U.S. patent application number 16/189550 was filed with the patent office on 2019-02-28 for polyconjugates for delivery of rnai triggers to tumor cells in vivo.
The applicant listed for this patent is Arrowhead Pharmaceuticals, Inc.. Invention is credited to Aaron M. Almeida, Andrei V. Blokhin, Jeffrey C. Carlson, Weijun Cheng, David B. Rozema, So Wong.
Application Number | 20190062748 16/189550 |
Document ID | / |
Family ID | 52449177 |
Filed Date | 2019-02-28 |
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United States Patent
Application |
20190062748 |
Kind Code |
A1 |
Rozema; David B. ; et
al. |
February 28, 2019 |
Polyconjugates for Delivery of RNAi triggers to Tumor Cells In
Vivo
Abstract
The present invention is directed compositions for delivery of
RNA interference (RNAi) triggers to integrin positive tumor cells
in vivo. The compositions comprise RGD ligand-targeted amphipathic
membrane active polyamines reversibly modified with enzyme
cleavable dipeptide-amidobenzyl-carbonate masking agents.
Modification masks membrane activity of the polymer while
reversibility provides physiological responsiveness. The reversibly
modified polyamines (dynamic polyconjugate or conjugate) are
further covalently linked to an RNAi trigger.
Inventors: |
Rozema; David B.; (Cross
Plains, WI) ; Wong; So; (Oregon, WI) ; Cheng;
Weijun; (Middleton, WI) ; Almeida; Aaron M.;
(Madison, WI) ; Blokhin; Andrei V.; (Fitchburg,
WI) ; Carlson; Jeffrey C.; (Madison, WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Arrowhead Pharmaceuticals, Inc. |
Pasadena |
CA |
US |
|
|
Family ID: |
52449177 |
Appl. No.: |
16/189550 |
Filed: |
November 13, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15278518 |
Sep 28, 2016 |
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16189550 |
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14452626 |
Aug 6, 2014 |
9487556 |
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15278518 |
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61863056 |
Aug 7, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 5/06078 20130101;
C12N 2320/32 20130101; C07K 5/06156 20130101; A61P 35/00 20180101;
C07K 5/06026 20130101; C07K 5/06052 20130101; C12N 2310/3535
20130101; A61K 31/713 20130101; C12N 15/113 20130101; C12N 2310/322
20130101; A61P 43/00 20180101; C07K 5/0817 20130101; C07K 5/06008
20130101; C07K 5/06043 20130101; C12N 2310/3513 20130101; C12N
2310/14 20130101 |
International
Class: |
C12N 15/113 20100101
C12N015/113; C07K 5/078 20060101 C07K005/078; A61K 47/18 20170101
A61K047/18; C07K 5/06 20060101 C07K005/06; C07K 5/062 20060101
C07K005/062; A61K 31/713 20060101 A61K031/713; C07K 5/065 20060101
C07K005/065 |
Claims
1. An RGD ligand-containing moiety comprising the structure
represented by: ##STR00067## wherein: R.sup.14 is ##STR00068## and
A comprises a linker, wherein the RGD ligand-containing moiety is
bound to a carbamate-containing moiety and an RNAi trigger.
2. The RGD ligand-containing moiety of claim 1, wherein A
comprises: ##STR00069## wherein n is 0, 1, 2, or 3, Y is absent or
##STR00070## Z is absent, ##STR00071## m is 0, 1, 2, 3, or 4, and
PEG is (CH.sub.2--CH.sub.2--O).sub.4-44.
3. The RGD ligand-containing moiety of claim 1, wherein R.sup.14 is
##STR00072##
4. The RGD ligand-containing moiety of claim 1, wherein the RGD
ligand-containing moiety is of the structure: ##STR00073## wherein
A' comprises a PEG-containing linker; R.sup.1 is a side group of an
alanine, phenylalanine, valine, leucine, isoleucine, and
tryptophan; R.sup.2 is a side chain of a citrulline, glycine,
threonine, asparagine, and glutamine; and the RGD ligand is bound
to an RNAi trigger.
5. The RGD ligand of claim 4, wherein R.sup.1 is the side group of
alanine.
6. The RGD ligand of claim 4, wherein R.sup.1 is the side group of
phenylalanine.
7. The RGD ligand of claim 4, wherein R.sup.1 is the side group of
leucine.
8. The RGD ligand of claim 4, wherein R.sup.1 is the side group of
isoleucine.
9. The RGD ligand of claim 4, wherein R.sup.1 is the side group of
tryptophan.
10. The RGD ligand of claim 4, wherein R.sup.2 is the side group of
citrulline.
11. The RGD ligand of claim 4, wherein R.sup.2 is the side group of
glycine.
12. The RGD ligand of claim 4, wherein R.sup.2 is the side group of
threonine.
13. The RGD ligand of claim 4, wherein R.sup.2 is the side group of
asparagine.
14. The RGD ligand of claim 4, wherein R.sup.2 is the side group of
glutamine.
15. A masking agent of the formula:
(R)-A.sup.1A.sup.2-amidobenzyl-carbonate, wherein, R is of the
formula: ##STR00074## R.sup.14 is ##STR00075## and A comprises a
linker; A.sup.1 is selected from alanine, phenylalanine, valine,
leucine, isoleucine, and tryptophan; and A.sup.2 is selected from
citrulline, glycine, threonine, asparagine, and glutamine.
16. The masking agent of claim 15, wherein R.sup.2 is
citrulline.
17. The masking agent of claim 15, wherein R.sup.14 is
##STR00076##
18. The masking agent of claim 15, wherein A comprises:
##STR00077## wherein n is 0, 1, 2, or 3, Y is absent or
##STR00078## Z is absent, ##STR00079## m is 0, 1, 2, 3, or 4, and
PEG is (CH.sub.2--CH.sub.2--O).sub.4-44.
19. The masking agent of claim 18, wherein n is 3.
20. The masking agent of claim 18, wherein Y is ##STR00080## and Z
is ##STR00081##
Description
PRIORITY
[0001] The present application is a continuation of U.S. patent
application Ser. No. 15/278,518, filed Sep. 28, 2016, which is a
continuation of Ser. No. 14/452,626, filed Aug. 6, 2014, now U.S.
Pat. No. 9,487,556, which claims the benefit of U.S. Provisional
Patent Application No. 61/863,056, filed Aug. 7, 2013, the contents
of each of which are incorporated herein in their entirety.
BACKGROUND OF THE INVENTION
[0002] The delivery of RNAi triggers and other substantially cell
membrane impermeable compounds into a living cell is highly
restricted by the complex membrane system of the cell. Drugs used
in antisense, RNAi, and gene therapies are relatively large
hydrophilic polymers and are frequently highly negatively charged.
Both of these physical characteristics severely restrict their
direct diffusion across the cell membrane. For this reason, the
major barrier to RNAi trigger delivery is the delivery of the RNAi
trigger across a cell membrane to the cell cytoplasm or
nucleus.
[0003] Numerous transfection reagents have also been developed that
achieve reasonably efficient delivery of polynucleotides to cells
in vitro. However, in vivo delivery of polynucleotides using these
same transfection reagents is complicated and rendered ineffective
by in vivo toxicity, adverse serum interactions, and poor
targeting. Transfection reagents that work well in vitro, cationic
polymers and lipids, typically form large cationic electrostatic
particles and destabilize cell membranes. The positive charge of in
vitro transfection reagents facilitates association with nucleic
acid via charge-charge (electrostatic) interactions thus forming
the nucleic acid/transfection reagent complex. Positive charge is
also beneficial for nonspecific binding of the vehicle to the cell
and for membrane fusion, destabilization, or disruption.
Destabilization of membranes facilitates delivery of the
substantially cell membrane impermeable polynucleotide across a
cell membrane. While these properties facilitate nucleic acid
transfer in vitro, they cause toxicity and ineffective targeting in
vivo. Cationic charge results in interaction with serum components,
which causes destabilization of the polynucleotide-transfection
reagent interaction, poor bioavailability, and poor targeting.
Membrane activity of transfection reagents, which can be effective
in vitro, often leads to toxicity in vivo.
[0004] For in vivo delivery, the vehicle (nucleic acid and
associated delivery agent) should be small, less than 100 nm in
diameter, and preferably less than 50 nm. Even smaller complexes,
less that 20 nm or less than 10 nm would be more useful yet.
Delivery vehicles larger than 100 nm have very little access to
cells other than blood vessel cells in vivo. Complexes formed by
electrostatic interactions tend to aggregate or fall apart when
exposed to physiological salt concentrations or serum components.
Further, cationic charge on in vivo delivery vehicles leads to
adverse serum interactions and therefore poor bioavailability.
Interestingly, high negative charge can also inhibit targeted in
vivo delivery by interfering with interactions necessary for
targeting, i.e. binding of targeting ligands to cellular receptors.
Thus, near neutral vehicles are desired for in vivo distribution
and targeting. Without careful regulation, membrane disruption or
destabilization activities are toxic when used in vivo. Balancing
vehicle toxicity with nucleic acid delivery is more easily attained
in vitro than in vivo.
[0005] Rozema et al., (U.S. Patent Publications 20080152661,
20110207799, 20120165393, and 20120172412) developed conjugates
suitable for in vivo delivery of polynucleotides. These conjugates
featured reversible regulation of membrane disruptive activity of a
membrane active polyamine using reversible physiologically labile
masking. Using uncharged galactose or cholesterol as targeting
ligands, Rozema et al. have shown in vivo delivery of
polynucleotides to hepatocytes using these conjugates. Adaptation
of these conjugates to target RNAi triggers to cancer cells would
provide another therapeutic in the fight against cancer.
[0006] Integrins are a group of cell surface glycoproteins which
mediate cell adhesion. Integrins are heterodimers composed of
.alpha. and .beta. polypeptide subunits. Currently eleven different
.alpha. subunits and six different .beta. subunits have been
identified. The various .alpha. subunits combine with various
.beta. subunits to form distinct integrins. The
.alpha..sub..nu..beta..sub.3 integrin (vitronectin receptor) has
been shown to play a role in tumor metastases, solid tumor growth
(neoplasia), and tumor angiogenesis. The integrin
.alpha..sub.v.beta..sub.3 plays an important role in angiogenesis.
It is expressed on tumoral endothelial cells as well as on some
tumor cells. Seftor et al. (Proc. Natl. Acad. Sci. USA, Vol. 89
(1992) 1557-1561), for example, have shown a role for
.alpha..sub..nu..beta..sub.3 integrin in melanoma cell invasion.
Brooks et al. (Cell, Vol. 79 (1994) 1157-1164) demonstrated that
systemic administration of .alpha..sub.v.beta..sub.3 antagonists
caused dramatic regression of various histologically distinct human
tumors.
[0007] Tumor cell expression of the integrins
.alpha..sub.v.beta..sub.3 is correlated with disease progression in
various tumor types. .alpha..sub.v.beta..sub.3 integrin is widely
expressed on blood vessels of human tumor biopsy samples but not on
vessels in normal tissues. In breast cancer, overexpression of
.alpha..sub.v.beta..sub.3 integrin is associated with bone
metastasis and induces increased tumor growth and invasion in
response to osteopontin. In glioblastoma, .alpha..sub.v.beta..sub.3
integrin is overexpressed at the invasive margins of the tumor and
levels of fibronectin are increased, which is associated with
enhanced cell motility and apoptosis resistance. In pancreatic
tumor, the increased expression of .alpha..sub.v.beta..sub.3
integrin is associated with increased activation of MMP-2 and lymph
node metastasis. In prostate carcinoma cell,
.alpha..sub.v.beta..sub.3 integrin is expressed resulting in
metastasis to bone because of an association between integrins and
processes of attachment and migration involving laminin,
fibronectin, and osteopontin.
[0008] .alpha..sub.v.beta..sub.3 integrins bind to a number of
Arg-Gly-Asp (RGD) containing matrix macromolecules. The RGD peptide
sequence has been linked to various other compounds to provide
.alpha..sub.v.beta..sub.3 integrin binding. Therefore, RGD peptides
have been examined for targeting of compounds to
.alpha..sub.v.beta..sub.3 integrin positive tumors. However, in
addition to relatively low affinity, many RGD peptides are also
relatively non-selective for RGD-dependent integrins. For example,
most RGD peptides which bind to .alpha..sub..nu..beta..sub.3 also
bind to .alpha..sub..nu..beta..sub.5, .alpha..sub..nu..beta..sub.1,
and .alpha..sub.IIb.beta..sub.3 integrins.
SUMMARY OF THE INVENTION
[0009] We describe compositions for delivering RNAi triggers to
tumor cells in mammals in vivo comprising: integrin-targeted
reversibly masked membrane active polyamines covalently linked to
RNAi triggers. The described compositions deliver RNAi triggers to
tumor cells where the RNAi triggers interact with the cells'
endogenous RNA interference pathways to inhibit expression of
target genes.
[0010] The invention features a composition for delivering an RNA
interference (RNAi) trigger to a tumor cell in vivo comprising: a
masked amphipathic membrane active polyamine (delivery polymer) and
an RNAi trigger wherein the RNAi trigger is covalently linked to
the delivery polymer. The delivery polymer comprises an amphipathic
membrane active polyamine masked by reversible modification of
polymer amines with one or more RGD dipeptide masking agents and
optionally one or more PEG dipeptide masking agents such that at
least 50% or at least 80% of the polymer amines are modified. A
preferred linkage for covalent attachment of the delivery polymer
to the RNAi trigger is a physiologically labile linkage. In one
embodiment, this linkage is orthogonal to the dipeptide masking
agent linkage. The delivery conjugate is administered to a mammal
in a pharmaceutically acceptable carrier or diluent.
[0011] In a preferred embodiment, we describe a composition
comprising: an amphipathic membrane active polyamine covalently
linked to: a) a plurality of RGD ligands and steric stabilizers via
dipeptide-amidobenzyl-carbamate reversible physiologically labile
linkages; and b) one or more RNAi triggers via one or more labile
covalent linkages. In one embodiment, the
dipeptide-amidobenzyl-carbamate is orthogonal to the labile
covalent linkage. The RNAi trigger-polymer conjugate is
administered to a mammal in a pharmaceutically acceptable carrier
or diluent.
[0012] In a preferred embodiment, a reversibly masked membrane
active polyamine (delivery polymer) comprises: an amphipathic
membrane active polyamine reversibly modified by reaction of amines
of the polyamine with RGD masking agents and steric stabilizer
masking agents. Reaction of a polymer amine with a masking agent
reversibly modifies the amine to form a reversible physiologically
labile covalent linkage. An amine is reversibly modified if
cleavage of the modifying group restores the amine. Reversible
modification of the membrane active polyamine reversibly inhibits
membrane activity of the membrane active polyamine, inhibits
interaction of the polyamine with serum components thereby
providing increased circulation properties, and targets the
polyamine to a tumor cell in vivo. In the masked state, the
reversibly masked membrane active polyamine does not exhibit
membrane disruptive activity. Membrane activity inhibition and/or
in vivo targeting of the membrane active polyamine requires
modification of >50% of the polymer amines. Reversible
modification of more than 50%, more than 55%, more than 60%, more
than 65%, more than 70%, more than 75%, more than 80%, more than
85%, or more than 90% of the amines on the polyamine with masking
agents may be required to form an optimal delivery polymer.
[0013] A modified polymer amine of the delivery polymers of the
invention is represented by:
##STR00001##
wherein R.sup.4 comprises an RGD ligand or steric stabilizer and
R.sup.1 and R.sup.2 are amino acid side chains. R.sup.1 is
preferably a side group of a hydrophobic amino acid. A preferred
hydrophobic amino acid is an alanine. R.sup.2 is preferably a side
chain of a hydrophilic uncharged amino acid at neutral pH. A
preferred hydrophilic uncharged amino acid is a citrulline. In vivo
enzymatic cleavage after the dipeptide, between the amino acid and
the amidobenzyl group, by removes R.sup.4 from the polymer and
initiates an elimination reaction in which the
amidobenzyl-carbamate undergoes a spontaneous rearrangement that
results in regeneration of the polymer amine.
[0014] The delivery polymer is further covalently linked to the
RNAi trigger. In one embodiment, the RNAi trigger is linked to the
delivery polymer via a physiologically labile bond. In a preferred
embodiment, the labile bond connecting the RNAi trigger to the
delivery polymer is orthogonal to the labile bond connected the
masking agents to the polyamine. Thus, conjugates of the invention
comprise: an RNAi trigger covalently linked to a reversibly
modified amphipathic membrane active polyamine having the general
form represented by:
##STR00002##
wherein N comprises an RNAi trigger, L.sup.2 is a reversible
physiologically labile linkage such as
A.sup.1A.sup.2-amidobenzyl-carbamate, P comprises an amphipathic
membrane active polyamine, R comprises an RGD ligand, each as
defined herein, PEG comprises a polyethylene glycol or other steric
stabilizer, L.sup.1 is a physiologically labile linker, y is an
integer greater than zero and z is an integer greater than zero
(0), wherein the value of the sum of y and z is greater than 50% of
the number of amines present on polyamine P as determine be the
number of amines in the unmodified membrane active polyamine.
[0015] The compounds according to the present invention can be
generally obtained using methods known to the person of ordinary
skill in the art of organic or medicinal chemistry. Further
objects, features, and advantages of the invention will be apparent
from the following detailed description when taken in conjunction
with the accompanying drawings.
[0016] In a preferred embodiment, polymer modifications -L.sup.2-R
and -L.sup.2-PEG have the general form:
R-A.sup.1A.sup.2-amidobenzyl-carbamate- (formula 2a)
and
PEG-A.sup.1A.sup.2-amidobenzyl-carbamate- (formula 2b).
wherein A.sup.1A.sup.2 is a dipeptide, A.sup.1 is an amino acid,
and A.sup.2 is an amino acid. An RGD ligand may be linked to the
dipeptide via a linker such as a PEG linker. A preferred steric
stabilizer is a polyethylene glycol (PEG). A.sup.1 is preferably a
hydrophobic amino acid. A.sup.2 is preferably a hydrophilic
uncharged amino acid. A.sup.1 and A.sup.2 are preferably linked via
an amide bond. A preferred amidobenzyl group is a p-amidobenzyl
group. The carbamate is formed by reaction of a carbonate with a
polymer amine. A preferred carbonate is an activated amine reactive
carbonate. The A.sup.1A.sup.2-amidobenzyl-carbamate linkage is
stable until the dipeptide is cleaved in vivo by an endogenous
protease, thus cleaving the steric stabilizer or RGD ligand from
the polyamine. Following enzymatic cleavage after the dipeptide
(between A.sup.2 and the amidobenzyl), the amidobenzyl-carbamate
undergoes a spontaneous rearrangement which results in regeneration
of the polymer amine.
[0017] In one embodiment, the RGD ligand is linked to the dipeptide
using a linker that aids in attachment of the RGD ligand to the
dipeptide and in solubility of the masking agent. A preferred
asking agent has the general form: RGD ligand-PEG1-diaryl
hydrazone-PEG2-dipeptide-amidobenzyl-carbonate. Each of the
components can be linked using standard methods in the art, such as
formation of amide linkages. The diaryl hydrazone can be formed by
reaction of a HyNic (hydrazino-nicotinamide) group with an aryl
aldehyde. PEG1 comprises (CH.sub.2--CH.sub.2--O).sub.n and PEG2
comprises (CH.sub.2--CH.sub.2--O).sub.n. n and m are independently
integers greater than or equal to 4 and the sum of n+m is 12-48.
The PEG groups aid in solubility and presentation of the RGD
ligand, thereby improving tumor targeting of the modified
polyamine. Surprisingly, the diaryl hydrazone also improves in vivo
function of the modified polyamine. In one embodiment, an aryl
aldehyde-PEG2-dipeptide-amidobenzyl-carbonate is first reacted with
a polyamine to form an aryl
aldehyde-PEG2-dipeptide-amidobenzyl-carbamate-polyamine. This
compound is then reacted with an RGD ligand-PEG1-HyNic to from: RGD
ligand-PEG1-diaryl
hydrazone-PEG2-dipeptide-amidobenzyl-carbomate-polyamine.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1. Illustrations showing the structure of (A) a
dipeptide masking agent or (B) a dipeptide masking agent linked to
a polyamine: R.sup.1 and R.sup.2 are the R groups of amino acids,
R.sup.4 comprises an RGD ligand or a steric stabilizer, --X-- is
--NH--, --O--, or --CH.sub.2--, --Y-- is --NH-- or --O--, --R.sup.5
is at position 2, 4, or 6 and is --CH.sub.2--O--C(O)--O--Z wherein
Z carbonate, and --R.sup.6 is independently hydrogen, alkyl, or
halide at each of positions 2, 3, 4, 5, or 6 except for the
position occupied by R.sup.5.
[0019] FIG. 2. Illustration showing the structures of various PEG
dipeptide masking agents: (A) PEG-GlyGly-PABC-PNP, (B)
PEG-AsnGly-PABC-PNP, (C) PEG-PheLys-PABC-PNP, (D)
PEG-ValCit-PABC-PNP, (E) PEG-AlaAsn-PABC-PNP, and (F)
PEG-PheLys(CH3)2-PABC-PNP.
[0020] FIG. 3. Illustration showing reversible modification of a
polyamine using a dipeptide masking agent: R comprises an RGD
ligand or a PEG, AA is a dipeptide (either with or without
protecting groups), R.sup.3 is an amine-reactive carbonate, and
polyamine is an amphipathic membrane active polyamine.
[0021] FIG. 4. Illustration showing the elimination reaction in
which the amidobenzyl-carbamate undergoes a spontaneous
rearrangement that results in regeneration of a polymer amine: AA
(A.sup.1A.sup.2) is a dipeptide, and R.sup.4 comprises an RGD
ligand or a steric stabilizer.
[0022] FIG. 5. Illustration showing synthesis of PEG dipeptide
masking agents: R comprises a PEG, and A.sup.1 and A.sup.2 are
amino acids (either protected or unprotected).
[0023] FIG. 6. Illustrations showing formation of (A) NHS esters of
dipeptides, (B) amino acids H-Asn(DMCP)-OH and H-Lys(MMT)-OH from
Fmoc-protected derivatives, and (C) Fmoc-A.sup.1A.sup.2-OH: A,
A.sup.1, and A.sup.2 are amino acids.
[0024] FIG. 7. Illustrations showing (A) formation of Fmoc-AA-PABA
and Fmoc-A-PABA and (B) coupling of H-Lys(CH.sub.3).sub.2-PABA with
Fmoc-Phe-NHS.
[0025] FIG. 8. Illustration showing formation of
H-A.sup.1A.sup.2-PABA and H-A.sup.1-PABA.
[0026] FIG. 9. Illustration showing formation of
PEG.sub.n-A.sup.1A.sup.2-PABA.
[0027] FIG. 10. Illustration showing formation of (A) and (B)
PEG-AA-PABC-PNP.
[0028] FIG. 11. Illustration showing RAFT copolymerization of
N-Boc-ethylethoxy acrylate and propyl methacrylate.
[0029] FIG. 12. Illustration showing terminal polymer modification
with azido-PEG-amine.
[0030] FIG. 13. Illustration showing a polyaminer modified by one
example of an RGD masking agent: RGD ligand-PEG1-diamine-diaryl
hydrazone-PEG2-dipeptide-amidobenzyl-carbomate-polyamine. Atoms not
explicitly indicated as being part of a unit by the (i.e. RGD
ligand, PEG1, etc.) are considered linking atoms and may be
considered to be part of the labeled unit to either side.
DETAILED DESCRIPTION OF THE INVENTION
[0031] The present invention provides conjugates and methods for
delivering RNA interference (RNAi) triggers into integrin
expressing tumor cells in vivo. The described conjugates comprise
integrin-targeted reversibly modified membrane active polyamines
covalently linked to the RNAi trigger to be delivered. Integrin
targeting is provided by RGD ligands described herein. Reversible
modification of the membrane active polyamine is provided by RNA
ligand and steric stabilizer peptidase cleavable masking agents
described herein. The peptidase cleavable linkages are stable to
hydrolysis in absence of protease, and provide extended stability
in storage and in in vivo circulation. Improved (longer) half-life
in circulation facilitates widening of the window of opportunity
for RGD ligand-mediated accumulation in tissue, such as tumor
tissue. In vivo delivery of RNAi triggers is useful for therapeutic
inhibition (knockdown) of gene expression.
[0032] The invention includes conjugate delivery systems of the
general structure:
##STR00003##
wherein N is an RNAi trigger, L.sup.1 is a physiologically labile
linkage, P is an amphipathic membrane active polyamine, M.sup.1
comprises an RGD ligand linked to P via a
dipeptide-amidobenzyl-carbamate linkage (RGD masking agent), and
M.sup.2 comprises a steric stabilizer linked to P via a
dipeptide-amidobenzyl-carbamate linkage (PEG masking agent). y and
z are each integers greater than zero provided the value of y+z has
a value greater than 50%, greater than 60%, greater than 70%,
greater than 80% or greater than 90% of the number of primary
amines on polyamine P, as determined by the quantity of amines on P
in the absence of any masking agents. In its unmodified state, P is
a membrane active polyamine. Delivery polymer
M.sup.1.sub.y-P-M.sup.2.sub.z is not membrane active. Reversible
modification of P primary amines, by attachment of M.sup.1 and
M.sup.2, reversibly inhibits or inactivates membrane activity of P.
It is noted that some small amphipathic membrane active polyamine,
such as melittin peptide, contain as few as 3-5 primary amines.
Modification of a percentage of amines is meant to reflect the
modification of a percentage of amines in a population of polymers.
Upon cleavage of M.sup.1 and M.sup.2, amines of the polyamine are
regenerated thereby reverting P to its unmodified, membrane active
state.
[0033] For tumor deliver, y has a value equal to 2-20% of the
number of primary amines on polymer P. More preferably, y has a
value equal to 2-10% of the number of primary amines on polymer P.
z therefore has a value equal to 80-98% of the number of primary
amines on polymer P. The ratio y (RGD):z (steric stabilizer) is
preferably about 1-12:50 and more preferably about 1:20.
[0034] In the masked state, the reversibly masked membrane active
polyamine does not exhibit membrane disruptive activity. Reversible
modification of more than 50%, more than 55%, more than 60%, more
than 65%, more than 70%, more than 75%, more than 80%, more than
85%, or more than 90% of the amines on the polyamine with dipeptide
masking agents may be required to inhibit membrane activity and
provide cell targeting function, i.e. form a reversibly masked
membrane active polymer (delivery polymer).
[0035] In one embodiment, the RNAi trigger is linked to the
delivery polymer of the invention via a physiologically labile
covalent linkage. By using a physiologically labile linkage, the
RNAi trigger can be cleaved from the polymer, releasing the RNAi
trigger to engage in functional interactions with cell
components.
[0036] Masking is accomplished through reversible attachment of the
described masking agents to the membrane active polyamine to form a
reversibly masked membrane active polymer, i.e. a delivery polymer.
In addition to inhibiting membrane activity, the masking agents
shield the polymer from non-specific interactions, reduce serum
interactions, increase circulation time, and/or provide
cell-specific interactions, i.e. targeting.
[0037] It is an essential feature of the masking agents that, in
aggregate, they inhibit membrane activity of the polymer. Masking
agents may shield the polymer from non-specific interactions
(reduce serum interactions, increase circulation time). The
membrane active polyamine is membrane active in the unmodified
(unmasked) state and not membrane active (inactivated) in the
modified (masked) state. A sufficient number of masking agents are
linked to the polymer to achieve the desired level of inactivation.
The desired level of modification of a polymer by attachment of
masking agent(s) is readily determined using appropriate polymer
activity assays. For example, if the polymer possesses membrane
activity in a given assay, a sufficient level of masking agent is
linked to the polymer to achieve the desired level of inhibition of
membrane activity in that assay. Masking requires modification of
.gtoreq.50%, .gtoreq.60%, .gtoreq.70%, .gtoreq.80% or .gtoreq.90%
of the primary amine groups on a population of polymer, as
determined by the quantity of primary amines on the polymer in the
absence of any masking agents. It is desirable that the masked
polymer retain aqueous solubility.
[0038] It is an essential feature of the RGD masking agents that,
in aggregate, they target the delivery polymer to
.alpha..sub..nu..beta..sub.3 integrin positive tumor cells. A
sufficient number of masking agents are linked to the polymer to
achieve the tumor cellular targeting. Targeting may require
modification of about 2% to about 20%, about 2% to about 10%, or
about 3% to about 6% of the primary amine groups on a population of
polymer, as determined by the number of primary amines on the
polymer in the absence of any masking agents.
[0039] In one embodiment, an RGD masking agent suitable for
modification of a polyamine to form an integrin-targeted delivery
polymer comprises: an RGD ligand covalently linked to a
dipeptide-amidobenzyl-carbonate (RGD dipeptide masking agent).
Similarly, a steric stabilizer dipeptide masking agent suitable for
modification of a polyamine to form an integrin-targeted delivery
polymer comprises: a steric stabilizer covalently linked to a
dipeptide-amidobenzyl-carbonate. The masking agents have the
general form:
(R or PEG)-A.sup.1A.sup.2-amidobenzyl-carbonate.
wherein R comprises an RGD ligand, PEG comprises a polyethylene
glycol of other steric stabilizer, A.sup.1A.sup.2 is a dipeptide
containing a first amino acid A.sup.1 and a second amino acid
A.sup.2, and carbonate is an activated amine-reactive carbonate.
Reaction of the masking agent carbonate with a polymer amine yields
a carbamate linkage. The RNA ligand or steric stabilizer may be
attached to the dipeptide prior to reaction of the carbonate with
the polymer amine or after formation of the carbamate linkage. The
masking agent is stably linked to the polymer until the dipeptide
is cleaved in vivo by an endogenous protease, thus cleaving the RGD
ligand or steric stabilizer from the polyamine. Following enzymatic
cleavage after the dipeptide (between A.sup.2 and the amidobenzyl),
the amidobenzyl-carbamate undergoes a spontaneous rearrangement
which results in regeneration of the polymer amine. An RGD ligand
may be linked to the dipeptide via a linker such as a PEG linker. A
preferred steric stabilizer masking agent is uncharged. A preferred
uncharged steric stabilizer is a polyethylene glycol (PEG). A
preferred dipeptide consists of a hydrophobic amino acid linked
(A.sup.1) to a hydrophilic uncharged amino acid (A.sup.2) via an
amide bond. A preferred amidobenzyl group is a p-amidobenzyl group.
A preferred carbonate is an activated amine reactive carbonate.
[0040] Masking agents suitable for formation of integrin-targeted
delivery polymers of the invention have the general structure:
##STR00004##
wherein R.sup.4 comprises an RGD ligand or steric stabilizer,
R.sup.3 comprises an amine reactive carbonate moiety, and R.sup.1
and R.sup.2 are amino acid side chains. R.sup.1 is preferably a
side group of a hydrophobic amino acid. A preferred hydrophobic
amino acid is an alanine. R.sup.2 is preferably a side chain of a
hydrophilic uncharged amino acid. A preferred hydrophilic uncharged
amino acid is a citrulline. A preferred activated carbonate is a
para-nitrophenol. However, other amine reactive carbonates known in
the art are readily substituted for the para-nitrophenol. Reaction
of the activated carbonate with an amine connects the RGD ligand or
steric stabilizer to the membrane active polyamine via a peptidase
cleavable dipeptide-amidobenzyl carbamate linkage as represented
by:
##STR00005##
wherein R.sup.4, R.sup.1, and R.sup.2 are as described above.
Enzyme cleavage after the dipeptide, between the amino acid and the
amidobenzyl group, removes R.sup.4 from the polymer and triggers an
elimination reaction in which the amidobenzyl-carbamate undergoes a
spontaneous rearrangement which results in regeneration of the
polymer amine.
[0041] In another embodiment, attachment of the RGD ligand to the
polyamine via a reversible physiologically labile linkage is
achieved by first reversibly modifying the amine with a
dipeptide-amidobenzyl-carbonate have the general structure:
##STR00006##
wherein R.sup.7 comprises a reactive group suitable for reaction
with an RGD ligand-containing moiety and less amine reactive than
the carbonate of R.sup.3. R.sup.1, R.sup.2, and R.sup.3 are as
defined above. In one embodiment, R.sup.7 further comprises a PEG
linking moiety (also termed PEG2 herein). We have found that
inserting a PEG linking moiety between the dipeptide and the
reactive group improves solubility and in vivo function of the
assembled RGD masking agent. A preferred PEG linking moiety is a
(CH.sub.2--CH.sub.2--O).sub.4-44. After modification of a polymer
amine with the reactive group (R.sup.7)-containing
dipeptide-amidobenzyl-carbonate, the RGD ligand-containing moiety
is covalently linked via reaction with reactive group R.sup.7.
Exemplary reactive group moieties suitable linking the dipeptide
and the RGD ligand-containing moiety include, but are not limited
to: HyNic and aldehyde (including aryl aldehyde), "Click" chemistry
crosslinkers (certain azides and alkynes). An exemplary molecule of
formula 3 is a (4-formylbenzaldehyde)-PEG-Ala-Cit-ara-aminobenzyl
carbonate.
[0042] Dipeptides of the dipeptide masking agents, represented
herein as A.sup.1A.sup.2 (or AA), are dimers of amino acids
connected via amide bonds. Amino acids, including .alpha. and
.beta. amino acids are well known in biology and chemistry and are
molecules containing an amine group, a carboxylic acid group and a
side-chain that varies between different amino acids. A preferred
amino acid is an L .alpha.-amino acid having the generic formula
H.sub.2NCHRCOOH, where R (R.sup.1 and R.sup.2 of formula 3) is an
organic substituent or side group. A preferred L .alpha. amino acid
is an uncharged naturally occurring amino acid. In a preferred
dipeptide, A.sup.1 is a hydrophobic amino acid and A.sup.2 is an
uncharged hydrophilic amino acid. A preferred hydrophobic amino
acid is phenylalanine, valine, isoleucine, leucine, alanine, or
tryptophan. A preferred uncharged hydrophilic amino acid is
asparagine, glutamine, or citrulline. A more preferred hydrophobic
amino acid is alanine or phenylalanine. A more preferred uncharged
hydrophilic amino acid is citrulline. While dipeptides are
preferred, it is possible to insert additional amino acids between
A.sup.1 and R. It is also possible to use a single amino acid
instead of a dipeptide by eliminating amino acid A.sup.1. Any
natural amino acids used in the present invention are referred to
herein by their common abbreviations.
[0043] In a preferred embodiment, an amphipathic membrane active
polyamine is reversibly modified by reaction with a described
dipeptide-amidobenzyl-carbonate masking agent to yield a membrane
inactive delivery polymer. The dipeptide masking agents shield the
polymer from non-specific interactions, increase circulation time,
enhance specific interactions, inhibit toxicity, or alter the
charge of the polymer.
[0044] Reversibly masked polymers of the invention comprise the
structure:
##STR00007##
wherein: [0045] X is --NH--, --O--, or --CH.sub.2-- [0046] Y is
--NH-- or --O-- [0047] R.sup.1 is preferably
--(CH.sub.2).sub.k-phenyl (k is 1, 2, 3, 4, 5, 6; k=1
phenylalanine), --CH--(CH.sub.3).sub.2 (valine),
--CH.sub.2--CH--(CH.sub.3).sub.2 (leucine),
--CH(CH.sub.3)--CH.sub.2--CH.sub.3 (isoleucine), --CH.sub.3
(alanine), or
[0047] ##STR00008## [0048] R.sup.2 is preferably hydrogen
(glycine), --(CH.sub.2).sub.3--NH--C(O)--NH.sub.2 (citrulline),
--CH.sub.2--C(O)--NH.sub.2 (asparagine),
--(CH.sub.2).sub.2--C(O)--NH.sub.2 (glutamine),
--(CH.sub.2).sub.4--N--(CH.sub.3).sub.2 (lysine(CH.sub.3).sub.2),
--(CH.sub.2).sub.k--C(O)--NH.sub.2; (k is 1, 2, 3, 4, 5, 6),
--CH.sub.2--C(O)--NR'R'' (aspartic acid amide),
--(CH.sub.2).sub.2--C(O)--NR'R'' (glutamic acid amide),
--CH.sub.2--C(O)--OR' (aspartic acid ester), or
--(CH.sub.2).sub.2--C(O)--OR' (glutamic acid ester), wherein R' and
R'' are alkyl groups [0049] R.sup.4 comprises an RGD ligand or a
polyethylene glycol; and [0050] the polyamine is an amphipathic
membrane active polyamine.
[0051] While the structure above indicates a single dipeptide
masking agent linked to the polymer, in practice of the invention,
50% to 100% of polymer amines are modified by dipeptide masking
agents.
[0052] In a preferred embodiment, a reversibly masked polymer of
the invention comprises the structure:
##STR00009##
wherein R.sup.1, R.sup.2, R.sup.4 and polyamine as described
above.
[0053] Reversibly masked polymers of the invention can be formed by
reaction of dipeptide masking agents of the invention with amines
on the polymer. Dipeptide masking agents of the invention have the
structure:
##STR00010##
wherein: [0054] X, Y, R.sup.1, R.sup.2, and R.sup.4 are as
described above [0055] R.sup.5 is at position 2, 4, or 6 and is
--CH.sub.2--O--C(O)--O--Z wherein Z is [0056] Halide,
##STR00011##
[0056] and [0057] R.sup.6 is independently hydrogen, alkyl,
--(CH.sub.2).sub.n--CH.sub.3 (wherein n=0-4),
--(CH.sub.2)--(CH.sub.3).sub.2, or halide at each of positions 2,
3, 4, 5, or 6 except for the position occupied by R.sup.5.
[0058] In a preferred embodiment, X is --NH--, Y is --NH--, R.sup.4
comprises an RGD ligand or PEG group, R.sup.5 is at position 4, and
R.sup.6 is hydrogen as shown by:
##STR00012##
[0059] In another embodiment, R.sup.4 of formula 4 is
R.sup.8--(O--CH.sub.2--CH.sub.2).sub.s--O--Y.sup.1--, wherein:
R.sup.8 is hydrogen, methyl, or ethyl; and s is an integer from 1
to 150, and Y.sup.1 is a linker suitable in the art for connecting
a PEG group to the dipeptide. Suitable linkers Y.sup.1 include, but
are not limited to: --(CH.sub.2).sub.1-3--C(O)--,
--Y.sup.2--NH--C(O)--(CH.sub.2).sub.2--C(O)-- (wherein Y.sup.2 is
--(CH.sub.2).sub.3--), and
--C(O)--N--(CH.sub.2--CH.sub.2--O).sub.p--CH.sub.2--CH.sub.2-- (p
is an integer from 1 to 20).
[0060] As used herein, an RGD ligand comprises a zwitterionic RGD
peptide or RGD mimic <1500 kDa in size that binds to (has
affinity for) the alpha v/beta 3 (.alpha.v.beta.3 or
.alpha..sub.v.beta..sub.3) integrin.
[0061] As used herein, an RGD peptide comprises an
arginine-glycine-aspartate tripeptide. An RGD peptide may further
comprise additional amino acids amino or carboxy terminal to the
RGD sequence. If additional amino acids are present, the contiguous
peptide sequence constitutes the RGD peptide. An RGD peptide may be
conformationally constrained. Conformational constraint is
typically accomplished by cyclization of the peptide, such as by
adding Cysteine amino acids amino and carboxy terminal of the RGD
sequence and forming a disulfide bond between the cysteine thiols.
A preferred constrained RGD peptide comprises the amino acid
sequence:
X.sub.n1C.sub.mX.sub.n2CX.sub.n3RGDX.sub.n4CX.sub.n5C.sub.mX.sub.n6
(SEQ ID 1) wherein X is a naturally occurring amino acid, m is zero
(0) or one (1), and n1-n6 are independently 0, 1, 2, or 3. If
present (n=1, 2, or 3), the one or more amino acids at each X are
independent of the selection of amino acid(s) at the other
positions. In one embodiment, m, n1, n2, and n5 are each one (1),
and n3, n4, and n6 are each zero (0). In another embodiment, m is
one (1), X.sub.n1 is Alanine, X.sub.n2 is Aspartate, X.sub.n5 is
Phenylalanine, and n3, n4, and n6 are each zero (0) (ACDCRGDCFC,
SEQ ID 2). An RGD peptide may have non-peptide components linked to
the RGD amino acid sequence. For example, the amino terminus of the
peptide may be acylated or a linker may be attached to the carboxy
terminus of the peptide. In another embodiment, m is one (1),
X.sub.n1 is acylated Alanine, X.sub.n2 is Aspartate, X.sub.n5 is
Phenylalanine, n3, n4, and n6 are each zero (0).
[0062] As used herein, an RGD mimic is a non-peptide synthetic
molecule other than an RDG peptide that biologically mimics the
active determinants of an RGD peptide, an integrin-binding RGD
portion of a integrin binding protein, or an
.alpha..sub.v.beta..sub.3 integrin binding RGD motif. An RGD mimic
may contain one or two naturally occurring amino acids linked via
amide bonds. An RGD mimetic may be a modified peptide, contain
non-standard amino acids or non-standard amino acid side chains. An
RGD mimic may have a peptide backbone represented by the
structure:
##STR00013##
wherein n is an integer.
[0063] In one embodiment, an RGD ligand comprises a guanidinium
group linked to a glycine-aspartate dipeptide via an amide bond.
Guanidinium groups of the invention have the structure represented
by:
##STR00014##
wherein R.sup.9 and R.sup.10 are independently hydrogen or alkyl
and may by connected to form a ring, and R.sup.11 is a linker
connecting the guanidinium group to the glycine-aspartate
dipeptide. The guanidinium group includes both the structure
represented above and its resonance structures. A preferred linker
is:
--(C.sup.1RR)--(C.sup.2RR)--(C.sup.3RR)-- or
--(C.sup.1RR)--(C.sup.2RR)--(C.sup.3RR)--(C.sup.4RR')--,
wherein: a) each R is independently optional and if present is
independently hydrogen, alkyl, or aryl, b) R' is hydrogen, alkyl,
aryl, or NH.sub.2, and c) C.sup.1, C.sup.2, and C.sup.3 may be
linked by single bonds, a single bond and a double bond, or
aromatic bonds.
[0064] While not explicitly shown in the structure RGD ligand
structures presented herein, is it well known and understood that
guanidinium groups are positively charged at neutral or near
neutral pH (pH 6.5-7.5):
##STR00015##
[0065] Similarly, while not explicitly shown in the RGD ligand
structures presented herein, is it well known and understood that
amino acid aspartic acid is negatively charged at neutral or near
neutral pH (pH 6.5-7.5):
##STR00016##
[0066] A phenoxy group attached to the aspartate amino acid of the
RGD ligand was found to improve targeting the polyamine to tumor
cells in vivo. A preferred RGD ligand comprises a
quanidinium-glycine-aspartate-4-aminophenoxy compound. A preferred
quanidinium-glycine-aspartate-4-aminophenoxy compound comprises the
structure represented by:
##STR00017##
wherein R.sup.13 is:
##STR00018##
[0067] A preferred guanidinium is
##STR00019##
and their resonance structures.
[0068] In another embodiment, an RGD ligand-containing moiety
comprises the structure represented by:
##STR00020##
wherein: [0069] R.sup.14 is
[0069] ##STR00021## [0070] A comprises a linker. The linker
connects the RGD mimic to another molecule such as a dipeptide
amidobenzyl-carbonate, provides for increased solubility, or
provides a means for covalent linkage to another molecule.
[0071] In one embodiment, linker A comprises:
##STR00022##
wherein [0072] n is 0, 1, 2, or 3, [0073] Y is absent or
[0073] ##STR00023## [0074] Z is absent,
[0074] ##STR00024## [0075] m is 0, 1, 2, 3, or 4, and [0076] PEG
(PEG1 in FIG. 13) is (CH.sub.2--CH.sub.2--O).sub.4-44, and [0077]
R.sup.12 comprises a reactive group capable of reacting with
R.sup.7 to from a covalent linkage.
[0078] Each of the separate components, PEG, reactive group, etc.
can be combined (covalently linked) using methods readily available
in the art, including, but not limited to formation of amide bonds.
In one embodiment, reactive group R.sup.12 can be linked to the PEG
via a diamine such as a lysine. The carboxyl group of the lysine
can be attached to a solid support to aid is synthesis of the R GD
ligand. The terminal and .epsilon.-amines are then used to link the
PEG group and reactive group. The reactive group R.sup.12 is
selected to readily reactive with reactive group R.sup.7 of formula
3 to forma covalently linkage. Pairs of reactive groups suitable
for use with R.sup.12 and R.sup.7 may be selected from the pairs
comprising: azide and phosphine, azide and alkyne, nitrone and
alkyne, tetrazine and octane, tetrazine and cyclopropene, tetrazine
and isonitrile, di-ene and alkene, aldehyde and hydrazine, aldehyde
and aminooxy, aldehyde and hydrazide, ketone and hydrazine, ketone
and aminooxy, and ketone and hydrazide. A preferred reactive group
is:
##STR00025##
[0079] In a preferred embodiment, an RGD ligand-containing moiety
comprises the structure represented by:
##STR00026## [0080] wherein: R.sup.14, n, Y, Z, m, PEG, and
R.sup.12 are each as defined above.
[0081] The reactive group R.sup.12 can be used to attached the RGD
ligand to a reversible physiologically labile linker such as a
dipeptide linker to yield an RGD masking agent. In one embodiment,
an RGD masking agent comprises the structure represented by:
##STR00027##
wherein R.sup.14 is a guanidinium-containing moiety as defined
above, A' comprises a PEG-containing linker, R.sup.1 is preferably
a side group of a hydrophobic amino acid, R.sup.2 is preferably a
side chain of a hydrophilic uncharged amino acid (at neutral pH),
and R.sup.3 is an amine-reactive carbonate. In one embodiment,
linker A' comprises a PEG group having 4-48 ethoxy units. In
another embodiment, linker A' comprises a first PEG (PEG1) group
having 4-44 ethylene units and a second PEG (PEG2) group having
4-44 ethylene groups separated by a diacyl hydrazine or other
linkage chemistry. In one embodiment, the diacyl hydrazone is
linked to the first PEG group via a diamine, such as a lysine. The
diaryl hydrazone can be formed by reaction of a HyNic
(hydrazino-nicotinamide) group with an aryl aldehyde.
[0082] In another embodiment, the linker A' comprises linkages form
be the reaction of: an azide with a phosphine, an azide with an
alkyne, a nitrone with an alkyne, a tetrazine with an octene, a
tetrazine with a cyclopropene, a tetrazine with an isonitrile, a
di-ene with an alkene, an aldehyde with a hydrazine, an aldehyde
with an aminooxy, an aldehyde with a hydrazide, a ketone with a
hydrazine, a ketone with an aminooxy, or a ketone with a
hydrazide.
[0083] Modification of a membrane active polyamine by attachment of
an RGD masking agent yields a reversibly modified polyamine. In one
embodiment, a membrane active polyamine modified by an RGD masking
agent comprises the structure represented by:
##STR00028##
wherein R.sup.14, R.sup.1, R.sup.2, and A' are as defined above. In
one embodiment, the RGD is attached to the dipeptide after the
dipeptide is linked to the amphipathic membrane active polyamine.
In one embodiment, an aryl
aldehyde-PEG2-dipeptide-amidobenzyl-carbonate is first reacted with
a polyamine to form an aryl
aldehyde-PEG2-dipeptide-amidobenzyl-carbamate-polyamine. This
compound is then reacted with an RGD ligand-PEG1-diamine-HyNic to
form: RGD ligand-PEG1-diamine-diaryl
hydrazone-PEG2-dipeptide-amidobenzyl-carbomate-polyamine (See FIG.
13).
[0084] As used herein, the term peptide has the usual meaning in
the art: a short chain of L .alpha. amino acid monomers linked by
peptide (amide) bonds, the covalent chemical bonds formed when the
carboxyl group of one amino acid reacts with the amino group of
another.
[0085] As used herein, the phrase naturally occurring amino acid
has the usual meaning in the art. As used herein, the phrase
standard amino acid has the usual meaning in the art: a naturally
occurring L .alpha. amino acid encoded directly by a triplet codon
in the genetic code.
[0086] Non-limiting examples of membrane active polymers suitable
for use with the invention have been previously described in US
Patent Publications US20080152661, US20090023890, US20080287630,
US20110207799, US20130121954, and US20130317079 (each of which is
incorporated herein by reference). Suitable amphipathic membrane
active polyamine can also be small peptides such as a melittin
peptide.
[0087] Polymer amines are reversibly modified using the peptidase
cleavable linkers described herein. An amine is reversibly modified
if cleavage of the modifying group results in regeneration of the
amine. Reaction of the activated carbonate of the masking agent
with a polymer amine connects an RGD ligand or steric stabilizer to
the polymer via a peptidase cleavable dipeptide-amidobenzyl
carbamate linkage as shown in FIG. 3.
[0088] Protecting groups may be used during synthesis and
conjugation of RGD ligands and dipeptide masking agents. If
present, protecting groups may be removed prior to or after
modification of the amphipathic membrane active polyamine.
[0089] Reversible modification of a sufficient percentage of the
polymer amines with the dipeptide masking agents inhibits membrane
activity of the membrane active polyamine. The
dipeptide-amidobenzyl-carbamate linkage is susceptible to protease
(or peptidase) cleavage. In presence of protease, the anilide bond
is cleaved, resulting in an intermediate which immediately
undergoes a 1,6 elimination reaction to release free polymer (FIG.
4). After the elimination reaction, the free polymer is unmodified
and membrane activity is restored.
[0090] The membrane active polyamine can be conjugated to masking
agents in the presence of an excess of masking agents. The excess
masking agent may be removed from the conjugated delivery polymer
prior to administration of the delivery polymer.
[0091] As used herein, a "steric stabilizer" is a non-ionic
hydrophilic polymer (either natural, synthetic, or non-natural)
that prevents or inhibits intramolecular or intermolecular
interactions of a polymer to which it is attached relative to the
polymer containing no steric stabilizer. A steric stabilizer
hinders a polymer to which it is attached from engaging in
electrostatic interactions. Electrostatic interaction is the
non-covalent association of two or more substances due to
attractive forces between positive and negative charges. Steric
stabilizers can inhibit interaction with blood components and
therefore opsonization, phagocytosis, and uptake by the
reticuloendothelial system. Steric stabilizers can thus increase
circulation time of molecules to which they are attached. Steric
stabilizers can also inhibit aggregation of a polymer. A preferred
steric stabilizer is a polyethylene glycol (PEG) or PEG derivative.
As used herein, a preferred PEG can have about 1-500 ethylene
glycol monomers, or 2-25. As used herein, a preferred PEG can also
have a molecular weight average of about 85-20,000 Daltons (Da),
about 85-1000 Da. As used herein, steric stabilizers prevent or
inhibit intramolecular or intermolecular interactions of a polymer
to which it is attached relative to the polymer containing no
steric stabilizer in aqueous solution.
[0092] "Ligands" enhance the pharmacokinetic or biodistribution
properties of a conjugate to which they are attached to improve
cell- or tissue-specific distribution and cell-specific uptake of
the conjugate. Ligands enhance the association of molecules with a
target cell. Thus, ligands can enhance the pharmacokinetic or
biodistribution properties of a conjugate to which they are
attached to improve cellular distribution and cellular uptake of
the conjugate. Binding of a ligand to a cell or cell receptor may
initiate endocytosis. Ligands may be monovalent, divalent,
trivalent, tetravalent, or have higher valency.
[0093] As used herein, membrane active polyamines are capable of
disrupting plasma membranes or lysosomal/endocytic membranes. This
membrane activity is an essential feature for cellular delivery of
the RNAi trigger. Membrane activity, however, leads to toxicity
when the polymer is administered in vivo. Polyamines also interact
readily with many anionic components in vivo, leading to undesired
bio-distribution. Therefore, reversible masking of membrane
activity of the polyamine is necessary for in vivo use.
[0094] In a one embodiment, the membrane active polyamine
comprises: an amphipathic polymer formed by random polymerization
of amine-containing monomers and hydrophobic group-containing
monomers. The amine-containing monomers contain pendant primary
amine groups. The hydrophobic monomers contain pendent hydrophobic
groups. The hydrophobic groups may be lower hydrophobic groups,
having 1-6 carbon atoms, or higher hydrophobic groups, having more
than 6 carbon atoms. Preferred hydrophobic group may be selected
from the list comprising: propyl, butyl, isopropyl, and isobutyl.
The ratio of amine groups to hydrophobic groups is selected to form
a water soluble polymer with membrane disruptive activity,
preferably .gtoreq.1 amine monomer per hydrophobic monomer. In one
embodiment the polymer will have 50-80% amine monomers and more
preferably 55-75% amine monomers. Hydrophobic groups may be
selected from the group consisting of: alkyl group, alkenyl group,
alkynyl group, aryl group, aralkyl group, aralkenyl group, and
aralkynyl group, each of which may be linear, branched, or cyclic.
Hydrophobic groups are preferably hydrocarbons, containing only
carbon and hydrogen atoms. However, substitutions or heteroatoms
which maintain hydrophobicity, and include, for example fluorine,
may be permitted.
[0095] "Amphipathic", or amphiphilic, polymers are well known and
recognized in the art and have both hydrophilic (polar,
water-soluble) and hydrophobic (non-polar, lipophilic,
water-insoluble) groups or parts.
[0096] "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. A hydrophilic group can be charged or uncharged.
Charged groups can be positively charged (anionic) or negatively
charged (cationic) or both (zwitterionic). Examples of hydrophilic
groups include the following chemical moieties: carbohydrates,
polyoxyethylene, certain peptides, oligonucleotides, amines,
amides, alkoxy amides, carboxylic acids, sulfurs, and
hydroxyls.
[0097] "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 form hydrogen bonds.
Lipophilic groups dissolve in fats, oils, lipids, and non-polar
solvents and have little to no capacity to form hydrogen bonds.
Hydrocarbons containing two (2) or more carbon atoms, certain
substituted hydrocarbons, cholesterol, and cholesterol derivatives
are examples of hydrophobic groups and compounds.
[0098] Hydrophobic groups are preferably hydrocarbons, containing
only carbon and hydrogen atoms. However, non-polar substitutions or
non-polar heteroatoms which maintain hydrophobicity, and include,
for example fluorine, may be permitted. The term includes aliphatic
groups, aromatic groups, acyl groups, alkyl groups, alkenyl groups,
alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and
aralkynyl groups, each of which may be linear, branched, or cyclic.
The term hydrophobic group also includes: sterols, steroids,
cholesterol, and steroid and cholesterol derivatives.
[0099] As used herein, with respect to amphipathic polymers, a part
is defined as a molecule derived when one covalent bond is broken
and replaced by hydrogen. For example, in butyl amine, a breakage
between the carbon and nitrogen bonds, and replacement with
hydrogens, results in ammonia (hydrophilic) and butane
(hydrophobic). If 1,4-diaminobutane is cleaved at nitrogen-carbon
bonds, and replaced with hydrogens, the resulting molecules are
again ammonia (2.times.) and butane. However, 1,4,-diaminobutane is
not considered amphipathic because formation of the hydrophobic
part requires breakage of two bonds.
[0100] As used herein, a surface active polymer lowers the surface
tension of water and/or the interfacial tension with other phases,
and, accordingly, is positively adsorbed at the liquid/vapor
interface. The property of surface activity is usually due to the
fact that the molecules of the substance are amphipathic or
amphiphilic.
[0101] As used herein, "membrane active" polymers are surface
active, amphipathic polymers that are able to induce one or more of
the following effects upon a biological membrane: an alteration or
disruption of the membrane that allows non-membrane permeable
molecules to enter a cell or cross the membrane, pore formation in
the membrane, fission of membranes, or disruption or dissolving of
the membrane. As used herein, a membrane, or cell membrane,
comprises a lipid bilayer. The alteration or disruption of the
membrane can be functionally defined by the polymer's activity in
at least one the following assays: red blood cell lysis
(hemolysis), liposome leakage, liposome fusion, cell fusion, cell
lysis, and endosomal release. Membrane active polymers that can
cause lysis of cell membranes are also termed membrane lytic
polymers. Polymers that preferentially cause disruption of
endosomes or lysosomes over plasma membrane are considered
endosomolytic. The effect of membrane active polymers on a cell
membrane may be transient. Membrane active possess affinity for the
membrane and cause a denaturation or deformation of bilayer
structures. Membrane active polymers may be synthetic or
non-natural amphipathic polymers.
[0102] As used herein, membrane active polymers are distinct from a
class of polymers termed cell penetrating peptides or polymers
represented by compounds such as the arginine-rich peptide derived
from the HIV TAT protein, the antennapedia peptide, VP22 peptide,
transportan, arginine-rich artificial peptides, small
guanidinium-rich artificial polymers and the like. While cell
penetrating compounds appear to transport some molecules across a
membrane, from one side of a lipid bilayer to other side of the
lipid bilayer, apparently without requiring endocytosis and without
disturbing the integrity of the membrane, their mechanism is not
understood.
[0103] Delivery of a RNAi trigger to a cell is mediated by the
membrane active polymer disrupting or destabilizing the plasma
membrane or an internal vesicle membrane (such as an endosome or
lysosome), including forming a pore in the membrane, or disrupting
endosomal or lysosomal vesicles thereby permitting release of the
contents of the vesicle into the cell cytoplasm.
[0104] Amphipathic membrane active polyamine copolymers of the
invention are the product of copolymerization of two or more
monomer species. In one embodiment, amphipathic membrane active
heteropolymers of the invention have the general structure:
-(A).sub.a-(B).sub.b-
wherein, A contains a pendent primary amine functional group and B
contains a pendant hydrophobic group. a and b are integers >0.
The polymers may be random, block, or alternating. The
incorporation of additional monomers is permissible.
[0105] As used herein, "endosomolytic polymers" are polymers that,
in response to an endosomal-specific environmental factors, such as
the presence of lytic enzymes, are able to cause disruption or
lysis of an endosome or provide for release of a normally cell
membrane impermeable compound, such as an RNAi trigger, from a
cellular internal membrane-enclosed vesicle, such as an endosome or
lysosome. Endosomolytic polymers undergo a shift in their
physico-chemical properties in the endosome. This shift can be a
change in the polymer's solubility or ability to interact with
other compounds or membranes as a result in a shift in charge,
hydrophobicity, or hydrophilicity. A reversibly masked membrane
active polyamine of the invention are considered to be
endosomolytic polymers.
[0106] As used herein, "melittin" is a small amphipathic membrane
active peptide which naturally occurs in bee venom (US patent
publication 20120165393). Melittin can be isolated from a
biological source or it can be synthetic. A synthetic polymer is
formulated or manufactured by a chemical process "by man" and is
not created by a naturally occurring biological process. As used
herein, melittin encompasses the naturally occurring bee venom
peptides of the melittin family that can be found in, for example,
venom of the species: Apis mellifera, Apis cerana, Vespula
maculifrons, Vespa magnifica, Vespa velutina nigrithorax, Polistes
sp. HQL-2001, Apis florae, Apis dorsata, Apis cerana cerana,
Polistes hebraeus. As used herein, melittin also encompasses
synthetic peptides having amino acid sequence identical to or
similar to naturally occurring melittin peptides. Specifically,
melittin amino acid sequence encompass those shown in Table 1.
Synthetic melittin peptides can contain naturally occurring L form
amino acids or the enantiomeric D form amino acids (inverso).
However, a melittin peptide should either contain essentially all L
form or all D form amino acids but may have amino acids of the
opposite stereocenter appended at either the amino or carboxy
termini. The melittin amino acid sequence can also be reversed
(reverso). Reverso melittin can have L form amino acids or D form
amino acids (retroinverso). Two melittin peptides can also be
covalently linked to form a melittin dimer. Melittin can have
modifying groups, other than masking agents, that enhance tissue
targeting or facilitate in vivo circulation attached to either the
amino terminal or carboxy terminal ends.
[0107] A linkage or "linker" is a connection between two atoms that
links one chemical group or segment of interest to another chemical
group or segment of interest via one or more covalent bonds. For
example, a linkage can connect a masking agent, polynucleotide, or
RNAi trigger to a polymer. A labile linkage contains a labile bond.
A linkage may optionally include a spacer that increases the
distance between the two joined atoms. A spacer may further add
flexibility and/or length to the linkage. Spacers may include, but
are not be limited to, alkyl groups, alkenyl groups, alkynyl
groups, aryl groups, aralkyl groups, aralkenyl groups, aralkynyl
groups; each of which can contain one or more heteroatoms,
heterocycles, amino acids, nucleotides, and saccharides. Spacer
groups are well known in the art and the preceding list is not
meant to limit the scope of the invention.
[0108] A "labile bond" is a covalent bond other than a covalent
bond to a hydrogen atom that is capable of being selectively broken
or cleaved under conditions that will not break or cleave other
covalent bonds in the same molecule. More specifically, a labile
bond is a covalent bond that is less stable (thermodynamically) or
more rapidly broken (kinetically) under appropriate conditions than
other non-labile covalent bonds in the same molecule. Cleavage of a
labile bond within a molecule may result in the formation of two
molecules. For those skilled in the art, cleavage or lability of a
bond is generally discussed in terms of half-life (t1/2) of bond
cleavage (the time required for half of the bonds to cleave). Thus,
labile bonds encompass bonds that can be selectively cleaved more
rapidly than other bonds a molecule.
[0109] As used herein, a "physiologically labile bond" is a labile
bond that is cleavable under conditions normally encountered or
analogous to those encountered within a mammalian body.
Physiologically labile linkage groups are selected such that they
undergo a chemical transformation (e.g., cleavage) when present in
certain physiological conditions.
[0110] As used herein, a cellular physiologically labile bond is a
labile bond that is cleavable under mammalian intracellular
conditions. Mammalian intracellular conditions include chemical
conditions such as pH, temperature, oxidative or reductive
conditions or agents, and salt concentration found in or analogous
to those encountered in mammalian cells. Mammalian intracellular
conditions also include the presence of enzymatic activity normally
present in a mammalian cell such as from proteolytic or hydrolytic
enzymes. A cellular physiologically labile bond may also be cleaved
in response to administration of a pharmaceutically acceptable
exogenous agent.
[0111] The term "polynucleotide", or nucleic acid or polynucleic
acid, is a term of art that refers to a polymer containing at least
two nucleotides. Nucleotides are the monomeric units of
polynucleotide polymers. Polynucleotides with less than 120
monomeric units are often called oligonucleotides. Natural nucleic
acids have a deoxyribose- or ribose-phosphate backbone. A
non-natural or synthetic polynucleotide is a polynucleotide that is
polymerized in vitro or in a cell free system and contains the same
or similar bases but may contain a backbone of a type other than
the natural ribose or deoxyribose-phosphate backbone.
Polynucleotides can be synthesized using any known technique in the
art. Polynucleotide backbones known in the art include: PNAs
(peptide nucleic acids), phosphorothioates, phosphorodiamidates,
morpholinos, and other variants of the phosphate backbone of native
nucleic acids. Bases include purines and pyrimidines, which further
include the natural compounds adenine, thymine, guanine, cytosine,
uracil, inosine, and natural analogs. Synthetic derivatives of
purines and pyrimidines include, but are not limited to,
modifications which place new reactive groups on the nucleotide
such as, but not limited to, amines, alcohols, thiols,
carboxylates, and alkylhalides. The term base encompasses any of
the known base analogs of DNA and RNA. A polynucleotide may contain
ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or
any suitable combination. Polynucleotides may be polymerized in
vitro, they may be recombinant, contain chimeric sequences, or
derivatives of these groups. A polynucleotide may include a
terminal cap moiety at the 5'-end, the 3'-end, or both the 5' and
3' ends. The cap moiety can be, but is not limited to, an inverted
deoxy abasic moiety, an inverted deoxy thymidine moiety, a
thymidine moiety, or 3' glyceryl modification.
[0112] RNAi triggers inhibit gene expression through the biological
process of RNA interference (RNAi). RNAi triggers comprise double
stranded RNA or RNA-like structures typically containing 15-50 base
pairs and preferably 18-25 base pairs and having a nucleobase
sequence identical (perfectly complementary) or nearly identical
(substantially complementary) to a coding sequence in an expressed
target gene within the cell. RNAi triggers include, but are not
limited to: short interfering RNAs (siRNAs), double-strand RNAs
(dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA),
meroduplexes, and dicer substrates (U.S. Pat. No. 8,084,599
8,349,809 and 8,513,207).
[0113] The RNAi trigger comprises at least two sequences that are
partially, substantially, or fully complementary to each other. In
one embodiment, the two RNAi trigger sequences comprise a sense
strand comprising a first sequence and an antisense strand
comprising a second sequence. In another embodiment, the two RNAi
trigger sequences comprise two sense strands which together
comprise a first sequence and an antisense strand comprising a
second sequence, wherein the sense strands and the antisense strand
together form a meroduplex (Tables 2 and 4). The sense strand may
be connected to the antisense strand via a linker molecule, such as
a polynucleotide linker or a non-nucleotide linker.
[0114] The antisense strand comprises a nucleotide sequence which
is complementary to a part of an mRNA encoding by a target gene,
and the region of complementarity is most preferably less than 30
nucleotides in length. The RNAi trigger sense strands comprise
sequences which have an identity of at least 90% to at least a
portion of an AAT mRNA. The RNAi trigger, upon delivery to a cell
expressing the target gene, inhibits the expression of said target
gene in vitro or in vivo.
[0115] RNAi trigger molecules may be comprised of naturally
occurring nucleotides or may be comprised of at least one modified
nucleotide or nucleotide mimic. The RNAi trigger sense and
antisense strands of the invention may be synthesized and/or
modified by methods well established in the art. RNAi trigger
molecules nucleosides, or nucleotide bases, may be linked by
phosphate-containing (natural) or non-phosphate-containing
(non-natural) covalent internucleoside linkages, i.e. the RNAi
trigger molecules may have natural or non-natural oligonucleotide
backbones. In another embodiment, at the RNAi trigger contains at
non-standard (non-phosphate) linkage between to nucleotide
bases.
[0116] Modified nucleotides include, but are not limited to: 2'
modifications, 2'-O-methyl nucleotide, 2'-deoxy-2'-fluoro
nucleotide, 2'-deoxy nucleotide, 2'-amino nucleotide, 2'-alkyl
nucleotide, terminal 3' to 3' linkages, inverted deoxythymidine, a
nucleotide comprising a 5'-phosphorothioate group, thiophosphate
linkages, phosphorodithioate group, non-natural base comprising
nucleotide, locked nucleotides, bridged nucleotides, peptide
nucleic acids, unlocked nucleotides (represented herein as
N.sub.UNA), morpholino nucleotides, and abasic nucleotide. It is
not necessary for all positions in a given compound to be uniformly
modified. Conversely, more than one modifications may be
incorporated in a single RNAi trigger compound or even in a single
nucleotide thereof. Ribose 2' modification may be combined with
modified nucleoside linkages.
[0117] RNAi trigger molecules may also comprise overhangs, i.e.
typically unpaired, overhanging nucleotides which are not directly
involved in the double helical structure normally formed by the
core sequences of the herein defined pair of sense strand and
antisense strand.
[0118] RNAi triggers may contain 3' and/or 5' overhangs of 1-5
bases independently on each of the sense strands and antisense
strands. In one embodiment, both the sense strand and the antisense
strand contain 3' and 5' overhangs. In one embodiment, one or more
of the 3' overhang nucleotides of one strand base pairs with one or
more 5' overhang nucleotides of the other strand. In another
embodiment, the one or more of the 3' overhang nucleotides of one
strand base do not pair with the one or more 5' overhang
nucleotides of the other strand. The sense and antisense strands of
an RNAi trigger may or may not contain the same number of
nucleotide bases. The antisense and sense strands may form a duplex
wherein the 5' end only has a blunt end, the 3' end only has a
blunt end, both the 5' and 3' ends are blunt ended, or neither the
5' end nor the 3' end are blunt ended. In another embodiment, one
or more of the nucleotides in the overhang contains a
thiophosphate, phosphorothioate, deoxynucleotide inverted (3' to 3'
linked) nucleotide or is a modified ribonucleotide or
deoxynucleotide.
[0119] Lists of known miRNA sequences can be found in databases
maintained by research organizations such as Wellcome Trust Sanger
Institute, Penn Center for Bioinformatics, Memorial Sloan Kettering
Cancer Center, and European Molecule Biology Laboratory, among
others. Known effective siRNA sequences and cognate binding sites
are also well represented in the relevant literature. RNAi
molecules are readily designed and produced by technologies known
in the art. In addition, there are computational tools that
increase the chance of finding effective and specific sequence
motifs (Pei et al. 2006, Reynolds et al. 2004, Khvorova et al.
2003, Schwarz et al. 2003, Ui-Tei et al. 2004, Heale et al. 2005,
Chalk et al. 2004, Amarzguioui et al. 2004).
[0120] An RNAi trigger modulates expression of RNA encoded by a
gene. Because multiple genes can share some degree of sequence
homology with each other, an RNAi trigger can be designed to target
a class of genes with sufficient sequence homology. Thus, an RNAi
trigger can contain a sequence that has complementarity to
sequences that are shared amongst different gene targets or are
unique for a specific gene target. Therefore, the RNAi trigger can
be designed to target conserved regions of an RNA sequence having
homology between several genes thereby targeting several genes in a
gene family (e.g., different gene isoforms, splice variants, mutant
genes, etc.). In another embodiment, the RNAi trigger can be
designed to target a sequence that is unique to a specific RNA
sequence of a single gene.
[0121] The term "complementarity" refers to the ability of a
polynucleotide to form hydrogen bond(s) with another polynucleotide
sequence by either traditional Watson-Crick or other
non-traditional types. In reference to the polynucleotide molecules
of the present invention, the binding free energy for a
polynucleotide molecule with its target (effector binding site) or
complementary sequence is sufficient to allow the relevant function
of the polynucleotide to proceed, e.g., enzymatic mRNA cleavage or
translation inhibition. Determination of binding free energies for
nucleic acid molecules is well known in the art (Frier et al. 1986,
Turner et al. 1987). A percent complementarity indicates the
percentage of bases, in a contiguous strand, in a first
polynucleotide molecule which can form hydrogen bonds (e.g.,
Watson-Crick base pairing) with a second polynucleotide sequence
(e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%,
and 100% complementary). Perfectly complementary means that all the
bases in a contiguous strand of a polynucleotide sequence will
hydrogen bond with the same number of contiguous bases in a second
polynucleotide sequence.
[0122] By inhibit, down-regulate, or knockdown gene expression, it
is meant that the expression of the gene, as measured by the level
of RNA transcribed from the gene or the level of polypeptide,
protein or protein subunit translated from the RNA, is reduced
below that observed in the absence of the RNAi trigger-conjugates
of the invention. Inhibition, down-regulation, or knockdown of gene
expression, with a RNAi trigger delivered by the compositions of
the invention, is preferably below that level observed in the
presence of a control inactive nucleic acid, a nucleic acid with
scrambled sequence or with inactivating mismatches, or in absence
of conjugation of the RNAi trigger to the masked polymer.
Linkage of an RNAi Trigger to Delivery Polymer
[0123] In one embodiment, the RNAi trigger is linked to the
delivery polymer via a physiologically labile bond or linker. The
physiologically labile linker is selected such that it undergoes a
chemical transformation (e.g., cleavage) when present in certain
physiological conditions, (e.g., disulfide bond cleaved in the
reducing environment of the cell cytoplasm). Release of the trigger
from the polymer, by cleavage of the physiologically labile
linkage, facilitates interaction of the trigger with the
appropriate cellular components for activity.
[0124] The RNAi trigger-polymer conjugate is formed by covalently
linking the trigger to the polymer. The polymer is polymerized or
modified such that it contains a reactive group A. The RNAi trigger
is also polymerized or modified such that it contains a reactive
group B. Reactive groups A and B are chosen such that they can be
linked via a reversible covalent linkage using methods known in the
art.
[0125] Conjugation of the RNAi trigger to the polymer can be
performed in the presence of an excess of polymer. Because the RNAi
trigger and the polymer may be of opposite charge during
conjugation, the presence of excess polymer can reduce or eliminate
aggregation of the conjugate. Alternatively, an excess of a carrier
polymer, such as a polycation, can be used. The excess polymer can
be removed from the conjugated polymer prior to administration of
the conjugate to the animal or cell culture. Alternatively, the
excess polymer can be co-administered with the conjugate to the
animal or cell culture.
In Vivo Administration
[0126] In pharmacology and toxicology, a route of administration is
the path by which a drug, fluid, poison, or other substance is
brought into contact with the body. In general, methods of
administering drugs and nucleic acids for treatment of a mammal are
well known in the art and can be applied to administration of the
compositions of the invention. The compounds of the present
invention can be administered via any suitable route, most
preferably parenterally, in a preparation appropriately tailored to
that route. Thus, the compounds of the present invention can be
administered by injection, for example, intravenously,
intramuscularly, intracutaneously, subcutaneously, or
intraperitoneally. Accordingly, the present invention also provides
pharmaceutical compositions comprising a pharmaceutically
acceptable carrier or excipient.
[0127] Parenteral routes of administration include intravascular
(intravenous, intraarterial), intramuscular, intraparenchymal,
intradermal, subdermal, subcutaneous, intratumor, intraperitoneal,
intrathecal, subdural, epidural, and intralymphatic injections that
use a syringe and a needle or catheter. 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, cerebrospinal fluid (CSF), lymphatic
fluid, or bile. Examples of vessels include arteries, arterioles,
capillaries, venules, sinusoids, veins, lymphatics, bile ducts, and
ducts of the salivary or other exocrine glands. The intravascular
route includes delivery through the blood vessels such as an artery
or a vein. The blood circulatory system provides systemic spread of
the pharmaceutical.
[0128] The described compositions are injected in pharmaceutically
acceptable carrier solutions. Pharmaceutically acceptable refers to
those properties and/or substances which are acceptable to the
mammal from a pharmacological/toxicological point of view. The
phrase pharmaceutically acceptable refers to molecular entities,
compositions, and properties that are physiologically tolerable and
do not typically produce an allergic or other untoward or toxic
reaction when administered to a mammal. Preferably, as used herein,
the term pharmaceutically acceptable means approved by a regulatory
agency of the Federal or a state government or listed in the U.S.
Pharmacopeia or other generally recognized pharmacopeia for use in
animals and more particularly in humans.
[0129] These carrier may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures, supra, and by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol, sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
Therapeutic Effect
[0130] RNAi triggers may be delivered for research purposes or to
produce a change in a cell that is therapeutic. In vivo delivery of
RNAi triggers is useful for research reagents and for a variety of
therapeutic, diagnostic, target validation, genomic discovery,
genetic engineering, and pharmacogenomic applications. We have
disclosed RNAi trigger delivery resulting in inhibition of
endogenous gene expression in hepatocytes. Levels of a reporter
(marker) gene expression measured following delivery of a RNAi
trigger indicate a reasonable expectation of similar levels of gene
expression following delivery of other RNAi triggers. Levels of
treatment considered beneficial by a person having ordinary skill
in the art differ from disease to disease. For example, Hemophilia
A and B are caused by deficiencies of the X-linked clotting factors
VIII and IX, respectively. Their clinical course is greatly
influenced by the percentage of normal serum levels of factor VIII
or IX: <2%, severe; 2-5%, moderate; and 5-30% mild. Thus, an
increase from 1% to 2% of the normal level of circulating factor in
severe patients can be considered beneficial. Levels greater than
6% prevent spontaneous bleeds but not those secondary to surgery or
injury. Similarly, inhibition of a gene need not be 100% to provide
a therapeutic benefit. A person having ordinary skill in the art of
gene therapy would reasonably anticipate beneficial levels of
expression of a gene specific for a disease based upon sufficient
levels of marker gene results. In the hemophilia example, if marker
genes were expressed to yield a protein at a level comparable in
volume to 2% of the normal level of factor VIII, it can be
reasonably expected that the gene coding for factor VIII would also
be expressed at similar levels. Thus, reporter or marker genes
serve as useful paradigms for expression of intracellular proteins
in general.
[0131] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, the route of administration, the time of administration,
the rate of excretion of the particular compound being employed,
the duration of the treatment, other drugs, compounds and/or
materials used in combination with the particular compositions
employed, the age, sex, weight, condition, general health and prior
medical history of the patient being treated, and like factors well
known in the medical arts.
[0132] As used herein, in vivo means that which takes place inside
an organism and more specifically to a process performed in or on
the living tissue of a whole, living multicellular organism
(animal), such as a mammal, as opposed to a partial or dead
one.
[0133] As used herein, "pharmaceutical composition" includes the
conjugates of the invention, a pharmaceutical carrier or diluent
and any other media or agent necessary for formulation.
[0134] As used herein, "pharmaceutical carrier" includes any and
all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like that are physiologically compatible. Preferably, the carrier
is suitable for intravenous, intramuscular, subcutaneous,
parenteral, spinal or epidermal administration (e.g. by injection
or infusion).
EXAMPLES
Example 1. Synthesis of PEG Protease (Peptidase) Cleavable Masking
Agents
[0135] All reactions, except coupling of amino acids in aqueous
NaHCO.sub.3 and silyl-group deprotection, were carried out in
anhydrous conditions using fresh anhydrous solvents. Column
purification was done on a silica gel using specified eluents.
Mass-spectra (MS) were taken using electrospray ionization.
[0136] In preparation of active p-nitrophenyl-p-acylamidobenzyl
carbonate derivatives of PEG PEG-AA-PABC-PNP) we utilized NHS ester
of PEG or to acylate amino terminus of dipeptido-p-acylaminobenzy
alcohol precursor. In the following steps benzylic hydroxyl group
was converted into p-nitrophenyl carbonate followed by removal of
protective groups from amino acids. In some applications, when
paranitrophenol (PNP)-carbonates were used for modification of
certain polymers, protective groups were removed prior to polymer
modification (see FIG. 5).
[0137] The synthesis starts from preparation of
H-A.sup.1A.sup.2-PABA (Table 2) derivatives. These adducts were
obtained utilizing synthetic scheme described by Dubowchik at al.
(2002) with some modifications. Fmoc-protected amino acids,
Fmoc-A.sup.1-OH, were activated by conversion into
N-hydroxycuccinimide esters, Fmoc-A.sup.1-NHS, in reaction with
dicyclohexylcarbodiimide (DCC) and N-hydroxycuccinimide (NHS).
These reactive NHS-esters were coupled with protected amino acids
A.sup.2 in presence of aqueous NaHCO.sub.3 added to keep amino
group reactive. For preparation of 1e and 1f (Table 2), instead of
NHS esters, commercially available pentafluorophenyl esters (OPfp)
for were used for coupling.
Synthesis of Fmoc Dipeptides 1a-h.
[0138] a) NHS esters of AA were prepared from respective amino
acids with NHS and DCC and used without additional purification
(FIG. 5A).
[0139] For Fmoc-Ala-NHS, DCC (286 mg, 1.38 mmol) was added to an
ice cold solution of Fmoc-Ala-OH (412 mg, 1.32 mmol) and NHS (160
mg, 1.38 mmol) in DCM (13 ml), stirred for 30 min, and then at
20.degree. C. for 16 h (hour). The solid dicyclohexylurea (DCU) was
filtered off and the solvent was removed in vacuo.
[0140] For Fmoc-Asn(DMCP)-NHS, DCC (148 mg, 0.72 mmol) was added to
an ice cold solution of Fmoc-Asn(DMCP)-OH (298 mg, 0.68 mmol) and
NHS (83 mg, 0.72 mmol) in DCM (13 ml), stirred for 30 min, and then
at 20.degree. C. for 16 h. The solid DCU was filtered off and the
solvent was removed in vacuo.
[0141] For Fmoc-Gly-NHS, Fmoc-Gly-OH (891 mg, 3 mmol) and NHS (380
mg, 3.3 mmol) were stirred in THF (10 ml) at 0.degree. C. for 5 min
and treated with a DCC solution (650 mg, 3.15 mmol) in THF (5 ml).
The cooling bath was removed in 30 min and the reaction mixture was
stirred at 20.degree. C. for 10 h. The solid DCU was filtered off,
washed with THF and the solvent was removed on the rotovap. The
product was weighed and dissolved in DME to make a 0.2 mM
solution.
[0142] For Fmoc-Glu(O-2PhiPr)-NHS, DCC (217 mg, 1.05 mmol) was
added to an ice cold solution of Fmoc-Glu(O-2PhiPr)-OH (487 mg, 1
mmol) and NHS (127 mg, 1.1 mmol) in THF (5 ml), stirred for 15 min
and then at 20.degree. C. for 10 h. The workup was done as
described for Fmoc-Gly-NHS.
[0143] For Fmoc-Phe-NHS, DCC (1.181 g, 5.72 mmol) was added to an
ice cold solution of Fmoc-Phe-OH (2.11 g, 5.45 mmol) and NHS (664
mg, 5.77 mmol) in DCM (50 ml), stirred for 30 min, and then at
20.degree. C. for 10 h. The solid DCU was filtered off and the
solvent was removed in vacuo.
[0144] For Fmoc-Val-NHS, DCC (227 mg, 1.1 mmol) was added to an ice
cold solution of Fmoc-Val-OH (339 mg, 1 mmol) and NHS (127 mg, 1.1
mmol) in DCM (13 ml), stirred for 30 min, and then at 20.degree. C.
for 16 h. The solid DCU was filtered off and the solvent was
removed in vacuo.
[0145] b) Amino acids H-Asn(DMCP)-OH and H-Lys(MMT)-OH were
prepared from available Fmoc-protected derivatives (see FIG.
5B).
[0146] H-Asn(DMCP)-OH. Fmoc-Asn(DMCP)-OH (576 mg, 1.32 mmol) was
stirred in DMF (9 ml) with Et.sub.3N (3.7 ml, 26.4 mmol) for 15 h.
All volatiles were removed on a rotovap at 40.degree. C./oil pump
vacuum. The residue was triturated with ether (30 ml) three times
and dried in vacuo. Yield 271 mg (96%). MS: 643.6 [3M+1].sup.+;
451.3 [2M+Na].sup.+; 429.5 [2M+1].sup.+; 236.7 [M+Na].sup.+; 215.3
[M+1].sup.+; 132.8 [M-DMCP+1].sup.+.
[0147] H-Lys(MMT)-OH. Fmoc-Lys(MMT)-OH (4.902 g, 7.65 mmol) was
stirred in DMF (100 ml) with Et.sub.3N (32 ml, 30 eq. 229.4 mmol)
for 10 h. All volatiles were removed on a rotovap at 40.degree.
C./oil pump vacuum. The residue was triturated with ether two times
and dried in vacuo. Yield 3.1 g (97%). MS (neg. mode): 455, 453.3
[M+Cl].sup.-; 417.8 [M-1].sup.-.
[0148] c) Synthesis of Fmoc-A.sup.1A.sup.2-OH. (FIG. 5C.) For
Fmoc-GlyGly-OH 1a, Glycine (75 mg, 1 mmol) and NaHCO.sub.3 (100 mg,
1.2 mmol) were dissolved in H.sub.2O (10 ml) and dimethoxyethane
(DME) (5 ml). Fmoc-Gly-NHS solution in DME (5 ml, 1 mmol) was
added. THF (2.5 ml) was added, the mixture was sonicated to make it
homogeneous and stirred for 20 h. All volatiles were removed on a
rotovap, the residue was treated with EtOAc and 5% KHCO.sub.3
solution in H.sub.2O. Product was extracted four times with EtOAc,
washed with brine at pH=3, dried (Na.sub.2SO.sub.4), concentrated
and dried in vacuo. Yield 321 mg (90%). MS: 775.0 [2M+2Na].sup.+;
377.4 [M+Na].sup.+; 355.1 [M+1].sup.+.
[0149] For Fmoc-Glu(O-2PhiPr)Gly-OH 1b, Glycine (75 mg, 1 mmol) and
NaHCO.sub.3 (84 mg, 1 mmol) were dissolved in a mixture of H.sub.2O
(2 ml), THF (4 ml) and DME (5 ml). Fmoc-Glu(O-2PhiPr)-NHS solution
in DME (5 ml, 1 mmol) was added and stirred for 10 h. All volatiles
was removed on a rotovap, 20 ml of 0.1M MES buffer (pH=5) was added
followed by EtOAc (25 ml). The reaction mixture was stirred on ice
and acidified to pH=5 with 5% solution of KHSO.sub.4. Product was
extracted four times with EtOAc, rinsed with brine at pH=5, dried
(Na.sub.2SO.sub.4), concentrated and dried in vacuo. Yield 528 mg
(96%). MS: 567 [M+Na].sup.+; 562 [M+NH.sub.4].sup.+; 545.0
[M+1].sup.+; 427.1 [M-2PhiPr].sup.+.
[0150] For Fmoc-Asn(DMCP)Gly-OH 1c was prepared from
Fmoc-Asn(DMCP)-NHS and H-Gly-OH as described above for 1b. Yield
96%. MS: 987.4 [2M+1].sup.+; 516.3 [M+Na].sup.+; 494.4 [M+1].sup.+;
412.2 [M-DMCP+1].sup.+.
[0151] For Fmoc-PheLys(MMT)-OH 1d was prepared from Fmoc-Phe-NHS
and H-Lys(MMT)-OH as described above for 1b. Yield 94%. MS: 788.5
[M+1].sup.+, 273.1 [M-MMT+1].sup.+.
[0152] For Fmoc-PheCit-OH 1e: [0153] i) To Fmoc-Phe-NHS (4.96 g,
10.26 mmol) in DME (40 ml) was added to a solution containing
L-citrulline (1.80 g, 10.26 mmol) and NaHCO.sub.3 (0.86 g, 10.26
mmol) in a mixture of H.sub.2O (40 ml) and THF (20 ml). The
reaction was stirred for 15 h. Residual DCC from activation was
filtered and the organic solvent was removed on a rotovap. To the
residue was added H.sub.2O (100 ml) and iPrOH (10 ml). The
suspension was acidified to pH=3 with 5% KHSO.sub.4, the product
was extracted with an EtOAc:iPrOH=9:1 solution (3.times., 500 ml),
washed with a mixture of brine:iPrOH=9:1 (2.times., 50 ml), dried
(Na.sub.2SO.sub.4), filtered and concentrated, and dried with oil
pump. Trituration with ether afforded the pure product 1e. Yield
3.84 g (68%). MS: 545.6 [M+Na].sup.+; 528.5 [M+H.sub.2O].sup.+;
306.3 [M-Fmoc+H.sub.2O].sup.+. [0154] ii) A solution of
Fmoc-Phe-OPfp (553 mg, 1 mmol) in THF (5 ml) was added to a
solution of H-Cit-OH (184 mg, 1.05 mmol) and NaHCO.sub.3 (88.2 mg,
1.05 mmol) in H.sub.2O (2.6 ml). THF (2 ml) was added to make the
solution homogeneous and stirred for 10 h. THF was removed on a
rotovap, the residue was diluted with H.sub.2O (10 ml) and iPrOH (1
ml) and acidified to pH=1 with 3% HCl. The product was extracted
five times with an EtOAc:iPrOH=9:1 solution, rinsed with a mixture
of brine:iPrOH=9:1, dried (Na.sub.2SO.sub.4) and concentrated in
vacuo. Trituration with ether afforded 313 mg of pure product 1e
(57%).
[0155] Fmoc-AlaCit-OH If was prepared from Fmoc-Ala-NHS and
H-Cit-OH as described above for 1e-(a). Yield 77%. MS: 959.8
[2M+Na].sup.+; 938.1 [2M+1].sup.+; 491.4 [M+Na].sup.+; 469.9
[M+1].sup.+.
[0156] Crude Fmoc-ValCit-OH 1g was prepared from Fmoc-Val-NHS and
H-Cit-OH as described above for 1b. The final purification was done
by trituration with ether. Total yield 76%. MS: 1060.3
[2M+3Na].sup.+; 1015.7 [2M+Na].sup.+; 519.7 [M+Na].sup.+; 497.9
[M+1].sup.+.
[0157] Fmoc-Ala-Asn(DMCP)-OH 1h was prepared from Fmoc-Ala-NHS and
H-Asn(DMCP)-OH as described above for 1b. Yield 95%. MS: 530.2
[M+Na].sup.+; 508.2 [M+1].sup.+; 426.0 [M-DMCP+1].sup.+.
Coupling with p-Aminobenzyl Alcohol, Preparation of Fmoc-AA-PABA
and Fmoc-A-PABA 2a-m.
[0158] Products 1a-h were coupled with p-aminobenzyl alcohol (PABA)
in presence of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline
(EEDQ) to form 2a-h. Four representatives 3 j-l with only one amino
acid attached to PABA moiety were also prepared (FIG. 7A).
[0159] For Fmoc-GlyGly-PABA 2a, a solution of 1a (318 mg, 0.9 mmol)
and PABA (220 mg, 1.8 mmol) in DCM (17 ml) and MeOH (6 ml) were
stirred with EEDQ (444 mg, 1.8 mmol) for 10 h. All volatiles were
removed on a rotovap, the residue was triturated with Et.sub.2O and
the product was filtered out and dried in vacuo. Yield 348 mg
(84%).
[0160] For Fmoc-Glu(O-2PhiPr)Gly-PABA 2b, a solution of 1b (524 mg,
0.96 mmol) and PABA (142 mg, 1.55 mmol) in DCM (10 ml) was stirred
with EEDQ (357 mg, 1.44 mmol) for 10 h. The workup was done as
described above for 2a. Yield 462 mg (74%).
[0161] Fmoc-Asn(DMCP)Gly-PABA 2c, was prepared as described above
for 2a. Yield 64%. MS: 621.5 [M+22].sup.+; 599.3 [M+1].sup.+.
[0162] Fmoc-PheLys(MMT)-PABA 2d, was prepared as described above
for 2b. Yield 70%.
[0163] For Fmoc-PheCit-PABA 2e, a solution of 1e (5.98 g, 10.97
mmol) and PABA (2.70 g, 21.95 mmol) in DCM (150 ml) and MeOH (50
ml) was treated with EEDQ (5.43 g, 21.95 mmol) and stirred for 15
h. The workup was done as described above for 2a. Yield 6.14 g
(86%). MS: 650.7 [M+1].sup.+; 527.3 [M-PABA+1].sup.+.
[0164] For Fmoc-AlaCit-PABA 2f, a solution of 1f (2.89 g, 6.17
mmol) and PABA (1.52 g, 12.34 mmol) in DCM (45 ml) and MeOH (15 ml)
was treated with EEDQ (3.05 g, 12.34 mmol) and stirred for 15 h.
The workup was done as described above for 2a. Yield 4.56 g (74%).
MS (ES, neg. mode): 307.4 [M-263.6-1].sup.-; 349.9
[M-Fmoc-1].sup.-; 610, 608.4 [M+HCl-1].sup.-.
[0165] Fmoc-ValCit-PABA 2g was prepared as described above for 2b.
(98%).
[0166] Fmoc-AlaAsn(DMCP)-PABA 2h was prepared as described above
for 2a. Yield 59%. MS: 613.2 [M+1].sup.+; 531.4 [M-DMCP+1].sup.+;
408.2 [M-205+1].sup.+.
[0167] For Fmoc-Lys(CH.sub.3).sub.2-PABA 2i,
Fmoc-Lys(CH.sub.3).sub.2--OHHCl salt (433 mg, 1 mmol) and PABA (246
mg, 2 mmol) were dissolved in DCM (10 ml) and MeOH (1.5 ml), cooled
to 5.degree. C. and EEDQ (495 mg, 2 mmol) was added. The cooling
bath was removed and the mixture was stirred for 10 h at RT (room
temperature). All volatiles were removed on a rotovap, the residue
was triturated with Et.sub.2O, and the crude product was filtered
off. It was redissolved in a mixture of DCM (2 ml) and MeOH (1 ml)
and precipitated again by adding dropwise into Et.sub.2O (40 ml).
Product was filtered and dried in vacuo. Yield 448 mg (83%).
[0168] For Fmoc-Leu-PABA 2j, a solution of Fmoc-Leu-OH (353 mg, 1
mmol), EEDQ (495 mg, 2 mmol) and PABA (222 mg, 1.8 mmol) in DCM (10
ml) was stirred for 10 h. All volatiles were removed on a rotovap,
the residue was dissolved in Et.sub.2O (40 ml), chilled on dry ice
for 2h and the solid was separated by centrifugation. The obtained
crude material was purified on a column, eluent gradient of MeOH
(1-2%) in CHCl.sub.3. Yield 444 mg (97%). MS: 459.4
[M+1].sup.+.
[0169] Fmoc-Asn(DMCP)-PABA 2k was prepared as described for 2j. In
workup instead of column purification after removing of DCM the
residue was triturated with Et.sub.2O, chilled to 0.degree. C. and
the crude product was filtered off. This treatment was repeated one
more time followed by drying in vacuo. Yield 77%. MS: 542.5
[M+1].sup.+.
[0170] For Fmoc-Cit-PABA 2l, a solution of Fmoc-Cit-OH (345.7 mg,
0.87 mmol) and PABA (214 mg, 1.74 mmol) in DCM (10 ml) and MeOH (4
ml) was treated with EEDQ (430 mg, 1.74 mmol) and stirred for 15 h.
The solid product was triturated three times with ether, and the
product was filtered and dried. Yield 288 mg (67%). MS: 502.3
[M+1].sup.+; 485.5 [M+H.sub.2O+1].sup.+; 263
[M-Fmoc-H.sub.2O+1].sup.+; 179.0 [M-306+1].sup.+; 120.2
[M-365.3+1].sup.+.
[0171] Product 2m was prepared using different scheme: coupling of
H-Lys(CH.sub.3).sub.2-PABA derivative 3 with Fmoc-Phe-NHS (FIG.
7B).
[0172] For Fmoc-PheLys(CH.sub.3)-PABA 2m,
Fmoc-Lys(CH.sub.3).sub.2-PABA (2i) (448 mg, 0.83 mmol) was Fmoc
deprotected by stirring with Et.sub.3N (3.5 ml) in DMF (11 ml) for
10 h. All volatiles were removed on a rotovap at 40.degree. C./oil
pump vacuum to obtain the crude product 3i. This product was
dissolved in DMF (7 ml), Fmoc-Phe-NHS (482 mg, 0.996 mmol) was
added followed by DIEA (0.42 ml, 2.2 mmol) and the mixture was
stirred for 10 h. The solvent with DIEA was removed on a rotovap at
40.degree. C./oil pump vacuum to obtain crude 2m which was used
without additional purification. MS: 549.4 [M+1].sup.+.
Preparation of H-AA-PABA 3a-h, m and H-A-PABA 3j-l (FIG. 7).
[0173] Fmoc-derivatives 2a-h, j-l were treated with Et.sub.3N in
DMF as described above for 3i followed by concentration and drying
in vacuo. The crude products were dissolved in DMF to make 0.1 M
solution and used without additional purification.
TABLE-US-00001 TABLE 2 Intermediates of H-A.sup.1A.sup.2-PABA (1-3)
A.sup.1 A.sup.2 1, 2, 3a Gly Gly 1, 2, 3b Glu(2PhiPr) Gly 1, 2, 3c
Asn(DMCP) Gly 1, 2, 3d Phe Lys(MMT) 1, 2, 3e Phe Cit 1, 2, 3f Ala
Cit 1, 2, 3g Val Cit 1, 2, 3h Ala Asn(DMCP) 1, 2, 3i
Lys(CH.sub.3).sub.2 1, 2, 3j Leu -- 1, 2, 3k Asn(DMCP) -- 1, 2, 3l
Cit -- 2, 3m Phe Lys(CH.sub.3).sub.2
##STR00029##
Preparation of Protease Cleavable PEG-Masking Reagents.
[0174] The amino group of any of H-AA-PABA 3b, e, g, h, j, k-m was
acylated with an NHS ester of PEG-acid (DIEA, DMF, 5-10h) to yield
22a-k. The hydroxyl group in product 22a-k was then converted into
p-nitrophenyl carbonate ((PNP).sub.2CO, dioxane or THF,
40-60.degree. C., 10h) to yield 23a-k. For 23a, d, g, protective
groups from Asn and Glu were removed by treatment with aqueous TFA
(TFA/H.sub.2O=3:1, 5.degree. C., 2-3h) to obtain desired products
24a-c.
Preparation of PEG.sub.n-AA-PABA 22a-k (FIG. 9).
[0175] Product 22a (n=11, AA=GluGly). A 0.1M solution of 3b in DMF
(3.5 ml, 0.35 mmol) was stirred for 10 h with PEG.sub.11-NHS ester
(240 mg, 0.35 mmol) and DIEA (0.061 ml, 0.35 mmol). All volatiles
were removed on a rotovap at 40.degree. C./oil pump and the product
was purified on a column, eluent: CHCl.sub.3:MeOH:AcOH=38:2:1.
Yield 274 mg (78%) MS: 1015.6 [M+NH.sub.4].sup.+, 998.7
[M+1].sup.+.
[0176] Product 22b (n=11, AA=PheCit). To a solution of 3e (0.88
mmol) and DIEA (167 .mu.l, 0.96 mmol) in DMF (3 ml) was added a
solution of PEG.sub.11-NHS ester (0.80 mmol) in DMF (3 ml). The
mixture was stirred for 16 h, filtered and all volatiles were
removed on a rotovap at 40.degree. C./oil pump vacuum. The crude
was precipitated into Et.sub.2O (45 ml) from CHCl.sub.3:MeOH (5 ml)
and purified on a column, eluent a gradient of MeOH (10-16%) in
CHCl.sub.3. Yield 420 mg (53%). MS: 1015.9 [M+H.sub.2O].sup.+;
998.8 [M+1].sup.+; 981.1 [M+H.sub.2O].sup.+.
[0177] Product 22c (n=1, AA=ValCit). Product 22f was prepared from
crude 3g (obtained from 300 mg, 0.5 mmol of 2g), PEG.sub.11-NHS
ester (298 mg, 0.435 mmol) and DIEA (0.09 ml, 0.522 mmol) as
described for 22a. Following concentration on a rotovap at
40.degree. C./oil pump the product was suspended in a MeOH:DCM=1:1
mixture (6 ml), sonicated, filtered and precipitated into Et.sub.2O
(50 ml). The solid was separated and the procedure repeated again.
The residual solvents were removed in vacuo. Yield 283 mg (60%).
MS: 951.5 [M+1].sup.+.
[0178] Product 22d (n=l 1, AA=AlaAsn(DMCP)). To a solution of 3h
(0.56 mmol) and DIEA (116 .mu.l, 0.67 mmol) in DMF (3 ml) was added
a solution of PEG.sub.11-NHS ester (0.56 mmol) in DMF (3 ml). The
mixture was stirred for 16 h, filtered and all volatiles were
removed on a rotovap at 40.degree. C./oil pump vacuum. The residue
was dissolved in a CHCl.sub.3:MeOH=1:1 mixture (5 ml) and
precipitated into chilled (0.degree. C.) Et.sub.2O (45 ml). The
solid was purified on a column, eluent gradient of MeOH (3-14%) in
DCM. Yield 261 mg (49%). MS: 983.7 [M+Na].sup.+; 979.1
[M+NH.sub.4].sup.+; 961.8 [M+1].sup.+; 943.9
[M+H.sub.2O+1].sup.+.
[0179] Product 22e (n=11, AA=PheLys(Me.sub.2)). Product 22e was
prepared as described for 22a. Purification was done using HPLC
column Nucleodur C-18, 250.times.4.6, eluent ACN-H.sub.2O (0.1%
TFA), ramp 15-30%. MS: 998.1 [M+1].sup.+. The isolated product was
desalted on Dowex 1.times.8 resin, eluent H.sub.2O. Yield 40%.
[0180] Product 22f (n=11, AA=Leu). Product 22f was prepared as
described for 22a and purified on a column, eluent:
CHCl.sub.3:EtOAc:MeOH:AcOH=9:7:2:0.04. Yield 48%. MS: 824.9
[M+NH.sub.4].sup.+.
[0181] Product 22g (n=l 1, AA=Asn(DMCP). Crude 3k (obtained from
419 mg, 0.77 mmol of 2k), PegiNHS ester (200 mg, 0.292 mmol) and
DIEA (0.06 ml, 0.35 mmol) were stirred in DCM (5 ml) for 10 h. The
solvent was removed on a rotovap and the product was purified on a
column, eluent CHCl.sub.3:EtOAc:MeOH AcOH=4.5:3.5:1:0.02. Yield 254
mg (37%). MS: 891.1 [M+1].sup.+.
[0182] Product 22h (n=1 AA=Cit). To a solution of 31 (0.50 mmol)
and DIEA (104 .mu.l, 0.60 mmol) in DMF (2.5 ml) was added a
solution of PEG.sub.11-NHS ester (0.50 mmol) in DMF (2.5 ml). The
mixture stirred for 16 h, filtered and all volatiles were removed
on a rotovap at 40.degree. C./oil pump vacuum. The residue was
dissolved in a CHCl.sub.3:MeOH=1:1 mixture (5 ml) and precipitated
into Et.sub.2O (45 ml). Precipitation was repeated two more times
and the product was used without additional purification. Yield 340
mg (80%). MS: 869.4 [M+NH.sub.4].sup.+; 851.9 [M+1].sup.+.
[0183] Product 22i (n=23, AA=PheCit). To a solution of 3e (0.72
mmol) and DIEA (130 .mu.l, 0.74 mmol) in DMF (3 ml) was added a
solution of PEG.sub.23-NHS ester (0.60 mmol) in DMF (3 ml). The
mixture was stirred for 16 h, filtered and all volatiles were
removed on a rotovap at 40.degree. C./oil pump vacuum. The residue
was dissolved in a CHCl.sub.3:MeOH=1:1 mixture (5 ml) and
precipitated into Et.sub.2O (45 ml). The solid product was purified
on a column, eluent gradient of MeOH (7-12%) in CHCl.sub.3. Yield
487 mg (53%). MS: 1555.2 [M+Na].sup.+; 1544.7 [M+NH.sub.4].sup.+;
1527.7 [M+1].sup.+.
[0184] Product 22j (PEG with average MW 1000. AA=PheCit). A mixture
of mPEG-1000-alcohol (Fluka) (0.173 g, 0.173 mmol),
N,N-disuccinimidyl carbonate (62 mg, 0.242 mmol), and TEA (0.101
ml, 0.726 mmol) were stirred in MeCN (1 ml) for 16 h. All volatiles
were removed on a rotovap and the crude residue was dissolved in
CHCl.sub.3 (10 ml). The organic layer was washed with H.sub.2O (1
ml, pH=5), then brine, dried over Na.sub.2SO.sub.4 and concentrated
to afford PEG-1000-NHS carbonate. This product was stirred for 16 h
with 3e (0.121 mmol) and DIEA (30 .mu.l, 0.173 mmol) in DMF (1 ml),
filtered and all volatiles were removed on a rotovap at 40.degree.
C./oil pump vacuum. The residue was dissolved in a
CHCl.sub.3:MeOH=1:1 mixture (5 ml) and precipitated into Et.sub.2O
(45 ml). Precipitation was repeated two more times and the product
was used without additional purification. Yield 134 mg (79%).
[0185] Product 22k (n=23, AA=ValCit). To a solution of 3g (1.0
mmol) and DIEA (183 .mu.l, 1.04 mmol) in DMF (4 ml) was added a
solution of PEG.sub.23-NHS ester (0.87 mmol) in DMF (4 ml). The
mixture was stirred for 16 h, filtered and all volatiles were
removed on a rotovap at 40.degree. C./oil pump vacuum. The residue
was dissolved in a CHCl.sub.3:MeOH=1:1 mixture (5 ml) and
precipitated into Et.sub.2O (45 ml). Precipitation was repeated two
more times and the product was used without additional
purification. Yield 1.0 g (77%). MS: 1496.1 [M+NH.sub.4].sup.+;
1479.3 [M+1].sup.+.
PEG-AA-PABC-PNP 23a-k (FIG. 10A)
[0186] For product 23a (n=1, AA=Glu(2PhiPr)Gly), product 22a (274
mg, 0.274 mmol) in DCM (15 ml) was stirred in the dark with
(PNP).sub.2CO (418 mg, 1.372 mmol) and DIEA (0.143 ml, 0.823 mmol)
for 15 h. The solvent was removed on a rotovap and the product was
purified on a column, eluent 4% MeOH, 0.2% AcOH in CHCl.sub.3.
Yield 260 mg (81%). MS: 1180.7 [M+NH.sub.4].sup.+.
[0187] For product 23b (n=11, AA=PheCit), a solution of 22b (419
mg, 0.42 mmol), (PNP).sub.2CO (766 mg, 2.52 mmol) and DIEA (263
.mu.l, 1.52 mmol) in dioxane (4 ml) was stirred in the dark at
50.degree. C. for 15 h and all volatiles were removed on a rotovap.
The residual DIEA was removed by two consecutive evaporations of
DMF on a rotovap at 40.degree. C./oil pump vacuum and the product
was purified on a column, eluent CHCl.sub.3:EtOAc:MeOH (4.5:5:0.5)
followed by CHCl.sub.3:MeOH (9:1). Yield 390 mg (80%). MS: 1181.2
[M+NH.sub.4].sup.+, 1164.2 [M+1].sup.+.
[0188] For product 23c (n=1, AA=ValCit), a solution of 22c (273 mg,
0.287 mmol), (PNP).sub.2CO (874 mg, 2.88 mmol) and DIEA (0.3 ml,
1.72 mmol) in 1,4-dioxane (22 ml) was stirred in the dark for 24 h
at 50.degree. C. The solvent was removed on rotovap at 40.degree.
C./oil pump and the product was purified on a column, eluent:
CHCl.sub.3:EtOAc:MeOH=16:3:1 followed by 12-15% MeOH in CHCl.sub.3
Yield 163 mg (51%). MS: 1116.0 [M+1].sup.+.
[0189] Product 23d (n=11, AA=AlaAsn(DMCP)) was prepared as
described in the preparation of 23b. The product was purified on a
column, eluent CHCl.sub.3:EtOAc:MeOH (9:2:1). Yield 77%. MS: 1144.0
[M+NH.sub.4].sup.+; 1127.3 [M+1].sup.+.
[0190] Product 23e (n=1, AA=PheLys(Me).sub.2) was prepared as
described for 23a and purified on a column, eluent: 10% MeOH, 0.2%
AcOH in CHCl.sub.3. Yield 63%. MS: 1163.1 [M+1].sup.+.
[0191] Product 23f (n=1, AA=Leu) was prepared as described for 23c
using only 5 equivalents of (PNP).sub.2CO and 3 equivalents of DIEA
applying heat for 24h. The product was purified on a column, eluent
gradient of MeOH (7-12%) in CHCl.sub.3. Yield 75%. MS: 972
[M+1].sup.+.
[0192] Product 23g (n=11, AA=Asn(DMCP)) was prepared as described
for 23f and the crude product was used in the following step
without additional purification. MS: 1073.4 [M+18].sup.+.
[0193] For product 23h (n=1, AA=Cit), solution of 22h (340 mg, 0.40
mmol), (PNP).sub.2CO (608 mg, 2.00 mmol) and DIEA (208 .mu.l, 1.20
mmol) in DCM (4 ml) was stirred in the dark at 30.degree. C. for
15h and all volatiles were removed on a rotovap. The residual DIEA
was removed by two consecutive evaporations of DMF on a rotovap at
40.degree. C./oil pump vacuum and the product was purified on a
column, eluent CHCl.sub.3:EtOAc:MeOH (7:2.5:0.5) followed by a
gradient of MeOH (8-14%) in CHCl.sub.3. Yield 390 mg (80%). MS:
1034.3 [M+NH.sub.4].sup.+; 1016.9 [M+1].sup.+.
[0194] Product 23i (n=23, AA=PheCit) was prepared as described in
the preparation of 23b and purified on a column, eluent
CHCl.sub.3:EtOAc:MeOH (4.5:5:0.5) followed by a gradient of MeOH
(6-12%) in CHCl.sub.3. Yield 86%. MS: 1711.4 [M+NH.sub.4].sup.+;
1694.4 [M+1].sup.+.
[0195] Product 23j (PEG 1000K AA=PheCit) was prepared as described
in the preparation of 23b and purified on a column, eluent
CHCl.sub.3:EtOAc:MeOH (4.5:5:0.5) followed by a gradient of MeOH
(6-12%) in CHCl.sub.3. Yield 72%.
[0196] Product 23k (n=23, AA=ValCit) was prepared as described in
the preparation of 23b, and the product purified with HPLC. Column:
Luna (Phenomenex) 5u, C-8, 100 A. Mobile phase: ACN-H.sub.2O
(F.sub.3CO.sub.2H 0.01%), ACN gradient 30-37%, 31 min. Yield: 530
mg (48%). MS: 1666.4 [M+Na].sup.+; 1644.2 [M+1].sup.+.
PEG-AA-PABC-PNP 24a-c, AA Deprotection (FIG. 10B).
[0197] Product 24a (n=11, AA=GluGly). Product 23a (250 mg, 0.215
mmol) was stirred in a 3% TFA solution of CHCl.sub.3 (16 ml) for 35
min, concentrated on a rotovap and dried in vacuo. Yield 224 mg
(100%) (MS: 1062.6 [M+NH.sub.4].sup.+; 1045.9 [M+1].sup.+.
[0198] Product 24b (n=11, AA=AlaAsn-PABC-PNP). Compound 23d was
stirred for 1.5 h in a mixture of TFA:DCM (3:1) and all volatiles
were removed on a rotovap at 20.degree. C. The product was purified
on a column, eluent gradient of MeOH (6-12%) in CHCl.sub.3. Yield
30%. MS: 1066.7 [M+Na].sup.+, 1062.0 [M+NH.sub.4].sup.+; 1045.2
[M+1].sup.+.
[0199] Product 24c (n=11, AA=Asn). A reaction flask with 23g (160
mg, 0.143 mmol) was chilled to 0.degree. C. and a cold mixture of
TFA:H.sub.2O (9:1) (12.5 ml) was added. The mixture was stirred for
1.5 h and was diluted with cold H.sub.2O (50 ml). The stirring was
continued for 20 min at 20.degree. C.
[0200] The precipitate was filtered off and rinsed with H.sub.2O.
All volatiles were removed on a rotovap at 40.degree. C. and the
product was purified on a column, eluent
CHCl.sub.3:EtOAc:MeOH:AcOH=4.5:3.5:1.2:0.02. Yield 43 mg (30%). MS:
974.0 [M+1].sup.+.
TABLE-US-00002 TABLE 3 Final PEG-L-A.sub.1A.sub.2-PABC used for
conjugate preparation. compound PEG.sub.n-AA- PEG.sub.n-AA- AA PABA
(PNP) A.sup.1 A.sup.2 size 22 23a Glu(2PhiPr) Gly n = 11 22 23b Phe
Cit n = 11 22 23c Val Cit n = 11 22 23d Ala Asn(DMCP) n = 11 22 23e
Phe Lys(CH.sub.3).sub.2 n = 11 22 23f Leu -- n = 11 22 23g
Asn(DMCP) -- n = 11 22 23h Cit -- n = 11 22 23i Phe Cit n = 23 22
23j Phe Cit 1 kDa 22 23k Val Cit n = 23 24a Glu Gly n = 11 24b Ala
Asn n = 11 24c Asn -- n = 11
Example 2. Synthesis or RGD Ligands
A. RGD-PEG-Thioate:
1. RGD Mimic #1-PEG.sub.8-Thioate, MW 982.1.
##STR00030##
[0201] 2. RGD Mimic #2-PEG.sub.8-Thioate, MW 1022.2.
##STR00031##
[0202] 3. RGD Mimic #3-PEG.sub.8-Thioate, MW 1080.2.
##STR00032##
[0203] 4. RGD Mimic #3-PEG.sub.12-Thioate, MW 1212.4.
##STR00033##
[0204] B. RGD-HyNic:
1. RGD Mimic #1-PEG.sub.12-HyNic, MW 1272.
##STR00034##
[0205] 2. RGD Mimic #1a-HyNic, MW 802.8.
##STR00035##
3. RGD Mimic #1b-HyNic, MW 830.9 (RGD).
##STR00036##
C. RGE-PEG.sub.12-HyNic (Control). MW 1282.
##STR00037##
[0206] D. RGD Peptide-HyNic: RGD4C,
[NH.sub.2-ACDCRGDCFCG-Lys(e-6-HyNic); SEQ ID 5], MW 1407.83.
##STR00038##
[0207] Example 3. Reversible Modification of Amphipathic Membrane
Active Polyamines
[0208] Linkage of protease cleavable masking agents to
amine-containing polymers--formation of p-acylamidobenzyl carbamate
linkages.
A. RGD-PEG-Thioate.
[0209] Cy3-labeled polymer was combined with
SPDP-PEG.sub.24-FCit-para-nitrophenol at desired ratios in 100 mM
HEPES pH 9.0 buffer for 1 h at RT. The
SPDP-PEG.sub.24-FCit-modified polymer was then reacted with
PEG.sub.12-FCit at a weight ratio of 1:8 (polymer:PEG.sub.12-FCit)
in 100 mM HEPES, pH 9.0 buffer for 1 h at RT. The modified polymer
was then purified using a sephadex G-50 spin column.
##STR00039##
[0210] RGD-PEG-Thioate mimic was deacetylated with hydroxylamine at
a molar ratio of 1:5 (RGD-PEG-Thioate mimic:hydroxylamine) in PBS
pH 7.4 at RT for 2 h.
[0211] Modified polymer was combined with deacetylated
RGD-PEG-Thioate mimic at a weight ratio of 1:1 (polymer:RGD) in PBS
pH 7.4 at RT for a minimum of 4 h to form the polymer RGD
conjugate. The polymer-RGD conjugate was purified using a sephadex
G-50 spin column.
[0212] Conjugation efficiency was quantified by measuring
Absorbance of the polymer-RGD conjugate at 343 nm using an
extinction coefficient of 8.08.times.10.sup.3 M.sup.-1 cm.sup.-1
for pyridine 2-thione.
Molar concentration of polymer ( mM ) = weight concentration of
polymer ( mg / ml ) molecular weight of polymer ( Dalton ) .times.
1000 ##EQU00001## Molar concentration of RGD ( mM ) = [ A 343 (
conj . - RGD * ) - A 343 ( conj . no RGD control ) ] 8.08
##EQU00001.2## ( * prior to sephadex G - 50 purification )
##EQU00001.3## Number of RGD per polymer = molar concentration of
RGD ( mM ) molar concentration of polymer ( mM ) ##EQU00001.4##
[0213] Polymer-RGD conjugates were diluted with isotonic glucose
solution to desired concentrations.
mg ml polymer = conj . Cy 3 fluorescence post - purification conj .
Cy 3 fluorescence pre - purification .times. mg ml conj . pre -
purification ##EQU00002##
B. RGD-PEG-HyNic.
[0214] Cy3-labeled polymer was combined with
aldehyde-PEG.sub.12/24-TFP (Quanta Biodesign #10082) at desired
ratios in 100 mM HEPES pH 9.0 buffer for 1 h at RT. The
aldehyde-PEG.sub.12/24-TFP-modified polymer was then reacted with
PEG.sub.12-FCit at a weight ratio of 1:8 (polymer:PEG.sub.12-FCit)
in 100 mM HEPES, pH 9.0 buffer for 1 h at RT. The modified polymer
was then purified using a sephadex G-50 spin column.
##STR00040##
[0215] Modified polymer was combined with RGD-HyNic mimic at a
weight ratio of 1:0.7 (polymer:RGD) in 50 mM MES, pH 5.0 buffer at
RT for a minimum of 4 h to form the polymer RGD conjugate. The
polymer-RGD conjugate was purified using a sephadex G-50 spin
column.
[0216] Conjugation efficiency was quantified by measuring
Absorbance of the polymer-RGD conjugate at 354 nm using an
extinction coefficient of 2.9.times.10.sup.1 M.sup.-1 cm.sup.-1 for
bis-aryl hydrazone bond.
Molar concentration of polymer ( mM ) = weight concentration of
polymer ( mg / ml ) molecular weight of polymer ( Dalton ) .times.
1000 ##EQU00003## Molar concentration of RGD ( mM ) = [ A 354 (
conj . - RGD * ) - A 354 ( conj . no RGD control ) ] 29
##EQU00003.2## Number of RGD per polymer = molar concentration of
RGD ( mM ) molar concentration of polymer ( mM ) ##EQU00003.3##
[0217] Polymer-RGD conjugates were diluted with isotonic glucose
solution to desired concentrations.
polymer ( mg ml ) = conj . Cy 3 fluorescence post - purification
conj . Cy 3 fluorescence pre - purification .times. mg ml conj .
pre - purification ##EQU00004##
Example 4. Modification of Polyamines with PEG Protease Cleavable
Masking Agents
[0218] Activated (amine reactive) carbonates of p-acylamidobenzyl
alcohol derivatives are reacted with amino groups of amphipathic
membrane active polyamines in H.sub.2O at pH>8 to yield a
p-acylamidobenzyl carbamate.
##STR00041##
R.sup.1 comprises a PEG, R.sup.2 is an amphipathic membrane active
polyamine, AA is a dipeptide (either protected or unprotected), and
Z is an amine-reactive carbonate.
[0219] To .times.mg polymer is added 12.times.mg of HEPES free base
in isotonic glucose. To the buffered polymer solution is added
2.times. to 16.times.mg 200 mg/ml dipeptide masking agent in DMF.
In some applications, the polymer is modified with 2.times.mg
dipeptide masking agent followed by attachment of siRNA. The
polymer-siRNA conjugate is then further modified with 6.times. to
8.times.mg dipeptide masking agent.
Example 5. Conjugate Formulation
[0220] A. Formation of siRNA Delivery Conjugate Using
RGD-PEG-Thioate and PEG-Dipeptide Masking Agents.
[0221] The indicated polymer was reacted with SMPT at a weight
ratio of 1:0.015 (polymer:SMPT) in 5 mM HEPES, pH 8.0 buffer for 1
h at RT.
[0222] The SMPT-modified polymer was then reacted with
SPDP-PEG.sub.24-FCit at desired ratios for 1 h at RT. The modified
polymer was then reacted with PEG.sub.12-FCit at a weight ratio of
1:2 (polymer:PEG.sub.12-FCit) in 100 mM HEPES, pH 9.0 buffer for 1
h at RT. The modified polymer was then reacted overnight with
SATA-siRNA at a weight ratio of 1:0.2 (polymer:SATA-siRNA) in 100
mM HEPES, pH 9.0 buffer at RT to attach the siRNA. Next, the
modified polymer was reacted with PEG.sub.12-FCit at a weight ratio
of 1:6 (polymer:PEG.sub.12-FCit) in 100 mM HEPES, pH 9.0 buffer for
1 h at RT. The resultant conjugate was purified using a sephadex
G-50 spin column.
[0223] Deacetylated RGD-PEG-thioate was conjugated to the modified
polymer to form the RGD-conjugate by reaction with the modified
polymer at a weight ratio of 1:1 (polymer:RGD-PEG-thiol) in PBS pH
7.4 at RT for a minimum of 4 h. The conjugate was purified using a
sephadex G-50 spin column. RGD targeting ligand conjugation
efficiency was determined as described above and found to be 3.5
and 21 RGD ligands per 42K unmodified polymer.
B. Formation of siRNA Delivery Conjugate Using RGD-PEG-HyNic and
PEG-Dipeptide Masking Agents.
1) Protocol 1.
[0224] The indicated polymer was reacted with SMPT at a weight
ratio of 1:0.015 (polymer: SMPT) in 5 mM HEPES, pH 8.0 buffer for 1
h at RT.
[0225] The SMPT-modified polymer was then reacted with
aldehyde-PEG-dipeptide masking agent (aldehyde-PEG.sub.12-FCit or
aldehyde-PEG.sub.24-ACit) at desired ratios for 1 h at RT. The
modified polymer was then reacted with PEG.sub.12-dipeptide masking
agent (PEG.sub.12-FCit, PEG.sub.12-ACit or PEG.sub.24-ACit) at a
weight ratio of 1:2 (polymer:PEG) in 100 mM HEPES, pH 9.0 buffer
for 1 h at RT. The modified polymer was then reacted overnight with
SATA-siRNA at a weight ratio of 1:0.2 (polymer: SATA-siRNA) in 100
mM HEPES, pH 9.0 buffer at RT to attach the siRNA. Next, the
modified polymer was reacted with protease cleavable PEG
(PEG.sub.12-FCit or PEG.sub.12-ACit or PEG.sub.24-ACit) at a weight
ratio of 1:6 (polymer:PEG) in 100 mM HEPES, pH 9.0 buffer for 1 h
at RT. The resultant conjugate was purified using a sephadex G-50
spin column.
[0226] RGD-HyNic (Example 2B) was attached to the modified polymer
to form the full delivery conjugate by reaction with the modified
polymer at a weight ratio of 1:0.7 (polymer:RGD-HyNic mimic) in 50
mM MES, pH 5.0 buffer for a minimum of 4 h at RT. The conjugate was
purified using a sephadex G-50 spin column. RGD ligand attachment
efficiency was determined as described above.
2) Protocol 2.
[0227] The indicated polymer was reacted with SMPT at a weight
ratio of 1:0.015 (polymer: SMPT) in 5 mM HEPES, pH 8.0 buffer for 1
h at RT.
[0228] The SMPT-modified polymer was then reacted with
aldehyde-PEG-dipeptide masking agent (aldehyde-PEG.sub.24-ACit) at
a weight ratio of 1:0.5 (polymer:PEG) and with PEG-dipeptide
masking agent (PEG.sub.12-FCit, PEG.sub.12-ACit or PEG.sub.24-ACit)
at a weight ratio of 1:2 (polymer:PEG) in 100 mM HEPES, pH 9.0
buffer for 1 h at RT. The modified polymer was then reacted
overnight with SATA-siRNA at a weight ratio of 1:0.2 (polymer:
SATA-siRNA) in 100 mM HEPES, pH 9.0 buffer at RT to attach the
siRNA. Next, the modified polymer was reacted with protease
cleavable-PEG (PEG.sub.12-FCit or PEG.sub.12-ACit or
PEG.sub.24-ACit) at a weight ratio of 1:6 (polymer:PEG) in 100 mM
HEPES, pH 9.0 buffer for 1 h at RT.
[0229] RGD-HyNic (Example 2B) was attached to the modified polymer
to form the full conjugate by reaction with the modified polymer at
a weight ratio of 1:0.7 (polymer:RGD-HyNic) in 69 mM hydrogen
chloride solution (HCl) overnight at RT. RGD ligand attachment
efficiency was determined as described above.
3) Protocol 3.
[0230] The indicated polymer was reacted with SMPT at a weight
ratio of 1:0.015 (polymer: SMPT) in 5 mM HEPES, pH 8.0 buffer for 1
h at RT.
[0231] The SMPT-modified polymer was then reacted with
aldehyde-PEG-dipeptide masking agent (aldehyde-PEG.sub.24-ACit) at
a weight ratio of 1:0.5 (polymer:PEG) and with PEG-dipeptide
masking agent (PEG.sub.12-FCit, PEG.sub.12-ACit or PEG.sub.24-ACit)
at a weight ratio of 1:2 (polymer:PEG) in 50 mM HEPES, pH 9.0
buffer for 1 h at RT. The modified polymer was then reacted
overnight with SATA-siRNA at a weight ratio of 1:0.2 (polymer:
SATA-siRNA) in 50 mM HEPES, pH 9.0 buffer at RT to attach the
siRNA. Next, the modified polymer was reacted with protease
cleavable-PEG (PEG.sub.12-FCit or PEG.sub.12-ACit or
PEG.sub.24-ACit) at a weight ratio of 1:6 (polymer:PEG) in 50 mM
HEPES, pH 9.0 buffer for 1 h at RT.
[0232] RGD-HyNic (Example 2B) was attached to the modified polymer
to form the full delivery conjugate by reaction with the modified
polymer at a weight ratio of 1:0.7 (polymer:RGD-HyNic mimic) in 100
mM MES free acid solution overnight at RT. RGD targeting ligand
conjugation efficiency was determined as described above.
4) Protocol 4.
[0233] The indicated polymer was reacted with Azido-PEG4-NHS at a
weight ratio of 1:0.015 (polymer:Azido) in 5 mM HEPES, pH 8.0
buffer for 1 h at RT.
[0234] The Azido-modified polymer was then reacted with
aldehyde-PEG-dipeptide masking agent (aldehyde-PEG.sub.24-ACit) at
a weight ratio of 1:0.5 (polymer:PEG) and with PEG-dipeptide
masking agent (PEG.sub.12-ACit) at a weight ratio of 1:2
(polymer:PEG) in 50 mM HEPES, pH 9.0 buffer for 1 h at RT. The
modified polymer was then reacted overnight with Alkyne-siRNA at a
weight ratio of 1:0.2 (polymer:Alkyne-siRNA) in 50 mM HEPES, pH 9.0
buffer at RT to attach the siRNA. Next, the modified polymer was
reacted with protease cleavable-PEG (PEG.sub.12-ACit) at a weight
ratio of 1:6 (polymer:PEG) in 50 mM HEPES, pH 9.0 buffer for 1 h at
RT.
[0235] RGD-HyNic (Example 2B) was attached to the modified polymer
to form the full delivery conjugate by reaction with the modified
polymer at a weight ratio of 1:0.7 (polymer:RGD-HyNic mimic) in 100
mM sodium acetate-acetic acid buffer solution, pH 5.0 overnight at
RT. RGD targeting ligand conjugation efficiency was determined as
described above.
5) Protocol 5.
[0236] The mono azide-polymer was reacted with
aldehyde-PEG-dipeptide masking agent (aldehyde-PEG.sub.24-ACit) at
a weight ratio of 1:0.5 (polymer:PEG) and with PEG-dipeptide
masking agent (PEG.sub.12-ACit) at a weight ratio of 1:2
(polymer:PEG) in 50 mM HEPES, pH 9.0 buffer for 1 h at RT. The
modified polymer was then reacted overnight with Alkyne-siRNA at a
weight ratio of 1:0.2 (polymer:Alkyne-siRNA) in 50 mM HEPES, pH 9.0
buffer at RT to attach the siRNA. Next, the modified polymer was
reacted with protease cleavable-PEG (PEG.sub.12-ACit) at a weight
ratio of 1:6 (polymer:PEG) in 50 mM HEPES, pH 9.0 buffer for 1 h at
RT.
[0237] RGD-HyNic (Example 2B) was attached to the modified polymer
to form the full delivery conjugate by reaction with the modified
polymer at a weight ratio of 1:0.7 (polymer:RGD-HyNic mimic) in 100
mM sodium acetate-acetic acid buffer solution, pH 5.0 overnight at
RT. RGD targeting ligand conjugation efficiency was determined as
described above.
Example 6. In Vitro Cell Binding
[0238] Tumor cells, at 50,000/ml in 2 ml, were seeded on glass
coverslips in 6-well plates for 24 h. 5 .mu.g/ml Cy3-labeled
RGD-PEG-Thioate or Cy3-labeled RGD-PEG-HyNic (protocols 1-5)
modified polymer (50 .mu.l of 0.2 mg/ml, RGD-PEG, RGD #1) was added
drop-wise to cells and the cells were incubated for 24 h at
37.degree. C. Cells were washed 2.times. with PBS and fixed in 10%
formalin solution or 30 min. After fixation, cells were washed
2.times. with PBS.
[0239] Cells were stained with Alexa Fluor 488 phalloidin (2
unit/ml) and ToPro-3 iodide (0.2 .mu.M) for 20 min to stain actin
and nuclei, respectively; followed by 2.times. wash with PBS.
Coverslips were mounted to slides with Vectashield mounting medium
and fluorescent signal was then captured with a Zeiss LSM 710 laser
scanning confocal microscope. Cellular uptake of RDG-modified
polyamines was determined by the presence of intracellular Cy3
fluorescence. Cy3 signal intensity in various types of tumor cells
is summarized in the following Table 4 (5: highest; 1: lowest).
TABLE-US-00003 TABLE 4 Tumor Cell Uptake of RDG-modified polyamines
RGD-polymer cell line origin internalization A498 kidney cancer 5
ACHN kidney cancer 5 CAKI-2 kidney cancer 5 769-P kidney cancer 5
786-O kidney cancer 3 A375 melanoma 3 U87MG glioblastoma 3 PANC-1
pancreatic cancer 4 H460 lung cancer 3 H661 lung cancer 3 H1573
lung cancer 4 H2126 lung cancer 3 HT29 Colon cancer 3 HCT116 colon
cancer 2 HepG2 liver cancer 3 Hep3B liver cancer 1 MCF7 breast
cancer 2 SK-BR3 breast cancer 2 DU145 prostate cancer 1 PC3
prostate cancer 1 LNCaP prostate cancer 1 MDA-PCa-2b prostate
cancer 1 KB oral cancer 1 CAL27 tongue cancer 2 SCC9 tongue cancer
1 Detroit562 pharynx cancer 2 OVCAR3 ovarian cancer 1 SKOV3 ovarian
cancer 2 A2780 ovarian cancer 2
Example 7. In Vivo Tumor Tracking
[0240] Cy3-labeled RGD (thiol-RGD #1)/PEG-modified Lau 1005-116C-1
(54% Ethylethoxyamine acrylate/46% Butyl acrylate RAFT Co-polymer,
Fraction 1, 104 k protected MW, 76 k deprotected MW, 1.2 PDI) in
isotonic glucose 100-200 .mu.g (200 .mu.l of 0.5 or 1 mg/ml) were
injected into immunodeficient mice bearing human tumor xenografts
via tail vein administration. 4 h after injection, animals were
sacrificed and tumors were harvested. Whole tumors were fixed by
incubation in 10% formalin solution for a minimum of 4 h. Tumors
were then submerged into 30% sucrose solution overnight or until
equilibrated. Saturated tumors were snap frozen in liquid nitrogen,
and cryosectioned into 7 m slices on glass slides (VWR superfrost
plus micro slides). Sections were then stained with Alexa Fluor 488
phalloidin (2 unit/ml) and ToPro-3 iodide (0.2 .mu.M) for 20 min at
RT. After staining, sections were washed 2.times. with PBS and
mounted with coverslips on top using Vectashield mounting medium
and viewed with a Zeiss LSM 710 laser scanning laser confocal
microscope. Representative images were taken to illustrate the
tissue and cellular distribution of Cy3 fluorescence. In A498 tumor
model, RGD-modified polymer penetrated deeply into tumor tissue and
entered tumor cells, regardless of the location of tumor
implantation (subcutaneous, liver, or kidney). Control polymers
(either no RGD (PEG only) or RGE-modified) remained primarily in
the vasculature, without entering tumor cells. In liver implanted
HepG2 tumor model, strong RGD-conjugate penetration into tumor
tissue was observed.
Example 8. In Vivo Tumor mRNA Knockdown
[0241] RGD-targeting conjugates in isotonic glucose (500 .mu.g; 300
.mu.l of 1.67 mg/ml) were injected into immunodeficient mice
bearing human tumors xenografts via tail vein administration. 72 h
after injection, animals were sacrificed and tumors were harvested.
Tumor tissue was homogenized in Tri reagent (Molecular Research
Center) to isolate total RNA. Relative mRNA knockdown by determined
using quantitative RT-PCR.
Example 9. Orthotopic Renal Cell Carcinoma (RCC) Tumor Mice
Model
[0242] A498 cells (ATCC) were grown in MEM (Invitrogen)
supplemented with 10% FBS (Invitrogen). 786-0 RCC cells (ATCC) were
grown in 1.times.MEM Non-Essential Amino Acids Solution
(Invitrogen) and RPMI (Invitrogen) supplemented with 10% FBS. Cells
were collected, counted, and mixed with matrigel matrix HC (BD
Biosciences, 30% by volume) on ice.
[0243] Female athymic nude mice were anesthetized with .about.3%
isoflourane and placed in the right lateral decubitus position. A
small, 0.5-1 cm, longitudinally abdominal incision in the left
flank was made. Using a moist cotton swab, the left kidney was
lifted out of peritoneum and gently stabilized. Just before
injection, a 1.0 ml syringe was filled with the cell/Matrigel
mixture and a 27 gauge needle catheter was attached to the syringe
tip. The filled syringe was then attached to a syringe pump
(Harvard Apparatus, model PHD2000) and primed to remove air. The
tip of a 27-gauge needle catheter attached to a syringe was
inserted just below the renal capsule near the caudal pole and the
tip of the needle was then carefully advanced cranially along the
capsule 3-4 mm. A 10 .mu.l aliquot of 2.5:1 (vol:vol) cell/matrigel
mixture containing about 200,000 cells was slowly injected into the
kidney parenchyma using a syringe pump. The needle was left in the
kidney for 15-20 seconds to ensure the injection was complete. The
needle was then removed from the kidney and a cotton swab was
placed over the injection site for 30 seconds to prevent leakage of
the cells or bleeding. The kidney was then gently placed back into
the abdomen and the abdominal wall was closed. Three (3) weeks
after implantation, tumor progression was evaluated by visual
examination and measurement after euthanasia. For most studies,
tumor mice were used 5-6 weeks after implantation, when tumor
measurements were typically around 4-8 mm.
Example 10. Subcutaneous (SQ) Tumors
[0244] For SQ tumor implantations, anesthesia was induced by
placing mice in an induction chamber with 3% isoflurane. Once
anesthetized, mice were placed on a drape and 3% isoflurane
anesthesia was supplemented through a nose cone. Mice were
positioned on their side (right or left) and injections were
performed into the opposite flank. Just before injection, a 1.0 ml
syringe was filled with the cell/Matrigel mixture and a 27 gauge
needle catheter was attached to the syringe tip. The filled syringe
was then attached to a syringe pump (Harvard Apparatus, model
PHD2000) and primed to remove air. The needle was inserted in the
flank just under the skin layer and cells (10 .mu.l or 20 .mu.l)
were injected at a rate of 250 .mu.l per minute. After the
injection, the needle was removed and the mouse was placed in a
recovery cage with heat provided by a water jacketed heating pad
placed under the cage. Animals were returned to their housing racks
once they have regained a normal level of activity.
Example 11. Liver Tumor Model
[0245] HepG2 cells were co-transfected with 2 expression vectors,
pMIR85 a human placental secreted alkaline phosphatase (SEAP)
vector and pMIR3 a neomycin/kanamycin-resistance gene vector, to
develop cell lines with stable SEAP expression. Cells were grown in
DMEM supplemented with 10% FBS and 300 .mu.g/ml G418. HT-29 colon
carcinoma cells were grown in McCoy's 5A medium supplemented with
10% FBS. For tumor implantation, cells were collected, counted, and
mixed with matrigel (BD Biosciences, 40% by volume). Athymic nude
mice were anesthetized with .about.3% isoflourane and placed in a
sternal recumbent position. A small, 1-2 cm, midline abdominal
incision was made just below the xyphoid. Using a moist cotton
swab, the left lobe of the liver was gently exteriorized. The left
lobe of the liver was gently retracted and a syringe needle was
inserted into the middle of the left lobe. Just before injection, a
1.0 ml syringe was filled with cell/Matrigel mixture and a 27 gauge
needle catheter was attached to the syringe tip. The filled syringe
was then attached to a syringe pump (Harvard Apparatus, model
PHD2000) and primed to remove air. The syringe needle was inserted
with the bevel down about 0.5 cm just under the capsule of the
liver. 10 .mu.l of cell/Matrigel mixture containing 100,000 cells,
was injected into the liver using a syringe pump. The needle was
left in the liver for 15-20 seconds to ensure the injection was
complete. The needle was then removed from the liver and a cotton
swab was placed over the injection site to prevent leakage of the
cells or bleeding. The Matrigel/cells mixture formed a mass that
was visible and did not disappear after removal of the needle. The
liver lobe was then gently placed back into the abdomen and the
abdominal wall was closed. For HepG2 tumor mice, sera were
collected once per week after tumor implantation and subjected to
SEAP assay to monitor tumor growth. For most studies, tumor mice
were used 4-5 weeks after implantation, when tumor measurements
were predicted to be around 4-8 mm based on SEAP values. For HT-29
tumor mice, based on historical observations, tumor mice were used
5-6 weeks after implantation when tumors typically reached 4-8 mm
in length and width.
Example 12. Quantitative Real-Time PCR Assay
[0246] In preparation for quantitative PCR, total RNA was isolated
from tissue samples homogenized in TriReagent (Molecular Research
Center, Cincinnati, Ohio) following the manufacturer's protocol.
Approximately 500 ng RNA was reverse-transcribed using the High
Capacity cDNA Reverse Transcription Kit (Life Technologies). For
human (tumor) Aha1 expression, pre-manufactured TaqMan gene
expression assays for human Aha1 (Assay ID: Hs00201602_m1) and CycA
(Part#: 4326316E) were used in biplex reactions in triplicate using
TaqMan Gene Expression Master Mix (Life Technologies) or VeriQuest
Probe Master Mix (Affymetrix). For human (tumor) EG5 expression,
pre-manufactured TaqMan gene expression assays for human EG5 (Assay
ID: Hs00189698_m1) and CycA (Part#: 4326316E) were used in biplex
reactions in triplicate using TaqMan Gene Expression Master Mix
(Life Technologies) or VeriQuest Probe Master Mix (Affymetrix).
Quantitative PCR was performed by using a 7500 Fast or StepOnePlus
Real-Time PCR system (Life Technologies). The .DELTA..DELTA.C.sub.T
method was used to calculate relative gene expression.
Example 13. Knockdown of Renal Cell Carcinoma Tumor Gene In
Vivo
[0247] RGD targeted siRNA delivery conjugates were formed using RGD
mimic #1-PEG-thioate or RGD mimic #1-PEG-HyNic. 500 .mu.g Lau
41648-106 polymer modified with 8.times.PEG.sub.12-FCit/0.5.times.
aldehyde-PEG.sub.24-FCit (with RGD mimic #1-PEG-HyNic using
protocol #1) or SPDP-PEG.sub.24-FCit (with RGD-PEG-thioate) and 100
.mu.g Aha1 siRNA. Kidney RCC tumor-bearing mice were generated as
described and treated with a single tail vein injection of Aha1
conjugates. Mice were euthanized 72 h after injection and total RNA
was prepared from kidney tumor using Trizol reagent following
manufacture's recommendation. Relative Aha1 mRNA levels were
determined by RT-qPCR as described compared to mice treated with
delivery buffer only. Conjugates formulated without targeting
ligand or RGE control ligand exhibited 25-35% reduction in gene
expression. RGD targeted conjugates exhibited 50-70% gene reduction
in tumor Aha1 expression (n=3 or 4).
TABLE-US-00004 TABLE 5 Relative tumor Aha1 mRNA levels in
orthotopic Kidney RCC tumor in mice following treatment with a
single dose of Aha1 siRNA conjugates. Relative human Aha1 Treatment
mRNA level in tumor (%) Delivery buffer (IG) 100 .+-. 14 No
ligand-conjugate 64 .+-. 2 HyNic-RGE mimic #1-conjugate 75 .+-. 5
Thiol-RGD mimic #1-conjugate 31 .+-. 4 HyNic-RGD mimic #1-conjugate
42 .+-. 8 HyNic-RGD4C-conjugate (CS Bio) 52 .+-. 14
[0248] Gene knockdown in animals treated with untargeted conjugates
(no ligand or RGE mimic (negative control ligand) was likely the
result of passive accumulation of conjugates in the tumor through
the enhanced permeability and retention (EPR) effect (Torchilin,
2011). Long serum stability and circulation of the conjugates
allows for significant accumulation in the tumor through EPR.
Still, RGD-targeted conjugates exhibited an additional 20-40%
reduction in gene expression compared to the passively targeted
conjugates.
Example 14. Knockdown of Renal Cell Carcinoma Tumor Gene In
Vivo
[0249] RGD mimic #1 targeted siRNA delivery conjugates were formed
using RGD mimic #1-PEG and protocol #1. 500 .mu.g Lau 41648-106
polymer was modified with 8.times.PEG.sub.12-FCit/0.5.times.
aldehyde-PEG.sub.24-FCit, and 100 .mu.g Aha1 siRNA. RGD mimic
#1-PEG-HyNic was used as targeting ligand for these samples. Mice
containing both metastatic (subcutaneous) and orthotopic kidney RCC
tumors were generated as described above and treated with a single
tail vein injection of Aha1 conjugates. Mice were euthanized 72 h
after injection and total RNA was prepared from kidney tumor using
Trizol reagent following manufacture's recommendation. Relative
human (tumor) Aha1 mRNA levels at tumor implantation sites were
determined by RT-qPCR as described above and normalized to mice
injected with delivery buffer only. RGD-targeted conjugate
exhibited 40-50% gene reduction in tumor Aha1 expression (n=3 or
4).
TABLE-US-00005 TABLE 6 Relative tumor Aha1 mRNA levels in
orthotopic kidney or metastatic subcutaneous RCC tumors in mice
following treatment with a single dose of Aha1 siRNA conjugates.
Relative tumor Aha1 Treatment Tumor mRNA level (%) Delivery buffer
kidney 100 .+-. 7 HyNic-RGD mimic #1-conjugate kidney 49 .+-. 14
Delivery buffer subcutaneous 100 .+-. 6 HyNic-RGD mimic
#1-conjugate subcutaneous 59 .+-. 3
Example 15. RDG-Targeted siRNA Delivery Conjugates Delivery siRNA
to Multiple Tumor Types
[0250] Cancers that affect the liver include cancers originating
from liver cells (e.g. hepatocellular carcinoma, HCC) and
metastatic cancers originating in other tissues as the colon, lung,
renal or breast. Various cancer cell types know to express
.alpha..sub.v.beta..sub.3 integrin and bind to RGD-conjugates in
vitro were implanted into the liver. RGD targeted siRNA delivery
conjugates were formed using RGD mimic #1-HyNic or RDG mimic
#1-PEG-thioate. 500 .mu.g Lau 41648-106 polymer was modified with
8.times.PEG.sub.12-FCit/0.5.times. aldehyde-PEG.sub.24-FCit
(protocol #1 or #5) or
8.times.PEG.sub.12-FCit-/0.5.times.SPDP-PEG.sub.24-FCit (using
RGD-PEG-Thioate protocol) and 100 .mu.g Aha1 siRNA. Tumor bearing
mice were then treated with a single dose of Aha1 siRNA delivery
conjugates administered by tail vein injection. Mice were
euthanized 72 h after injection and total RNA was prepared from
liver tumors using Trizol reagent following manufacture's
recommendation. For each tumor type, tumor Aha1 mRNA levels were
normalized tumor in mice receiving delivery buffer only.
RGD-targeted conjugates exhibited 50-60% reduction in tumor Aha1
gene expression (n=3 or 4).
TABLE-US-00006 TABLE 7 Relative tumor Aha1 mRNA levels in various
liver tumors in mice following treatment with a single dose of Aha1
siRNA conjugates. Relative tumor Aha1 Treatment Tumor cell type
mRNA level (%) Delivery buffer 100 .+-. sd Thiol-RGD mimic
#1-conjugate RCC (A498) 50 .+-. 15 HyNic-RGD4C-conjugate HCC
(HepG2) 60 .+-. 5 HyNic-RGD mimic #1-conjugate Colon (HT-29) 63
.+-. 10 HyNic-RGD mimic/click siRNA Lung (H460) 63 .+-. 8
Example 16. RGD-Conjugate Formulation with Different Polymer
Classes
[0251] Polymer Emi 1034-68C-1 [0252] Polymer Emi 1034-81F-1 [0253]
Polymer LH 1073-20C-1
[0254] RGD mimic #1 targeted siRNA delivery conjugates were formed
using RGD mimic #1b and protocol #2, 400 .mu.g Emi 1034-68C-1, Emi
1034-81F-1 or LH 1073-20C-1 polymer was modified with
8.times.PEG.sub.12 ACit/0.5.times. aldehyde-PEG.sub.24-ACit and 80
.mu.g Aha1 siRNA. RGD mimic #1b was used as targeting ligand for
these formulations. Orthotopic RCC tumor bearing mice were then
treated with a single dose of Aha1 conjugates administered by tail
vein injection. Mice were euthanized 72 h after injection and total
RNA was prepared from liver tumors using Trizol reagent following
manufacture's recommendation. Tumor Aha1 mRNA levels were
normalized to tumor in mice receiving delivery buffer only.
TABLE-US-00007 TABLE 8 Relative tumor Aha1 mRNA levels in
orthotopic kidney RCC tumor in mice following treatment with a
single dose of Aha1 siRNA conjugates. Relative tumor Aha1 Treatment
mRNA level (%) Delivery buffer 100 .+-. 9.8 RGD/PEG-Emi1034-68C-1
conjugate 49 .+-. 4.9 RGD/PEG-Emi1034-81F-1 conjugate 42 .+-. 6.3
RGD/PEG-Lor1073-20C-1 conjugate 56 .+-. 4.5
Example 17. RGD-Conjugate Formulation with ACit or FCit Dipeptide
Masking Reagents
[0255] RGD mimic #1 targeted siRNA delivery conjugates were formed
using RGD mimic #1b and protocol #2. 400 .mu.g Emi 1034-68C-1
polymer was modified with 8.times.PEG.sub.12-FCit/0.5.times.
aldehyde-PEG.sub.12-FCit (for FCit dipeptide masked delivery
conjugate) or 8.times.PEG.sub.12-ACit/0.5.times.
aldehyde-PEG.sub.24-ACit (for ACit dipeptide masked delivery
conjugate) and 80 .mu.g Aha1 siRNA. RGD mimic #1b-HyNic was used as
targeting ligand for these formulations. Orthotopic RCC tumor
bearing mice were then treated with a single dose of Aha1 siRNA
delivery conjugates administered by tail vein injection. Mice were
euthanized 72 h after injection and total RNA was prepared from
liver tumors using Trizol reagent following manufacture's
recommendation. Tumor Aha1 mRNA levels were normalized to tumor in
mice receiving delivery buffer only. There was no significant
difference in gene knockdown efficacy between ACit or FCit siRNA
delivery conjugates. RGD-targeted conjugates exhibited 51% or 53%
reduction in tumor Aha1 gene expression (n=3) for ACit or FCit
conjugates, respectively.
TABLE-US-00008 TABLE 9 Relative tumor Aha1 mRNA levels in
orthotopic kidney RCC tumor in mice following treatment with a
single dose of Aha1 siRNA conjugates. Relative tumor Aha1 mRNA
Treatment level (%) Delivery buffer 100 .+-. 9.8 ACit RGD mimic
#1b-conjugate 49 .+-. 4.9 FCit RGD mimic #1b-conjugate 47 .+-.
7.3
Example 18. siRNAs
[0256] siRNAs had the following sequences:
TABLE-US-00009 AhaI siRNA: sense: (SEQ ID 3)
(NH.sub.2C.sub.6)GfgAfuGfaAfgUfgGfaGfaUfuAfgUf(invdT) antisense:
(SEQ ID 4) pdAsCfuAfaUfcUfcCfaCfuUfcAfuCfcdTsdT
[0257] p=phosphate [0258] d before nucleotide=2'-deoxy [0259]
s=phosphorothioate linkage [0260] f after nucleotide=2'-F
substitution [0261] lower case=2'-O--CH.sub.3 substitution
[0262] RNA synthesis was performed on solid phase by conventional
phosphoramidite chemistry on an AKTA Oligopilot 100 (GE Healthcare,
Freiburg, Germany) or Mermade 12 (Bioautomation, Plano Tex.) and
controlled pore glass (CPG) as solid support.
Example 19. Synthesis of Alkyne Disulfide Sense-Strand RNA
Conjugate
[0263] Crude sense-strand RNA (3.4 mg, 506 nmol) with a 5' C-6
amino modification, was precipitated using sodium acetate (0.3M) in
EtOH at -80.degree. C., lyophilized, and dissolved in 300 .mu.L
0.2M NaHCO.sub.3, pH 8-9. Dibenzocyclooctyne-N-hydroxysuccinimidyl
(Dibenzocyclooctyne-S--S--N-hydroxysuccinimidyl ester (DBCO-NHS),
item #761532 Aldrich) (2.86 mg, 5060 nmol) was dissolved in 286
.mu.L DMF and added to the RNA solution. The reaction mixture was
mixed well and allowed to proceed for 2 h at RT. The reaction was
monitored using RP-HPLC. After reaction completion, the reaction
mixture was dried down and purified using RP-HPLC. The RNA
conjugate was prepared in 45% yield (229 nmol). The purity of the
RNA conjugate was determined by RP-HPLC (purity: 96.6%) and the
identity was confirmed by MALDI-TOF/TOF (Mass calculated: 7164.0;
Mass observed: 7164.8).
Example 20. Synthesis of 4-(Fmoc-4-Aminophenoxy) Butyric Acid
##STR00042##
[0264] A. Synthesis of 3
[0265] p-nitro-phenol (2) (7.5 g, 53.9 mmol) was combined with
ethyl 4-bromobutyrate (8.45 ml, 5.9 mmol) and K.sub.2CO.sub.3 (7.5
g, 5.4 mmol) in DMF (75 ml) and stirred for 2 h at 100.degree. C.
DMF was removed and the crude was diluted in a mixture of 3:1
mixture of 2 N NaOH and methanol and stirred 4 h at RT. The
reaction mixture was acidified with 6M HCl. The white precipitate
was collected (10.9 g 90% yield). [.sup.1H-NMR (400 MHz, DMSO)
.delta.: 12.165 (bs, 1H) .delta.: 8.175 (AA', J=Hz, 2H) .delta.:
7.120 (AA', J=Hz, 2H), .delta.: 4.122 (t, J=6.8 Hz, 2H), .delta.:
2.379 (t, J=6.8 Hz, 2H), .delta.: 1.975 (p, J=6.8 Hz, 2H)]
B. Synthesis of 4
[0266] 3 (37.1 g, mmol) was dissolved in MeOH (1 L) with ammonium
formate (35 g, mmol) and 10% Pd/C (Degussa Type) (3.5 g) was added.
The mixture was refluxed at 65.degree. C. overnight. The reaction
was filtered with celite to yield a reddish brown solid (30.5 g,
95% yield). [.sup.1H-NMR (400 MHz, DMSO), .delta.: 6.609 (AA',
J=Hz, 2H) .delta.: 6.470 (AA', J=Hz, 2H), .delta.: 3.790 (t, J=6.8
Hz, 2H), .delta.: 2.288 (t, J=7.2 Hz, 2H), .delta.: 1.832 (p, J=7.2
Hz, 2H)]
C. Synthesis of 4-(Fmoc-4-aminophenoxy) butyric acid (1)
[0267] 4 (5.1 g, 26 mmol) was dissolved in a 6:4 a mixture of
aqueous saturated sodium bicarbonate solution and THF (300 ml) to
make a white slurry. Fmoc-OSu (8.82 g, 26.1 mmol) was added and the
reaction was stirred for 4 h. The acetone was removed, the reaction
was acidified and the off-white precipitate was collected and
triturated in 1N HCl to yield 9.6 g product (88% yield, molecular
weight 389.40066). [.sup.1H-NMR (400 MHz, DMSO) .delta.: 9.508 (bs,
1 Hz), .delta.: 7.885 (d, J=7.6, 2 Hz, 2H), .delta.: 7.727 (d,
J=6.8 Hz, 2H), .delta.: 7.389-7.32 (bm, 7H), .delta.: 7.328 (dd,
J=6.8, 6.4 Hz, 2H), .delta.: 6.828 (d, J=7.6 Hz, 2H), .delta.:
4.435 (d, J=6.0 Hz, 2H), .delta.: 4.275 (t, J=6.4 Hz, 1H), .delta.:
3.897 (t, J=6.0 Hz, 2H), .delta.: 2.335 (t, J=7.2 Hz, 2H), .delta.:
1.885 (p, J=7.2 Hz)]
[0268] Reagents used: Dimethylformamide (DMF), Dichloromethane
(DCM), Methanol (MeOH), H.sub.2O (HPLC grade), Acetone,
p-Nitrophenol, Ethyl 4-Bromobutyrate, Potassium Carbonate, Pd/C
(Degussa type) were purchased from Sigma Aldrich and used as is.
Fmoc-OSu was purchased from Novabiochem.
Example 21. Synthesis of
Guanadino-Gly-Asp(OH)-APOA-PEG.sub.12-HyNic-Boc
TABLE-US-00010 [0269] Fmoc-Gly-Asp(O-2PhiPr)-OH 2
[0270] A. Fmoc-Gly-Asp(O-2PhiPr)-OH (2). Fmoc-Gly-OH (CAS
29022-11-5) (1.57 g, 5.28 mmol) was dissolved in THF (30 ml) and
set to stir in an ice water bath. NHS (0.668 g, 5.81 mmol) and DCC
(1.2 g, 5.81 mmol) were added to the solution, stirred for 5 min,
and the ice bath was removed. The reaction mixture was stirred for
16 h at 20.degree. C., cooled to 0.degree. C. for 1 h, then
filtered, concentrated and dried in vacuum. The crude product was
dissolved in THF (20 ml) and added to a solution of
H-Asp(2-PhiPr)-OH (CAS 200336-86-3) (1.328 g, 5.28 mmol) and
NaHCO.sub.3 (600 mg, 7.14 mmol) in H.sub.2O (30 ml).
1,2-Dimethoxyehtane (DME, 30 ml) was added to make the solution
homogeneous and stirred for 16 h. THF and DME were removed on a
rotovap, the residue was diluted with H.sub.2O (150 ml) and
acidified to pH=3 with 3% HCl to yield Fmoc-Gly-Asp(O-2PhiPr)-OH
(2). Product was extracted 5 times with EtOAc, rinsed with brine,
dried (Na.sub.2SO.sub.4) and concentrated and dried in vacuum.
Yield 2.54 g. Crude product was used in the next step assuming 100%
yield.
##STR00043##
[0271] B. Fmoc-Gly-Asp(O-2PhiPr)-BAPOA-Boc (3). Dipeptide 2 (1.168
g, 2.2 mmol) was dissolved in DCM (20 ml) and set to stir in an ice
water bath. NHS (291 mg, 2.53 mmol) and DCC (522 mg, 2.53 mmol)
were added to the solution, stirred for 5 min at 0.degree. C. and
then 16 h at 20.degree. C. The reaction mixture was cooled on an
ice bath for 1 h, filtered and concentrated and dried in vacuum.
The obtained NHS derivative was dissolved in DCM (15 ml) and added
to a solution of 4-[3-(Boc-amino)propan-1-yloxy]-aniline (APOA-Boc,
644 mg, 2.42 mmol) (Quelever, Frederic. and Kraus Organic &
Biomolecular Chemistry, 1(10), 1676-1683; 2003) and TEA (175 .mu.l,
1.25 mmol). The reaction was stirred at 20.degree. C. for 3 h and
1,4-dioxane (10 ml) was added. All volatiles were removed in vacuo.
The residue was dissolved in EtOAc (200 ml), washed with 5%
KHSO.sub.4 (2.times.40 ml), water (1.times.40 ml), concentrated
sodium bicarbonate solution (1.times.40 ml), and brine (1.times.40
ml). The organic layer was dried (Na.sub.2SO.sub.4), than
concentrated and dried in vacuum. Yield 1.714 g. Crude product,
Fmoc-Gly-Asp(O-2PhiPr)-BAPOA-Boc (3), was used in the next step
assuming 100% yield.
##STR00044##
[0272] C. Fmoc-Gly-Asp(OH)-APOA HCl salt (4). Compound 3 (800 mg,
1.02 mmol) was treated with an ice cold solution of HCl in dioxane
(4M, 22 ml) and stirred at 0.degree. C. for 45 min. Cooling bath
was removed and the suspension was concentrated. The residue was
suspended in CHCl.sub.3 (2.5 ml), diethyl ether (45 ml) was added,
and the solid was separated. The reprecipitation procedure of HCl
salt of 4-[3-(amino)propan-1-yloxy]-aniline (APOA) derivative was
repeated twice and the product was dried in vacuo. Yield: 557 mg
(92%).
##STR00045##
[0273] D. Fmoc-Gly-Asp(OH)-APOA-PEG.sub.12-NH-Boc (5). To a
solution containing Boc-Peg.sub.12-CO.sub.2H (Quanta Biodesign
Limited 10761, 803 mg, 1.12 mmol) in DCM (8 ml) at 0.degree. C. was
added NHS (193 mg, 1.68 mmol) followed by DCC (346 mg, 1.68 mmol).
Cooling was removed and stirring continued for 24 h. The reaction
mixture was chilled to -20.degree. C., filtered and concentrated.
The NHS derivative was treated with a solution of compound 4 (667
mg, 1.12 mmol) and DIEA (154 .mu.l, 0.89 mmol) in DMF (14 ml),
stirred for 16 h, filtered, and concentrated. The crude residue
obtained was purified by flash SiO.sub.2 chromatography eluting
DCM:MeOH:Acetic Acid (92.5:7:0.5). Yield: 575 mg (41%).
##STR00046##
[0274] E. Fmoc-Gly-Asp(OH)-APOA-PEG.sub.12-NH.sub.2 (6). Compound 5
(140 mg, 0.11 mmol) was treated with TFA (3 ml) and H.sub.2O (1
ml). The mixture was stirred for 20 min, diluted with cold H.sub.2O
and all volatiles were removed on a rotovap. The residue was
triturated 3.times. with Et.sub.2O, dried in vacuo and used without
further purification. Yield: 115 mg (90%).
##STR00047##
[0275] F. H.sub.2N-Gly-Asp(OH)-APOA-PEG.sub.12-HyNic-Boc (7). To a
solution containing 6 (105 mg, 0.091 mmol) and DIEA (31 .mu.l, 0.18
mmol) in DCM (3 ml) was added Boc-HyNic-NHS (Abrams, M. J., Juweid,
M., Tenkate, C. I., Schwartz, D. A., Hauser, M. M. Gaul, F. E., 15
Fuccello, A. J., Rubin, R. H., Strauss, H. W., Fischmann, A. J. J.
Nucl. Med. 31, 2022-2028, 1990.) (35 mg, 0.10 mmol).
##STR00048##
[0276] The mixture was stirred for 16 hr, treated with ethyl amine
(30 .mu.l, 2.0M) in MeOH, and stirred for an additional 3 h (to
quench the excess of unreacted Boc-HyNic-NHS). All volatiles were
removed on a rotovap. The crude residue was dissolved in DMF (3
ml), then treated with TEA (400 .mu.l) and stirred for 16 h. All
volatiles removed on a rotovap with an oil pump vacuum. The crude
product was purified with preparative HPLC using a Thermo Aquasil
C18 column (5u, 100 .ANG.) eluting a gradient (18-43%) of ACN (0.1%
formic acid) in H.sub.2O (0.1% formic acid) over 30 min. Yield: 36
mg (34%).
##STR00049##
[0277] G. 3-Guanadinobenzoic acid-NHS ester (1). NHS (589 mg, 5.12
mmol) was added into a stirring solution of 3-guanidinobenzoic acid
(1 g, 4.65 mmol; Chandrakumar, N., et al. U.S. Pat. No. 5,773,646)
in DMF (25 ml), stirred for 5 min, then DCC (1.056 g, 5.12 mmol)
was added into the reaction mixture. The stirring was continued for
2h then the reaction mixture was cooled to -20.degree. C. for 30
min, the product was filtered off, washed with cold DMF, and dried
in vacuo using an oil pump. Yield: 1.283 g (100%).
##STR00050##
[0278] H. Guanadino-Gly-Asp(OH)-APOA-PEG.sub.12-HyNic-Boc (8). A
mixture of H.sub.2O (1 ml) and THF (0.9 ml) containing 7 (36 mg,
0.031 mmol) and NaHCO.sub.3 (13 mg, 0.155 mmol) was treated with
NHS ester of 3-guanadinobenzoic acid (1) (19.5 mg, 0.071 mmol) and
stirred for 22 h. The mixture was then diluted with H.sub.2O and
THF was removed on a rotovap. The residue was suspended in water (3
ml) at pH=3 (1% HCl) and solid impurities were filtered off.
Product was concentrated in vacuo, the crude residue was treated
with a mixture of H.sub.2O (0.8 ml), acetone (0.8 ml), and TFA (1.8
ml) and stirred for 40 min. Upon completion the solution was
diluted with acetone (2 ml) and all volatiles were removed on a
rotovap. The crude product obtained was purified with preparative
HPLC using a Thermo Aquasil C18 column (5u, 100 .ANG.) eluting a
gradient (18-43%) of ACN (0.1% formic acid) in H.sub.2O (0.1%
formic acid) over 30 min. Yield: 34 mg (89%).
Example 22. Solid Phase Synthesis of RGD Ligand
##STR00051##
[0280] Dimethylformamide (DMF), piperidine, dichloromethane (DCM),
Diethyl ether (Et.sub.2O), H.sub.2O (HPLC grade), acetonitrile
(ACN) (HPLC grade), triethylamine (TEA), F.sub.3CCO.sub.2H (TFA),
and triisopropyl silane (TIPS). PyBOP
(benzotriazol-1-yl-oxytripyrrolidino-phosphonium
hexafluorophosphate) was purchased from Oakwood Products Inc. Rink
Amide MBHA resin, Fmoc-Gly-OH, and Fmoc-Asp(tBu)-OH were purchased
from Novabiochem. 4-(N-Fmoc para-aminophenoxy)-butyric acid and
di-boc m-guanidino benzoic acid were synthesized using standard
techniques in the art. Fritted polypropylene syringes were
purchased from Torviq. Solvent A is H.sub.2O:F.sub.3CCO.sub.2H
100:0.1 v/v. Solvent B is CH.sub.3CN:F.sub.3CCO.sub.2H 100:0.1 v/v.
Fmoc-Lys(HyNic)-OH was synthesized as previously described.
##STR00052##
[0281] A. Peptide Synthesis.
[0282] Rink Amide MBHA resin was placed in fritted polypropylene
syringe and agitated in DCM for 2 h prior to use. The following
standard solid phase peptide synthesis conditions were used. Fmoc
deprotections were carried out by soaking 10 ml (per 1 mmol of
resin) of a piperidine:DMF solution (20:80 v/v) for 20 min. Amide
couplings were carried out by soaking the resin with 4 eq.
Fmoc-amino acid, 4 eq. PyBOP and 10 eq. Triethylamine in DMF at 0.1
M concentration in DMF for 40 min.
[0283] Fmoc-Lys(HyNic)-OH, 4-(N-Fmoc para-aminophenoxy)-butyric
acid, Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, and Fmoc-3-aminobenzoic acid
were sequentially coupled onto the resin. Progress of amide
couplings were checked using MALDI-TOF analysis. Fmoc deprotection
of 4-(N-Fmoc para-aminophenoxy)-butyric acid was carried out for 40
min to ensure complete Fmoc-deprotection. The Fmoc-Asp(tBu)-OH
residue was double coupled to ensure amide formation between the
Fmoc-aspartate acid and the amino moiety of the
4-(aminophenoxy)-butyric acid residue. Cleavage from the resin was
carried out in a TFA:H.sub.2O:TIPS:Acetone (92.5:2.5:2.5:2.5
v/v/v/v) solution for 2 h. Blowing air was used to reduce TFA
solution to .about.10% volume, and the peptide was precipitated in
Et.sub.2O. The precipitate was redissolved in A:B (1:1 v/v) solvent
mixture and purified using reverse phase HPLC.
[0284] B. Peptide Purification.
[0285] Purification (to >90% homogeneity) was carried out on a
preparative scale Shimadzu HPLC equipped with a Phenomenex Gemini
C18 (250.times.21.2 mm, 5 .mu.m particle) column using a 10-30% B
solvent gradient over 20 min. Purity was assessed using a 10-60% B
gradient over 50 min on an analytical Shimadzu HPLC equipped with a
Waters xBridge C18 (4.6.times.250 mm, 5 .mu.m particle) column.
Lyophilization of the HPLC fractions yielded the purified peptide
as a TFA salt.
Example 23
A. Synthesis of PEG.sub.12/24-FCit-PABOC-PNP.
##STR00053##
[0286] Preparation of Dipeptide Precursors:
a) Fmoc-FCit-OH
##STR00054##
[0288] A solution of Fmoc-Phe-OPfp (EMD NovaBiochem 852226) (553
mg, 1 mmol) in THF (5 ml) was added to a solution of H-Cit-OH
(Sigma-Aldrich C7629) (184 mg, 1.05 mmol) and NaHCO.sub.3 (88.2 mg,
1.05 mmol) in H.sub.2O (2.6 ml). THF (2 ml) was added to make the
solution homogeneous and stirred for 10 h. THF was removed on a
rotavap, the residue was diluted with H.sub.2O (10 ml) and iPrOH (1
ml) and acidified to pH=1 with 3% HCl. Product was extracted
5.times. with 9:1 EtOAc:iPrOH solution, rinsed with a 9:1 mixture
of brine:iPrOH, dried (Na.sub.2SO.sub.4) and concentrated and dried
in vacuo. Trituration with ether afforded 313 mg of pure product
(57%). Similar conditions can be used for the preparation of
Fmoc-ACit-OH.
b) Fmoc-FCit-PAB-OH
##STR00055##
[0290] A solution of Fmoc-FCit-OH (5.98 g, 10.97 mmol) and PABA
(2.70 g, 21.95 mmol) in DCM (150 ml) and MeOH (50 ml) was treated
with EEDQ (5.43 g, 21.95 mmol) and let to stir at 20.degree. C. for
15 h. All volatiles were removed on a rotavap, the residue was
triturated with Et.sub.2O, and product was filtered out and dried
in vacuo. Yield 6.14 g (86%). Similar conditions can be used for
the preparation of Fmoc-ACit-PAB-OH.
c) H.sub.2N-FCit-PAB-OH (Same Conditions for Preparation of
H.sub.2N-A-Cit-PAB-OH)
##STR00056##
[0291] Fmoc-FCit-PAB-OH (0.83 mmol) was Fmoc deprotected by
stirring with Et.sub.3N (3.5 ml) in DMF (11 ml) for 10 h. All
volatiles were removed on a rotavap at 40.degree. C./oil pump
vacuum to obtain crude product which was used without additional
purification.
B. Preparation of PEG.sub.12-FCit-PABC-PNP. (FCit
Phenylalanine-Citrulline)
Quanta Biodesign Product Number: 10262
##STR00057##
[0292] Quanta Biodesign Product Number: 10304
##STR00058##
[0293] 1. PEG.sub.12-FCit-PAB-OH
##STR00059##
[0294] To a solution of H.sub.2N-FCit-PAB-OH (0.88 mmol) and DIEA
(167 .mu.l, 0.96 mmol) in DMF (3 ml) was added a solution of
PEG.sub.12-NHS (Quanta Biodesign 10262) or PEG.sub.24-NHS (Quanta
Biodesign 10304) (0.8 mmol) in DMF (3 ml). The mixture was stirred
for 16 h, filtered and concentrated. The crude was precipitated
into Et.sub.2O (45 ml) from CHCl.sub.3:MeOH (1:1, 5 ml) and used
without additional purification. Yield: 420 mg (53%).
ii) PEG.sub.12-FCit-PABC-PNP
##STR00060##
[0296] A solution containing PEG.sub.12/24-FCit-PAB-OH (419 mg,
0.42 mmol), (PNP).sub.2CO (Sigma-Aldrich 161691) (766 mg, 2.52
mmol) and DIEA (263 .mu.l, 1.52 mmol) in dioxane (4 ml) was stirred
in the dark at 50.degree. C. for 15 h. All volatiles were removed
on a rotavap and residual DIEA was removed by evaporation from DMF.
The product was purified on a column, eluent
CHCl.sub.3:EtOAc:MeOH=4.5:5:0.5 followed by a gradient of MeOH
(12-15%) in CHCl.sub.3. Yield: 390 mg (80%).
Example 24. Amphipathic Membrane Active Polyamine Syntheses
A.
[0297] RAFT copolymerization of N-Boc-ethylethoxy acrylate and
propyl methacrylate (FIG. 11). For other membrane active polymers,
A can be also be protected ethyl, propyl, or butyl amino acrylate.
B can be higher hydrophobic (10-24 carbon atoms, C18 shown)
acrylate, lower hydrophobic (1-6 carbon atoms, C4 shown) acrylate,
or a combination of lower an higher hydrophobic acrylates.
[0298] Copolymers consisting of Amine acrylate/C3 methacrylate were
synthesized as follows. The monomers and RAFT agent were weighed
and brought up into butyl acetate at the indicated ratios. AIBN
(azobis-isobutyronitrile) was added and nitrogen was bubbled
through the reaction at RT for 1 h. The reaction mixture was then
placed into an oil bath at 80.degree. C. for 15 h. The polymer was
then precipitated with hexane, and further fractionally
precipitated using a DCM/Hexane solvent system (see below). The
polymer was then dried under reduced pressure. The polymer was
deprotected with 7 ml 2M HCl in Acetic Acid for 30 min at RT. After
30 min, 15 ml of water was added to the reaction mixture, and the
mixture was transferred into 3.5 kDa MWCO dialysis tubing. The
polymer was dialyzed overnight against NaCl and then another day
against dH.sub.2O. The water was then removed through
lyophilization, and the polymer was dissolved in dH.sub.2O.
B. Random Copolymerization of N-Boc-Ethylethoxy Acrylate and Propyl
Methacrylate.
[0299] Copolymers consisting of Amine acrylate/C.sub.n methacrylate
were synthesized as follows. The monomers were weighed brought up
into dioxane at the indicated ratios. AIBN
(azobis-isobutyronitrile) was added and nitrogen was bubbled
through the reaction at RT for 1 h. The reaction mixture was then
placed into an oil bath at 60.degree. C. for 3h. The polymer was
then dried under reduced pressure. The polymer was purified by GPC.
After which the polymer fractions were deprotected with 7 ml 2M HCl
in Acetic Acid for 30 min at RT. After 30 min, 15 ml of water was
added to the reaction mixture, and the mixture was transferred into
3.5 kDa MWCO dialysis tubing. The polymer was dialyzed overnight
against NaCl and then another day against dH.sub.2O. The water was
then removed through lyophilization, and the polymer was dissolved
in dH.sub.2O.
C. Synthesis of Water-Soluble, Amphipathic, Membrane Active
Poly(Vinyl Ether) Polyamine Terpolymers.
[0300] X mol % amine-protected vinylether (e.g., 2-Vinyloxy Ethyl
Phthalimide) is added to an oven dried round bottom flask under a
blanket of nitrogen in anhydrous dichloromethane. To this solution
Y mol % lower hydrophobic group (e.g., propyl, butyl) vinylether
and optionally Z mol % higher hydrophobic group (e.g., dodecyl,
octadecyl) vinylether are added (FIG. 1). The solution is placed in
a -50 to -78.degree. C. bath, and the 2-vinyloxy ethyl phthalimide
is allowed to precipitate. To this solution 10 mol %
BF.sub.3--(OCH.sub.2CH.sub.3).sub.2 is added and the reaction is
allowed to proceed for 2-3 h at -50 to -78.degree. C.
Polymerization is terminated by addition of ammonium hydroxide in
methanol solution. The polymer is brought to dryness under reduced
pressure and then brought up in 1,4-dioxane/methanol (2/1). 20 mol
eq. of hydrazine per phthalimide is added to remove the protecting
group from the amine. The solution is refluxed for 3 h and then
brought to dryness under reduced pressure. The resulting solid is
dissolved in 0.5 mol/L HCl and refluxed for 15-min to form the
hydrochloride salt of the polymer, diluted with distilled water,
and refluxed for an additional hour. The solution is then
neutralized with NaOH, cooled to RT, transferred to molecular
cellulose tubing, dialyzed against distilled water, and
lyophilized. The polymer can be further purified using size
exclusion or other chromatography. The molecular weight of the
polymers is estimated using columns according to standard
procedures, including analytical size-exclusion chromatography and
size-exclusion chromatography with multi-angle light scattering
(SEC-MALS).
D. Polymer Ant-41658-111:
1) Monomer Synthesis.
[0301] 2,2'-Azobis(2-methylpropionitrile) (AIBN, radical
initiator), 4-Cyano-4-(phenylcarbonothioylthio) pentanoic acid
(CPCPA, RAFT Agent) and butyl acetate were purchased from Sigma
Aldrich. Propyl Methacrylate monomer (Alfa Aesar) was filtered to
remove inhibitors.
[0302] In a 2 L round-bottom flask equipped with a stir bar,
2-(2-aminoethoxy) ethanol (21.1 g, 202.9 mmol, Sigma Aldrich) was
dissolved in 350 ml dichloromethane. In a separate 1 L flask, BOC
anhydride (36.6 g, 169.1 mmol) was dissolved in 660 ml
dichloromethane. The 2 L round-bottom flask was fitted with an
addition funnel and BOC anhydride solution was added to the flask
over 6 h. The reaction was left to stir overnight. In a 2 L
separatory funnel, the product was washed with 300 ml each of 10%
citric acid, 10% K.sub.2CO.sub.3, sat. NaHCO.sub.3, and sat. NaCl.
The product, BOC protected 2-(2-aminoethoxy) ethanol, was dried
over Na.sub.2SO.sub.4, gravity filtered, and DCM was evaporated
using rotary evaporation and high vacuum.
[0303] In a 500 ml round bottom flask equipped with a stir bar and
flushed with argon, BOC protected 2-(2-aminoethoxy) ethanol (27.836
g, 135.8 mmol) was added, followed by 240 ml anhydrous
dichloromethane. Diisopropylethyl amine (35.5 ml, 203.7 mmol) was
added, and the system was placed in a dry ice/acetone bath.
Acryloyl Chloride (12.1 ml, 149.4 mmol) was diluted using 10 ml of
dichloromethane, and added drop-wise to the argon flushed system.
The system was kept under argon and left to come to RT and stirred
overnight. The product was washed with 100 ml each of dH.sub.2O,
10% citric acid, 10% K.sub.2CO.sub.3, sat. NaHCO.sub.3, and
saturated NaCl. The product, BOC-amino ethyl ethoxy acrylate
(BAEEA), was dried over Na.sub.2SO.sub.4, gravity filtered, and DCM
was evaporated using rotary evaporation. The product was purified
through column chromatography on 29 cm silica using a 7.5 cm
diameter column. The solvent system used was 30% ethyl acetate in
hexane. Rf: 0.30. Fractions were collected and solvent was removed
using rotary evaporation and high vacuum. BAEEA, was obtained with
74% yield. BAEEA was stored in the freezer.
##STR00061##
2) Polymerization:
[0304] Solutions of AIBN (1.00 mg/ml) and RAFT agent
(4-Cyano-4(phenylcarbonothioylthio)pentanoic acid (CPCPA), 10.0
mg/ml) in butyl acetate were prepared. Monomer molar feed ratio was
75 BAEEA:25 propyl methacrylate (CAS:2210-28-8) with 0.108 CPCPA
RAFT agent and 0.016 AIBN catalyst (0.00562 total mol).
[0305] BAEEA (1.09 g, 4.21 mmol) (A), propyl methacrylate (0.180 g,
1.41 mmol) (B), CPCPA solution (0.170 ml, 0.00609 mmol) (C), AIBN
solution (0.150 ml, 0.000915 mmol), and butyl acetate (5.68 ml)
were added to a 20 ml glass vial with stirrer bar. The vial was
sealed with a rubber cap and the solution was bubbled with nitrogen
using a long syringe needle with a second short syringe needle as
the outlet for 1 h. The syringe needles were removed and the system
was heated to 80.degree. C. for 15 h using an oil bath. The
solution was allowed to cool to RT and transferred to a 50 ml
centrifuge tube before hexane (35 ml) was added to the solution.
The solution was centrifuged for 2 min at 4,400 rpm. The
supernatant layer was carefully decanted and the bottom (solid or
gel-like) layer was rinsed with hexane. The bottom layer was then
re-dissolved in DCM (7 ml), precipitated in hexane (35 ml) and
centrifuged once more. The supernatant was decanted and the bottom
layer rinsed with hexane before the polymer was dried under reduced
pressure for several hours. Molecular weight obtained through MALS:
73,000 (PDI 1.7); Polymer composition obtained using H.sup.1NMR:
69:31 Amine:Alkyl.
Fractional Precipitation.
[0306] The dried, precipitated product was dissolved in DCM (100
mg/ml). Hexane was added until just after the cloud point was
reached (.about.20 ml). The resulting milky solution was
centrifuged. The bottom layer (thick liquid representing .about.60%
of polymer) was extracted and fully precipitated into hexane. The
remaining upper solution was also fully precipitated by further
addition of hexane. Both fractions were centrifuged, after which
the polymer was isolated and dried under vacuum. Fraction 1: Mw
87,000 (PDI 1.5); Fraction 2: Mw 52,000 (PDI 1.5-1.6).
MALS Analysis.
[0307] Approximately 10 mg of the polymer was dissolved in 0.5 ml
89.8% dichloromethane, 10% tetrahydrofuran, 0.2% triethylamine. The
molecular weight and polydispersity (PDI) were measured using a
Wyatt Helos II multiangle light scattering detector attached to a
Shimadzu Prominence HPLC using a Jordi 5.mu. 7.8.times.300 Mixed
Bed LS DVB column. Crude Polymer: MW: 73,000 (PDI 1.7), Fraction 1:
MW 87,000 (PDI: 1.5), Fraction 2: MW 52,000 (PDI 1.5-1.6)
[0308] The purified BOC-protected polymer was reacted 2M HCl in
Acetic Acid (7 ml) for 0.5 h to remove the BOC protecting groups
and produce the amines. 15 ml dH.sub.2O were added to the reaction,
the solution was transferred to 3500 MW cutoff cellulose tubing,
dialyzed against high salt for 24 h, then against dH.sub.2O for 18
h. The contents were lyophilized, then dissolved in DI H.sub.2O at
a concentration of 20 mg/ml. The polymer solution was stored at
2-8.degree. C.
E. Polymer Lau24B was prepared as above except the monomer feed
ratio was 72.5 BAEEA:27.5 propyl methacrylate. F. Ant-129-1 was
made as essentially as described above except the following
monomers were used:
##STR00062##
TABLE-US-00011 TABLE 10 Ant-129-1 polymer synthesis reactants. MW
mass volume reaction (g/mol) mol % moles (g) (ml) moles Monomers
N-Boc-amino-propyl acrylate 229.27 70 3.94 .times. 10.sup.-3 0.9031
0.005627 butyl methacrylate 142.2 25 1.41 .times. 10.sup.-3 0.2000
0.224 0.005627 C18 methacrylate 338.54 5 2.81 .times. 10.sup.-4
0.0952 0.005627 ethylene glycol diacrylate 170.16 5 2.81 .times.
10.sup.-4 0.0479 0.44 0.005627 other reagents CPCPA (RAFT reagent)
279.38 0.213 1.2 .times. 10.sup.-5 0.0033 0.335 0.005627 AIBN
(initiator) 164.21 0.032 1.8 .times. 10.sup.-6 0.0003 0.295
0.005627 butyl acetate 5.272 target molecular weight 100000 total
units per CTA 469.56 % CTA 0.213
[0309] For N-Boc-Amino-Propyl-Acrylate (BAPA), In a 500 ml round
bottom flask equipped with a stir bar and flushed with argon,
3-(BOC-amino).sub.1-propanol (TCI) (135.8 mmol) was added, followed
by 240 ml anhydrous dichloromethane. Diisopropylethyl amine (203.7
mmol) was added, and the system was placed in a dry ice/acetone
bath. Acryloyl Chloride (149.4 mmol) was diluted using 10 ml of
dichloromethane, and added drop-wise to the argon flushed system.
The system was kept under argon and left to come to RT and stirred
overnight. The product was washed with 100 ml each of dH.sub.2O,
10% citric acid, 10% K.sub.2CO.sub.3, sat. NaHCO.sub.3, and
saturated NaCl. The product, BOC-amino propyl acrylate (BAPA), was
dried over Na.sub.2SO.sub.4, gravity filtered, and DCM was
evaporated using rotary evaporation. The product was purified
through column chromatography on 29 cm silica using a 7.5 cm
diameter column. The solvent system used was 30% ethyl acetate in
hexane. Rf: 0.30. Fractions were collected and solvent was removed
using rotary evaporation and high vacuum. BAPA was obtained with
74% yield. BAPA was stored in the freezer.
G. Polymer Lau41648-106.
[0310] Monomer molar feed ratio was 80 BAEEA:20 propyl methacrylate
(CAS:2210-28-8) and 3% AIBN catalyst based on total monomer moles.
BAEEA (6.53 g, 25.2 mmol) (A), propyl methacrylate (0.808 g, 6.3
mmol) (B), AIBN (0.155 g, 0.945 mmol), and dioxane (34.5 ml) were
added to a 50 ml glass tube with stir bar. Compounds A and B were
prepared described above in Example 16Ai. The reaction was set up
in triplicate. Each solution was bubbled with nitrogen using a long
pipette for 1 h. The pipette was removed and each tube carefully
capped. Then each solution was heated at 60.degree. C. for 3 h
using an oil bath. Each solution was allowed to cool to RT and
combined in a round bottom. The crude polymer was dried under
reduced pressure. Molecular weight obtained through MALS: 55,000
(PDI 2.1); Polymer composition obtained using H.sup.1NMR: 74:26
Amine:Alkyl.
##STR00063##
GPC Fractionation.
[0311] The dried crude polymer was brought up at 50 mg/ml in 75%
dichloromethane, 25% tetrahydrafuran, and 0.2% triethylamine. The
polymer was then fractionated on a Jordi Gel DVB 10.sup.4 .ANG.-500
mm/22 mm column using a flow rate of 5 ml/min and 10 ml injections.
An earlier fraction was collected from 15-17 min, and a later
fraction was collected from 17-19 min. Fraction 15-17: Mw 138,000
(PDI 1.1); Fraction 17-19: Mw 64,000 (PDI 1.2).
MALS Analysis.
[0312] Approximately 10 mg of the polymer was dissolved in 0.5 ml
89.8% dichloromethane, 10% tetrahydrofuran, 0.2% triethylamine. The
molecular weight and polydispersity (PDI) were measured using a
Wyatt Helos II multiangle light scattering detector attached to a
Shimadzu Prominence HPLC using a Jordi 5.mu. 7.8.times.300 Mixed
Bed LS DVB column. Crude Polymer: MW: 55,000 (PDI 2.1), Fraction
15-17: MW 138,000 (PDI:1.1), Fraction 17-19: MW 64,000 (PDI
1.2)
The purified BOC-protected polymer was reacted 2M HCl in Acetic
Acid (7 ml) for 0.5 h to remove the BOC protecting groups and
produce the amines. 15 ml dH.sub.2O were added to the reaction, the
solution was transferred to 3500 MW cutoff cellulose tubing,
dialyzed against high salt for 24 h, then against dH.sub.2O for 18
h. The contents were lyophilized, then dissolved in DI H.sub.2O at
a concentration of 20 mg/ml. The polymer solution was stored at
2-8.degree. C.
H. Polymer DW1360.
[0313] An amine/butyl/octadecyl poly(vinyl ether) terpolymer, was
synthesized from 2-vinyloxy ethyl phthalimide (5 g, 23.02 mmol),
butyl vinylether (0.665 g, 6.58 mmol), and octadecyl vinylether
(0.488 g, 1.64 mmol) monomers. 2-vinyloxy ethyl phthalimide was
added to a 200 ml oven dried round bottom flask containing a
magnetic stir bar under a blanket of Argon in 36 ml anhydrous
dichloromethane. To this solution was added butyl vinyl ether and
n-octadecyl vinyl ether. The monomers were fully dissolved at RT
(RT) to obtain a clear, homogenous solution. The reaction vessel
containing the clear solution was then placed into a -50.degree. C.
bath generated by addition of dry ice to a 1:1 solution of ACS
grade denatured alcohol and ethylene glycol and a visible
precipitation of phthalimide monomer was allowed to form. After
cooling for about 1.5 min, BF.sub.3--(OCH.sub.2CH.sub.3).sub.2
(0.058 g, 0.411 mmol) was added to initiate the polymerization
reaction. The phthalimide monomer dissolved upon initiation of
polymerization. The reaction was allowed to proceed for 3 h at
-50.degree. C. The polymerization was stopped by the addition of 5
ml of 1% ammonium hydroxide in methanol. The solvents were then
removed by rotary evaporation.
[0314] The polymer was then dissolved in 30 ml of
1,4-dioxane/methanol (2/1). To this solution was added hydrazine
(0.147 g, 46 mmol) and the mixture was heated to reflux for 3 h.
The solvents were then removed by rotary evaporation and the
resulting solid was then brought up in 20 ml of 0.5 mol/L HCl and
refluxed for 15 min, diluted with 20 ml distilled water, and
refluxed for an additional hour. This solution was then neutralized
with NaOH, cooled to RT, transferred to 3,500 molecular weight
cellulose tubing, dialyzed for 24 h (2.times.20 L) against
distilled water, and lyophilized.
I. Polymer Emi 1034-68C.
[0315] Monomer molar feed ratio was 52.5 BAEAA:47.5 propyl acrylate
(CAS: 925-60-0) and 6.66:1 ratio of CTA (CPCPA) to Initiator
(AIBN). BAEAA (2.6851 g, 10.35 mmol), propyl acrylate (1.0742 g,
9.41 mmol), CPCPA (0.0105 g, 0.0375 mmol), AIBN (0.000924 g,
0.00563 mmol), and butyl acetate (15.9 ml) were added to a 40 ml
glass vial with stir bar. The solution was bubbled with nitrogen
using a long hypodermic needle in a septum cap for 1 h. The needle
was removed and the solution was heated at 80.degree. C. for 16 h
using an oil bath. Each solution was allowed to cool to RT. The
crude polymer was precipitated out using hexane (.about.8.times.
vol.) and centrifuged. The solvent was decanted and the polymer was
rinsed with hexane and dissolved in DCM. The dissolved polymer was
precipitated again with hexane (.about.8.times. vol.). After
centrifugation, the solvent was decanted and the polymer was dried
under reduced pressure. Molecular weight obtained through MALS:
59,640 (PDI 1.328); Polymer composition obtained using H.sup.1NMR:
55.4:44.6 Amine:Alkyl.
##STR00064##
Fractional Precipitation.
[0316] In a 50 ml centrifuge tube, samples were dissolved in 60%
heptane/40% dioxane at 25 mg sample/ml solvent using sonication.
The samples were vortexed for 10 seconds and allowed to sit for 4
h. The solvent was pipetted off the top and the polymer that
precipitated out was rinsed twice with hexane. The samples were
dried using high vacuum.
MALS Analysis.
[0317] A small amount of the polymer was dissolved at 10 mg/ml in
75% DCM, 20% THF, and 5% ACN. The molecular weight and
polydispersity (PDI) were measured using a Wyatt Helos II
multiangle light scattering detector attached to a Shimadzu
Prominence HPLC using a Phenogel 5.mu. Linear (2) 7.8.times.300
column. Crude Polymer: MW: 59,640 (PDI 1.328), Fraction 1: MW
80,540 (PDI:1.120).
Boc-Deprotection.
[0318] The purified BOC-protected polymer was reacted 2M HCl in
Acetic Acid (7 ml) for 0.5 h to remove the BOC protecting groups
and produce the amines. 15 ml dH.sub.2O were added to the reaction,
the solution was transferred to 3500 MW cutoff cellulose tubing,
dialyzed against high salt for 24 h, then against dH.sub.2O for 18
h. The contents were lyophilized, then dissolved in DI H.sub.2O at
a concentration of 20 mg/ml. The polymer solution was stored at
2-8.degree. C.
J. Polymer Emi1034-81F.
[0319] Monomer molar feed ratio was 65 BAEAA:35 butyl acrylate
(CAS: 141-32-2) and 6.66:1 ratio of CTA (CPCPA) to Initiator
(AIBN). BAEAA (0.9876 g, 3.809 mmol), butyl acrylate (0.2607 g,
2.034 mmol), CPCPA (0.0035 g, 0.0125 mmol), AIBN (0.000308 g,
0.00188 mmol), and butyl acetate (5.3 ml) were added to a 20 ml
glass vial with stir bar. The solution was bubbled with nitrogen
using a long hypodermic needle in a septum cap for 1 h. The needle
was removed and the solution was heated at 80.degree. C. for 16 h
using an oil bath. Each solution was allowed to cool to RT. The
crude polymer was precipitated out using hexane (.about.8.times.
vol.) and centrifuged. The solvent was decanted and the polymer was
rinsed with hexane and dissolved in DCM. The dissolved polymer was
precipitated again with hexane (.about.8.times. vol.). After
centrifugation, the solvent was decanted and the polymer was dried
under reduced pressure. Molecular weight obtained through MALS:
63,260 (PDI 1.318); Polymer composition obtained using H.sup.1NMR:
68.7:31.3 Amine:Alkyl.
##STR00065##
Fractional Precipitation.
[0320] In a 50 ml centrifuge tube, samples were dissolved in 60%
heptane/40% dioxane at 25 mg sample/ml solvent using sonication.
The samples were vortexed for 10 seconds and allowed to sit for 4
h. The solvent was pipetted off the top and the polymer that
precipitated out was rinsed twice with hexane. The samples were
dried using high vacuum.
MALS Analysis.
[0321] A small amount of the polymer was dissolved at 10 mg/ml in
75% DCM, 20% THF, and 5% ACN. The molecular weight and
polydispersity (PDI) were measured using a Wyatt Helos II
multiangle light scattering detector attached to a Shimadzu
Prominence HPLC using a Phenogel 5 Linear (2) 7.8.times.300 column.
Crude Polymer: MW: 63,260 (PDI 1.318), Fraction 1: MW 65,990
(PDI:1.246).
Boc-Deprotection.
[0322] The purified BOC-protected polymer was reacted 2M HCl in
Acetic Acid (7 ml) for 0.5 h to remove the BOC protecting groups
and produce the amines. 15 ml dH.sub.2O were added to the reaction,
the solution was transferred to 3500 MW cutoff cellulose tubing,
dialyzed against high salt for 24 h, then against dH.sub.2O for 18
h. The contents were lyophilized, then dissolved in DI H.sub.2O at
a concentration of 20 mg/ml. The polymer solution was stored at
2-8.degree. C.
K. Polymer LH1073-20C-1:
[0323] Monomer feed ratio was 55 BAPVE (Boc-amino propyl vinyl
ester):45 vinyl butyrate (CAS: 123-20-6), and 10:1 ratio of Chain
Transfer Agent (MDPD) to initiator (AIBN). BAPVE (0.8 g, 3.72
mmol), MDPD (9.26 mg, 0.0229 mmol), AIBN (0.21 mg, 0.00229 mmol),
and butyl acetate (0.5 ml) were added to a 20 ml glass vial with
stir bar. This solution was bubbled with nitrogen using a long
hypodermic needle in a septum cap for 1 h. A separate vial of
excess vinyl butyrate was degassed similarly. Needles/nitrogen were
removed and vinyl butyrate (0.35 g, 3.04 mmol) was added to the
reaction solution with a Hamilton syringe. The solution was stirred
for 4 h at 80.degree. C. and then allowed to cool to RT. The
resulting viscous solution was dissolved in 5 ml DCM and the
polymer was precipitated by addition of 40 ml hexane. After
centrifugation, the upper solvent was decanted and the polymer was
rinsed with 5 ml of hexane. The rinsed polymer was re-dissolved in
5 ml DCM, and precipitated once more with 40 ml hexane,
centrifuged, and decanted upper solvent. The polymer was then dried
under high vacuum. Molecular weight obtained through MALS: 42,230
(PDI 1.205); Polymer composition obtained using H.sup.1NMR: 57.5:
42.5 Amine: Alkyl.
Fractional Precipitation:
[0324] In a 50 ml centrifuge tube, polymer was dissolved in 10 ml
DCM and enough hexane was added to take the solution past the cloud
point (approximately 30 ml). The cloudy mixture was centrifuged,
forming two liquid layers. The more viscous bottom layer was
removed, diluted with 5 ml DCM and fully precipitated with 40 ml of
hexane to yield fraction one. After centrifugation of fraction the
solvent was decanted and the polymer dried under reduced pressure.
Molecular weight obtained through MALS: 61,350 (PDI 1.205).
MALS Analysis:
[0325] A small amount of the polymer was dissolved to afford a 10
mg/ml in 75% DCM, 20% THF, and 5% ACN. The molecular weight and
polydispersity (PDI) were measured using a Wyatt Helos II
multiangle light scattering detector attached to a Shimadzu
Prominence HPLC with a Phenogel 5u Linear (2) 7.8.times.300 column.
See molecular weight above.
Boc-Deprotection:
[0326] The fractionated BOC-protected polymer was reacted with 2N
HCl in Acetic Acid (5 ml) and stirred for 1 h to remove the BOC
groups and produce the amines. The solution was diluted with water
(30 ml) and dialyzed (3500 MWCO cellulose tubing) against an
aqueous NaCl solution and then deionized water over two days. The
contents were lyophilized, and then dissolved in DI water at a
concentration of 20 mg/ml. The polymer was stored at 2-8.degree.
C.
##STR00066##
L. Melittin.
[0327] All melittin peptides were made using peptide synthesis
techniques standard in the art. Suitable melittin peptides can be
all L-form amino acids, all D-form amino acids (inverso).
Independently of L or D form, the melittin peptide sequence can be
reversed (retro).
Example 25. Terminal Polymer Modification with Azido-PEG-Amine
[0328] In a 40 ml scintillation vial equipped with a septa cap and
stir bar, polymer (Emi 1034-68C class, 1 g, 0.0143 mmol) was
dissolved in 20 ml anhydrous dichloromethane (Sigma).
Pentafluorophenol (Sigma, 26.3 mg, 0.143 mmol) and
N,N'-Dicyclohexylcarbodiimide (Sigma, 29.5 mg, 0.143 mmol) were
added to the flask with stirring. Using a N.sub.2 gas line and a
needle for venting, the system was purged with N.sub.2 for -10 min.
The reaction was left to stir at RT overnight. Additional
Pentafluorophenol (Sigma, 26.3 mg, 0.143 mmol) and
N,N'-Dicyclohexylcarbodiimide (Sigma, 29.5 mg, 0.143 mmol) were
added to the flask, the system was purged with N.sub.2 gas, and the
reaction was stirred for 3 h at RT. The polymer was precipitated
with hexane (.about.10.times. volume, Sigma), centrifuged, and the
solvent was decanted. The polymer was dissolved in minimal
dichloromethane, precipitated with hexane (10.times. volume),
centrifuged, and the solvent was decanted. The polymer was
dissolved in minimal ethyl acetate (Sigma), precipitated with
hexane (.about.10.times. volume), centrifuged, and the solvent was
decanted. The polymer precipitate was dried under high vacuum until
the solid reached a constant weight.
[0329] In a 40 ml scintillation vial equipped with a septa cap and
stir bar, polymer from the previous step (Emi 1034-68C class, 1 g,
0.0143 mmol) was dissolved in 20 ml anhydrous dichloromethane.
Azido PEG.sub.4 Amine (PurePeg, 37.5 mg, 0.143 mmol) and
N,N-Diisopropylethylamine (Sigma, 20.3 mg, 0.157 mmol) were added
to the flask with stirring. The system was purged with N.sub.2 gas
for -10 min, and the reaction was left to stir at RT overnight.
Additional Azido PEG.sub.4 Amine (PurePeg, 37.5 mg, 0.143 mmol) and
N,N-Diisopropylethylamine (Sigma, 20.3 mg, 0.157 mmol) were added
to the flask, the system was purged with N.sub.2 gas, and the
reaction was stirred for 3 h at RT. The polymer was precipitated
with hexane (.about.10.times. volume), centrifuged, and the solvent
was decanted. The polymer was dissolved in minimal dichloromethane,
precipitated with hexane (10.times. volume), centrifuged, and the
solvent was decanted. The polymer precipitate was dried under high
vacuum until the solid reached a constant weight (FIG. 12).
Sequence CWU 1
1
5113PRTArtificial SequenceRGD integrin binding
peptidemisc_feature1, 3, 5, 9, 11, 13Xaa can be any naturally
occurring amino acid 1Xaa Cys Xaa Cys Xaa Arg Gly Asp Xaa Cys Xaa
Cys Xaa1 5 10210PRTArtificial SequenceRGD integrin binding peptide
2Ala Cys Asp Cys Arg Gly Asp Cys Phe Cys1 5 10 320DNAArtificial
SequenceMouse Aha1 siRNA sense strand sequencemodified_base1, 3, 5,
7, 9, 11, 13, 15, 17, 19/mod_base = "2'-deoxy-2'-flouro
corresponding nucleoside"modified_base2, 4, 6, 8, 10, 12, 14, 16,
18/mod_base = "2'-O-methyl corresponding
nucleoside"modified_base20/mod_base = "3'-3'-linked deoxythymidine"
3ggaugaagug gagauuagut 20421DNAArtificial Sequencemouse Aha1 siRNA
antisense strand sequencemodified_base2, 4, 6, 8, 10, 12, 14, 16,
18/mod_base = "2'-deoxy-2'-flouro corresponding
nucleoside"modified_base2, 21/mod_base = "5'-phosphorothioate
nucleoside"modified_base3, 5, 7, 9, 11, 13, 15, 17, 19/mod_base =
"2'-O-methyl corresponding nucleoside" 4acuaaucucc acuucaucct t
21512PRTArtificial SequenceRGD integrin binding peptide 5Ala Cys
Asp Cys Arg Gly Asp Cys Phe Cys Gly Lys1 5 10
* * * * *