U.S. patent application number 13/610150 was filed with the patent office on 2013-08-22 for amphiphilic polymers and methods of use thereof.
This patent application is currently assigned to Massachusetts Institute of Technology. The applicant listed for this patent is Clark K. Colton, Robert Fisher, Jayant Kumar, Rajesh Kumar, Virinder S. Parmar, Arthur Watterson. Invention is credited to Clark K. Colton, Robert Fisher, Jayant Kumar, Rajesh Kumar, Virinder S. Parmar, Arthur Watterson.
Application Number | 20130216480 13/610150 |
Document ID | / |
Family ID | 37115837 |
Filed Date | 2013-08-22 |
United States Patent
Application |
20130216480 |
Kind Code |
A1 |
Colton; Clark K. ; et
al. |
August 22, 2013 |
AMPHIPHILIC POLYMERS AND METHODS OF USE THEREOF
Abstract
The present invention relates to amphiphilic polymers, and
micelles and compositions comprising the same, and their use in a
variety of biological settings, including imaging, targeting drugs,
or a combination thereof for diagnostic and therapeutic
purposes.
Inventors: |
Colton; Clark K.; (Newton,
MA) ; Watterson; Arthur; (Lowell, MA) ; Kumar;
Rajesh; (Dracut, MA) ; Parmar; Virinder S.;
(Lowell, MA) ; Fisher; Robert; (West Roxbury,
MA) ; Kumar; Jayant; (Westford, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Colton; Clark K.
Watterson; Arthur
Kumar; Rajesh
Parmar; Virinder S.
Fisher; Robert
Kumar; Jayant |
Newton
Lowell
Dracut
Lowell
West Roxbury
Westford |
MA
MA
MA
MA
MA
MA |
US
US
US
US
US
US |
|
|
Assignee: |
Massachusetts Institute of
Technology
Cambridge
MA
|
Family ID: |
37115837 |
Appl. No.: |
13/610150 |
Filed: |
September 11, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11405012 |
Apr 17, 2006 |
8349991 |
|
|
13610150 |
|
|
|
|
60672533 |
Apr 19, 2005 |
|
|
|
60672856 |
Apr 20, 2005 |
|
|
|
60732633 |
Nov 3, 2005 |
|
|
|
Current U.S.
Class: |
424/1.85 ;
424/9.37; 435/34; 435/6.1; 435/7.23 |
Current CPC
Class: |
A61K 49/0043 20130101;
A61K 49/126 20130101; A61K 38/10 20130101; C08G 63/672 20130101;
A61K 51/06 20130101; C08G 2261/126 20130101; A61K 49/0054 20130101;
A61K 49/0032 20130101; G01N 33/57492 20130101; A61K 49/0002
20130101; A61K 47/6907 20170801; A61K 49/0082 20130101; A61K
49/0041 20130101; A61K 9/51 20130101; A61K 47/60 20170801; A61K
31/785 20130101; A61K 51/1237 20130101; C08G 63/6854 20130101; A61K
47/593 20170801; C08G 73/028 20130101; A61K 49/1818 20130101; C08G
69/40 20130101; C08G 63/668 20130101; A61K 49/14 20130101; A61K
49/0056 20130101; C08L 79/02 20130101; C08G 63/6826 20130101; C08G
63/914 20130101; B82Y 5/00 20130101; C08L 77/00 20130101; A61K
49/1809 20130101; G01N 33/5091 20130101 |
Class at
Publication: |
424/1.85 ;
424/9.37; 435/7.23; 435/34; 435/6.1 |
International
Class: |
A61K 49/12 20060101
A61K049/12; G01N 33/574 20060101 G01N033/574; G01N 33/50 20060101
G01N033/50; A61K 51/06 20060101 A61K051/06 |
Claims
1-166. (canceled)
167. A method of imaging a cell, the method comprising contacting a
cell with an amphiphilic polymer characterized by the structure of
the general formula I: ##STR00051## wherein R is a hydroxyl,
O-alkyl, O-Acyl, O-Activating group, SH, S-alkyl, or an acid
activating group including halogen, O-vinyl, O-allyl, O-aryl,
OCOalkyl, OCOaryl, OCH.sub.2CF.sub.3, NH.sub.2, a fluorochrome, an
indole-containing compound, an antibody or antibody fragment, a
peptide, an oligonucleotide, a drug, a ligand for a biological
target, an immunoconjugate, a chemomimetic functional group, a
glycolipid, a labelling agent, an enzyme, a metal ion chelate, an
enzyme cofactor, a cytotoxic compound, a growth factor, a hormone,
a cytokine, a toxin, a prodrug, an antimetabolite, a microtubule
inhibitor, a radioactive material, a targeting moiety; R' is OH,
NH.sub.2, SH; each R.sub.1 group is, independently, H, ##STR00052##
a fluorochrome, an indole-containing compound, an antibody or
antibody fragment, a peptide, an oligonucleotide, a drug, a ligand
for a biological target, an immunoconjugate, a chemomimetic
functional group, a glycolipid, a labelling agent, an enzyme, a
metal ion chelate, an enzyme cofactor, a cytotoxic compound, a
growth factor, a hormone, a cytokine, a toxin, a prodrug, an
antimetabolite, a microtubule inhibitor, a radioactive material, a
perfluorocarbon, a perfluorocarbon-R.sub.4, a
perfluorocarbon-OR.sub.4, ##STR00053## each R.sub.2 group is,
independently, a fluorochrome, an indole-containing compound, an
antibody or antibody fragment, a peptide, an oligonucleotide, a
drug, a ligand for a biological target, an immunoconjugate, a
chemomimetic functional group, a glycolipid, a labelling agent, an
enzyme, a metal ion chelate, an enzyme cofactor, a cytotoxic
compound, a growth factor, a hormone, a cytokine, a toxin, a
prodrug, an antimetabolite, a microtubule inhibitor, a radioactive
material, a perfluorocarbon, a perfluorocarbon-R.sub.4, a
perfluorocarbon-OR.sub.4, ##STR00054## each R.sub.3 group is,
independently, ##STR00055## a hydrogen, a hydroxyl, O-alkyl, SH,
S-alkyl, or an acid activating group including halogen, O-vinyl,
O-allyl, O-aryl, OCOalkyl, OCOaryl, OCH.sub.2CF.sub.3, NH.sub.2, a
fluorochrome, an indole-containing compound, an antibody or
antibody fragment, a peptide, an oligonucleotide, a drug, a ligand
for a biological target, an immunoconjugate, a chemomimetic
functional group, a glycolipid, a labelling agent, an enzyme, a
metal ion chelate, an enzyme cofactor, a cytotoxic compound, a
growth factor, a hormone, a cytokine, a toxin, a prodrug, an
antimetabolite, a microtubule inhibitor, a radioactive material, a
targeting moiety; each R.sub.4 group is, independently, an alkyl
group, an alkylene group, a carboxylate group, a carboxylic acid
group, an amino group, an ammonium group, an alkoxyl group, a
hydroxyl group or another nitrogen, oxygen or sulfur-containing
group; each A group is, independently, O, NH, S, a fluorochrome,
##STR00056## an indole-containing compound, an antibody or antibody
fragment, a peptide, an oligonucleotide, a drug, a ligand for a
biological target, an immunoconjugate, a chemomimetic functional
group, a glycolipid, a labeling agent, an enzyme, a metal ion
chelate, an enzyme cofactor, a cytotoxic compound, a growth factor,
a hormone, a cytokine, a toxin, a prodrug, an antimetabolite, a
microtubule inhibitor, a radioactive material, a targeting moiety,
an acyl group, an aryl group, a linear or branched alkenyl group, a
linear or branched alkyl group, wherein said alkyl, alkenyl or aryl
group is substituted with a perfluorocarbon,
perfluorocarbon-R.sub.4, perfluorocarbon-OR.sub.4,
perfluorocarbon-OR.sub.4, or ##STR00057## n, m, p, p' and x are
integers; and q is an integer between 0-10, wherein the amphiphilic
polymer further comprises a targeting agent, the method further
comprising imaging said cell, whereby said polymer enables the
imaging of said cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 60/672,533, filed Apr. 19, 2005, U.S.
Provisional Application Ser. No. 60/672,856, filed Apr. 20, 2005
and U.S. Provisional Application Ser. No. 60/732,633 filed Nov. 3,
2005, all of which are hereby incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0002] This invention provides amphiphilic polymers, processes for
producing the same and methods of use thereof. Polymers of this
invention may be used in diagnostics and imaging, as well as
treatments of diseases and disorders including cancer and gene
therapy applications.
BACKGROUND OF THE INVENTION
[0003] One of the most fundamental limitations to reducing
mortality due to a number of diseases, including cancer, is the
fact that current medical imaging techniques, such as CT and MRI,
provide detailed anatomical snapshots of the body but fail to
provide accurate, basic information necessary to manage the
patient's disease optimally.
[0004] The limitations are manifested in several ways, such as for
example in cancer: (1) Small primary tumors go undetected. Even
under the best conditions, tumors smaller than 2 mm (roughly
500,000 cells) cannot be seen. (2) Metastatic disease is grossly
underdiagnosed, and patients with negative scans for metastases at
initial presentation routinely go on to develop, and die, from
metastatic cancer. (3) Treatment response to therapy is poorly
measured. "Measurable disease" is absent after surgical excision of
many tumors. The standard of care is to blindly treat with
chemotherapy selected by convention using prior retrospective
studies and to consider this treatment a success or failure only in
retrospect (e.g., failure is when a relapse occurs in less than 5
years). Residual metastatic disease can expand undetected. When
metastatic disease leads to a tumor that is large enough to be
detected (stage 4), it is often too late for anything but a modest
extension in patient lifetime with available treatments.
[0005] How can conventional imaging be so far off the mark? One
reason is that conventional radiologic approaches produce their
images based upon bulk structural and anatomical features of the
tissue. For example, the image displayed in MRI is that of protons
in water or fat as modified by relative concentration and
environment. The degree to which, for example, a tumor can be
visualized on conventional CT or MRI is merely a function of the
ability of that tumor to differentially scatter, absorb, or emit
radiation as compared to the surrounding tissue and inherent
background noise. It is not surprising that this signal has little
sensitivity and specificity for the detection of a tumor.
[0006] The signal can be enhanced, however, through the use of
targeted probes. Supramolecular assemblies that can be made to form
nanospherical structures for carrying contrast agent, such as
liposomes and polymer micelles, offer potential for improving
various imaging modalities. Results with such liposomes, however,
have essentially been disappointing.
[0007] Moreover, equally frustrating is a lack of versatile
delivery systems for therapeutics, targeted delivery, and a
reliable means of proper dosing and tissue distribution of the
therapeutic.
SUMMARY OF THE INVENTION
[0008] The invention provides, in one embodiment, an amphiphilic
polymer, characterized by the structure of the general formula
I:
##STR00001##
wherein [0009] R is a hydroxyl (OH), O-alkyl, O-Acyl, O-Activating
group, SH, S-alkyl, or an acid activating group such as halogen
(Cl, Br, I), O-vinyl, O-allyl, O-aryl, OCOalkyl, OCOaryl,
OCH.sub.2CF.sub.3, NH.sub.2, a fluorochrome, an indole-containing
compound, an antibody or antibody fragment, a peptide, an
oligonucleotide, a drug, a ligand for a biological target, an
immunoconjugate, a chemomimetic functional group, a glycolipid, a
labelling agent, an enzyme, a metal ion chelate, an enzyme
cofactor, a cytotoxic compound, a growth factor, a hormone, a
cytokine, a toxin, a prodrug, an antimetabolite, a microtubule
inhibitor, a radioactive material, a targeting moiety; [0010] R' is
OH, NH.sub.2, SH; [0011] each R.sub.1 group is, independently,
H,
##STR00002##
[0011] a fluorochrome, an indole-containing compound, an antibody
or antibody fragment, a peptide, an oligonucleotide, a drug, a
ligand for a biological target, an immunoconjugate, a chemomimetic
functional group, a glycolipid, a labelling agent, an enzyme, a
metal ion chelate, an enzyme cofactor, a cytotoxic compound, a
growth factor, a hormone, a cytokine, a toxin, a prodrug, an
antimetabolite, a microtubule inhibitor, a radioactive material, a
perfluorocarbon, a perfluorocarbon-R.sub.4, a
perfluorocarbon-OR.sub.4,
##STR00003## [0012] each R.sub.2 group is, independently, a
fluorochrome, an indole-containing compound, an antibody or
antibody fragment, a peptide, an oligonucleotide, a drug, a ligand
for a biological target, an immunoconjugate, a chemomimetic
functional group, a glycolipid, a labelling agent, an enzyme, a
metal ion chelate, an enzyme cofactor, a cytotoxic compound, a
growth factor, a hormone, a cytokine, a toxin, a prodrug, an
antimetabolite, a microtubule inhibitor, a radioactive material, a
perfluorocarbon, a perfluorocarbon-R.sub.4, a
perfluorocarbon-OR.sub.4,
[0012] ##STR00004## [0013] each R.sub.3 group is,
independently,
##STR00005##
[0013] a hydrogen, a hydroxyl (OH), O-alkyl, SH, S-alkyl, or an
acid activating group such as halogen (Cl, Br, I), O-vinyl,
O-allyl, O-aryl, OCOalkyl, OCOaryl, OCH.sub.2CF.sub.3, NH.sub.2, a
fluorochrome, an indole-containing compound, an antibody or
antibody fragment, a peptide, an oligonucleotide, a drug, a ligand
for a biological target, an immunoconjugate, a chemomimetic
functional group, a glycolipid, a labelling agent, an enzyme, a
metal ion chelate, an enzyme cofactor, a cytotoxic compound, a
growth factor, a hormone, a cytokine, a toxin, a prodrug, an
antimetabolite, a microtubule inhibitor, a radioactive material, a
targeting moiety; [0014] each R.sub.4 group is, independently, an
alkyl group, an alkylene group, a carboxylate group, a carboxylic
acid group, an amino group, an ammonium group, an alkoxyl group, a
hydroxyl group or another nitrogen, oxygen or sulfur-containing
group; [0015] each A group is, independently, O, NH, S, a
fluorochrome,
##STR00006##
[0015] an indole-containing compound, an antibody or antibody
fragment, a peptide, an oligonucleotide, a drug, a ligand for a
biological target, an immunoconjugate, a chemomimetic functional
group, a glycolipid, a labeling agent, an enzyme, a metal ion
chelate, an enzyme cofactor, a cytotoxic compound, a growth factor,
a hormone, a cytokine, a toxin, a prodrug, an antimetabolite, a
microtubule inhibitor, a radioactive material, a targeting moiety,
an acyl group, an aryl group, a linear or branched alkenyl group, a
linear or branched alkyl group, wherein said alkyl, alkenyl or aryl
group is substituted with a perfluorocarbon,
perfluorocarbon-R.sub.4, perfluorocarbon-OR.sub.4,
perfluorocarbon-OR.sub.4, or
##STR00007## [0016] n, m, p, p' and x are integers; and [0017] q is
an integer between 0-10.
[0018] In another embodiment, this invention provides a polymer is
characterized by the structure of the general formula II:
##STR00008##
wherein
[0019] R'=OH, NH.sub.2, SH;
[0020] R=OH, OAlkyl, OAryl, OAcyl, OActivating group;
[0021] R.sub.1 and R.sub.3 are H; and
[0022] A=O, NH, S.
[0023] In another embodiment, this invention provides an
amphiphilic polymer, characterized by the structure of the general
formula III:
##STR00009##
wherein [0024] each R group is, independently: a fluorochrome, an
indole-containing compound, an antibody or antibody fragment, a
peptide, an oligonucleotide, a drug, a ligand for a biological
target, an immunoconjugate, a chemomimetic functional group, a
glycolipid, a labelling agent, an enzyme, a metal ion chelate, an
enzyme cofactor, a cytotoxic compound, a growth factor, a hormone,
a cytokine, a toxin, a prodrug, an antimetabolite, a microtubule
inhibitor, a radioactive material, a targeting moiety, an acyl
group, an aryl group, a linear or branched alkenyl group, a linear
or branched alkyl group, wherein said alkyl, alkenyl or aryl group
is substituted with a perfluorocarbon, perfluorocarbon-R.sub.4,
perfluorocarbon-OR.sub.4, or
[0024] ##STR00010## [0025] each R' group is, independently, a
hydrogen, a hydroxyl (OH), O-alkyl, SH, S-alkyl, or an acid
activating group such as halogen (Cl, Br, I), O-vinyl, O-allyl,
O-aryl, OCOalkyl, OCOaryl, OCH.sub.2CF.sub.3NH.sub.2, SH, an acyl
group, a fluorochrome, an indole-containing compound, an antibody
or antibody fragment, a peptide, an oligonucleotide, a drug, a
ligand for a biological target, an immunoconjugate, a chemomimetic
functional group, a glycolipid, a labelling agent, an enzyme, a
metal ion chelate, an enzyme cofactor, a cytotoxic compound, a
growth factor, a hormone, a cytokine, a toxin, a prodrug, an
antimetabolite, a microtubule inhibitor, a radioactive material, a
targeting moiety, or
##STR00011##
[0025] wherein [0026] R''' is a hydroxyl group, an alkoxyl group or
a primary or secondary amino group, O activating group, SH and
S-alkyl; [0027] R.sub.4 is independently an alkyl group, an
alkylene group, a carboxylate group, a carboxylic acid group, and
amino group, an ammonium group, an alkoxyl group, a hydroxyl group
or another nitrogen, oxygen or sulfur-containing group, a halogen;
[0028] A is a fluorochrome, an indole-containing compound, an
antibody or antibody fragment, a peptide, an oligonucleotide, a
drug, a ligand for a biological target, an immunoconjugate, a
chemomimetic functional group, a glycolipid, a labeling agent, an
enzyme, a metal ion chelate, an enzyme cofactor, a cytotoxic
compound, a growth factor, a hormone, a cytokine, a toxin, a
prodrug, an antimetabolite, a microtubule inhibitor, a radioactive
material, a targeting moiety, an acyl group, an aryl group, a
linear or branched alkenyl group, a linear or branched alkyl group,
wherein said alkyl, alkenyl or aryl group is substituted with a
perfluorocarbon, perfluorocarbon-R.sub.4, perfluorocarbon-OR.sub.4,
or
[0028] ##STR00012## [0029] p and p' are integers; [0030] n is at
least 1; and [0031] m is at least 1.
[0032] In another embodiment, this invention provides an
amphiphilic polymer, characterized by the structure of the general
formula IV:
##STR00013##
wherein [0033] each R group, independently, is a hydroxyl (OH),
OCH, CF.sub.3, NH.sub.2, SH, S, a fluorochrome, an
indole-containing compound, an antibody or antibody fragment, a
peptide, an oligonucleotide, a drug, a ligand for a biological
target, an immunoconjugate, a chemomimetic functional group, a
glycolipid, a labelling agent, an enzyme, a metal ion chelate, an
enzyme cofactor, a cytotoxic compound, a growth factor, a hormone,
a cytokine, a toxin, a prodrug, an antimetabolite, a microtubule
inhibitor, a radioactive material, a targeting moiety, a halogen,
an aryl group, a linear or branched alkenyl group, a linear or
branched alkyl group, wherein said alkyl, alkenyl or aryl group is
substituted with a perfluorocarbon, perfluorocarbon-R.sub.4,
perfluorocarbon-OR.sub.4, or
[0033] ##STR00014## [0034] R.sub.4 is independently an alkyl group,
an alkylene group, a carboxylate group, a carboxylic acid group,
and amino group, an ammonium group, an alkoxyl group, a hydroxyl
group or another nitrogen, oxygen or sulfur-containing group [0035]
B or B' is, independently: alkyl, substituted alkyl, aryl,
substituted aryl, OH, NH.sub.2, OR, NHR; [0036] x=0-6; [0037]
y=0-6; [0038] p, p' are integers; [0039] n is at least 1; and
[0040] m is at least 1.
[0041] In another embodiment, this invention provides an
amphiphilic polymer, characterized by the structure of the general
formula V:
##STR00015##
wherein [0042] R is a hydroxyl (OH), O-alkyl, SH, S-alkyl, or an
acid activating group such as halogen (Cl, Br, I), O-vinyl,
O-allyl, O-aryl, OCOalkyl, OCOaryl, OCH.sub.2CF.sub.3NH.sub.2, NH,
SH, an acyl group, a fluorochrome, an indole-containing compound,
an antibody or antibody fragment, a peptide, an oligonucleotide, a
drug, a ligand for a biological target, an immunoconjugate, a
chemomimetic functional group, a glycolipid, a labelling agent, an
enzyme, a metal ion chelate, an enzyme cofactor, a cytotoxic
compound, a growth factor, a hormone, a cytokine, a toxin, a
prodrug, an antimetabolite, a microtubule inhibitor, a radioactive
material, a targeting moiety, an aryl group, a linear or branched
alkenyl group, a linear or branched alkyl group, wherein said
alkyl, alkenyl or aryl group is substituted with a perfluorocarbon,
perfluorocarbon-R.sub.4, perfluorocarbon-OR.sub.4, or
[0042] ##STR00016## [0043] R.sub.4 is independently an alkyl group,
an alkylene group, a carboxylate group, a carboxylic acid group,
and amino group, an ammonium group, an alkoxyl group, a hydroxyl
group or another nitrogen, oxygen or sulfur-containing group [0044]
A is, independently: O, S or NH [0045] B or B' is, independently:
alkyl, substituted alkyl, aryl, substituted aryl, OH, NH.sub.2, OR,
NHR; [0046] n is an integer from 1-10,000 [0047] Each m,
independently, is an integer from 1-1,000; [0048] y or y'
independently, is an integer from 1-10.
[0049] In another embodiment, this invention provides an
amphiphilic polymer, characterized by the structure of the general
formula VI:
##STR00017##
wherein [0050] R is a hydroxyl (OH), O-alkyl, SH, S-alkyl, or an
acid activating group such as halogen (Cl, Br, I), O-vinyl,
O-allyl, O-aryl, OCOalkyl, OCOaryl, OCH, CF.sub.3NH.sub.2, NH, SH,
an acyl group, a fluorochrome, an indole-containing compound, an
antibody or antibody fragment, a peptide, an oligonucleotide, a
drug, a ligand for a biological target, an immunoconjugate, a
chemomimetic functional group, a glycolipid, a labelling agent, an
enzyme, a metal ion chelate, an enzyme cofactor, a cytotoxic
compound, a growth factor, a hormone, a cytokine, a toxin, a
prodrug, an antimetabolite, a microtubule inhibitor, a radioactive
material, a targeting moiety, an aryl group, a linear or branched
alkenyl group, a linear or branched alkyl group, wherein said
alkyl, alkenyl or aryl group is substituted with a perfluorocarbon,
perfluorocarbon-R.sub.4, perfluorocarbon-OR.sub.4, or
[0050] ##STR00018## [0051] R.sub.4 is independently an alkyl group,
an alkylene group, a carboxylate group, a carboxylic acid group,
and amino group, an ammonium group, an alkoxyl group, a hydroxyl
group or another nitrogen, oxygen or sulfur-containing group;
[0052] T, independently is:
[0052] ##STR00019## [0053] z is, independently, a halogen, a nitro
group, a hydroxy group, an amino group, an alkyl group, a
substituted alkyl group, an aryl group, a substituted aryl group,
wherein said substituted alkyl or aryl group is substituted with a
perfluorocarbon, perfluorocarbon-R.sub.4, perfluorocarbon-OR.sub.4,
or
[0053] ##STR00020## [0054] A is, independently O, S, NH; [0055] p,
p' are integers; [0056] each x, independently, is an integer from
1-1000; and [0057] y is an integer from 1-10,000.
[0058] In another embodiment, this invention provides an
amphiphilic polymer, characterized by the structure of the general
formula VII:
##STR00021##
wherein [0059] R is a hydroxyl (OH), O-alkyl, SH, S-alkyl, or an
acid activating group such as halogen (Cl, Br, I), O-vinyl,
O-allyl, O-aryl, OCOalkyl, OCOaryl, OCH.sub.2CF.sub.3NH.sub.2, NH,
SH, an acyl group, a fluorochrome, an indole-containing compound,
an antibody or antibody fragment, a peptide, an oligonucleotide, a
drug, a ligand for a biological target, an immunoconjugate, a
chemomimetic functional group, a glycolipid, a labelling agent, an
enzyme, a metal ion chelate, an enzyme cofactor, a cytotoxic
compound, a growth factor, a hormone, a cytokine, a toxin, a
prodrug, an antimetabolite, a microtubule inhibitor, a radioactive
material, a targeting moiety, an aryl group, a linear or branched
alkenyl group, a linear or branched alkyl group, wherein said
alkyl, alkenyl or aryl group is substituted with a perfluorocarbon,
perfluorocarbon-R.sub.4, perfluorocarbon-OR.sub.4, or
[0059] ##STR00022## [0060] R.sub.4 is independently an alkyl group,
an alkylene group, a carboxylate group, a carboxylic acid group,
and amino group, an ammonium group, an alkoxyl group, a hydroxyl
group or another nitrogen, oxygen or sulfur-containing group;
[0061] T, independently is:
[0061] ##STR00023## [0062] z is, independently, H, alkyl, aryl,
NH2, NH-alkyl, NH-acyl, NH-aryl, OH, O-acyl, O-alkyl, O-aryl, a
halogen, a nitro group, a hydroxy group, a substituted alkyl group,
a substituted aryl group, wherein said substituted alkyl or aryl
group is substituted with a perfluorocarbon,
perfluorocarbon-OR.sub.4, or
[0062] ##STR00024## [0063] A is, independently O, S, NH; [0064] p,
p' are integers; [0065] each y, independently, is an integer from
1-1000; and [0066] n is an integer from 1-10,000.
[0067] In another embodiment, this invention provides a composition
or a micelle comprising a polymer of this invention.
[0068] In another embodiment, this invention provides a process for
producing an amphiphilic polymer comprising perfluorocarbons, the
process comprising the steps of: [0069] contacting a dialkyl
5-hydroxy-isophthalate, a dialkyl 5-alkoxy-isophthalate, a dialkyl
5-amino-isophthalate, any derivative thereof or any combination
thereof with a polyethylene glycol to form an amphiphilic
copolymer; and [0070] linking a perfluorocarbons to said
amphiphilic copolymer, thereby being a process for producing
amphiphilic polymers comprising perfluorocarbons.
[0071] In another embodiment, this invention provides a method of
imaging a cell, the method comprising the steps of contacting a
cell with an amphiphilic polymer of this invention and imaging said
cell, whereby said polymer enables the imaging of said cell.
[0072] In another embodiment, this invention provides a method of
targeted delivery of at least one agent in a subject comprising the
steps of administering to said subject an amphiphilic polymer of
this invention, wherein said polymer comprises said agent and a
targeting agent.
[0073] In another embodiment, this invention provides a method for
detecting neoplastic cells in a subject, comprising contacting a
cell in, or a cell derived from said subject with an effective
tumor-detecting amount of an amphiphilic polymer of this invention,
wherein said polymer comprises a targeting moiety specific for
neoplastic cells; and detecting any of said polymer associated with
neoplastic cells present in said subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0074] FIG. 1 depicts a synthetic scheme for the preparation of a
basic copolymer structure.
[0075] FIGS. 2a and 2b depict schemes for the formation of
self-assembling alternating copolymer micelles.
[0076] FIG. 3 depicts micelle nanoparticles with perfluorocarbon
side chains and cargo and .sup.19F Spectra from perfluorocarbon
encapsulated 1,1,2,2-tetrahydro perfluorodecanol particles.
[0077] FIG. 4 shows cellular uptake of the particles. Cryo
transmission electron microscopy (FIG. 4A) and confocal microscopy
(FIGS. 4D and 4E) were used to qualitatively evaluate cellular
uptake. Cellular uptake was also evaluated quantitatively (FIGS.
4B, 4C and 4F). INS-1 cells were incubated at 37.degree. C., with
the compound (1 mg/mL), and the uptake was measured.
[0078] FIG. 5 shows cellular cytotoxicity, following exposure of
the cells to some polymers of the invention.
[0079] FIG. 6 describes the kinetics of cellular uptake and
intracellular localization of some polymers of the invention.
[0080] FIG. 7 depicts a crosslinked iron oxide (CLIO)-EPPT
multi-modal imaging probe. (A) The core protein of the MUC-1 tumor
antigen. The immunodominant region of the tandem repeat is
recognized by the EPPT1 peptide derived from an ASM2 monoclonal
antibody (45). (B) Synthesis (left) and scheme of the probe
(right). (C) The absorption spectrum of CLIO-EPPT showed the
presence of three peaks corresponding to FITC, Cy5.5, and iron
oxide nanoparticles. (D) Cell binding assay: cells expressing
underglycosylated mucin-1 accumulate significantly more CLIO-EPPT
(P<0.05) than uMUC-1-negative tumor or normal cells. (E)
Fluorescence-activated cell sorting analysis of the set of
underglycosylated mucin-1 antigen (uMUC-1)-positive tumor cell
lines (BT-20, CAPAN-2, ChaGo-K-1, HT-29, LS174T) showed a shift in
fluorescence in the FL1 and FL4 channels and no shift in the
control uMUC-1-negative cell line U87. Fluorescence microscopy
showed colocalization of the FITC and Cy5.5 signal within the set
of the same cell lines after incubation with the CLIO-EPPT probe.
Left, overlay of the bright field and FITC channel; middle, overlay
of the bright field and Cy5.5 channel; right, overlay of the FITC
and Cy5.5 channels. Note that no fluorescence was observed in FITC
or Cy5.5 channels in the U87 cell line. Magnification bars=10
.mu.m.
[0081] FIG. 8 demonstrates results of imaging of the animals
bearing underglycosylated mucin-1 antigen (uMUC-1)-negative (U87)
and uMUC-1-positive (LS174T) tumors. (A) Transverse (top) and
coronal (bottom) images showed a significant (52%; P<0.0001)
decrease in signal intensity in uMUC-1-positive tumors 24 h after
administration of the CLIO-EPPT probe. (B) White light (left),
near-infrared fluorescent (NIRF) (middle) images, and a color-coded
map (right) of mice bearing bilateral underglycosylated mucin-1
antigen uMUC-1-negative (U87) and uMUC-1-positive (LS174T) tumors.
NIRF imaging was performed immediately after the MRI session. (C)
White light (top) and NIRF (bottom) images of LS174T- and
U87-excised tumors and muscle tissue. uMUC-1-positive LS tumor
produced a strong NIRF signal. (D) Dual channel fluorescence
microscopy of the frozen LS174T tumor section. Green channel
fluorescence from the FITC-labeled EPPT peptide (left) colocalized
with Cy5.5 fluorescence derived from Cy5.5-labeled cross-linked
iron oxides (middle). The combination image shows colocalization of
two signals (right). Magnification bar=10 .mu.m.
[0082] FIG. 9 schematically depicts a first stage of synthesis of
the amphiphilic polymer I, accomplished via enzymatic
polymerization.
[0083] FIG. 10 depicts some embodiments of the alternations for
perfluorocarbon side chains that may be synthesized or used
according to this invention.
[0084] FIG. 11 depicts structures of the polymers with various
substituents. Structure of nanospheres and positions available for
iodination (11a). A scheme for the fluorescent labeling of
perfluorinated side chains (11b).
[0085] FIG. 12 schematically depicts some embodiments of polymer
structures which may be prepared according to the methods of this
invention, which may find application as probes for multi-modal
imaging.
DETAILED DESCRIPTION OF THE INVENTION
[0086] The invention provides, in one embodiment, an amphiphilic
polymer, characterized by the structure of the general formula
I:
##STR00025##
wherein [0087] R is a hydroxyl (OH), O-alkyl, O-Acyl, O-Activating
group, SH, S-alkyl, or an acid activating group such as halogen
(Cl, Br, I), O-vinyl, O-allyl, O-aryl, OCOalkyl, OCOaryl,
OCH.sub.2CF.sub.3, NH.sub.2, a fluorochrome, an indole-containing
compound, an antibody or antibody fragment, a peptide, an
oligonucleotide, a drug, a ligand for a biological target, an
immunoconjugate, a chemomimetic functional group, a glycolipid, a
labelling agent, an enzyme, a metal ion chelate, an enzyme
cofactor, a cytotoxic compound, a growth factor, a hormone, a
cytokine, a toxin, a prodrug, an antimetabolite, a microtubule
inhibitor, a radioactive material, a targeting moiety; [0088] R' is
OH, NH.sub.2, SH, OR'', NHR'', SR''; [0089] Where W' is a
fluorochrome, an indole-containing compound, an antibody or
antibody fragment, a peptide, an oligonucleotide, a drug, a ligand
for a biological target, an immunoconjugate, a chemomimetic
functional group, a glycolipid, a labelling agent, an enzyme, a
metal ion chelate, an enzyme cofactor, a cytotoxic compound, a
growth factor, a hormone, a cytokine, a toxin, a prodrug, an
antimetabolite, a microtubule inhibitor, a radioactive material, a
targeting moiety; [0090] each R.sub.1 group is, independently,
H,
##STR00026##
[0090] a fluorochrome, an indole-containing compound, an antibody
or antibody fragment, a peptide, an oligonucleotide, a drug, a
ligand for a biological target, an immunoconjugate, a chemomimetic
functional group, a glycolipid, a labelling agent, an enzyme, a
metal ion chelate, an enzyme cofactor, a cytotoxic compound, a
growth factor, a hormone, a cytokine, a toxin, a prodrug, an
antimetabolite, a microtubule inhibitor, a radioactive material, a
perfluorocarbon, [0091] a perfluorocarbon-R.sub.4, a
perfluorocarbon-OR.sub.4,
##STR00027##
[0091] OH, NH2, NH, S, SH, O-alkyl;
[0092] each R.sub.2 group is, independently, a fluorochrome, an
indole-containing compound, an antibody or antibody fragment, a
peptide, an oligonucleotide, a drug, a ligand for a biological
target, an immunoconjugate, a chemomimetic functional group, a
glycolipid, a labelling agent, an enzyme, a metal ion chelate, an
enzyme cofactor, a cytotoxic compound, a growth factor, a hormone,
a cytokine, a toxin, a prodrug, an antimetabolite, a microtubule
inhibitor, a radioactive material, a perfluorocarbon, a
perfluorocarbon-R.sub.4, a perfluorocarbon-OR.sub.4,
[0092] ##STR00028## [0093] each R.sub.3 group is,
independently,
##STR00029##
[0093] a hydrogen, a hydroxyl (OH), O-alkyl, SH, S-alkyl, or an
acid activating group such as halogen (Cl, Br, I), O-vinyl,
O-allyl, O-aryl, OCOalkyl, OCOaryl, OCH.sub.2CF.sub.3, NH.sub.2, a
fluorochrome, an indole-containing compound, an antibody or
antibody fragment, a peptide, an oligonucleotide, a drug, a ligand
for a biological target, an immunoconjugate, a chemomimetic
functional group, a glycolipid, a labelling agent, an enzyme, a
metal ion chelate, an enzyme cofactor, a cytotoxic compound, a
growth factor, a hormone, a cytokine, a toxin, a prodrug, an
antimetabolite, a microtubule inhibitor, a radioactive material, a
targeting moiety; [0094] each R.sub.4 group is, independently, an
alkyl group, an alkylene group, a carboxylate group, a carboxylic
acid group, an amino group, an ammonium group, an alkoxyl group, a
hydroxyl group or another nitrogen, oxygen or sulfur-containing
group, a halogen; [0095] each A group is, independently, O, NH, S,
a fluorochrome,
##STR00030##
[0095] an indole-containing compound, an antibody or antibody
fragment, a peptide; an oligonucleotide, a drug, a ligand for a
biological target, an immunoconjugate, a chemomimetic functional
group, a glycolipid, a labeling agent, an enzyme, a metal ion
chelate, an enzyme cofactor, a cytotoxic compound, a growth factor,
a hormone, a cytokine, a toxin, a prodrug, an antimetabolite, a
microtubule inhibitor, a radioactive material, a targeting moiety,
an acyl group, an aryl group, a linear or branched alkenyl group, a
linear or branched alkyl group, wherein said alkyl, alkenyl or aryl
group is substituted with a perfluorocarbon,
perfluorocarbon-R.sub.4, perfluorocarbon-OR.sub.4,
perfluorocarbon-OR.sub.4, or
##STR00031## [0096] n, m, p, p' and x are integers; and [0097] q is
an integer between 0-10.
[0098] In another embodiment, this invention provides a polymer is
characterized by the structure of the general formula II:
##STR00032##
wherein
[0099] R'=OH, NH.sub.2, SH, OAlkyl, OAryl, OAcyl, OActivating
group;
[0100] R=OH, NH.sub.2, SH, OAlkyl, OAryl, OAcyl, OActivating
group;
[0101] R.sub.1=H;
[0102] R.sub.3=H, a hydroxyl (OH), O-alkyl, SH, S-alkyl, or an acid
activating group such as halogen (Cl, Br, I), O-vinyl, O-allyl,
O-aryl, OCOalkyl, OCOaryl, OCH.sub.2CF.sub.3, NH.sub.2, (a
fluorochrome, an indole-containing compound, an antibody or
antibody fragment, a peptide, an oligonucleotide, a drug, a ligand
for a biological target, an immunoconjugate, a chemomimetic
functional group, a glycolipid, a labelling agent, an enzyme, a
metal ion chelate, an enzyme cofactor, a cytotoxic compound, a
growth factor, a hormone, a cytokine, a toxin, a prodrug, an
antimetabolite, a microtubule inhibitor, a radioactive material, a
targeting moiety); and
[0103] A=O, NH, S.
[0104] In another embodiment, this invention provides an
amphiphilic polymer, characterized by the structure of the general
formula III:
##STR00033##
wherein [0105] each R group is, independently: a fluorochrome, an
indole-containing compound, an antibody or antibody fragment, a
peptide, an oligonucleotide, a drug, a ligand for a biological
target, an immunoconjugate, a chemomimetic functional group, a
glycolipid, a labelling agent, an enzyme, a metal ion chelate, an
enzyme cofactor, a cytotoxic compound, a growth factor, a hormone,
a cytokine, a toxin, a prodrug, an antimetabolite, a microtubule
inhibitor, a radioactive material, a targeting moiety, an acyl
group, an aryl group, a linear or branched alkenyl group, a linear
or branched alkyl group, wherein said alkyl, alkenyl or aryl group
is substituted with a perfluorocarbon, perfluorocarbon-R.sub.4,
perfluorocarbon-OR.sub.4, or
[0105] ##STR00034## [0106] each R' group is, independently, a
hydrogen, a hydroxyl (OH), O-alkyl, SH, S-alkyl, or an acid
activating group such as halogen (Cl, Br, I), O-vinyl, O-allyl,
O-aryl, OCOalkyl, OCOaryl, OCH.sub.2CF.sub.3NH.sub.2, SH, an acyl
group, a fluorochrome, an indole-containing compound, an antibody
or antibody fragment, a peptide, an oligonucleotide, a drug, a
ligand for a biological target, an immunoconjugate, a chemomimetic
functional group, a glycolipid, a labelling agent, an enzyme, a
metal ion chelate, an enzyme cofactor, a cytotoxic compound, a
growth factor, a hormone, a cytokine, a toxin, a prodrug, an
antimetabolite, a microtubule inhibitor, a radioactive material, a
targeting moiety, or OR", NHR", SR'' [0107] wherein R'' is a
fluorochrome, an indole-containing compound, an antibody or
antibody fragment, a peptide, an oligonucleotide, a drug, a ligand
for a biological target, an immunoconjugate, a chemomimetic
functional group, a glycolipid, a labelling agent, an enzyme, a
metal ion chelate, an enzyme cofactor, a cytotoxic compound, a
growth factor, a hormone, a cytokine, a toxin, a prodrug, an
antimetabolite, a microtubule inhibitor, a radioactive material, a
targeting moiety; [0108] wherein R''' is a hydroxyl group, an
alkoxyl group or a primary or secondary amino group, O activating
group, SH and S-alkyl; [0109] R.sub.2 is, independently, a
fluorochrome, an indole-containing compound, an antibody or
antibody fragment, a peptide, an oligonucleotide, a drug, a ligand
for a biological target, an immunoconjugate, a chemomimetic
functional group, a glycolipid, a labelling agent, an enzyme, a
metal ion chelate, an enzyme cofactor, a cytotoxic compound, a
growth factor, a hormone, a cytokine, a toxin, a prodrug, an
antimetabolite, a microtubule inhibitor, a radioactive material, a
perfluorocarbon, a perfluorocarbon-R.sub.4, a
perfluorocarbon-OR.sub.4,
[0109] ##STR00035## [0110] R.sub.4 is independently an alkyl group,
an alkylene group, a carboxylate group, a carboxylic acid group,
and amino group, an ammonium group, an alkoxyl group, a hydroxyl
group or another nitrogen, oxygen or sulfur-containing group, a
halogen; [0111] A is a fluorochrome, an indole-containing compound,
an antibody or antibody fragment, a peptide, an oligonucleotide, a
drug, a ligand for a biological target, an immunoconjugate, a
chemomimetic functional group, a glycolipid, a labeling agent, an
enzyme, a metal ion chelate, an enzyme cofactor, a cytotoxic
compound, a growth factor, a hormone, a cytokine, a toxin, a
prodrug, an antimetabolite, a microtubule inhibitor, a radioactive
material, a targeting moiety, an acyl group, an aryl group, a
linear or branched alkenyl group, a linear or branched alkyl group,
wherein said alkyl, alkenyl or aryl group is substituted with a
perfluorocarbon, perfluorocarbon-R.sub.4, perfluorocarbon-OR.sub.4,
or
[0111] ##STR00036## [0112] p and p' are integers; [0113] n is at
least 1; and [0114] m is at least 1.
[0115] In another embodiment, this invention provides an
amphiphilic polymer, characterized by the structure of the general
formula IV:
##STR00037##
wherein [0116] each R group, independently, is a hydroxyl (OH),
OCH.sub.2CF.sub.3, NH.sub.2, SH, S, a fluorochrome, an
indole-containing compound, an antibody or antibody fragment, a
peptide, an oligonucleotide, a drug, a ligand for a biological
target, an immunoconjugate, a chemomimetic functional group, a
glycolipid, a labelling agent, an enzyme, a metal ion chelate, an
enzyme cofactor, a cytotoxic compound, a growth factor, a hormone,
a cytokine, a toxin, a prodrug, an antimetabolite, a microtubule
inhibitor, a radioactive material, a targeting moiety, a halogen,
an aryl group, a linear or branched alkenyl group, a linear or
branched alkyl group, wherein said alkyl, alkenyl or aryl group is
substituted with a perfluorocarbon, perfluorocarbon-R.sub.4,
perfluorocarbon-OR.sub.4, or
[0116] ##STR00038## [0117] R.sub.4 is independently an alkyl group,
an alkylene group, a carboxylate group, a carboxylic acid group,
and amino group, an ammonium group, an alkoxyl group, a hydroxyl
group, a halogen or another nitrogen, oxygen or sulfur-containing
group [0118] B or B' is, independently: alkyl, substituted alkyl,
aryl, substituted aryl, OH, NH.sub.2, OR.sub.1, NHR.sub.1;
OCOR.sub.1, NHCOR.sub.1 [0119] Where R.sub.1 is alkyl, substituted
alkyl, aryl, substituted aryl, wherein the said alkyl or aryl group
is either perfluorinated or substituted with perfluorinated
compound. [0120] x=0-10; [0121] y=0-10; [0122] p, p' are integers;
[0123] n is at least 1; and [0124] m is at least 1.
[0125] In another embodiment, this invention provides an
amphiphilic polymer, characterized by the structure of the general
formula V:
##STR00039##
wherein [0126] R is a hydroxyl (OH), O-alkyl, SH, S-alkyl, or an
acid activating group such as halogen (Cl, Br, I), O-vinyl,
O-allyl, O-aryl, OCOalkyl, OCOaryl, OCH.sub.2CF.sub.3NH.sub.2, NH,
SH, an acyl group, a fluorochrome, an indole-containing compound,
an antibody or antibody fragment, a peptide, an oligonucleotide, a
drug, a ligand for a biological target, an immunoconjugate, a
chemomimetic functional group, a glycolipid, a labelling agent, an
enzyme, a metal ion chelate, an enzyme cofactor, a cytotoxic
compound, a growth factor, a hormone, a cytokine, a toxin, a
prodrug, an antimetabolite, a microtubule inhibitor, a radioactive
material, a targeting moiety, an aryl group, a linear or branched
alkenyl group, a linear or branched alkyl group, wherein said
alkyl, alkenyl or aryl group is substituted with a perfluorocarbon,
perfluorocarbon-R.sub.4, perfluorocarbon-OR.sub.4, or
[0126] ##STR00040## [0127] R' is hydrogen, a fluorochrome, an
indole-containing compound, an antibody or antibody fragment, a
peptide, an oligonucleotide, a drug, a ligand for a biological
target, an immunoconjugate, a chemomimetic functional group, a
glycolipid, a labelling agent, an enzyme, a metal ion chelate, an
enzyme cofactor, a cytotoxic compound, a growth factor, a hormone,
a cytokine, a toxin, a prodrug, an antimetabolite, a microtubule
inhibitor, a radioactive material, a targeting moiety, an aryl
group, a linear or branched alkenyl group, a linear or branched
alkyl group, wherein said alkyl, alkenyl or aryl group is
substituted with a perfluorocarbon, perfluorocarbon-R.sub.4,
perfluorocarbon-OR.sub.4; [0128] R.sub.4 is independently an alkyl
group, an alkylene group, a carboxylate group, a carboxylic acid
group, and amino group, an ammonium group, an alkoxyl group, a
hydroxyl group or another nitrogen, oxygen or sulfur-containing
group [0129] A is, independently: O, S or NH [0130] B or B' is,
independently, alkyl, substituted alkyl, aryl, substituted aryl,
OH, NH.sub.2, OR.sub.1, NHR.sub.1; Where R.sub.1 is alkyl,
substituted alkyl, aryl, substituted aryl, wherein the said alkyl
or aryl group is either perfluorinated or substituted with
perfluorinated compound, NHR; [0131] n is an integer from 1-10,000
[0132] Each m, independently, is an integer from 1-1,000; [0133] y
or y' independently, is an integer from 1-10.
[0134] In another embodiment, this invention provides an
amphiphilic polymer, characterized by the structure of the general
formula VI:
##STR00041##
wherein [0135] R is a hydroxyl (OH), O-alkyl, SH, S-alkyl, or an
acid activating group such as halogen (Cl, Br, I), O-vinyl,
O-allyl, O-aryl, OCOalkyl, OCOaryl, OCH.sub.2CF.sub.3NH.sub.2, NH,
SH, an acyl group, a fluorochrome, an indole-containing compound,
an antibody or antibody fragment, a peptide, an oligonucleotide, a
drug, a ligand for a biological target, an immunoconjugate, a
chemomimetic functional group, a glycolipid, a labelling agent, an
enzyme, a metal ion chelate, an enzyme cofactor, a cytotoxic
compound, a growth factor, a hormone, a cytokine, a toxin, a
prodrug, an antimetabolite, a microtubule inhibitor, a radioactive
material, a targeting moiety, an aryl group, a linear or branched
alkenyl group, a linear or branched alkyl group, wherein said
alkyl, alkenyl or aryl group is substituted with a perfluorocarbon,
perfluorocarbon-R.sub.4, perfluorocarbon-OR.sub.4, or
[0135] ##STR00042## [0136] R' is hydrogen, a fluorochrome, an
indole-containing compound, an antibody or antibody fragment, a
peptide, an oligonucleotide, a drug, a ligand for a biological
target, an immunoconjugate, a chemomimetic functional group, a
glycolipid, a labelling agent, an enzyme, a metal ion chelate, an
enzyme cofactor, a cytotoxic compound, a growth factor, a hormone,
a cytokine, a toxin, a prodrug, an antimetabolite, a microtubule
inhibitor, a radioactive material, a targeting moiety, an aryl
group, a linear or branched alkenyl group, a linear or branched
alkyl group, wherein said alkyl, alkenyl or aryl group is
substituted with a perfluorocarbon, perfluorocarbon-R4,
perfluorocarbon-OR4. [0137] R.sub.4 is independently an alkyl
group, an alkylene group, a carboxylate group, a carboxylic acid
group, and amino group, an ammonium group, an alkoxyl group, a
hydroxyl group or another nitrogen, oxygen or sulfur-containing
group; [0138] T, independently is:
[0138] ##STR00043## [0139] z is, independently, a halogen, a nitro
group, a hydroxy group, an amino group, an alkyl group, a
substituted alkyl group, an aryl group, a substituted aryl group,
wherein said substituted alkyl or aryl group is substituted with a
perfluorocarbon, perfluorocarbon-R.sub.4, perfluorocarbon-OR.sub.4,
or
[0139] ##STR00044## [0140] A is, independently O, S, NH; [0141] p,
p' are integers; [0142] each x, independently, is an integer from
1-1000; and [0143] y is an integer from 1-10,000.
[0144] In another embodiment, this invention provides an
amphiphilic polymer, characterized by the structure of the general
formula VII:
##STR00045##
wherein [0145] R is a hydroxyl (OH), O-alkyl, SH, S-alkyl, or an
acid activating group such as halogen (Cl, Br, I), O-vinyl,
O-allyl, O-aryl, OCOalkyl, OCOaryl, OCH.sub.2CF.sub.3NH.sub.2, NH,
SH, an acyl group, a fluorochrome, an indole-containing compound,
an antibody or antibody fragment, a peptide, an oligonucleotide, a
drug, a ligand for a biological target, an immunoconjugate, a
chemomimetic functional group, a glycolipid, a labelling agent, an
enzyme, a metal ion chelate, an enzyme cofactor, a cytotoxic
compound, a growth factor, a hormone, a cytokine, a toxin, a
prodrug, an antimetabolite, a microtubule inhibitor, a radioactive
material, a targeting moiety, an aryl group, a linear or branched
alkenyl group, a linear or branched alkyl group, wherein said
alkyl, alkenyl or aryl group is substituted with a perfluorocarbon,
perfluorocarbon-R.sub.4, perfluorocarbon-OR.sub.4, or
[0145] ##STR00046## [0146] R' is hydrogen, a fluorochrome, an
indole-containing compound, an antibody or antibody fragment, a
peptide, an oligonucleotide, a drug, a ligand for a biological
target, an immunoconjugate, a chemomimetic functional group, a
glycolipid, a labelling agent, an enzyme, a metal ion chelate, an
enzyme cofactor, a cytotoxic compound, a growth factor, a hormone,
a cytokine, a toxin, a prodrug, an antimetabolite, a microtubule
inhibitor, a radioactive material, a targeting moiety, an aryl
group, a linear or branched alkenyl group, a linear or branched
alkyl group, wherein said alkyl, alkenyl or aryl group is
substituted with a perfluorocarbon, perfluorocarbon-R.sub.4,
perfluorocarbon-OR.sub.4, [0147] R.sub.4 is independently an alkyl
group, an alkylene group, a carboxylate group, a carboxylic acid
group, and amino group, an ammonium group, an alkoxyl group, a
hydroxyl group or another nitrogen, oxygen or sulfur-containing
group; [0148] T, independently is:
[0148] ##STR00047## [0149] z is, independently, H, alkyl, aryl,
NH2, NH-alkyl, NH-acyl, NH-aryl, OH, O-acyl, O-alkyl, O-aryl, a
halogen, a nitro group, a hydroxy group, a substituted alkyl group,
a substituted aryl group, wherein said substituted alkyl or aryl
group is substituted with a perfluorocarbon,
perfluorocarbon-R.sub.4, perfluorocarbon-OR.sub.4, or
[0149] ##STR00048## [0150] A is, independently O, S, NH; [0151] p,
p' are integers; [0152] each y, independently, is an integer from
1-1000; and [0153] n is an integer from 1-10,000.
[0154] In one embodiment, the polymers with a structure
characterized by formula I or II of this invention will be such
that the weight of a fraction of the polymer ranges between 0-5%
or, in another embodiment, 6-99% of the weight of said polymer, or,
in another embodiment, 5-10% of the weight of said polymer, or in
another embodiments, x represents about 10-25% of the weight of
said polymer, or in another embodiment, x represents from about
30-75% of the weight of said polymer, or in another embodiment, x
represents from about 50-100% of the weight of said polymer,
wherein the fraction is represented by the structure:
##STR00049##
[0155] In one embodiment, the polymers with a structure
characterized by formula I or II of this invention will be such
that the weight of a fraction of the polymer ranges between 1-94%
or, in another embodiment, 0% of the weight of said polymer, or, in
another embodiment, 5-10% of the weight of said polymer, or in
another embodiments, x represents about 10-25% of the weight of
said polymer, or in another embodiment, x represents from about
30-75% of the weight of said polymer, or in another embodiment, x
represents from about 50-90% of the weight of said polymer, wherein
the fraction is represented by the structure:
##STR00050##
[0156] The polymers of this invention are amphiphilic. In one
embodiment, the term "amphiphilic" refers to a molecule that
contains both hydrophilic and lipophilic (or, synonymously,
hydrophobic) moieties.
[0157] In one embodiment, the term "alkyl" refers to C.sub.1-32
straight-chain or C.sub.1-32 branched hydrocarbons, e.g. methyl,
isobutyl, hexyl, etc. In another embodiment, the term "alkyl" (or
"lower alkyl") refers to both "unsubstituted alkyls" and
"substituted alkyls", the latter of which refers to alkyl moieties
having substituents replacing a hydrogen on one or more carbons of
the hydrocarbon backbone. Such substituents can include, for
example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an
ester, a formyl, or a ketone), a thiocarbonyl (such as a thioester,
a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a
phosphonate, a phosphinate, an amino, an amido, an amidine, an
imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a
sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a
heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety.
It will be understood by those skilled in the art that the moieties
substituted on the hydrocarbon chain can themselves be substituted,
if appropriate. For instance, the substituents of a substituted
alkyl may include substituted and unsubstituted forms of amino,
azido, imino, amido, phosphoryl (including phosphonate and
phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl
and sulfonate), and silyl groups, as well as ethers, alkylthios,
carbonyls (including ketones, aldehydes, carboxylates, and esters),
--CF.sub.3, --CN and the like.
[0158] In one embodiment, the term "alkoxy" refers to an alkyl
group connected to a main chain or backbone through an oxygen atom.
In another embodiment, the term "alkoxyl" or "alkoxy" are
interchangeable, and representative alkoxyl groups include methoxy,
ethoxy, propyloxy, tert-butoxy and the like.
[0159] In one embodiment, the term "aryl" refers to aromatic rings
such as phenyl, pyridinyl, thienyl, thiazolyl, or furyl, optionally
substituted with one or more groups, such as a halo group, a
haloalkyl group, an amino group, or an alkyl group. In one
embodiment, the term "aryl" includes 5-, 6- and 7-membered
single-ring aromatic groups that may include from zero to four
heteroatoms, for example, benzene, pyrrole, furan, thiophene,
imidazole, oxazole, thiazole, triazole, pyrazole, pyridine,
pyrazine, pyridazine and pyrimidine, and the like. Those aryl
groups having heteroatoms in the ring structure may also be
referred to as "aryl heterocycles" or "heteroaromatics". The
aromatic ring can be substituted at one or more ring positions with
such substituents as described above, for example, halogen, azide,
alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino,
nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,
carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido,
ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic
moieties, --CF.sub.3, --CN, or the like. The term "aryl" also
includes polycyclic ring systems having two or more rings in which
two or more carbons are common to two adjoining rings (the rings
are "fused") wherein at least one of the rings is aromatic, e.g.,
the other rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,
aryls and/or heterocyclyls. In one embodiment, the term "aryloxy"
refers to aryl groups attached to a main chain or backbone through
an oxygen atom.
[0160] In one embodiment, the term "amine" refers to any amine,
including primary, secondary, tertiary, quaternary, or a
combination thereof, as applicable herein.
[0161] In one embodiment, the term "acid activating group" refers
to a group which facilitates conjugation of the polymers with a
desired substance, via a suitable reactive derivative of a
carboxylic acid, which may comprise inter-alia, an acyl halide, for
example an acyl chloride formed by the reaction of the acid and an
inorganic acid chloride, for example thionyl chloride; a mixed
anhydride, for example an anhydride formed by the reaction of the
acid and a chloroformate such as isobutyl chloroformate; an active
ester, for example an ester formed by the reaction of the acid and
a phenol such as pentafluorophenol, an ester such as
pentafluorophenyl trifluoroacetate or an alcohol such as methanol,
ethanol, isopropanol, butanol or N-hydroxybenzotriazole; an acyl
azide, for example an azide formed by the reaction of the acid and
azide such as diphenylphosphoryl azide; an acyl cyanide, for
example a cyanide formed by the reaction of an acid and a cyanide
such as diethylphosphoryl cyanide; or the product of the reaction
of the acid and a carbodiimide such as
dicyclohexylcarbodiimide.
[0162] In one embodiment, the term "fluorochrome" refers to a
fluorescent substance and may comprise, inter-alia, DAPI, FITC,
Cy3, Cy3.5, Cy5, Cy5.5, Cy7, GFP, and others as will be appreciated
by one skilled in the art, each selected for specific properties,
for example, as described by Waggoner, A. (Methods in Enzymology
246:362-373 (1995) herein incorporated by reference).
[0163] In one embodiment, the term "antibody or antibody fragment"
refers to intact antibody molecules as well as functional fragments
thereof, such as Fab, F(ab')2, and Fv that are capable of binding
to an epitope. In one embodiment, an Fab fragment refers to the
fragment which contains a monovalent antigen-binding fragment of an
antibody molecule, which can be produced by digestion of whole
antibody with the enzyme papain to yield an intact light chain and
a portion of one heavy chain. In one embodiment, Fab' fragment
refers to a part of an antibody molecule that can be obtained by
treating whole antibody with pepsin, followed by reduction, to
yield an intact light chain and a portion of the heavy chain. Two
Fab' fragments may be obtained per antibody molecule. In one
embodiment, (Fab').sub.2 refers to a fragment of an antibody that
can be obtained by treating whole antibody with the enzyme pepsin
without subsequent reduction. In another embodiment, F(ab').sub.2
is a dimer of two Fab' fragments held together by two disulfide
bonds. In one embodiment, Fv, may refer to a genetically engineered
fragment containing the variable region of the light chain and the
variable region of the heavy chain expressed as two chains. In one
embodiment, the antibody fragment may be a single chain antibody
("SCA"), a genetically engineered molecule containing the variable
region of the light chain and the variable region of the heavy
chain, linked by a suitable polypeptide linker as a genetically
fused single chain molecule.
[0164] Methods of making these fragments are known in the art. (See
for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York, 1988, incorporated herein by
reference).
[0165] In one embodiment, the antibody will recognize an epitope,
which in another embodiment, refers to antigenic determinant on an
antigen to which the paratope of an antibody binds. Epitopic
determinants may, in other embodiments, consist of chemically
active surface groupings of molecules such as amino acids or
carbohydrate side chains and in other embodiments, may have
specific three dimensional structural characteristics, and/or in
other embodiments, have specific charge characteristics.
[0166] Antibody fragments according to the present invention can be
prepared by proteolytic hydrolysis of the antibody or by expression
in E. coli or mammalian cells (e.g. Chinese hamster ovary cell
culture or other protein expression systems) of DNA encoding the
fragment.
[0167] In other embodiments, antibody fragments can be obtained by
pepsin or papain digestion of whole antibodies by conventional
methods. For example, antibody fragments can be produced by
enzymatic cleavage of antibodies with pepsin to provide a 5S
fragment denoted F(ab')2. This fragment can be further cleaved
using a thiol reducing agent, and optionally a blocking group for
the sulfhydryl groups resulting from cleavage of disulfide
linkages, to produce 3.5S Fab' monovalent fragments. Alternatively,
an enzymatic cleavage using pepsin produces two monovalent Fab'
fragments and an Fc fragment directly. These methods are described,
for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647,
and references contained therein, which patents are hereby
incorporated by reference in their entirety. See also Porter, R.
R., Biochem. J., 73: 119-126, 1959. Other methods of cleaving
antibodies, such as separation of heavy chains to form monovalent
light-heavy chain fragments, further cleavage of fragments, or
other enzymatic, chemical, or genetic techniques may also be used,
so long as the fragments bind to the antigen that is recognized by
the intact antibody.
[0168] Fv fragments comprise an association of VH and VL chains.
This association may be noncovalent, as described in Inbar et al.,
Proc. Nat'l Acad. Sci. USA 69:2659-62, 1972. Alternatively, the
variable chains can be linked by an intermolecular disulfide bond
or cross-linked by chemicals such as glutaraldehyde. Preferably,
the Fv fragments comprise VH and VL chains connected by a peptide
linker. These single-chain antigen binding proteins (sFv) are
prepared by constructing a structural gene comprising DNA sequences
encoding the VH and VL domains connected by an oligonucleotide. The
structural gene is inserted into an expression vector, which is
subsequently introduced into a host cell such as E. coli. The
recombinant host cells synthesize a single polypeptide chain with a
linker peptide bridging the two V domains. Methods for producing
sFvs are described, for example, by Whitlow and Filpula, Methods,
2: 97-105, 1991; Bird et al., Science 242:423-426, 1988; Pack et
al., Bio/Technology 11:1271-77, 1993; and Ladner et al., U.S. Pat.
No. 4,946,778, which is hereby incorporated by reference in its
entirety.
[0169] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing
cells. See, for example, Larrick and Fry, Methods, 2: 106-10,
1991.
[0170] In one embodiment, the term "peptide" refers to native
peptides (either degradation products, synthetically synthesized
peptides or recombinant peptides) and/or peptidomimetics
(typically, synthetically synthesized peptides), such as peptoids
and semipeptoids which are peptide analogs, which may have, for
example, modifications rendering the peptides more stable while in
a body or more capable of penetrating into cells. Such
modifications include, but are not limited to N terminus
modification, C terminus modification, peptide bond modification,
including, but not limited to, CH.sub.2--NH, CH.sub.2--S,
CH.sub.2--S.dbd.O, O.dbd.C--NH, CH.sub.2--O, CH.sub.2--CH.sub.2,
S.dbd.C--NH, CH.dbd.CH or CF.dbd.CH, backbone modifications, and
residue modification. Methods for preparing peptidomimetic
compounds are well known in the art and are specified, for example,
in Quantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F.
Choplin Pergamon Press (1992), which is incorporated by reference
as if fully set forth herein. Further details in this respect are
provided hereinunder.
[0171] Peptide bonds (--CO--NH--) within the peptide may be
substituted, for example, by N-methylated bonds
(--N(CH.sub.3)--CO--), ester bonds (--C(R)H--C--O--O--C(R)--N--),
ketomethylen bonds (--CO--CH.sub.2--), *-aza bonds
(--NH--N(R)--CO--), wherein R is any alkyl, e.g., methyl, carba
bonds (--CH.sub.2--NH--), hydroxyethylene bonds
(--CH(OH)--CH.sub.2--), thioamide bonds (--CS--NH--), olefinic
double bonds (--CH.dbd.CH--), retro amide bonds (--NH--CO--),
peptide derivatives (--N(R)--CH.sub.2--CO--), wherein R is the
"normal" side chain, naturally presented on the carbon atom.
[0172] These modifications can occur at any of the bonds along the
peptide chain and even at several (2-3) at the same time. Natural
aromatic amino acids, Trp, Tyr and Phe, may be substituted for
synthetic non-natural acid such as TIC, naphthylelanine (Nol),
ring-methylated derivatives of Phe, halogenated derivatives of Phe
or o-methyl-Tyr.
[0173] In addition to the above, the peptides of the present
invention may also include one or more modified amino acids or one
or more non-amino acid monomers (e.g. fatty acids, complex
carbohydrates etc).
[0174] In one embodiment, the term "amino acid" or "amino acids" is
understood to include the 20 naturally occurring amino acids; those
amino acids often modified post-translationally in vivo, including,
for example, hydroxyproline, phosphoserine and phosphothreonine;
and other unusual amino acids including, but not limited to,
2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine,
nor-leucine and ornithine. Furthermore, the term "amino acid" may
include both D- and L-amino acids.
[0175] Peptides or proteins of this invention may be prepared by
various techniques known in the art, including phage display
libraries [Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991);
Marks et al., J. Mol. Biol. 222:581 (1991)].
[0176] In one embodiment, the term "oligonucleotide" is
interchangeable with the term "nucleic acid", and may refer to a
molecule, which may include, but is not limited to, prokaryotic
sequences, eukaryotic mRNA, cDNA from eukaryotic mRNA, genomic DNA
sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic
DNA sequences. The term also refers to sequences that include any
of the known base analogs of DNA and RNA.
[0177] Nucleic acid sequences, of which the polymers, micelles
and/or compositions of this invention may be comprised, may include
their being a part a particular vector, depending, in one
embodiment, upon the desired method of introduction of the sequence
within cells. In one embodiment, such vectors may be encapsulated
within the micelles of this invention. In one embodiment,
polynucleotide segments encoding sequences of interest can be
ligated into commercially available expression vector systems
suitable for transducing/transforming mammalian cells and for
directing the expression of recombinant products within the
transduced cells. It will be appreciated that such commercially
available vector systems can easily be modified via commonly used
recombinant techniques in order to replace, duplicate or mutate
existing promoter or enhancer sequences and/or introduce any
additional polynucleotide sequences such as for example, sequences
encoding additional selection markers or sequences encoding
reporter polypeptides.
[0178] The efficacy of a particular expression vector system and
method of introducing nucleic acid into a cell can be assessed by
standard approaches routinely used in the art. For example, DNA
introduced into a cell can be detected by a filter hybridization
technique (e.g., Southern blotting) and RNA produced by
transcription of introduced DNA can be detected, for example, by
Northern blotting, RNase protection or reverse
transcriptase-polymerase chain reaction (RT-PCR). The gene product
can be detected by an appropriate assay, for example by
immunological detection of a produced protein, such as with a
specific antibody, or by a functional assay to detect a functional,
activity of the gene product, such as an enzymatic assay. If the
gene product of interest to be expressed by a cell is not readily
assayable, an expression system can first be optimized using a
reporter gene linked to the regulatory elements and vector to be
used. The reporter gene encodes a gene product, which is easily
detectable and, thus, can be used to evaluate efficacy of the
system. Standard reporter genes used in the art include genes
encoding .beta.-galactosidase, chloramphenicol acetyl transferase,
luciferase and human growth hormone, or any of the marker proteins
listed herein.
[0179] As will be appreciated by one skilled in the art, a fragment
or derivative of a nucleic acid sequence or gene that encodes for a
protein or peptide can still function in the same manner as the
entire, wild type gene or sequence. Likewise, forms of nucleic acid
sequences can have variations as compared to wild type sequences,
nevertheless encoding the protein or peptide of interest, or
fragments thereof, retaining wild type function exhibiting the same
biological effect, despite these variations. Each of these
represents a separate embodiment of this present invention.
[0180] The nucleic acids can be produced by any synthetic or
recombinant process such as is well known in the art. Nucleic acids
can further be modified to alter biophysical or biological
properties by means of techniques known in the art. For example,
the nucleic acid can be modified to increase its stability against
nucleases (e.g., "end-capping"), or to modify its lipophilicity,
solubility, or binding affinity to complementary sequences.
[0181] DNA according to the invention can also be chemically
synthesized by methods known in the art. For example, the DNA can
be synthesized chemically from the four nucleotides in whole or in
part by methods known in the art. Such methods include those
described in Caruthers (1985). DNA can also be synthesized by
preparing overlapping double-stranded oligonucleotides, filling in
the gaps, and ligating the ends together (see, generally, Sambrook
et al. (1989) and Glover et al. (1995)). DNA expressing functional
homologues of the protein can be prepared from wild-type DNA by
site-directed mutagenesis (see, for example, Zoller et al. (1982);
Zoller (1983); and Zoller (1984); McPherson (1991)). The DNA
obtained can be amplified by methods known in the art. One suitable
method is the polymerase chain reaction (PCR) method described in
Saiki et al. (1988), Mullis et al., U.S. Pat. No. 4,683,195, and
Sambrook et al. (1989).
[0182] Methods for modifying nucleic acids to achieve specific
purposes are disclosed in the art, for example, in Sambrook et al.
(1989). Moreover, the nucleic acid sequences of the invention can
include one or more portions of nucleotide sequence that are
non-coding for the protein of interest. Variations in the DNA
sequences, which are caused by point mutations or by induced
modifications (including insertion, deletion, and substitution) to
enhance the activity, half-life or production of the polypeptides
encoded thereby, are also encompassed in the invention.
[0183] In another embodiment, the agent that inhibits gene
expression, activity or function comprises a nucleic acid. The
nucleic acid may, in one embodiment, be DNA, or in another
embodiment, the nucleic acid is RNA. In other embodiments, the
nucleic acid may be single or double stranded.
[0184] In another embodiment, the agent is a nucleic acid that is
antisense in orientation to a sequence encoding for a caspase.
[0185] In one embodiment, the polymers, micelles or compositions of
this invention may be used for gene silencing applications. In one
embodiment, the activity or function of a particular gene is
suppressed or diminished, via the use of antisense
oligonucleotides, which are chimeric molecules, containing two or
more chemically distinct regions, each made up of at least one
nucleotide. In one embodiment, the antisense molecules may be
conjugated to the polymers of this invention, as described, or in
another embodiment, encapsulated within micelles of this invention,
much as any of the respective groups listed herein, applicable in
the methods of this invention, in another embodiment, may be
conjugated to the polymers of this invention, or encapsulated
within micelles of this invention.
[0186] Antisense oligonucleotides, in one embodiment, may be
chimeric oligonucleotides, which contain at least one region
wherein the oligonucleotide is modified so as to confer upon the
oligonucleotide an increased resistance to nuclease degradation,
increased cellular uptake, and/or increased binding affinity for
the target polynucleotide. An additional region of the
oligonucleotide may serve as a substrate for enzymes capable of
cleaving RNA:DNA or RNA:RNA hybrids, which according to this aspect
of the invention, serves as a means of gene silencing via
degradation of specific sequences. Cleavage of the RNA target can
be routinely detected by gel electrophoresis and, if necessary,
associated nucleic acid hybridization techniques known in the
art.
[0187] The chimeric antisense oligonucleotides may, in one
embodiment, be formed as composite structures of two or more
oligonucleotides and/or modified oligonucleotides, as is well
described in the art (see, for example, U.S. Pat. Nos. 5,013,830;
5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133;
5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922), and
can, in another embodiment, comprise a ribozyme sequence.
[0188] Inhibition of gene expression, activity or function is
effected, in another embodiment, via the use of small interfering
RNAs, which provides sequence-specific inhibition of gene
expression. Administration of double stranded/duplex RNA (dsRNA)
corresponding to a single gene in an organism can silence
expression of the specific gene by rapid degradation of the mRNA in
affected cells. This process is referred to as gene silencing, with
the dsRNA functioning as a specific RNA inhibitor (RNAi). RNAi may
be derived from natural sources, such as in endogenous virus and
transposon activity, or it can be artificially introduced into
cells (Elbashir S M, et al (2001). Nature 411:494-498) via
microinjection (Fire et al. (1998) Nature 391: 806-11), or by
transformation with gene constructs generating complementary RNAs
or fold-back RNA, or by other vectors (Waterhouse, P. M., et al.
(1998). Proc. Natl. Acad. Sci. USA 95, 13959-13964 and Wang, Z., et
al. (2000). J. Biol. Chem. 275, 40174-40179). The RNAi mediating
mRNA degradation, in one embodiment, comprises duplex or
double-stranded RNA, or, in other embodiments, include
single-stranded RNA, isolated RNA (partially purified RNA,
essentially pure RNA, synthetic RNA, recombinantly produced RNA),
as well as altered RNA that differs from naturally occurring RNA by
the addition, deletion and/or alteration of one or more
nucleotides.
[0189] When referring to nucleic acid sequences utilized as
modulators in this invention, it is to be understood that such
reference allows for the incorporation of non-nucleotide material,
which may be added, for example, to the end(s) of the nucleotide
sequence, including for example, terminal 3' hydroxyl groups, or
internal additions, at one or more nucleotides. Nucleic acids may,
in another embodiment, incorporate non-standard nucleotides,
including non-naturally-occurring nucleotides. Alterations may also
include the construction of blunt and/or overhanging ends.
Collectively all such altered nucleic acids may be referred to as
analogs, and represent contemplated embodiments of the
invention.
[0190] In another embodiment, gene expression can be
inhibited/downregulated simply by "knocking out" the gene.
Typically this is accomplished by disrupting the gene, the promoter
regulating the gene or sequences between the promoter and the gene.
Such disruption can be specifically directed to a particular gene
by homologous recombination where a "knockout construct" contains
flanking sequences complementary to the domain to which the
construct is targeted. Insertion of the knockout construct (e.g.
into the gene of interest) results in disruption of that gene. The
phrases "disruption of the gene" and "gene disruption" refer to
insertion of a nucleic acid sequence into one region of the native
DNA sequence (in some embodiments, in one or more exons) and/or the
promoter region of a gene so as to decrease or prevent expression
of that gene in the cell as compared to the wild-type or naturally
occurring sequence of the gene.
[0191] Knockout constructs can be produced by standard methods
known to those of skill in the art. The knockout construct can be
chemically synthesized or assembled, e.g., using recombinant DNA
methods. The DNA sequence to be used in producing the knockout
construct is digested with a particular restriction enzyme selected
to cut at a location(s) such that a new DNA sequence encoding a
marker gene can be inserted in the proper position within this DNA
sequence. The proper position for marker gene insertion is that
which will serve to prevent expression of the native gene; this
position will depend on various factors such as the restriction
sites in the sequence to be cut, and whether an exon sequence or a
promoter sequence, or both is (are) to be interrupted (i.e., the
precise location of insertion necessary to inhibit promoter
function or to inhibit synthesis of the native exon).
[0192] It is to be understood that the above nucleic acids may be
delivered to any tissue or cells in one embodiment, in their native
form, or, in another embodiment within an expression vector that is
competent to transfect cells in vitro and/or in vivo, and comprise
an embodiment of this invention.
[0193] In another embodiment, this invention provides a method of
nucleic acid delivery, comprising contacting a cell with a polymer,
micelle or composition of this invention, comprising a nucleic acid
of interest. In one embodiment, the nucleic acid encodes for a
compound, which stimulates organogenesis, for example, the compound
is osteogenic, chondrogenic or angiogenic. In another embodiment,
the nucleic acid encodes for an antibacterial, antiviral,
antifungal or antiparasitic peptide or protein. In another
embodiment, the nucleic acid encodes for a peptide or protein with
cytotoxic or anti-cancer activity. In another embodiment, the
nucleic acid encodes for an enzyme, a receptor, a channel protein,
a hormone, a cytokine or a growth factor. In another embodiment,
the nucleic acid encodes for a peptide or protein, which is
immunostimulatory. In another embodiment, the nucleic acid encodes
for a peptide or protein, which inhibits inflammatory or immune
responses. In another embodiment, release of the nucleic acid
occurs over a period of time.
[0194] In one embodiment, the polymers, micelles or compositions of
this invention are targeted to cells. In one embodiment, the cell
may be any responsive cell, such as, in one embodiment, an
epithelial cell, a lung cell, a kidney cell, a liver cell, a
cardiocyte, an astrocyte, a glial cell, a prostate cell, a
professional antigen presenting cell, a lymphocyte, an M cell, a
pancreatic cell, a stem cell, a myoblast, a hepatocyte, an
osteoblast, an osteocyte, an osteoclast, a chondrocyte, a
chodroblast, or other bone or cartilage cells and may be used for
applications as described in, for example, Wilson, J. M et al.
(1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano, D. et
al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Wolff, J. A. et
al. (1990) Science 247:1465-1468; Chowdhury, J. R. et al. (1991)
Science 254:1802-1805; Ferry, N. et al. (1991) Proc. Natl. Acad.
Sci. USA 88:8377-8381; Wilson, J. M. et al. (1992) J. Biol. Chem.
267:963-967; Quantin, B. et al. (1992) Proc. Natl. Acad. Sci. USA
89:2581-2584; Dai, Y. et al. (1992) Proc. Natl. Acad. Sci. USA
89:10892-10895; van Beusechem, V. W. et al. (1992) Proc. Natl. Acad
Sci. USA 89:7640-7644; Rosenfeld, M. A. et al. (1992) Cell
68:143-155; Kay, M. A. et al. (1992) Human Gene Therapy 3:641-647;
Cristiano, R. J. et al. (1993) Proc. Natl. Acad Sci. USA
90:2122-2126; Hwu, P. et al. (1993) J. Immunol. 150:4104-4115; and
Herz, J. and Gerard, R. D. (1993) Proc. Natl. Acad Sci. USA
90:2812-2816.
[0195] In one embodiment, the polymers, micelles or compositions of
this invention comprise a drug. In one embodiment, the term "drug"
refers to a substance applicable for use in the diagnosis, or in
another embodiment, cure, or in another embodiment, mitigation, or
in another embodiment, treatment, or in another embodiment,
prevention of a disease, disorder, condition or infection. In one
embodiment, the term "drug" refers to any substance which affect
the structure or function of the target to which it is applied.
[0196] In another embodiment, the term "drug" refers to a molecule
that alleviates a symptom of a disease or disorder when
administered to a subject afflicted thereof. In one embodiment, a
drug is a synthetic molecule, or in another embodiment, a drug is a
naturally occurring compound isolated from a source found in
nature.
[0197] In one embodiment, drugs may comprise antihypertensives,
antidepressants, antianxiety agents, anticlotting agents,
anticonvulsants, blood glucose-lowering agents, decongestants,
antihistamines, antitussives, anti-inflammatories, antipsychotic
agents, cognitive enhancers, cholesterol-reducing agents,
antiobesity agents, autoimmune disorder agents, anti-impotence
agents, antibacterial and antifungal agents, hypnotic agents,
anti-Parkinsonism in agents, antibiotics, antiviral agents,
anti-neoplastics, barbituates, sedatives, nutritional agents, beta
blockers, emetics, anti-emetics, diuretics, anticoagulants,
cardiotonics, androgens, corticoids, anabolic agents, growth
hormone secretagogues, anti-infective agents, coronary
vasodilators, carbonic anhydrase inhibitors, antiprotozoals,
gastrointestinal agents, serotonin antagonists, anesthetics,
hypoglycemic agents, dopaminergic agents, anti-Alzheimer's Disease
agents, anti-ulcer agents, platelet inhibitors and glycogen
phosphorylase inhibitors.
[0198] In one embodiment, examples of the drugs conjugated to the
polymers of this invention, or in another embodiment, encapsulated
within a micelle of this invention, comprise, inter-alia,
antihypertensives including prazosin, nifedipine, trimazosin,
amlodipine, and doxazosin mesylate; the antianxiety agent
hydroxyzine; a blood glucose lowering agent such as glipizide; an
anti-impotence agent such as sildenafil citrate; anti-neoplastics
such as chlorambucil, lomustine or echinomycin; anti-inflammatory
agents such as betamethasone, prednisolone, piroxicam, aspirin,
flurbiprofen and
(+)-N-{4-[3-(4-fluorophenoxy)phenoxy]-2-cyclopenten-1-yl}-N-hyroxyurea;
antivirals such as acyclovir, nelfinavir, or virazole;
vitamins/nutritional agents such as retinol and vitamin E; emetics
such as apomorphine; diuretics such as chlorthalidone and
spironolactone; an anticoagulant such as dicumarol; cardiotonics
such as digoxin and digitoxin; androgens such as
17-methyltestosterone and testosterone; a mineral corticoid such as
desoxycorticosterone; a steroidal hypnotic/anesthetic such as
alfaxalone; an anabolic agent such as fluoxymesterone or
methanstenolone; antidepression agents such as fluoxetine,
pyroxidine, venlafaxine, sertraline, paroxetine, sulpiride,
[3,6-dimethyl-2-(2,4,6-trimethyl-phenoxy)-pyridin-4-yl]-(lethylpropyl)-am-
ine or
3,5-dimethyl-4-(3'-pentoxy)-2-(2',4',6'-trimethylphenoxy)pyridine;
an antibiotic such as ampicillin and penicillin G; an
anti-infective such as benzalkonium chloride or chlorhexidine; a
coronary vasodilator such as nitroglycerin or mioflazine; a
hypnotic such as etomidate; a carbonic anhydrase inhibitor such as
acetazolamide or chlorzolamide; an antifungal such as econazole,
terconazole, fluconazole, voriconazole or griseofulvin; an
antiprotozoal such as metronidazole; an imidazole-type
anti-neoplastic such as tubulazole; an anthelmintic agent such as
thiabendazole or oxfendazole; an antihistamine such as astemizole,
levocabastine, cetirizine, or cinnarizine; a decongestant such as
pseudoephedrine; antipsychotics such as fluspirilene, penfluridole,
risperidone or ziprasidone; a gastrointestinal agent such as
loperamide or cisapride; a serotonin antagonist such as ketanserin
or mianserin; an anesthetic such as lidocaine; a hypoglycemic agent
such as acetohexamide; an anti-emetic such as dimenhydrinate; an
antibacterial such as cotrimoxazole; a dopaminergic agent such as
L-DOPA; anti-Alzheimer agents such as THA or donepezil; an
anti-ulcer agent/H2 antagonist such as famotidine; a
sedative/hypnotic such as chlordiazepoxide or triazolam; a
vasodilator such as alprostadil; a platelet inhibitor such as
prostacyclin; an ACE inhibitor/antihypertensive such as enalaprilic
acid or lisinopril; a tetracycline antibiotic such as
oxytetracycline or minocycline; a macrolide antibiotic such as
azithromycin, clarithromycin, erythromycin or spiramycin; and
glycogen phosphorylase inhibitors such as
[R--(R*S*)]-5-chloro-N-[2-hydroxy-3{methoxymethylamino}-3-oxo-1-(phenylme-
thyl)-propyl]-1H-indole-2-carboxamide or
5-chloro-1-Hindole-2-carboxylic acid
[(IS)-benzyl(2R)-hydroxy-3-((3R,4S)dihydroxy-pyrrolidin-1-yl-)-oxypr-
opyl]amide.
[0199] Further examples of drugs deliverable by the invention are
the glucose-lowering drug chlorpropamide, the anti-fungal
fluconazole, the anti-hypercholesterolemic atorvastatin calcium,
the antipsychotic thiothixene hydrochloride, the anxiolytics
hydroxyzine hydrochloride or doxepin hydrochloride, the
anti-hypertensive amlodipine besylate, the antiinflammatories
piroxicam and celicoxib and valdicoxib, and the antibiotics
carbenicillin indanyl sodium, bacampicillin hydrochloride,
troleandomycin, and doxycycline hyclate.
[0200] In another embodiment a drug of this invention may comprise
other antineoplastic agents such as platinum compounds (e.g.,
spiroplatin, cisplatin, and carboplatin), methotrexate,
fluorouracil, adriamycin, mitomycin, ansamitocin, bleomycin,
cytosine arabinoside, arabinosyl adenine, mercaptopolylysine,
vincristine, busulfan, chlorambucil, melphalan (e.g., PAM, L-PAM or
phenylalanine mustard), mercaptopurine, mitotane, procarbazine
hydrochloride dactinomycin (actinomycin D), daunorubicin
hydrochloride, doxorubicin hydrochloride, paclitaxel and other
taxenes, rapamycin, manumycin A, TNP-470, plicamycin (mithramycin),
aminoglutethimide, estramustine phosphate sodium, flutamide,
leuprolide acetate, megestrol acetate, tamoxifen citrate,
testolactone, trilostane, amsacrine (m-AMSA), asparaginase
(L-asparaginase) Erwina asparaginase, interferon .alpha.-2a,
interferon .alpha.-2b, teniposide (VM-26), vinblastine sulfate
(VLB), vincristine sulfate, bleomycin sulfate, hydroxyurea,
procarbazine, and dacarbazine; mitotic inhibitors such as
etoposide, colchicine, and the vinca alkaloids,
radiopharmaceuticals such as radioactive iodine and phosphorus
products; hormones such as progestins, estrogens and antiestrogens;
anti-helmintics, antimalarials, and antituberculosis drugs;
biologicals such as immune serums, antitoxins and antivenoms;
rabies prophylaxis products; bacterial vaccines; viral vaccines;
respiratory products such as xanthine derivatives theophylline and
aminophylline; thyroid agents such as iodine products and
anti-thyroid agents; cardiovascular products including chelating
agents and mercurial diuretics and cardiac glycosides; glucagon;
blood products such as parenteral iron, hemin, hematoporphyrins and
their derivatives; biological response modifiers such as
muramyldipeptide, muramyltripeptide, microbial cell wall
components, lymphokines (e.g., bacterial endotoxin such as
lipopolysaccharide, macrophage activation factor), sub-units of
bacteria (such as Mycobacteria, Corynebacteria), the synthetic
dipeptide N-acetyl-muramyl-L-alanyl-D-isoglutamine; anti-fungal
agents such as ketoconazole, nystatin, griseofulvin, flucytosine
(5-fc), miconazole, amphotericin B, ricin, cyclosporins, and
.beta.-lactam antibiotics (e.g., sulfazecin); hormones such as
growth hormone, melanocyte stimulating hormone, estradiol,
beclomethasone dipropionate, betamethasone, betamethasone acetate
and betamethasone sodium phosphate, vetamethasone disodium
phosphate, vetamethasone sodium phosphate, cortisone acetate,
dexamethasone, dexamethasone acetate, dexamethasone sodium
phosphate, flunisolide, hydrocortisone, hydrocortisone acetate,
hydrocortisone cypionate, hydrocortisone sodium phosphate,
hydrocortisone sodium succinate, methylprednisolone,
methylprednisolone acetate, methylprednisolone sodium succinate,
paramethasone acetate, prednisolone, prednisolone acetate,
prednisolone sodium phosphate, prednisolone tebutate, prednisone,
triamcinolone, triamcinolone acetonide, triamcinolone diacetate,
triamcinolone hexacetonide, fludrocortisone acetate, oxytocin,
vassopressin, and their derivatives; vitamins such as
cyanocobalamin neinoic acid, retinoids and derivatives such as
retinol palmitate, and .alpha.-tocopherol; peptides, such as
manganese super oxide dismutase; enzymes such as alkaline
phosphatase; anti-allergic agents such as amelexanox;
anti-coagulation agents such as phenprocoumon and heparin;
circulatory drugs such as propranolol; metabolic potentiators such
as glutathione; antituberculars such as para-aminosalicylic acid,
isoniazid, capreomycin sulfate cycloserine, ethambutol
hydrochloride ethionamide, pyrazinamide, rifampin, and streptomycin
sulfate; antivirals such as amantadine azidothymidine (AZT, DDI,
Foscarnet, or Zidovudine), ribavirin and vidarabine monohydrate
(adenine arabinoside, ara-A); antianginals such as diltiazem,
nifedipine, verapamil, erythritol tetranitrate, isosorbide
dinitrate, nitroglycerin (glyceryl trinitrate) and pentaerythritol
tetranitrate; anticoagulants such as phenprocoumon, heparin;
antibiotics such as dapsone, chloramphenicol, neomycin, cefaclor,
cefadroxil, cephalexin, cephradine erythromycin, clindamycin,
lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin,
dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin,
nafcillin, oxacillin, penicillin including penicillin G and
penicillin V, ticarcillin rifampin and tetracycline;
antiinflammatories such as diflunisal, ibuprofen, indomethacin,
meclofenamate, mefenamic acid, naproxen, oxyphenbutazone,
phenylbutazone, piroxicam, sulindac, tolmetin, aspirin and
salicylates; antiprotozoans such as chloroquine,
hydroxychloroquine, metronidazole, quinine and meglumine
antimonate; antirheumatics such as penicillamine; narcotics such as
paregoric; opiates such as codeine, heroin, methadone, morphine and
opium; cardiac glycosides such as deslanoside, digitoxin, digoxin,
digitalin and digitalis; neuromuscular blockers such as atracurium
mesylate, gallamine triethiodide, hexafluorenium bromide,
metocurine iodide, pancuronium bromide, succinylcholine chloride
(suxamethonium chloride), tubocurarine chloride and vecuronium
bromide; sedatives (hypnotics) such as amobarbital, amobarbital
sodium, aprobarbital, butabarbital sodium, chloral hydrate,
ethchlorvynol, ethinamate, flurazepam hydrochloride, glutethimide,
methotrimeprazine hydrochloride, methyprylon, midazolam
hydrochloride, paraldehyde, pentobarbital, pentobarbital sodium,
phenobarbital sodium, secobarbital sodium, talbutal, temazepam and
triazolam; local anesthetics such as bupivacaine hydrochloride,
chloroprocaine hydrochloride, etidocaine hydrochloride, lidocaine
hydrochloride, mepivacaine hydrochloride, procaine hydrochloride
and tetracaine hydrochloride; general anesthetics such as
droperidol, etomidate, fentanyl citrate with droperidol, ketamine
hydrochloride, methohexital sodium and thiopental sodium; and
radioactive particles or ions such as strontium, iodide rhenium and
yttrium.
[0201] In one embodiment, the term "drug" refers to a therapeutic
compound. In one embodiment, the therapeutic compound is a peptide,
a protein or a nucleic acid. In another embodiment, the therapeutic
compound is organogenic, such as osteogenic, chondrogenic or
angiogenic. In another embodiment, the therapeutic compound is an
antibacterial, antiviral, antifungal or antiparasitic compound. In
another embodiment, the therapeutic compound has cytotoxic or
anti-cancer activity. In another embodiment, the therapeutic
compound is an enzyme, a receptor, a channel protein, a hormone, a
cytokine or a growth factor. In another embodiment, the therapeutic
compound is immunostimulatory. In another embodiment, the
therapeutic compound inhibits inflammatory or immune responses.
[0202] In one embodiment, the term "therapeutic", refers to a
molecule, which when provided to a subject in need, provides a
beneficial effect. In some cases, the molecule is therapeutic in
that it functions to replace an absence or diminished presence of
such a molecule in a subject. In one embodiment, the molecule is a
nucleic acid coding for the expression of a protein is absent, such
as in cases of an endogenous null mutant being compensated for by
expression of the foreign protein. In other embodiments, the
endogenous protein is mutated, and produces a non-functional
protein, compensated for by the expression of a heterologous
functional protein. In other embodiments, expression of a
heterologous protein is additive to low endogenous levels,
resulting in cumulative enhanced expression of a given protein. In
other embodiments, the molecule stimulates a signalling cascade
that provides for expression, or secretion, or others of a critical
element for cellular or host functioning. In one embodiment, the
therapeutic compound is a protein or polypeptide.
[0203] In one embodiment, the therapeutic protein may include
cytokines, such as interferons or interleukins, or their receptors.
Lack of expression of cytokines, or of the appropriate ones, has
been implicated in susceptibility to diseases, and enhanced
expression may lead to resistance to a number of infections.
Expression patterns of cytokines may be altered to produce a
beneficial effect, such as for example, a biasing of the immune
response toward a Th1 type expression pattern, or a Th2 pattern in
infection, or in autoimmune disease, wherein altered expression
patterns may prove beneficial to the host.
[0204] In another embodiment, the therapeutic protein may comprise
an enzyme, such as one involved in glycogen storage or breakdown.
In another embodiment, the therapeutic protein comprises a
transporter, such as an ion transporter, for example CFTR, or a
glucose transporter, or other transporters whose deficiency, or
inappropriate expression, results in a variety of diseases.
[0205] In another embodiment, the therapeutic protein comprises a
tumor suppressor, or pro-apoptotic compound, which alters
progression of cancer-related events.
[0206] In another embodiment, the therapeutic compound of the
present invention may comprise an immunomodulating protein. In one
embodiment, the immunomodulating protein comprises cytokines,
chemokines, complement or components, such as interleukins 1 to 15,
interferons alpha, beta or gamma, tumour necrosis factor,
granulocyte-macrophage colony stimulating factor (GM-CSF),
macrophage colony stimulating factor (M-CSF), granulocyte colony
stimulating factor (G-CSF), chemokines such as neutrophil
activating protein (NAP), macrophage chemoattractant and activating
factor (MCAF), RANTES, macrophage inflammatory peptides MIP-1a and
MIP-1b, or complement components.
[0207] In another embodiment, a therapeutic compound of this
invention may comprise a growth factor, or tissue-promoting factor.
In one embodiment, the therapeutic compound is a bone morphogenetic
protein, or OP-1, OP-2, BMP-5, BMP-6, BMP-2, BMP-3, BMP-4, BMP-9,
DPP, Vg-1, 60A, or Vgr-1. In another embodiment, the therapeutic
compound facilitates nerve regeneration or repair, and may include
NGF, or other growth factors.
[0208] In one embodiment, drug may also refer to a nucleic acid, or
construct comprising a nucleic acid, whose expression ameliorates
or abrogates symptoms of a disease or a disorder, or diminishes,
suppresses or inhibits a disease, disorder or condition. In one
embodiment, the nucleic acid or construct comprising the same, is
used for gene therapy, for providing or replacing endogenous
expression, or in another embodiment, suppressing endogenous
expression.
[0209] In another embodiment, the therapeutic molecule may be
natural or non-natural insulins, amylases, proteases, lipases,
kinases, phosphatases, glycosyl transferases, trypsinogen,
chymotrypsinogen, carboxypeptidases, hormones, ribonucleases,
deoxyribonucleases, triacylglycerol lipase, phospholipase A2,
elastases, amylases, blood clotting factors, UDP glucuronyl
transferases, ornithine transcarbamoylases, cytochrome p450
enzymes, adenosine deaminases, serum thymic factors, thymic humoral
factors, thymopoietins, growth hormones, somatomedins,
costimulatory factors, antibodies, colony stimulating factors,
erythropoietin, epidermal growth factors, hepatic erythropoietic
factors (hepatopoietin), liver-cell growth factors, interleukins,
interferons, negative growth factors, fibroblast growth factors,
transforming growth factors of the .alpha. family, transforming
growth factors of the .beta. family, gastrins, secretins,
cholecystokinins, somatostatins, serotonins, substance P,
transcription factors or combinations thereof.
[0210] In one embodiment, the polymers, micelles or compositions of
this invention may further comprise a ligand for a biological
target, which in another embodiment, provides for directional
specificity as to which cells or tissues are provided the polymers,
micelles or compositions of this invention. In one embodiment, the
term "ligand for a biological target" refers to a molecule which
enables the specific delivery of the polymer, micelle or
composition of this invention to a particular site in vivo. In one
embodiment, such a ligand may be referred to as an "anti-receptor",
which functions to direct the polymer or micelle to, for example,
virally infected cells, via anti-receptor binding to viral proteins
expressed on infected cell surfaces. In this case, antireceptors to
promote fusion with virally-infected cells, will recognize and bind
to virally expressed surface proteins. For example, HIV-1 infected
cells may express HIV-associated proteins, such as gp120, and
therefore the presence of CD4 on the polymer or micelle surface
promotes targeting to HIV infected cells, via CD4-gp120
interaction.
[0211] The anti-receptor proteins or polypeptide fragments thereof
may be designed to enhance fusion with cells infected with members
of the following viral families: Arenaviridae, Bunyaviridae,
Coronaviridae, Filoviridae, Flaviviridae, Herpesviridae,
Hepadnaviridae, Orthomyxoviridae, Paramyxoviridae, Poxviridae,
Retroviridae, and Rhabdoviridae. Additional viral targeting agents
may be derived from the following: African Swine Fever Virus, Boma
Disease Virus, Hepatitis X, HIV-1, Human T Lymphocyte virus type-I
(HTLV-1), HTLV-2, 1 5 lentiviruses, Epstein-Barr Virus, papilloma
viruses, herpes simplex viruses, hepatitis B and hepatitis C.
[0212] In another embodiment, targeting virally-infected cells may
be accomplished through the additional expression of viral
co-receptors on an exposed surface of the polymers/micelles of this
invention, for enhanced fusion facilitation with infected cells. In
one embodiment, the polymers/micelles of this invention comprise an
HIV co-receptor such as CXCR4 or CCR5, for example.
[0213] Bacterial proteins expressed during intracellular infection
are also potential targets contemplated for therapeutic
intervention by polymers/micelles of this invention. The
intracellular bacteria may include, amongst others: Shigella,
Salmonella, Legionella, Streptococci, Mycobacteria, Francisella and
Chlamydiae (See G. L. Mandell, "Introduction to Bacterial Disease"
IN CECIL TEXTBOOK OF MEDICINE, (W.B. Saunders Co., 1996) 1556-7).
These bacteria would be expected to express a bacteria-related
protein on the surface of the infected cell to which the
polymers/micelles of this invention would attach.
[0214] In another embodiment, the targeting moieties may include
integrins or class II molecules of the MHC, which may be
upregulated on infected cells such as professional antigen
presenting cells.
[0215] Proteins of parasitic agents, which reside intracellularly,
also are targets contemplated for targeting by the
polymers/micelles of this invention. The intracellular parasites
contemplated include for example, Protozoa. Protozoa, which infect
cells, include: parasites of the genus Plasmodium (e.g., Plasmodium
falciparum, P. Vivax, P. ovale and P. malariae), Trypanosoma,
Toxoplasma, Leishmania, and Cryptosporidium.
[0216] Diseased and/or abnormal cells may be targeted using the
polymers/micelles of this invention by the methods described above.
The diseased or abnormal cells contemplated include: infected
cells, neoplastic cells, pre-neoplastic cells, inflammatory foci,
benign tumors or polyps, cafe au lait spots, leukoplakia, other
skin moles, self-reactive cells, including T and/or NK cells, etc.
Any cell, to which specific delivery of an agent to modulate its
activity is contemplated for the methods of this invention, and
represents an embodiment thereof.
[0217] The polymers/micelles of this invention may be targeted
using an anti-receptor that will recognize and bind to its cognate
receptor or ligand expressed on a diseased or abnormal cell, in
another embodiment.
[0218] In one embodiment, the targeting agent specifically binds,
or preferentially binds only diseased cells, for delivery of a
therapeutic agent, or in another embodiment, a cytotoxic agent. In
one embodiment, the targeting agent is an antibody, or fragment
thereof. Examples of antibodies include those antibodies, which
react with malignant prostatic epithelium but not with benign
prostate tissue (e.g., ATCC No. HB-9119; ATCC HB-9120; and ATCC No.
HB-1 1430) or react with malignant breast cancer cells but not with
normal breast tissue (e.g., ATCC No. HB-8691; ATCC No. HB-10807;
and 21HB-108011). Other antibodies or fragments thereof, which
react with diseased tissue and not with normal tissue, would be
apparent to the skilled artisan.
[0219] A wide variety of tumor-specific antibodies are known in the
art, such as those described in U.S. Pat. Nos. 6,197,524,
6,191,255, 6,183,971, 6,162,606, 6,160,099, 6,143,873, 6,140,470,
6,139,869, 6,113,897, 6,106,833, 6,042,829, 6,042,828, 6,024,955,
6,020,153, 6,015,680, 5,990,297, 5,990,287, 5,972,628, 5,972,628,
5,959,084, 5,951,985, 5,939,532, 5,939,532, 5,939,277, 5,885,830,
5,874,255, 5,843,708, 5,837,845, 5,830,470, 5,792,616, 5,767,246,
5,747,048, 5,705,341, 5,690,935, 5,688,657, 5,688,505, 5,665,854,
5,656,444, 5,650,300, 5,643,740, 5,635,600, 5,589,573, 5,576,182,
5,552,526, 5,532,159, 5,525,337, 5,521,528, 5,519,120, 5,495,002,
5,474,755, 5,459,043, 5,427,917, 5,348,880, 5,344,919, 5,338,832,
5,298,393, 5,331,093, 5,244,801, and 5,169,774. See also The
Monoclonal Antibody Index Volume 1: Cancer (3rd edition).
Accordingly, the polymers, micelles and/or compositions of this
invention may comprise tumor-specific antibodies which may
recognize tumors derived from a wide variety of tissue types,
including, but not limited to, breast, prostate, colon, lung,
pharynx, thyroid, lymphoid, lymphatic, larynx, esophagus, oral
mucosa, bladder, stomach, intestine, liver, pancreas, ovary,
uterus, cervix, testes, dermis, bone, blood and brain.
[0220] In another embodiment, the polymers, micelles or
compositions of this invention will incorporate an antibody which
possesses tumoricidal activity. Antibodies that possess tumoricidal
activity are also known in the art, including IMC-C225, EMD 72000,
OvaRex Mab B43.13, anti-ganglioside G(D2) antibody ch 14.18,
CO17-1A, trastuzumab, rhuMAb VEGF, sc-321, AF349, BAF349, AF743,
BAF743, MAB743, AB1875, Anti-Flt-4AB3127, FLT41-A, rituximab, 2C3,
CAMPATH 1H, 2G7, Alpha IR-3, ABX-EGF, MDX-447, anti-p75 IL-2R,
anti-p64 IL-2R, and 2A11.
[0221] Epitopes to which tumor-specific antibodies bind are also
well known in the art. For example, epitopes bound by the
tumor-specific antibodies of the invention include, but are not
limited to, those known in the art to be present on CA-125,
gangliosides G(D2), G(M2) and G(D3), CD20, CD52, CD33, Ep-CAM, CEA,
bombesin-like peptides, PSA, HER2/neu, epidermal growth factor
receptor, erbB2, erbB3, erbB4, CD44v6, Ki-67, cancer-associated
mucin, VEGF, VEGFRs (e.g., VEGFR3), estrogen receptors, Lewis-Y
antigen, TGF/31, IGF-1 receptor, EGFa, c-Kit receptor, transferrin
receptor, IL-2R and CO17-1A. It is to be understood that antibodies
to these, and other epitopes, may be designed by methods well known
in the art, such as, for example, as described in Harlow and Lane
(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, New York (Harlow and Lane, 1988), or "Current Protocols
in Immunology" (Coligan, 1991), and may be incorporated in the
polymers, micelles and/or compositions of this invention, and
represents embodiments thereof.
[0222] In one embodiment, the targeting moiety is a peptide, an
antibody, an antibody fragment, a receptor, Protein A, Protein G,
biotin, avidin, streptavidin, a metal ion chelate, an enzyme
cofactor, a nucleic acid or a ligand.
[0223] In another embodiment, the targeting moiety is a peptide
which binds to an underglycosylated mucin-1 protein. In one
embodiment, the peptide is an EPPT1 peptide.
[0224] Mucin-1 (MUC-1) is a transmembrane molecule, which is
overexpressed on the cell surface and in intracellular compartments
of almost all human epithelial cell adenocarcinomas, including more
than 90% of human breast cancers, ovarian, pancreatic, colorectal,
lung, prostate, colon and gastric carcinomas. Expression has been
demonstrated in non-epithelial cancer cell lines (for example,
astrocytoma, melanoma, and neuroblastoma), as well as in
hematological malignancies such as multiple myeloma and some B-cell
non-Hodgkin lymphomas, constituting more than 50% of all cancers in
humans.
[0225] In one embodiment, the synthetic peptide EPPT1, also known
as alpha-M2 peptide (YCAREPPTRTFAYWG--SEQ ID NO: 1), derived from
the CDR3 Vh region of a monoclonal antibody (ASM2) raised against
human epithelial cancer cells, is used in the polymers/micelles
and/or methods of this invention.
[0226] In one embodiment, the targeting moiety enhances attachment
to a molecule, or, in another embodiment, a cell in low abundance,
which is of interest. In another embodiment, the targeting moiety
enhances attachment following supply of an energy source. In one
embodiment, the targeting moiety is chemically attached to the
polymers via a chemical cross-linking group, or in another
embodiment, forms a stable association with the polymer, or, in
another embodiment, forms an association with the polymer, which
readily dissociates following changes in solution conditions, such
as, for example, salt concentration or pH.
[0227] In one embodiment, the targeting moiety may be an antibody,
which specifically recognizes a molecule of interest, such as a
protein or nucleic acid. In another embodiment, the antibody may
specifically recognize a reporter molecule attached to a molecule
of interest. In another embodiment, the targeting moiety may be an
antibody fragment, Protein A, Protein G, biotin, avidin,
streptavidin, a metal ion chelate, an enzyme cofactor, or a nucleic
acid. In another embodiment, the targeting moiety may be a
receptor, which binds to a cognate ligand of interest, or
associated with a cell or molecule of interest, or in another
embodiment, the targeting moiety may be a ligand which is used to
attach to a cell via interaction with its cognate receptor.
[0228] In one embodiment, the term "immunoconjugate" refers to an
antibody bound to a compound. In one embodiment, the conjugation of
an antibody as described, with a polymer or encapsulated within a
micelle of this invention represents the immunoconjugates
comprising the invention. In another embodiment, the compound to
which the antibody is bound, is conjugated to a polymer or
encapsulated within a micelle of this invention, and is to be
considered as part of this invention, or in another embodiment, the
antibody, to which a compound is bound, is further conjugated to a
polymer, or encapsulated within a micelle of this invention.
[0229] In one embodiment, the term "a labeling agent" refers to a
molecule which renders readily detectable that which is contacted
with a labeling agent. IN one embodiment, the labeling agent is a
marker polypeptide. The marker polypeptide may comprise, for
example, green fluorescent protein (GFP), DS-Red (red fluorescent
protein), secreted alkaline phosphatase (SEAP), beta-galactosidase,
luciferase, or any number of other reporter proteins known to one
skilled in the art. In another embodiment, the labeling agent may
be conjugated to another molecule which provides greater
specificity for the target to be labeled. For example, and in one
embodiment, the labeling agent is a fluorochrome conjugated to an
antibody which specifically binds to a given target molecule, or in
another embodiment, which specifically binds another antibody bound
to a target molecule, such as will be readily appreciated by one
skilled in the art.
[0230] In one embodiment, the polymer may be conjugated to a
quantum dot. In one embodiment, the term "quantum dot" refers to a
semiconductor nanocrystal with size-dependent optical and
electronic properties. In particular, the band gap energy of a
semiconductor nanocrystal varies with the diameter of the crystal.
"Semiconductor nanocrystal" includes, for example, inorganic
crystallites between about 1 nm and about 1000 nm in diameter, or
in one embodiment, between about 2 nm and about 50 nm, or in
another embodiment, between about 5 nm to about 20 nm (such as
about 5. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
nm) that includes a "core" of one or more first semiconductor
materials, and which can be surrounded by a "shell" of a second
semiconductor material. A semiconductor nanocrystal core surrounded
by a semiconductor shell is referred to as a "core/shell"
semiconductor nanocrystal. The surrounding "shell" material may, in
another embodiment, have a bandgap greater than the bandgap of the
core material and can be chosen so to have an atomic spacing close
to that of the "core" substrate. The core and/or the shell can be a
semiconductor material including, but not limited to, those of the
group II-VI (e.g., ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe,
HgTe, MgTe and the like) and III-V (e.g., GaN, GaP, GaAs, GaSb,
InN, InP, InAs, InSb, AlAs, AlP, AlSb, AlS, and the like) and IV
(e.g., Ge, Si, Pb and the like) materials, and an alloy thereof, or
a mixture, including ternary and quaternary mixtures, thereof.
[0231] In one embodiment, the term "toxin" refers to a molecule
which results in toxic effects in cells and/or tissue exposed to
the toxin. In one embodiment, the toxin results in cell death, or
in another embodiment, cell damage. In one embodiment, the toxin is
a natural product of cells, such as bacterial cells, wherein the
toxin is used, in one embodiment, when specifically targeted to
disease cells as a means of selective cell killing of diseased
cells. In one embodiment, the toxin may comprise any known in the
art, such as, for example that produced by cholera, tetanus, or any
other appropriate species, as will be appreciated by one skilled in
the art.
[0232] In another embodiment, this invention also comprises
incorporation of any toxic substance for therapeutic purpose. In
one embodiment, the polymers/micelles of this invention may
incorporate an oligonucleotide encoding a suicide gene, which when
in contact with diseased cells or tissue, is expressed within such
cells. In one embodiment, the term "suicide gene" refers to a
nucleic acid coding for a product, wherein the product causes cell
death by itself or in the presence of other compounds. A
representative example of a suicide gene is one, which codes for
thymidine kinase of herpes simplex virus. Additional examples are
thymidine kinase of varicella zoster virus and the bacterial gene
cytosine deaminase, which can convert 5-fluorocytosine to the
highly cytotoxic compound 5-fluorouracil.
[0233] Suicide genes may produce cytotoxicity by converting a
prodrug to a product that is cytotoxic. In one embodiment, the term
"prodrug" means any compound that can be converted to a toxic
product for cells. Representative examples of such a prodrug is
gancyclovir which is converted in vivo to a toxic compound by
HSV-thymidine kinase. The gancyclovir derivative subsequently is
toxic to cells. Other representative examples of prodrugs include
acyclovir, FIAU
[1-(2-deoxy-2-fluoro-.beta.-D-arabinofuranosyl)-5-iodouracil],
6-methoxypurine arabinoside for VZV-TK, and 5-fluorocytosine for
cytosine deaminase.
[0234] In another embodiment, the polymers/micelles or compositions
of this invention may comprise at least one molecule, which in
another embodiment, is a protein, which is immunogenic.
[0235] In one embodiment, the term "immunogenic", refers to an
ability to elicit an immune response. Immune responses that are
cell-mediated, or immune responses that are classically referred to
as "humoral", referring to antibody-mediated responses, or both,
may be elicited by the polymers/micelles or compositions of this
invention of the present invention.
[0236] Polymers/micelles or compositions of this invention may, in
one embodiment, be used for vaccine purposes, as a means of
preventing infection.
[0237] In another embodiment, the polymers/micelles or compositions
of this invention are utilized, to provide an immunogenic protein
or polypeptide eliciting a "Th1" response, in a disease where a
so-called "Th2" type response has developed, when the development
of a so-called "Th1" type response is beneficial to the subject.
Introduction of the immunogenic protein or polypeptide results in a
shift toward a Th1 type response.
[0238] As used herein, the term "Th2 type response" refers to a
pattern of cytokine expression, elicited by T Helper cells as part
of the adaptive immune response, which support the development of a
robust antibody response. Typically Th2 type responses are
beneficial in helminth infections in a subject, for example.
Typically Th2 type responses are recognized by the production of
interleukin-4 or interleukin 10, for example.
[0239] As used herein, the term "Th1 type response" refers to a
pattern of cytokine expression, elicited by T Helper cells as part
of the adaptive immune response, which support the development of
robust cell-mediated immunity. Typically Th1 type responses are
beneficial in intracellular infections in a subject, for example.
Typically Th1 type responses are recognized by the production of
interleukin-2 or interferon .gamma., for example.
[0240] In another embodiment, the reverse occurs, where a Th1 type
response has developed, when Th2 type responses provide a more
beneficial outcome to a subject, where introduction of the
immunogenic protein or polypeptide via the polymers/micelles or
compositions of this invention provides a shift to the more
beneficial cytokine profile. One example would be in leprosy, where
the polymers/micelles or compositions of the present invention
express an antigen from M. leprae, where the antigen stimulates a
Th1 cytokine shift, resulting in tuberculoid leprosy, as opposed to
lepromatous leprosy, a much more severe form of the disease,
associated with Th2 type responses.
[0241] It is to be understood that any use of the polymers/micelles
or compositions of this invention comprising an immunogenic protein
for purposes of immunizing a subject to prevent disease, and/or
ameliorate disease, and/or alter disease progression are to be
considered as part of this invention.
[0242] Examples of infectious virus to which stimulation of a
protective immune response is desirable include: Retroviridae
(e.g., human immunodeficiency viruses, such as HIV-1 (also referred
to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other
isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses,
hepatitis A virus; enteroviruses, human coxsackie viruses,
rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause
gastroenteritis); Togaviridae (e.g., equine encephalitis viruses,
rubella viruses); Flaviridae (e.g., dengue viruses, encephalitis
viruses, yellow fever viruses); Coronaviridae (e.g.,
coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses,
rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae
(e.g., parainfluenza viruses, mumps virus, measles virus,
respiratory syncytial virus); Orthomyxoviridae (e.g. influenza
viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses,
phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever
viruses); Reoviridae (erg., reoviruses, orbiviurses and
rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus);
Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses,
polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae
(herpes simplex virus (HSV) 1 and 2, varicella zoster virus,
cytomegalovirus (CMV), herpes viruses'); Poxyiridae (variola
viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g.
African swine fever virus); and unclassified viruses (e.g., the
etiological agents of Spongiform encephalopathies, the agent of
delta hepatities (thought to be a defective satellite of hepatitis
B virus), the agents of non-A, non-B hepatitis (class 1=internally
transmitted; class 2=parenterally transmitted (i.e., Hepatitis C);
Norwalk and related viruses, and astroviruses).
[0243] Examples of infectious bacteria to which stimulation of a
protective immune response is desirable include: Helicobacter
pylori, Borellia burgdorferi, Legionella pneumophilia, Mycobacteria
sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansaii,
M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae,
Neisseria meningitidis, Listeria monocytogenes, Streptococcus
pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B
Streptococcus), Streptococcus (viridans group), Streptococcus
faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.),
Streptococcus pneumoniae, pathogenic Campylobacter sp.,
Enterococcus sp., Chlamydia sp., Haemophilus influenzae, Bacillus
antracis, corynebacterium diphtheriae, corynebacterium sp.,
Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium
tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella
multocida, Bacteroides sp., Fusobacterium nucleatum,
Streptobacillus moniliformis, Treponema pallidium, Treponema
pertenue, Leptospira, Actinomyces israelli and Francisella
tularensis.
[0244] Examples of infectious fungi to which stimulation of a
protective immune response is desirable include: Cryptococcus
neoformans, Histoplasma capsulatum, Coccidioides immitis,
Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.
Other infectious organisms (i.e., protists) include: Plasmodium
sp., Leishmania sp., Schistosoma sp. and Toxoplasma sp.
[0245] In another embodiment, the polymers/micelles or compositions
of this invention comprising an immunogenic protein further
comprise additional immunomodulating proteins.
[0246] Examples of useful immunomodulating proteins include
cytokines, chemokines, complement components, immune system
accessory and adhesion molecules and their receptors of human or
non-human animal specificity. Useful examples include GM-CSF, IL-2,
IL-12, OX40, OX40L (gp34), lymphotactin, CD40, and CD40L. Further
useful examples include interleukins for example interleukins 1 to
15, interferons alpha, beta or gamma, tumour necrosis factor,
granulocyte-macrophage colony stimulating factor (GM-CSF),
macrophage colony stimulating factor (M-CSF), granulocyte colony
stimulating factor (G-CSF), chemokines such as neutrophil
activating protein (NAP), macrophage chemoattractant and activating
factor (MCAF), RANTES, macrophage inflammatory peptides MIP-1a and
MIP-1b, complement components and their receptors, or an accessory
molecule such as B7.1, B7.2, TRAP, ICAM-1, 2 or 3 and cytokine
receptors. OX40 and OX40-ligand (gp34) are further useful examples
of immunomodulatory proteins.
[0247] In another embodiment, the immunomodulatory proteins may be
of human or non-human animal specificity, and may comprise
extracellular domains and/or other fragments with comparable
binding activity to the naturally occurring proteins.
Immunomodulatory proteins may, in another embodiment, comprise
mutated versions of the embodiments listed, or comprise fusion
proteins with polypeptide sequences, such as immunoglobulin heavy
chain constant domains. Multiple immunomodulatory proteins may be
incorporated within a single construct, and as such, represents an
additional embodiment of the invention.
[0248] It is to be understood that the polymers/micelles or
compositions of this invention may comprise multiple immunogenic
proteins. In one embodiment, the immunogenic proteins or peptides
are derived from the same or related species. Vaccine incorporation
of multiple antigens has been shown to provide enhanced
immunogenicity.
[0249] The polymers/micelles or compositions of this invention
comprising an immunogenic protein or peptide fragment may generate
immune responses of a variety of types that can be stimulated thus,
including responses against the protein or peptide itself, other
antigens that are now immunogenic via a "by-stander" effect,
against host antigens, and others, and represent additional
embodiments of the invention. It is envisioned that methods of the
present invention can be used to prevent or treat bacterial, viral,
parasitic or other disease states, including tumors, in a
subject.
[0250] Combination vaccines have been shown to provide enhanced
immunogenicity and protection, and, as such, in another embodiment,
the immunogenic proteins or peptides are derived from different
species.
[0251] In one embodiment, the incorporated groups described herein,
which are to comprise the micelles, polymers and/or compositions of
this invention, may be conjugated to the polymer, or in another
embodiment, encapsulated within.
[0252] In another embodiment, this invention provides a composition
or a micelle comprising a polymer of this invention.
[0253] This invention provides amphiphilic polymers, which in one
embodiment, are terpolymers. In one embodiment, amphiphilic
polymers allow for the formation of spherical nanoparticles, which,
in another embodiment, self-assemble into nanospheres. The polymers
of this invention, in some embodiments, offer a number of
advantages as delivery systems, as compared to other such systems
described in the art, as a result of the unique chemical structure
of the polymers of this invention.
[0254] In one embodiment, the fundamental unit of the polymers of
this invention comprises a hydrophilic segment, typically
polyethylene glycol (PEG) coupled to a multifunctional, hydrophobic
linker molecule. In one embodiment, the PEG ranges in size from
600-4,400 Daltons.
[0255] In one embodiment, the multifunctional hydrophobic linker
molecule is a trifunctional linker molecule. In one embodiment, the
linker is 5-amino dimethylphthalate or 5-hydroxydimethylphthalate.
In one embodiment, a hydrophobic side chain is attached to one of
the functional groups of the linker via an ether, ester, or amide
bond, and the side chain is terminated by a hydrogen or by a
functional group such as amino, hydroxyl, or carboxyl. This basic
unit is, in another embodiment, further polymerized to yield a base
polymer with a molecular size of 150-200,000 Da.
[0256] The polymers of this invention may assume any structural
configuration, which will be a function of, in some embodiments,
the chemical makeup of the polymers, and the environment to which
the polymer is exposed. In some embodiments, the polymers of this
invention may assume a particle configuration, comprising a core
and shell, or in another embodiment, a micelle configuration.
[0257] In one embodiment, when the polymer is dissolved in water
above the critical micelle concentration, about 8 to 12 polymeric
units may self assemble into a spherical micelle consisting of a
compact core of side chains covered by linkers with an external
corona of deformable PEG loops.
[0258] Depending upon chemical composition, the micelles have, in
some embodiments, a molecular weight of about 100-200,000 Da and a
diameter (twice the radius of gyration) of about 10 to 300 nm.
[0259] In other embodiments, additional agents can be encapsulated
in the core by dissolving the polymer and agent in a solvent,
evaporating the solvent, and dissolving the resulting viscous
mixture in water, with appropriate choice of the side chain
terminal group. According to this aspect, and in other embodiments
of this invention, a wide variety of compounds, such as, for
example, various drugs or therapeutic agents (such as, for example,
aspirin, naproxen, celebrex, inulin, insulin, and others, as
described herein, and as will be known by one skilled in the art)
are encapsulated as cargo within the micelles. In one embodiment,
incorporation of these compounds may increase micelle size up to
300 nm in diameter.
[0260] The micelle structure may be stabilized, in some
embodiments, by the water-soluble PEG at the exterior surface and
by hydrophobic interactions between the side chains and linkers.
When agents with a hydrophilic character are to be encapsulated
within the micelle, in some embodiments, the side chains are chosen
to retain sufficient hydrophobic character, as to keep the micelle
intact. The stability of the micelles to intracellular conditions,
for example, in lysozymes, can be varied by selection of coupling
between linker and side chain to obtain more or less resistance to
low pH, esterases, and other enzymes, in other embodiments.
[0261] In other embodiments, the polymers and/or micelles of this
invention may comprise a targeting agent. In one embodiment, the
polymers and/or micelles of this invention may contain a
therapeutic agent as described, and additionally comprise a
targeting agent, such that the targeting agent serves to deliver
the therapeutic agent to a desired location, for therapeutic
applications. In another embodiment, the targeting agent serves for
diagnostic and/or imaging purposes, where an agent is delivered to
a particular site, where verification of delivery is desired. In
another embodiment, the targeting agent serves to provide a
sensitive means of detection of a particular molecule at a
particular site, for example, the targeting agent directs a micelle
or polymer of this invention to a tissue which expresses a
preneoplastic marker, or a cancer associated antigen, wherein the
molecule which is being detected is available in low concentration,
and in some embodiments, is not detectable by existing methods in
the art.
[0262] In some embodiments, the targeting agent may be coupled to a
free PEG hydroxyl at an end of a base polymer chain.
[0263] In some embodiments, through the use of various PEG lengths,
linkers, side chains, and side chain terminal groups, great
flexibility in polymer/micelle chemical composition, size,
structure, and function can be obtained. In some embodiments, such
polymers/micelles may be constructed via multiple-step reaction
pathways that involve synthesis of a suitable monomer with a
protected functional group prior to the polymerization step,
followed by deprotection. In other embodiments, the synthesis may
be carried out with a chemical/enzymatic/chemo-enzymatic approach
as exemplified and described further herein.
[0264] In one embodiment, the polymers/micelles of this invention
incorporate a perfluorocarbon. In one embodiment, the
perfluorocarbon is a linear, cyclic or branched fluoroalkyl,
preferably perfluoroalkyl, radical optionally containing one or
more oxygen, nitrogen, chlorine, phosphorous, hydrogen and/or
sulfur atoms and/or one or more sulfonyl or carbonyl groups, or a
sulfonyl or carbonyl-containing fluoropolymeric group.
[0265] In one embodiment, the perfluorocarbon may be derived from
at least one fluorine-containing polymerizable monomer such as
vinyl fluoride, hexafluoropropylene, vinylidene fluoride,
trifluoroethylene, trifluorostyrene, chlorotrifluoroethylene,
perfluoro(alkyl vinyl ether), tetrafluoroethylene, or cyclic
monomers such as --CF.dbd.C(OCF.sub.3)O(CF.sub.2).sub.20-- or
--CF.dbd.CFOC(CF.sub.2).sub.20-- or mixtures thereof.
[0266] In another embodiment, sulfonyl fluoride containing monomers
are used, and may include, inter-alia,
CF.sub.2.dbd.CFOCF.sub.2CF.sub.2SO.sub.2F,
CF.sub.2.dbd.CFOCF.sub.2CFOCF.sub.2CF.sub.2SO.sub.2F,
CF.sub.2.dbd.CFOCF.sub.2CFOCF.sub.2CFOCF.sub.2CF.sub.2SO.sub.2F,
CF.sub.2.dbd.CFCF.sub.2CF.sub.2SO.sub.2F, or
CF.sub.2.dbd.CFOCF.sub.2CFOCF.sub.2CF.sub.2SO.sub.2F. In other
embodiments, fluorocarbon polymer precursors may comprise polymers
containing one or more monomers lacking sulfonyl or carbonyl halide
functional groups, but which can be modified to include sulfonyl or
carbonyl halide groups before or after forming the polymer.
Suitable monomers for such use may include trifluorostyrene,
trifluorostyrenesulfonic acid or the like.
[0267] In one embodiment, fluorocarbon polymer precursors having
pendant carbonyl-based functional groups can be prepared in any
suitable conventional manner such as in accordance with U.S. Pat.
No. 4,151,052 or Japanese patent application No. 52 (1977)38486,
which are incorporated herein by reference or polymerized from a
carbonyl functional group containing a monomer derived from a
sulfonyl group containing monomer by a method such as is shown in
U.S. Pat. No. 4,151,051 which is incorporated herein by reference.
Once prepared, such polymers may then be utilized to form the
polymers of this invention, as will be appreciated by one skilled
in the art.
[0268] A sulfonic acid form of the fluorocarbon polymer precursor
can be converted to the sulfonyl or carbonyl halide form of the
fluorocarbon polymer precursor by a process, such as described, for
example, in U.S. Pat. No. 4,209,367 which is incorporated herein by
reference
[0269] Reaction of the fluorocarbon polymer precursors with amide
or sulfonamide-containing reactants or salt thereof can be carried
out with the fluorocarbon polymer precursor being in solid form,
solvent-swollen form or in solution with the appropriate reactants
in the solid, liquid or gas phase. When the fluorocarbon polymer
precursor is in the solid form, the reaction is carried out under
anhydrous conditions by contacting it with the substituted or
unsubstituted amide or sulfonamide-containing reactant or salt
thereof in a solvent that is non-reactive with the starting
reactants. Representative suitable solvents include anhydrous polar
aprotic solvents such as acetonitrile, tetrahydrofuran, dioxane, or
the like, halogenated solvents such as chloroform, or the like. The
reaction is carried out in the presence of an organic
non-nucleophilic base in order to scavenge the halide-containing
byproduct of the reaction. Representative suitable non-nucleophilic
bases include alkylamines such as triethylamine, trimethylamine, or
the like, pyridines, alkyl pyridines, alkyl piperidines, N-alkyl
pyrrolidines, or the like, The reaction can be carried out in the
absence of a solvent under conditions where there is enough
mobility of the reactants to interact with each other such as when
the non-nucleophilic base functions as a medium for the reaction.
Other suitable halide-containing byproduct scavengers include KF,
Na.sub.2CO.sub.3, Zn powder, CsF, or the like. Reaction is effected
under anhydrous conditions such as under an inert atmosphere such
as argon, nitrogen or the like in a vessel or a glove box at a
temperature between about 0 and about 200.degree. C., or in another
embodiment, between about 25 and about 125.degree. C. Reaction
times may be, in other embodiments, between about 5 minutes and
about 72 hours, in some embodiments, between about 1 hour and about
24 hours. The reaction can be effected while mixing.
[0270] When the fluorocarbon polymer precursor is in solution, it
is contacted with the substituted or unsubstituted amide or
sulfonamide-containing reactant or salt thereof under the
conditions set forth above. The product is recovered as a solid
such as by precipitation or by removing the solvent. Representative
suitable solvents for the fluorocarbon polymer precursor include
halogenated solvents such as polychlorotrifluoroethylene, for
example Halocarbon oil, perfluoroalkylamines, for example
Fluorinert FC-70, or the like.
[0271] In one embodiment, the perfluorocarbon comprises .sup.19F.
In one embodiment, polymers comprising .sup.19F are particularly
useful in applications of this invention in imaging and
diagnostics, and offer several advantages over traditionally used
agents in such applications, in particular in magnetic resonance
imaging (MRI). .sup.19F is a magnetically active nucleus with a
relative intrinsic sensitivity 83% of .sup.1H. The normal
concentration of MRI-observable fluorine in tissue is extremely
low. Most tissue fluorine is concentrated in bone mineral as ionic
fluoride and therefore exhibits an NMR signal with solid state
(broad line) characteristics (extremely short T2) that does not
contribute to the image brightness using conventional MRI
techniques. As a consequence, use of polymers/micelles of this
invention, comprising .sup.19F in MRI will result in a
contrast-to-noise ratio that is very high as compared to the
gray-scale images typical of .sup.1H-MRI, with the quality of
.sup.19F-MRI limited only by the signal-to-noise ratio of the
acquired image.
[0272] Another useful property of .sup.19F for MRI imaging is the
linear relationship between the .sup.19F spin-lattice relaxation
rate (R.sub.1=1/T.sub.1) and local oxygen partial pressure, which
provides a means for non-invasive pO.sub.2 measurement using
.sup.19F-MRI. The increasing R.sub.1 with increasing pO2 also leads
to an increase in pixel brightness in T.sub.1-weighted .sup.19F-MR
images. This property may be exploited in various applications
using the polymers/micelles of this invention, such as, for
example, in assessing tumor growth and development (see, for
example, Song, Y., et al., NIR spectoscopy. In: Dunn and Swartz
(eds.), Oxygen Transport to Tissue XXIV, pp. 225-236: Kluwer
Academic/Plenum Publishers, 2003), in evaluating respiratory
function (see, for example, Thomas, S. R., et al. Investigative
Radiology, 32: 29-38, 1997), in ventilation (see, for example,
Laukemper-Ostendorf, S., et al. Magnetic Resonance in Medicine, 47:
82-89, 2002), and other applications (see, for example, Noth, U.,
et al., Magnetic Resonance in Medicine, 42: 1039-1047, 1999;
Williams, S. N. O., et al., Biotechnology and Bioengineering, 56:
56-61, 1997). In another embodiment, the polymers/micelles of this
invention may further find application in cancer imaging, wherein a
subject may breathe oxygen-enriched air during .sup.19F-MRI imaging
of the perfluorocarbon-containing polymers/micelles of this
invention, where increased O.sub.2 inspiration leads to a local
pO.sub.2 enhancement, or find application in measuring gastric
emptying and gastrointestinal transit time in by gavage, and/or
imaging pulmonary pathways with fluorinated gases.
[0273] In other embodiments, the polymers/micelles of this
invention and compositions comprising the same may find application
in .sup.19F-MR spectroscopy (MRS), imaging (MRI), and spectroscopic
imaging (MRSI) for in vivo quantitative metabolic mapping, as a
tool for pharmacokinetic studies, such as, for example, uptake with
the chemotherapeutic agent 5-fluorouracil and the selective
serotonin reuptake inhibitors fluvoxamine and fluoxetine and their
metabolites.
[0274] In one embodiment, .sup.19F MRI may have a conservative
detection limit of about 20 .mu.M with a 3T magnet (assuming a
linear variation of signal to noise ratio with field strength and
inversely with coil diameter), dropping to about 10 .mu.M in a 7T
magnet. Moreover, it is expected that the very short .sup.19F T1 of
140 ms (which increases the signal to noise ratio achievable in a
given scanning time) reported by Kimura, et al. (Magnetic Resonance
Imaging, 22: 855-860, 2004) will not occur in vivo, in using the
polymers/micelles of this invention. Further, in one embodiment of
this invention, additional loading of .sup.19F may be accomplished
using the micelles and compositions of this invention, enhancing
the signal.
[0275] In another embodiment, the sensitivity of MRI detection of
the .sup.19F containing polymers/micelles of this invention can
potentially be increased several fold by other approaches. The
large chemical shifts of fluorine generally result in
perfluorocarbons having complex chemical shift spectra, yielding
groups of widely separated resonances corresponding to the
different chemical environments of fluorine in these molecules.
Within the chemical shift bands, there are resolved or unresolved
isotropic homonuclear J-coupling patterns. .sup.19F images may be
plagued with multiple, at times overlapping, ghost images that
result from the convolution of the ideal images with the chemical
shift spectra. The phase modulation due to the J-coupling, which is
not refocused by 180 degree RF pulses, creates additional
artifacts. Typically, this situation is addressed, by using
chemical shift selective pulses to image only one resonance band,
thereby wastefully discarding the bulk of the potentially usable
fluorine signal.
[0276] In one embodiment, a means of overcoming the chemical shift
artifact is to use weak imaging gradients such that the projections
of different chemical shift lines do not overlap; the separate
projections may then be combined to form a single image of full
signal to noise ratio. In one embodiment, this technique is useful
with very high field magnets where the chemical shift frequency
differences are very large, or in another embodiment, in situations
where the sensitivity is low and therefore weak gradients and low
spatial resolution (which preserve the signal to noise ratio) are
needed.
[0277] In another embodiment, a means of overcoming the chemical
shift artifact is via deconvolving the chemical shift spectrum from
raw image data, as described (Busse, L. J., et al. Medical Physics,
13: 518-524, 1986).
[0278] Advantages of optical imaging methods, as described herein,
include the use of non-ionizing low energy radiation, high
sensitivity with the possibility of detecting micron-sized objects,
continuous data acquisition, and others. At the near infrared
region between 700 and 900 nm, absorption by intrinsic photoactive
biomolecules is low and allows light to penetrate several
centimeters into the tissue. Moreover, imaging in the near-infrared
(NIR) region has minimal tissue autofluorescence, which
dramatically improves the target/background ratio. Optical imaging
can be carried out at different resolutions and depth penetrations.
Fluorescence-mediated tomography (FMT) can three-dimensionally
localize and quantify fluorescent probes in deep tissues at high
sensitivity, and NIR fluorochromes may be coupled to affinity
molecules, which may serve, in other embodiments, as targeting
agents (see, for example, Becker, A., et al. Nature Biotechnology,
19: 327-331, 2001; Folli, S., et al. Cancer Research, 54:
2643-2649, 1994).
[0279] In another embodiment, the polymers/micelles of this
invention allow for the combination of different imaging
modalities.
[0280] In another embodiment, the polymers, micelles, compositions,
or combinations thereof of this invention may comprise halogens, as
described herein, such as, for example, fluorine or iodine. In one
embodiment, any isotope of the halogen may be used in the polymers,
micelles, compositions, or combinations thereof of this invention,
and according to the methods of this invention, and may find
application in various imaging means, which make use of specific
isotopes, as will be appreciated by one skilled in the art.
[0281] In one embodiment, this invention provides for the
combination of two imaging modalities which enable MR imaging using
.sup.19F or iron oxide, for example, as a contrast agent and a
fluorescent label, such as the Cy5.5 dye as a near-infrared
fluorescent (NIRF) probe. Cy5.5 can be coupled to one functional
group on the trifunctional linking molecule in place of a side
chain. Combined MR/optical probes may be used, in some embodiments,
for imaging enzymatic activity, such as for example, protease
activity as described (Josephson, L., et al. Bioconjugate
Chemistry, 13: 554-560, 2002; Kircher, M., et al. Molecular
Imaging, 1: 89-95, 2002). In some embodiments, such combination
polymers/micelles enable specific recognition of a desired tissue,
for example, produce a high resolution signal on MR images, and
allow for real-time continuous data acquisition by NIRF
imaging.
[0282] In another embodiment, the polymers are synthesized
enzymatically. In one embodiment, the enzymes used to synthesize
the polymers or micelles of this invention comprise lipases, such
as, for example Candida antarctica lipase, or in another
embodiment, lipase A, or in another embodiment, lipase B. In
another embodiment, the enzyme may comprise an esterase, or in
another embodiment, a protease, such as, for example papain or
chymotrypsin. In one embodiment, molecular weight of the
hydrophilic units is chosen such that its ability to affect
polymerization is considered. In one embodiment, the polymer is
functionalized with for example, an alkyl group of varying chain
length, comprising a polar functionality at the end of the
chain.
[0283] Polymers obtained by methods as described herein can be
characterized by methods well known in the art. For example, the
molecular weight and molecular weight distributions can be
determined by gel permeation chromatography (GPC), matrix assisted
laser desorption ionization (MALDI), and static or dynamic light
scattering. Physical and thermal properties of the polymer products
can be evaluated by thermal gravemetric analysis (TGA),
differential scanning calorimetry (DSC), or surface tensiometer;
the chemical structures of the polymers can be determined by, e.g.,
NMR (1H, 13C NMR, 1H-1H correlation, or 1H-13C correlation), IR,
UV, Gas Chromatography-Electron Impact Mass Spectroscopy (GC-EIMS),
EIMS, or Liquid Chromatography Mass Spectroscopy (LCMS).
[0284] In another embodiment, incorporation of perfluorocarbons
within the polymers, micelles and compositions of this invention
allows for the following advantages, in applications of
.sup.19F-MRI imaging: such use facilitates much higher
signal-to-noise ratio and greater sensitivity compared to protons
because of the absence of 19F background signals; fluorine is
prepared at high concentration in the form of a perfluorocarbon
contained within a unique self-assembling polymeric micelle that is
small enough to be taken up by cells; and if the micelle exterior
is functionalized with a ligand that binds to a receptor found on
most solid tumors but not on normal cells, the resulting
receptor-mediated endocytosis greatly enhances selectivity for
tumor tissue.
[0285] The structure of an embodiment of this invention, a
self-assembling, alternating copolymer micelle, is shown
schematically in FIG. 1. Each polymer, in this embodiment, consists
of a hydrophilic polyethylene glycol (PEG) segment (molecular
weight main chain 60-10,000) bound to a linker (aromatic or peptide
bond) to which a hydrophobic side chain is bound (via ether or
ester linkages) that is terminated by a hydrophobic or hydrophilic
group. When dissolved in water above the critical micelle
concentration, about 8 to 12 polymeric units self assemble into a
spherical micelle consisting of a compact core surrounded by an
outer envelope of PEG loops that provide biocompatibility. The
micelles have a molecular weight of about 100-200,000 and a
hydraulic radius ranging from about 10 to 30 nm. Additional agents
can be encapsulated in the core. These micelles can be taken up
intact by cells, as demonstrated by with micelles fluorescently
labeled on the main chain and on the cargo. Micelle synthesis in
which the side chain is a perfluorocarbon was accomplished, using
perfluoroctyl bromide, and micelles were formed having a 30 nm
radius, and containing 28% (w/v) fluorine. Additional
perfluorocarbon cargo can be encapsulated inside each micelle,
substantially increasing the fluorine content. Only 10.sup.5 of
these micelles are estimated to be needed in a cell to achieve a
concentration on the order of 1 mM, which is the minimum required
for effective .sup.19F imaging. It has been shown that
intravenously administered perfluorocarbon emulsions with diameters
3 to 4 times larger preferentially accumulate in the interstitial
space of solid tumors and can be detected using .sup.19F NMR
spectroscopy and imaging.
[0286] In another embodiment, the polymers form micelles or
nanoparticles, which range in size from 5-1000 nm. In one
embodiment, the size range is from 25-200 nm. In one embodiment,
the size range is from 30-200 nm, or in another embodiment, the
size range is from 35-200 nm, or in another embodiment, the size
range is from 40-200 nm, or in another embodiment, the size range
is from 45-200 nm, or in another embodiment, the size range is from
50-200 nm, or in another embodiment, the size range is from 75-200
nm, or in another embodiment, the size range is from 100-200 nm, or
in another embodiment, the size range is from 125-200 nm, or in
another embodiment, the size range is from 150-200 nm, or in
another embodiment, the size range is from 175-200 nm, or in
another embodiment, the size range is from 35-75 nm, or in another
embodiment, the size range is from 50-100 nm, or in another
embodiment, the size range is from 75-200 nm, or in another
embodiment, the size range is from 75-150 nm, or in another
embodiment, the size range is from 50-125 nm, or in another
embodiment, the size range is from 20-100 nm, or in another
embodiment, the size range is from 20-125 nm.
[0287] In another embodiment, the hydrophilic polymer molecular
weight may be varied. In one embodiment, the molecular weight of
the hydrophilic polymer may range from 150-200,000 Da.
[0288] In one embodiment, the compositions of this invention, which
comprise a polymer and/or micelle of this invention is
biocompatible, and in another embodiment, may comprise
pharmaceutically acceptable carriers or excipients, such as
disclosed in Remington's Pharmaceutical Sciences, Mack Publishing
Company, Easton, Pa., USA, 1985. The polymers, micelles and/or
compositions of this invention may be used in the treatment or
diagnosis of certain conditions such as in tagging, detecting
and/or removing cancer cells for example from a sample or
tissue.
[0289] In another embodiment, this invention provides a process for
producing an amphiphilic polymer comprising perfluorocarbons, the
process comprising the steps of:
[0290] contacting a dialkyl 5-hydroxy-isophthalate, a dialkyl
5-alkoxy-isophthalate, a dialkyl 5-amino-isophthalate, any
derivative thereof or any combination thereof with a polyethylene
glycol to form an amphiphilic copolymer; and
[0291] linking a perfluorocarbon to said amphiphilic copolymer,
thereby being a process for producing amphiphilic polymers
comprising perfluorocarbons.
[0292] In one embodiment, a chemo-enzymatic approach for the
synthesis is used. In one embodiment, the processes of this
invention may further comprise the step of protecting the amino
group of dialkyl 5-amino-isophthalate with an amino protecting
group.
[0293] The phrase "protecting group" as used herein means temporary
modifications of a potentially reactive functional group which
protect it from undesired chemical transformations. Examples of
such protecting groups include esters of carboxylic acids, silyl
ethers of alcohols, and acetals and ketals of aldehydes and
ketones, respectively. The field of protecting group chemistry has
been reviewed (Greene, T. W.; Wuts, P. G. M. Protective Groups in
Organic Synthesis, 2nd ed.; Wiley: New York, 1991).
[0294] In another embodiment, the processes of this invention may
further comprise the step of protecting the hydroxy group of a
dialkyl 5-hydroxy-isophthalate with an hydroxy protecting
group.
[0295] In one embodiment, enzymatic polymerization of a hydrophilic
with a multifunctional linking molecule to form the copolymer
backbone is conducted initially. In one embodiment, the linking
moiety is dissolved in the hydrophillic liquid without any
additional solvent, enzyme is added, and polymerization is carried
out at high temperature, for example at about 90.degree. C., under
vacuum.
[0296] In one embodiment, the process of this invention comprises
synthesis of a polymer comprising a perfluorocarbon, wherein the
perfluorocarbon is linked to a hydroxyl group, amino group, or
combination thereof of the isophthalate, and in one embodiment, the
attachment of perfluorocarbon to isophthalate is via an enteric
bond, an amide bond, or a combination thereof.
[0297] In one embodiment, the synthesis of the basic polymer takes
place in two steps, which comprise, inter-alia, attachment of a
targeting agent and/or labeling agent subsequent to the basic
polymer formation.
[0298] In one embodiment, the hydrophilic moiety is a PEG oligomer
(n=10-34) and the multifunctional linker is dimethyl
5-hydroxyisophthalate.
[0299] According to this aspect, and in one embodiment, the
reaction is a trans-esterification, and the methanol formed during
the reaction is removed under vacuum. In one embodiment, the method
employs the use of lipase B from Candida antartica, and takes
advantage of the regioselectivity of the enzyme, such that the
phenolic group does not take part in the polymerization, thereby
giving a polymer with a reactive functional group. This reactive
functional group, in turn, may be used for further chemical
reactions, in this case attachment of a hydrophobic group with
either an ether or ester linkage using standard group replacement
chemistry, as will be appreciated by one skilled in the art.
[0300] In one embodiment, the enzyme may be immobilized within
porous poly(methyl methacrylate) beads, such as, for example, that
available as Novozyme 435 from Novozyme A/S).
[0301] In other embodiments, any number of multifunctional linkers
may be used, such as, for example, those with hydroxy or amino
functional group and a variety of hydrophobic moieties may be
attached with and without an additional terminal functional group.
For example, and in other embodiments, polymers comprising linkers
such as dimethyl 5-amino isophthalate, amino malonic acid,
aspartatic and glutamic acid as linkers, have been attached to
hydrocarbon chains which have a functionality of hydroxy, carboxy,
amino, or guanidinyl groups at the end of the chain, by methods
well known in the art [see for example, Kumar, R., et al. Journal
of the American Chemical Society, 126: 10640-10644, 2004; Kumar,
R., et al., Green Chemistry, 6: 516-520, 2004; Kumar, R., et al.,
Journal of Macromolecular Science: Pure and Applied Chemistry, A40:
1283, 2003; Tyagi, R., et al., Polymer Preprint, 44: 778, 2003;
Sharma, S. K., et al. Polymer Preprint, 44: 791, 2003; Sharma, S.
K., et al., Journal of Macromolecular Science: Pure and Applied
Chemistry, A41: 1459, 2004, all of which are incorporated herein by
reference].
[0302] In one embodiment, the linkage at the aromatic oxygen may be
an ester or ether linkage. For example, if an aminophthalate is
used, the connection of the side chain is an amide link.
[0303] In other embodiments, the length of the PEG or hydrophilic
segment may be varied over a wide range, such as disclosed, for
example in Kumar, R., et al. Journal of Macromolecular Science,
A39: 1137-1149, 2002, such that the hydrophilicity/hydrophobicity
ratio for the polymer may be controlled. In other embodiments, the
polymerization conditions may be controlled, such that a structure
is obtained in which hydroxyl groups of the PEG component are
available on both ends of the polymer chain, which in other
embodiments, may be used for subsequent chemical modification.
These hydroxyl groups may be used, in other embodiments, to attach
a targeting agent, such as for example, the peptide EPPT1, as
exemplified herein, or, in another embodiment, a labeling agent,
such as, for example, a fluorescent compound.
[0304] The polymers may form micelles, when in solution.
Characterization of the polymers in aqueous solution with light
scattering techniques, demonstrates formation of nanoparticles via
a self-assembly process with a PEG external surface and a
hydrophobic internal cavity. The ratio of the radius of gyration,
Rg, (static light scattering) to the hydrodynamic radius, Rh,
(dynamic light scattering) is about 1.75, indicating that the
nanoparticles correspond to a hollow spheroidal structure.
Attachment of functional groups at the end of the hydrophobic
chains allows modification of the cavity of these nanospheres,
which affects their size and stability as well as the nature of
cargo that can be encapsulated. Static light Scattering of the
nanospheres gives Rg in the range of 10-80 nm. The size of the
nanospheres and their stability are influenced by the length of the
PEG oligomers and the nature of the hydrophobic group. The
incorporation of hydrophobic side chains may add to the stability
of the micelles.
[0305] In one embodiment, the polymers will have a molecular weight
of around 200,000 Da and contain 10-12 copolymer chains per
nanosphere, each about 20,000 Da in molecular weight.
[0306] A wide variety of small molecules including drugs may be
encapsulated within the micelles of this invention. Larger
molecules such as proteins (insulin) and polysaccharides (inulin)
have also been encapsulated since the nanospheres may adjust to the
size of the encapsulant molecule in the self assembly process. In
order to encapsulate smaller molecules, a protocol as described
(Kumar, R., et al., Journal of the American Chemical Society, 126:
10640-10644, 2004; Sharma, S. K., et al., Chemical Communication
23: 2689-2691 2004) may be used. The polymer and cargo are
dissolved together in an organic solvent, such as chloroform, and
then the solvent is evaporated to dryness. The residue is dissolved
in water and any unencapsulated material removed by filtration. The
aqueous solution is freeze-dried, kept until needed, and then
reconstituted with water to give clear solutions of the
encapsulated material. The amount of encapsulant as a fraction by
weight may be determined by several methods. When the UV
absorptivity of the encapsulant is sufficiently different from the
polymer, UV spectroscopy may be used. In other cases, .sup.1H-NMR
may be used, as described in Sharma, supra. Typically, a ratio of
1:4 or 1:5 cargo to polymer weight ratios are used. As the ratio
increases, the fractional mass of the cargo increases, and the
nanoparticle size increases until a maximum is reached.
[0307] The choice of starting reagents used to construct the
polymers of this invention may be tailored, for example, for the
attachment of different types of pendant groups, for example to a
hydroxyl group, including alkyl or alkenyl chains, aryl groups,
carboxyl-containing groups, amino groups, ammonium groups, and/or
additional hydroxyl groups. In another embodiment, appropriate
choice of the pendant group functionalities, enables enhanced
polymer interaction with incorporated molecules, such as
therapeutic compounds, fluorochromes, perfluorocarbons, etc., for
optimal conjugation of the various functional groups herein
described.
[0308] For example, a carboxyl-containing functional pendant group
can interact with nitrogen bases (e.g., primary, secondary, or
heterocyclic amines), and can form Schiff bases under appropriate
conditions. By choosing appropriate encapsulation conditions, the
resulting structure can be formed in such a way that the drug is
well held in the core of the micelle, protected from the
physiological milieu. As another example, a carboxylic acid group
on the drug can be ion-paired with a pendant amine (e.g., a
secondary or tertiary amine). The resulting ion pair can be formed
in such a fashion that it resides substantially within the core of
the micelle. Such pendant groups can be incorporated into the
polymer with relative ease, using well-known synthesis methods.
Thus, the polymers can be readily tailored to create vehicles that
meet the specific requirements of a given guest drug molecule, for
example, and in one embodiment, or any other molecule for delivery,
using the polymers and/or micelles of this invention.
Synthesis of Fluorine-Containing Nanoparticles
[0309] Fluorine incorporation into the base copolymer may be via
any number of standard methods of formation of ester or ether
linkages to attach a perfluorinated chain. For example, and in one
embodiment of this invention, the amphiphilic copolymer (with PEG,
n=15) is mixed with perfluoro octanoyl chloride under basic
conditions to attach an acyl perfluoro group to a phenolic moiety
as the hydrophobic side chain. The attachment may be confirmed with
IR spectroscopy and .sup.19F-NMR. A fluorine-modified polymer thus
formed demonstrated nanoparticles with an Rg of about 75 nm, as
determined by static light scattering. It contained 28% (w/w)
fluorine, corresponding to about 3,800 .sup.19F atoms per
nanoparticle.
[0310] The amphiphilic copolymers with perfluorocarbon side chains
were then used to further encapsulate 1,1,2,2,-tetrahydro
perfluorododecanol (20% w/w) using the same procedure as described
above. The amount of perfluorocarbon cargo encapsulated by the
fluorinated polymer was determined by integration of fluorine NMR
spectra. The entire particle contained 42% (w/w) fluorine,
corresponding to almost 6,000 .sup.19F atoms per nanoparticle.
Loading may be increased by at least a factor of two to 12,000
.sup.19F atoms per nanoparticle. Assuming a cell volume of 10.sup.3
.mu.m.sup.3, uptake of 10.sup.5, 10.sup.6, or 10.sup.7 of the
nanoparticles per cell is obtainable, and result in cellular
fluorine concentrations of about 2, 20, or 200 mM, respectively,
amounts sufficient for efficient imaging, in clinical settings.
[0311] The physical and chemical properties of the
polymers/microspheres of this invention may readily be determined
with standard techniques such as IR spectroscopy, NMR spectroscopy,
gel permeation chromatography, and light scattering (dynamic and
static).
[0312] In one embodiment, the targeting agent is a peptide, which
in one embodiment binds to an underglycosylated mucin-1 protein,
which in one embodiment is EPPT1, as described herein. In one
embodiment, the EPPT1 peptide is based on the CDR 3 VH and
framework regions of the idiotype of a murine antitumor monoclonal
antibody ASM2 directed against the polymorphic epithelial human
mucin epitope (Hussain, R., et al. Peptides: Chemistry, Structure,
and Biology. Proceedings of the 14th American Peptide Symposium,
England, 1996, pp. 808-809).
[0313] In one embodiment, synthesis of the polymer comprising the
EPPT1 peptide will comprise polymerization with two linkers, one of
which will be in a small amount (1-5%) to give the polymer as shown
in FIG. 6. The synthesis is conducted such that PEG hydroxy groups
are at the ends of the chain, and perfluorocarbon side chains are
attached to the linker hydroxyls by standard acylation procedures
to form the ester linkage with the polymer backbone. Numerous
fluorine-containing polymers may be prepared via this route,
including the formation of (CF.sub.2).sub.8CF.sub.3,
(CF.sub.2).sub.6CF.sub.3, (CF.sub.2).sub.3CF.sub.3,
CH.sub.2OCH.sub.2(CF.sub.2).sub.8CF.sub.3,
CH.sub.2OCH.sub.2(CF.sub.2).sub.6CF.sub.3,
CH.sub.2OCH.sub.2(CF.sub.2).sub.4CF.sub.3 or
CH.sub.2OCH.sub.2CH.sub.2(CF.sub.2).sub.11CF.sub.3. In other
embodiments, the synthetic processes of this invention are highly
flexible, enabling the variation of the chain length (from 5 to 13
carbons) and the relative number of fluorine atoms to alter the
hydrophobicity of the side chain.
[0314] The effect of any of the parameters on
polymer/micelle-loading and stability may be evaluated, by any
number of methods known to one skilled in the art, and a number of
encapsulating materials may be evaluated concurrently, including
perfluorodecalin, bromo-perfluoroheptane, and perfluoro-crown
ether. Cy5.5 may be attached to the polymers to enable NIRF
determination, EPPT1 peptides for targeting, and radioiodine for
cell binding and biodistribution studies.
[0315] In one embodiment, the process of this invention comprises
linking a perfluorocarbon to the amphiphilic copolymer via,
inter-alia, converting the amino group (--NH2) of the isophthalate
to --NH--R1, wherein R1 is as defined herein.
[0316] In one embodiment, the process of this invention comprises
linking a perfluorocarbon to an amphiphilic copolymer via,
inter-alia, alkylating the hydroxy group (--OH) of the isophthalate
to produce --(CH.sub.2).sub.qCO--R.sub.2, wherein R.sub.2 is as
defined herein.
[0317] In another embodiment, this invention provides a polymer or
micelle, or composition comprising a product of a process of this
invention.
[0318] In another embodiment, this invention provides a method of
imaging a cell, the method comprising the steps of contacting a
cell with an amphiphilic polymer of this invention and imaging said
cell, whereby said polymer enables the imaging of said cell.
Attachment of Fluorescent Probe
[0319] Fluorochromes may readily be attached to a polymer of this
invention, and represent an embodiment thereof. As exemplified
herein, Rhodamine B was converted to its acid chloride using oxalyl
chloride. Treatment of the polymer (substituted with a decane chain
as the hydrophobic group) with the acid chloride and base formed an
ester linkage with the CH.sub.2OH groups at the ends of the polymer
chains, binding it covalently to the polymer, and attachment did
not interfere with nanosphere formation as determined by light
scattering.
[0320] Neuroblastoma cells incubated with nanospheres with
Rhodamine B attached to the polymer, and brilliant green loaded
within the spheres, showed nanosphere polymer and cargo penetrated
cells, as evidenced by colocalization of the two fluorescent
signals, indicating that the nanoparticles entered the cell with
its cargo intact.
[0321] In one embodiment, a fluorescent molecule is attached to a
polymer of this invention. In one embodiment, the fluorescent
molecule is Cy5.5. In one embodiment, Cy5.5 is attached to amine
groups of the base polymer and non-reacted dye may be removed by
any number of conventional means, such as, for example, via column
chromatography.
[0322] In another embodiment, the fluorescent molecule may be
introduced within a targeting moiety which is coupled to a polymer
of this invention. For example, an EPPT1 peptide
(YCAREPPTRTFAYWG-SEQ ID NO: 1) is modified to introduce a FITC
label, to produce a final peptide with the following sequence:
Y-C(ACM)-A-R-E-P-P-T-R-T-F-A-Y-W-G-K(FITC)K (SEQ ID NO: 2).
[0323] In one embodiment, peptides of this invention may be
purified from appropriate sources, or in other embodiments, may be
synthesized, by means well known in the art. In one embodiment,
peptides may be synthesized on an automatic synthesizer using Fmoc
chemistry with HBTU and HOBT. They may be further purified by C18
reverse phase HPLC. Molecular weight may be determined by MALDI
mass spectroscopy.
[0324] In one embodiment, both the targeting moiety and the polymer
may be labeled with fluorescent markers, or, in another embodiment,
any other agent, as described. In one embodiment, such conjugation
may be accomplished by any number of methods known in the art, such
as, for example, that of Zalipsky, et. al. (Advanced Drug Delivery
Reviews, 54: 459-476, 2002), or Roberts, M. J. et al. Advanced Drug
Delivery Reviews, 54: 459-476, 2002).
[0325] In one embodiment, polymers or micelles of this invention
may be radiolabeled. For example, incorporation of Na .sup.125I may
be accomplished using the Iodogen method (Pierce, Rockford, Ill.)
using available Tyr within the peptide sequence, in conjugated
polymers. In another embodiment, the basic polymer backbone may be
radiolabeled with the same procedure via substitution of the
isophthalate ring similar to that of the tyrosine aromatic ring, in
peptide or protein-conjugated polymers.
[0326] In another embodiment, the methods of this invention are
directed to the imaging of individual cells, a group of cells, a
tissue, an organ or a combination thereof.
[0327] In one embodiment, imaging is accomplished with computed
tomography, computed radiography, magnetic resonance imaging,
fluorescence microscopy, angiography, arteriography, or a
combination thereof. In one embodiment, a cell is contacted with a
polymer of this invention, ex-vivo, and is subsequently implanted
in a subject. In one embodiment, the cell is inter-alia, labeled
with a labeling agent as described herein, and may further comprise
a therapeutic compound, and/or in another embodiment, the
therapeutic compound is labeled with a labeling agent, and in one
embodiment, the delivery of the cell and/or therapeutic compound
may be verified by imaging the labeling agent.
[0328] In one embodiment, the imaging methods of this invention are
conducted on a subject. In another embodiment, the imaging methods
are conducted on a sample taken from a subject. In one embodiment,
the subject has or is suspected of having cancer, or in another
embodiment, atherosclerotic lesions, or in another embodiment, is
infected, or in another embodiment, has ischemica.
[0329] In one embodiment, the imaging methods as described herein
may comprise near infrared fluorescence imaging. In one embodiment,
an advantages of such optical imaging methods may include the use
of non-ionizing low energy radiation, high sensitivity with the
possibility of detecting micron-sized objects, continuous data
acquisition, and the development of potentially cost-effective
equipment. Optical imaging can be carried out at different
resolutions and depth penetrations. Fluorescence-mediated
tomography (FMT) can three-dimensionally localize and quantify
fluorescent probes in deep tissues at high sensitivity. Several NIR
fluorochromes have recently been coupled to affinity molecules
(Becker, A., et al. Nature Biotechnology, 19: 327-331, 2001; Folli,
S., et al Cancer Research, 54: 2643-2649, 1994, and can be adapted
to comprise the polymers or micelles of this invention, as will be
appreciated by one skilled in the art.
[0330] In one embodiment, the imaging methods as described herein
may comprise nuclear imaging methods. Nuclear imaging is based on
labeling molecules with a radioactive atom before their release in
the system under study. Since photons of relatively high energy
(>80 keV) can escape from the human body, it is possible to
follow over time the 3D spatial distribution of the radioactive
tracer through detection of the emitted radiation. A large variety
of isotopes can be imaged. Their broadest classification is perhaps
that in gamma and positron emitters: the former family is at the
basis of single photon emission methods (such as planar
scintigraphy and tomography, or SPECT), and the latter is used in
Positron Emission Tomography (PET). Unlike in MRI or computed
tomography (CT), the signal detected in nuclear imaging techniques
is the radioactive emission of a single atom. Because these
emissions are specific to the radioisotope used, and because it is
possible with standard physics instrumentation to detect the
emission of a single atom, nuclear imaging enjoys the advantages of
both high specificity and sensitivity. Structural information,
however, may be obtained only as far as the radiotracer
redistributes following anatomical structures. Resolution of
clinical scanners may be limited to about 5-6 mm for PET and
.about.1 cm for SPECT, thus, nuclear imaging methods are often used
to complement the information provided by CT and/or MRI scans in
the context of multimodality imaging, and may be applied in this
manner herein, representing an embodiment of this invention. In one
embodiment, nuclear imaging is used in particular because of its
sensitivity to extremely small quantities of matter. For example,
it has recently been estimated that PET can detect as few as a
cluster of 250 cells each bearing 30 Bq of .sup.18F, which
corresponds to 2.1 fg.
[0331] While PET techniques achieve good resolution with high
sensitivity (2-4%), common positron emitters such as .sup.18F has a
relatively short half-life, which may affect it's widespread
applicability. In one embodiment, however, nanoparticle
encapsulation as described herein, may lengthen this half-life and
enhance it's applicability.
[0332] In another embodiment, different iodine isotopes can be
chosen for radioactive labeling of compounds. In one embodiment,
.sup.123I, .sup.125I and .sup.131I can be used to obtain molecules
with the same chemical and biological characteristics but different
imaging and dosimetric properties. .sup.131I In one embodiment, the
isotope for imaging is .sup.123I (159 keV), or in another
embodiment, 37 MBq of .sup.123I-MIBG, which results in an exposure
to a radiation dose no higher than 1.8 MBq of .sup.131I-MIBG.
[0333] In radioimmunotherapy (RIT), cytotoxic radiation from
therapeutic radioisotopes is delivered to tumors via antibodies or
peptides that bind to tumor-specific or tumor-associated antigens
(116). Radioactive metal ions can be attached to an antibody
through a metal chelating agent (117). One advantage for RIT over
other immunotherapies, such as immunotoxins, is that there is no
need to target every tumor cell to cause an antitumor effect at the
cellular level because nontargeted cells can be irradiated and
often killed by radiation from targeted neighboring cells. With
immunotoxins, each tumor cell must be targeted for the antitumor
effect to occur at the cellular level (116).
[0334] In another embodiment, some of the radioisotopes may serve a
dual purpose, such as, in one embodiment, for imaging the sites to
which the radioisotope is delivered, and in another embodiment, as
part of radiotherapy, including radioimmunotherapy. In one
embodiment, .sup.131I and .sup.90Y are used. .sup.131I, in one
embodiment, may be attached to an antibody or peptide by simple
techniques (such as the IODOGEN or chloramine-T methods), and may
be imaged by instrumentation which detects .gamma.-emission, while
.beta.-emission serves for therapeutic application in the
subject.
Delivery of Therapeutic Compounds
[0335] The micelles of this invention may be used to encapsulate
any number of therapeutic agents, individually or in combination.
Some examples of therapeutic compounds are described herein, such
as, for example, non-steroidal anti-inflammatory drugs such as
aspirin and naproxen, or others as described hereinabove. In one
embodiment, the terms "drugs" and "therapeutic compound" are
interchangeable, and refer, in some embodiments to compounds
producing symptom palliative effects, delay in severity of symptoms
or disease progression, inhibition of disease, or any positive
effect attributable to the therapy, or a combination thereof.
[0336] Delivery to a subject through various routes, for example,
intravenously, intramuscularly, topically, etc., which may vary, in
some embodiments, as a function of the desired site of delivery, or
timing, or combination thereof.
[0337] Any number of assays may be utilized in order to verify that
the drugs are delivered to the appropriate site, and are
functional, and such assays will be tailored for the particular
drug utilized As an example, a human cell line such as OM10.1
(Butera et al., AIDS Res. Hum. Retroviruses, 8:991-995, 1992),
which is chronically infected with HIV-1, may be used to test
antiviral activities of polymer encapsulated anti-HIV drugs, which
is one embodiment of this invention. Such an assay may be conducted
as described, in for example, Critchfield et al., AIDS Res. Hum.
Retroviruses, 12:39-46, 1996). Anti-viral effects can be determined
through a variety of assays, including measuring HIV-1 p24 antigen
levels, for example, using a commercially available ELISA kit
(Coulter), and for reverse transcriptase (RT) activity, using a
commercially available chemiluminescent ELISA RT assay such as that
sold by Boehringer Mannheim, each according to the manufacturer's
instructions. Inhibition of viral cell-to-cell spread may be
measured, in another embodiment, serving as an indicator of
anti-viral efficacy, using a model system, for example, as
described (Rabin et al, 1996; Sato et al., 1992).
[0338] It is to be understood that any assay for measuring a
particular activity which is modulated by the therapeutic compound
may be employed, as a means of determining the efficacy of the
compound, in one embodiment, optimal loading of the compound, in
another embodiment, timing and dosage, in another embodiment, or a
combination thereof.
Targeting of Specific Agents Using the Polymers and Micelles of
this Invention
[0339] FITC-labeled EPPT1 peptide-conjugated micelles were
exemplified herein. Any number of cells or cell lines may be
incubated with the tagged molecules and targeting of desired cells
and/or uptake may be demonstrated by conventional means, including
microscopy, FACS analysis, western blot analysis, and others.
[0340] In vivo imaging can be readily performed on subjects exposed
to labeled polymers/micelles. MR-imaging or NIRF analysis may be
used, as well as fluorescence microscopy of excised target tissue,
the images of which may be compared to those obtained by MIR or
NIRF.
[0341] In another embodiment, this invention provides a method of
targeted delivery of at least one agent in a subject comprising the
steps of administering to said subject an amphiphilic polymer of
this invention, wherein said polymer comprises said agent and a
targeting agent.
[0342] In another embodiment, this invention provides a method for
detecting neoplastic cells in a subject, comprising contacting a
cell in, or a cell derived from said subject with an effective
tumor-detecting amount of an amphiphilic polymer of this invention,
wherein said polymer comprises a targeting moiety specific for
neoplastic cells; and detecting any of said polymer associated with
neoplastic cells present in said subject.
[0343] In another embodiment, this invention provides a method of
imaging a cell, the method comprising the steps of contacting a
cell with an amphiphilic polymer of this invention and imaging said
cell, whereby said polymer enables the imaging of said cell.
[0344] In another embodiment, this invention provides a method of
targeted delivery of at least one agent in a subject comprising the
steps of administering to said subject an amphiphilic polymer of
this invention, wherein said polymer comprises said agent and a
targeting agent.
[0345] In one embodiment, multiple targeting moieties, may be
incorporated in the polymers or micelles of this invention. In one
embodiment, multiples of the same targeting moiety will be
incorporated, or in another embodiment, multiple targeting
moieties, which target the same cell or tissue, may be
incorporated.
[0346] In another embodiment, this invention provides a method for
detecting neoplastic cells in a subject, comprising contacting a
cell in, or a cell derived from said subject with an effective
tumor-detecting amount of an amphiphilic polymer of this invention,
wherein said polymer comprises a targeting moiety specific for
neoplastic cells; and detecting any of said polymer associated with
neoplastic cells present in said subject.
[0347] As used herein, the term "contacting a target cell" refers
to both direct and indirect exposure of the target cell to a
polymer, micelle or composition of this invention. In one
embodiment, contacting a cell may comprise direct injection of the
cell through any means well known in the art, such as
microinjection. It is also envisaged, in another embodiment, that
supply to the cell is indirect, such as via provision in a culture
medium that surrounds the cell.
[0348] Protocols for introducing the polymers, micelles or
compositions of the invention to cells and subject may comprise,
for example: direct uptake techniques, injection, receptor-mediated
uptake (for further detail see, for example, "Methods in
Enzymology" Vol. 1-317, Academic Press, Current Protocols in
Molecular Biology, Ausubel F. M. et al. (eds.) Greene Publishing
Associates, (1989) and in Molecular Cloning: A Laboratory Manual,
2nd Edition, Sambrook et al. Cold Spring Harbor Laboratory Press,
(1989), or other standard laboratory manuals), and others, as will
be appreciated by one skilled in the art. It is to be understood
that any direct means or indirect means of intracellular access of
polymers, micelles or compositions of the invention is contemplated
herein, and represents an embodiment thereof.
[0349] In one embodiment, the cell which is targeted for uptake of
a polymer, micelle or composition of this invention may include any
epithelial cell, muscle cell, nerve cell, lung cell, kidney cell,
liver cell, astrocyte, glial cell, prostate cell, professional
antigen presenting cell, lymphocyte, M cell, or any other cell in
the body, where the polymers or micelles or compositions of this
invention may be useful.
[0350] In one embodiment, the polymers or micelles or compositions
of this invention may be administered in any effective, convenient
manner including, for instance, administration by intravascular
(i.v.), intramuscular (i.m.), intranasal (i.n.), subcutaneous
(s.c.), oral, rectal, intravaginal delivery, or by any means in
which the polymers or micelles or compositions of this invention
can be delivered to tissue (e.g., needle or catheter).
Alternatively, topical administration may be desired for insertion
into epithelial cells. Another method of administration is via
aspiration or aerosol formulation.
[0351] For administration to mammals, and particularly humans, it
is expected that the physician will determine the actual dosage and
duration of treatment, which will be most suitable for an
individual and can vary with the age, weight and response of the
particular individual.
[0352] According to this aspect of the invention, the disease for
which the subject is thus treated may comprise, but is not limited
to: muscular dystrophy, cancer, cardiovascular disease,
hypertension, infection, renal disease, neurodegenerative disease,
such as alzheimer's disease, parkinson's disease, huntington's
chorea, Creurtfeld-Jacob disease, autoimmune disease, such as
lupus, rheumatoid arthritis, endocarditis, Graves' disease or ALD,
respiratory disease such as asthma or cystic fibrosis, bone
disease, such as osteoporosis, joint disease, liver disease,
disease of the skin, such as psoriasis or eczema, ophthalmic
disease, otolaryngeal disease, other neurological disease such as
Turret syndrome, schizophrenia, depression, autism, or stoke, or
metabolic disease such as a glycogen storage disease or diabetes.
It is to be understood that any disease whereby expression of a
particular protein, provision of a therapeutic protein, provision
of a drug, inhibition of expression of a particular protein, etc.,
which can be accomplished via the use of the polymers, micelles or
compositions of this invention is sought, is to be considered as
part of this invention.
[0353] The following examples are presented in order to more fully
illustrate some embodiments of the invention. They should, in no
way be construed, however, as limiting the scope of the
invention.
EXAMPLES
Example 1
Synthesis and Characterization of Novel Multi-Modal Micelle
Nanoparticle
[0354] A chemo-enzymatic approach (Kumar, R., et al. Journal of
Macromolecular Science, A39: 1137-1149, 2002; Kumar, R., et al.
Journal of the American Chemical Society, 126: 10640-10644, 2004)
was developed for the design and synthesis of a complex polymer
structure that forms nanospheres, which in one embodiment possesses
targeted multi-modal imaging capability. The polymer self-assembles
into spherical nanoparticles. Synthesis of the basic polymer takes
place in two steps, following which, in one embodiment of the
invention, attachment of a targeting peptide and fluorescent moiety
may occur subsequent to the basic polymer formation.
[0355] Enzymatic polymerization of a PEG oligomer (n=10-34) with a
trifunctional linking molecule (dimethyl 5-hydroxyisophthalate) is
conducted and results in the formation of a copolymer backbone
(FIG. 1). The phthalate is dissolved in liquid PEG without any
additional solvent, enzyme is added, and the polymerization is
carried out at 90.degree. C. under vacuum, as described (Kumar, R.,
et al. Journal of Macromolecular Science, A39: 1137-1149, 2002;
Kumar, R., et al. Journal of the American Chemical Society, 126:
10640-10644, 2004). The reaction is a transesterification, with the
methanol formed removed under vacuum. The method takes advantage of
the regioselectivity of the enzyme (lipase B from Candida antartica
immobilized within porous poly(methyl methacrylate) beads,
available as Novozyme 435 from Novozyme A/S) such that the phenolic
group does not take part in the polymerization, thereby giving a
polymer with a reactive functional group. This reactive functional
group may be used for further chemical reactions, in this case,
attachment of a hydrophobic group with either an ether or ester
linkage using standard group replacement chemistry.
[0356] The generality of the method enables utilization of a number
of trifunctional linkers with hydroxy or amino groups as the
remaining functional group and attaching a variety of hydrophobic
moieties with and without an additional terminal functional group.
Specifically, dimethyl 5-amino isophthalate (Kumar, R., et al.
Biocatalytic "Green" synthesis of PEG-based aromatic polyesters:
optimization of the substrate and reaction conditions. Green
Chemistry, 6: 516-520, 2004), amino malonic acid (Kumar, R., et al.
Journal of Macromolecular Science: Pure and Applied Chemistry, A40:
1283, 2003), and aspartatic and glutamic acid (Tyagi, R., et al.
Polymer Preprint, 44: 778, 2003) have been used as linkers, and
hydrocarbon chains which have a functionality of hydroxy (Kumar,
R., et al. Journal of the American Chemical Society, 126:
10640-10644, 2004), carboxy (Kumar, R., et al. Journal of the
American Chemical Society, 126: 10640-10644, 2004), amino (Sharma,
S. K., et al. Polymer Preprint, 44: 791, 2003), or guanidinyl
(Sharma, S. K., et al. Journal of Macromolecular Science: Pure and
Applied Chemistry, A41: 1459, 2004) groups have been attached at
the end of the chain. The linkage at the aromatic oxygen may be an
ester or ether. If the aminophthalate is used, the connection of
the side chain would be an amide link. The length of the PEG or
hydrophilic segment may be varied over a wide range (Kumar, R., et
al. Journal of Macromolecular Science, A39: 1137-1149, 2002), thus
providing control of the hydrophilicity/hydrophobicity ratio as
well as functionality. The polymerization conditions can be
controlled such as to obtain a structure with the hydroxyl group of
the PEG component available on both ends of the polymer chain for
subsequent chemical modification. These hydroxyl groups are used to
attach the peptide EPPT1 or to attach a fluorescent moiety if the
peptide is absent.
[0357] Characterization of these polymers in aqueous solution with
light scattering techniques (Chen, M. H., et al. Polymer Preprints,
44: 1199-1200, 2003) has shown that they form nanoparticles via a
self-assembly process with a PEG external surface and a hydrophobic
internal cavity (FIG. 2). The ratio of the radius of gyration, Rg,
(static light scattering) to the hydrodynamic radius, Rh, (dynamic
light scattering) is about 1.75, which indicates that the
nanoparticles correspond to a spheroidal structure. Attachment of
functional groups at the end of the hydrophobic chains allows
modification of the cavity of these nanospheres, which affects
their size and stability as well as the nature of cargo that can be
encapsulated. Static light scattering of these nanospheres gives Rg
in the range of 10-80 nm. The size of the nanospheres and their
stability are influenced by the length of the PEG oligomers and the
nature of the hydrophobic group. The nanospheres are quite stable
as long as the side chains have significant hydrophobic character.
They have a molecular weight around 200,000 Da and contain 10-12
copolymer chains per nanosphere, each about 20,000 Da in molecular
weight.
Example 2
Encapsulation of Cargo Materials
[0358] A wide variety of small molecules including drugs may be
encapsulated by the self-assembly process described in Example 1.
Larger molecules such as proteins (insulin) and polysaccharides
(inulin) have been encapsulated, since the nanospheres may adjust
to the size of the encapsulant molecule in the self-assembly
process. In order to encapsulate smaller molecules, a simple
protocol is used (Kumar, R., et al. Journal of the American
Chemical Society, 126: 10640-10644, 2004, Sharma, S. K., et al.
Chemical Communication 23: 2689-2691, 2004). The polymer and cargo
are dissolved together in an organic solvent, such as chloroform,
and then the solvent is evaporated to dryness. The residue is
dissolved in water and any unencapsulated material removed by
filtration. The aqueous solution is freeze-dried, kept until
needed, and then reconstituted with water to give clear solutions
of the encapsulated material. The amount of encapsulant as a
fraction by weight has been determined by several methods. When the
UV absorptivity of the encapsulant is sufficiently different from
the polymer, UV spectroscopy is used. In other cases, .sup.1H-NMR
(Sharma, S. K., et al., supra) is used. Typically, a ratio of 1:4
or 1:5 cargo to polymer weight ratios has been used. As the ratio
increases, the fractional mass of the cargo increases, and the
nanoparticle size increases until a maximum is reached.
[0359] The attachment of targeting peptides was also evaluated. The
chemistry of attachment of peptides was straight forward with
peptides/proteins attached at one at the ends of the polymeric
chains which, upon choosing the proper polymerization procedure,
have the PEG chain at both ends. Since in one embodiment,
nanospheres have 8-10 chains per particle, this should give 16-20
peptide units per particle.
[0360] A monomethoxy PEG chain was used to simulate the chemistry
at the end of a long PEG chain. The terminal hydroxy group was
activated by a succinimidyl ester according to the method of Miron
and Wilchek (Miron, T. and Wilchek, M. Bioconjugate Chemistry, 4,
1993) as shown in the first scheme, FIG. 2B, which demonstrates the
attachment of amino acids to Monomethoxy PEG 2000.
[0361] In each of the amino acid attachments, the same peaks in the
NMR spectra changed. The peaks on the proton NMR spectra for the
methylene protons (x) of PEG at 4.45 ppm and the succinimidyl ester
at 2.56 ppm disappeared over a period of 60 hours. The same PEG
methylene protons (x) appeared at 4.1 ppm in the final amino acid
attachment product. All of the amino acids, arginine, alanine,
glycine and 2-fluorophenyl glycine, exhibited the same changes in
the NMR (data not shown).
[0362] The same reactions were carried out with the basic polymer
and the same amino acids, which provided the same results yielding
the structures shown in Scheme 2, of FIG. 2B. The attachment of the
EPPT1 peptide to the polymer using the same chemistry, was then
conducted.
[0363] An alternative attachment for increasing the percentage of
targeting peptides is to utilize the same chemistry and attach a
small percentage (5%) of PEG units to the linker via a selective
process to give additional reactive PEG hydroxy end groups. The
structure of this selective process is shown in FIG. 2B, third
scheme. The PEG units are located on the outside of the particle
since they are highly hydrophilic. This would give a much greater
number of peptide units per particle and yet would not disrupt the
particle formation.
Example 3
Synthesis of Fluorine-Containing Nanoparticles
[0364] Fluorine incorporation into the base copolymer was
accomplished using standard methods of formation of ester or ether
linkages to attach a perfluorinated chain. Each polymer consists of
a hydrophilic polyethylene glycol (PEG) segment (molecular weight
main chain 600-1500) bound to a linker (aromatic or peptide bond)
to which a hydrophobic side chain is bound (via ether or ester
linkages) that is terminated by a hydrophobic or hydrophilic group.
When dissolved in water above the critical micelle concentration,
about 8 to 12 polymeric units self assemble into a spherical
micelle consisting of a compact core surrounded by an outer
envelope of PEG loops that provide biocompatibility. The micelles
have a molecular weight of about 100-200,000 and a hydraulic radius
ranging from about 10 to 30 nm.
[0365] The amphiphilic copolymer (with PEG, n=15) was mixed with
perfluoro octanoyl chloride under basic conditions to attach the
acyl perfluoro group to the phenolic moiety as the hydrophobic side
chain (FIG. 3A). The attachment was confirmed with IR spectroscopy
and .sup.19F-NMR. This fluorine-modified polymer formed
nanoparticles with Rg about 75 nm as determined by static light
scattering. It contained 28% (w/w) fluorine, corresponding to about
3,800 .sup.19F atoms per nanoparticle.
[0366] Additional agents can be encapsulated in the core. Micelles,
in which the side chain is a perfluorocarbon synthesized from
perfluoroctyl bromide that forms a micelle with 30 nm radius
containing 28% (w/v) fluorine has been synthesized. Additional
perfluorocarbon cargo can be encapsulated inside each micelle to
substantially increase fluorine content.
[0367] This proof of concept confirmed that fluorine-containing
polymers can be produced, and that they form nanospheres.
[0368] Other syntheses attaching fluorine groups are possible. The
amphiphilic copolymers with perfluorocarbon side chains were used
to further encapsulate 1,1,2,2,-tetrahydro perfluorododecanol (20%
w/w) (Schmatized also in FIG. 3B) using the same procedure. The
amount of perfluorocarbon cargo encapsulated by the fluorinated
polymer was determined by integration of fluorine NMR spectra. The
entire particle contained 42% (w/w) fluorine, corresponding to
almost 6,000 .sup.19F atoms per nanoparticle. We anticipate that we
can increase the loading by at least a factor of two to 12,000
.sup.19F atoms per nanoparticle. By assuming a cell volume of
10.sup.3 .mu.m.sup.3, we estimate that uptake of 10.sup.5,
10.sup.6, 10.sup.7 or 10.sup.8 of these nanoparticles per cell
would lead to cellular fluorine concentrations of about 2, 20, 200,
or 2,000 mM, respectively. The lower level would be adequate for
.sup.19F-MRI in the mouse; the upper levels would be more than
enough for imaging humans.
[0369] FIG. 3C shows the .sup.19F Spectra from perfluorocarbon
encapsulated by 1,1,2,2-tetrahydro perfluorodecanol (referred to
herein as base polymer), as an example of data obtained with one
embodiment of an amphiphilic copolymer of this invention. A
Bruker/Magnex 600 MHz spectrometer (Martinos Center), with
microimaging probe (not spinning) was used. Sample 2a was that of a
failed covalent synthesis while sample 3 represents encapsulated
20% (w/w) 1,1,2,2-tetrahydro perfluorodecanol, with 19F=15% (w/w)
and [19F] in solution=17.3 mg polymer/mL water=140 mM
Example 4
Biological Characterization of Nanospheres
Attachment of Fluorescent Probe
[0370] Rhodamine B was converted to its acid chloride using oxalyl
chloride. Treatment of the polymer (substituted with a decane chain
as the hydrophobic group) with this acid chloride and base gave a
reaction to form an ester linkage with the CH.sub.2OH groups at the
ends of the polymer chains, binding it covalently to the polymer.
This attachment did not interfere with nanosphere formation as
shown by light scattering. Cy5.5 and the targeting peptide
attachment may be accomplished as well, as described
hereinbelow.
Size Measurement of Nanoparticles
[0371] Multi-angle dynamic light scattering (MADLS) (in addition to
90.degree. DLS) was conducted, together with cryo transmission
electron microscopy (cryo-TEM) measurements to characterize the
size of the probes. Two types of formulations, as shown in Table 1,
were conducted. Formulation (1) is the base polymer producing
unmodified nanoparticles. Formulation (2) is the fluorine-loaded
nanoparticles containing 43% (w/w) fluorine.
TABLE-US-00001 TABLE 1 Polymer Components for MADLS and cryo-TEM
Side Chain - Cargo:Polymer Formulation Main Chain Linker Side Chain
Linker Linkage Cargo by weight (1) PEG-600 Isophthalate Decane
Ether None NA (2) PEG-600 Isophthalate Perflurocdane Ester
1H,1H,2H,2H- 1:4 Perfluorododecanol
[0372] Dynamic light scattering data were collected using a
BI-200SM Brookhaven laser light scattering instrument (equipped
with a Brookhaven BI 9000 AT digital correlator) at seven different
angles (45.degree., 60.degree., 75.degree., 90.degree.,
105.degree., 120.degree., and 135.degree.) with multiple runs at
each angle per sample (3.about.6 runs). Both samples were measured
at 25.degree. C. at a concentration of 2.0 mg/mL. The
autocorrelation function and the sampling time produced by the BI
9000 AT digital correlator were used to fit into an exponential
decay function so that the characteristic decay time (.GAMMA.) was
obtained for each run at each angle per sample (Brookhaven Website.
http://www.bic.com/DLSBasics.html, 2005). Then the characteristic
decay times at each angle were averaged, and the average decay
times were plotted as a function of the square of the scattering
vector q, where q=[4.pi.n sin(.theta./2)]/.lamda., n=the refractive
index of the solution, .theta.=scattering angle, .lamda.=the laser
wavelength. The slope of the .GAMMA.-q2 plot (R2>0.97) gave the
diffusion coefficient (D) of the particles in the solution, and the
hydrodynamic particle diameter d was evaluated from Stokes'
equation D=kBT/(3.pi..eta.d), where is kB=Boltzmann constant,
T=temperature in Kelvin, and .eta.=liquid viscosity. By this method
(Cipelletti, L. and Weitz, D. A. Review of Scientific Instruments,
70: 3214-3221, 1999. Kirsch, S., et al. Journal of Chemical
Physics, 104: 1758-1761, 1996), the particle diameter of
formulation (1) was 10.2.+-.1.0 nm, of formulation (2) was
34.3.+-.1.4 nm.
[0373] Cryo-transmission electron microscopy (TEM) images were
collected by a JEOL JEM-2200FS Field Emission Electron Microscope.
A 2.5 .mu.L aliquot of sample solution was dropped on a holey
carbon film coated copper-carbon grid using a micro pipette. The
grid was then blotted with filter paper for five seconds and
immediately dipped into liquid ethane. Then the grid was
transferred into the electron microscope for image collection using
liquid nitrogen to maintain the temperature. Examples of images are
shown in FIG. 4 for formulation 1 (A) at a concentration of 44.5
mg/mL and formulation 2 (B) at 48.5 mg/mL, respectively. The
nanoparticles accumulate at the interface of the ice and the carbon
substrate. The fine-grained light gray background is the amorphous
ice crystal. The dark gray background is the carbon substrate. FIG.
4B shows values obtained for the base polymer alone, and that with
encapsulated fluorocarbon.
Uptake of Fluorescently Labeled Nanoparticles
[0374] To demonstrate the ability of the nanoparticles to carry a
cargo into cells, INS-1 cells were incubated in vitro with labeled
nanospheres, containing Rhodamine B chemically attached to the
amphiphilic copolymer at the free ends of the PEG chains, at
37.degree. C. for 35 minutes, washed, and fixed. The cells were
then examined with a Zeiss LSM 510 Meta high resolution laser
scanning confocal microscope equipped with a 100.times. oil
emersion objective lens. Data was gathered using both the
appropriate laser for imaging Rhodamine B and transmitted light for
imaging the cells, and the images were merged into one picture
(FIGS. 4D and 4E). Staining may represent localization of the
polymer/nanoparticles within endosomes of the cell. Uptake was
quantitated and is presented graphically in FIGS. 4C and 4F. INS-1
cells were incubated at 37.degree. C., with the compound (1 mg/mL),
and the uptake was found to be temperature-independent, with a
maximum non-selective uptake of 2.times.10.sup.8 nanoparticles/cell
seen.
Drug Delivery with Nanoparticles
[0375] In order to determine the capability of the nanospheres to
carry drug cargo into a cell, in vivo studies were conducted which
demonstrated the efficacy of encapsulated non-steroidal
anti-inflammatory drugs such as aspirin and naproxen (Kumar, R., et
al. Journal of the American Chemical Society, 126: 10640-10644,
2004), in a transdermal application, indicating that the
nanospheres were able to carry cargo through the skin.
Acute Systemic Toxicity
[0376] Acute oral toxicity testing of intact nanospheres and
individual components was carried out to determine the LD50 (median
lethal dose) with C57Bl/6 (Table 2). For all but one case, the LD50
was far above 2 g/Kg, the limit for essentially non-toxic
substances (Botham, P. A. Toxicology in Vitro, 18: 227-230, 2004;
NIH Guidance Document on Using In Vitro Data to Estimate In Vivo
Starting Doses for Acute Systemic Toxicity, NIH Publ 01-4500. pp.
48. Research Triangle Park, N.C., USA: NIEHS, 2001). Even the most
toxic nanospheres in this study, (isophthalate linker, ether
linkage), gave an LD50 of 1 g/Kg, which is higher than that for
many food additives on the list of those generally regarded as safe
(GRAS) (NIH Guidance Document, supra; EAFUS: A Food Additive
Database. FDA/Center for Food Safety & Applied Nutrition,
2004).
TABLE-US-00002 TABLE 2 Acute oral toxicity tests Hydrophilic
Linkage Group Linker Bond Side Chain LD50 (g/Kg) PEG 600 38
Aspartic Acid 20 PEG 600 Aspartic Acid Amide Nonyl 60 PEG 600
Isophthalate Ester Decane 60 PEG 600 Isophthalate Ether Nonyl 1
[0377] Cellular cytotoxicity following exposure to the polymers and
reagents: INS-1 cells were incubated with the polymers and/or
reagents (1 mg/mL polymer or 0.05 mg/mL dye) at 37.degree. C., and
cellular viability was determined by MTS (FIG. 5). Base polymer or
encapsulated rhodamine exposure did not induce any observable
cytotoxicity, up to 48 hours post-exposure, though cytotoxic
effects were readily observed even 3 hours following exposure to
Rhodamine alone.
[0378] INS cells incubated with 0.2 mg/mL free or encapsulated
doxorubicin at 37.degree. C., showed a comparable rate of cell
death, with the death rate decreasing with the presence of PFC side
chains, as measured by MTS. Encapsulation of doxorubicin resulted
in reduced cytotoxicity in U87 cells, with BP, but not PFC side
chains.
[0379] The kinetics and sensitivity of detection of cellular uptake
were evaluated as well (FIG. 6). INS-1 cells were incubated with 1
mg/mL polymer at 37.degree. C. and evaluated by the indicated
reader. The sensitivity of the plate reader was low as the
background created too much noise. Data obtained using Cellomics
ArrayScan was less sensitive to background noise and allowed for
quantification of cellular uptake, which increased rapidly, then
levelled out after 4 hours (FIG. 6A). Confocal microscopic
evaluation of INS-1 cells incubated with 0.2 mg/mL BP-RB for 30 min
or 1 mg/mL BP-PFC-FITC for 14 hr, washed 3.times. (2 min), and
fixed (20 min) (FIG. 6B), showed that the fluorescent polymer was
essentially confined to cytoplasmic vesicles and not the
nucleus.
[0380] Uptake of free and encapsulated Doxorubicin was evaluated by
confocal microscopy as well (FIG. 6C). Cells were incubated with
0.2 mg/mL free or encapsulated doxorubicin for 5 hr at 37.degree.
C., washed 3.times. (2 min), and fixed (20 min). Doxorubicin was
largely confined to the nucleus. Therefore, doxorubicin must be
released from the polymer, since the polymer is too large to enter
the nucleus.
Example 5
Targeting of uMUC-1 Antigen with a Multi-Modal Imaging Probe
[0381] Nanoparticles are more easily taken up by a tumor as
compared to normal tissue, because of the generally greater
vascularization and interstitial volume of a tumor. However, in
order to determine whether it is possible to greatly enhance
selectivity, a ligand may be coupled to the free ends (16-24 per
micelle) of the PEG-linker segments and, in another embodiment, to
functional groups introduced into the PEG chains.
[0382] The ligand initially chosen was a 15-amino acid synthetic
peptide designated EPPT1 that is derived from the binding site of a
monoclonal antibody raised against human epithelial cancer cells
displaying the underglycosylated mucin-1 antigen (uMUC-1). MUC-1 is
a transmembrane molecule expressed over the cell surface and in
internal compartments by most glandular epithelial cells. It is
overexpressed on almost all human epithelial cell adenocarcinomas
as well as some nonepithelial and hematological malignancies
(altogether accounting for more than 70% of all newly diagnosed
cancer cases) in an underglycosylated form, which exposes an
immunogenic epitope that is normally masked. The synthetic peptide
EPPT1 has a reasonably high binding constant (K.sub.d=20 .mu.M),
and nanoparticles bound to the epitope would either remain bound to
the cell surface or be internalized by receptor-mediated
endocytosis. In this way, selectivity is determined primarily by
the specific ligand-receptor interaction rather than by the ease of
perfusion through the tumor.
[0383] A multi-modal imaging probe targeting uMUC-1 tumor antigen
was synthesized and tested both in vitro and in vivo (Moore, A., et
al. Cancer Research, 64: 1821-1827, 2004). The probe consisted of
cross-linked iron oxide as an MR-imaging contrast agent that
carried Cy5.5 dye as a NIRF optical probe and EPPT1 peptides
produced by solid-phase synthesis, both attached to amino groups
linked to the CLIO dextran coat. A FITC label was added to the NH,
terminus of the peptide for subsequent fluorescence microscopy
analysis, and the probe was radioiodinated by attachment to the
peptide tyrosines by the Iodogen method for cell binding analysis
and biodistribution studies (FIG. 7).
[0384] A terpolymer (FIG. 7, Series 1) was obtained via enzymatic
polymerization using novozyme-435, 5-amino dimethylphthalate,
5-hydroxy dimethylphthalate and polyethylene glycol. FITC and Cy5.5
were attached to the polymer by stirring separately in
dimethylformamide for four hours at room temperature with the
terpolymer. The resultant polymer was dialysed and used for'partial
o-alkylation using alpha bromo acetyl triethyleneglycol in
K.sub.2CO.sub.3 and acetonitrile on refluxing them together. The
free hydroxyl group at the end of the triethyleneglycol unit of the
partially alkylated polymer was activated by stirring
disuccinimidyl carbonate in acetonitrile with DMAP and used for
peptide attachment. The activated hydroxyl polymer was stirred with
peptide in phosphate buffer (pH-7.2) for 12 hours to obtain the
desired polymer. A hydrocarbon chain was then introduced by
stirring the halogenated hydrocarbon chain with polymer in triethyl
amine to make the carrier molecule suitable for micelle
formation.
[0385] To attach FITC to the Terpolymer, FITC was added to a three
necked round bottom flask containing terpolymer dissolved in
anhydrous DMF under the environment of nitrogen. The resulting
mixture was stirred at room temperature for four hours, after which
DMF was washed out using an excess of hexane. The remaining residue
was dried under vacuum. Residue was then subjected to dialysis
(6000-8000 Mw dialysis bag) to remove unreacted FITC in the
product. The resultant product was characterized from its
.sup.1HNMR and UV spectra.
[0386] To attach Cy5.5 to the Terpolymer, Cy5.5 was added to a
three necked round bottom flask containing terpolymer dissolved in
anhydrous DMF under the environment of nitrogen. The resulting
mixture was stirred at room temperature for four hours. After the
completion of the reaction DMF was removed by washing several times
with an excess of hexane. The residue was further dried under
vacuum. Residue was then subjected to dialysis (6000-8000 Mw) to
get rid of unreacted Cy5.5 in the obtained product. The resultant
product was characterized from its .sup.1HNMR and UV spectra.
[0387] For the esterification of free hydroxyl on the terpolymer,
nonanoyl chloride dissolved in dichloromethane was added dropwise
in reaction mixture containing dye-attached terpolymer and
triethylamine, under nitrogen with constant stirring at room
temperature. The resulting mixture was stirred for six hours. After
completion of the reaction, solvent was removed under vacuum and
THF was added and then filtered to remove salt formed in the
reaction. Unreacted nananoyl chloride was removed by washing with
hexane. Obtained product was dried under vacuum and was
characterized on the basis of its .sup.1HNMR and UV spectrum. A
similar method was used for polymers with FITC and polymers with
Cy5.5.
[0388] For O-alkylation, the polymer was dissolved in anhydrous
acetonitrile and added to three neck round bottom flask under
nitrogen with constant stirring containing fused K.sub.2CO.sub.3,
followed by dropwise addition of bromoester of TEG dissolved in
acetonitrile. The resulting mixture was refluxed for eight hours.
After completion of the reaction K.sub.2CO.sub.3 was filtered off
and filtrate obtained was concentrated under vacuum to get the
desired product.
[0389] For activation of the free hydroxyl group, disuccinimidyl
carbonate and the polymer dissolved in acetonitrile in the presence
of DMAP were stirred in a nitrogen environment, and the resulting
mixture was stirred for 12 hours. Solvent was then removed under
vacuum at room temperature. Separated salts and N-hydroxyl
succinamides were removed by repeated precipitation with diethyl
ether in acetonitrile. The thus obtained pure compound was vacuum
dried, characterized, and used for peptide attachment.
[0390] For peptide attachment, an activated hydroxyl polymer was
stirred with peptide in phosphate buffer (pH-7.2) for 12 hours to
get the desired peptide attached polymer. The product was
characterized by NMR, IR and UV spectroscopy. After the peptide was
attached, the hydrocarbon chain was attached in order to make the
polymer suitable for micelle formation. This was achieved by
stirring the halogenated hydrocarbon chain with polymer in
triethylamine.
[0391] To extend these studies using perfluoro labeled polymer, the
same FITC-labeled EPPT1 peptide was used. The crosslinked iron
oxide (CLIO)-EPPT probes had a hydrodynamic diameter measured by
dynamic light scattering of about 36 nm, slightly smaller than
fluorine-containing micelle nanoparticles, and contained 14
peptides and 5 Cy5.5 molecules per particle, comparable to what is
anticipated with the micelles. The study with the CLIO-EPPT
nanoparticles established test procedures which will be used.
[0392] EPPT1 peptides were attached to the free PEG terminal
hydroxyls using carbodiimide chemistry: a stoichiometric excess of
N-hydroxysuccinimide and carbodiimide are added to the micelles in
water, reacted for 15-30 minutes at room temperature, and purified
by dialysis. The EPPT1 peptides were added to the modified polymer
and reacted overnight at room temperature. The reaction occurs
between --OH on the PEG unit and the N terminus (--NH.sub.2) on the
peptide. Purification of the probe is accomplished by dialysis or
column separation.
[0393] Cell binding assays (FIG. 7D) were carried out with a
variety of human uMUC-1-positive tumor cell lines: ZR-75-1
(breast), BT-20 (breast), HT29 (colon), CAPAN-2 (pancreas), LS174T
(colon), and ChaGo-K-1 (lung) as well as human control
uMUC-1-negative tumor and normal cell lines. Cell lines were
incubated with varying amount of .sup.125I-labeled CLIO-EPPT for 1
hour. uMUC-1-negative and normal cell lines had much lower
nanoparticle uptake than that of the uMUC-1-positive tumor cell
lines, which bound on the order of 10.sup.7-10.sup.8 CLIO-EPPT
nanoparticles per cell.
[0394] The same lines and protocols will be used for determining
fluorine-loaded micelle nanoparticle uptake, with 10-1000 or higher
mM fluorine concentration uptake in the uMUC-1-positive cells being
the concentration sought. Similar binding assays will be conducted,
and binding as a function of both concentration and time will be
determined, in order to explore conditions that provide maximum
selectivity between uMUC-1-positive tumors and normal cells.
[0395] In vitro specificity of CLIO-EPPT for adenocarcinomas was
further characterized by flow cytometric analysis of probe binding
to selected adenocarcinoma and control cell lines (FIG. 7E). A
control cell line showed no cell binding, whereas adenocarcinoma
cell lines bound the probe and displayed diverse staining
intensities, consistent with variable glycosylation. Fluorescence
microscopy experiments, in which adenocarcinoma and control cells
were incubated with the probe, confirmed the fluorescence-activated
cell sorting data. All of the adenocarcinoma cell lines stained
strongly and showed colocalization of the FITC and Cy5.5
signals.
[0396] In vivo .sup.1H-MR imaging was performed on animals bearing
bilateral uMUC-1-positive and uMUC-1-negative tumors before and 24
hours after probe injection. No significant change in signal
intensity of T2-weighted images was observed in uMUC-1-negative
tumors, whereas significant signal reduction was observed in some
regions of uMUC-1-positive tumors (a 52% decrease for LS174T tumors
versus a 13-18% decrease in control tumors, FIG. 8A). The same
animals were subjected to optical imaging immediately after the
MR-imaging session. A high intensity NIRF signal was obtained from
the uMUC-1-positive tumors, whereas no significant signal was
observed from the control tumors (FIGS. 8B and 8C). Fluorescence
microscopy of excised tumors and muscle tissue gave results
consistent with NIRF images. From biodistribution studies with
.sup.125I-CLIO-EPPT, on average uMUC-1-positive tumors accumulated
3.4 times more of the probe than uMUC-1-negative tumors.
Correlative dual channel fluorescence microscopy of excised tumors
showed colocalization of FITC and Cy5.5 signals in uMUC-1-positive
tumors but no signal in control tumors (FIG. 8D).
[0397] A multi-modal probe for MR and NIRF imaging was herein
characterized and tested in vitro and in vivo in animal models of
human cancer, indicating specific accumulation in uMUC-1-expressing
tumors and providing in vivo imaging results.
[0398] Similarly, nanoparticles carrying perfluorocarbons will be
used for .sup.19F-MR imaging, which has some advantages over
.sup.1H-MR imaging. Cell-associated fluorine concentrations
necessary to make use of these advantages are obtainable, and
versatile imaging probes can be developed according to the methods
and processes of this invention, that may be used for the detection
of cancer, inter-alia, and, in other embodiments, monitoring the
progression of intervention in afflicted subjects.
Example 6
Synthesis and Characterization of Multi-Modal Probes for In Vivo
Cancer Imaging
[0399] Probes comprising a perfluorinated polymer with Cy5.5
attached for NIR study as well as attachment of the targeting
peptide EPPT1 to the hydroxyl groups at the free ends of the PEG
chains may be synthesized as described herein.
[0400] Synthesis of the polymer backbone will be carried out using
a well-established enzymatic method (Kumar, R., et al., Journal of
Macromolecular Science, 2002, supra; Kumar, R., et al. Journal of
the American Chemical Society, 2004, supra). The synthetic scheme
is shown in FIG. 9. Polymerization involves two linkers with linker
2 present in small amounts (1-5%) to produce the polymer shown. The
synthesis is conducted such that PEG hydroxy groups are present at
the ends of the chain.
[0401] Perfluorocarbon side chains are then attached to the linker
hydroxyls using standard acylation procedures to form ester
linkages with the polymer backbone. Other linkers, such as, for
example, ether amide, may similarly be utilized.
[0402] A variety of fluorine-containing polymers may be prepared
via this scheme, as exemplified in FIG. 10. The synthetic schemes
may be conducted using amine-modified base polymer, while the
inclusion of small amounts of the amine moiety should not interfere
with nanosphere formation.
[0403] In addition, chain length may be varied (from 5 to 13
carbons), and the relative number of fluorine atoms may be varied,
which may alter the hydrophobicity of the side chain.
[0404] The synthesis and in vitro characterization studies
described hereinabove may be used for optimization of nanoparticle
formulation. The effect of synthesis parameters on loading and
stability may also be studied, and a number of encapsulating
materials may be investigated, including perfluorodecalin,
bromo-perfluoroheptane, and perfluoro-crown ether. Cy5.5 may be
attached, in order to perform NIRF determination, EPPT1 peptides
may be used for targeting, and radioiodine may be incorporated for
cell binding and biodistribution studies. A number of formulations
may be tested to determine specificity and cellular uptake.
[0405] Synthesis of the polymer labeled with Cy5.5 dye may be
conducted as described (Josephson, L., et al. Bioconjugate
Chemistry, 10: 186-191, 1999), using aminated CLIO nanoparticles,
or partially aminated basic polymer. The attachment of the Cy5.5
monofunctional dye to amine groups of the base polymer is performed
by adding 100 mg of polymer in 0.5 M sodium bicarbonate with the pH
adjusted to 9.6 to 1 mg of Cy5.5 dye (Amersham-Pharmacia, Cat.
#Q15408). The mixture is incubated on a rotator overnight at room
temperature. After incubation, the mixture is purified from
non-reacted dye on a G-25 Sephadex column equilibrated with 20 mM
sodium citrate buffer with 0.15 M NaCl, pH 8.0. Incubation times
are varied to achieve different polymer:Cy5.5 ratios.
[0406] The EPPT1 peptide, YCAREPPTRTFAYWG (SEQ ID NO: 1) may be
modified to introduce a FITC label for subsequent fluorescence
microscopy/FACS analysis. The FITC label will be introduced by
adding FITC-labeled Lys on the C-terminus followed by a second
unlabeled lysine to serve as an attachment point. The Cys thiol
group will be protected with an acetoxy methyl group. The final
peptide will have the following sequence:
Y-C(ACM)-A-R-E-P-P-T-R-T-F-A-Y-W-G-K(FITC)K (SEQ ID NO: 2) (Italic
font indicates the original sequence). The peptide is synthesized
on an automatic synthesizer (PS3, Rainin, Woburn, Mass.) using Fmoc
chemistry with HBTU and HOBT. Peptides are cleaved from the Rink
amide HBHA resin (Novabiochem, San Diego, Calif.) with 5 ml of
TFA/thioanisole/ethanedithiol/anisole (90/5/3/2) and purified by
C18 reverse phase HPLC. Molecular weight is determined by MALDI
mass spectroscopy.
[0407] The synthesis of Cy5.5-EPPT1 nanoparticles is accomplished
via the attachment of the FITC-labeled peptides to the
Cy5.5-modified basic polymer, via the procedure of Zalipsky, et.
al. (Zalipsky, S., et al. Biotechnology and Applied Biochemistry,
15: 100-114, 1992), which attaches proteins through succinimidyl
activation of the PEG hydroxy group to react with the N-terminus of
the peptide, in this case, the added lysine group.
[0408] A number of other methods for attaching the peptide are also
envisioned (see, for example, Roberts, et. al. (Roberts, M. J., et
al. Advanced Drug Delivery Reviews, 54: 459-476, 2002).
[0409] In order to determine cell binding and biodistribution, the
probe may be radiolabeled with Na .sup.125I with the Iodogen method
(Pierce, Rockford, Ill.) using available Tyr within the peptide
sequence. The basic polymer backbone itself may be radiolabeled
with the same procedure because the isophthalate ring is
substitutable in the same manner as the tyrosine aromatic ring.
Nanospheres may have a substituted phenolic aromatic ring as the
linker in polymeric backbone which has the positions ortho and para
to the substituted hydroxy group available for substitution, as
shown in FIG. 11A. The positions indicated by the arrows would be
available to substitute radiolabeled iodine. Utilization of
commercially available kits would give single or multiple
substitutions on these aromatic rings. The EPPT1 peptide also has a
tyrosine moiety which would also be susceptible to iodine
substitution via the same procedures. The number of iodines bound
per polymer chain (i.e. specific activity) is controlled by the
radioiodine concentration and reaction time. Substitution at many
of these active sites would provide a relatively "hot" sample which
could be utilized for in vivo imaging or RIT studies in mice, if
desired.
[0410] It is also possible to encapsulate chelated yttrium and
indium by appropriately modifying the nanospheres. These
radionuclides may be utilized with the nanospheres containing the
targeting peptides to further enhance the sensitivity of the
imaging with radioactive nuclei. Technetium may also be similarly
used.
[0411] In order to track the encapsulated cargo of the
nanoparticles, fluorescent labeling of perfluorocarbon is
desirable. In one embodiment, Rhodamine B is converted to its acid
chloride form and treated with 1,1,2,2 tetrahydro
perfluorododecanol to form the esterified perfluoro compound. This
will then be encapsulated with the perfluorinated basic polymer.
This modification will allow tracking of the cargo by fluorescence
microscopy.
[0412] It is also possible to synthesize polymers with labeled
perfluorinated side chains as shown in FIG. 11B. The synthesis of
Rhodamine B-labeled perfluorinated bromo compounds utilizes
enzymatic synthesis involving the lipase Novozyme-435. It is
possible to selectively monoacylate a number of compounds with
bromoacetic acid. Alkylation of the phenolic hydroxyl group of the
polymer with the Rhodamine B-labeled perfluororinated bromo
compound results in the desired polymer with labeled perfluorinated
side chains. This sequence represents another embodiment for a
method of attaching the fluorinated groups to the polymer
backbone.
[0413] The final polymer structure(s) is, in one embodiment, the
sum of the above syntheses as shown in FIG. 11.
[0414] The final structures may then be analyzed for (1) elemental
analysis (Galbraith, Knoxville, Tenn.) of loaded and not-loaded
nanoparticles to determine fluorine content, (2) number of Cy5.5
molecules, (3) number of peptides, (4) particle size (N4-MD,
Coulter), and (5) isotope binding yield. Absorption spectra of the
probe will be taken using a Hitachi 3500 spectrophotometer (Amax
for Cy5.5 is at 675 nm). The syntheses described may be adjusted in
order to optimize these parameters.
[0415] The morphology of the micelles and the size of the corona
and core may be visualized by cryogenic transmission electron
microscopy according to the method described (Lam, Y. M., et al.
Molecular Simulation, 30: 239-247, 2004). In order to form a
monolayer of micelles, which is optimal for observation under a
transmission electron microscope, aqueous solutions of varying
concentrations is vitrified. Approximately 2 .mu.L of solution is
applied to a carbon grid while the system is maintained at
40.degree. C. in a high humidity freezing apparatus for 30 s. The
grid is blotted with a double layer of filter paper for 5-10
seconds and then plunged into liquid ethane. The vitrified sample
is placed in a cryotransfer holder maintained at liquid nitrogen
temperature .about.170.degree. C. TEM images of the sample will
then be recorded on a high resolution cryo-electron microscope
(JEOL 2200 FS) available at the Whitehead-MIT Bioimaging Center.
The size and shape of the micelles are observed and qualitative and
quantitative assessments made.
Example 7
Specificity and Cellular Uptake of the Candidate Probes by
uMUC-1-Positive and uMUC-1-Negative Tumor Cell Lines
[0416] For targeted multi-modal imaging probes labeling of tumor
cells to be "visible" for the imaging systems using .sup.19F-MR
imaging, it is necessary to attain a sufficiently high
cell-associated probe concentration, a large number of binding
sites for the EPPT1 peptide, a rapid rate of accumulation, high
specificity, etc.
[0417] In order to measure the amount of cell-associated
nanoparticle probes (and by inference the amount of fluorine) as a
function of the probe solution concentration and incubation time,
cell lines having different disseminating potential may be used,
since the probe should accumulate not only in primary tumors but
also in metastatic sites. Probe specificity may be determined by
comparing probe accumulation results for normal cells,
uMUC-1-positive and uMUC-1-negative tumor cells. Cell viability
assays may be performed with cells having maximal cellular
accumulation of probes to investigate effects on cell
viability.
[0418] The probe is radiolabelled with Na .sup.125I using the
Iodogen method (Pierce, Rockford, Ill.) using available Tyr within
the EPPT1 peptide. Cell lines used may be, for example, those
listed in Table 3.
TABLE-US-00003 TABLE 3 Cell lines used for measurement of cellular
accumulation of probes Cell line Tissue MUC-1 expression CAPAN-2
pancreas + LS174T colon + ChaGo-K-1 lung + NCI-H661 lung;
metastatic site: lymph node - ZR-75-1 breast + BT-20 breast +
MCF10-A normal breast; fibrocystic disease - RF-1 stomach + RF-48
stomach; metastatic site: ascites - OVCAR-3 ovary + DU-145 prostate
+ LNCaP prostate - U87 glioma - 293 primary embryonic kidney -
primary; peritoneum - macrophage
Cells are incubated with increasing concentrations of
.sup.125I-labeled probe for different time periods at 37.degree. C.
in a humidified CO.sub.2 atmosphere, followed by extensive washing
with HBSS. After the final wash, cells are lysed with 0.1% Triton
X, and cell lysates are counted in a gamma counter (1289 Compugamma
LS; Wallac, Turku, Finland). The relationship between .sup.125I
radioactivity and probe are measured using solutions with known
probe concentrations. Cell number or cell concentration are
estimated by flow cytometry to calculate the amount of probes
associated per cell after each measurement
[0419] Once the amount of fluorine accumulation in 10 million cells
is within the detection limit of MRI, then .sup.19F-MRI phantoms
are prepared using cell pellets. Selected cells may be subjected to
cell viability assays with MIT (mitochondrial function), caspase
activation (apoptosis), and 7-AAD (membrane integrity).
[0420] Differential binding of targeted nanoparticles to
adenocarcinoma (CAPAN-2, HT-29, LS174T, BT-20, and ChaGo-K-1)
versus control (MCF10A, 293, and U87) cell lines may also evaluated
using fluorescence microscopy. Cells are grown overnight on
coverslips, fixed in 4% paraformaldehyde, incubated with targeted
probes, and washed. Cells are identified under a bright-field
microscope and then subjected to correlative dual-channel
fluorescence microscopy in the green (for FITC detection) and NIR
(for Cy5.5 detection) channels, using an inverted fluorescent
microscope (Zeiss Axiovert 100TV, Zeiss, Wetzlar, Germany). Images
are collected using a cooled charge-coupled device (Photometrics,
Tucson, Ariz.) with appropriate excitation and emission filters
(Omega Optical, Brattleboro, Vt.).
[0421] The Cy5.5 and FITC fluorescence intensity in cell lysates
and in known samples containing known probe concentrations are
measured, for example, with a plate reader (BMG PolarStar, BMG
Labtech, Offenburg, Germany). Scatchard plot analysis is used to
estimate R.sub.T, the number of receptors per cell, and
K.sub.D,eff, the effective affinity of the probe for the cell.
Optimization of these procedures such that probe accumulation is
sufficient for MR and/or NIRF imaging, with the Scatchard plot
analysis providing an estimate of the effective affinity of the
multivalent probe for the cell (Schwartz, A. L., et al. Journal of
Biological Chemistry, 257: 4230-4237, 1982).
Example 8
Temporal Cellular Distribution of the Candidate Probes
[0422] In order to optimize nanoparticle uptake, retaining their
high specificity, fluorescently labeled perfluorocarbon-containing
nanoparticles are evaluated for nanoparticle distribution and
nanoparticle disintegration in uMUC-1-positive and uMUC-1-negative
tumor cell lines and normal cells.
Cells are seeded in 35 mm cell culture dishes and six-well plates
containing 22 mm-diameter glass cover slips at a density of
1.5.times.10.sup.5 cell/mL and incubated with nanoparticle
concentrations that give maximal uptake, as described in Example 7.
Laser scanning confocal micrographs will be recorded using, for
example, a Zeiss LSM 510 Meta high-resolution laser scanning
confocal microscope (Carl Zeiss AG). Scanning speed and laser
intensity will be adjusted to avoid photobleaching of the
fluorescent probes and damage or morphological changes of the
cells. The microscope will be equipped with a microcultivation
system (Incubator S, CTI controller 3700 digital, Zeiss) to control
temperature, humidity and CO.sup.2 for maintaining physiological
conditions during experiments. Image analysis and fluorescence
signal quantification will be performed using, for example, Zeiss
LSM software.
[0423] In order to investigate subcellular probe distribution,
selective labeling of different cellular organelles will be
conducted as described (Savic, R., et al. Science, 300: 615-618,
2003): (1) plasma membrane with 5-dodecanoylaminofluorescein (DAF,
green), (2) nuclei with Hoechst 33342 (blue), (3) lysosomes with
lysotracker DND-26 (green), (4) mitochondria with Mito-Tracker
Green FM (green), (5) Golgi apparatus and endoplasmic reticulum
(ER) with Brefeldin A BODIPY FL conjugate (green), and (6)
mitochondria and ER with 3,3'-dihexyloxacarbocyanine iodide
(green). When using green dyes for labeling organelles, it will be
necessary to remove FITC from the EPPT1 peptide. Probes with no
peptides will serve as controls for all experiments.
[0424] A spinning-disk confocal microscope, such as, for example,
the Perkin Elmer PE Ultraview RS100, equipped with a high speed
digital cooled CCD camera with a 1.5 s scan time for 3-D images
that is suitable for producing time-lapse video of rapid events may
similarly be used.
Example 9
Biological Characterization of Multi-Modal Imaging Probes
[0425] In order to determine the accumulation of the probe in
different organs, and/or the signal to noise ratio for MR imaging
of the tumors a bi-lateral tumor propagation method (Weissleder,
R., et al. Nature Medicine, 6: 351-354, 2000; Moore, A., et al.
Radiology, 221: 751-758, 2001) will be used, where uMUC-1-positive
tumor will be injected in one flank of the mouse and
uMUC-1-negative tumor will be injected in the opposite flank, and
include tumors with different metastatic potential. The
accumulation of the probe at primary and metastatic sites is
evaluated.
[0426] Animals are anesthetized with an intraperitoneal injection
of ketamine/xylazine (80 mg/kg/12 mg/kg, Parke-Davis. Morris
Plains, N.J./Miles Inc., Shawnee Mission, Kans.). Tumor cells
(uMUC-1+ and uMUC-1-) are injected in the flanks of nu/nu mice
(n=5/pair; approximately 5.times.10.sup.6 cells/flank depending on
the tumor doubling time). Tumors are allowed to grow to 0.5 cm in
size, and animals are injected intravenously with .sup.125I-labeled
.sup.19F nanoparticle probes. Animals are sacrificed, for example,
24, 48 and 72 hours later by lethal IV injection of sodium
pentobarbital (200 mg/kg). Tumors, tumor metastasis, the pancreas,
spleen, liver, heart, intestine, lung, lymph nodes, thymus, blood,
bone, muscle, brain and fat are excised, weighed and radioactivity
is measured in a gamma counter. Aliquots of the probes are counted
simultaneously to correct for radioactive decay and to calculate
the dose in each organ. Biodistribution results are expressed as
the percentage of the injected dose per gram of tissue (%
ID/g).
[0427] .sup.125I-labeled probe in tumor bearing animals (n=5) is
assessed. Blood half-life of the probe is determined after
intravenous injection of 50 mCi/animal of the .sup.125I-labeled
.sup.19F nanoparticles. Blood samples are withdrawn over several
time points from the tail vein, weighed, and radioactivity in the
blood is counted in a gamma-counter. Blood half-life is calculated
as described (Ritschel, W. Handbook on Basic Pharmacokinetics, 3d
edition, p. 168-190. Hamilton, Ill.: Drug Intelligence
Publications, Inc., 1986). The preparation of Cy5.5-labeled 19F
nanoparticles with no peptides attached and/or with nonsense
peptide serve as controls for all studies.
[0428] Results with acute oral toxicity testing indicated that the
nanospheres with a hydrocarbon side chain have little or no
toxicity. The nanoparticles for .sup.19F-MR imaging contain
perfluorocarbons, which have been used extensively in artificial
blood applications, and most are generally viewed as biologically
inert. An acute lethality (LD50) test may be used to determine if
the greater concentrations used in the applications of this
invention result in toxicity.
[0429] A basal cytotoxicity test may be initially conducted to
predict a starting dose for an in vivo lethality test. The neutral
red uptake (NRU) test may be undertaken with BALB/c 3T3 cells (NIH
Guidance Document on Using In Vitro Data to Estimate In Vivo
Starting Doses for Acute Systemic Toxicity, NIH Publ 01-4500. pp.
48. Research Triangle Park, N.C., USA: NIEHS, 2001). NR is a weak
cationic dye that readily penetrates cell membranes by non-ionic
diffusion and accumulates intracellularly in lysosomes. Alterations
of the cell surface or the sensitive lysosomal membrane lead to
lysosomal fragility and other changes that gradually become
irreversible, leading to a decreased uptake and binding of NR. It
is thus possible to distinguish between viable, damaged, or dead
cells, which is the basis of this assay. Healthy BALB/c 3T3 cells,
when maintained in culture, continuously divide and multiply over
time. A toxic chemical, regardless of site or mechanism of action,
will interfere with this process and result in a reduction of the
growth rate as reflected by cell number. Cytotoxicity is expressed
as a concentration dependent reduction of the uptake of the vital
dye, NR, after one day (one cell cycle) of chemical exposure, thus
providing a sensitive, integrated signal of both cell integrity and
growth inhibition.
[0430] BALB/c 3T3 cells are seeded into 96-well plates and
maintained in culture for 24 hours to form a semi-confluent
monolayer. Cells are exposed to the nanoparticles over a range of
concentrations. After 24 hours exposure, NRU is determined for each
treatment concentration and compared to that of control cultures.
For each concentration of the test chemical, the percent inhibition
of growth is calculated. The IC.sub.50 (the concentration producing
50% reduction of NR uptake) is calculated from the
concentration-response and used to estimate the starting dose for
lethality test.
[0431] In one embodiment, an acute lethality test may comprise
targeted nanoparticle administration into the tail vein of 20-25
gram ICR mice. Different dose levels are used, and a number of
animals are given each dose. The animals are observed for, for
example, 14 days, with the LD.sub.50 determined by the Reed-Muench
method (Reed, L. J. and Muench, H. Am J Hyg, 27: 493-494, 1938),
and the safety factor calculated as the ratio of LD.sub.50 to the
effective dose.
Example 10
In Vivo Imaging of uMUC-1-Expressing Human Tumors
[0432] .sup.19F MR spectral characteristics of nanoparticles taken
up in cells such that optimal imaging is achieved, may nonetheless
be influenced by changes in the .sup.19F MR spectrum, which
negatively affect image formation (for example, resonance
line-widths must not be substantially broadened on binding such
that the T2* becomes too short for imaging).
[0433] Toward this end, uMUC-1-positive tumors are grown in animals
as described hereinabove, after which animals are injected
intravenously with non-radiolabeled .sup.19F nanoparticle probes
and sacrificed. Tumors and metastases are harvested and analyzed ex
vivo by .sup.19F NMR spectroscopy at 14 T field strength,
37.degree. C. For each specimen, single pulse static (non-spinning)
and 2.5 kHz magic angle spinning (MAS) spectra are obtained. T1 is
measured by inversion recovery and T2 measured by CPMG in MAS
spectra. The static spectra yields the best estimate of the
appearance of the in vivo spectrum, and is used to calculate T2*
(=1/.pi..smallcircle.FWHH) for each resolved chemical shift
band.
[0434] By tabulating T2, T2* and T1, an early predictor for the
performance of the nanoparticles under in vivo imaging conditions
is obtained. The T2* (a measure of the inverse line-width) of any
resonance must not be below the order of a few ms in order for
imaging to be successful based on that resonance. The T2* will be
affected by the degree of molecular motion, with nanoparticle
binding potentially creating T2* values which are too short for
particular resonances to be used for image creation. Shortening of
T1 on binding or aggregation, improves image signal to noise ratio,
making it useful to identify resonances with advantageously short
T1s. Although chemical shift selective pulses have been used in
.sup.19F MRI in order to select out of a complicated chemical shift
spectrum just a single resonance that is used for image creation,
this results in much (or most) of the potentially available
fluorine signal to be discarded.
[0435] The determination of T2, T2* and T1 for each resonance band
enables an assessment of which resonances in the chemical shift
spectrum can be profitably used in image creation.
[0436] The magic angle spinning spectra of the tissue specimens
yield the highest spectral resolution and lowest detection limits
because the spinning eliminates isotropic magnetic susceptibility
broadening effects (Cheng, L. L., et al. Magnetic Resonance in
Medicine, 36: 653-658, 1996). MAS spectroscopy has become the
standard for measurement of proton NMR spectra of tissue specimens.
Each resonance in the PFC chain is resolvable with this technique,
and hence amenable to assessment for its contribution to image
creation. Interestingly, despite the availability of this technique
in tissue NMR spectroscopy for almost a decade, there have been no
reports of its use for .sup.19F spectroscopy of tissue specimens.
Although .sup.19F generally has much larger chemical shifts than
protons, making the elimination of susceptibility broadening
potentially less important for most applications of .sup.19F tissue
spectroscopy, the severe crowding and complexity of the CF2
resonances in the nanoparticle spectra may accrue substantial
benefits from MAS spectroscopy.
[0437] In order to determine whether the nanoparticle molecular
probes of the invention can successfully label tumors for detection
by .sup.19F-MRI, in vivo imaging is conducted. uMUC-1-positive
tumors grown in animals to 0.5 cm in size, are injected
intravenously with nonradiolabeled .sup.19F nanoparticle probes and
scanned by .sup.1H and .sup.19F-MRI, at, for example, 24, 48 and 72
hours post injection. Scanning is performed with, for example, a
multinuclear Bruker (Karlsruhe, Germany and Billerica, Mass.)
Avance NMR console interfaced to a 14 T (600 MHz proton frequency)
Magnex (Oxford, UK) actively shielded 89 mm vertical
superconducting magnet. Animals are physiologically supported (if
necessitated by the vertical positioning and long exam time) with a
ventilation system and monitored for respiration, core temperature,
and ECG. The animals are suspended head-up (by a bite bar
arrangement) in a Bruker Micro 2.5 microimaging probe. A
Teflon-free dedicated Bruker 19F 30 mm cylindrical RF resonator is
used for excitation and detection of both .sup.1H and .sup.19F
images. This resonator, although optimized for .sup.19F MRI, will
also tune to .sup.1H without removal of the animal from the magnet
and performs proton MRI.
[0438] The anesthetized animal is positioned in the magnet,
stabilized, with a triplane proton scout image used to roughly
locate the tumor area. Multi-slice proton T1-weighted spin echo and
T2-weighted gradient echo images (256.times.256 matrix size, 0.5 mm
slice thickness) are obtained to delineate the tumor and
surrounding area. The resonator is tuned to the .sup.19F frequency
(564 MHz) and imaged by .sup.19F MRI as described below. The
.sup.19F images (showing the nanoparticle distribution) are
overlaid on the .sup.1H images (which delineate detailed
anatomy).
[0439] Because of the lower signal to noise ratio of the .sup.19F
signal and the broad chemical shift range, all .sup.19F images are
non-slice selective (projective). Several MRI techniques are
explored. In order to capture as much of the available .sup.19F
signal as possible, chemical shift selective pulses may not be
used. The most direct method for total-.sup.19F MRI is to use small
magnetic field gradients, which do not cause overlap of projections
from different chemical shift bands. In the case of the
nanoparticle probe spectra obtained at 14 T under in vivo-like
conditions (using the imaging probe and a low resolution sample
holder), the linewidths of the CF3 and CF2 bands are about 0.5 ppm
(based on spectra we obtained with the nanoparticles). This
linewidth arises primarily from unresolved J-couplings in the CF3
band and the bands from CF2 adjacent to either CF3 or the polymer
linkage (at 85, 40 and 49 ppm from C6F6 respectively). The
remaining CF2 band centered at 44 ppm contains multiple chemical
shifts as well as the couplings. A 5 ppm projection width will just
barely avoid overlap of these bands, and will provide 10 pixels of
linear image resolution across the subject, a spatial resolution
comparable to the true resolution obtained in other in vivo
.sup.19F applications in the literature. Use of a larger gradient
results in higher spatial resolution, but lower signal to noise
ratio. This method can be applied to both projection (radial)
reconstruction and phase encoded approaches toward building a 2D
image.
[0440] A second approach may use mathematical deconvolution to
remove the resolution-degrading effects of the chemical shift
spectrum from the image. It requires that the chemical shift
spectrum be constant in shape (although it may vary in intensity)
at every position in the field of view, which is applicable for the
nanoparticles of this invention. An approximate reconstruction is
possible, and a high quality reconstruction can be obtained. Sharp,
high quality projection reconstruction .sup.19F images may be
obtained from perfluorocarbon systems by deconvolving the spectrum
from the projections prior to reconstruction (Busse, L. J., et al.
Medical Physics, 13: 518-524, 1986). The deconvolution is most
efficiently carried out in the time/k-space domain. A complex
reference FID (if the spatial reconstruction is performed on FIDs)
corresponding to the spectrum (with no gradients applied) is
inverted, and then multiplied by a window function to avoid the
blowup at long times where the FID is small in magnitude. The
window function is constructed as a Weiner filter to maintain an
optimal adaptive approach that takes into account the instantaneous
relative magnitudes of signal and noise power in the FID so that
random noise is not unduly amplified. The method is equally
applicable to both FID and echo based reconstructions. Because the
chemical shift and J-coupling have different behavior as a function
of field strength, it is essential that the reference data be
obtained at the field at which imaging is carried out. Both FID and
gradient echo imaging require an FID reference function, whereas
spin echo imaging requires a spin echo reference function. The
projection (or frequency encoded) data are multiplied by the
inverted/windowed reference on a pixel by pixel basis to yield the
deconvolved projections which may then be used in a conventional
reconstruction.
[0441] Although most imaging today employs spin echoes, there is an
advantage to using FIDs for the input to the reconstruction.
Because perfluorocarbons exhibit significant J-coupling (which is
not refocused by a 180 degree RF pulse), in the creation of a spin
echo (by means of an RF pulse) the chemical shift and other
resonance offset interactions (e.g., static magnetic
susceptibility) are refocused at the echo, whereas the J-coupling
is not. Therefore, significant oscillatory dephasing still occurs
at the echo, and this J-modulation varies strongly with the echo
time TE. The optimum approach to eliminating this source of
artifact and signal loss is to use reconstruction from projections
(radial imaging), such as is done for solid state MRI (Wu, Y., et
al. Calcified Tissue International, 62: 512-518, 1998; Wu, Y., et
al. Proceedings of the National Academy of Sciences of the United
States of America, 96: 1574-1578, 1999; Wu, Y., et al. Magnetic
Resonance in Medicine, 50: 59-68, 2003; Ramanathan, C. and
Ackerman, J. L. Magnetic Resonance in Medicine, 41: 1214-1220,
1999). We have extensive experience with projection reconstruction
in two and three dimensions, and can use software developed in
house for this work.
[0442] Images obtained are analyzed for spatial resolution (in
phantoms), for signal to noise ratio and contrast to noise ratio.
These results may be correlated with NIRF, immunohistochemical, and
histological image data acquired from the same animals.
[0443] In vivo NIRF imaging of uMUC-1-expressing tumors in mouse
models of human cancer may also be performed. Tumor bearing mice
injected with the probe above are used. Near-infrared reflectance
optical imaging is performed using a whole mouse imaging system as
described in (Mahmood, U., et al. Radiology, 213: 866-870, 1999).
Cy5.5 fluorescence is measured with the appropriate filters, and
mice are monitored for a period of 3 to 4 weeks post injection,
with probe assessed as a function of NIRF signal intensity. As the
tumors grows, signal intensity is plotted as a function of tumor
volume, which is obtained from caliper measurements and an indirect
calculation of tumor volume based on the doubling time for the
particular tumor. Animals are sacrificed after the completion of MR
and NIRF imaging sessions; tumors are excised and subjected to NIRF
imaging as described.
[0444] .sup.125I nuclear imaging may also be used, for example for
imaging of uMUC-1-expressing tumors in mouse models of human
cancer. The uptake ratio and the time course of the probe
accumulation may be collected, with imaging at successive time
points.
[0445] Probes are prepared to inject approximately 3.7 GBq of
activity in each experiment. Measurements may be performed using a
low-energy high-resolution collimator on a large field of view
Isocam II (Isocam Technologies Inc., Castana, Iowa) gamma camera.
Subjects are placed flat on the surface of the collimator and
imaged. Subsequently, pinhole SPECT may be performed using the same
camera. Subjects are placed in a vertical position in front of a
pinhole collimator mounted on one of the heads of the Isocam H
(Isocam Technologies Inc., Castana, Iowa) gamma camera available at
the MIT Nuclear Science and Engineering Department. The subject may
be positioned at a distance from the pinhole sufficient for the
acquisition of a whole body 2D image, and 2d images will be
corrected for parallax. The subject is then moved axially to the
height of the lesion. Fiducial markers will be placed next to the
subject, to aid the estimate of the axial shift if necessary. The
support is moved closer to the pinhole (.about.3 cm radius of
rotation) and rotated in a step and shoot protocol for the
acquisition of 60 to 180 angular projections. Literature methods
will be used for a careful registration of the imaging parameters,
notably the center of rotation. All these techniques are
well-established and proven in the nuclear imaging field. For
examples of pinhole SPECT see (Acton, P. D., et al. European
Journal of Nuclear Medicine, 29: 691-698, 2002; Schramm, N. U., et
al. IEEE Transactions on Nuclear Science, 50: 315-320, 2003; Moore,
R. H., et al. Cancer, 32: 987, 1991; Strand, S.-E. et al. Cancer,
73: 981-984, 1994; Weber, D. A. et al. Journal of Nuclear Medicine,
35: 342-348, 1994; Jaszczak, R. J., et al. Physics in Medicine and
Biology, 39: 425-437, 1994; Ishizu, K. et al. Journal of Nuclear
Medicine, 36: 2282-2287, 1995; Booij, J., et al. European Journal
of Nuclear Medicine, 29: 1221-1224, 2002; Acton, P. D. and Kung, H.
F. Nucl Med Biol, 30: 889-895, 2003.).
[0446] Alternatives/Data analysis. In the very unlikely event that
2D and 3D pinhole imaging should fail, parallel hole collimator
images will be acquired to obtain low-resolution estimates of the
biodistribution of the radiotracer. Given the large Fov of the
camera, several animals can be evaluated at the same time. Pinholes
of different diameters (e.g. 0.25, 0.5, 1 and 2 mm) will be
designed and fabricated. The availability of several pinholes will
allow the optimization of the resolution-sensitivity trade-off for
image quality. The best pinhole diameter will be chosen in
preliminary phantom experiments with phantoms representative of the
expected uptake ratios. Phantom images will be evaluated for
resolution and contrast recovery via region of interest analysis.
Projection data will be reconstructed with an Ordered Subset
Expectation Maximization (OSEM) iterative algorithm. Regions of
interest will be drawn in the reconstructed image to evaluate
uptake ratios from the ratio of counts in a target and in a
background region. To increase sensitivity, data can be acquired on
opposite sides of the animal by using the second head of the
camera, for which a second set of pinholes will be fabricated.
[0447] In order to follow intra-tumoral probe distribution at the
microscopic level, immunohistochemistry and histologic evaluation
of the tumors will be correlated with results of MR, NI and NIRF
studies.
[0448] Colocalization of the MR, NI and NIRF signals with staining
for uMUC-1, FITC, and Cy5.5 fluorescence may be assessed. After
imaging sessions, tumors are excised and snap frozen in liquid
nitrogen. Immunohistochemistry probing sections with mouse
monoclonal antibodies B423 (VU4H5) against uMUC-1 60-mer tandem
repeat (Biomeda, Foster City, Calif.) is followed by incubation
with PE-labeled rabbit anti-mouse antibody (Pharmingen, San Diego,
Calif.). Dual channel fluorescence microscopy is performed on
consecutive sections.
[0449] Microscopic sections are digitized using, for example, a
Polaroid SprintScan 35-mm scanner (Polaroid Corporation, Cambridge,
Mass.) and a PathScan Enabler System (Meyer Instruments, Inc.,
Houston, Tex.). MR and NIRF images and corresponding digitized
histological sections are displayed in a graphics package, such as,
for example, Photoshop.TM. and matching structures are identified,
as described (Benveniste, H., et al. Proceedings of the National
Academy of Sciences of the United States of America, 96:
14079-14084, 1999). The shape and arrangement of blood vessels
within the tissue may be used as landmarks. Approximation between
histological sections and MR images may be further improved by
modifying the imaging plane in the volumetric MR data. The volumes
of individual hypointense spots on the 3D MR images are measured
and correlated to corresponding staining/fluorescence
microscopy.
Example 11
Correlating In Vivo Imaging Data with Biological Function
[0450] The in vitro cytotoxicity of therapeutic probes in
uMUC-1-positive cell lines and normal cells may also be examined.
Incubating the target uMUC-1-positive and uMUC-1-negative tumor
cells and normal cells with empty nanoparticles and nanoparticles
containing the anti-cancer agent, with and without targeting
peptide, may be assessed for effects on cell death, which may be
determined by various assays.
[0451] To determine cell death, methods may include incubating
uMUC-1-positive, uMUC-1-negative, and normal cells with increasing
concentrations and various nanoparticle types for time periods
ranging from 6 hours to 3 days at 37.degree. C. in a humidified
CO.sub.2 atmosphere, followed by extensive washing with media. Cell
samples at each time point and for each particle concentration may
be subjected to cell viability assays with MTT (mitochondrial
function), caspase activity (apoptosis), and 7-AAD (membrane
integrity). The MTT Assay (Molecular Probes, Vybrant.RTM. MTT Cell
Proliferation Assay Kit V-13154) is a measure of the reducing
environment in cells. The MTT reagent is a water soluble
tetrazolium compound
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) that
is reduced to an insoluble formazan product. A known concentration
of cells, for example, is resuspended in DPBS containing 20 mM
glucose. 20 .mu.l of WIT reagent (5 mg/mL) and 100 ml of cell
suspension will be added to each well and the microplate will be
placed in an incubator at 37.degree. C. for 4 hours. A 100 .mu.l
aliquot of a 10% (w/v) SDS and 0.01 M HCl solution is added to each
well and incubated at 37.degree. C. for 4-18 hours. The absorbance
at 570 nm is determined on an ELISA plate reader and compared with
controls. Caspase activity is measured, for example, with the Guava
MultiCaspase Kit, which determines the fractions of a cell
population that are live, early apoptotic, late apoptotic, and
dead. This assay measures caspase activity as well as membrane
integrity. The assay kit contains SR-VAD-FMK
(sulforhodamine-valyl-alanyl-aspartyl-fluoromethylketone), a
fluorochrome-conjugated inhibitor of caspases which binds to
apoptotic cells, and 7-AAD, a membrane integrity dye which stains
late apoptotic and dead cells. SR-VAD-FMK penetrates all cells but
only binds to active caspases and can be washed away from
non-apoptotic cells. 100 ml of cell suspension in Apoptosis Wash
Buffer will be stained with SR-VAD-FMK and incubated for 1 hour at
37.degree. C. The cells are washed three times with Apoptosis Wash
Buffer, after which 5 ml of 7-AAD reagent is added to each sample
and incubated for 10 minutes at room temperature. The samples are
analyzed using the Guava Personal Cell Analysis System (Guava
Technologies), a novel flow cytometer.
[0452] Other methods may be used to assess cytotoxic effects of
specific agents, for example as outlined herein, when doxorubicin
and .sup.131I are incorporated. Once internalized by the cell
.sup.131I remains attached to the polymer to which it is covalently
bound, thus .sup.131I may be conjugated to the polymer. Since the
antitumor activity of doxorubicin requires direct interactions with
DNA or DNA topoisomerase, doxorubicin should be released from
polymer, in order to gain access to the nucleus of the cell.
Nanoparticles/micelles loaded with radiolabel and doxorubicin may
be diluted in PBS (1:1 by volume), loaded in a dialysis cassette
(molecular weight cutoff of 10,000 Da), and dialyzed against 50%
PBS at 37.degree. C. At different time points, aliquots may be
measured for doxorubicin concentration. The results will be
expressed as t.sub.1/2 (time in which 50% of drug exits the
nanoparticle/micelle).
[0453] Pharmacokinetic data (biodistribution, blood half-life) in
terms of drug affinity and accumulation in uMUC-1-positive primary
tumors and metastasis in vivo may be assessed.
[0454] In vivo cytotoxicity studies in for example, a mouse model
of human cancer may be conducted as well. Survival time and tumor
volume following administration of targeted therapeutic probes
containing doxorubicin may be evaluated. For example, nu/nu mice
injected subcutaneously with uMUC-1-positive and uMUC-1-negative
tumor cell suspensions are assessed for tumor volume via calipers
(0.5 cm in diameter). Single- and multiple-dose treatment studies
may be conducted with the drug, given intravenously. Tumor growth
may be assessed twice a week by caliper measurements. Tumor volume
may be calculated using the equation for the volume of a prolate
ellipsoid: (a.times.b.sup.2).times..pi./6, where a is the larger
and b is the smaller dimension of the tumor. The results may be
expressed as relative tumor volume, Vt/Vo, where Vo is the tumor
volume at the start of the treatment and Vt is the tumor volume at
any given time point.
[0455] Results from in vivo cytotoxicity experiments may be
correlated with in vivo .sup.19F MR, NIRF, and/or nuclear imaging.
The time course of nanoparticle/micelle accumulation by imaging
during treatment may be evaluated. In addition, histology of
excised tumors may be correlated with apoptosis and/or differential
uMUC-1 expression in vivo in response to therapy. For the latter,
quantitative RT-PCR may be used to determine changes in uMUC-1
expression. The following primers and probes specific for the MUC-1
5' non-repeat region may be used:
TABLE-US-00004 Forward primer, (SEQ ID NO: 3)
5'-ACAGGTTCTGGTCATGCAAGC-3'; Reverse primer, (SEQ ID NO: 4)
5'-CTCACAGCATTCTTCTCAGTAGAGCT-3' TaqMan Probe, (SEQ ID NO: 5)
5'-FAM-TGGAGAAAAGGAGACTTCGGCTACCCAGA-TAMRA-3'.
[0456] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art, any of which are to be considered as part
of this invention.
Sequence CWU 1
1
5115PRTHomo sapiens 1Tyr Cys Ala Arg Glu Pro Pro Thr Arg Thr Phe
Ala Tyr Trp Gly1 5 10 15224PRTHomo sapiens 2Tyr Cys Ala Cys Met Ala
Arg Glu Pro Pro Thr Arg Thr Phe Ala Tyr1 5 10 15Trp Gly Lys Phe Ile
Thr Cys Lys 20321DNAHomo sapiens 3acaggttctg gtcatgcaag c
21426DNAHomo sapiens 4ctcacagcat tcttctcagt agagct 26529DNAHomo
sapiens 5tggagaaaag gagacttcgg ctacccaga 29
* * * * *
References