleic anhydride in 50 .mu.L ethanol with rapid vortexing.
For cis-aconitic anhydride 250 .mu.g anhydride, 1.25 mg of HEPES,
and 250 mg of NaOH were used. Measurement of amine content by TNBS
revealed complete acylation of melittin by all of the
anhydrides.
[0057] The membrane activity of the modified peptides was measured
using a red blood cell (RBC) hemolysis assay. Porcine whole blood
was isolated in heparin-containing vacutainers. RBC's were isolated
by centrifugation at 2,500 rpm for 5 min and washed three times
with 100 mM phosphate buffer. 20 .mu.L of the washed RBC suspension
(.about.10.sup.8 cells) was added to 500 .mu.L phosphate buffer. To
this solution was added various amounts of peptide and the samples
were incubated for 1.5 h at 37.degree. C. Samples were then
centrifuged for 1 min at 14,000 rpm. Lysis of the RBCs was
determined by measuring the absorbance of the supernatant at 541
nm. Modification by all of the anhydrides except dimethylmaleic
anhydride resulted in a complete loss of membrane activity.
[0058] Melittin modified with either CDM or cis-aconitic anhydride
was incubated at pH 5. At various times, the pH was raised by the
addition of pH 7.5 buffer and the hemolytic activity of each sample
was measured. The membrane activity for CDM-melittin returned to
100% within 25 minutes. Kinetics of the return of activity
(plotting of In[1 -(A.sub.t/A.sub.0)] revealed a rate constant of
0.07/min (t.sub.1/2=10 minutes). Incubation of cis-aconitylated
melittin at pH 5 for 27 h resulted in only a 30% return in
activity. Analysis of the kinetics of its cleavage revealed a rate
constant of 0.015/hr (t.sub.1/2=47 hours). These results indicate
that CDM is able to inhibit activity of a membrane active peptide.
Furthermore, the inhibition is reversed at a physiologically
relevant pH in timeframe amenable to delivery of material to cells
via endocytosis.
[0059] 6. CDM-Melittin mediated release of fluorescent polyethylene
glycol from the endocytic compartment. HeLa cells grown on glass
coverslips were incubated with 1 mg/ml fluorescein-PEG3000 either
with or without 400 .mu.g/ml modified melittin in 1 ml DMEM for 10
min at 37.degree. C. After this pulse, cells were washed and chased
for an additional 35 min at 37.degree. C. in DMEM+10% bovine serum.
Cells were then washed three times with PBS (Sigma), fixed for 30
min in PBS+4% formaldehyde (Sigma) at 4.degree. C., and washed
three times in PBS. Coverslips were then mounted onto glass slides
for fluorescent microscopy. Images of the samples were collected by
confocal microscopy on a Zeiss LSM510 confocal microscope (Zeiss,
Germany). In the absence of CDM-melittin, the fluorescein-PEG had a
punctuate appearance, indicative of localization in endosomes
and/or lysosomes. In contrast, when CDM-melittin was included
diffuse fluorescence was observed throughout the cell, indicating
release of fluorescein-PEG from internal endosomes/lysosomes.
Co-incubation with cis-aconitic modified melittin resulted in
punctate fluorescence indistinguishable from fluorescein-PEG alone
control. Similar results were observed when fluorescein-labeled 10
kDa dextran was used as the marker molecule. In addition,
bafilomycin, an inhibitor of endosomal acidification, inhibited
release of the fluorescein-PEG.
[0060] Incubation of cells with 400 .mu.g/ml CDM-melittin for 10
min had no visually apparent cytotoxic effect. In contrast, 10
.mu.g/ml unmodified melittin completely destroyed cells in less
than 10 minutes. Additionally, propidium iodide staining was used
to provide a more sensitive indication of cellular toxicity. In the
cells exposed to CDM-melittin for 10 min, only .about.1% of cells
subsequently showed nuclear staining with propidium iodide, similar
to control samples. Thus, CDM-melittin enabled endosomal release
while substantially reducing its cellular toxicity.
[0061] 7. Cytoplasmic/nuclear delivery of an oligonucleotide with
CDM-modified membrane active peptide: HeLa-LUC/705 cells carry a
stably integrated mutant luciferase gene that has a defective
splice site (Gene-Tools, Philomath, Oreg.). This mutant splice site
results in production of a mRNA coding for a truncated, inactive
luciferase protein. The presence of an appropriate
phosphorodiamidate morpholino oligonucleotide (PMO, sequence CCT
CTT ACC TCA GTT ACA ATT TAT A, SEQ ID 6) blocks this splice site
leading to correct splicing and expression of the full-length
active enzyme. Therefore, luciferase activity in this cell line is
directly proportional to the amount of PMO present in the
cytoplasm/nucleus (released from the endosomal compartment).
[0062] HeLa-LUC cells were incubated with 2.5 .mu.M PMO+2.5 nM
CDM-melittin, citraconyl-melittin, or aconityl-melittin. Cells were
incubated for 4 hours in a humidified, 5% CO.sub.2 incubator at
37.degree. C. The media was then replaced with DMEM containing 10%
fetal bovine serum and cells were incubated for an additional 48 h.
The cells were harvested and lysates were then assayed for
luciferase expression using a Lumat LB 9507 (EG&G Berthold,
Bad-Wildbad, Germany) luminometer. Co-incubation of PMO with
CDM-melittin resulted in 5-12 fold increase in luciferase
expression above incubation with PMO alone. Neither
cis-aconitic-modified melittin nor citraconic-modified melittin
resulted in any increase in luciferase expression/PMO delivery.
2 Condition Luciferase Induction PMO alone 1.0 +CDM-melittin 8.5
.+-. 3.0 +citraconyl-melittin 1.2 .+-. 0.3 +aconityl-melittin 1.2
.+-. 0.4
[0063] These data indicate that CDM-melittin, but not
citraconyl-melittin or aconityl-melittin regains activity on a time
scale that enables delivery of oligonucleotides to cells.
[0064] 8. Reversible attachment of an oligonucleotide to a polymer.
In order to deliver a biologically active molecule to a cell in
vivo, the reversibly-modified membrane active compound must be
complexed with the molecule to be delivered. To link PMO's with
CDM-modified melittin, we have developed coupling techniques based
upon native chemical ligation in which a thioester and an
N-terminal cysteine group react to form an amide bond. A benefit of
this procedure is that the acid (of CDM) which is to be converted
to an amide may be preactivated as a thioester. The thioester
itself is relatively stable when compared to activated esters such
as N-hydroxysuccinimidyl esters, but will couple readily and
selectively with N-terminal cysteine groups. The reaction mixture
may contain other amines and other thiols, but only an
amino-terminal cysteine couples to form a stable amide (thiol
within 2-3 bonds of an amine).
[0065] The stability of the thioester allows reaction of the
anhydride of CDM-thioester with an amine on the PMO to form
maleamic acid groups without reaction between amine and thioester.
The resultant thioester is then able to react with an
amino-terminal cysteine group that has been placed onto a
polycation such as polyethyleneimine (PEI-Cys). In this way, we are
able to attach the PMO to the polycation through the acid-labile
maleamic group. Following the attachment of the PMO to PEI-Cys,
CDM-thioester modified melittin is coupled to the PEI-Cys-PMO
conjugate to form a single molecule complex that contains PEI-Cys,
PMO, and CDM-modified melittin.
[0066] To synthesis cysteine-modified polyethyleneimine,
N.sub..alpha.-Fmoc-S-tert-butylthio-L-cysteine (20 mg) was
dissolved in 5 mL of acetonitrile. To this solution was added
dicyclohexylcarbodiimide (10 mg) and N-hydroxysuccinimide (5 mg).
The succinimidyl ester was allowed to form overnight. The reacted
dicyclohexylurea was then filtered off and the ester was added to a
solution of polyethyleneimine (20 mg) in 10 mL of methanol. After
10 min, the derivatized polymer was precipitated out of solution by
addition of 45 mL diethylether. The polymer was then dissolved in 2
mL piperidine to remove the FMOC groups. The polymer was again
precipitated out of solution by addition of diethylether. The
modified polymer was dissolved in 2 mL water and the solution was
adjusted to pH 4 by addition of hydrochloric acid.
[0067] Oligonucleotide-polycation complex delivery assay. 0.5 nmol
amine-modified PMO (SEQ ID 6, Gene Tools, Philomath, Oreg.) was
reacted with 1 nmol CDM-thioester in the presence of 500 mg HEPES
buffer pH 7.9. The modified PMO was then added to a 50 .mu.L
solution containing 10 .mu.g/mL PEI-Cys, 1 mM dithiothreitol, and 5
mM HEPES buffer pH 7.5. Melittin was modified with CDM-thioester by
reaction with 0.5 wt equivalent CDM thioester in the presence of 3
wt equivalents HEPES base. After 1 h of conjugation, CDM-thioester
modified melittin was added to the PEI-Cys-PMO complex to a final
concentration of 80 .mu.g/ml melittin. Similar conditions were used
to attach either citraconyl-Melittin or aconityl-Melittin to
PEI-Cys-PMO.
[0068] HeLa Luc/705 cells were grown under conditions used for HeLa
cells. The cells were plated in 24-well culture dishes at a density
of 3.times.10.sup.6 cells/well and incubated for 24 hours. Media
was replaced with 0.5 ml DMEM and the PMO complexes were added. The
cells were incubated for 4 hours in a humidified, 5% CO.sub.2
incubator at 37.degree. C. The media was then replaced with DMEM
containing 10% fetal bovine serum. The cells were then incubated
for an additional 48 h. The cells were then harvested and the
lysates were then assayed for luciferase expression.
[0069] PEI-Cys-PMO-CDM-Melittin addition was effective in delivery
of PMO, while PEI-Cys -PMO-citraconyl-melittin and
PEI-Cys-PMO-aconityl-melittin additions were not.
3 Condition Luciferase Induction PMO alone 1
PEI-Cys-PMO-CDM-melittin 67
[0070] 9. Reversible inhibition of a cell penetrating compound. The
cationic import peptide cysteine-TAT (CGRKKRRQRRR,SEQ ID 7) was
labeled with the fluorophore Texas Red. This modification does not
affect the import properties of the peptide and provides a visual
assay for internalization. To a solution of 1 mg cysteine-modified
TAT (SEQ ID 7) (10 mg/mL) and 10 mM HEPES buffer pH 7.5 was added
0.2 mg of Texas Red C5 bromoacetamide (Molecular Probes) in 500
.mu.L of ethanol. After reacting overnight at room temperature, the
ethanol was removed by speed evaporation to yield the labeled
peptide.
[0071] The labeled peptide was then acylated with the disubstituted
maleic anhydrides, dimethylmaleic and CDM. TAT-Texas Red (50 .mu.g,
10 mg/mL) was modified by addition of dimethylmaleic or CDM
anhydrides (50 .mu.g, 20 mg/mL in ethanol) in the presence of 100
mM HEPES buffer pH 7.9.
[0072] For analysis of TAT-mediated delivery, HeLa cells were
plated in 6-well plates containing glass coverslips at a density of
50,000-100,000 cells per well. After 24-48 hours, when cells
reached 40-75% confluency, growth media was aspirated off and
replaced with 1.0 ml DMEM, either pre-cooled to 4.degree. C. or
pre-warmed to 37.degree. C. TAT conjugates were then added and
cells were incubated at either 4.degree. C. or 37.degree. C. At
4.degree. C. endocytosis was completely blocked and the
TAT-conjugates entered the cell only directly through the plasma
membrane. At 37.degree. C., endocytic internalization was also
possible. After incubation at the indicated temperature for 1-2
hours, cells were washed three times with PBS, fixed for 30 min at
4.degree. C. in PBS containing 4% formaldehyde, and again washed
three times with PBS. Coverslips were then mounted onto slides for
viewing in a Zeiss LSM510 confocal microscope. For fluorescein
labeled conjugates, a 488 nm wavelength argon laser was used for
excitation and a long pass 505 filter was used for detection. For
rhodamine or texas red conjugates, a 543 nm wavelength HeNe laser
was used for excitation and a long pass 585 filter was used for
detection.
[0073] Addition of the modified peptides to cells at 4.degree. C.
resulted in no transport of the peptide The unmodified peptide
showed intense intracellular localization under these conditions.
Upon shift to 37.degree. C., dimethylmaleamate-modified TAT
regained its activity at while the CDM-modified peptide did not.
Presumably, dimethylmaleamate-modified TAT was internalized into
the acidic environment of the endosome at 37.degree. C. Once in
this acidic environment the anhydride was cleaved, restoring
activity to the TAT peptide. In contrast, the CDM-modified TAT was
not significantly endocytosed at 37.degree. C., and therefore did
not experience an acidic environment. The ability to regain
transport activity thus appears to be dependent upon endocytosis
into an acidic environment and cleavage of the dimethylmaleamate
group to regenerate the active TAT peptide.
[0074] 10. Reversible attachment of PEG using CDM. We have
demonstrated the facile synthesis of CDM and CDM-thioester. The
ease with which CDM and its derivatives may be synthesized enables
us to easily make other acid cleavable materials. In particular,
polyethyleneglycol (PEG) may be conjugated to CDM via the acid
chloride of CDM. To a solution of 2-propionic-3-methylmaleic
anhydride (30 mg, 0.16 mmol) in 5 mL methylene chloride was added
oxalyl chloride (200 mg, 10 eq) and dimethylformamide (1 .mu.L).
The reaction was allowed to proceed overnight at which time the
excess oxalyl chloride and methylene chloride were removed by
rotary evaporation to yield the acid chloride, a clear oil. The
acid chloride was dissolved in 1 mL of methylene chloride. To this
solution was added polyethyleneglycol monomethyl ether, molecular
weight average of 5,000 (815 mg, 1 eq) and pyridine (20 .mu.l, 1.5
eq) in 10 mL of methylene chloride. The solution was then stirred
overnight. The solvent was then removed and the resulting solid was
dissolved in 8.15 mL water. In contrast, Garman and Kalindjian made
a PEG-containing maleic anhydride using
2-bromomethyl-3-methylmaleic anhydride [Garman and Kalindjian1987].
Attempts in our lab to duplicate this synthesis were unsuccessful
and it has been reported that this intermediate is "difficult to
prepare in good yield and high purity" [Greenwald et al. 2000]. The
difficulty in synthesis makes the contribution of CDM to the
synthesis of acid cleavable PEG derivatives nontrivial. Plasmid DNA
labeled with Cy3 Label IT(Mirus Corporation, Madison, Wis.) was
compacted into a particle with a 1.2 fold charge excess of
poly-L-lysine (mw:52,000). The particles were then reacted with
either a non-reactive Polyethylene Glycol (mw: 5000) or with
amine-reactive CDM-PEG at a 0.5 molar equivalent to amines on the
poly-L-lysine. Particles, containing 50 .mu.g DNA, were injected
into the tail vein of 20 gram male ICR mice. Blood was taken at one
hour and the smears were inspected for Cy3 fluorescence still in
circulation. The animals were then sacrificed and the liver, lung,
kidney and spleen were harvested and snap frozen for cryosectioning
and the resulting slices were inspected for Cy3 fluorescence. It
was found that the PEG-CDM modification increased circulation times
dramatically over unmodified PLL-DNA particles. The animal injected
with the fluorescent particles treated with non-reactive
Polyethylene Glycol showed no fluorescence in circulation in the
blood at one hour and very little fluorescence in the liver, kidney
or spleen, leaving the significant portion of fluorescence in the
lung. The animal injected with the fluorescent particles treated
with CDM-PEG showed a high level of fluorescence still in
circulation in the blood at one hour and also had a high level of
fluorescence evenly spread throughout the liver, kidney and spleen,
with little fluorescence in the lung.
[0075] 11. Reversible Modification of anticancer drug doxorubicin.
To a 1 mM solution of doxorubicin (Dox) in 50 mM HEPES buffer pH
7.9 is added 3 equivalents CDM adduct (such as CDM or a CDM-polymer
conjugate i.e. PEG-CDM). The modified DOX is then added to cells in
tissue culture or injected in vivo.
[0076] 12. Reversible Inhibition of a Membrane Permeable Peptide
for Targeted Delivery: Streptavidin was modified with maleimide
groups by reaction with 6 molar equivalents of Succinimidyl
4-[N-maleimidomethyl]cy- clohexane-1-carboxylate (SMCC from
Pierce). The liver targeting peptide
Cys-PEG4-KNESSTNATNTKQWRDETKGFRDEAKRFKNTAG (SEQ ID 8) was
conjugated to streptavidin by reaction with the maleimide groups.
70,000 MW amino dextran with 18 amine groups/dextran (Molecular
Probes) was reacted with 5 molar equivalents of Cy3 NHS ester
(Amersham). The Cy3-labeled dextran was then reacted with 5
equivalents of NHS-activated biotin. The Cy3 and biotin labeled
dextran was then reacted with 20 molar equivalents of SMCC. The
Cy3/biotin/SMCC-modified dextran was isolated by size exclusion
chromatography using sephadex G25. Cys-TAT peptide (prepared as
described above) was then conjugated to the dextran via
maleimide-thiol coupling and the dextran was isolated by size
exclusion chromatography. 45 .mu.g of Cy3/biotin/TAT-dextran was
reacted with 80 .mu.g of CDM in the presence of 4 mg HEPES
base.
[0077] Unmodified and CDM-modified Cy3/biotin/TAT-dextran (45
.mu.g) was added to 36 .mu.g of liver-targeting
peptide-streptavidin in 250 .mu.L of PBS. These samples were
injected into the tail vein of mice. 10 minutes postinjection, the
mice were sacrificed, their livers harvested, frozen and sectioned
for microscopy. A broader, more even distribution of labeled
dextran was observed in the liver when the TAT cell permeable
peptide was reversibly modified with CDM.
[0078] In a similar manner, a toxin, such as diphtheria toxin, or
an anticancer drug can be conjugated to a cell permeable molecule
such as the TAT peptide (SEQ ID 1). Inactivation of the TAT peptide
by modification with CDM would prevent non-specific toxicity.
Activation of the TAT in the reduced pH environment of a tumor
would lead to entry of the toxin into the cancer cells.
[0079] 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.
REFERENCES
[0080] Blattler WA, Kuenzi B S, Lambert J M, Senter P D. New
Heterobifunctional Protein Cross-Linking Reagent That Forms an Acid
Labile Link. Biochemistry. 1985. 24:1517-1524.
[0081] Blattler, W. A., Kuenzi, B. S., Lambert, J. M., and Senter,
P. D. New Heterobifunctional Protein Cross-Linking Reagent That
Forms an Acid-Labile Link. Biochemistry. 1985. 24:1517-1524.
[0082] Dixon HBF, Perham RN. Reversible Blocking of AAmino Groups
with Citraconic Anhydride. Biochemical Journal. 1968.
109:312-313.
[0083] Futaki S. Arginine-rich peptides: potential for
intracellular delivery of macromolecules and the mystery of the
translocation mechanisms. Int J Pharm. 2002. 245(1-2): 1-7.
[0084] Garman A J, Kalindjian S B. The Preparation and Properties
of Novel Reversible Polymer-Protein Conjugates. FEBS Letters. 1987.
223(2):364-365.
[0085] Garman, A. J. and Kalindjian, S. B. The preparation and
properties of novel reversible polymer-protein conjugates. FEBS
Lett. 1987. 223:361-365.
[0086] Greenwald, R. B., Conover, C. D., and Choe, Y. H.
Poly(ethylene glycol) Conjugated Drugs and Prodrugs: A
Comprehensive Review. Critical Reviews in Therapeutic Drug Carrier
Systems 2000. 17:101-161.
[0087] Kirby, A. J. and Lancaster, P. W. Structure and Efficiency
in Intramolecular and Enzymic Catalysis. Catalysis of Amide
Hydrolysis by the Carboxy-group of Substituted Maleamic Acids. J
Chem Soc Perkin 1972.11:1206-1214.
[0088] Kratz F, Beyer U, Thomas-Schutte M. Drug-Polymer Conjugates
Containing Acid-Cleavable Bonds. Critical Reviews in Therapeutic
Drug Carrier Systems. 1999. 16(3): 245-289.
[0089] Lee, H. J. and Pardridge, W. M. Pharmacokinetics and
delivery of tat and tat-protein conjugates to tissues in vivo.
Bioconjug Chem 2001. 12:995-999.
[0090] Lindgren, M., Hallbrink, M., Prochiantz, A., and Langel, U.
Cell-penetrating peptides. Trends Pharmacol Sci 2000.
21:99-103.
[0091] Naganawa A, Ichikawa Y, Isobe MT. Synthetic Studies on
Tautomycin Synthesis of 2,3-Disubstituted Maleic Anhydride Segment.
Tetrahedron 1994. 50:8969.
[0092] Nieto M A, Palacian E. Effects of Temperature and pH on the
Regeneration of the Amino Groups of Ovalbumin After Modification
with Citraconic and Dimethylmaleic Anhydrides. Biochimica et
Biophysica Acta. 1983. 749:204-210.
[0093] Nieto, M. A. and Palacian, E. Effects of temperature and pH
on the regeneration of the amino groups of ovalbumin after
modification with citraconic and dimethylmaleic anhydrides.
Biochemica Biophysica Acta 1983. 749:204-210.
[0094] Reddy J A, Low P S. Enhanced folate receptor mediated gene
therapy using a novel pH-sensitive lipid formulation. Journal of
Controlled Release. 2000. 64:27-37.
[0095] Reddy, J. A. and Low, P. S. Enhanced folate receptor
mediated gene therapy using a novel pH-sensitive lipid formulation.
J Control Release 2000. 64:27-37.
[0096] Schwarze, S. R., Ho, A., Vocero-Akbani, A., and Dowdy, S. F.
In vivo protein transduction: delivery of a biologically active
protein into the mouse. Science 1999. 285:1569-72.
[0097] Shah D, Shen W C. Transcellular delivery of an
insulin-transferrin conjugate in enterocyte-like Caco-2 cells. J
Pham Sci. 1996. 85(12):1306-1311.
[0098]
Sequence CWU 1
1
8 1 10 PRT Human immunodeficiency virus 1 Gly Arg Lys Lys Arg Arg
Gln Arg Arg Arg 1 5 10 2 16 PRT Drosophila melanogaster 2 Arg Gln
Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 3
4 PRT Homo sapiens 3 Lys Asp Glu Leu 1 4 26 PRT Apis florea 4 Gly
Ile Gly Ala Ile Leu Lys Val Leu Ala Thr Gly Leu Pro Thr Leu 1 5 10
15 Ile Ser Trp Ile Lys Asn Lys Arg Lys Gln 20 25 5 21 PRT
Artificial synthetic amphipathic sequence 5 Lys Leu Leu Lys Leu Leu
Leu Lys Leu Trp Leu Lys Leu Leu Lys Leu 1 5 10 15 Leu Leu Lys Leu
Leu 20 6 25 DNA Photinus pyralis 6 cctcttacct cagttacaat ttata 25 7
11 PRT Human immunodeficiency virus 7 Cys Gly Arg Lys Lys Arg Arg
Gln Arg Arg Arg 1 5 10 8 33 PRT Bacteriophage T7 8 Lys Asn Glu Ser
Ser Thr Asn Ala Thr Asn Thr Lys Gln Trp Arg Asp 1 5 10 15 Glu Thr
Lys Gly Phe Arg Asp Glu Ala Lys Arg Phe Lys Asn Thr Ala 20 25 30
Gly
* * * * *