U.S. patent application number 15/403809 was filed with the patent office on 2017-08-03 for cxcr4 inhibiting carriers for nucleic acid delivery.
The applicant listed for this patent is Wayne State University. Invention is credited to Jing Li, David Oupicky.
Application Number | 20170216445 15/403809 |
Document ID | / |
Family ID | 47178897 |
Filed Date | 2017-08-03 |
United States Patent
Application |
20170216445 |
Kind Code |
A1 |
Oupicky; David ; et
al. |
August 3, 2017 |
CXCR4 INHIBITING CARRIERS FOR NUCLEIC ACID DELIVERY
Abstract
The present invention generally relates to carriers including
polymers and lipids that comprise a CXCR4 inhibiting moiety. More
specifically, these carriers are biodegradable and can be
bioreducible polymers that comprise a CXCR4 inhibiting moiety.
These carriers can be suitable for delivery of nucleic acids to
cells. These carriers and pharmaceutical compositions comprising
these carriers can be used to treat various conditions including
cancers and inflammation conditions.
Inventors: |
Oupicky; David; (Canton,
MI) ; Li; Jing; (Madison Heights, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Wayne State University |
Detroit |
MI |
US |
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|
Family ID: |
47178897 |
Appl. No.: |
15/403809 |
Filed: |
January 11, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14351789 |
Apr 14, 2014 |
9545453 |
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PCT/US2012/060292 |
Oct 15, 2012 |
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15403809 |
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61547490 |
Oct 14, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 51/06 20130101;
C07D 257/02 20130101; A61K 51/0482 20130101; A61K 47/64 20170801;
A61K 38/12 20130101; A61K 47/60 20170801; A61K 31/395 20130101;
A61K 48/0041 20130101; A61K 38/12 20130101; A61K 31/7105 20130101;
A61K 47/59 20170801; C08G 73/028 20130101; C08G 73/06 20130101;
A61K 31/713 20130101; C08G 69/26 20130101; A61K 47/55 20170801;
C08G 63/685 20130101; A61K 47/543 20170801; C12N 15/87 20130101;
A61K 2300/00 20130101 |
International
Class: |
A61K 51/08 20060101
A61K051/08; A61K 31/713 20060101 A61K031/713; C08G 73/06 20060101
C08G073/06; A61K 48/00 20060101 A61K048/00; A61K 47/48 20060101
A61K047/48 |
Claims
1. A polymer comprising structural units of a CXCR4 inhibiting
moiety and either (i) a structural unit of Formula 11, (ii) a
structural unit of Formula 22, (iii) structural units of Formulae
11 and 22, (vi) structural units of Formulae 11 and 88, (vii)
structural units of Formulae 22 and 88, (viii) structural units of
Formulae 11, 22, and 77, (ix) structural units of Formulae 11, 22,
and 88, or (x) structural units of Formulae 11, 22, 77, and 88);
the structural units of Formulae 11, 22, 77, and 88 corresponding
to the following structures: ##STR00029## wherein X.sub.11 and
X.sub.22 are independently --NH--C(O)--CH.sub.2CH.sub.2--,
--O--C(O)--CH.sub.2CH.sub.2--, --C(O)O--, --C(O)--, or
--NH--C(O)--; R.sub.77 is hydrogen or alkyl; R.sub.12, R.sub.13,
R.sub.14, R.sub.15 are independently hydrogen, alkyl, or
substituted alkyl; R.sub.88 and R.sub.89 are independently alkyl or
substituted alkyl; n.sub.1 is independently an integer from 1 to 4;
and n.sub.2 is an integer from 1 to 8.
2. The polymer of claim 1 wherein the structural unit corresponds
to Formula 11.
3. The polymer of claim 2 wherein X.sub.11 is
--NH--C(O)--CH.sub.2CH.sub.2-- or --O--C(O)--CH.sub.2CH.sub.2-- and
R.sub.12, R.sub.13, R.sub.14, R.sub.15 are independently
hydrogen.
4. The polymer of claim 1 wherein R.sub.12 and R.sub.14 are
hydrogen and R.sub.13 and R.sub.15 are --C(O)O-alkyl.
5. The polymer of claim 3 wherein X.sub.11 is
--NH--C(O)--CH.sub.2CH.sub.2-- and n.sub.1 is 2.
6. The polymer of claim 1 wherein the structural unit corresponds
to Formula 22.
7. The polymer of claim 6 wherein X.sub.22 is
--NH--C(O)--CH.sub.2CH.sub.2-- or
--O--C(O)--CH.sub.2CH.sub.2--.
8. The polymer of claim 7 wherein X.sub.22 is
--NH--C(O)--CH.sub.2CH.sub.2-- and n.sub.2 is an integer from 4 to
6.
9. The polymer of claim 1 wherein the structural units correspond
to Formulae 11 and 22.
10-15. (canceled)
16. The polymer of claim 1 wherein CXCR4 inhibiting moiety is
derived from a cyclam compound and the cyclam compound corresponds
to either Formula 5 or Formula 6, wherein Formulae 5 and 6
correspond to the following structures: ##STR00030## wherein
R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are independently hydrogen
or --R.sub.8--NH.sub.2; R.sub.5, R.sub.6, and R.sub.7 are
independently hydrogen or --R.sub.8--NH.sub.2; and R.sub.8 is
independently C.sub.2 to C.sub.12 alkylene, arylene, or C.sub.2 to
C.sub.12 alkylene wherein one or more of the --CH.sub.2-- groups of
the alkylene group is replaced with an amide, an amine, a carbonyl,
an ether, an ester, a cycloalkyl, an aryl, or a heterocyclo
functional group.
17-46. (canceled)
47. The polymer of claim 16 wherein the cyclam monomer has a
structure corresponding to Formula 5 and at least one of R.sub.1,
R.sub.2, R.sub.3, or R.sub.4 is --R.sub.8--NH.sub.2.
48. The polymer of claim 16 wherein the cyclam monomer has a
structure corresponding to Formula 6 and at least one of R.sub.5,
R.sub.6, or R.sub.7 is --R.sub.8--NH.sub.2.
49. The polymer of claim 16 wherein R.sub.8 is independently
--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--,
--(CH.sub.2).sub.2--O--CH.sub.2).sub.2--O--(CH.sub.2).sub.2--,
--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--(CH.sub.-
2).sub.2--,
--(CH.sub.2).sub.2--N(CH.sub.3)--(CH.sub.2).sub.2--N(CH.sub.3)--(CH.sub.2-
).sub.2--,
--CH.sub.2--C.sub.6H.sub.4--CH.sub.2--N(CH.sub.3)--(CH.sub.2).s-
ub.2--N(CH.sub.3)--(CH.sub.2).sub.2--,
--CH.sub.2--C.sub.6H.sub.4--CH.sub.2--N(C(O)Ot-Bu)-(CH.sub.2).sub.3--,
--CH.sub.2--C.sub.6H.sub.4--CH.sub.2--N(C(O)Ot-Bu)-(CH.sub.2).sub.3--N(C(-
O)Ot-Bu)-(CH.sub.2).sub.3--, or
--CH.sub.2--C.sub.6H.sub.4--CH.sub.2--N(CH.sub.3)--CH.sub.2--C.sub.5H.sub-
.3N--CH.sub.2--.
50. The polymer of claim 49 wherein R.sub.8 is independently
--CH.sub.2--C.sub.6H.sub.4--CH.sub.2--N(C(O)Ot-Bu)-(CH.sub.2).sub.3--
or
--CH.sub.2--C.sub.6H.sub.4--CH.sub.2--N(C(O)Ot-Bu)-(CH.sub.2).sub.3--N(C(-
O)Ot-Bu)-(CH.sub.2).sub.3--.
51. The polymer of claim 49 wherein R.sub.8 is independently
--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--,
--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--,
--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--(CH.sub.-
2).sub.2--,
--(CH.sub.2).sub.2--N(CH.sub.3)--(CH.sub.2).sub.2--N(CH.sub.3)--(CH.sub.2-
).sub.2--,
--CH.sub.2--C.sub.6H.sub.4--CH.sub.2--N(CH.sub.3)--(CH.sub.2).s-
ub.2--N(CH.sub.3)--(CH.sub.2).sub.2--, or
--CH.sub.2--C.sub.6H.sub.4--CH.sub.2--N(CH.sub.3)--CH.sub.2--C.sub.5H.sub-
.3N--CH.sub.2--.
52. The polymer of claim 51 wherein R.sub.8 is independently
--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--,
--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--, or
--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--(CH.sub.-
2).sub.2--.
53-55. (canceled)
56. The polymer of claim 16 wherein the weight average molecular
weight is from about 1.5 kDa to about 20 kDa.
57-68. (canceled)
69. A polyplex comprising a polymer of claim 16 and a nucleic
acid.
70. The polyplex of claim 69 wherein the nucleic acid is plasmid
DNA, shRNA, siRNA or microRNA.
71. A pharmaceutical composition comprising a pharmaceutically
acceptable excipient and a polymer of claim 16.
72-96. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. National patent
application Ser. No. 14/351,789 filed on Apr. 14, 2014, which
claims priority to PCT Patent Application Ser. No.
PCT/US2012/060292 filed on Oct. 15, 2012, which claims priority to
U.S. Provisional Patent Application Ser. No. 61/547,490 filed on
Oct. 14, 2011, the disclosure of which is incorporated herein by
reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention generally relates to carriers
including polymers and lipids that comprise a CXCR4 inhibiting
moiety. More specifically, these carriers are bioreducible,
biodegradable, or non-biodegradable carriers that comprise a CXCR4
inhibiting moiety and are suitable for delivery of nucleic acids to
cells. These carriers and pharmaceutical compositions comprising
the carriers can be used to treat various conditions including
cancers and inflammation conditions.
BACKGROUND
[0003] There are numerous types of nucleic acid carriers that can
be used to deliver genetic material inside cells. Transfection can
be achieved using viral methods (ex: viruses, bacteriophages),
physical methods (ex: electroporation, lasers, heat, injected
nanoparticles) or through chemical based methods such as combining
DNA with nanoparticles, cyclodetrins, liposomes, dendrimers or
polymers that are then encapsulated by target cells.
Polyelectrolyte complexes of nucleic acids with polycations
(polyplexes) can be used for delivery of nucleic acids.
[0004] The main benefits of bioreducible polycation polymers (BRP)
are reduced toxicity and, compared to hydrolytically degradable
polycations, better spatial control of disassembly and release of
DNA that is localized predominantly to the cytoplasm and nucleus.
Improved spatial control of polyplex disassembly has been shown to
enhance transfection of several types of nucleic acids (plasmid
DNA, mRNA, siRNA) in a number of cancer cell lines. Bioreducible
polycations are degraded selectively in the reducing intracellular
space. The CXCR4 receptor is expressed on multiple cell types
including lymphocytes, hematopoietic stem cells, endothelial and
epithelial cells and cancer cells. CXCR4 is a trans-membrane
chemokine receptor protein specific for a ligand known as CXCL12.
Therapeutics that can act as antagonists and inhibit or block the
CXCR4/CXCL12 pathway are important drug targets. Incorporation of
known CXCR4 inhibiting moieties into carriers should allow
targeting of cells expressing CXCR4. Cyclam compound derivatives
that act as CXCR4 inhibitors have been developed, notably the AIDS
drug AMD-3100. In addition cyclam derivatives form highly stable
complexes with virtually all transition metal ions, particularly,
cyclam (1,4,8,11-tetraazacyclotetradecane) a well-known macrocyclic
ligand.
[0005] While there are many classes of BRP known, a need still
exists for polymers that can act as CXCR4 inhibitors. Bioreducible
polymers that are suitable for applications where biodegradability
is required (i.e., systemic delivery of nucleic acids) and can
simultaneously act as CXCR4 inhibitors present a promising dual
function approach.
SUMMARY OF INVENTION
[0006] Among the various aspects of the invention is a carrier
comprising a CXCR4 inhibiting moiety. Specifically, the carrier can
be a bioreducible polymer comprising a disulfide group that imparts
the capability for the polymer to biodegrade in the reducing
environment of a cell. The carrier can also be a biodegradable
polymer, or a non-biodegradable polymer comprising a CXCR4
inhibiting moiety. The carrier can also be a lipid comprising a
CXCR4 inhibiting moiety. These polymers can act as CXCR4 inhibitors
and in some cases can deliver nucleic acids for gene therapy.
[0007] One aspect of the invention is a polymer that comprises
structural units of a CXCR4 inhibiting moiety and either (i) a
structural unit of Formula 11, (ii) a structural unit of Formula
22, (iii) structural units of Formulae 11 and 22, (vi) structural
units of Formulae 11 and 88, (vii) structural units of Formulae 22
and 88, (viii) structural units of Formulae 11, 22, and 77, (ix)
structural units of Formulae 11, 22, and 88, or (x) structural
units of Formulae 11, 22, 77, and 88). The structural units of
Formulae 11, 22, 77, and 88 correspond to the following
structures:
##STR00001##
wherein X.sub.11 and X.sub.22 are independently
--NH--C(O)--CH.sub.2CH.sub.2--, --O--C(O)--CH.sub.2CH.sub.2--,
--C(O)O--, --C(O)--, or --NH--C(O)--; R.sub.77 is hydrogen or
alkyl; R.sub.12, R.sub.13, R.sub.14, R.sub.15 are independently
hydrogen, alkyl, or substituted alkyl; R.sub.88 and R.sub.89 are
independently alkyl or substituted alkyl; n.sub.1 is independently
an integer from 1 to 4; and n.sub.2 is an integer from 1 to 8.
[0008] The CXCR4 inhibiting monomer can comprise a cyclam monomer
and the cyclam monomer can correspond to either Formula 5 or
Formula 6, wherein Formulae 5 and 6 correspond to the following
structures:
##STR00002##
wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are independently
hydrogen or -R.sub.8-NH.sub.2; R.sub.5, R.sub.6, and R.sub.7 are
independently hydrogen or --R.sub.8--NH.sub.2; and R.sub.8 is
independently C.sub.2 to C.sub.12 alkylene, arylene, or C.sub.2 to
C.sub.12 alkylene wherein one or more of the --CH.sub.2-- groups of
the alkylene group is replaced with an amide, an amine, a carbonyl,
an ether, an ester, a cycloalkyl, an aryl, or a heterocyclo
functional group.
[0009] Another aspect of the invention is a polymer comprising a
reaction product of a polymerization mixture comprising a CXCR4
inhibiting monomer and either (i) a monomer of Formula 1, (ii) a
monomer of Formula 2, (iii) a monomer of Formula 12; (iii) monomers
of Formulae 1 and 2; (iv) monomers of Formulae 1 and 7, (v)
monomers of Formulae 2 and 7, (vi) monomers of Formulae 1 and 8,
(vii) monomers of Formulae 2 and 8, (viii) monomers of Formulae 1,
2, and 7, (ix) monomers of Formulae 1, 2, and 8, or (x) monomers of
Formulae 1, 2, 7, and 8) the monomers of Formulae 1, 2, 7, and 8
corresponding to the following structures:
##STR00003##
wherein X.sub.1 and X.sub.2 are independently
--NH--C(O)--CH.dbd.CH.sub.2, --O--C(O)--CH.dbd.CH.sub.2, --C(O)OH,
--C(O)Cl, or --N.dbd.C.dbd.O; R.sub.70 is hydrogen or alkyl;
R.sub.80 and R.sub.81 are independently alkyl or substituted alkyl;
R.sub.12, R.sub.13, R.sub.14, R.sub.15 are independently hydrogen,
alkyl, or substituted alkyl; n.sub.1 is independently an integer
from 1 to 4; and n.sub.2 is an integer from 1 to 8.
[0010] Yet another aspect of the invention is a polyplex comprising
a polymer described herein and a nucleic acid. The nucleic acid can
be plasmid DNA, mRNA, antisense oligonucleotide, shRNA, siRNA or
microRNA.
[0011] A further aspect is pharmaceutical composition comprising a
pharmaceutically acceptable excipient and a polymer or a polyplex
described herein.
[0012] Yet a further aspect is a method for treating breast cancer
in a patient, the method comprising administering to the patient a
therapeutically effective amount of a polymer, polyplex, or
pharmaceutical composition described herein.
[0013] Another aspect of the invention is a method for treating
prostate cancer in a male patient, the method comprising
administering to the male patient a therapeutically effective
amount of a polymer, polyplex, or pharmaceutical composition
described herein.
[0014] Still another aspect is a method for treating lung cancer in
a patient, the method comprising administering to the patient a
therapeutically effective amount of a polymer, polyplex, or
pharmaceutical composition described herein.
[0015] Yet another aspect is a method for treating inflammatory
bowel disease (IBD) in a patient, the method comprising
administering to the patient a therapeutically effective amount of
a polymer, polyplex, or pharmaceutical composition described
herein.
[0016] A further aspect of the invention is a method for inhibiting
or reducing metastasis in a patient, the method comprising
administering to the patient a polymer, a polyplex, or a
pharmaceutical composition described herein.
[0017] Yet a further aspect is an imaging method comprising imaging
a tissue of a patient using a polymer, a pharmaceutical
composition, or a polyplex, wherein the tissue comprises a CXCR4
receptor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a schematic representation of the mechanism of
action of CBRP-based polyplexes. Polyplex carriers are assembled
from DNA(example: shRNA plasmid) and CBRP polymers and introduced
as pharmaceutical formulations. The CXCR4 inhibiting moiety acts as
an antagonist to the CXCR4 receptor blocking the CXCL12 ligand and
resulting signal cascade pathway. For example, CXCR4 antagonism
results in inhibition of cell invasion and metastatic spread of
cancer cells. The carrier polyplexes undergo endocytosis and are
degraded in the reducing environment of cytoplasm and release the
DNA. The released DNA product is then transcribed, (e.g., the shRNA
loop is removed by DICER, resulting in processed siRNA). Cells
expressing the CXCR4 receptor have increased carrier concentrations
surrounding them resulting in enhanced transfection efficiency or
more successful gene therapy.
[0019] FIG. 2 is a .sup.1H NMR of a polymer prepared from
1,1'-[1,4-Phenylenebis
(methylene)]bis[1,4,8,11-tetraazacyclotetradecane] (AMD3100) and
N,N'-Cystamine bisacrylamide (CBA).
[0020] FIG. 3 is a graph of relative fluorescence intensity versus
weight/weight ratio that shows pDNA condensation of a polymer
prepared from 1,1'-[1,4-Phenylenebis
(methylene)]bis[1,4,8,11-tetraazacyclotetradecane] (AMD3100) and
N,N'-Cystamine bisacrylamide (CBA) (P(AMD-CBA or RPA) and AMD3100
by ethidium bromide exclusion assay.
[0021] FIG. 4 is an electrophoresis gel that shows polyplex
disassembly and pDNA release with 20 mM glutathione (GSH) -/+150 mM
NaCl. The polyplexes were prepared at w/w 5. Lane 1: polyplexes
alone, Lane 2: +20 mM GSH, Lane 3: +20mM GSH+NaCl.
[0022] FIG. 5 is a graph of the relative light units (RLU)/mg
protein versus weight/weight ratio that shows transfection
efficiency of P(AMD-CBA) polyplexes prepared using different w/w
ratio in B16F10 cells.
[0023] FIG. 6 is a .sup.1H NMR of one of the polymers prepared from
the polymerization of cyclam (Cyc) and N,N'-Cystamine bisacrylamide
(CBA) (P(Cyc-CBA)).
[0024] FIG. 7 is a graph of relative fluorescence intensity versus
weight/weight ratio that shows pDNA condensation of P(Cyc-CBA)
polymer and Cyclam by EtBr exclusion assay.
[0025] FIG. 8 shows a series of electrophoresis gels that show
polyplex disassembly and pDNA release with heparin -/+20 mM GSH or
DTT. The polyplexes were prepared at w/w 5.
[0026] FIG. 9A and FIG. 9B are graphs of the relative light units
(RLU)/mg protein versus weight/weight ratio that show transfection
efficiency of P(Cyc-CBA) polyplexes prepared using different w/w
ratio in (A) B16F10 cells and (B) MDA-MB-231 cells.
[0027] FIG. 10 is a graph or the percent cell viability versus the
Log of the concentration for P(Cyc-CBA) by MTS assay. IC.sub.50
values for P(Cyc-CBA)/3, P(Cyc-CBA)/2 and P(Cyc-CBA)/1.8 in
MDA-MB-231 cells are 247.6.+-.15.9 .mu.g/ml, 113.6.+-.12.0 .mu.g/ml
and 53.3.+-.3.9 .mu.g/ml, respectively
[0028] FIG. 11A, FIG. 11B, and FIG. 11C show a series of graphs of
absorbance versus wavelength (nm) for metal complexation of
P(Cyc-CBA). (A) Copper(II) complexation; (B) Zn(II) complexation;
(C) Co(II) complexation. The absorption spectrums were obtained by
UV-vis spectroscopy.
[0029] FIG. 12 shows a series of electrophoresis gels showing
stability of metal complexes of P(Cyc-CBA) against heparin
disassembly using P(Cyc-CBA) with 50% metal complexation.
[0030] FIG. 13A, FIG. 13B, and FIG. 13C show a series of graphs of
the RLU/mg protein versus weight/weight ratio showing transfection
efficiency of metal complexes of P(Cyc-CBA) in B16F10 cells. (A)
P(Cyc-CBA)/3; (B) P(Cyc-CBA)/2; (C) P(Cyc-CBA)/1.8.
[0031] FIG. 14 shows a series of graphs of the Ca.sup.2+ release
versus time showing that P(AMD-CBA) and AMD3100 are CXCL12
antagonists.
[0032] FIG. 15 is a series of graphs showing decreased toxicity of
bioreducible polycations based on polymers prepared from
CBAP(AMD-CBA).
[0033] FIG. 16 is a graph of the size of polyplexes versus time
that shows the colloidal stability of the polyplexes.
[0034] FIG. 17 is a series of graphs of absorbance versus
wavelength (nm) for metal complexation of AMD3100, P(DMADP-CBA)
control, P(AMD-CBA), and CuCl.sub.2. The absorption spectrums were
obtained by UV-vis spectroscopy.
[0035] FIG. 18 is electrophoresis gel showing DNA condensation by
Cu--P(AMD-CBA).
[0036] FIG. 19 is a series of graphs showing the effect of copper
complexation on toxicity (left) and transfection activity
(right).
[0037] FIG. 20 is a comparison graph of cytotoxicity of RPA and PEI
25 kDa in HepG2 cells (RPA: o, PEI: ) and CXCR4+U2OS cells (RPA:
.DELTA., PEI: .tangle-solidup.) determined by MTS.
[0038] FIG. 21A and FIG. 21B depict (a) DNA condensation by EtBr
Exclusion assay; (b) reduction triggered DNA release from RPA/DNA
polyplexes (polyplexes were prepared at w/w 5).
[0039] FIG. 22 depicts CXCR4 antagonism of RPA and RPA/DNA
polyplexes. CXCR4 receptor redistribution assay was conducted in
U2OS cells expressing GFP tagged CXCR4 (a). Before stimulation with
10 nM CXCL12, the cells were treated for 30 min with (b) no drug;
(c) 0.24 .mu.g/mL AMD3100.8HCl; (d) 1.5 .mu.g/ml RPA.HC1; (e) 2.5
.mu.g/ml RPA.HCl; (f) 1.5 .mu.g/ml RHB.HCl; (g) RPA/DNA polyplexes
(w/w 5, total RPA conc. 2.5 .mu.g/ml); (h) RPA/DNA polyplexes (w/w
1, total RPA conc. 0.5 .mu.g/ml); and (i) RHB/DNA polyplexes (w/w
5, total RHB conc. 2.5 .mu.g/ml). The scale bars for all the images
are 200 .mu.m.
[0040] FIG. 23A and FIG. 23B depict dose-dependent CXCR4
antagonistic ability of AMD3100 and RPA.HCl. (a) Representative
images of redistribution of CXCR4 receptors on U2OS cells treated
with increasing concentrations of AMD3100 and RPA; (b)
Dose-response curve of CXCR4 inhibition (% receptor translocation)
and calculated EC50 value based on images obtained from (a).
[0041] FIG. 24A and FIG. 24B depict inhibition of cancer cell
invasion by RPA and RPA/DNA polyplexes. (a) Cell invasion assay
with CXCR4+U2OS cells treated with 1) no drug; 2) 0.24 .mu.g/mL
AMD3100.8HCl; 3) 2 .mu.g/ml RHB.HCl; 4) 2 .mu.g/ml RPA.HCl; 5) 5
.mu.g/ml RPA.HCl and 6) RPA/DNA polyplexes (w/w 5, total RPA conc,
5 .mu.g/ml). Cells were seeded in Matrigel-coated inserts and
allowed to invade towards CXCL12-containing medium for 16 h before
fixation and imaging. (b) Average number of invaded cells in
20.times. imaging area.
[0042] FIG. 25A and FIG. 25B show transfection activity of RPA/DNA
polyplexes prepared at w/w 5, 15 and 25 in the absence (white bars)
and the presence (black bars) of 10% FBS in (a) B16F10 and (b)
CXCR4+U2OS cells. (Results are shown as mean luciferase expression
in RLU/mg protein.+-.SD, n=3).
[0043] FIG. 26 shows intracellular distribution of RPA/DNA
polyplexes in CXCR4+U2OS cells (red fluorescence: CX-Rhodamine
labeled plasmid DNA; green fluorescence: GFP-CXCR4 receptor).
[0044] FIG. 27 depicts the effect of CXCR4 stimulation/inhibition
on RPA/DNA transfection. CXCR4+U2OS cells were pre-treated with 300
nM AMD3100 for 15 min before adding polyplexes prepared at
different w/w ratios. The cells were then stimulated with 10 nM
CXCL12 and co-incubated with the polyplexes during
transfection.
[0045] FIG. 28 depicts AMD3100 and RPA do not inhibit
phorbol-stimulated CXCR4 internalization. CXCR4+U2OS cells were
treated with AMD3100.8HCl (0.24 .mu.g/mL) and then stimulated with
a) 10 nM CXCL12 or b) 100 ng/ml of phorbol myristate acetate. c)
CXCR4+U2OS cells were treated with RPA/DNA (w/w 5) polyplexes
(i.e., 0.5 .mu.g/mL RPA, 0.1 .mu.g/mL DNA) and then stimulated with
100 ng/ml of phorbol myristate acetate.
[0046] FIG. 29 depicts the effect of phorbol myristate (+/-PMA)
treatment on transfection activity of RPA/DNA prepared at w/w 5, 15
and 25.
[0047] FIG. 30A and FIG. 30B show simultaneous transfection and
CXCR4 inhibition by RPA/DNA polyplexes in CXCR4+U2OS cells. a)
Cells treated with RPA/DNA polyplexes (RPA/DNA w/w=5, 10 and 15)
showed CXCR4 inhibition both at 0 h and, a weaker one, at 24 h
after polyplex incubation. In contrast, RHB/DNA polyplexes (RHB(5))
showed no CXCR4 antagonism at any time. b) Simultaneously, RPA/DNA
polyplexes exhibit similar transfection (luciferase expression) as
control RHB polyplexes at 24 h after polyplex incubation.
DETAILED DESCRIPTION
[0048] The present invention is directed to polymers that comprise
a CXCR4 inhibiting moiety. When these polymers are bioreducible,
they are generally called CXCR4 inhibiting bioreducible polymers
(CBRPs). These CBRPs can be suitable for delivery of nucleic acids
to cells. In addition, polymers that are biodegradable, but are not
bioreducible can comprise a CXCR4 inhibiting moiety and can be
referred to as NPA. These polymers do not contain a disulfide
group. Preferably, the CXCR4 inhibiting moiety is a cyclam
derivative. When used for delivery of nucleic acids to cell, the
polymers preferably comprise, in addition to the nucleic acids a
cRGD for targeting of the polymers to cells. These polymers and
their pharmaceutical compositions can be used to treat various
conditions including cancers and inflammation conditions, such as
breast cancer, prostate cancer, lung cancer, metastasis, and
inflammatory bowel disease (IBD). Furthermore, the polymers wherein
a metal ion, such as copper(II), zinc(II), cobalt(II) or nickel is
complexed with the cyclam monomer or cyclam compound can be used
for imaging a tissue of a patient where the tissue comprises a
CXCR4 receptor.
[0049] The polymer can comprise a reaction product of a
polymerization mixture comprising a CXCR4 inhibiting monomer and
either (i) a monomer of Formula 1, (ii) a monomer of Formula 2,
(iii) monomers of Formulae 1 and 2; (iv) monomer of Formulae 1 and
7, (v) monomers of Formulae 2 and 7, (vi) monomers of Formulae 1
and 8, (vii) monomers of Formulae 2 and 8, (viii) monomers of
Formulae 1, 2, and 7, (ix) monomers of Formulae 1, 2, and 8, or (x)
monomers of Formulae 1, 2, 7, and 8. The monomers of Formulae 1, 2,
7, and 8 corresponding to the following structures:
##STR00004##
[0050] wherein X.sub.1 and X.sub.2 are independently
--NH--C(O)--CH.dbd.CH.sub.2, --O--C(O)--CH.dbd.CH.sub.2, --C(O)OH,
--C(O)Cl, or --N.dbd.C.dbd.O; R.sub.70 is hydrogen or alkyl;
R.sub.80 and R.sub.81 are independently alkyl or substituted alkyl;
R.sub.12, R.sub.13, R.sub.14, R.sub.15 are independently hydrogen,
alkyl, or substituted alkyl; n.sub.1 is independently an integer
from 1 to 4; and n.sub.2 is an integer from 1 to 8.
[0051] The polymer can comprise a reaction product of a
polymerization mixture comprising a CXCR4 inhibiting monomer and
either (i) a monomer of Formula 1, (ii) a monomer of Formula 2, or
(iii) monomers of Formulae 1 and 2. When the polymers comprise a
monomer of Formula 1, R.sub.12, R.sub.13, R.sub.14, R.sub.15 are
hydrogen.
[0052] The polymers can be a bioreducible polymer that is the
reaction product of a polymerization mixture that comprises a
monomer corresponding to Formula 1. These polymers have an X.sub.1
of --NH--C(O)--CH.dbd.CH.sub.2 or --O--C(O)--CH.dbd.CH.sub.2;
preferably, X.sub.1 is --NH--C(O)--CH.dbd.CH.sub.2. In these
polymers, n.sub.1 can be 1 to 3, 1 to 2, or 2. Particularly,
n.sub.1 is 2.
[0053] Further, for the monomers of Formula 1, R.sub.12 and
R.sub.14 are hydrogen and R.sub.13 and R.sub.15 are --C(O)O-alkyl.
For these monomers, n.sub.i can be 1. Also, the alkyl group can be
methyl, ethyl, propyl, butyl, pentyl, or hexyl; preferably, the
alkyl group is methyl.
[0054] The polymer can also be the reaction product of a
polymerization mixture comprising a monomer corresponding to
Formula 2. The polymers can have an X.sub.2 of
--NH--C(O)--CH.dbd.CH.sub.2 or --O--C(O)--CH.dbd.CH.sub.2;
preferably, X.sub.2 is --NH--C(O)--CH.dbd.CH.sub.2. In these
polymers, n.sub.2 is an integer from 2 to 8, 3 to 8, 3 to7, 4 to 7,
5 to 7, or 4 to 6. Particularly, n.sub.2 is 6.
[0055] Polymers can also comprise monomers corresponding to
Formulae 1 and 2.
[0056] The polymers described herein can further comprise a monomer
of Formula 7. When the polymer comprises a monomer of Formula 7,
R.sub.70 can be hydrogen, methyl, ethyl, or propyl; preferably,
R.sub.70 is hydrogen. R.sub.70 can also be methyl.
[0057] When the polymers comprise a repeat unit derived from a
monomer of Formula 8, R.sub.80 can be methyl, ethyl, propyl, butyl,
pentyl, or hexyl. Particularly, R.sub.80 is methyl. Further,
R.sub.81 can be methyl, ethyl, propyl, butyl, pentyl, hexyl, or
substituted methyl, ethyl, propyl, butyl, pentyl, or hexyl.
Particularly, R.sub.81 can be 2-hydroxy propyl.
[0058] When the polymer includes a repeat unit derived from a
monomer of Formula 7 or 8, the monomers combine to form a block of
repeat units. This block of repeat units can comprise from 5 to 60,
from 10 to 50, from 20 to 50, from 30 to 50, from 40 to 50, and 45
repeat units. Particularly, the block of repeat units comprises 45
repeat units.
[0059] When the polymers of the invention comprise a monomer of
Formula 7 or 8, the structural unit derived from the monomer of
Formula 7 or 8 can be linked to the structural unit derived from
the monomer of Formula 1 or Formula 2 by a linking group. The
linking group can comprise a heterocyclo or heteroaryl group. The
heterocyclo or heteroaryl group can be benzofuranyl,
benzo[d]thiazolyl, benzo[d]thiazolium, isoquinolinyl,
isoquinolinium, quinolinyl, quinolinium, thiophenyl, imidazolyl,
imidazolium, oxazolyl, oxazolium, furanyl, thiazolyl, thiazolium,
pyridinyl, pyridinium, furyl, thienyl, pyridyl, pyrrolyl,
pyrrolidinium, indolyl, indolinium, pyrrolidino, pyrrolidinium,
piperidino, piperidinium, morpholino, morpholinium, piperazino,
piperazinium, succinimide, or a combination thereof.
[0060] Further, the linking group can comprise the following
structures:
##STR00005##
[0061] The polymers of the invention can have the CXCR4 inhibiting
monomer corresponding to one or more of the following peptide
(5-14[C9W, F13-14f] dimer, SDF-1; 1-9[P2G] dimer, SDF-1; V1 1-9
vMIP-II; T22; T140; T134; ALX40-4C; CGP64222; FC131) or cyclam
(AMD3100 or AMD3465) structures:
##STR00006## ##STR00007## ##STR00008## ##STR00009##
##STR00010##
[0062] In other polymers, the CXCR4 inhibiting monomer is a cyclam
monomer. In these polymers, the cyclam monomer corresponds to
either Formula 5 or Formula 6, wherein Formulae 5 and 6 correspond
to the following structures:
##STR00011##
wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are independently
hydrogen or --R.sub.8--NH.sub.2; R.sub.5, R.sub.6, and R.sub.7 are
independently hydrogen or --R.sub.8--NH.sub.2; and R.sub.8 is
independently C.sub.2 to C.sub.12 alkylene, arylene, or C.sub.2 to
C.sub.12 alkylene wherein one or more of the --CH.sub.2-- groups of
the alkylene group is replaced with an amide, an amine, a carbonyl,
an ether, an ester, a cycloalkyl, an aryl, or a heterocyclo
functional group.
[0063] These polymers can include a cyclam monomer having a
structure corresponding to Formula 5 and at least one of R.sub.1,
R.sub.2, R.sub.3, or R.sub.4 is -R.sub.8-NH.sub.2. Other polymers
have a cyclam monomer having a structure corresponding to Formula 6
and at least one of R.sub.5, R.sub.6, or R.sub.7 is
--R.sub.8--NH.sub.2. In these polymers having a cyclam monomer
corresponding to Formula 5 or 6, R.sub.8 is independently
--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--,
--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--,
--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--(CH.sub.-
2).sub.2--,
--(CH.sub.2).sub.2--N(CH.sub.3)--(CH.sub.2).sub.2--N(CH.sub.3)--(CH.sub.2-
).sub.2--,
--CH.sub.2--C.sub.6H.sub.4--CH.sub.2--N(CH.sub.3)--(CH.sub.2).s-
ub.2--N(CH.sub.3)--(CH.sub.2).sub.2--, or
--CH.sub.2--C.sub.6H.sub.4--CH.sub.2--N(CH.sub.3)--CH.sub.2--C.sub.5H.sub-
.3N--CH.sub.2--. In these polymers having a cyclam monomer
corresponding to Formula 5 or 6, R.sub.8 is independently
--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--,
--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--, or
--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--(CH.sub.-
2).sub.2--.
[0064] The polymer of the invention can comprise a structure
corresponding to Formula 5 or 6 wherein R.sub.8 is independently
--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--,
--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--,
--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--(CH.sub.-
2).sub.2--,
--(CH.sub.2).sub.2--N(CH.sub.3)--(CH.sub.2).sub.2--N(CH.sub.3)--(CH.sub.2-
).sub.2--,
--CH.sub.2--C.sub.6H.sub.4--CH.sub.2--N(CH.sub.3)--(CH.sub.2).s-
ub.2--N(CH.sub.3)--(CH.sub.2).sub.2--,
--CH.sub.2--C.sub.6H.sub.4--CH.sub.2--N(C(O)Ot-Bu)--(CH.sub.2).sub.3--,
--CH.sub.2--C.sub.6H.sub.4--CH.sub.2--N(C(O)Ot-Bu)--(CH.sub.2).sub.3--N(C-
(O)Ot-Bu)--(CH.sub.2).sub.3--, or
--CH.sub.2--C.sub.6H.sub.4--CH.sub.2--N(CH.sub.3)--CH.sub.2--C.sub.5H.sub-
.3N--CH.sub.2--. Further, R.sub.8 can independently be
--CH.sub.2--C.sub.6H.sub.4--CH.sub.2--N(C(O)Ot-Bu)--(CH.sub.2).sub.3--
or
--CH.sub.2--C.sub.6H.sub.4--CH.sub.2--N(C(O)Ot-Bu)-(CH.sub.2).sub.3--N(C(-
O)Ot-Bu)-(CH.sub.2).sub.3--.
[0065] In a particularly preferred polymer, the polymer is a
reaction product of a polymerization mixture comprising a monomer
corresponding to Formula 1 wherein X.sub.1 is
--NH--C(O)--CH.dbd.CH.sub.2 and n.sub.1 is 2, and the CXCR4
inhibiting moiety is a cyclam monomer having a structure
corresponding to Formula 6, wherein R.sub.5, R.sub.6, and R.sub.7
are hydrogen.
[0066] The polymers can further comprise an amine monomer of
Formula 3, the amine monomer of Formula 3 corresponding to the
following structure:
R.sub.11R.sub.12N--R.sub.10--NH.sub.2 Formula 3
wherein R.sub.10 is C.sub.2 to C.sub.12 alkylene, arylene, or
C.sub.2 to C.sub.12 alkylene wherein one or more of the
--CH.sub.2-- groups of the alkylene group is replaced with an
amine; R.sub.11 and R.sub.12 are independently hydrogen, alkyl or
aryl. In these polymers, R.sub.11 and R.sub.12 are alkyl;
preferably, R.sub.11 and R.sub.12 are methyl. In these polymers,
R.sub.10 can be butylene. Also, R.sub.10 can be
--CH.sub.2CH.sub.2CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2--.
[0067] The polymers can comprise structural units of a CXCR4
inhibiting moiety and either (i) a structural unit of Formula 11,
(ii) a structural unit of Formula 22, (iii) structural units of
Formulae 11 and 22, (vi) structural units of Formulae 11 and 88,
(vii) structural units of Formulae 22 and 88, (viii) structural
units of Formulae 11, 22, and 77, (ix) structural units of Formulae
11, 22, and 88, or (x) structural units of Formulae 11, 22, 77, and
88); the structural units of Formulae 11, 22, 77, and 88 structural
units of Formulae 11, 22, 77, and 88 correspond to the following
structures:
##STR00012##
[0068] wherein X.sub.11 and X.sub.22 are independently
--NH--C(O)--CH.sub.2CH.sub.2--, --O--C(O)--CH.sub.2CH.sub.2--,
--C(O)O--, --C(O)--, or --NH--C(O)--; R.sub.77 is hydrogen or
alkyl; R.sub.12, R.sub.13, R.sub.14, R.sub.15 are independently
hydrogen, alkyl, or substituted alkyl; R.sub.88 and R.sub.89 are
independently alkyl or substituted alkyl; n.sub.1 is independently
an integer from 1 to 4; and n.sub.2 is an integer from 1 to 8.
[0069] The polymers can comprise (i) a structural unit of Formula
11, (ii) a structural unit of Formula 22, or (iii) structural units
of Formulae 11 and 22. When the polymers comprise a structural unit
of Formula 11, R.sub.12, R.sub.13, R.sub.14, R.sub.15 are
hydrogen.
[0070] Further, for the structural units of Formula 11, R.sub.12
and R.sub.14 are hydrogen and R.sub.13 and R.sub.15 are
--C(O)O-alkyl. For these monomers, n.sub.1 can be 1. Also, the
alkyl group can be methyl, ethyl, propyl, butyl, pentyl, or hexyl;
preferably, the alkyl group is methyl.
[0071] The structural units of Formulae 11, 22, 77, and 88 can also
be represented by the structural units of Formulae 11, 22, 77, and
88, which correspond to the following structures:
##STR00013##
wherein X.sub.11, X.sub.22, R.sub.12, R.sub.13, R.sub.14, R.sub.15,
R.sub.77, R.sub.88, R.sub.89, n.sub.1, and n.sub.2 are defined in
connection with Formulae 11, 22, 77, and 88; p.sub.1 and p.sub.2
are independently integers equal to or greater than 2, 3, 4, 5, 10,
15, 20, 25, 30, 32, 34 or more; and p.sub.3 and p.sub.4 are
independently integers from 5 to 60. Further, p.sub.1 and p.sub.2
are independently from 2 to 35, from 3 to 35, from 4 to 35, from 5
to 35, from 6 to 35, or from 7 to 35. Also, p.sub.3 and p.sub.4 are
independently integers from 10 to 50, from 20 to 50, from 30 to 50,
from 40 to 50, and 45.
[0072] Many polymers are bioreducible polymers and comprise a
structural unit corresponding to Formula 11. In these polymers,
preferably, X.sub.11 is --NH--C(O)--CH.sub.2CH.sub.2-- or
--O--C(O)--CH.sub.2CH.sub.2--; more preferably, X.sub.1 is
--NH--C(O)--CH.sub.2CH.sub.2--. In these polymers, n.sub.1 can be 1
to 3, 1 to 2, or 2. Particularly, n.sub.1 is 2.
[0073] Some polymers comprise a structural unit corresponding to
Formula 22. In these polymers, preferably, X.sub.22 is
--NH--C(O)--CH.sub.2CH.sub.2-- or --O--C(O)--CH.sub.2CH.sub.2--;
more preferably, X.sub.22 is --NH--C(O)--CH.sub.2CH.sub.2--. In
these polymers, n.sub.2 is an integer from 2 to 8, 3 to 8, 3 to7, 4
to 7, 5 to 7, 4 to 6, or 6. Particularly, n.sub.2 is 6.
[0074] Polymers of the invention can also comprise structural units
corresponding to Formulae 11 and 22.
[0075] The polymers described herein can further comprise a
structural unit of Formula 77. When the polymers comprise a monomer
of Formula 77, R.sub.77 can be hydrogen, methyl, ethyl, or propyl;
preferably, R.sub.77 is hydrogen. R.sub.77 can also be methyl.
[0076] When the polymers comprise a repeat unit of Formula 88,
R.sub.88 can be methyl, ethyl, propyl, butyl, pentyl, or hexyl.
Particularly, R.sub.80 is methyl. Further, R.sub.89 can be methyl,
ethyl, propyl, butyl, pentyl, hexyl, or substituted methyl, ethyl,
propyl, butyl, pentyl, or hexyl. Particularly, R.sub.89 can be
2-hydroxy propyl.
[0077] When the polymer includes a repeat unit of Formulae 77 or
88, the monomers combine to form a block of repeat units. This
block of repeat units can comprise from 5 to 60, from 10 to 50,
from 20 to 50, from 30 to 50, from 40 to 50, and 45 repeat units.
Particularly, the block of repeat units comprises 45 repeat units
of Formula 77 or 88.
[0078] When the polymers of the invention comprise a structural
unit of Formula 77 or 88, the structural unit of Formula 77 or 88
can be linked to the structural unit of Formula 11 or Formula 22 by
a linking group. The linking group can comprise a heterocyclo or
heteroaryl group. The heterocyclo or heteroaryl group can be
benzofuranyl, benzo[d]thiazolyl, benzo[d]thiazolium, isoquinolinyl,
isoquinolinium, quinolinyl, quinolinium, thiophenyl, imidazolyl,
imidazolium, oxazolyl, oxazolium, furanyl, thiazolyl, thiazolium,
pyridinyl, pyridinium, furyl, thienyl, pyridyl, pyrrolyl,
pyrrolidinium, indolyl, indolinium, pyrrolidino, pyrrolidinium,
piperidino, piperidinium, morpholino, morpholinium, piperazino,
piperazinium, succinimide, or a combination thereof.
[0079] Further, the linking group can comprise the following
structures:
##STR00014##
[0080] When preparing polymers incorporating a structural unit of
Formula 77 or 88, the block of repeat units can be reacted in its
polymeric form with the reactive end groups of the structural units
of Formulae 11 or 22 or the block of repeat units can be prepared
by reacting the monomeric units of Formulae 7 and 8 to form the
block of repeat units of Formula 77 or 88.
[0081] These polymers can have a molecular weight of from 4 to 20
kilodalton (kDa).
[0082] In the polymers described herein, the CXCR4 inhibiting
moiety can be derived from one or more of peptide (5-14[C9W,
F13-14f] dimer, SDF-1; 1-9[P2G] dimer, SDF-1; V1 1-9 vMIP-II; T22;
T140; T134; ALX40-4C; CGP64222; FC131) or cyclam (AMD3100 or
AMD3465) structures described herein above.
[0083] For these polymers, the CXCR4 inhibiting moiety is derived
from a cyclam compound. The cyclam compound corresponds to the
structure of either Formula 5 or Formula 6 as described herein.
[0084] Particularly, the polymers comprising a CXCR4 inhibiting
moiety can be prepared as follows:
##STR00015##
* could be any secondary amine and could have multiple attachments
to the same ring. Further, with respect to the synthetic scheme
above, the AMD3100 could be substituted with a cyclam of formula 5
as described herein.
[0085] For example, a specific polymer known as CopCX can be
prepared according to the following synthetic scheme using Michael
addition conducted in methanol or methanol/water (7/3 v/v) at 37 C.
Molar ratio of PEG to Cl and C2-containing block can be 1:1 or
2:1.
##STR00016##
The PEG conjugation can incorporate a polyethylene glycol (PEG)
polymer that is already prepared or the polyethylene glycol could
be synthesized from an epoxide monomer.
[0086] In a particularly preferred polymer, the polymer comprises
structural units of Formula 11 wherein X.sub.1 is
--NH--C(O)--CH.sub.2CH.sub.2-- and n.sub.1 is 2, and the CXCR4
inhibiting moiety is derived from a cyclam monomer having a
structure corresponding to Formula 66, wherein R.sub.65, R.sub.66,
and R.sub.67 are hydrogen.
[0087] The polymers can further comprise an amine structural unit
of Formula 33. The amine structural unit of Formula 33 corresponds
to the following structure:
##STR00017##
wherein R.sub.30 is C.sub.2 to C.sub.12 alkylene, arylene, or
C.sub.2 to C.sub.12 alkylene wherein one or more of the
--CH.sub.2-- groups of the alkylene group is replaced with an
amine; and R.sub.31 and R.sub.32 are independently hydrogen, alkyl,
or aryl.
[0088] In preferred polymers, R.sub.31 and R.sub.32 are alkyl;
preferably, R.sub.31 and R.sub.32 are methyl. For these preferred
polymers, R.sub.30 can be butylene. In other polymers, R.sub.30 is
--CH.sub.2CH.sub.2CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2--.
[0089] The polymers of the invention have a weight average
molecular weight from about 1.5 kDa to about 20 kDa; preferably,
from about 4 kDa to about 15 kDa. For the polymers of the
invention, the molar ratio of the CXCR4 inhibiting monomer or CXCR4
inhibiting moiety to the monomer of Formulae 1 or 2 or the
structural unit of Formulae 11 or 22 is from about 2:1 to about
1:2; preferably, the molar ratio of the CXCR4 inhibiting monomer or
CXCR4 inhibiting moiety to the monomer of Formulae 1 or 2 or the
structural unit of Formulae 11 or 22 is from about 1.5:1 to about
1:1.5.
[0090] The polymers can further comprise a cyclic RGD peptide. The
cyclic RGD peptide can comprise cyclo(Arg-Gly-Asp-D-Phe-Cys),
cyclo(Arg-Gly-Asp-D-Phe-Lys),
H-Glu[cyclo(Arg-Gly-Asp-D-Phe-Lys)].sub.2,
DOTA-Glu-[cyclo(Arg-Gly-Asp-D-Phe-Lys)].sub.2,
H-Arg-Gly-Asp-Ser-Lys-OH, cyclo(Arg-Gly-Asp-D-Tyr-Lys), or a
combination thereof. Preferably, the cyclic RGD peptide comprises
cyclo(Arg-Gly-Asp-D-Phe-Cys).
[0091] Additionally, a lipid can comprise a CXCR4 inhibiting moiety
and an amine moiety of Formula 9, the amine moiety of Formula 9
corresponding to the following structure:
##STR00018##
wherein R.sub.81, R.sub.82, and R.sub.83 are independently alkyl
and at least one of R.sub.81, R.sub.82, and R.sub.83 is a C.sub.10
to C.sub.50 alkyl. For these lipids, at least one of R.sub.81,
R.sub.82, and R.sub.83 can be C.sub.10 to C.sub.30 alkyl; at least
two of R.sub.81, R.sub.82, and R.sub.83 is a C.sub.10 to C.sub.50
alkyl. Preferably, at least two of R.sub.81, R.sub.82, and R.sub.83
is a C.sub.10 to C.sub.30 alkyl.
[0092] For the lipid comprising an amine moiety of Formula 9,
R.sub.81 can be methyl, ethyl, propyl, or butyl; and R.sub.82 and
R.sub.83 can be independently C.sub.10 to C.sub.30 alkyl.
Preferably, R.sub.81 is methyl and R.sub.82 and R.sub.83 are
independently C.sub.14 to C.sub.20 alkyl.
[0093] Specifically, the lipid can have the following formula known
as CXLip synthesized by step-wise alkylation of the cyclam with the
corresponding oligoamine linker and lipid moiety:
##STR00019##
[0094] Further, a lipid can comprise a CXCR4 inhibiting moiety, an
amine moiety of Formula 10, and a linker. The amine moiety of
Formula 10 corresponds to the following structure:
##STR00020##
wherein R.sub.84 and R.sub.85 are independently alkyl and at least
one of R.sub.84 and R.sub.85 is a C.sub.10 to C.sub.50 alkyl, the
linker being a C.sub.6 to C.sub.15 alkylene wherein one or more of
the --CH.sub.2-- groups is replaced by an aryl, an amine, a
--C(O)-- group, or a combination thereof
[0095] For the lipid comprising Formula 10, R.sub.84 and R.sub.85
can independently be C.sub.10 to C.sub.50 alkyl. Additionally, the
linker is a C.sub.6 to C.sub.15 alkylene wherein two or more of the
--CH.sub.2-- groups is replaced by an amine. Specifically, the
linker can be
--CH.sub.2--C.sub.6H.sub.4--CH.sub.2--NR.sub.86--(CH.sub.2).sub.o--NR.sub-
.86--(CH.sub.2).sub.o-- wherein R.sub.86 can be hydrogen or alkyl
and o can be an integer of 2 or 3. Further, the linker can be
--CH.sub.2--C.sub.6H.sub.4--CH.sub.2--NR.sub.86--(CH.sub.2).sub.o--NR.sub-
.86--(CH.sub.2).sub.o--NR.sub.86--(CH.sub.2).sub.o-- wherein
R.sub.86 can be hydrogen or alkyl and o can be an integer of 2 or
3. Preferably, R.sub.86 is hydrogen. When the linker is substituted
with an aryl group, the aryl group can be substituted as a
para-phenylene group.
[0096] The polymers and lipids can further comprise a polyethylene
glycol linking moiety between the cyclic RGD peptide and the
polymer or lipid. This polyethylene glycol linking moiety can be
derived from a PEG crosslinking moiety having a structure
corresponding to Formula 4
##STR00021##
wherein L.sub.1 and L.sub.2 are independently derived from a
sulfhydryl-reactive group or an amine-reactive group. The
sulfhydryl-reactive group can be a maleimide group and the
amine-reactive group can be a N-hydroxysuccinimide group.
[0097] Concerning the above methods, the polymer or lipid and the
cyclic RGD peptide can be linked either directly or indirectly. In
cases where linking is indirect, a polyethylene glycol (PEG)
linking moiety can be used. It is especially useful to use PEG when
administration of pharmaceutical compositions is systemic.
[0098] The polymers or lipids can further comprise a metal ion
complexed with the cyclam monomer or compound. The metal ion can be
copper(II), zinc(II), cobalt(II), nickel(II), manganese(II) or a
combination thereof. Preferably, the metal ion comprises
.sup.64Cu.sup.2+.
[0099] The invention is further directed to a polyplex comprising a
polymer or lipid described herein and a nucleic acid. The nucleic
acid can be plasmid DNA, messenger RNA, antisense oligonucleotides,
shRNA, siRNA or microRNA.
[0100] The carriers (e.g., polymers or lipids) described herein can
also be combined with a pharmaceutically acceptable excipient to
form a pharmaceutical composition.
[0101] The polymers described herein can generally be synthesized
by Michael addition of a CXCR4 inhibiting monomer to a monomer of
Formulae 1 or 2 or a combination of Formulae 1 and 2. The monomers
are weighed and dissolved in a polar solvent such as methanol/water
and allowed to react for up to 48 hours at 37.degree. C. in the
dark.
[0102] Particularly, a cyclam compound of Formulae 5 or 6 is
dissolved in a polar solvent with a monomer of Formula 1 and
allowed to react for up to 48 hours at 37.degree. C. in the dark.
Once reaction is completed, hydrochloric acid in ethanol (1.25 M)
is added to form the HC1 salt of the polymers. The precipitated
products are centrifuged and washed with ethanol twice to remove
extra acid. The products are dried using a vacuum pump and
redissolved in water. After dialysis against water for 2 days (MWCO
3,500), the polymers are lyophilized and ready to use.
Specifically, the poly(AMD-CBA) can be prepared as described in
Scheme 2.
##STR00022##
*could be any secondary amine and could have multiple attachments
to the same ring
[0103] Some of the cyclam monomers are commercially available and
can be modified according to the following scheme wherein the
cyclam reacts with a protecting group (PG) then reacts with an
alkyl halide, optionally carrying a functional group such as an
amine and then followed by a deprotection reaction.
##STR00023##
[0104] Specifically, these cyclams can be modified as described in
more detail in Scheme 3 below.
[0105] When modifying the cyclam compounds it is useful to consider
the amine groups that are needed for binding to the CXCR4 receptor
site. These amines are indicated in the following structure in bold
blue.
##STR00024##
[0106] For these polymers there can be different polymer
architectures such as hyper-branched cyclam bioreducible polymers
(HB-CBRP), linear side chain functionalized CBRP (LSC-CBRP), and
linear-terminus-functionalized CBRP (LT-CBRP).
Nucleic Acid Delivery
[0107] Synthetic delivery vectors based on self-assembly of nucleic
acids and polycations (polyplexes) continue to gain strength as
viable alternatives to viral vectors. Significant effort has been
devoted to the synthesis of safe and efficient biodegradable
polycations. The polymers of the present invention are bioreducible
polycations (BRPs) having the benefits of reduced toxicity compared
to polycations and better spatial control of disassembly compared
to hydrolytically degradable polycations. Improved spatial control
of polyplex disassembly and release of DNA that is localized
predominantly to the cytoplasm and nucleus have been shown to
enhance transfection of several types of nucleic acids (plasmid
DNA, mRNA, siRNA) in a number of cancer cell lines. Bioreducible
polycations are degraded selectively in the reducing intracellular
space (Christensen et al., Bioconjugate Chem, (2006) 17: 1233-1240,
Zhang et al., J Controlled Rel, (2010) 143: 359-366). The
degradation is mediated by thiol/disulfide exchange reactions with
small redox molecules like GSH; possibly with the help of redox
enzymes (Biaglow et al., Anal Biochem, (2000) 281: 77-86). GSH is
the most abundant intracellular thiol present in mM concentrations
inside the cell but only in .mu.M concentrations in the blood
plasma (Jones et al., Clin Chim Acta, (1998) 275: 175-84). The
majority of GSH is usually found in the cytoplasm (1-11 mM), which
is also the principal site of GSH biosynthesis. The most reducing
environment is usually found within the nucleus, where it is
required for DNA synthesis and repair and to maintain a number of
transcription factors in reduced state. Metastatic cancer cells
have been shown to have significantly elevated levels of GSH. BRPs
are thus particularly promising for nucleic acid delivery to
metastatic cancers because significantly elevated levels of GSH are
often associated with high metastatic potential of cells.
[0108] CXCR4 is a highly conserved transmembrane G-protein-coupled
receptor that binds exclusively its ligand CXCL12. It has been
shown that common metastatic sites for prostate and breast cancers
have high levels of CXCL12 and that metastatic cancer cells
overexpress CXCR4 (Muller et al., Nature, (2001) 410: 50-6,
Taichman et al., Cancer Res, (2002) 62: 1832-7, Chinni et al.,
Prostate, (2006) 66: 32-48, Rhodes et al., Cancer Res, (2011) 71:
603-613). CXCR4 expression increases during progression of prostate
cancer (PC), and localized prostate carcinoma and bone metastasis
tissue express significantly higher levels than benign prostate
tissue (Sun et al., J Cell Biochem, (2003) 89: 462-73, Mochizuki et
al., Biochem Biophys Res Commun, (2004) 320: 656-63). CXCR4
expression in PC is associated with poor survival (Akashi et al.,
Cancer Sci, (2008) 99: 539-42) and aggressive types of cancer
(Wallace et al., Cancer Res, (2008) 68: 927-36). The chemokine
CXCL12 is also over-expressed in PC metastatic tissue compared to
normal tissue (Sun et al., supra). At the tumor cellular level,
osteoblasts, stromal cells and endothelial cells all express CXCL12
(Taichmann et al., supra, Chinni et al., supra), and contribute to
bone metastasis of PC cells. The CXCL12/CXCR4 binding has been
shown to play an important role in PC cell proliferation, migration
and invasion. In addition to prostate cancer, CXCR4 plays a role in
metastasis of various tumor types, including breast cancer.
[0109] Cyclams are known to bind to CXCR4 and act as antagonists
thereof. While not being bound to a particular theory, it is
believed that the multivalent nature of cyclam-based BRPs (CBRPs)
results in increased residence time of binding with the CXCR4,
which in turn results in enhanced anti-CXCR4 activity. Furthermore,
since all CBRPs of the present invention are synthesized to provide
polycations with biodegradability in the intracellular reducing
environment, they find use not only in reducing or inhibiting
metastasis but in increasing the efficiency of transfection of DNA
into cells.
[0110] Polyplexes are nucleic acids condensed with polycations,
which can be used to transfect the nucleic acids into cells. CBRPs
of the present invention are particularly useful for forming
polyplexes. A vast majority of published reports confirm that
polyplexes must be formulated with excess polycations in order to
achieve efficient transfection. One of the advantages of using the
CBRPs of the present invention to form polyplexes is that the
polycation excess provided by CBRPs also has its own pharmacologic
function, namely antagonism of CXCR4.
[0111] Ability to condense DNA is a prerequisite for successful
polyplex gene delivery. FIG. 1 schematically represents this
process. While not being bound to a particular theory, it is
believed that the accessibility of amines in CBRPs of the present
invention allows for efficient interaction with nucleic acids,
resulting in their ability to condense DNA and allow for efficient
transfection. By way of example, complexes based on cyclam or low
molecular weight drug AMD3100 and nucleic acids mediated only
background levels of transfection, which was reflective of their
poor DNA condensing ability.
[0112] Polyethylene glycol (PEG) has been shown to improve
colloidal stability of CBRP polyplexes and to reduce non-specific
interactions that will enable selective targeting to PC (Pun et
al., Bioconjugate Chem, (2002) 13: 630-639). Accordingly, PEG can
be attached to CBRPs as a linking moiety between CBRP and a cyclic
RGD peptide. For example, substituting the content of CBRP with at
least about 5%-30% PEG-BRP and preferably with about 20% of PEG-BRP
in the formulation is effective to decrease the rate of aggregation
of the polyplexes. The use of PEG also shields the positive surface
charge and allows specific targeting of polyplexes when equipped
with appropriate targeting ligand, such as a cyclic RGD peptide.
While not being bound to a particular theory, it is believed that
PEG shielding prevents binding of polyplexes to CXCR4 and that only
free polycations (i.e., not complexed with DNA) will be available
for CXCR4 binding and inhibition.
[0113] The selection of cRGD as the targeting ligand for performing
transfections is particularly advantageous as CXCL12 has been shown
to stimulate an increase in the expression of activated
.alpha..sub.v.beta..sub.3 integrin receptors in metastatic prostate
cells C4-2B and PC3 (but not in LNCaP, the non-metastatic cell line
from which C4-2B is derived) (Sun et al., The Prostate, (2007) 67:
61-73). Integrins are receptors that mediate attachment between a
cell and the tissues surrounding it, which may be other cells or
the extracellullar matrix (ECM). There are many types of integrins,
and many cells have multiple types on their surface. All five
.alpha.V integrins, two .crclbar.1 integrins (.alpha.5, .alpha.8)
and .alpha.IIb.beta.3 share the ability to recognize ligands
containing an RGD tripeptide active site. The RGD-binding integrins
are among the most promiscuous in the family, with 133 integrins in
particular binding to a large number of extracellular matrix and
soluble vascular ligands. Accordingly, the use of RGD peptides
allows for transfection of polyplexes described herein into
numerous types of cells, including breast cancer cells, prostate
cancer cells, endothelial cells, etc.
[0114] When the carriers (including polymers or lipids) of the
invention comprise a cRGD, the cyclic RGD peptide can be selected
from the group consisting of cyclo(Arg-Gly-Asp-D-Phe-Cys),
cyclo(Arg-Gly-Asp-D-Phe-Lys),
H-Glu[cyclo(Arg-Gly-Asp-D-Phe-Lys)].sub.2,
DOTA-Glu-[cyclo(Arg-Gly-Asp-D-Phe-Lys)].sub.2,
H-Arg-Gly-Asp-Ser-Lys-OH, cyclo(Arg-Gly-Asp-D-Tyr-Lys), or a
combination thereof. Preferably, the cyclic RGD peptide is
cyclo(Arg-Gly-Asp-D-Phe-Cys).
[0115] Another advantage of the present invention is that it allows
for the use of a wide variety of nucleic acids to be condensed with
CBRPs of the present invention. Examples include plasmid DNA,
shRNA, siRNA, microRNA, mRNA, and antisense oligonucleotides. Also,
the nucleic acids can be plasmid DNA sequences. The nucleic acids
can also be double-stranded (ds) RNA sequences involved in RNA
interference, such as shRNA, siRNA and microRNA. The amount of DNA
used in polyplexes is variable, and is determined by the content of
CBRP. Preferably, the molar ratio between the protonizable amines
of CBRP and the DNA phosphate groups is at least 0.9:1.
[0116] For the polymers of the invention, the cRGD-PEG-CBRP can
have the following structure:
Uses
[0117] The carriers (including polymers and lipids) of the present
invention can be used for a number of therapeutic applications. For
such purposes, they can be formulated as pharmaceutical
compositions with a pharmaceutically acceptable excipient.
[0118] Pharmaceutical compositions of the present invention are
characterized as being at least sterile and pyrogen-free. Methods
for preparing pharmaceutical compositions of the invention are
within the skill in the art, for example as described in
Remington's Pharmaceutical Science, 17th ed., Mack Publishing
Company, Easton, Pa., (1985).
[0119] The pharmaceutical compositions of the present invention can
comprise any of the bioreducible polycation polymers described
herein coupled to a CXCR4 inhibiting moiety, wherein a CBRP can
also include a cyclic RGD peptide, an optional PEG linker, and can
also be condensed with nucleic acids to forma polyplex. The various
combinations of these polymers are described in the foregoing
sections.
[0120] The present pharmaceutical formulations can comprise the
polymers, lipids, or combinations thereof disclosed herein.
[0121] Preferred physiologically acceptable excipients are water,
buffered water, saline solutions (e.g., normal saline or balanced
saline solutions such as Hank's or Earle's balanced salt
solutions), 0.4% saline, 0.3% glycine, hyaluronic acid and the
like.
[0122] The pharmaceutical composition of the present invention can
be administered orally, nasally, parenterally, intrasystemically,
intraperitoneally, topically (as by drops or transdermal patch),
bucally, sublingually or as an oral or nasal spray, or as a
pulmonary inhalation.
[0123] A pharmaceutical composition of the present invention for
parenteral injection can comprise pharmaceutically acceptable
sterile aqueous or nonaqueous solutions, dispersions, suspensions
or emulsions as well as sterile powders for reconstitution into
sterile injectable solutions or dispersions just prior to use.
Examples of suitable aqueous and nonaqueous excipients, diluents,
solvents or vehicles include water, ethanol, polyols (such as
glycerol, propylene glycol, polyethylene glycol, and the like),
carboxymethylcellulose and suitable mixtures thereof, vegetable
oils (such as olive oil), and injectable organic esters such as
ethyl oleate. Proper fluidity can be maintained, for example, by
the use of coating materials such as lecithin or PEG, by the
maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0124] Injectable depot forms are made by forming microencapsule
matrices of the drug in biodegradable polymers such as
polylactide-polyglycolide. Depending upon the ratio of drug to
polymer and the nature of the particular polymer employed, the rate
of drug release can be controlled. Examples of other biodegradable
polymers include poly(orthoesters) and poly(anhydrides). Depot
injectable formulations are also prepared by entrapping the drug in
liposomes or microemulsions which are compatible with body
tissues.
[0125] The injectable formulations can be sterilized, for example,
by filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium just prior to use.
[0126] In some cases, to prolong the effect of the pharmaceutical
compositions, it is desirable to slow the absorption from
subcutaneous or intramuscular injection. This can be accomplished
by the use of a liquid suspension of crystalline or amorphous
material with poor water solubility. Alternatively, delayed
absorption of a parenterally administered pharmaceutical
composition form is accomplished by dissolving or suspending the
composition in an oil vehicle. Prolonged absorption of the
injectable pharmaceutical form can be brought about by the
inclusion of agents which delay absorption such as aluminum
monostearate and gelatin.
[0127] Solid dosage forms for oral administration include, but are
not limited to, capsules, tablets, pills, powders, and granules. In
such solid dosage forms, the active compounds are mixed with at
least one item pharmaceutically acceptable excipient or carrier
such as sodium citrate or dicalcium phosphate and/or (a) fillers or
extenders such as starches, lactose, sucrose, glucose, mannitol,
and silicic acid, (b) binders such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,
sucrose, and acacia, (c) humectants such as glycerol, (d)
disintegrating agents such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate, (e) solution retarding agents such as paraffin, (0
absorption accelerators such as quaternary ammonium compounds, (g)
wetting agents such as, for example, acetyl alcohol and glycerol
monostearate, (h) absorbents such as kaolin and bentonite clay, and
(i) lubricants such as talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof. In the case of capsules, tablets and pills, the dosage
form can also comprise buffering agents.
[0128] Solid compositions of a similar type can also be employed as
fillers in soft and hard filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like.
[0129] The solid dosage forms of tablets, dragees, capsules, pills,
and granules can be prepared with coatings and shells such as
enteric coatings and other coatings well known in the
pharmaceutical formulating art. They can optionally contain
opacifying agents and can also be of a composition that they
release the active ingredient(s) only, or preferentially, in a
certain part of the intestinal tract, optionally, in a delayed
manner. Examples of embedding compositions which can be used
include polymeric substances and waxes.
[0130] The pharmaceutical compositions of the present invention can
also be in a hydrogel, in a micro-encapsulated form, and the like,
if appropriate, with one or more of the above-mentioned
excipients.
[0131] Liquid dosage forms for oral administration include, but are
not limited to, pharmaceutically acceptable emulsions, solutions,
suspensions, syrups and elixirs. In addition to the active
compounds, the liquid dosage forms can contain inert diluents
commonly used in the art such as, for example, water or other
solvents, solubilizing agents and emulsifiers such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
dimethyl formamide, oils (in particular, cottonseed, groundnut,
corn, germ, olive, castor, and sesame oils), glycerol,
tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid
esters of sorbitan, and mixtures thereof.
[0132] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, and perfuming agents.
[0133] Suspensions can contain suspending agents as, for example,
ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and
sorbitan esters, microcrystalline cellulose, aluminum
metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures
thereof. Alternatively, the composition can be pressurized and
contain a compressed gas, such as nitrogen or a liquefied gas
propellant. The liquefied propellant medium and indeed the total
composition are preferably such that the active ingredients do not
dissolve therein to any substantial extent. The pressurized
composition can also contain a surface active agent. The surface
active agent can be a liquid or solid non-ionic surface active
agent or can be a solid anionic surface active agent. It is
preferred to use the solid anionic surface active agent in the form
of a sodium salt.
[0134] Pharmaceutical compositions of the invention can also
comprise conventional pharmaceutical excipients and/or additives.
Suitable pharmaceutical excipients include stabilizers,
antioxidants, osmolality adjusting agents, buffers, and pH
adjusting agents. Suitable additives include physiologically
biocompatible buffers (e.g., tromethamine hydrochloride), additions
of chelants (such as, for example, DTPA or DTPA-bisamide) or
calcium chelate complexes (as for example calcium DTPA,
CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium
salts (for example calcium chloride, calcium ascorbate, calcium
gluconate or calcium lactate).
[0135] One of ordinary skill in the art will appreciate that
effective amounts of the agents of the invention can be determined
empirically. It will be understood that, when administered to a
human patient, the total daily usage of the agents or composition
of the present invention will be decided by the attending physician
within the scope of sound medical judgment. The specific
therapeutically effective dose level for any particular patient
will depend upon a variety of factors: the type and degree of the
cellular or physiological response to be achieved; activity of the
specific agent or composition employed; the specific agents or
composition employed; the age, body weight, general health, sex and
diet of the patient; the time of administration, route of
administration, and rate of excretion of the agent; the duration of
the treatment; drugs used in combination or coincidental with the
specific agent; and like factors well known in the medical arts.
For example, it is well within the skill of the art to start doses
of the agents at levels lower than those required to achieve the
desired therapeutic effect and to gradually increase the dosages
until the desired effect is achieved.
[0136] One skilled in the art can also readily determine an
appropriate dosage regimen for administering the pharmaceutical
compositions of the invention to a given subject. For example, they
can be administered to the subject once, such as by a single
injection or deposition. Alternatively, they can be administered to
a subject multiple times daily or weekly, and for prolonged periods
of time, if required.
[0137] The pharmaceutical compositions of the present invention
find use in many different therapeutic applications, such as
treatment of breast cancer and prostate cancer. The basis for the
therapeutic applications lies in the inventors' discovery that
polymers of the present invention once containing a cyclam
compound, which acts as a CXCR4 inhibiting moiety, and to a cyclic
RGD or another targeting ligand can form complexes with nucleic
acids, and allow for efficient transfections of these complexes
into cells. While not being bound to a particular theory, CXCR4
antagonism is thought to result in inhibition of cell invasion and
metastatic spread of cancer cells. The additional benefit of using
cyclam-based bioreducible polycations (CBRPs) is that they provide
excess polycations, which have been show to increase the efficiency
of transfections. Furthermore, RGD peptides bind to
.alpha..sub.v.beta..sub.3 integrin receptors expressed on breast
and prostate cancer cells, allowing for the complexes to be
endocytosed. CBRPs allow for nucleic acids to be released in the
cytoplasm or nucleus. Any nucleic acids can be used; however,
nucleic acids capable of RNA interference (RNAi) such as microRNAs,
siRNAs and shRNAs find particular uses. These short RNA molecules
can bind to complementary mRNA transcripts in the cell, and prevent
translation of proteins encoded by such mRNAs.
[0138] The process of RNAi begins by the presence of a long dsRNA
in a cell, wherein the dsRNA comprises a sense RNA having a
sequence homologous to the target gene mRNA and antisense RNA
having a sequence complementary to the sense RNA. The presence of
dsRNA stimulates the activity of a ribonuclease III enzyme referred
to as Dicer. Dicer is involved in the processing of the dsRNA into
short pieces of dsRNA known as short interfering RNAs (siRNAs)
(Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs
derived from Dicer activity are typically about 21 to about 23
nucleotides in length and comprise about 19 base pair duplexes
(Elbashir et al., 2001, Genes Dev., 15, 188). siRNAs in turn
stimulate the RNA-induced silencing complex (RISC) by incorporating
one strand of siRNA into the RISC and directing the degradation of
the homologous mRNA target.
[0139] In research laboratories, two types of siRNA have been
widely used to suppress exogenous as well as endogenous gene
expression: synthetic siRNA and vector-based siRNA (i.e. in vivo
transcribed siRNA). The vector based siRNA is usually generated
through short hairpin RNA (shRNA). In this system, RNA polymerase
III promoters, such as H1 promoter and U6 promoter are used to
drive transcription of shRNA. The shRNA transcript consists of a
19- to 29-bp RNA stem, with the two strands joined by a tightly
structured loop. shRNA is processed in the cell into siRNA through
the action of the Dicer family of enzymes. Thus, the transcribed
products mimic the synthetic siRNA duplexes and are as effective as
the synthetic siRNA for suppressing their corresponding genes. In
addition to the above-mentioned nucleic acids, antisense
oligonucleotides can also be delivered to the cells using
RGD-linked CBRPs as described herein.
[0140] Accordingly, the present invention provides a method for
treating breast cancer in a patient by administering to the patient
a therapeutically effective amount of a pharmaceutical composition
comprising a bioreducible polycation polymer of the present
invention, wherein the CXCR4 inhibiting moiety is a cyclam
compound, wherein the polymer is linked to a cyclic RGD peptide,
and further comprises a shRNA, siRNA or microRNA directed against
survivin RNA.
[0141] Survivin, also called baculoviral inhibitor of apoptosis
repeat-containing 5 or BIRC5, is a protein that, in humans, is
encoded by the BIRC5 gene (Altieri DC, J. Biol. Chem. 269 (5):
3139-42, Feb. 1994). Survivin is a member of the inhibitor of
apoptosis (IAP) family. The survivin protein functions to inhibit
caspase activation, thereby leading to negative regulation of
apoptosis or programmed cell death. This has been shown by
disruption of survivin induction pathways leading to increase in
apoptosis and decrease in tumor growth. The survivin protein is
expressed highly in most human tumors and fetal tissue, but is
completely absent in terminally differentiated cells (Sah et al.,
Cancer Lett. 244 (2): 164-71, December 2006). This fact therefore
makes survivin an ideal target for breast and prostate cancer
therapy as cancer cells are targeted while normal cells are not
affected by survivin inhibition.
[0142] The present invention provides also provides a method for
treating breast cancer in a patient by administering to the patient
a therapeutically effective amount of a pharmaceutical composition
comprising a polymer of the present invention, wherein the CXCR4
inhibiting moiety is a cyclam compound, wherein the polymer is
linked to a cyclic RGD peptide, and which further comprises a
shRNA, siRNA or microRNA directed against Bcl-2 RNA.
[0143] Bcl-2 protein is associated with membranes and membrane
activity. Bcl-2 derives its name from B-cell lymphoma 2, as it is
the second member of a range of proteins initially described in
chromosomal translocations involving chromosomes 14 and 18 in
follicular lymphomas. The Bcl-2 protein is a part of a complex
system of signaling that controls apoptosis. Apoptosis (cell death)
may be induced by a variety of signals including irreparable DNA
damage. This form of cellular suicide prevents the expansion of
damaged cells. Bcl-2 works to prevent apoptosis. Therefore, its
overexpression can prevent apoptosis in cells that are damaged.
This can lead to the continued division of the mutated cells lines
and eventually cancer. Bcl-2 is localized to the luminal cells of
the normal breast, which are considered to be the origin of
malignant breast disease.
[0144] The present invention can also provide a method for treating
breast cancer in a patient by administering to the patient a
therapeutically effective amount of a pharmaceutical composition
comprising a polymer of the present invention, wherein the CXCR4
inhibiting moiety is a cyclam compound, wherein the polymer is
linked to a cyclic RGD peptide, and which further comprises a
shRNA, siRNA or microRNA directed against Her2 RNA.
[0145] HER2/neu (also known as ErbB-2) stands for "Human Epidermal
growth factor Receptor 2" and is a protein giving higher
aggressiveness in breast cancers. It is a member of the ErbB
protein family, more commonly known as the epidermal growth factor
receptor family. HER2/neu has also been designated as CD340
(cluster of differentiation 340) and p185. It is encoded by the
ERBB2 gene. HER2 is a cell membrane surface-bound receptor tyrosine
kinase and is normally involved in the signal transduction pathways
leading to cell growth and differentiation. It is encoded within
the genome by HER2/neu, a known proto-oncogene. Approximately 30%
of breast cancers have an amplification of the HER2/neu gene or
overexpression of its protein product. Overexpression of this
receptor in breast cancer is associated with increased disease
recurrence and worse prognosis. Accordingly, inhibiting HER2
expression in breast cancer is of great value for treatment
success.
[0146] The present invention can further provide a method for
treating prostate cancer (PC) in a male patient by administering to
the male patient a therapeutically effective amount of a
pharmaceutical composition comprising a polymer of the present
invention, wherein the CXCR4 inhibiting moiety is a cyclam
compound, wherein the polymer is linked to a cyclic RGD peptide,
and which further comprises a shRNA, siRNA or microRNA directed
against survivin RNA.
[0147] The present invention can additionally provide a method for
treating prostate cancer in a male patient by administering to the
patient a therapeutically effective amount of a pharmaceutical
composition comprising a polymer of the present invention, wherein
the polymer is linked to a cyclic RGD peptide, and which further
comprises a shRNA, siRNA or microRNA directed against Bcl-2 RNA.
Preferably, the CXCR4 inhibiting moiety is a cyclam compound.
[0148] The present invention is also directed to a method for
treating prostate cancer in a male patient by administering to the
patient a therapeutically effective amount of a pharmaceutical
composition comprising a polymer of the present invention, wherein
the polymer is linked to a cyclic RGD peptide, and which further
comprising a shRNA, siRNA or microRNA directed against Her-2 RNA.
Preferably, the CXCR4 inhibiting moiety is a cyclam compound. In
addition to breast cancer, HER-2 has also been indicated in other
cancers including prostate cancer. While not being bound to a
theory, when prostate cancers progress from an androgen-dependent
to an androgen-independent phenotype, epidermal growth factor
pathways are frequently activated, potentially resulting in Her-2
activation. Prostate cancer, like most hormone dependent cancers
becomes refractory to treatment after one to three years, and
resumes growth despite hormone therapy. Previously considered
"hormone-refractory prostate cancer" or "androgen-independent
prostate cancer", the term castration-resistant has replaced
"hormone refractory" because while it is no longer responsive to
castration treatment (reduction of available
androgen/testosterone/DHT by chemical or surgical means), prostate
cancer still show reliance upon hormones for androgen receptor
activation. Thus, inhibiting HER2 can be especially beneficial for
use in castration-resistant prostate cancer.
[0149] Also, a therapeutically effective amount of a pharmaceutical
composition comprising a polymer of the present invention and which
further comprises a shRNA, siRNA or microRNA directed against akt2,
PARP or STAT3 can be administered to a patient to treat breast
cancer or to a male patient to treat prostate cancer.
[0150] The akt2 gene is a putative oncogene that plays an important
role in balancing cell survival and apoptosis. Studies have shown
that Akt2 overexpression leads to increased metastasis. Akt is the
direct downstream effector of PI3K signaling pathway involved in
CXCR4-mediated tumor progression and metastasis (Vlahakis et al.,
"G protein-coupled chemokine receptors induce both survival and
apoptotic signaling pathways." J Immunol, 2002, 169(10): p.
5546-54). Akt activation by SDF-1 is required for CXCR4-mediated
chemotaxis of breast cancer cells (Zhao, M., B. M. Mueller, R. G.
DiScipio, and I. U. Schraufstatter, "Akt plays an important role in
breast cancer cell chemotaxis to CXCL12." Breast cancer research
and treatment, 2008, 110(2): p. 211-22). CXCR4/SDF-1 axis also
promotes VEGF-mediated tumor angiogenesis through Akt signaling
pathway (Liang, Z., J. Brooks, M. Willard, K. Liang, Y. Yoon, S.
Kang, and H. Shim, "CXCR4/CXCL12 axis promotes VEGF-mediated tumor
angiogenesis through Akt signaling pathway." Biochemical and
biophysical research communications, 2007, 359(3): p. 716-22).
Reduction of Akt expression by siRNA inhibits invasiveness of
multiple breast cancer cell lines (Wang, J., W. Wan, R. Sun, Y.
Liu, X. Sun, D. Ma, and N. Zhang, "Reduction of Akt2 expression
inhibits chemotaxis signal transduction in human breast cancer
cells." Cellular signaling, 2008, 20(6): p. 1025-34).
Poly(ADP-ribose) polymerase (PARP) is a protein involved in DNA
repair, and its overexpression was observed in breast cancer
(Goncalves et al., "Poly(ADP-ribose) polymerase-1 mRNA expression
in human breast cancer: a meta-analysis." Breast Cancer Res Treat,
2011, 127(1): p. 273-81). Inhibition of PARP has shown promising
efficacy for breast cancer treatment (Fogelman et al., "Evidence
for the Efficacy of Iniparib, a PARP-1 Inhibitor, in
BRCA2-associated Pancreatic Cancer." Anticancer Res, 2011, 31(4):
p. 1417-20; Perkins et al., "Novel inhibitors of poly(ADP-ribose)
polymerase/PARP1 and PARP2 identified using a cell-based screen in
yeast." Cancer Res, 2001, 61(10): p. 4175-83; Yuant al., "Novel
targeted therapeutics: inhibitors of MDM2, ALK and PARP." J Hematol
Oncol, 2011, 4(1): p. 16; Goldberg et al. , "Nanoparticle-mediated
delivery of siRNA targeting Parpl extends survival of mice bearing
tumors derived from Brcal-deficient ovarian cancer cells."
Proceedings of the National Academy of Sciences, 2011, 108(2): p.
745-750). Signal transducer and activator of transcription 3
(STAT3) is a transcription factor that is involved in a variety of
physiological processes. Constitutive activation of STAT3 is
associated with many human cancers, including breast cancer
(Buettner et al., "Activated signal transducers and activators of
transcription 3 signaling induces CD46 expression and protects
human cancer cells from complement-dependent cytotoxicity." Mol
Cancer Res, 2007, 5(8): p. 823-32; Calo et al., "STAT proteins:
from normal control of cellular events to tumorigenesis." J Cell
Physiol, 2003, 197(2): p. 157-68; Klampfer, L., "The role of signal
transducers and activators of transcription in colon cancer." Front
Biosci, 2008, 13: p. 2888-99; Nikitakis et al., "Targeting the STAT
pathway in head and neck cancer: recent advances and future
prospects." Curr Cancer Drug Targets, 2004, 4(8): p. 637-51)).
STAT3 is a novel target in cancer therapy (Turkson, J., "STAT
proteins as novel targets for cancer drug discovery." Expert Opin
Ther Targets, 2004, 8(5): p. 409-22),and its siRNA inhibition has
shown promising effects on suppression of cell growth and induction
of apoptosis (Gao et al., "Inhibition of STAT3 expression by siRNA
suppresses growth and induces apoptosis in laryngeal cancer cells."
Acta Pharmacologica Sinica, 2005, 26(3): p. 377-383; Lee et al.,
"RNA interference targeting Stat3 inhibits growth and induces
apoptosis of human prostate cancer cells." Prostate, 2004, 60(4):
p. 303-309; Klosek et al, "Stat3 as a molecular target in RNA
interference-based treatment of oral squamous cell carcinoma."
Oncology Reports, 2008, 20(4): p. 873-878).
[0151] Further, the present invention provides a method for
treating lung cancer in a patient by administering to the patient a
therapeutically effective amount of a pharmaceutical composition
comprising a polymer of the present invention, wherein the CXCR4
inhibiting moiety is a cyclam compound, wherein the polymer is
linked to a cyclic RGD peptide, and which further comprises a
shRNA, siRNA or microRNA directed against any of the RNAs selected
from akt2, survivin, PARP, STAT3 and EGFR (epidermal growth factor
receptor). The lung cancer can be either a small cell lung cancer
(SCLC) or non-small cell lung cancer (NSCLC).
[0152] Still further, the present invention is directed to a method
for treating inflammatory bowel disease (IBD) in a patient by
administering to the patient a therapeutically effective amount of
a pharmaceutical composition comprising a polymer of the present
invention and further comprises a shRNA, siRNA or microRNA directed
against TNF-alpha RNA. Preferably, the CXCR4 inhibiting moiety is a
cyclam compound. IBD is a bowel disorder characterized by chronic
abdominal pain, discomfort, bloating, and alteration of bowel
habits in the absence of any detectable organic cause. There is no
specific laboratory or imaging test that can be performed to
diagnose IBD. Diagnosis of IBD involves excluding conditions that
produce IBD-like symptoms, and then following a procedure to
categorize the patient's symptoms. Ruling out parasitic infections,
lactose intolerance, small intestinal bacterial overgrowth and
celiac disease is recommended for all patients before a diagnosis
of irritable bowel syndrome is made. While the cause of IBD is
unknown, it has been shown that proinflammatory cytokines, such as
TNF-alpha are higher in patient with IBD than in control subjects
(Liebregts et al., Gastroenterology 2007 March; 132(3):913-20).
Accordingly, while not being bound to a theory, inhibiting
TNF-alpha could result in improvement or cure of IBD.
[0153] Additionally, the present invention is directed to a method
for inhibiting or reducing metastasis, the method comprising
administering to a patient a polymer linked to a CXCR4 inhibiting
moiety; preferably, the CXCR4 inhibiting moiety is a cyclam
compound. As noted in the foregoing sections, CXCR4 is a highly
conserved transmembrane G-protein-coupled receptor that binds
exclusively its ligand CXCL12. It has also been shown that common
metastatic sites for prostate and breast cancers have high levels
of CXCL12 and that metastatic cancer cells overexpress CXCR4. Since
CXCR4 plays a role in metastasis in a large number of different
tumor types, cyclam-based BRPs of the present invention can be used
to inhibit or reduce metastasis, regardless of the cancer cell
where it originated. For purposes of inhibiting or reducing
metastasis, CBRPs of the present invention can be formulated as
pharmaceutical compositions.
[0154] In all of the above methods, a patient is preferably a
human. The pharmaceutical compositions used in the above methods
can be administered parenterally. Alternatively, the pharmaceutical
compositions can be administered enterally. It may be desirable to
administer pharmaceutical compositions used for prostate cancer,
breast cancer and for reducing or inhibiting metastasis
parenterally whereas it may be desirable to administer compositions
used for treating IBD enterally. As mentioned in the previous
sections, a PEG linking moiety can be used between the cyclic RGD
peptide and the BRP polymer for parenteral administrations.
[0155] The invention is also directed to a method for positron
emission tomography (PET) or magnetic resonance imaging using the
polymers that further comprise a metal ion complexed with the
cyclam monomer or compound. PET is a nuclear medicine imaging
technique that produces a three-dimensional image or picture of
functional processes in the body. The system detects pairs of gamma
rays emitted indirectly by a positron-emitting radionuclide
(tracer), which is introduced into the body on a biologically
active molecule. Three-dimensional images of tracer concentration
within the body are then constructed by computer analysis. In
modern scanners, three dimensional imaging is often accomplished
with the aid of a CT X-ray scan performed on the patient during the
same session, in the same machine.
[0156] The radioisotopes that can be used for PET imaging are
.sup.68Ga, .sup.64Cu, .sup.48V, .sup.71As, .sup.72As, .sup.76Br, or
other polyvalent, cationic radiometals that decay by positron
emission.
General Methods
[0157] Molecular biological techniques, biochemical techniques, and
microorganism techniques as used herein are well known in the art
and commonly used, and are described in, for example, Sambrook J.
et al. (1989), Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor and its 3rd Ed. (2001); Ausubel, F. M. (1987), Current
Protocols in Molecular Biology, Greene Pub. Associates and
Wiley-interscience; Ausubel, F. M. (1989), Short Protocols in
Molecular Biology: A Compendium of Methods from Current Protocols
in Molecular Biology, Greene Pub. Associates and
Wiley-interscience; Innis, M. A. (1990), PCR Protocols: A Guide to
Methods and Applications, Academic Press; Ausubel, F. M. (1992),
Short Protocols in Molecular Biology: A Compendium of Methods from
Current Protocols in Molecular Biology, Greene Pub. Associates;
Ausubel, F. M. (1995), Short Protocols in Molecular Biology: A
Compendium of Methods from Current Protocols in Molecular Biology,
Greene Pub. Associates; Innis, M. A. et al. (1995), PCR Strategies,
Academic Press; Ausubel, F. M. (1999), Short Protocols in Molecular
Biology: A Compendium of Methods from Current Protocols in
Molecular Biology, Wiley, and annual updates; Sninsky, J. J. et al.
(1999), PCR Applications: Protocols for Functional Genomics,
Academic Press; Special issue, and the like. Relevant portions (or
possibly the entirety) of each of these publications are herein
incorporated by reference.
[0158] Any technique may be used herein for introduction of a
nucleic acid molecule into cells, including, for example,
transformation, transduction, transfection, and the like. Such a
nucleic acid molecule introduction technique is well known in the
art and commonly used, and is described in, for example, Ausubel F.
A. et al., editors, (1988), Current Protocols in Molecular Biology,
Wiley, New York, N.Y.; Sambrook J. et al. (1987) Molecular Cloning:
A Laboratory Manual, 2nd Ed. and its 3rd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N. Y.; Special issue, and the
like. Gene introduction can be confirmed by method as described
herein, such as Northern blotting analysis and Western blotting
analysis, or other well-known, common techniques.
Definitions and Abbreviations
[0159] "Treatment" or "treating" refers to a therapeutic
intervention that ameliorates a sign or symptom of a disease or
pathological condition, such a sign or symptom of cancer. Treatment
can also induce remission or cure of a condition.
[0160] The term "patient" includes any human or animal subject who
is in need of treatment for an indication as claimed herein.
[0161] The phrase "therapeutically-effective" is intended to
qualify the amount of each agent which will achieve the goal of
improvement in disorder severity and the frequency of incidence
over no treatment.
[0162] "RGD peptide" refers to an amino acid sequence
Arginine-Glycine-Aspartic acid ("RGD" is the one-letter amino acid
code, as is standardly expressed in the art).
[0163] "Small interfering RNA" (siRNA) refers to double-stranded
RNA molecules from about 10 to about 30 nucleotides long that are
named for their ability to specifically interfere with protein
expression. The length of the siRNA molecule is based on the length
of the antisense strand of the siRNA molecule.
[0164] A "shRNA" is an abbreviation for short hairpin RNA.
[0165] "Transfection" is the term used to describe the introduction
of foreign material such as foreign DNA into eukaryotic cells. It
is used interchangeably with "transformation" and "transduction"
although the latter term, in its narrower scope refers to the
process of introducing DNA into cells by viruses, which act as
carriers. Thus, the cells that undergo transfection are referred to
as "transfected," "transformed" or "transduced" cells.
[0166] A "CBRP" is a CXCR4 inhibiting bioreducible polymer and
examples of those polymers are referred to herein as P(AMD-CBA),
RPA, and the like.
[0167] Biodegradable, but not bioreducible polymers are known as
NPA and examples of these polymers are P(AMD-HMBA), CopCX, and the
like.
[0168] A "RHB" polymer is a control polymer that is a bioreducible
polymer that does not comprise a CXCR4 inhibiting moiety.
[0169] Unless otherwise indicated, the alkyl groups described
herein are preferably lower alkyl containing from one to eight
carbon atoms in the principal chain and up to 20 carbon atoms.
Alkyls may be substituted or unsubstituted and straight or branched
chain. Examples of unsubstituted alkyls include methyl, ethyl,
n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl,
i-pentyl, s-pentyl, t-pentyl, and the like. The term "substituted,"
as in "substituted alkyl," means that various heteroatoms such as
oxygen, nitrogen, sulfur, phosphorus, and the like can be attached
to the carbon atoms of the alkyl group either in the main chain or
as pendant groups. For example, the substituted alkyl groups can
have --C--X--C-- fragments in the main chain wherein the X is a
heteroatom. Further, the substituted alkyl groups can have at least
one hydrogen atom bound to a carbon atom replaced with one or more
substituent groups such as hydroxy, alkoxy, alkylthio, phosphino,
amino, halo, silyl, nitro, esters, ketones, heterocyclics, aryl,
and the like.
[0170] The term "aryl" as used herein alone or as part of another
group denotes an optionally substituted monovalent aromatic
hydrocarbon radical, preferably a monovalent monocyclic or bicyclic
group containing from 6 to 12 carbons in the ring portion, such as
phenyl, biphenyl, naphthyl, substituted phenyl, substituted
biphenyl or substituted naphthyl. Phenyl and substituted phenyl are
the more preferred aryl groups. The term "aryl" also includes
heteroaryl.
[0171] The term "-ene" as used as a suffix as part of another group
denotes a bivalent radical in which a hydrogen atom is removed from
each of two terminal carbons of the group, or if the group is
cyclic, from each of two different carbon atoms in the ring. For
example, alkylene denotes a bivalent alkyl group such as methylene
(--CH.sub.2--) or ethylene (--CH.sub.2CH.sub.2--), and arylene
denotes a bivalent aryl group such as o-phenylene, m-phenylene, or
p-phenylene. For clarity, addition of the -ene suffix is not
intended to alter the definition of the principal word other than
denoting a bivalent radical. Thus, continuing the example above,
alkylene denotes an optionally substituted linear saturated
bivalent hydrocarbon radical.
[0172] The term "hydrocarbon" as used herein describes a compound
or radical consisting exclusively of the elements carbon and
hydrogen.
[0173] The term "substituted" as in "substituted aryl,"
"substituted alkyl," and the like, means that in the group in
question (i.e., the alkyl, aryl or other group that follows the
term), at least one hydrogen atom bound to a carbon atom is
replaced with one or more substituent groups such as hydroxy
(--OH), alkylthio, phosphino, amido (--CON(R.sub.A)(R.sub.B),
wherein R.sub.A and R.sub.B are independently hydrogen, alkyl, or
aryl), amino(--N(R.sub.A)(R.sub.B), wherein R.sub.A and R.sub.B are
independently hydrogen, alkyl, or aryl), halo (fluoro, chloro,
bromo, or iodo), silyl, nitro (--NO.sub.2), an ether (--OR.sub.A
wherein R.sub.A is alkyl or aryl), an ester (--OC(O)R.sub.A wherein
R.sub.A is alkyl or aryl), keto (--C(O)R.sub.A wherein R.sub.A is
alkyl or aryl), heterocyclo, and the like. When the term
"substituted" introduces a list of possible substituted groups, it
is intended that the term apply to every member of that group. That
is, the phrase "optionally substituted alkyl or aryl" is to be
interpreted as "optionally substituted alkyl or optionally
substituted aryl."
[0174] The term "heteroaryl," as used herein alone or as part of
another group, denotes an optionally substituted monovalent
monocyclic or bicyclic aromatic radical of 5 to 10 ring atoms in
protonated or unprotonated form, where one or more, preferably one,
two, or three, ring atoms are heteroatoms independently selected
from N, O, and S, and the remaining ring atoms are carbon.
Exemplary heteroaryl moieties include benzofuranyl,
benzo[d]thiazolyl, benzo[d]thiazolium, isoquinolinyl,
isoquinolinium, quinolinyl, quinolinium, thiophenyl, imidazolyl,
imidazolium, oxazolyl, oxazolium, furanyl, thiazolyl, thiazolium,
pyridinyl, pyridinium, furyl, thienyl, pyridyl, pyrrolyl,
pyrrolidinium, indolyl, indolinium, and the like.
[0175] The term "heterocyclo," as used herein alone or as part of
another group, denotes a saturated or unsaturated monovalent
monocyclic group of 4 to 8 ring atoms in protonated or unprotonated
form, in which one or two ring atoms are heteroatom(s),
independently selected from N, O, and S, and the remaining ring
atoms are carbon atoms. Additionally, the heterocyclic ring may be
fused to a phenyl or heteroaryl ring, provided that the entire
heterocyclic ring is not completely aromatic. Exemplary heterocyclo
groups include the heteroaryl groups described above, pyrrolidino,
pyrrolidinium, piperidino, piperidinium, morpholino, morpholinium,
piperazino, piperazinium, succinimide, and the like. In some cases,
the heterocyclo can be a bivalent radical wherein the hydrogen is
removed from each of two atoms in the heterocyclo group.
[0176] Having described the invention in detail, it will be
apparent that modifications and variations are possible without
departing from the scope of the invention defined in the appended
claims.
EXAMPLES
[0177] The following non-limiting examples are provided to further
illustrate the present invention.
Example 1
Synthesis of Cyclam Monomer with Ethyleneoxide
##STR00025##
[0179] Tri Boc cyclam. A solution of t-butyl dicarbonate
(Boc.sub.2O) (2.96 g, 13.1 mmol) in 80 mL of methylene chloride
(CH.sub.2Cl.sub.2) was added dropwise over a period of 2 hours to a
solution of cyclam (0.88 g, 4.4 mmol) in CH.sub.2Cl.sub.2 at
0.degree. C. The mixture was allowed to warm to room temperature
and stirred overnight. The mixture was concentrated and purified by
chromatography (ethyl acetate (AcOEt).fwdarw.10:1 AcOEt:methanol
(CH.sub.3OH)) to give cyclam-Boc.sub.4 (0.91 g, 35%) as a white set
foam and cyclam-Boc.sub.3 (1.22 g, 56%) as a light yellow set foam.
Tri Boc cyclam: .sup.1H NMR (CDCl.sub.3) .delta.1.46 (s, 27H),
1.65-1.76 (m, 2H), 2.00-1.80 (m, 2H), 2.62 (bt, J=5.6 Hz, 2H), 2.79
(t, J=4.8 Hz, 2H), 3.31 (t, J=6.4 Hz, 8H), 3.47-3.34 (m, 4H). (see
Schickaneder, C.; Heinemann, F. W.; Alsfasser, R. "Copper II
Complexes of the Tetraazamacrocyclic Tertiary Amide Ligand
Alanyl-Cyclam," Eur. J Chem. 2006, 2357-2363)
[0180] N-(2-(2-(Chloroethoxy)ethoxy)ethyl)phthalimide. A mixture of
potassium phthalimide (3.00 g, 15.9 mmol) and
1,2-bis(2-chloroethoxy)ethane (25 mL, 30 g, 160 mmol) was heated at
130.degree. C. overnight. The excess 1,2-bis(2-chloroethoxy)ethane
was distilled under high vacuum to leave a yellow semi-solid. This
was mixed with CH.sub.2C1.sub.2 (50 mL) and the insoluble material
removed by filtration. The filtrate was concentrated to give a
yellow liquid and purified by chromatography (2:1 Hexanes:AcOEt) to
give (4.40 g, 93%) as a colorless liquid. .sup.1H NMR (CDCl.sub.3)
.delta.3.55 (t, J=6.0 Hz, 2H), 3.68-3.61 (m, 4H), 3.69 (t, J=6.0
Hz, 2H), 3.76 (t, J=6.0 Hz, 2H), 3.92 (t, J=6.0 Hz, 2H), 7.73 (dd,
J=5.2, 2.4 Hz, 2H), 7.86 (dd, J=5.2, 2.4 Hz, 2H). (see Lukyanenko,
N. G.; Kirichenko, T. I.; Shcherbakov, S. V. "Synthesis of Lariat
Diazacrown Ethers with Terminal Amino Groups in the Side Chains,"
Chem. Heterocycl. Compd. 2004, 40: 343-350.)
[0181] N-(2-(2-(Iodoethoxy)ethoxy)ethyl)phthalimide. A mixture of
N-(2-(2- (chloroethoxy)ethoxy)ethyl)phthalimide (4.0 g, 13 mmol),
NaI (4.6 g, 31 mmol) and acetonitrile (CH.sub.3CN) (30 mL) was
refluxed overnight. After cooling it was concentrated to give a
brown orange semi solid that was mixed with CH.sub.2Cl.sub.2 (50
mL) and the insoluble material removed by filtration. The filtrate
was washed with 5% sodium thiosulfate (Na.sub.2S.sub.2O.sub.3) (10
mL) until all of the brown color disappeared. The layers were
separated and the aqueous layer extracted with CH.sub.2Cl.sub.2.
The combined CH.sub.2Cl.sub.2 layers were dried with anhydrous
magnesium sulfate (MgSO.sub.4) and concentrated to give a yellow
liquid. This was purified by chromatography (2:1 Hexanes:AcOEt) to
give (5.15 g, 100%) as a light orange liquid. .sup.1H NMR
(CDCl.sub.3) .delta.3.15 (t, J=6.4 Hz, 2H), 3.63-3.56 (m, 4H), 3.66
(t, J=6.8 Hz, 2H), 3.74 (t, J=6.0 Hz, 2H), 3.89 (t, J=5.6 Hz, 2H),
7.70 (dd, J=5.2, 2.4 Hz, 2H), 7.8 (dd, J=5.2, 2.4 Hz, 2H).
[0182] Tri Boc cyclam N-(2-(2-ethoxyethoxy)ethyl)phthalimide. A
mixture of tri Boc cyclam (0.50 g, 1.0 mmol),
N-(2-(2-(Iodoethoxy)ethoxy)ethyl)phthalimide (0.78 g, 2.0 mmol),
anhydrous potassium carbonate (K.sub.2CO.sub.3) (0.34 g, 2.5 mmol)
and CH.sub.3CN (10 mL) was refluxed overnight. After cooling it was
concentrated to give a light yellow semi solid that was mixed with
hot AcOEt (25 mL) and the insoluble material removed by filtration.
The filtrate was concentrated to give a yellow liquid and purified
by chromatography (AcOEt.fwdarw.10:1 AcOEt:CH.sub.3OH) to give
(0.45 g, 98% based on 60% conversion) as a colorless liquid and
(0.20 g) of unreacted tri Boc cyclam. .sup.1H NMR (CDCl.sub.3)
.delta.1.45 (s, 18H), 1.46 (s, 9H), 1.72-1.59 (m, 2H), 1.94-1.82
(m, 2H), 2.45-2.36 (m, 2H), 2.58 (t, J=6.4 Hz, 4H), 3.21-3.15 (m,
2H), 3.41-3.22 (m, 10H), 3.45 (t, J=6.4 Hz, 2H), 3.51 (t, J=4.8 Hz,
2H), 3.64-3.60 (m, 2H), 3.73 (t, J=6.0 Hz, 2H), 3.89 (t, J=6.0 Hz,
2H), 7.72 (dd, J=5.6, 2.4 Hz, 2H), 7.85 (dd, J=4.8, 2.4 Hz,
2H).
[0183] Tri Boc cyclam 2-(2-ethoxyethoxy)ethanamine. To a solution
of triBoc cyclam phthalimide (0.45 g, 0.59 mmol) in CH.sub.3OH (10
mL), hydrazine (NH.sub.2NH.sub.2) (0.19 mL, 0.19 g, 6.0 mmol) was
added and stirred overnight. The mixture was concentrated to give a
white solid that was mixed with hot CH.sub.2Cl.sub.2 (25 mL) and
the insoluble material removed by filtration. The filtrate was
concentrated to give a yellow liquid and purified by chromatography
(10:1 CH.sub.2Cl.sub.2:CH.sub.3OH+0.5% NH.sub.3) to give (0.36 g,
97%) as a yellow set liquid. .sup.1H NMR (CDCl.sub.3) .delta.1.46
(s, 27H), 1.72-1.60 (m, 2H), 1.94-1.82 (m, 2H), 2.53-2.41 (m, 2H),
2.72-2.53 (m, 4H), 3.12-3.02 (m, 2H), 3.25-3.16 (m, 2H), 3.45-3.25
(m, 10H), 3.55- 3.50 (m, 2H), 3.60-3.55 (m, 2H), 3.65-3.60 (m, 2H),
3.70-3.65 (m, 2H), 5.11 (bs, 2H).
Example 2
Synthesis of P(AMD-CBA) (Also Referred to Herein as RPA)
##STR00026##
[0185] P(AMD-CBA) was synthesized by Michael addition of
1,1'-[1,4-Phenylenebis
(methylene)]bis[1,4,8,11-tetraazacyclotetradecane] (AMD3100) and
N',N'-cystamine bisacrylamide (CBA) at molar ratio of 1:1.
Calculated amounts of AMD3100 (200.8 mg, 0.4 mmol) and CBA (104 mg,
0.4 mmol) were weighed and dissolved in methanol/water mixture (4
mL, v/v 7:3) to make soluble RPA. Polymerization was allowed to
proceed under nitrogen protection in the dark at 37.degree. C. for
72 hours before gelation. Then, an additional 20 mg of AMD3100 was
added to the reaction mixture to consume any residual acrylamide
groups and the mixture was stirred for another 6 hours. The
reaction mixture was added dropwise to an excess 1.25 M HCl in
ethanol and the pH of the mixture was kept at about 3. The
resulting precipitated HCL salt of the RPA was centrifuged and
washed with ethanol twice to remove the extra acid. The products
were then dried using a vacuum pump and redissolved in water. After
dialysis against water for 2 days (MWCO 3,500), the polymers were
freeze dried and ready to use.
Example 3
Characterization of P(AMD-CBA)
[0186] The composition of the polymers was analyzed by .sup.1NMR.
Molecular weight of the polymers was determined by Gel Permeation
Chromatography (GPC) (Malvern Instruments). Sodium acetate (0.3 M,
pH 5) was used as an eluent at flow rate of 0.3 mL/minute. Number
average molecular weight (M.sub.n) of P(AMD-CBA) was 12,553, Weight
average (M.sub.w) molecular weight of P(AMD-CBA) was 13,756, and
PDI was 1.096. See FIG. 2.
Example 4
Condensation of Plasmid DNA to Show That P(AMD-CBA) Forms Complexes
with DNA
[0187] The ability of P(AMD-CBA) to condense pDNA was determined by
ethidium bromide exclusion (EtBr) assay by measuring the changes in
EtBr/pDNA fluorescence. pDNA solution at a concentration of 20
.mu.g/mL was mixed with EtBr (1 .mu.g/mL) and fluorescence was
measured and set to 100% using an excitation wavelength of 540 nm
and an emission wavelength of 590 nm. Fluorescence readings were
taken following a stepwise addition of the polycation solution, and
the condensation curve for each polycation was constructed. See
FIG. 3.
Example 5
Size and Zeta Potential of P(AMD-CBA) Complexes with Plasmid DNA
(Polyplexes)
[0188] Luciferase plasmid DNA (gWiz-Luc pDNA, Aldevron) solution at
a concentration 20 .mu.g/mL was prepared in 10 mM HEPES buffer (pH
7.4). Polyplexes were formed by adding a predetermined volume of
polymer to achieve the desired weight/weight ratio (polymer/pDNA)
and mixed by vigorous vortexing for 10 seconds. Polyplexes were
further allowed to stand for 30 minutes prior to use. The
determination of hydrodynamic diameters and zeta potentials of
polyplexes was performed by Dynamic Light Scattering (DLS). Results
were expressed as mean.+-.standard deviation (SD) of three
independent experiments with 3 runs each.
TABLE-US-00001 TABLE 1 Size and zeta-potential of P(AMD-CBA)
polyplexes at different polymer/DNA w/w ratio. w/w Size(nm)
Zeta-potential (mV) 5 55.8 .+-. 0.4 25.2 .+-. 4.6 10 69.9 .+-. 5.5
18.4 .+-. 4.4 15 56.7 .+-. 1.1 30.6 .+-. 4.1 20 61.7 .+-. 4.2 33.0.
.+-. 3.2 25 57.5 .+-. 0.2 25.5 .+-. 3.9
Example 6
Ability of P(AMD-CBA)DNA Polyplex to Disassemble and Release
Plasmid DNA in a Reducing Environment
[0189] The redox-sensitivity and stability of the polyplexes were
examined by agarose gel electrophoresis. Polyplexes were prepared
at P(AMD-CBA)/pDNA w/w ratio=5 and incubated under indicated
conditions of 20 mM GSH with or without the presence of 150 mM NaCl
at 37.degree. C. for 1 hour. Samples were then loaded onto a 0.8%
agarose gel containing 0.5 .mu.g/mL ethyl bromide (EtBr) and run
for 75 minutes at 120 V in 0.5X Tris/Borate/ethylene diamine
tetraacetic acid (EDTA) (TBE) running buffer. The gel was
visualized under UV. See FIG. 4.
Example 7
Transfection Efficiency of the Complexes of P(AMD-CBA) with
Luciferase Reporter Gene Plasmid DNA
[0190] B16F10 cells were seeded in 48 well plate at a density of
40,000 cells/well 24 hours prior to transfection. The cells were
incubated with the polyplexes (DNA dose: 0.5 .mu.g/well) in 175
.mu.L of medium with or without 10% v/v fetal bovine serum (FBS).
Wherever indicated, 100 .mu.M of chloroquine was present in the
media to improve the endosomal escape. After 4 hours incubation,
polyplexes were completely removed and the cells were cultured in
complete culture medium for 24 hours. The medium was then discarded
and the cells were lysed in 100 .mu.L of 0.5X cell culture lysis
reagent buffer (Promega, Madison, Wis.) for 30 minutes. To measure
the luciferase content, 100 .mu.L of 0.5 mM luciferin solution was
automatically injected into each well of 20 .mu.L of cell lysate
and the luminescence was integrated over 10 seconds using BioTek
Synergy 2 Microplate Reader. Total cellular protein in the cell
lysate was determined by the BCA protein assay using calibration
curve constructed with standard bovine serum albumin solutions
(Pierce, Rockford, Ill.). See FIG. 5.
Example 8
Synthesis of P(Cyc-CBA)
[0191] A series of three P(Cyc-CBA) was synthesized by Michael
addition of different molar ratio of cyclam and N',N'-Cystamine
bisacrylamide (CBA). Calculated amounts of cyclam and CBA were
weighed and dissolved in methanol/water mixture (v/v 7:3).
Polymerization was allowed to proceed in the dark at 37.degree. C.
for 24-72 hours before gelation. HCl in ethanol (1.25 M) was then
added to form the HCl salt of the polymers. The precipitated
products were centrifuged down and washed with ethanol twice to
remove the extra acid. The products were then dried using vacuum
pump and redissolved in water. After deep dialysis against water
for 2 days (MWCO 1,000 for P(Cyc-CBA)/3 and MWCO 3,500 for
P(Cyc-CBA/2 and 1.8), the polymers were lyophilized and ready to
use.
##STR00027##
Example 9
Characterization of P(Cyc-CBA)
[0192] The composition of the polymers was analyzed by .sup.1H NMR.
Molecular weight of the polymers was determined by Gel Permeation
Chromatography (GPC) (Malvern Instruments). Sodium acetate (0.3 M,
pH 5) was used as an eluent at a flow rate of 0.3 mL/minute. See
FIG. 6.
TABLE-US-00002 TABLE 2 Characterization of P(Cyc-CBA). Molar
Feeding Cyclam Ratio Ratio % Mn Mw PDI P(Cyc-CBA)/3 1.5 3 56.6
3,131 4,420 1.412 P(Cyc-CBA)/2 1 2 49.6 12,030 13,714 1.14
P(Cyc-CBA)/1.8 0.9 1.8 47.8 12,912 13,751 1.065
Example 10
Condensation of Plasmid DNA by P(Cyc-CBA)
[0193] The ability of P(Cyc-CBA) to condense pDNA was determined by
ethidium bromide exclusion (EtBr) assay by measuring the changes in
EtBr/pDNA fluorescence. pDNA solution at a concentration of 20
.mu.g/mL was mixed with EtBr (1 .mu.g/mL) and fluorescence was
measured and set to 100% using an excitation wavelength of 540 nm
and an emission wavelength of 590 nm. Fluorescence readings were
taken following a stepwise addition of the polycation solution, and
the condensation curve for each polycation was constructed. See
FIG. 7.
Example 11
Preparation and Physical Characterization of P(Cyc-CBA) Polyplexes
with Plasmid DNA
[0194] gWiz-Luc pDNA solution (Aldevron) at a concentration 20
.mu.g/mL was prepared in 10 mM HEPES buffer (pH 7.4). Polyplexes
were formed by adding predetermined volume of polymer to achieve
the desired weight/weight ratio (polymer/pDNA) and mixed by
vigorous vortexing for 10 seconds. Polyplexes were further allowed
to stand for 30 minutes prior to use. The determination of
hydrodynamic diameters and zeta potentials of polyplexes was
performed by Dynamic Light Scattering (DLS). Results were expressed
as mean.+-.standard deviation (SD) of three independent experiments
with 3 runs each.
TABLE-US-00003 TABLE 3 Size and zeta-potential of P(Cyc-CBA)
polyplexes at different w/w ratio. Polymer w/w Size(nm)
Zeta-potential (mV) P(Cyc-CBA)/3 5 101.7 .+-. 1.7 13.3 .+-. 1.3 10
124.4 .+-. 6.6 13.9 .+-. 1.9 15 111.0 .+-. 1.1 17.0 .+-. 5.4 20
113.8 .+-. 3.6 14.7 .+-. 3.2 25 125.3 .+-. 1.8 17.3 .+-. 2.7
P(Cyc-CBA)/2 5 119.2 .+-. 1.9 21.4 .+-. 3.7 10 124.9 .+-. 2.2 26.1
.+-. 2.1 15 204.9 .+-. 9.0 16.0 .+-. 2.0 20 167.0 .+-. 2.6 17.0
.+-. 2.1 25 163.0 .+-. 2.8 15.5 .+-. 2.7 P(Cyc-CBA)/1.8 5 87.4 .+-.
1.5 16.3 .+-. 2.3 10 89.5 .+-. 3.3 22.7 .+-. 3.8 15 121.3 .+-. 3.3
28.6 .+-. 2.8 20 80.9 .+-. 2.9 19.8 .+-. 1.8 25 79.9 .+-. 1.1 17.1
.+-. 1.8
Example 12
Disassembly and pDNA Release from P(Cyc-CBA) Polyplexes
[0195] The redox-sensitivity and stability of the polyplexes were
examined by agarose gel electrophoresis. Polyplexes were prepared
at P(Cyc-CBA)/pDNA w/w ratio=5 and incubated under indicated
conditions of different concentrations of heparin with or without a
reducing agent (either GSH or DTT) at 37.degree. C. for 1 hour.
Samples were then loaded onto a 0.8% agarose gel containing 0.5
.mu.g/mL EtBr and run for 75 minutes at 120 V in 0.5X
Tris/Borate/EDTA (TBE) running buffer. The gel was visualized under
UV. See FIG. 8.
Example 13
Transfection Efficiency of the Complexes of P(Cyc-CBA) with
Luciferase Reporter Plasmid
[0196] Human breast cancer cell line MDA-MB-231 was a kind gift
from Dr. Jing Li, Karmanos Cancer Institute (Detroit, Mich.). The
cells were maintained in RPMI1640 medium supplemented with 10% FBS.
Murine melanoma cell line B16F10 and human hepatocellular carcinoma
cell line Hep G2 were purchased from ATCC (Manassas, Va.). B16F10
cells were maintained in DMEM media supplemented with 10% FBS and
Hep G2 cells were maintained in MEM media supplemented with 10%
FBS. All the cells were cultured at 37.degree. C. in 5% CO2
atmosphere.
[0197] All transfection experiments were conducted in 48-well
plates with cells at logarithmic growth phase following a
previously published protocol (Read, Singh et al. 2005). Cells were
seeded at a density of 40,000 cells/well 24 h prior to
transfection. On the day of transfection, cells were incubated with
the polyplexes (DNA conc. 2.35 .mu.g/mL) in 170 .mu.L of serum-free
or 10% FBS-containing media. After 4 h incubation, polyplexes were
completely removed and the cells were cultured in complete culture
medium for 24 h prior to measuring luciferase expression. The
medium was discarded and the cells were lysed in 100 .mu.L of 0.5x
cell culture lysis reagent buffer (Promega, Madison, Wis.) for 30
min. To measure the luciferase content, 100 .mu.L of 0.5 mM
luciferin solution was automatically injected into each well of 20
.mu.L of cell lysate and the luminescence was integrated over 10 s
using Synergy 2 Microplate Reader (BioTek, Vt.). Total cellular
protein in the cell lysate was determined by the Bicinchoninic acid
protein assay using calibration curve constructed with standard
bovine serum albumin solutions (Pierce, Rockford, Ill.).
Transfection activity was expressed as relative light units
(RLU)/mg cellular protein.+-.SD of quadruplicate samples.
[0198] B16F10 cells were seeded in 48 well plate at a density of
40,000 cells/well 24 hours prior to transfection. The cells were
incubated with the polyplexes (DNA dose: 0.5 .mu.g/well) in 175
.mu.L of medium with or without 10% v/v FBS. Wherever indicated,
100 .mu.M of chloroquine was present in the media to improve the
endosomal escape. After 4 hours incubation, polyplexes were
completely removed and the cells were cultured in complete culture
medium for 24 hours. The medium was then discarded and the cells
were lysed in 100 .mu.L of 0.5X cell culture lysis reagent buffer
(Promega, Madison, Wis.) for 30 minutes. To measure the luciferase
content, 100 .mu.L of 0.5 mM luciferin solution was automatically
injected into each well of 20 .mu.L of cell lysate and the
luminescence was integrated over 10 seconds using BioTek Synergy 2
Microplate Reader. Total cellular protein in the cell lysate was
determined by the BCA protein assay using calibration curve
constructed with standard bovine serum albumin solutions (Pierce,
Rockford, Ill.). See FIG. 9.
Example 14
Cytotoxicity of P(Cyc-CBA)
[0199] Cytotoxicity of P(Cyc-CBA) in MDA-MB-231 cells was
determined by MTS assay using a commercially available kit
(CellTiter 96.RTM. Aqueous Cell Proliferation Assay, Promega).
20,000 cells were seeded per well in 96-well plates 24 hours ahead.
The culturing medium was first removed and then replaced with 150
.mu.L of medium containing increasing concentration of the
polycations. After 24 hours, the incubation medium was removed and
a mixture of 100 .mu.L of fresh serum-free medium and 20 .mu.L of
MTS reagent solution was added to each well. The cells were
incubated for at 37.degree. C. in CO.sub.2 incubator for 2 hours.
The absorbance at wavelength 505 nm was then measured to determine
cell viability. IC.sub.50 values were calculated by Prism Graphpad
Software. See FIG. 10.
[0200] Toxicity of polycations was also evaluated by MTS assay in
Hep G2 cells and CXCR4+U2OS cells. HepG2 were purchased from ATCC
(Manassas, Va.). Hep G2 cells were maintained in MEM supplemented
with 10% FBS. Human epithelial osteosarcoma U2OS cells stably
expressing human CXCR4 receptor fused to the N-terminus of enhanced
green fluorescent protein were purchased form Fisher Scientific.
The cells were cultured in DMEM supplemented with 2 mM L-Glutamine,
10% FBS, 1% Pen-Strep and 0.5 mg/ml G418.The cells were plated into
96-well microtiter plates at a density of 20,000 cells/well. After
24 h, culture medium was replaced by 150 .mu.l of serial dilutions
of a polymer in serum-supplemented medium and the cells were
incubated for 24 h. Polymer solutions were aspirated and replaced
by a mixture of 100 .mu.l serum-free media and 20 .mu.l of MTS
reagent (CellTiter 96.RTM. AQueous Non-Radioactive Cell
Proliferation Assay, Promega). After 2 h incubation, the absorbance
was measured spectrophotometrically in Synergy 2 Microplate Reader
(BioTek, Vt.) at a wavelength of 490 nm. The relative cell
viability (%) was calculated as [A]sample/[A]untreated.times.100%.
The IC50 were calculated as polymer concentration, which inhibits
growth of 50% of cells relative to untreated cells. The IC50 values
were calculated based on "log(inhibitor) vs. response-absolute
IC50" curve fitting procedure in GraphPad Prism, with constrains of
Fifty=50, Top=100 and a formula Y=Bottom+(Top-Bottom)/(1+10 ((Log
IC50- X)*HillSlope+Log((Top-Bottom)/(Fifty-Bottom)-1))). In both
cell lines RPA had remarkably low toxicity compared with 25-kDa
poly(ethyleneimine) (PEI) control. The IC50 of RPA was almost 50
times higher than that of PEI in Hep G2 cells (599 vs. 12 .mu.g/mL)
and 116 times higher in U2OS cells (464 vs. 4 .mu.g/mL). The IC50
of control polymer RHB was 57 .mu.g/mL in Hep G2 cells.
Example 15
Transition Metal Complexes of P(Cyc-CBA)
[0201] Metal complexes of P(Cyc-CBA) polymers were formed by
incubating polymer solutions with 10 mM CuCl.sub.2, ZnCl.sub.2 or
CoCl.sub.2 at 37.degree. C. for 1 hours. For Cu(II) complexation,
cyclam (1.25 M) or P(Cyc-CBA) polymers (0.5 mg/ml) were incubated
with different concentration of CuCl.sub.2 (1 equivalent=1.25 M) in
sodium acetate buffer (pH 6) at room temperature for 1 h. For
Zn(II) complexation, cyclam (1.25 M) or P(Cyc-CBA) polymers (0.5
mg/mL) were incubated with different concentration of ZnCl.sub.2 (1
equivalents=1.25 M) in cacodylate buffer (pH 7.4) at room
temperature for 1 hours and then 1 equivalent of CuCl.sub.2 were
added. For Co(II) complexation, cyclam (1.25 M) or P(Cyc-CBA)
polymers (0.5 mg/mL) were incubated with different concentration of
CoCl.sub.2 (1 equivalent=1.25 M) in HEPES buffer (pH 7.4) at room
temperature for 1 hours. All the absorption spectrums were obtained
by UV-vis spectroscopy. See FIG. 11.
##STR00028##
Example 16
Stability of Plasmid DNA Polyplexes of the P(Cyc-CBA) Metal
Complexes
[0202] pDNA polyplexes were prepared at w/w 5 and incubated under
indicated conditions of different concentrations of heparin at
37.degree. C. for 1 hour. Samples were then loaded onto a 0.8%
agarose gel containing 0.5 .mu.g/mL EtBr and run for 75 minutes at
120 V in 0.5X Tris/Borate/EDTA (TBE) running buffer. The gel was
visualized under UV. See FIG. 12.
Example 17
Transfection Efficiency of the Polyplexes of Luciferase Plasmid DNA
with the Metal Complexes of P(Cyc-CBA)
[0203] B16F10 cells were seeded in 48 well plate at a density of
40,000 cells/well 24 hours prior to transfection. The cells were
incubated with the polyplexes of luciferase plasmid DNA with the
metal complexes of P(Cyc-CBA) (DNA dose: 0.5 .mu.g/well) in 175
.mu.L of medium with or without 10% v/v FBS. Wherever indicated,
100 .mu.M of chloroquine was present in the media to improve the
endosomal escape. After 4 hours incubation, polyplexes were
completely removed and the cells were cultured in complete culture
medium for 24 hours. The medium was then discarded and the cells
were lysed in 100 .mu.L of 0.5X cell culture lysis reagent buffer
(Promega, Madison, Wis.) for 30 minutes. To measure the luciferase
content, 100 .mu.L of 0.5 mM luciferin solution was automatically
injected into each well of 20 .mu.L of cell lysate and the
luminescence was integrated over 10 seconds using BioTek Synergy 2
Microplate Reader. Total cellular protein in the cell lysate was
determined by the BCA protein assay using calibration curve
constructed with standard bovine serum albumin solutions (Pierce,
Rockford, Ill.). See FIG. 13.
Example 18
Cytotoxicity of the Metal Complexes of P(Cyc-CBA)
[0204] Cytotoxicity of metal complexes of P(Cyc-CBA) in MDA-MB-231
cells was determined by MTS assay using a commercially available
kit (CellTiter 96.RTM. Aqueous Cell Proliferation Assay, Promega).
20,000 cells were seeded per well in 96-well plates 24 hours ahead.
The culturing medium was first removed and then replaced with 150
.mu.L of medium containing increasing concentration of the
polycations. After 24 hours, the incubation medium was removed and
a mixture of 100 .mu.L of fresh serum-free medium and 20 .mu.L of
MTS reagent solution was added to each well. The cells were
incubated for at 37 .degree. C. in CO.sub.2 incubator for 2 hours.
The absorbance at wavelength 505 nm was then measured to determine
cell viability. IC.sub.50 values were calculated by Prism Graphpad
Software.
TABLE-US-00004 TABLE 4 IC50 values of metal complexes of
P(Cyc-CBA). w/w P(Cyc-CBA)/3 P(Cyc-CBA)/2 P(Cyc-CBA)/1.8 Metal Free
247.6 .+-. 15.9 113.6 .+-. 12.0 53.31 .+-. 3.9 50% Cu 319.2 .+-.
15.1 132.9 .+-. 13.2 64.37 .+-. 4.4 100% Cu 143.8 .+-. 10.9 83.9
.+-. 3.2 34.7 .+-. 2.6 50% Zn 204.0 .+-. 20.1 106.6 .+-. 8.8 36.8
.+-. 4.7 100% Zn 166.4 .+-. 30.8 104.4 .+-. 11.2 41.4 .+-. 1.0 50%
Co 138.1 .+-. 18.8 110.4 .+-. 16.9 71.0 .+-. 4.3 100% Co 129.8 .+-.
9.7 74.4 .+-. 5.4 53.0 .+-. 2.2
Example 19
P(AMD-CBA) Antagonizes CXCL12
[0205] Binding of chemokine CXCL12 to its receptor CXCR4 triggers
an intracellular signal transduction cascade comprising a transient
increase in cytosolic free calcium. The antagonist AMD3100 is
unable to trigger the calcium flux and therefore inhibits the
chemokine-induced calcium signaling..sup.71 We used fluorescent
calcium indicator Fluo-3 to monitor the intracellular calcium flux
induced by CXCL12 (FIG. 14). Cells untreated with any of the tested
agents exhibited a rapid increase in intracellular calcium after
CXCL12 stimulation, confirming activation of the CXCR4 by CXCL12
binding. Treatment with AMD3100 and P(AMD-CBA) had a strong
antagonistic effect on this signaling pathway. In contrast, the
control non-cyclam polycation P(DMADP-CBA) failed to block the
CXCR4-mediated signaling pathway showing similar levels of calcium
flux as control cells. This demonstrates that P(AMD-CBA) is CXCR4
antagonist.
Example 20
Low Toxicity of P(AMD-CBA)
[0206] The cytotoxicity of P(AMD-CBA) was determined by MTS assay
as described above in example 14. See FIG. 15. Using BRP with
different disulfide content, synthesized as described in Chen, J.,
C. Wu, and D. Oupicky, "Bioreducible Hyperbranched Poly(amido
amine)s for Gene Delivery." Biomacromolecules, (2009) 10:
2921-2927), the toxicity of BRP is a direct function of the
disulfide content and intracellular GSH concentration. This is
shown by the increasing IC.sub.50 with increasing disulfide content
and by steeper IC.sub.50 vs. disulfide content dependence in cells
with higher GSH content. See FIG. 15 middle. Non-degradable nBRP
induce apoptosis as soon as 12 hours after incubation, no
significant apoptosis induction was observed for BRP for up to 36
hours. See FIG. 15 right. This study provides additional
information about the safety of bioreducible polycations.
Example 21
Colloidal Stabilization of P(AMD-CBA) Polyplexes
[0207] The colloidal stability of P(AMD-CBA) polyplexes was
increased by formulating the polyplexes with a mixture of
P(AMD-CBA) and PEG-BRP copolymer See FIG. 16. The polyplexes were
formed generally using the procedure described in example 11.
Increasing the content of PEG-BRP in the formulation decreased the
rate of aggregation of the polyplexes in 0.15 M NaCl. The colloids
were stable for at least 3 hours when the formulation included 20%
PEG-BRP.
Example 22
Evaluation of P(AMD-CBA) Complexes with Copper
[0208] Complexes of AMD3100 with positron emitting .sup.64Cu
(t.sub.1/2 12.7 h) have been used to image CXCR4 positive cancers
using PET. (Nimmagadda, S., M. Pullambhatla, K. Stone, G. Green, Z.
M. Bhujwalla, and M. G. Pomper, "Molecular Imaging of CXCR4
Receptor Expression in Human Cancer Xenografts with [Cu-64]AMD3100
Positron Emission Tomography." Cancer Res, (2010) 70: 3935-3944).
We first compared the copper binding ability of P(AMD-CBA) to
AMD3100 and control non-cyclam polycation P(DMADP-CBA) in a
titration experiment with CuCl.sub.2. See FIG. 17. The procedure
was similar to that described in example 15. Absorption at 550 nm
showed formation of the copper complexes with AMD3100 and
P(AMD-CBA) but not with the control P(DMADP-CBA) where DMADP is
N,N-dimethyldipropylenetriamine. Analysis of the titration data
that not all cyclam rings in the polycation can bind copper (most
likely due to reduced accessibility of some cyclam rings in the
polymer and electrostatic repulsion). Since P(AMD-CBA) form copper
complexes, they are suitable for PET imaging studies.
Example 23
Condensation of Plasmid DNA by Copper Complexes of P(AMD-CBA)
[0209] Copper complexation increased the overall charge of
P(AMD-CBA) and provided more effective DNA condensation using the
procedure generally described in example 10. See FIG. 18. However,
this increased charge also increased toxicity of the polycation
with increasing copper content as determined using the procedure
described in example 14. See FIG. 19. No toxicity was seen for
equivalent concentration of CuCl.sub.2, confirming that the
toxicity was the result of increased cationic character of
P(AMD-CBA). The luciferase transfection of the copper complexes of
P(AMD-CBA) increased with increasing copper content and reached
maximum when 75% of the cyclam moieties were complexed with copper
as determined using the procedure of example 13. See FIG. 19.
However, since the amount of copper needed for microPET imaging is
negligible, no adverse effects on toxicity are anticipated.
Example 24
Formation and Characterization of RPA Polyplexes
[0210] gWiz-Luc DNA solution in 10 mM HEPES (pH 7.4) was prepared
to give a DNA concentration in the final polyplexes=20 .mu.g/mL.
Polyplexes were formed by adding predetermined volume of polymer to
achieve the desired polycation/DNA weight/weight (w/w) ratio and
mixed by vigorous vortexing for 10 seconds. Polyplexes were further
allowed to stand for 30 min prior to use. The determination of
hydrodynamic diameters and zeta potentials of polyplexes was
performed by Dynamic Light Scattering following previously
published method. Results were expressed as mean.+-.standard
deviation (S.D.) of 3-10 experimental runs.
Example 25
CXCR4 Redistribution Assay
[0211] CXCR4+U2OS cells were plated in 96-well plate 18-24 h before
the experiment at a seeding density of 8,000 cells per well. The
cells were first washed with 100 .mu.L assay buffer (DMEM
supplemented with 2 mM L-Glutamine, 1% FBS, 1% Pen-Strep and 10 mM
HEPES) twice and then incubated with different concentrations of
the polycations or AMD3100 in assay buffer containing 0.25% DMSO at
37.degree. C. for 30 min. In experiments with RPA/DNA and RHB/DNA
polyplexes (wherein RPA is P(AMD-CBA) and RHB is P(DMADP-CBA), DNA
concentration was 0.5 .mu.g/mL. Human
SDF-1.alpha..quadrature.(CXCL-12) was then added to each well to
make final concentration 10 nM. DMSO alone was used as the negative
control, and hSDF-1.alpha..quadrature. alone was used as the
positive control. After 1 h incubation at 37.degree. C., the cells
were fixed with 4% formaldehyde at room temperature for 20 min
followed by 4- time washing with PBS. All the images were taken by
EVOS fl microscope at 20.times..
[0212] The quantification of the receptor redistribution was
conducted by ImageXpress.quadrature.Micro high throughput imaging
system by Molecular Devices (Sunnyvale, Calif.). The system enables
high-quality imaging of 96-well plates based on automatic focusing
of fluorescently labeled cell nuclei (by DAPI or Hoechst dye)
followed by image analysis by MetaXpress software (High Throughput
Mode) based on the average green fluorescent granule intensity
(internalized GFP-CXCR4). Untreated cells U2OS cells stimulated
with 10 nM CXCL12 were used as negative control (100% CXCR4
translocation) and 300 nM AMD3100 treated cells were used as
positive controls (0% CXCR4 translocation). The method was verified
by establishing a dose response curve of AMD3100 and its calculated
EC50 was comparable with cell line data sheet from Fisher
Scientific. The operation of the instrument and analysis of the
data were conducted with the help of Steve Swaney at the Center for
Chemical Genomics of Life Sciences Institute, University of
Michigan (Ann Arbor, Mich.).
Example 26
Cell Invasion Assay
[0213] The upper sides of the transwell inserts were coated with 40
.mu.l Matrigel diluted in serum-free medium (v/v 1:3) per insert.
The 24-well plates with coated inserts were then placed in
37.degree. C. incubator for 2 h. CXCR4+U2OS cells were trypsinized
and resuspended in different concentrations of drugs in serum-free
medium for 30 min before adding to the inserts at a final
concentration of 10,000 cells in 300 .mu.l medium per insert. 20 nM
CXCL12 in serum-free medium as the chemo-attractant was then added
to corresponding wells in the companion plate. After 16 h, the
non-invaded cells on the upper surface of the inserts were removed
with a cotton swab. The invaded cells were then fixed and stained
by dipping the inserts into Diff-Quick solution. The images were
taken by EVOS xl microscope. Five 20.times. imaging areas were
randomly selected for each insert and each sample was conducted in
triplicate. Statistical significance of the observed differences in
cell invasion was analyzed using non-parametric ANOVA with Dunn's
multiple comparison test using GraphPad InStat (v. 3.10). P<0.05
was considered significant.
[0214] All transfection experiments were conducted in 48-well
plates with cells at logarithmic growth phase. Cells were seeded at
a density of 40,000 cells/well 24 h prior to transfection. On a day
of transfection, the cells were incubated with the polyplexes (DNA
conc. 2.35 .mu.g/ml) in 170 .mu.L of serum-free or 10%
FBScontaining media. After 4 h incubation, polyplexes were
completely removed and the cells were cultured in complete culture
medium for 24 h prior to measuring luciferase expression. The
medium was discarded and the cells were lysed in 100 .mu.L of 0.5x
cell culture lysis reagent buffer (Promega, Madison, Wis.) for 30
min. To measure the luciferase content, 100 .mu.L of 0.5 mM
luciferin solution was automatically injected into each well of 20
.mu.L of cell lysate and the luminescence was integrated over 10 s
using Synergy 2 Microplate Reader (BioTek, Vt.). Total cellular
protein in the cell lysate was determined by the Bicinchoninic acid
protein assay using calibration curve constructed with standard
bovine serum albumin solutions (Pierce, Rockford, Ill.).
Transfection activity was expressed as RLU/mg cellular
protein.+-.SD of quadruplicate samples.
Example 27
Intracellular Distribution of RPA/DNA Polyplexes
[0215] Luciferase DNA was labeled with Label IT-Tracker.TM.
CX-Rhodamine Kit (Mirus, Madison, Wis.) according to manufacturer's
protocol. 120,000 CXCR4+U2OS cells were plated in glass-bottom dish
(MatTek P35GC-0-14-C) 24 h before the experiment. The cells were
incubated with RPA/DNA polyplexes prepared at w/w 5 (2.35 .mu.g/mL
DNA) for 3 h before adding 10 nM hCXCL12. The cells were incubated
for another 1 h before a PBS wash, fixation and imaging by Perkin
Elmer Spinning Disk confocal microscope.
Example 28
Physicochemical Characterization of RPA/DNA Polyplexes
[0216] DNA condensation ability of RPA was first compared with PEI,
RHB, and AMD3100 by EtBr exclusion assay (FIG. 21a). The
condensation curves for all three polycations displayed typical
sigmoidal shape, characteristic of DNA condensation by polycations.
At pH 7.4, a w/w ratio above 2 was required for RPA to fully
condense the DNA, which was higher than that required in case of
RHB (w/w 1) and PEI (w/w 0.5). AMD3100 has six secondary amines and
two tertiary amines and is thus, to a very limited extent, also
able to condense DNA as demonstrated by a decrease in EtBr
fluorescence by about 30%. The redox stability of RPA/DNA
polyplexes was tested by agarose gel electrophoresis after GSH
treatment. As shown in FIG. 21b, 20 mM GSH triggered DNA release
form RPA/DNA polyplexes due to the depolymerization of RPA, which
decreased its affinity to DNA.
Example 29
CXCR4 Antagonism of RPA
[0217] When CXCL12 binds to CXCR4 it induces downstream signaling
through multiple pathways, including Ras and P13 kinase. Treatment
with CXCR4 antagonists not only prevents the CXCL12-induced
downstream signaling but it also inhibits endocytosis of the
receptor (Forster, Kremmer et al. 1998; Orsini, Parent et al. 1999;
Hatse, Princen et al. 2002; Dar, Goichberg et al. 2005). To
evaluate CXCR4 antagonism by RPA and RPA/DNA, CXCR4 receptor
redistribution assay was conducted (FIG. 22). The assay used U2OS
cells stably expressing human CXCR4 receptor fused to the
N-terminus of enhanced green fluorescent protein (EGFP). The assay
monitors cellular translocation of the GFP-CXCR4 receptors in
response to stimulation with human CXCL12. Here, the
internalization of the CXCR4 receptors into endosomes in
CXCL12-stimulated cells was observed, as suggested by the punctate
distribution of the GFP fluorescence (FIG. 22b) away from the
original diffuse pattern in non-stimulated cells (FIG. 22a). To
exclude the possibility that the observed effect was caused by
nonspecific electrostatic binding of RPA to the negatively charged
binding site of the CXCR4 receptor, control polycation RHB without
AMD3100 moiety was also tested, but no CXCR4 antagonistic
properties was observed (FIG. 22f). Next, a study was done to
determine whether polyplexes themselves exhibit CXCR4 antagonism.
CXCR4 internalization was inhibited more efficiently by RPA/DNA
prepared at w/w 5 (2.5 .mu.g/mL total RPA) than at w/w 1 (FIG. 22g
and h). DNA was not fully condensed in polyplexes at w/w 1 (FIG.
21a); thus, the formulation contained only a minimum amount of free
RPA. The findings at w/w 1 thus suggest that the polyplexes
themselves may inhibit CXCR4 to some extent. Similar to RHB
polymer, no CXCR4 antagonism was observed with RHB/DNA polyplexes
(FIG. 22i), confirming that the specific CXCR4 antagonism of RPA
and RPA/DNA is due to the AMD3100 moiety in RPA.
[0218] To determine the half-inhibitory (EC50) concentrations of
RPA, the CXCR4+U2OS cells were treated with increasing
concentrations of RPA.HCl before stimulating them with human
CXCL12. AMD3100 was used as positive control. The level of CXCR4
antagonism was evaluated by quantifying the fluorescent intensity
of granules (endocytosed GFP-CXCR4) in the individual images. The
dose-response curves for AMD3100 and RPA.HCl were established based
on % CXCR4 translocation and EC50 values were calculated
accordingly (FIG. 23). Based on the results of elemental analysis
(data not shown), the equivalent AMD3100 content in RPA could be
obtained (60% weight of RPA.HCl).
Example 30
Antimetastatic Ability of RPA by Cell Invasion Assay
[0219] The CXCR4/CXCL12 axis plays a critical role in cancer
metastasis due to its function in trafficking and homing of cancer
cells to organs that express high levels of CXCL12. Blocking the
CXCR4/CXCL12 interactions with small-molecule antagonists
suppresses metastasis in a variety of cancers (Yoon, Liang et al.
2007; Liang, Zhan et al. 2012). To further confirm the CXCR4
antagonism of RPA and RPA/DNA polyplexes, the anti-metastatic
ability was evaluated by a Matrigel cell invasion assay. As shown
in FIG. 24, RPA and RPA/DNA polyplexes effectively blocked
CXCL12-mediated invasion of CXCR4+U2OS cells. Both free RPA and
RPA/DNA blocked invasion of 71-77% of cells, similar to that of
AMD3100 (75%). The DNA dose used in the experiment with the
polyplexes (1 .mu.g/mL DNA) was in the range of typical doses used
in transfection experiments. The observed decrease in cell invasion
with control RHB/DNA polyplexes was not statistically significant
(p>0.05). At the same time, the differences between RPA and
RPA/DNA polyplexes vs. untreated controls were highly significant
with P<0.001, based on non-parametric ANOVA analysis with Dunn's
multiple comparison test. The slight decrease in the number of
invaded cells with RHB treatment could also be attributed by higher
toxicity of RHB compared with RPA. The membrane damage caused by
the treatment with RHB may affect the motility of the cells and
thus decrease their ability to invade through the extracellular
matrix.
Example 31
Transfection of RPA/DNA Polyplexes
[0220] Having confirmed CXCR4 antagonism and inhibition of cancer
cell invasion of the synthesized RPA, its gene delivery capability
was evaluated (FIG. 25). A routine luciferase transfection
experiment was conducted. RPA/DNA polyplexes exhibited high in
vitro transfection efficiency that was comparable with that of
control PEI/DNA polyplexes and RHB/DNA in B16F10 and U2OS cell
lines at a DNA dose of 2.35 .mu.g/mL. It is interesting that
AMD3100 itself was able to mediate some transfection, especially in
B16F10 cells when compared with naked DNA only. As shown in FIG.
21, the partial DNA condensation is the most likely reason for the
observed transfection, which is nevertheless several orders of
magnitude below transfection of the polymers.
Example 32
Simultaneous CXCR4 Antagonism and Gene Delivery
[0221] As shown in the experiment below, the RPA/DNA polyplexes use
an alternative uptake pathway that does not require CXCR4. This is
documented by the lack of signal from RPA/DNA polyplexes with
fluorescently labeled DNA colocalized with the membrane-present
CXCR4 receptor. As shown in FIG. 26, after 3 h incubation with
RPA/DNA polyplexes, the CXCR4+U2OS cells were stimulated with
hCXCL12 and the confocal image (taken in the middle of the Z-stack)
showed more clearly that the GFP-CXCR4 receptors were mostly
presented in the cell membrane. At the same time, labeled RPA/DNA
polyplexes (red fluorescence) were shown internalized into the
cells and not bound with the membrane-localized CXCR4 receptors.
While not being bound to a theory, it is believed that the
polyplexes are internalized through a different endocytic pathway
that does not involve CXCR4.
[0222] To further study if CXCR4 inhibition affects the gene
delivery function of RPA/DNA polyplexes, AMD3100 was used to block
the cell surface CXCR4 receptors before conducting transfection. No
significant difference was observed in transfection efficiency in
CXCR4+U2OS cells either with or without CXCL12 simulation (FIG.
27). The results suggest that the uptake of RPA/DNA polyplexes is
not dependent on binding with CXCR4 receptors.
[0223] It has been reported that CXCL12 and phorbol esters trigger
CXCR4 internalization through entirely different uptake pathway
(Signoret, Oldridge et al. 1997). AMD3100 only inhibits
CXCL12-induced CXCR4 endocytosis, but does not affect phorbol
ester-induced receptor internalization (Hatse, Princen et al.
2002). Here, CXCR4+U2OS cells were treated with RPA/DNA polyplexes
or AMD3100 before incubation with 100 ng/mL of phorbol 12-myristate
13-acetate (PMA) and the cells were imaged by fluorescence
microscope (FIG. 28). The results show that internalization of
CXCR4 receptor was not inhibited by RPA/DNA polyplexes similarly to
AMD3100 when the cells were stimulated with phorbol myristate
(PMA).
[0224] To further confirm if CXCR4 was involved in the transfection
process of the polyplexes, the effect of PMA treatment was
evaluated on transfection activity of RPA/DNA polyplexes. The
experiment was conducted using the same conditions as described
above, except that the cells were co-incubated with polyplexes and
100 ng/mL of PMA in serum-free medium for 4 h. No cytotoxicity of
PMA was observed under the used experimental conditions. The
results show that PMA did not enhance transfection of RPA
polyplexes despite its ability to trigger internalization of the
CXCR4 receptor by an alternative pathway from CXCL12 (FIG. 29).
This finding provides further support for the lack of involvement
of the CXCR4 receptor in transfection activity of RPA/DNA.
[0225] FIG. 30 also shows concurrent CXCR4 inhibition and
transfection with RPA/DNA polyplexes. CXCR4+U2OS cells were plated
in black 96-well plate with optical bottom 24 h before the
experiment at a seeding density of 8,000 cells per well. The cells
were incubated with RPA/DNA polyplexes prepared at w/w 5, 10 and 15
(2.35 .mu.g/mL DNA) or RHB/DNA polyplexes (negative control)
prepared at w/w 5 in serum-free media. The polyplexes were removed
after 4 h incubation and the cells were continued to grow in fresh
complete culture media. The luciferase transfection was measured
after 24 h. The CXCR4 antagonism was evaluated in the same cells at
0 h and 24 h after polyplex incubation by stimulating the cells
with 10 nM hCXCL12. The results show that RPA/DNA polyplexes
simultaneously inhibited CXCR4 (FIG. 30a) and mediated effective
transfection (FIG. 30b). Additionally, RPA/DNA polyplexes
maintained their CXCR4 inhibiting properties even after 24 hours
(although the inhibition was not as complete as in the early time
point as judged by the reappearance of the punctate fluorescence
distribution of the CXCR4 receptor at 24 h in FIG. 30a). In
contrast, the negative control (RHB/DNA polyplexes) shows no CXCR4
antagonism at any time point, while mediating similar transfection
activity as RPA/DNA. These findings support the mechanism of action
in which the free RPA inhibits CXCR4 while the rest of the RPA/DNA
polyplex formulation participates in transfection, most likely
through nonspecific charge-mediated uptake.
Example 33
Breast Cancer Study Design
[0226] A panel of pH-sensitive biodegradable block copolymers
(CopCX), such as P(AMD-CBA)DNA polyplex with CXCR4 antagonistic
properties can be synthesized. Cytotoxicity, CXCR4 antagonism,
CXCR4 receptor binding specificity and gene silencing capability in
mouse breast cancer cells 4T1.Luc can be evaluated in vitro using
CopCX/siRNA nanocarriers. The best performing nanocarriers can be
used to identify therapeutic siRNA that will provide maximum
synergy with CXCR4 inhibition in anticancer activity and in
inhibition of breast cancer cell invasiveness in vitro. Therapeutic
siRNA candidates will include, for example, akt2, HER2, survivin,
PARP, and STAT3. The following controls will be used in all in
vitro studies: FDA-approved CXCR4 antagonist AMD3100, polycation
with no CXCR4 inhibiting activity (poly(ethyleneimine) (PEI)), and
scrambled control siRNA. The best performing CopCX/siRNA
nanocarrier will be advanced to in vivo studies in metastatic
breast cancer model 4T1.Luc. Two experimental setups (with and
without primary tumor removal) will be used to test the anti-cancer
and anti-metastatic activity of CopCX/siRNA in vivo. The mice will
be treated with multiple intravenous doses of CopCX/siRNA. Control
animals will be treated using the same administration regimen with
CopCX/scrambled siRNA, free CopCX, PEI/siRNA, free PEI, and saline.
Tumor growth and metastasis will be monitored by bioluminescence
imaging. Antitumor efficacy will be evaluated using tumor growth
delay and inverse of tumor growth inhibition analysis.
[0227] For example, two cyclam monomers with different side chains
can be synthesized and used for the synthesis of CopCX. Stabilizing
poly(ethylene glycol) (PEG) block can be conjugated via a
reversible linkage to take advantage of acidic tumor
microenvironment for tumor-selective PEG removal.
Structure-activity relationships (SAR) studies with the assembled
CopCX/siRNA nanocarriers will identify those with maximum CXCR4
antagonism and siRNA silencing activity in mouse mammary carcinoma
cells stably expressing luciferase (4T1.Luc).
[0228] While not being bound to a particular theory, it is believed
that CXCR4 antagonism of CopCX will depend on the surface
presentation and accessibility of the cyclam moieties and on the
molecular weight of the polymers. Thus, CopCX with several
different molecular weights (4-20 kDa) using the two monomers with
different side chains can be tested for cyclam accessibility. The
cationic block can be prepared first with terminal acrylate groups
for subsequent PEG 2 kDa conjugation as previously shown (Chen, J.,
C. Wu, and D. Oupicky, "Bioreducible Hyperbranched Poly(amido
amine)s for Gene Delivery." Biomacromolecules, 2009, 10(10): p.
2921-2927; Wu et al., "2A(2)+BB' B'' approach to hyperbranched
poly(amino ester)s." Macromolecules, 2005, 38(13): p. 5519-5525).
The cyclam Boc- protecting groups will be removed and PEG will be
conjugated using thiol addition to the terminal acrylate via a
linker containing either hydrazone or orthoester groups. Both
linkers are well established as rapidly degradable in mildly acidic
conditions. Alternatively, PEG may be grafted directly to CopCX
backbone by reaction with the cyclam amines to achieve .about.1-2
PEG/CopCX substitution using the same X linkers.
[0229] IC.sub.10 of CopCX in 4T1.Luc cells can be obtained in MTS
assays and used to obtained information to establish non-toxic
working concentration range (defined as concentrations
<IC.sub.10) for the subsequent experiments. CXCR4 antagonism of
CopCX will be studied using SDF-1-mediated CXCR4 receptor
redistribution using a commercially available assay (Li et al.,
"Dual-Function CXCR4 Antagonist Polyplexes To Deliver Gene Therapy
and Inhibit Cancer Cell Invasion." Angew. Chem. Int. Ed. Engl.,
2012). Specificity of CopCX binding to CXCR4 receptor will be then
evaluated from the ability of CopCX to displace bound anti-CXCR4
mAb using flow cytometry (Khan et al., "Fluorescent CXCR4 chemokine
receptor antagonists: metal activated binding." Chem. Commun.,
2007(4): p. 416-418; Nimmagadda e tal., "Molecular Imaging of CXCR4
Receptor Expression in Human Cancer Xenografts with [Cu-64]AMD3100
Positron Emission Tomography." Cancer Res., 2010, 70(10): p.
3935-3944). A negative control for nonspecific background of
isotype control mAb will be used. The silencing activity of the
CopCX/siRNA nanocarriers will be evaluated using anti-Luc siRNA in
4T1.Luc cells using previously published study (Manickam et al.,
"Effect of innate glutathione levels on activity of
redox-responsive gene delivery vectors." J. Controlled Rel., 2010,
141(1): p. 77-84). Simultaneous siRNA transfection and CXCR4
antagonism of the best CopCX will be confirmed and the composition
of CopCX/siRNA nanocarriers will be optimized in experiments that
will evaluate siRNA silencing and CXCR4 antagonism in 4T1.Luc.
CopCX will be rank-ordered based on their silencing and CXCR4
inhibition activities.
[0230] Anticancer activity of CopCX/siRNA nanocarriers formulated
with the proposed siRNAs will be determined by MTS assay. The goal
will be to identify active dose ranges of the nanocarriers and to
adjust relative content of CopCX and siRNA to maximize the
combination effect with CXCR4 inhibition. The extent and
specificity of silencing of individual siRNAs will be verified by
western blot.
[0231] To determine the maximum tolerated dose (MTD), CopCX will be
administered intravenously (i.v.) to tumor-free mice at increasing
doses. The MTD will be defined as the dose which causes less than
20% body weight loss with an overall projected lethality under 10%.
At defined endpoints (morbidity, 20% weight loss, or tissue
harvest), mice will be humanely euthanized with appropriate tissues
(liver, kidneys, lungs, heart, spleen) and serum harvested for
further analyses: histopathology, cytokine induction (TNF, IL-6,
IFN-.alpha.), and blood levels of the liver enzymes alanine
aminotransferase and aspartate aminotransferase.
[0232] The antitumor activity of CopCX/siRNA nanocarriers against
4T1.Luc tumor will be tested in two types of experiments. First,
orthotopic 4T1.Luc tumors will be established by mammary fat pad
cell injection in Balb/c mice (female, 7-8 wks, 22-24 g) using
previously published protocols (Lelekakis et al., "A novel
orthotopic model of breast cancer metastasis to bone." Clin Exp
Metastasis, 1999, 17(2): p. 163-170; Aslakson, C. J. and F. R.
Miller, "Selective events in the metastatic process defined by
analysis of the sequential dissemination of subpopulations of a
mouse mammary tumor." Cancer Res, 1992, 52(6): p. 1399-405;
Olkhanud et al., "Breast Cancer Lung Metastasis Requires Expression
of Chemokine Receptor CCR4 and Regulatory T Cells." Cancer Res.,
2009, 69(14): p. 5996-6004; Tao et al., "Imagable 4T1 model for the
study of late stage breast cancer." BMC Cancer, 2008, 8(1): p.
228). CopCX/siRNA nanocarriers will be prepared with the best
performing siRNA as identified above. The treatment can commence,
e.g., 3 days after cell injection. This experimental setup will
allow to evaluate activity of the nanocarriers against primary
tumor and in preventing metastatic dissemination. In the second
experimental setup, the primary tumors will be established and then
surgically removed by en-bloc excision when they are upstaged to
.about.500 mg and metastases are detected in the lung by BLI.
Treatment with CopCX will commence after primary tumor removal,
which will help to evaluate activity of the nanocarriers against
established metastasis after primary tumor removal.
[0233] In both of the above types of experiments, the mice will be
formally randomized and treated every two to five days (3-5 courses
in total) with i.v. injection of three different doses of
CopCX/siRNA nanocarriers using a dose range determined from the MTD
study. Control animals will be treated using the same
administration regimen with (i) CopCX/siRNA nanocarrier prepared
with scrambled siRNA control, (ii) free CopCX, (iii) PEI/siRNA
nanocarrier with therapeutic siRNA, (iv) PEI, and (v) saline. Total
of about 200 Balb/c mice will be used: [3 doses*5 mice/group*5
treatments]+[3 doses*7 mice/group*5 treatments=180+20 for
experimental complications and untreated controls =200]. Group size
can be increased to, e.g., 7 mice in the second type of
experimental setup to account for primary tumor regrowth and
complications due to tumor removal surgery. Animal weight, tumor
growth and total tumor load will be monitored, and growth curves
will be constructed from the bioluminescence intensity of the
metastatic lesions and by measuring the size of the primary tumors
by calipers. All animals in the study will be necropsied and
remaining tumor (if any) and liver, spleen, lung, and adjacent
lymph nodes will be harvested. Tissue sections will be used for (i)
H&E staining and histopathological evaluations, (ii)
immunohistochemical (IHC) staining with anti-Ki-67 to detect
proliferating tumor cells, (iii) TUNEL assay and IHC of activated
caspase-3 to detect cells undergoing apoptosis, and (iv) counting
of metastasis nodules in tissue sections. The specificity of siRNA
silencing will be verified in tumor homogenates by western blot.
Antitumor efficacy of the CopCX/siRNA nanocarriers will be analyzed
using the following quantitative endpoints: (i) tumor growth delay
(T-C), where T is median days for the treatment group to reach a
pre-determined size, and C is median days for the control group
tumors to reach the same size (tumor-free survivors are excluded
and tabulated separately); (ii) %T/C (inverse of tumor growth
inhibition), where treated/control tumors are measured when control
group tumors reach .about.700-1200 mg. The median for each group is
determined as a non-quantitative measure of antitumor
effectiveness. T/C<42% is considered significant activity by the
NCI; T/C<10% is highly significant activity. The Kaplan-Meier
method will be used to analyze the survival curves.
Example 34
Lung Cancer (LCa) Study Design
[0234] Many preclinical and clinical studies observed significant
correlation between expression of CXCR4 chemokine receptor and
metastasis in LCa. CXCR4 expression is associated with poor
survival and aggressive type of cancer both in small cell lung
cancer (SCLC) and nonsmall cell lung cancer (NSCLC). Consistent
with the seed-and-soil hypothesis of metastatic dissemination
(Burger, J. A. and T. J. Kipps, "CXCR4: a key receptor in the
crosstalk between tumor cells and their microenvironment." Blood,
(2006) 107: 1761-1767), LCa cells utilize CXCR4 and its ligand
CXCL12 to metastasize to distant sites. Thus as expected, the
primary sites of LCa metastasis (lymph nodes, bone, liver) are also
sites with high levels of CXCL12 expression (Gangadhar, T., S.
Nandi, and R. Salgia, "The role of chemokine receptor CXCR4 in lung
cancer." Cancer Biology & Therapy, (2010) 9: 409-416).
CXCR4/CXCL12 axis regulates survival, proliferation, migration and
invasion of LCa cells by activating signaling pathways such as MAPK
and PI3K pathways (Burger et al. , "Functional expression of CXCR4
(CD184) on small-cell lung cancer cells mediates migration,
integrin activation, and adhesion to stromal cells." Oncogene,
(2003) 22: 8093-8101).
[0235] COPCX formulations with anti-EGFR siRNA will be tested. Four
other candidate siRNAs (akt2, survivin, PARP, and STAT3) that have
been validated as promising in LCa treatment will be tested
too.
[0236] Lewis lung carcinoma stably expressing luciferase (LL/2-luc)
will be used to test the anticancer and anti-metastatic activity of
COPCX/siRNA in vivo. LL/2-luc has the capability to spontaneously
metastasize after subcutaneous and intravenous (i.v.)
administration in SCID-bg mice. Tumor growth and metastatic spread
can be easily monitored by whole-body bioluminescence imaging
(BLI). BLI will be advantageously used for longitudal noninvasive
studies of the COPCX activity. Maximum tolerated dose (MTD) of
COPCX will be determined using 6 mice before testing anticancer
activity.
[0237] Activity of the optimized COPCX/siRNA against subcutaneously
implanted LL/2-luc tumor and in preventing its metastatic
dissemination will be tested in SCID-bg mice. The mice will be
treated every two to five days (3-5 courses in total) with i.v.
injection of three different doses of COPCX/siRNA using a dose
range determined from the MTD study. Control animals will be
treated using the same administration regimen with (i) COPCX/siRNA
prepared with scrambled siRNA control, (ii) free CXLip, (iii)
DOTAP/siRNA, (iv) saline. Seventy five SCID-bg mice will be used:
(3 doses.times..quadrature.5 mice/group.times..quadrature.4
treatments)=60+15 for experimental complications and untreated
controls. Animal weight, tumor growth and total tumor load will be
monitored, and growth curves will be constructed from the
bioluminescence imaging (BLI) intensity of the metastatic lesions
and by measuring the size of the primary tumors by calipers.
Animals will be necropsied and tissue sections will be used for (i)
H&E staining and histopathological evaluations, (ii)
immunohistochemical (IHC) staining with anti-Ki-67 to detect
proliferating tumor cells, (iii) TUNEL assay and
immunohistochemistry (IHC) of activated caspase-3 to detect cells
undergoing apoptosis, and (iv) counting of metastasis nodules in
tissue sections. The specificity of siRNA silencing will be
verified in tumor homogenates by western blot. Antitumor efficacy
of the COPCX/siRNA nanoparticles will be analyzed using the
following quantitative endpoints: (i) tumor growth delay (T-C),
where T is median days for the treatment group to reach a
predetermined size, and C is median days for the control group
tumors to reach the same size (tumor-free survivors are excluded
and tabulated separately); (ii) %T/C (inverse of tumor growth
inhibition), where treated/control tumors are measured when control
group tumors reach .about.700-1200 mg. The median for each group is
determined as a non-quantitative measure of antitumor
effectiveness. T/C <42% is considered significant activity by
the National Cancer Institute (NCI); T/C <10% is highly
significant activity.
* * * * *