U.S. patent application number 14/490357 was filed with the patent office on 2015-01-01 for mmp-targeted therapeutic and/or diagnostic nanocarriers.
The applicant listed for this patent is MALLINCKRODT LLC. Invention is credited to John N. Freskos, Thomas E. Rogers.
Application Number | 20150004097 14/490357 |
Document ID | / |
Family ID | 47430071 |
Filed Date | 2015-01-01 |
United States Patent
Application |
20150004097 |
Kind Code |
A1 |
Rogers; Thomas E. ; et
al. |
January 1, 2015 |
MMP-TARGETED THERAPEUTIC AND/OR DIAGNOSTIC NANOCARRIERS
Abstract
The present invention provides targeted delivery compositions
and methods of using the compositions for treating and diagnosing a
disease state in a subject.
Inventors: |
Rogers; Thomas E.; (Ballwin,
MO) ; Freskos; John N.; (Clayton, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MALLINCKRODT LLC |
Hazelwood |
MO |
US |
|
|
Family ID: |
47430071 |
Appl. No.: |
14/490357 |
Filed: |
September 18, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13689694 |
Nov 29, 2012 |
8871189 |
|
|
14490357 |
|
|
|
|
61565461 |
Nov 30, 2011 |
|
|
|
Current U.S.
Class: |
424/1.65 ;
424/9.3; 424/9.4; 424/9.6; 514/89 |
Current CPC
Class: |
A61P 43/00 20180101;
B82Y 5/00 20130101; A61K 49/126 20130101; A61K 47/60 20170801; A61K
49/00 20130101; A61K 49/0054 20130101; A61K 49/085 20130101; A61K
31/337 20130101; A61K 47/6911 20170801; A61K 31/282 20130101; A61K
51/0497 20130101; A61K 47/545 20170801; A61P 35/00 20180101; A61K
49/04 20130101 |
Class at
Publication: |
424/1.65 ;
424/9.6; 424/9.3; 424/9.4; 514/89 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 51/04 20060101 A61K051/04; A61K 31/337 20060101
A61K031/337; A61K 49/12 20060101 A61K049/12; A61K 49/04 20060101
A61K049/04; A61K 31/282 20060101 A61K031/282; A61K 49/00 20060101
A61K049/00 |
Claims
1-73. (canceled)
74. A method of treating or diagnosing a cancerous condition in a
subject comprising: (a) administering a targeted delivery
composition, comprising: (i) a nanocarrier including a therapeutic
or diagnostic agent or a combination thereof; and (ii) a conjugate
having the formula: A-(LPEG)-MMP.sup.i; wherein, A is an attachment
component for attaching said conjugate to said nanocarrier; (LPEG)
is selected from: 1. a linking group having a linear assembly of
from 1 to 3 polyethylene glycol components, 2. a linking group
having the formula [(EG)(P)].sub.m wherein each EG is an ethylene
glycol group independently selected from the group consisting of
triethylene glycol, tetraethylene glycol, pentaethylene glycol,
hexaethylene glycol, heptaethylene glycol and octaethylene glycol,
P is a phosphoryl or thiophosphoryl group, and m is an integer of
from 1 to 20; or 3. a linking group having the formula
--Z.sup.1--Z.sup.2--Z.sup.3--, wherein Z.sup.1 and Z.sup.3 are
independently selected from the group consisting of a PEG component
having a defined length and W.sub.n, wherein W is an amino acid and
the subscript n is an integer from 0 to 3; and Z.sup.2 is selected
from the group consisting of a PEG component having a defined
length and a coupling group selected from an amide, thioamide,
ester, carbamate or urea for connecting Z.sup.1 and Z.sup.3; and
MMP.sup.i is a MMP enzyme inhibitor having the formula;
##STR00006## wherein X is a member selected from the group
consisting of O and S; Y is a member selected from the group
consisting of pyridyl and phenyl, wherein said phenyl is optionally
substituted with OH, OCH.sub.3, OCF.sub.3 and CH.sub.3; and the
wavy line indicates the point of attachment to (LPEG); wherein the
therapeutic or diagnostic agent is sufficient to treat or diagnose
the condition.
75. The method of claim 74, wherein MMP.sup.i is selected from the
group consisting of ##STR00007##
76. The method of claim 74, wherein MMP.sup.i is selected from the
group consisting of ##STR00008##
77. The method of claim 74, wherein said nanocarrier has embedded
in, encapsulated in, or tethered to a therapeutic agent selected
from the group consisting of doxorubicin, cisplatin, oxaliplatin,
carboplatin, 5-fluorouracil, gemcitibine and a taxane.
78. The method of claim 74, wherein said nanocarrier has embedded
in, encapsulated in, or tethered to a diagnostic agent selected
from the group consisting of a radioactive agent, a fluorescent
agent, and a contrast agent.
79. A method of treating a cancerous condition in a subject
comprising: (a) administering to the subject a targeted delivery
composition, comprising: (i) a nanocarrier including a therapeutic
or diagnostic agent or a combination thereof; and (ii) a conjugate
having the formula: A-(LPEG)-MMP.sup.i; wherein, A is an attachment
component for attaching said conjugate to said nanocarrier; (LPEG)
is selected from: 1. a linking group having a linear assembly of
from 1 to 3 polyethylene glycol components, 2. a linking group
having the formula [(EG)(P)].sub.m wherein each EG is an ethylene
glycol group independently selected from the group consisting of
triethylene glycol, tetraethylene glycol, pentaethylene glycol,
hexaethylene glycol, heptaethylene glycol and octaethylene glycol,
P is a phosphoryl or thiophosphoryl group, and m is an integer of
from 1 to 20; or 3. a linking group having the formula
--Z.sup.1--Z.sup.2--Z.sup.3--, wherein Z.sup.1 and Z.sup.3 are
independently selected from the group consisting of a PEG component
having a defined length and W.sub.n, wherein W is an amino acid and
the subscript n is an integer from 0 to 3; and Z.sup.2 is selected
from the group consisting of a PEG component having a defined
length and a coupling group selected from an amide, thioamide,
ester, carbamate or urea for connecting Z.sup.1 and Z.sup.3; and
MMP.sup.i is a MMP enzyme inhibitor wherein the therapeutic agent
is sufficient to treat the condition; and wherein the
administration of the composition inhibits growth of the tumor
cells by at least 25%.
80. A method of treating a cancerous condition in a subject
comprising: (a) administering to the subject a targeted delivery
composition, comprising: (i) a nanocarrier including a therapeutic
or diagnostic agent or a combination thereof; and (ii) a conjugate
having the formula: A-(LPEG)-MMP.sup.i; wherein, A is an attachment
component for attaching said conjugate to said nanocarrier; (LPEG)
is selected from: 1. a linking group having a linear assembly of
from 1 to 3 polyethylene glycol components, 2. a linking group
having the formula [(EG)(P)].sub.m wherein each EG is an ethylene
glycol group independently selected from the group consisting of
triethylene glycol, tetraethylene glycol, pentaethylene glycol,
hexaethylene glycol, heptaethylene glycol and octaethylene glycol,
P is a phosphoryl or thiophosphoryl group, and m is an integer of
from 1 to 20; or 3. a linking group having the formula
--Z.sup.1--Z.sup.2--Z.sup.3--, wherein Z.sup.1 and Z.sup.3 are
independently selected from the group consisting of a PEG component
having a defined length and W.sub.n, wherein W is an amino acid and
the subscript n is an integer from 0 to 3; and Z.sup.2 is selected
from the group consisting of a PEG component having a defined
length and a coupling group selected from an amide, thioamide,
ester, carbamate or urea for connecting Z.sup.1 and Z.sup.3; and
MMP.sup.i is a MMP enzyme inhibitor wherein the therapeutic agent
is sufficient to treat the condition; and wherein the
administration of the composition reduces the tumor burden by at
least 25%.
81. A method of treating a cancerous condition in a subject
comprising: (a) administering to the subject a targeted delivery
composition, comprising: (i) a nanocarrier including a therapeutic
or diagnostic agent or a combination thereof; and (ii) a conjugate
having the formula: A-(LPEG)-MMP.sup.i; wherein, A is an attachment
component for attaching said conjugate to said nanocarrier; (LPEG)
is selected from: 1. a linking group having a linear assembly of
from 1 to 3 polyethylene glycol components, 2. a linking group
having the formula [(EG)(P)].sub.m wherein each EG is an ethylene
glycol group independently selected from the group consisting of
triethylene glycol, tetraethylene glycol, pentaethylene glycol,
hexaethylene glycol, heptaethylene glycol and octaethylene glycol,
P is a phosphoryl or thiophosphoryl group, and m is an integer of
from 1 to 20; or 3. a linking group having the formula
--Z.sup.1--Z.sup.2--Z.sup.3--, wherein Z.sup.1 and Z.sup.3 are
independently selected from the group consisting of a PEG component
having a defined length and W.sub.n, wherein W is an amino acid and
the subscript n is an integer from 0 to 3; and Z.sup.2 is selected
from the group consisting of a PEG component having a defined
length and a coupling group selected from an amide, thioamide,
ester, carbamate or urea for connecting Z.sup.1 and Z.sup.3; and
MMP.sup.i is a MMP enzyme inhibitor wherein the therapeutic agent
is sufficient to treat the condition; and wherein the composition
is administered at a dosage range of about 15 mg/kg to about 60
mg/kg.
82. The method of claim 79, wherein MMP.sup.i has an IC.sub.50 of
about 13 nM or lower.
83. The method of claim 80, wherein MMP.sup.i has an IC.sub.50 of
about 13 nM or lower.
84. The method of claim 81, wherein MMP.sup.i has an IC.sub.50 of
about 13 nM or lower.
85. The method of claim 79, wherein the administration of the
composition inhibits growth of the tumor cells by at least 50%.
86. The method of claim 80, wherein the administration of the
composition reduces the tumor burden by at least 50%.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional
Patent Application No. 61/565,461, filed Nov. 30, 2011, the
entirety of which is incorporated herein by reference.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] NOT APPLICABLE
REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED ON A COMPACT DISK
[0003] NOT APPLICABLE
BACKGROUND OF THE INVENTION
[0004] Cancer is a class of diseases that can affect people of all
ages. Accordingly, there is considerable effort to provide
therapies that can treat or diagnose cancer in patients. Targeted
delivery of nanocarriers in the body has been discussed recently as
a potential new avenue in drug delivery and diagnostic imaging
techniques. Unfortunately, obstacles still exist in making
nanocarrier based-products that can effectively treat or diagnose
cancer.
[0005] Many if not all solid tumors either express matrix
metalloproteinase (MMP) enzymes on their surface or excrete it into
the surrounding matrix or cause MMP enzymes to be produced via
angiogenesis (see, Y. Chau, F. E. Tan, and R. Langer, Bioconjugate
Chem, 2004, 15:931-941 and A. Matter, `Tumor Angiogenesis as a
Therapeutic Target`, DRUG DISCOVERY TODAY, 6:1005-1024 (2001)).
Thus, the tumor environment is particularly rich in MMP 2, 9, and
13 enzyme content as well as others, such as members of the
membrane bound family, MMP 14-17. The activity of MMP enzymes in a
mouse tumor model has been exquisitely revealed by use of a
FRET-based MMP enzyme assay where fluorescent dye is released in
vivo once the dye-bearing molecule is transported into the tumor
(L. Zhu, J. Xie, M. Swierczewska, F. Zhang, Q. Quan, Y. Ma, X.
Fang, K. Kim, S. Lee, X. Chen, Theranostics, 2011, 1:18-27).
[0006] Nanoparticles, such as liposomes, are commonly modified to
incorporate polyethylene glycol (PEG) groups on their surface to
enhance in vivo performance. It would be advantageous to target the
liposomal nanoparticle to a tumor cell related receptor or enzyme
within the tumor and also have it targeted for cellular uptake of
the cytotoxic payload (or other cargo) by endocytosis (or other
internalization mechanism) driven by enzyme/receptor recognition
and binding events.
[0007] There remains a need for new targeted delivery approaches
that can treat or diagnose cancer and provide ways to facilitate
personalized care for a patient. The present disclosure addresses
this need.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention provides targeted delivery
compositions and their methods of use in treating and diagnosing a
disease state, such as a cancerous condition, in a subject.
[0009] In one aspect of the invention, the targeted delivery
compositions can include a nanocarrier including a therapeutic
agent, a diagnostic agent, or a combination thereof, and a
conjugate having the formula:
A-(LPEG)-MMP.sup.i;
wherein, [0010] A is an attachment component for attaching said
conjugate to said nanocarrier; [0011] (LPEG) is a linking group
selected from a linear assembly of from 1 to 3 polyethylene glycol
components; an [(EG)(P)].sub.m linking group as defined herein; and
a --Z.sup.1--Z.sup.2--Z.sup.3-- linking group as defined herein;
and [0012] MMP.sup.i is a MMP inhibitor.
[0013] The targeted delivery compositions and methods of making and
using such compositions provide a number of unique advantages to
the areas of drug delivery and diagnostic imaging. For example, the
targeted delivery compositions linking groups can be synthesized to
have a discrete number of monomers, which can be tailored to, e.g.,
provide a specific length and/or chemical property. Furthermore,
the linking groups are fully customizable and can be prepared to
include only one type of monomer or multiple types of monomers in
any order. The linking groups can also be synthesized on a solid
phase support, which allows for simple, automated syntheses. In
addition to the linking groups, the targeted delivery compositions
can be used to treat diseases more effectively by utilizing lower
doses of agents that if administered with normal dosage amounts
might otherwise be toxic to a patient.
[0014] A further understanding of the nature and advantages of the
present invention can be realized by reference to the remaining
portions of the specification and the drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows a mass spectrum for Conjugate 1.
[0016] FIG. 2 shows the synthesis of 1-tert-butyl 4-ethyl
4-(4-(pyridin-3-yloxy)phenylsulfonyl)piperidine-1,4-dicarboxylate.
[0017] FIG. 3 shows the synthesis of
1-(tert-butoxycarbonyl)-4-(4-(pyridin-3-yloxy)phenylsulfonyl)piperidine-4-
-carboxylic acid.
[0018] FIG. 4 shows the synthesis of tert-butyl
4-(benzyloxycarbamoyl)-4-(4-(pyridin-3-yloxy)phenylsulfonyl)piperidine-1--
carboxylate.
[0019] FIG. 5 shows the synthesis of
N-(benzyloxy)-4-(4-(pyridin-3-yloxy)phenylsulfonyl)piperidine-4-carboxami-
de.
[0020] FIG. 6 shows the synthesis of a PEG 1000 piperidine amido
amine derivative of
N-(benzyloxy)-4-(4-(pyridin-3-yloxy)phenylsulfonyl)piperidine-4-carboxami-
de.
[0021] FIG. 7 shows the deprotection of the PEG 1000 piperidine
amido amine derivative of
N-(benzyloxy)-4-(4-(pyridin-3-yloxy)phenylsulfonyl)piperidine-4-carboxami-
de.
[0022] FIG. 8 shows the synthesis of a protected
N-(benzyloxy)-4-(4-(pyridin-3-yloxy)phenylsulfonyl)piperidine-4-carboxami-
de-PEG1000-PEG5000 conjugate.
[0023] FIG. 9 shows the synthesis of an
N-hydroxy-4-(4-(pyridin-3-yloxy)phenyl-sulfonyl)piperidine-4-carboxamide--
PEG1000-PEG5000 conjugate (Conjugate 1).
[0024] FIG. 10 shows the synthesis of 1-benzyl 4-methyl
4-((4-phenoxyphenyl)sulfonyl)piperidine-1,4-dicarboxylate.
[0025] FIG. 11 shows the synthesis of
1-((benzyloxy)carbonyl)-4-((4-phenoxyphenyl)sulfonyl)piperidine-4-carboxy-
lic acid.
[0026] FIG. 12 shows the synthesis of benzyl
4-((4-phenoxyphenyl)sulfonyl)-4-(((tetrahydro-2H-pyran-2-yl)oxy)carbamoyl-
)piperidine-1-carboxylate.
[0027] FIG. 13 shows the synthesis of
4-((4-phenoxyphenyl)sulfonyl)-4-(((tetrahydro-2H-pyran-2-yl)oxy)carbamoyl-
)piperidine.
[0028] FIG. 14 shows the synthesis of benzyl tert-butyl
((5S)-6-oxo-6-(4-((4-phenoxyphenyl)sulfonyl)-4-(((tetrahydro-2H-pyran-2-y-
l)oxy)carbamoyl)piperidin-1-yl)hexane-1,5-diyl)dicarbamate.
[0029] FIG. 15 shows the synthesis of tert-butyl
((2S)-6-amino-1-oxo-1-(4-((4-phenoxyphenyl)sulfonyl)-4-(((tetrahydro-2H-p-
yran-2-yl)oxy)carbamoyl)piperidin-1-yl)hexan-2-yl)carbamate.
[0030] FIG. 16 shows the synthesis of a protected
4-((4-phenoxyphenyl)sulfonyl)-4-(((tetrahydro-2H-pyran-2-yl)oxy)carbamoyl-
)piperidine-PEG5000-DSPE conjugate.
[0031] FIG. 17 shows the synthesis of an
N-hydroxy-4-((4-phenoxyphenyl)-sulfonyl)piperidine-4-carboxamide-PEG5000--
DSPE conjugate (Conjugate 2).
[0032] FIG. 18 shows mass spectra observed for Conjugate 2.
[0033] FIG. 19A shows mean tumor volume observed in mice bearing
BxPC3 pancreatic tumors treated with MMP-targeted liposomal
oxaliplatin, as compared to mice treated with untargeted liposomal
oxaliplatin and non-liposomal oxaliplatin. FIG. 19B shows percent
survival rates for test groups treated with MMP-targeted liposomal
oxaliplatin, untargeted liposomal oxaliplatin, and non-liposomal
oxaliplatin.
[0034] FIG. 20A shows the body weight changes observed in mice
bearing BxPC3 pancreatic tumors treated with MMP-targeted liposomal
oxaliplatin, as compared to mice treated with untargeted liposomal
oxaliplatin and non-liposomal oxaliplatin. FIG. 20B shows rates of
survival, moribundity, weight loss, death, ulcerated tumors, and
tumor burden for test groups treated with MMP-targeted liposomal
oxaliplatin, untargeted liposomal oxaliplatin, and non-liposomal
oxaliplatin.
[0035] FIG. 21A shows mean tumor volume observed in nude mice
bearing human fibrosarcoma HT1080 tumors overexpressing MMP14,
treated with MMP-targeted liposomal oxaliplatin, as compared to
mice treated with untargeted liposomal oxaliplatin and
non-liposomal oxaliplatin. FIG. 21B shows percent survival rates
for test groups treated with MMP-targeted liposomal oxaliplatin,
untargeted liposomal oxaliplatin, and non-liposomal
oxaliplatin.
[0036] FIG. 22A shows the observed activity of MMP2 in the presence
of MMP-targeted liposomal oxaliplatin at varying concentrations.
FIG. 22B shows the observed activity of MMP14 in the presence of
MMP-targeted liposomal oxaliplatin at varying concentrations.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0037] As used herein, the term "targeted delivery composition"
refers to a composition of a nanocarrier attached to a conjugate
having the formula: A-(LPEG)-MMP.sup.i, as further described
herein. The compositions of the present invention can be used as
therapeutic compositions, as diagnostic compositions, or as both
therapeutic and diagnostic compositions. In certain embodiments,
the compositions can be targeted to a specific MMP-expressing
tissue within a subject or a test sample, as described further
herein.
[0038] As used herein, the term "nanocarrier" refers to particles
of varied size, shape, type and use, which are further described
herein. As will be appreciated by one of ordinary skill in the art,
the characteristics of the nanocarriers, e.g., size, can depend on
the type and/or use of the nanocarrier as well as other factors
generally well known in the art. In general, nanocarriers can range
in size from about 1 nm to about 1000 nm. In other embodiments,
nanocarriers can range in size from about 10 nm to about 200 nm. In
yet other embodiments, nanocarriers can range in size from about 50
nm to about 150 nm. In certain embodiments, the nanocarriers are
greater in size than the renal excretion limit, e.g., greater than
about 6 nm in diameter. In other embodiments, the nanocarriers are
small enough to avoid clearance from the bloodstream by the liver,
e.g., smaller than 1000 nm in diameter. Nanocarriers can include
spheres, cones, spheroids and other shapes generally known in the
art. Nanocarriers can be hollow (e.g., solid outer core with a
hollow inner core) or solid or be multilayered with hollow and
solid layers or a variety of solid layers. For example, a
nanocarrier can include a solid core region and a solid outer
encapsulating region, both of which can be cross-linked.
Nanocarriers can be composed of one substance or any combination of
a variety of substances, including lipids, polymers, silica,
magnetic materials, or metallic materials, such as gold, iron
oxide, and the like. Lipids can include fats, waxes, sterols,
cholesterol, fat-soluble vitamins, monoglycerides, diglycerides,
phospholipids, sphingolipids, glycolipids, cationic or anionic
lipids, derivatized lipids, cardiolipin and the like. Polymers can
include block copolymers generally, poly(lactic acid),
poly(lactic-co-glycolic acid), polyethylene glycol, acrylic
polymers, cationic polymers, as well as other polymers known in the
art for use in making nanocarriers. In some embodiments, the
polymers can be biodegradable and/or biocompatible. Nanocarriers
can include a liposome, a micelle, a lipoprotein, a lipid-coated
bubble, a block copolymer micelle, a polymersome, a niosome, a
quantum dot, an iron oxide particle, a gold particle, a dendrimer,
or a silica particle. In certain embodiments, a lipid monolayer or
bilayer can fully or partially coat a nanocarrier composed of a
material capable of being coated by lipids, e.g., polymer
nanocarriers. In some embodiments, liposomes can include
multilamellar vesicles (MLV), large unilamellar vesicles (LUV), and
small unilamellar vesicles (SUV).
[0039] As used herein, the term "therapeutic agent" refers to a
compound or molecule that, when present in an effective amount,
produces a desired therapeutic effect on a subject in need thereof.
The present invention contemplates a broad range of therapeutic
agents and their use in conjunction with the targeted delivery
compositions, as further described herein.
[0040] As used herein, the term "diagnostic agent" refers to a
component that can be detected in a subject or test sample and is
further described herein.
[0041] As used herein, the term "conjugate" refers generally to a
molecule that includes a linking group. In some embodiments, a
conjugate of the present invention has the formula:
A-(LPEG)-MMP.sup.i. A is an attachment component that can attach
(covalently or non-covalently) the conjugate to a nanocarrier. The
conjugate can be covalently bonded to any part of a nanocarrier
including the surface or an internal region. Covalent attachment
can be achieved through a functional group using a linking
chemistry well known in the art, which is further described herein.
In other embodiments, a non-covalent attachment can include
interactions that are generally well known in the art and further
described herein. The conjugates of the present invention can
further include a linking group having the formula (LPEG) and a
targeting agent, MMP.sup.i, each being described further
herein.
[0042] As used herein, the term "linking group" refers to part of a
conjugate that links two components, e.g., an attachment component
and a targeting agent. Depending on the conjugate being prepared
and the properties desired for the conjugate, the linking group can
be assembled from readily available monomeric components to achieve
an appropriate separation of targeting agent and nanocarrier or
agent.
[0043] As used herein, the term "targeting agent" refers to a
molecule that is specific for a target, such as a matrix
metalloproteinase (MMP). In certain embodiments, a targeting agent
can include a small molecule mimic or inhibitor of the target
enzyme. MMP inhibitors (MMP.sup.i) can bind a wide variety of MMPs,
including targets in organs, tissues, cells, extracellular matrix
components, and/or intracellular compartments that can be
associated with a specific developmental stage of a disease. In
some embodiments, targets can include cancer cells, particularly
cancer stem cells. Targets can further include antigens on a
surface of a cell, or a tumor marker that is an antigen present or
more prevalent on a cancer cell as compared to normal tissue.
[0044] As used herein, the term "stealth agent" refers to a
molecule that can modify the surface properties of a nanocarrier. A
stealth agent can prevent nanocarriers from sticking to each other
and to blood cells or vascular walls. In certain embodiments,
stealth nanocarriers, e.g., stealth liposomes, can reduce
immunogenicity and/or reactogenicity when the nanocarriers are
administered to a subject. Stealth agents can also increase blood
circulation time of a nanocarrier within a subject. In some
embodiments, a nanocarrier can include a stealth agent such that,
for example, the nanocarrier is partially or fully composed of a
stealth agent or the nanocarrier is coated with a stealth agent.
Stealth agents for use in the present invention can include those
generally well known in the art. In certain embodiments, a stealth
agent can include "polyethylene glycol," which is well known in the
art and refers generally to an oligomer or polymer of ethylene
oxide. Polyethylene glycol (PEG) can be linear or branched, wherein
branched PEG molecules can have additional PEG molecules emanating
from a central core and/or multiple PEG molecules can be grafted to
the polymer backbone. PEG can include low or high molecular weight
PEG, e.g., PEG500, PEG2000, PEG3400, PEG5000, PEG6000, PEG9000,
PEG10000, PEG20000, or PEG50000 wherein the number, e.g., 500,
indicates the average molecular weight. In certain embodiments,
PEGylated-lipids are present in a bilayer of the nanocarrier, e.g.,
a liposome, in an amount sufficient to make the nanocarrier
"stealth," wherein a stealth nanocarrier shows reduced
immunogenicity. Other suitable stealth agents can include but are
not limited to dendrimers, polyalkylene oxide, polyvinyl alcohol,
polycarboxylate, polysaccharides, and/or hydroxyalkyl starch.
Stealth agents can be attached to the targeted delivery
compositions of the present invention through covalent and/or
non-covalent attachment, as described further herein.
[0045] As used herein, the term "embedded in" refers to the
location of an agent on or in the vicinity of the surface of a
nanocarrier. Agents embedded in a nanocarrier can, for example, be
located within a bilayer membrane of a liposome or located within
an outer polymer shell of a nanocarrier so as to be contained
within that shell.
[0046] As used herein, the term "encapsulated in" refers to the
location of an agent that is enclosed or completely contained
within the inside of a nanocarrier. For liposomes, for example,
therapeutic and/or diagnostic agents can be encapsulated so as to
be present in the aqueous interior of the liposome. Release of such
encapsulated agents can then be triggered by certain conditions
intended to destabilize the liposome or otherwise effect release of
the encapsulated agents.
[0047] As used herein, the term "tethered to" refers to attachment
of one component to another component so that one or more of the
components has freedom to move about in space. In certain exemplary
embodiments, an attachment component can be tethered to a
nanocarrier so as to freely move about in solution surrounding the
nanocarrier. In some embodiments, an attachment component can be
tethered to the surface of a nanocarrier, extending away from the
surface.
[0048] As used herein, the term "lipid" refers to lipid molecules
that can include fats, waxes, sterols, cholesterol, fat-soluble
vitamins, monoglycerides, diglycerides, phospholipids,
sphingolipids, glycolipids, cationic or anionic lipids, derivatized
lipids, and the like. Lipids can form micelles, monolayers, and
bilayer membranes. In certain embodiments, the lipids can
self-assemble into liposomes. In other embodiments, the lipids can
coat a surface of a nanocarrier as a monolayer or a bilayer.
[0049] As used herein, the term "subject" refers to any mammal, in
particular human, at any stage of life.
[0050] As used herein, the terms "administer," "administered," or
"administering" refers to methods of administering the targeted
delivery compositions of the present invention. The targeted
delivery compositions of the present invention can be administered
in a variety of ways, including topically, parenterally,
intravenously, intradermally, intramuscularly, colonically,
rectally or intraperitoneally. Parenteral administration and
intravenous administration are the preferred methods of
administration. The targeted delivery compositions can also be
administered as part of a composition or formulation.
[0051] As used herein, the terms "treating" or "treatment" of a
condition, disease, disorder, or syndrome includes (i) inhibiting
the disease, disorder, or syndrome, i.e., arresting its
development; and (ii) relieving the disease, disorder, or syndrome,
i.e., causing regression of the disease, disorder, or syndrome. As
is known in the art, adjustments for systemic versus localized
delivery, age, body weight, general health, sex, diet, time of
administration, drug interaction and the severity of the condition
may be necessary, and will be ascertainable with routine
experimentation by one of ordinary skill in the art.
[0052] As used herein, the term "formulation" refers to a mixture
of components for administration to a subject. Formulations
suitable for parenteral administration, such as, for example, by
intraarticular (in the joints), intravenous, intramuscular,
intratumoral, intradermal, intraperitoneal, and subcutaneous
routes, include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives. Injection
solutions and suspensions can also be prepared from sterile
powders, granules, and tablets. The formulations of a targeted
delivery composition can be presented in unit-dose or multi-dose
sealed containers, such as ampoules and vials. A targeted delivery
composition, alone or in combination with other suitable
components, can be made into aerosol formulations (i.e., they can
be "nebulized") to be administered via inhalation through the mouth
or the nose. Aerosol formulations can be placed into pressurized
acceptable propellants, such as dichlorodifluoromethane, propane,
nitrogen, and the like. Suitable formulations for rectal
administration include, for example, suppositories, which comprise
an effective amount of a targeted delivery composition with a
suppository base. Suitable suppository bases include natural or
synthetic triglycerides or paraffin hydrocarbons. In addition, it
is also possible to use gelatin rectal capsules which contain a
combination of the targeted delivery composition with a base,
including, for example, liquid triglycerides, polyethylene glycols,
and paraffin hydrocarbons. In certain embodiments, formulations can
be administered topically or in the form of eye drops.
EMBODIMENTS OF THE INVENTION
II. General
[0053] The present invention provides targeted delivery
compositions and methods for using the compositions for treating
and diagnosing a disease state in a subject. The disclosed
compositions and methods provide a number of beneficial features
over currently existing approaches. For example, the targeted
delivery compositions include linking groups that can be
synthesized to have a discrete number of monomers, which can be
tailored to, e.g., provide a specific length and/or chemical
property. Furthermore, the linking groups are fully customizable
and can be prepared to include only one type of monomer or multiple
types of monomers in any order. The linking groups can also be
synthesized on a solid phase support, which allows for simple,
automated syntheses. The targeted delivery compositions can be used
to treat diseases more effectively by utilizing lower doses of
agents that can be toxic to patients if administered with normal
dosage amounts.
[0054] Entry into this solid tumor microenvironment can be achieved
by allowing MMP.sup.i targeted liposomes access through systemic
blood supply. As a significant percentage of tumor blood vessels
are deficiently formed and `leaky` and a MMP.sup.i targeted
liposome now has contact with tumor stroma MMP enzyme and can
partition toward the enzyme gradient. This and the EPR effect will
effectively deliver the nanoparticle liposomes to the tumor stroma.
Once in the stroma the MMP.sup.i targeted liposome can contact
membrane bound MMP enzyme and be internalized by endocytosis and
deliver liposomal encapsulated drug to the cell. Thus, a suitably
anchored and linked MMP enzyme inhibitor (MMP.sup.i) molecule can
bind the tumor stroma and, if properly designed, be internalized
into cells expressing membrane bound MMP enzymes, thus delivering
nanoparticle/cytotoxic drug into the tumor or tumor stromal
cell.
III. Targeted Delivery Compositions
[0055] a. Targeted Delivery Compositions Including a
Nanocarrier
[0056] In one aspect, the targeted delivery compositions of the
present invention can include a targeted delivery composition,
comprising: (a) a nanocarrier including a therapeutic or diagnostic
agent or a combination thereof; and (b) a conjugate having the
formula: A-(LPEG)-MMP.sup.i. For such conjugates, A is an
attachment component for attaching the conjugate to the nanocarrier
and MMP.sup.i in an inhibitor of MMP. (LPEG) is selected from: i) a
linking group having from one to three polyethylene glycol
components; ii) a linking group having the formula [(EG)(P)].sub.m;
and iii) a linking group having the formula
--Z.sup.1--Z.sup.2--Z.sup.3--. For linking groups having the
formula [(EG)(P)].sub.m, EG represents an ethylene glycol component
(e.g., ethylene glycol, triethylene glycol, tetraethylene glycol,
hexaethylene glycol, and the like) and P represents a phosphoryl or
thiophosphoryl group, and the subscript m is an integer of from 1
to 15. For linking groups having the formula
--Z.sup.1--Z.sup.2--Z.sup.3--, Z.sup.1 and Z.sup.3 are
independently selected from the group consisting of a PEG component
having a defined length and W.sub.n, wherein W is an amino acid and
the subscript n is an integer from 0 to 3; and Z.sup.2 is selected
from the group consisting of a PEG component having a defined
length and a coupling group selected from an amide, thioamide,
ester, carbamate or urea for connecting Z' and Z.sup.3.
Nanocarriers
[0057] A wide variety of nanocarriers can be used in constructing
the targeted delivery compositions. As will be appreciated by one
of ordinary skill in the art, the characteristics of the
nanocarriers, e.g., size, can depend on the type and/or use of the
nanocarrier as well as other factors generally well known in the
art. Suitable particles can be spheres, spheroids, flat,
plate-shaped, tubes, cubes, cuboids, ovals, ellipses, cylinders,
cones, or pyramids. Suitable nanocarriers can range in size of
greatest dimension (e.g., diameter) from about 1 nm to about 1000
nm, from about 10 nm to about 200 nm, and from about 50 nm to about
150 nm.
[0058] Suitable nanocarriers can be made of a variety of materials
generally known in the art. In some embodiments, nanocarriers can
include one substance or any combination of a variety of
substances, including lipids, polymers, silica, or metallic
materials, such as gold, iron oxide, and the like. Examples of
nanocarriers can include but are not limited to a liposome, a
micelle, a lipoprotein, a lipid-coated bubble, a block copolymer
micelle, a polymersome, a niosome, an iron oxide particle, a gold
particle, a silica particle, a dendrimer, or a quantum dot.
[0059] In some embodiments, the nanocarriers are liposomes composed
partially or wholly of saturated or unsaturated lipids. Suitable
lipids can include but are not limited to fats, waxes, sterols,
cholesterol, fat-soluble vitamins, monoglycerides, diglycerides,
phospholipids, sphingolipids, glycolipids, derivatized lipids, and
the like. In some embodiments, suitable lipids can include
amphipathic, neutral, non-cationic, anionic, cationic, or
hydrophobic lipids. In certain embodiments, lipids can include
those typically present in cellular membranes, such as
phospholipids and/or sphingolipids. Suitable phospholipids include
but are not limited to phosphatidylcholine (PC), phosphatidic acid
(PA), phosphatidylethanolamine (PE), phosphatidylglycerol (PG),
phosphatidylserine (PS), and phosphatidylinositol (PI). Suitable
sphingolipids include but are not limited to sphingosine, ceramide,
sphingomyelin, cerebrosides, sulfatides, gangliosides, and
phytosphingosine. Other suitable lipids can include lipid extracts,
such as egg PC, heart extract, brain extract, liver extract, and
soy PC. In some embodiments, soy PC can include Hydro Soy PC
(HSPC). Cationic lipids include but are not limited to
N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC),
N,N-distearyl-N,N-dimethylammonium bromide (DDAB),
N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium
chloride (DOTMA), and N,N-dimethyl-2,3-dioleyloxy)propylamine
(DODMA). Non-cationic lipids include but are not limited to
dimyristoyl phosphatidyl choline (DMPC), distearoyl phosphatidyl
choline (DSPC), dioleoyl phosphatidyl choline (DOPC), dipalmitoyl
phosphatidyl choline (DPPC), dimyristoyl phosphatidyl glycerol
(DMPG), distearoyl phosphatidyl glycerol (DSPG), dioleoyl
phosphatidyl glycerol (DOPG), dipalmitoyl phosphatidyl glycerol
(DPPG), dimyristoyl phosphatidyl serine (DMPS), distearoyl
phosphatidyl serine (DSPS), dioleoyl phosphatidyl serine (DOPS),
dipalmitoyl phosphatidyl serine (DPPS), dioleoyl phosphatidyl
ethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine (POPE) and
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE),
1,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE), and
cardiolipin. In certain embodiments, the lipids can include
derivatized lipids, such as PEGlyated lipids. Derivatized lipids
can include, for example, DSPE-PEG2000, cholesterol-PEG2000,
DSPE-polyglycerol, or other derivatives generally well known in the
art.
[0060] Any combination of lipids can be used to construct a
nanocarrier such as a liposome. In certain embodiments, the lipid
composition of a targeted delivery composition, such as a liposome,
can be tailored to affect characteristics of the liposomes, such as
leakage rates, stability, particle size, zeta potential, protein
binding, in vivo circulation, and/or accumulation in tissue, such
as a tumor, liver, spleen or the like. For example, DSPC and/or
cholesterol can be used to decrease leakage from the liposomes.
Negatively or positively lipids, such as DSPG and/or DOTAP, can be
included to affect the surface charge of a liposome. In some
embodiments, the liposomes can include about ten or fewer types of
lipids, or about five or fewer types of lipids, or about three or
fewer types of lipids. The molar percentage (mol %) of a specific
type of lipid present typically ranges from about 0% to about 10%,
from about 10% to about 30%, from about 30% to about 50%, from
about 50% to about 70%, from about 70% to about 90%, or from about
90% to 100% of the total lipid present in a nanocarrier such as a
liposome. The lipids described herein can be included in a
liposome, or the lipids can be used to coat a nanocarrier of the
invention, such as a polymer nanocarrier. Coatings can be partially
or wholly surrounding a nanocarrier and can include monolayers
and/or bilayers. In one embodiment, liposomes can be composed of
about 50.6 mol % HSPC, about 44.3 mol % cholesterol, and about 5.1
mol % DSPE-PEG2000.
[0061] In other embodiments, a portion or all of a nanocarrier can
include a polymer, such as a block copolymer or other polymers
known in the art for making nanocarriers. In some embodiments, the
polymers can be biodegradable and/or biocompatible. Suitable
polymers can include but are not limited to polyethylenes,
polycarbonates, polyanhydrides, polyhydroxyacids,
polypropylfumerates, polycaprolactones, polyamides, polyacetals,
polyethers, polyesters, poly(orthoesters), polycyanoacrylates,
polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates,
polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,
polyamines, and combinations thereof. In some embodiments,
exemplary particles can include shell cross-linked knedels, which
are further described in the following references: Becker et al.,
U.S. application Ser. No. 11/250,830; Thurmond, K. B. et al., J.
Am. Chem. Soc., 119 (28) 6656-6665 (1997)); Wooley, K. L., Chem.
Eur. J., 3 (9): 1397-1399 (1997); Wooley, K. L., J. Poly. Sci.:
Part A: Polymer Chem., 38: 1397-1407 (2000). In other embodiments,
suitable particles can include poly(lactic co-glycolic acid) (PLGA)
(Fu, K. et al., Pharm Res., 27:100-106 (2000).
Conjugates for Attaching to a Nanocarrier
[0062] In certain embodiments, the targeted delivery compositions
including a nanocarrier also can include a conjugate having the
formula: A-(LPEG)-MMP.sup.i, wherein the attachment component A can
be used to attach the conjugate to a nanocarrier. The attachment
component can attach to any location on the nanocarrier, such as on
the surface of the nanocarrier. The attachment component can attach
to the nanocarrier through a variety of ways, including covalent
and/or non-covalent attachment. As described further below, the
conjugate also includes a linking group (LPEG) and an MMP.sup.i
targeting agent.
[0063] In certain embodiments, the attachment component A can
include a functional group that can be used to covalently attach
the attachment component to a reactive group present on the
nanocarrier. The functional group can be located anywhere on the
attachment component, such as the terminal position of the
attachment component. A wide variety of functional groups are
generally known in the art and can be reacted under several classes
of reactions, such as but not limited to nucleophilic substitutions
(e.g., reactions of amines and alcohols with acyl halides or active
esters), electrophilic substitutions (e.g., enamine reactions) and
additions to carbon-carbon and carbon-heteroatom multiple bonds
(e.g., Michael reaction or Diels-Alder addition). These and other
useful reactions are discussed in, for example, March, Advanced
Organic Chemistry, 3rd Ed., John Wiley & Sons, New York, 1985;
and Hermanson, Bioconjugate Techniques, Academic Press, San Diego,
1996. Suitable functional groups can include, for example: (a)
carboxyl groups and various derivatives thereof including, but not
limited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole
esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl
esters, alkyl, alkenyl, alkynyl and aromatic esters; (b) hydroxyl
groups which can be converted to esters, ethers, aldehydes, etc.
(c) haloalkyl groups wherein the halide can be later displaced with
a nucleophilic group such as, for example, an amine, a carboxylate
anion, thiol anion, carbanion, or an alkoxide ion, thereby
resulting in the covalent attachment of a new group at the site of
the halogen atom; (d) dienophile groups which are capable of
participating in Diels-Alder reactions such as, for example,
maleimido groups; (e) aldehyde or ketone groups for derivatization
via formation of carbonyl derivatives such as, for example, imines,
hydrazones, semicarbazones or oximes, or via such reactions as
Grignard addition or alkyllithium addition; (f) sulfonyl halide
groups for subsequent reaction with amines, for example, to form
sulfonamides; (g) thiol groups, which can be converted to
disulfides or reacted with acyl halides or Michael acceptors; (h)
amine or sulfhydryl groups, which can be, for example, acylated,
alkylated or oxidized; (i) alkenes, which can undergo, for example,
cycloadditions, acylation, Michael addition, etc.; and (j)
epoxides, which can react with, for example, amines and hydroxyl
compounds. In some embodiments, click chemistry-based platforms can
be used to attach the attachment component to a nanocarrier (Kolb,
H. C. et al. M. G. Finn and K. B. Sharpless, Angew. Chem. Int'l.
Ed. 40 (11): 2004-2021 (2001)). In some embodiments, the attachment
component can include one functional group or a plurality of
functional groups that result in a plurality of covalent bonds with
the nanocarrier.
[0064] Table 1 provides an additional non-limiting, representative
list of functional groups that can be used in the present
invention.
TABLE-US-00001 TABLE 1 Exemplary Functional Group Pairs for
Conjugation Chemistry Functional Groups: Reacts with: Ketone and
aldehyde groups Amino, hydrazido and aminooxy Imide Amino,
hydrazido and aminooxy Cyano Hydroxy Alkylating agents (such as
haloalkyl Thiol, amino, hydrazido, groups and maleimido
derivatives) aminooxy Carboxyl groups (including activated Amino,
hydroxyl, hydrazido, carboxyl groups) aminooxy Activated sulfonyl
groups (such as Amino, hydroxyl, hydrazido, sulfonyl chlorides)
aminooxy Sulfhydryl Sulfhydryl His-tag (such as 6-His tagged Nickel
nitriloacetic acid peptide or protein)
[0065] In other embodiments, an attachment component can be
attached to a nanocarrier by non-covalent interactions that can
include but are not limited to affinity interactions, metal
coordination, physical adsorption, hydrophobic interactions, van
der Waals interactions, hydrogen bonding interactions, magnetic
interactions, electrostatic interactions, dipole-dipole
interactions, antibody-binding interactions, hybridization
interactions between complementary DNA, and the like. In some
embodiments, an attachment component can be present in a lipid
bilayer portion of a nanocarrier such as a liposome. For example,
an attachment component can be a lipid that interacts partially or
wholly with the hydrophobic and/or hydrophilic regions of the lipid
bilayer. In some embodiments, the attachment component can include
one group that allows non-covalent interaction with the
nanocarrier, but a plurality of groups is also contemplated. For
example, a plurality of ionic charges can be used to produce
sufficient non-covalent interaction between the attachment
component and the nanocarrier. In alternative embodiments, the
attachment component can include a plurality of lipids such that
the plurality of lipids interacts with a bilayer membrane of a
liposome or bilayer or monolayer coated on a nanocarrier. In
certain embodiments, surrounding solution conditions can be
modified to disrupt non-covalent interactions thereby detaching the
attachment component from the nanocarrier.
Linking Groups
[0066] Linking groups designated (LPEG) are another feature of the
targeted delivery conjugates used in the compositions provided
herein. One of ordinary skill in the art can appreciate that a
variety of linking groups are known in the art and can be found,
for example, in the following reference: Hermanson, G. T.,
Bioconjugate Techniques, 2.sup.nd Ed., Academic Press, Inc. (2008).
Linking groups of the present invention can be used to provide
additional properties to the composition, such as providing spacing
between different portions of a conjugate, e.g., A and MMP.sup.i.
This spacing can be used, for example, to overcome steric hindrance
issues caused by the nanocarrier, e.g., when a targeting agent
binds to a target. In some embodiments, linking groups can be used
to change the physical properties of the targeted delivery
composition.
[0067] In one group of embodiments, the linking group (LPEG) has
the formula:
--Z.sup.1--Z.sup.2--Z.sup.3--.
[0068] In some embodiments, Z.sup.1 and Z.sup.3 are independently
selected from the group consisting of a PEG component having a
defined length and W.sub.n, wherein W is an amino acid and the
subscript n is an integer from 0 to 3; and Z.sup.2 is selected from
the group consisting of a PEG component having a defined length and
a coupling group selected from an amide, thioamide, ester,
carbamate or urea for connecting Z.sup.1 and Z.sup.3. In some
embodiments, (LPEG) is --Z.sup.1--Z.sup.2--Z.sup.3--. In some
embodiments, Z.sup.1 is W.sub.n; Z.sup.2 is selected from an amide,
thioamide, ester, carbamate urea, or combination thereof; and
Z.sup.3 is a PEG component having a defined length. In some
embodiments, the subscript n is 1. In some embodiments, the
subscript n is 2. In some embodiments, the subscript n is 3. In
some embodiments, the subscript n is 0. In those embodiments where
the subscript n is other than 0, the amino acid W can be an
.alpha.-amino acid. The linking groups can contain any suitable
.alpha.-amino acid. Examples of suitable .alpha.-amino acids
include, but are not limited to, alanine, cysteine, aspartic acid,
glutamic acid, phenylalanine, glycine, histidine, isoleucine,
lysine, leucine, methionine, asparagine, proline, glutamine,
arginine, serine, threonine, valine, tryptophan, and tyrosine. In
some embodiments, the .alpha.-amino acid is selected from the group
consisting of aspartic acid, glutamic acid, lysine, arginine, and
glycine. In some embodiments, the .alpha.-amino acid is selected
from the group consisting of glutamic acid and lysine. In some
embodiments, the .alpha.-amino acid is lysine.
[0069] In some embodiments, each of Z.sup.1 and Z.sup.3 are a PEG
component having a defined length, and Z.sup.2 is a coupling group
(e.g., an amide, thioamide, ester, carbamate, urea or combination
linkage group) for connecting the two PEG components. One of skill
in the art will appreciate that the coupling group (Z.sup.2) will
often be an alkylene group having functional groups on each end
which can be the same or different to facilitate assembly of
--Z.sup.1--Z.sup.2--Z.sup.3--. For example, in one group of
embodiments, Z.sup.1 is a PEG component attached to an attachment
component (A, preferably a lipid such as a phospholipid or
cardioleptin molecule). Similarly, Z.sup.3 is a PEG component that
is attached to MMP.sup.i. A number of PEG components having known
lengths and the requisite functional groups for use in linkage
assemblies are commercially available or can be prepared by known
methods. For example, a PEG component having the formula:
HO.sub.2C--CH.sub.2CH.sub.2--(OCH.sub.2CH.sub.2).sub.24NH--BOC is
readily available and has functional groups that can be selectively
reacted to prepare a suitable linkage assembly. In one group of
embodiments, Z.sup.1 is a PEG 3400 or PEG 5000 component (77 or 140
polyethylene glycol units, respectively). In other embodiments,
Z.sup.3 is a PEG 1000 component (24 polyethylene glycol units). In
certain selected embodiments, (LPEG) has the formula:
--C(O)-PEG.sub.3400-5000-OCH.sub.2CH.sub.2CH.sub.2NHC(O)CH.sub.2CH.sub.2-
CH.sub.2C(O)NH-PEG.sub.1000-C(O)--.
[0070] In some embodiments, (LPEG) has the formula:
--C(O)-PEG.sub.3400-5000-OCH.sub.2CH.sub.2CH.sub.2NHC(O)CH.sub.2CH.sub.2-
CH.sub.2C(O)NH--CH.sub.2CH.sub.2--CH.sub.2CH(NH.sub.2)--C(O)--.
[0071] In one group of embodiments, the targeted delivery
compositions can include a linking group (LPEG) having the formula:
[(EG)(P)].sub.m, wherein each EG is an ethylene glycol group
independently selected from triethylene glycol, tetraethylene
glycol, pentaethylene glycol, hexaethylene glycol, heptaethylene
glycol, and octaethylene glycol; and P is independently selected
from a group consisting of phosphate and thiophosphate. In some
embodiments, m can be equal to a number sufficient to make the
linking group longer than a poly(ethylene glycol) moiety extending
from a nanocarrier. In some embodiments, m can be greater than 1.
In other embodiments, m can be an integer from 1 to 10, 1 to 20, 1
to 30, or 1 to 40. In yet other embodiments, m can be an integer
from 2 to 12, 3 to 12, 4 to 12, 5 to 12, 6 to 12, 7 to 12, 8 to 12,
9 to 12, 10 to 12 and 11 to 12. In yet other embodiments, m can
range from 4 to 20, 6 to 20, 8 to 20, 10 to 20, 12 to 20, 14 to 20,
16 to 20, and 18 to 20. In one embodiment, m can be 8. In yet other
embodiments, m can be 4, 5, 6, 7, 8, 9, 10, 11 or 12. With respect
to EG and P, any combination of both can be used in the linking
group. For example, the linking group can be composed of one type
of ethylene glycol, such as hexaethylene glycol with only phosphate
(HEGp). In other embodiments, different ethylene glycols can be
used and combined with any combination of phosphate or
thiophosphate. In an exemplary embodiment, the linking group can be
tetraethylene glycol-phosphate-hexaethylene
glycol-thiophosphate-hexaethylene glycol-phosphate-triethylene
glycol-phosphate. One of ordinary skill in the art will appreciate
the vast number of combinations available for the linking groups of
the present invention.
[0072] Illustrated below are a few variations of the described
linking groups:
##STR00001##
Linking group A shows an octaethylene glycol phosphate. In A, m can
be, e.g., between 1 to 20. A can, also, optionally be part of
another linking group, or A can be attached to another linking
group. Similarly, linking group B shows a hexaethylene glycol
phosphate (also described herein as HEGp). B can include a number
of repeat units, e.g., m can be between 1 to 20, or preferably
about 8. As shown in linking group C, m can equal a specific
integer, e.g., m=2, as depicted by an exemplary dimer of
triethylene glycol phosphate. Alternatively, linking groups can,
e.g., be described using additional subscripts, x and y, such that
x+y=m. Linking group D, for example, shows a tetraethylene glycol
phosphate linked to a triethylene glycol phosphate. In certain
embodiments, the ethylene glycol portions (EG) within the
subscripted brackets of x and y can be independently selected from
a group consisting of triethylene glycol, tetraethylene glycol,
pentaethylene glycol, hexaethylene glycol, heptaethylene glycol,
and octaethylene glycol.
Therapeutic Agents
[0073] The nanocarriers used in the targeted therapeutic or
diagnostic delivery compositions of the present invention include a
therapeutic agent, diagnostic agent, or a combination thereof. The
therapeutic agent and/or diagnostic agent can be present anywhere
in, on, or around the nanocarrier. In some embodiments, the
therapeutic agent and/or diagnostic agent can be embedded in,
encapsulated in, or tethered to the nanocarrier. In certain
embodiments, the nanocarrier is a liposome and the diagnostic
and/or therapeutic agent is encapsulated in the liposome.
[0074] A therapeutic agent used in the present invention can
include any agent directed to treat a condition in a subject. In
general, any therapeutic agent known in the art can be used,
including without limitation agents listed in the United States
Pharmacopeia (U.S.P.), Goodman and Gilman's The Pharmacological
Basis of Therapeutics, 10.sup.th Ed., McGraw Hill, 2001; Katzung,
Ed., Basic and Clinical Pharmacology, McGraw-Hill/Appleton &
Lange, 8th ed., Sep. 21, 2000; Physician's Desk Reference (Thomson
Publishing; and/or The Merck Manual of Diagnosis and Therapy,
18.sup.th ed., 2006, Beers and Berkow, Eds., Merck Publishing
Group; or, in the case of animals, The Merck Veterinary Manual,
9.sup.th ed., Kahn Ed., Merck Publishing Group, 2005; all of which
are incorporated herein by reference.
[0075] Therapeutic agents can be selected depending on the type of
disease desired to be treated. For example, certain types of
cancers or tumors, such as carcinoma, sarcoma, leukemia, lymphoma,
myeloma, and central nervous system cancers as well as solid tumors
and mixed tumors, can involve administration of the same or
possibly different therapeutic agents. In certain embodiments, a
therapeutic agent can be delivered to treat or affect a cancerous
condition in a subject and can include chemotherapeutic agents,
such as alkylating agents, antimetabolites, anthracyclines,
alkaloids, topoisomerase inhibitors, and other anticancer agents.
In some embodiments, the agents can include antisense agents,
microRNA, siRNA and/or shRNA agents.
[0076] In some embodiments, a therapeutic agent can include an
anticancer agent or cytotoxic agent including but not limited to
avastin, doxorubicin, cisplatin, oxaliplatin, carboplatin,
5-fluorouracil, gemcitibine or taxanes, such as paclitaxel and
docetaxel. Additional anti-cancer agents can include but are not
limited to 20-epi-1,25 dihydroxyvitamin D3,4-ipomeanol,
5-ethynyluracil, 9-dihydrotaxol, abiraterone, acivicin,
aclarubicin, acodazole hydrochloride, acronine, acylfulvene,
adecypenol, adozelesin, aldesleukin, all-tk antagonists,
altretamine, ambamustine, ambomycin, ametantrone acetate, amidox,
amifostine, aminoglutethimide, aminolevulinic acid, amrubicin,
amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis
inhibitors, antagonist D, antagonist G, antarelix, anthramycin,
anti-dorsalizing morphogenetic protein-1, antiestrogen,
antineoplaston, antisense oligonucleotides, aphidicolin glycinate,
apoptosis gene modulators, apoptosis regulators, apurinic acid,
ARA-CDP-DL-PTBA, arginine deaminase, asparaginase, asperlin,
asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2,
axinastatin 3, azacitidine, azasetron, azatoxin, azatyrosine,
azetepa, azotomycin, baccatin III derivatives, balanol, batimastat,
benzochlorins, benzodepa, benzoylstaurosporine, beta lactam
derivatives, beta-alethine, betaclamycin B, betulinic acid, BFGF
inhibitor, bicalutamide, bisantrene, bisantrene hydrochloride,
bisaziridinylspermine, bisnafide, bisnafide dimesylate, bistratene
A, bizelesin, bleomycin, bleomycin sulfate, BRC/ABL antagonists,
breflate, brequinar sodium, bropirimine, budotitane, busulfan,
buthionine sulfoximine, cactinomycin, calcipotriol, calphostin C,
calusterone, camptothecin derivatives, canarypox IL-2,
capecitabine, caracemide, carbetimer, carboplatin,
carboxamide-amino-triazole, carboxyamidotriazole, carest M3,
carmustine, cam 700, cartilage derived inhibitor, carubicin
hydrochloride, carzelesin, casein kinase inhibitors,
castanospermine, cecropin B, cedefingol, cetrorelix, chlorambucil,
chlorins, chloroquinoxaline sulfonamide, cicaprost, cirolemycin,
cisplatin, cis-porphyrin, cladribine, clomifene analogs,
clotrimazole, collismycin A, collismycin B, combretastatin A4,
combretastatin analog, conagenin, crambescidin 816, crisnatol,
crisnatol mesylate, cryptophycin 8, cryptophycin A derivatives,
curacin A, cyclopentanthraquinones, cyclophosphamide, cycloplatam,
cypemycin, cytarabine, cytarabine ocfosfate, cytolytic factor,
cytostatin, dacarbazine, dacliximab, dactinomycin, daunorubicin
hydrochloride, decitabine, dehydrodidemnin B, deslorelin,
dexifosfamide, dexormaplatin, dexrazoxane, dexverapamil,
dezaguanine, dezaguanine mesylate, diaziquone, didemnin B, didox,
diethylnorspermine, dihydro-5-azacytidine, dioxamycin, diphenyl
spiromustine, docetaxel, docosanol, dolasetron, doxifluridine,
doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene
citrate, dromostanolone propionate, dronabinol, duazomycin,
duocarmycin SA, ebselen, ecomustine, edatrexate, edelfosine,
edrecolomab, eflomithine, eflomithine hydrochloride, elemene,
elsamitrucin, emitefur, enloplatin, enpromate, epipropidine,
epirubicin, epirubicin hydrochloride, epristeride, erbulozole,
erythrocyte gene therapy vector system, esorubicin hydrochloride,
estramustine, estramustine analog, estramustine phosphate sodium,
estrogen agonists, estrogen antagonists, etanidazole, etoposide,
etoposide phosphate, etoprine, exemestane, fadrozole, fadrozole
hydrochloride, fazarabine, fenretinide, filgrastim, finasteride,
flavopiridol, flezelastine, floxuridine, fluasterone, fludarabine,
fludarabine phosphate, fluorodaunorunicin hydrochloride,
fluorouracil, fluorocitabine, forfenimex, formestane, fosquidone,
fostriecin, fostriecin sodium, fotemustine, gadolinium texaphyrin,
gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors,
gemcitabine, gemcitabine hydrochloride, glutathione inhibitors,
hepsulfam, heregulin, hexamethylene bisacetamide, hydroxyurea,
hypericin, ibandronic acid, idarubicin, idarubicin hydrochloride,
idoxifene, idramantone, ifosfamide, ilmofosine, ilomastat,
imidazoacridones, imiquimod, immunostimulant peptides, insulin-like
growth factor-1 receptor inhibitor, interferon agonists, interferon
alpha-2A, interferon alpha-2B, interferon alpha-N1, interferon
alpha-N3, interferon beta-IA, interferon gamma-IB, interferons,
interleukins, iobenguane, iododoxorubicin, iproplatin, irinotecan,
irinotecan hydrochloride, iroplact, irsogladine, isobengazole,
isohomohalicondrin B, itasetron, jasplakinolide, kahalalide F,
lamellarin-N triacetate, lanreotide, lanreotide acetate,
leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole,
leukemia inhibiting factor, leukocyte alpha interferon, leuprolide
acetate, leuprolide/estrogen/progesterone, leuprorelin, levamisole,
liarozole, liarozole hydrochloride, linear polyamine analog,
lipophilic disaccharide peptide, lipophilic platinum compounds,
lissoclinamide 7, lobaplatin, lombricine, lometrexol, lometrexol
sodium, lomustine, lonidamine, losoxantrone, losoxantrone
hydrochloride, lovastatin, loxoribine, lurtotecan, lutetium
texaphyrin, lysofylline, lytic peptides, maitansine, mannostatin A,
marimastat, masoprocol, maspin, matrilysin inhibitors, matrix
metalloproteinase inhibitors, maytansine, mechlorethamine
hydrochloride, megestrol acetate, melengestrol acetate, melphalan,
menogaril, merbarone, mercaptopurine, meterelin, methioninase,
methotrexate, methotrexate sodium, metoclopramide, metoprine,
meturedepa, microalgal protein kinase C inhibitors, MIF inhibitor,
mifepristone, miltefosine, mirimostim, mismatched double stranded
RNA, mitindomide, mitocarcin, mitocromin, mitogillin, mitoguazone,
mitolactol, mitomalcin, mitomycin, mitomycin analogs, mitonafide,
mitosper, mitotane, mitotoxin fibroblast growth factor-saporin,
mitoxantrone, mitoxantrone hydrochloride, mofarotene, molgramostim,
monoclonal antibody, human chorionic gonadotrophin, monophosphoryl
lipid a/myobacterium cell wall SK, mopidamol, multiple drug
resistance gene inhibitor, multiple tumor suppressor 1-based
therapy, mustard anticancer agent, mycaperoxide B, mycobacterial
cell wall extract, mycophenolic acid, myriaporone,
n-acetyldinaline, nafarelin, nagrestip, naloxone/pentazocine,
napavin, naphterpin, nartograstim, nedaplatin, nemorubicin,
neridronic acid, neutral endopeptidase, nilutamide, nisamycin,
nitric oxide modulators, nitroxide antioxidant, nitrullyn,
nocodazole, nogalamycin, n-substituted benzamides,
06-benzylguanine, octreotide, okicenone, oligonucleotides,
onapristone, ondansetron, oracin, oral cytokine inducer,
ormaplatin, osaterone, oxaliplatin, oxaunomycin, oxisuran,
paclitaxel, paclitaxel analogs, paclitaxel derivatives, palauamine,
palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene,
parabactin, pazelliptine, pegaspargase, peldesine, peliomycin,
pentamustine, pentosan polysulfate sodium, pentostatin, pentrozole,
peplomycin sulfate, perflubron, perfosfamide, perillyl alcohol,
phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil,
pilocarpine hydrochloride, pipobroman, piposulfan, pirarubicin,
piritrexim, piroxantrone hydrochloride, placetin A, placetin B,
plasminogen activator inhibitor, platinum complex, platinum
compounds, platinum-triamine complex, plicamycin, plomestane,
porfimer sodium, porfiromycin, prednimustine, procarbazine
hydrochloride, propyl bis-acridone, prostaglandin J2, prostatic
carcinoma antiandrogen, proteasome inhibitors, protein A-based
immune modulator, protein kinase C inhibitor, protein tyrosine
phosphatase inhibitors, purine nucleoside phosphorylase inhibitors,
puromycin, puromycin hydrochloride, purpurins, pyrazofurin,
pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene
conjugate, RAF antagonists, raltitrexed, ramosetron, RAS farnesyl
protein transferase inhibitors, RAS inhibitors, RAS-GAP inhibitor,
retelliptine demethylated, rhenium RE 186 etidronate, rhizoxin,
riboprine, ribozymes, RII retinamide, RNAi, rogletimide,
rohitukine, romurtide, roquinimex, rubiginone B 1, ruboxyl,
safingol, safingol hydrochloride, saintopin, sarcnu, sarcophytol A,
sargramostim, SDI 1 mimetics, semustine, senescence derived
inhibitor 1, sense oligonucleotides, signal transduction
inhibitors, signal transduction modulators, simtrazene, single
chain antigen binding protein, sizofuran, sobuzoxane, sodium
borocaptate, sodium phenylacetate, solverol, somatomedin binding
protein, sonermin, sparfosate sodium, sparfosic acid, sparsomycin,
spicamycin D, spirogermanium hydrochloride, spiromustine,
spiroplatin, splenopentin, spongistatin 1, squalamine, stem cell
inhibitor, stem-cell division inhibitors, stipiamide,
streptonigrin, streptozocin, stromelysin inhibitors, sulfinosine,
sulofenur, superactive vasoactive intestinal peptide antagonist,
suradista, suramin, swainsonine, synthetic glycosaminoglycans,
talisomycin, tallimustine, tamoxifen methiodide, tauromustine,
tazarotene, tecogalan sodium, tegafur, tellurapyrylium, telomerase
inhibitors, teloxantrone hydrochloride, temoporfin, temozolomide,
teniposide, teroxirone, testolactone, tetrachlorodecaoxide,
tetrazomine, thaliblastine, thalidomide, thiamiprine, thiocoraline,
thioguanine, thiotepa, thrombopoietin, thrombopoietin mimetic,
thymalfasin, thymopoietin receptor agonist, thymotrinan, thyroid
stimulating hormone, tiazofurin, tin ethyl etiopurpurin,
tirapazamine, titanocene dichloride, topotecan hydrochloride,
topsentin, toremifene, toremifene citrate, totipotent stem cell
factor, translation inhibitors, trestolone acetate, tretinoin,
triacetyluridine, triciribine, triciribine phosphate, trimetrexate,
trimetrexate glucuronate, triptorelin, tropisetron, tubulozole
hydrochloride, turosteride, tyrosine kinase inhibitors,
tyrphostins, UBC inhibitors, ubenimex, uracil mustard, uredepa,
urogenital sinus-derived growth inhibitory factor, urokinase
receptor antagonists, vapreotide, variolin B, velaresol, veramine,
verdins, verteporfin, vinblastine sulfate, vincristine sulfate,
vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate
sulfate, vinleurosine sulfate, vinorelbine, vinorelbine tartrate,
vinrosidine sulfate, vinxaltine, vinzolidine sulfate, vitaxin,
vorozole, zanoterone, zeniplatin, zilascorb, zinostatin, zinostatin
stimalamer, or zorubicin hydrochloride or suitable prodrugs of the
aforementioned drugs.
[0077] In some embodiments, the therapeutic agents can be part of
cocktail of agents that includes administering two or more
therapeutic agents. For example, a liposome having both cisplatin
and oxaliplatin can be administered. In addition, the therapeutic
agents can be delivered before, after, or with immune stimulatory
adjuvants, such as aluminum gel or salt adjuvants (e.g., alumimum
phosphate or aluminum hydroxide), calcium phosphate, endotoxins,
toll-like receptor adjuvants and the like.
[0078] Therapeutic agents of the present invention can also include
radionuclides for use in therapeutic applications. For example,
emitters of Auger electrons, such as .sup.111In, can be combined
with a chelate, such as diethylenetriaminepentaacetic acid (DTPA)
or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),
and included in a targeted delivery composition, such as a
liposome, to be used for treatment. Other suitable radionuclide
and/or radionuclide-chelate combinations can include but are not
limited to beta radionuclides (.sup.177Lu, .sup.153Sm, .sup.88/90Y)
with DOTA, .sup.64Cu-TETA, .sup.188/186Re(CO).sub.3-IDA;
.sup.188/186Re(CO)triamines (cyclic or linear),
.sup.188/186Re(CO).sub.3-Enpy2, and
.sup.188/186Re(CO).sub.3-DTPA.
[0079] As described above, the therapeutic agents used in the
present invention can be associated with the nanocarrier in a
variety of ways, such as being embedded in, encapsulated in, or
tethered to the nanocarrier. Loading of the therapeutic agents can
be carried out through a variety of ways known in the art, as
disclosed for example in the following references: de Villiers, M.
M. et al., Eds., Nanotechnology in Drug Delivery, Springer (2009);
Gregoriadis, G., Ed., Liposome Technology: Entrapment of drugs and
other materials into liposomes, CRC Press (2006). In a group of
embodiments, one or more therapeutic agents can be loaded into
liposomes. Loading of liposomes can be carried out, for example, in
an active or passive manner. For example, a therapeutic agent can
be included during the self-assembly process of the liposomes in a
solution, such that the therapeutic agent is encapsulated within
the liposome. In certain embodiments, the therapeutic agent may
also be embedded in the liposome bilayer or within multiple layers
of multilamellar liposome. In alternative embodiments, the
therapeutic agent can be actively loaded into liposomes. For
example, the liposomes can be exposed to conditions, such as
electroporation, in which the bilayer membrane is made permeable to
a solution containing therapeutic agent thereby allowing for the
therapeutic agent to enter into the internal volume of the
liposomes.
Diagnostic Agents
[0080] A diagnostic agent used in the present invention can include
any diagnostic agent known in the art, as provided, for example, in
the following references: Armstrong et al., Diagnostic Imaging,
5.sup.th Ed., Blackwell Publishing (2004); Torchilin, V. P., Ed.,
Targeted Delivery of Imaging Agents, CRC Press (1995);
Vallabhajosula, S., Molecular Imaging: Radiopharmaceuticals for PET
and SPECT, Springer (2009). A diagnostic agent can be detected by a
variety of ways, including as an agent providing and/or enhancing a
detectable signal that includes, but is not limited to,
gamma-emitting, radioactive, echogenic, optical, fluorescent,
absorptive, magnetic or tomography signals. Techniques for imaging
the diagnostic agent can include, but are not limited to, single
photon emission computed tomography (SPECT), magnetic resonance
imaging (MRI), optical imaging, positron emission tomography (PET),
computed tomography (CT), x-ray imaging, gamma ray imaging, and the
like.
[0081] In some embodiments, a diagnostic agent can include
chelators that bind, e.g., to metal ions to be used for a variety
of diagnostic imaging techniques. Exemplary chelators include but
are not limited to ethylenediaminetetraacetic acid (EDTA),
[4-(1,4,8,11-tetraazacyclotetradec-1-yl)methyl]benzoic acid (CPTA),
Cyclohexanediaminetetraacetic acid (CDTA),
ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA),
diethylenetriaminepentaacetic acid (DTPA), citric acid,
hydroxyethyl ethylenediamine triacetic acid (HEDTA), iminodiacetic
acid (IDA), triethylene tetraamine hexaacetic acid (TTHA),
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylene phosphonic
acid) (DOTP), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic
acid (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid (DOTA), and derivatives thereof.
[0082] A radioisotope can be incorporated into some of the
diagnostic agents described herein and can include radionuclides
that emit gamma rays, positrons, beta and alpha particles, and
X-rays. Suitable radionuclides include but are not limited to
.sup.225Ac, .sup.72As, .sup.211At, .sup.11B, .sup.128Ba,
.sup.212Bi, .sup.75Br, .sup.77Br, .sup.14C, .sup.109Cd, .sup.62Cu,
.sup.64Cu, .sup.67Cu, .sup.18F, .sup.67Ga, .sup.68Ga, .sup.3H,
.sup.123I, .sup.125I, .sup.130I, .sup.131I, .sup.111In, .sup.177Lu,
.sup.13N, .sup.15O, .sup.32P, .sup.33P, .sup.212Pb, .sup.103Pd,
.sup.186Re, .sup.188Re, .sup.47Sc, .sup.153Sm, .sup.89Sr,
.sup.99mTc, .sup.88Y and .sup.90Y. In certain embodiments,
radioactive agents can include .sup.111In-DTPA,
.sup.99mTc(CO).sub.3-DTPA, .sup.99mTC(CO).sub.3-ENPY2,
.sup.62/64/67Cu-TETA, .sup.99Tc(CO).sub.3-IDA, and
.sup.99mTc(CO).sub.3triamines (cyclic or linear). In other
embodiments, the agents can include DOTA and its various analogs
with .sup.111In, .sup.177Lu, .sup.153Sm, .sup.88/90Y,
.sup.62/64/67Cu, or .sup.67/68Ga. In some embodiments, the
liposomes can be radiolabeled, for example, by incorporation of
lipids attached to chelates, such as DTPA-lipid, as provided in the
following references: Phillips et al., Wiley Interdisciplinary
Reviews: Nanomedicine and Nanobiotechnology, 1(1): 69-83 (2008);
Torchilin, V. P. & Weissig, V., Eds. Liposomes 2nd Ed.: Oxford
Univ. Press (2003); Elbayoumi, T. A. & Torchilin, V. P., Eur.
J. Nucl Med. Mol. Imaging 33:1196-1205 (2006); Mougin-Degraef, M.
et al., Int'l J. Pharmaceutics 344:110-117 (2007).
[0083] In other embodiments, the diagnostic agents can include
optical agents such as fluorescent agents, phosphorescent agents,
chemiluminescent agents, and the like. Numerous agents (e.g., dyes,
probes, labels, or indicators) are known in the art and can be used
in the present invention. (See, e.g., Invitrogen, The Handbook--A
Guide to Fluorescent Probes and Labeling Technologies, Tenth
Edition (2005)). Fluorescent agents can include a variety of
organic and/or inorganic small molecules or a variety of
fluorescent proteins and derivatives thereof. For example,
fluorescent agents can include but are not limited to cyanines,
phthalocyanines, porphyrins, indocyanines, rhodamines,
phenoxazines, phenylxanthenes, phenothiazines, phenoselenazines,
fluoresceins, benzoporphyrins, squaraines, dipyrrolo pyrimidones,
tetracenes, quinolines, pyrazines, corrins, croconiums, acridones,
phenanthridines, rhodamines, acridines, anthraquinones,
chalcogenopyrylium analogues, chlorins, naphthalocyanines, methine
dyes, indolenium dyes, azo compounds, azulenes, azaazulenes,
triphenyl methane dyes, indoles, benzoindoles, indocarbocyanines,
benzoindocarbocyanines, and BODIPY.TM. derivatives having the
general structure of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene,
and/or conjugates and/or derivatives of any of these. Other agents
that can be used include, but are not limited to, for example,
fluorescein, fluorescein-polyaspartic acid conjugates,
fluorescein-polyglutamic acid conjugates, fluorescein-polyarginine
conjugates, indocyanine green, indocyanine-dodecaaspartic acid
conjugates, indocyanine-polyaspartic acid conjugates, isosulfan
blue, indole disulfonates, benzoindole disulfonate,
bis(ethylcarboxymethyl)indocyanine,
bis(pentylcarboxymethyl)indocyanine, polyhydroxyindole sulfonates,
polyhydroxybenzoindole sulfonate, rigid heteroatomic indole
sulfonate, indocyaninebispropanoic acid, indocyaninebishexanoic
acid,
3,6-dicyano-2,5-[(N,N,N',N'-tetrakis(carboxymethyl)amino]pyrazine,
3,6-[(N,N,N',N'-tetrakis(2-hydroxyethyDamino]pyrazine-2,5-dicarboxylic
acid, 3,6-bis(N-azatedino)pyrazine-2,5-dicarboxylic acid,
3,6-bis(N-morpholino)pyrazine-2,5-dicarboxylic acid,
3,6-bis(N-piperazino)pyrazine-2,5-dicarboxylic acid,
3,6-bis(N-thiomorpholino)pyrazine-2,5-dicarboxylic acid,
3,6-bis(N-thiomorpholino)pyrazine-2,5-dicarboxylic acid S-oxide,
2,5-dicyano-3,6-bis(N-thiomorpholino)pyrazine S,S-dioxide,
indocarbocyaninetetrasulfonate, chloroindocarbocyanine, and
3,6-diaminopyrazine-2,5-dicarboxylic acid.
[0084] One of ordinary skill in the art will appreciate that
particular optical agents used can depend on the wavelength used
for excitation, depth underneath skin tissue, and other factors
generally well known in the art. For example, optimal absorption or
excitation maxima for the optical agents can vary depending on the
agent employed, but in general, the optical agents of the present
invention will absorb or be excited by light in the ultraviolet
(UV), visible, or infrared (IR) range of the electromagnetic
spectrum. For imaging, dyes that absorb and emit in the near-IR
(.about.700-900 nm, e.g., indocyanines) are preferred. For topical
visualization using an endoscopic method, any dyes absorbing in the
visible range are suitable.
[0085] In some embodiments, the non-ionizing radiation employed in
the process of the present invention can range in wavelength from
about 350 nm to about 1200 nm. In one exemplary embodiment, the
fluorescent agent can be excited by light having a wavelength in
the blue range of the visible portion of the electromagnetic
spectrum (from about 430 nm to about 500 nm) and emits at a
wavelength in the green range of the visible portion of the
electromagnetic spectrum (from about 520 nm to about 565 nm). For
example, fluorescein dyes can be excited with light with a
wavelength of about 488 nm and have an emission wavelength of about
520 nm. As another example, 3,6-diaminopyrazine-2,5-dicarboxylic
acid can be excited with light having a wavelength of about 470 nm
and fluoresces at a wavelength of about 532 nm. In another
embodiment, the excitation and emission wavelengths of the optical
agent may fall in the near-infrared range of the electromagnetic
spectrum. For example, indocyanine dyes, such as indocyanine green,
can be excited with light with a wavelength of about 780 nm and
have an emission wavelength of about 830 nm.
[0086] In yet other embodiments, the diagnostic agents can include
but are not limited to magnetic resonance (MR) and x-ray contrast
agents that are generally well known in the art, including, for
example, iodine-based x-ray contrast agents, superparamagnetic iron
oxide (SPIO), complexes of gadolinium or manganese, and the like.
(See, e.g., Armstrong et al., Diagnostic Imaging, 5.sup.th Ed.,
Blackwell Publishing (2004)). In some embodiments, a diagnostic
agent can include a magnetic resonance (MR) imaging agent.
Exemplary magnetic resonance agents include but are not limited to
paramagnetic agents, superparamagnetic agents, and the like.
Exemplary paramagnetic agents can include but are not limited to
Gadopentetic acid, Gadoteric acid, Gadodiamide, Gadolinium,
Gadoteridol, Mangafodipir, Gadoversetamide, Ferric ammonium
citrate, Gadobenic acid, Gadobutrol, or Gadoxetic acid.
Superparamagnetic agents can include but are not limited to
superparamagnetic iron oxide and Ferristene. In certain
embodiments, the diagnostic agents can include x-ray contrast
agents as provided, for example, in the following references: H. S
Thomsen, R. N. Muller and R. F. Mattrey, Eds., Trends in Contrast
Media, (Berlin: Springer-Verlag, 1999); P. Dawson, D. Cosgrove and
R. Grainger, Eds., Textbook of Contrast Media (ISIS Medical Media
1999); Torchilin, V. P., Curr. Pharm. Biotech. 1:183-215 (2000);
Bogdanov, A. A. et al., Adv. Drug Del. Rev. 37:279-293 (1999);
Sachse, A. et al., Investigative Radiology 32(1):44-50 (1997).
Examples of x-ray contrast agents include, without limitation,
iopamidol, iomeprol, iohexol, iopentol, iopromide, iosimide,
ioversol, iotrolan, iotasul, iodixanol, iodecimol, ioglucamide,
ioglunide, iogulamide, iosarcol, ioxilan, iopamiron, metrizamide,
iobitridol and iosimenol. In certain embodiments, the x-ray
contrast agents can include iopamidol, iomeprol, iopromide,
iohexol, iopentol, ioversol, iobitridol, iodixanol, iotrolan and
iosimenol.
[0087] Similar to therapeutic agents described above, the
diagnostic agents can be associated with the nanocarrier in a
variety of ways, including for example being embedded in,
encapsulated in, or tethered to the nanocarrier. Similarly, loading
of the diagnostic agents can be carried out through a variety of
ways known in the art, as disclosed for example in the following
references: de Villiers, M. M. et al., Eds., Nanotechnology in Drug
Delivery, Springer (2009); Gregoriadis, G., Ed., Liposome
Technology: Entrapment of drugs and other materials into liposomes,
CRC Press (2006).
Targeting Agents
[0088] The targeted delivery compositions of the present invention
also include MMP.sup.i, a targeting agent. Generally, MMP.sup.i
refers to any matrix metalloproteinase inhibitor. In certain
embodiments, MMP.sup.i is an inhibitor having the formula:
##STR00002##
wherein [0089] X is a member selected from the group consisting of
O and S; [0090] Y is a member selected from the group consisting of
pyridyl and phenyl, wherein said phenyl is optionally substituted
with OH, OCH.sub.3, OCF.sub.3 and CH.sub.3; and the wavy line
indicates the point of attachment to (LPEG).
[0091] In certain specific embodiments, MMP.sup.i is selected
from:
##STR00003##
[0092] In certain specific embodiments, MMP.sup.i is selected
from:
##STR00004##
B. Individual Components of the Targeted Delivery Compositions
Including a Nanocarrier
[0093] In another aspect, the present invention provides individual
components of the targeted delivery compositions disclosed herein.
In particular, the present invention includes a conjugate having
the formula: A-(LPEG)-MMP.sup.i; wherein, A is an attachment
component; (LPEG) is a linking group as described above; and,
MMP.sup.i is a MMP inhibitor.
[0094] It will be appreciated by one of ordinary skill in the art
that components of the targeted delivery compositions similarly
include each of the specific embodiments described above.
IV. Methods of Preparing Targeted Delivery Compositions and
Components
A. Targeted Delivery Compositions Including a Nanocarrier
[0095] The targeted delivery compositions of the present invention
can be produced in a variety of ways. In one aspect, targeted
delivery compositions of the present invention can be prepared by
attaching a nanocarrier to a conjugate having the formula:
A-(LPEG)-MMP.sup.i; wherein, A is an attachment component for
attaching said conjugate to said nanocarrier; (LPEG) is a linking
group; and, MMP.sup.i is a MMP inhibitor. The nanocarrier can be
contacted with the conjugate either as a loaded nanocarrier (e.g.,
having incorporated a therapeutic or diagnostic agent) or an
unloaded nanocarrier.
Nanocarriers
[0096] Nanocarriers can be produced by a variety of ways generally
known in the art and methods of making such nanocarriers can depend
on the particular nanocarrier desired. Any measuring technique
available in the art can be used to determine properties of the
targeted delivery compositions and nanocarriers. For example,
techniques such as dynamic light scattering, x-ray photoelectron
microscopy, powder x-ray diffraction, scanning electron microscopy
(SEM), transmission electron microscopy (TEM), and atomic force
microscopy (AFM) can be used to determine average size and
dispersity of the nanocarriers and/or targeted delivery
compositions.
[0097] Liposomes used in the targeted delivery compositions of the
present invention can be made using a variety of techniques
generally well known in the art. (See, e.g., Williams, A. P.,
Liposomes: A Practical Approach, 2.sup.nd Edition, Oxford Univ.
Press (2003); Lasic, D. D., Liposomes in Gene Delivery, CRC Press
LLC (1997)). For example, liposomes can be produced by but are not
limited to techniques such as extrusion, agitation, sonication,
reverse phase evaporation, self-assembly in aqueous solution,
electrode-based formation techniques, microfluidic directed
formation techniques, and the like. In certain embodiments, methods
can be used to produce liposomes that are multilamellar and/or
unilamellar, which can include large unilamellar vesicles (LUV)
and/or small unilamellar vesicles (SUV). Similar to self-assembly
of liposomes in solution, micelles can be produced using techniques
generally well known in the art, such that amphiphilic molecules
will form micelles when dissolved in solution conditions sufficient
to form micelles. Lipid-coated bubbles and lipoproteins can also be
constructed using methods known in the art (See, e.g., Farook, U.,
J. R. Soc. Interface, 6(32): 271-277 (2009); Lacko et al.,
Lipoprotein Nanocarriers as Delivery Vehicles for Anti-Cancer
Agents in Nanotechnology for Cancer Therapy, CRC Press (2007)).
[0098] Methods of making polymeric nanocarriers that can be used in
the present invention are generally well known in the art (See,
e.g., Sigmund, W. et al., Eds., Particulate Systems in Nano- and
Biotechnologies, CRC Press LLC (2009); Karnik et al., Nano Lett.,
8(9): 2906-2912 (2008)). For example, block copolymers can be made
using synthetic methods known in the art such that the block
copolymers can self-assemble in a solution to form polymersomes
and/or block copolymer micelles. Niosomes are known in the art and
can be made using a variety of techniques and compositions (Baillie
A. J. et al., J. Pharm. Pharmacol., 38:502-505 (1988)). Magnetic
and/or metallic particles can be constructed using any method known
in the art, such as co-precipitation, thermal decomposition, and
microemulsion. (See also Nagarajan, R. & Hatton, T. A., Eds.,
Nanocarriers Synthesis, Stabilization, Passivation, and
Functionalization, Oxford Univ. Press (2008)). Gold particles and
their derivatives can be made using a variety of techniques
generally known in the art, such as the Turkevich method, Brust
method, Perraut Method or sonolysis (See also, Grzelczak et al.,
Chem. Soc. Rev., 37: 1783-1791 (2008)). In some embodiments, the
attachment component can be attached through sulfur-gold tethering
chemistry. Quantum dots or semiconductor nanocrystals can be
synthesized using any method known in the art, such as colloidal
synthesis techniques.
Conjugates for Attaching to a Nanocarrier
[0099] The conjugates having the formula
A-[(EG)(P)].sub.m-MMP.sup.i, as described herein, can be
manufactured using a variety of techniques. In some embodiments,
the entire conjugate can be synthesized in oligonucleotide
synthesizers well known in the art. In certain embodiments,
incorporation of [(EG)(P)].sub.m, such as (HEGp).sub.m, can be
performed using modified synthesis cycles for more effective
incorporation. In particular, increased amidite equivalents and
extended wash cycles can incorporate multiple [(EG)(P)] units as
linking groups in the conjugates of the present invention. In
certain embodiments, an attachment component, such as cholesterol
or a cholesterol derivative (e.g., cholesterol-tetraethylene
glycol) can then be added using standard or modified synthesis
cycles, which can include doubling the coupling recycle step to
insure effective incorporation. In certain embodiments, the
conjugates can be synthesized using solid phase approaches, such as
silica-based or polystyrene-based supports.
[0100] In other embodiments, the [(EG)(P)].sub.m linking group can
be attached to an attachment component, such as a cholesterol
derivative (cholesterol-tetraethylene glycol), using conventional
chemistry known in the art. The [(EG)(P)].sub.m linking group can
be synthesized using the methods described above. Next, the linking
group and the attachment component can be mixed and reacted under
conditions sufficient to form a portion of the conjugate,
A-[(EG)(P)].sub.m. Subsequently, a targeting agent, MMP.sup.i, can
be attached to the other end of the [(EG)(P)].sub.m linking group.
Alternatively, the targeting agent can be attached to the
[(EG)(P)].sub.m linking group first, followed by the attachment
component. As will be appreciated by one of ordinary skill in the
art, targeting agents of the present invention can be attached to
the [(EG)(P)].sub.m linking group by a variety of ways that can
depend on the characteristics of the specific MMP.sup.i
component.
V. Methods of Administering Targeted Delivery Compositions
[0101] As described herein, the targeted delivery compositions and
methods of the present invention can be used for treating and/or
diagnosing any disease, disorder, and/or condition associated with
a subject. In one embodiment, the methods of the present invention
include a method for treating or diagnosing a cancerous condition
in a subject, comprising administering to the subject a targeted
delivery composition of the present invention that includes a
nanocarrier, wherein the therapeutic or diagnostic agent is
sufficient to treat or diagnose the condition. In certain
embodiments, the cancerous condition can include cancers that
sufficiently express (e.g., on the cell surface or in the
vasculature) a receptor that is being targeted by a targeting agent
of a targeted delivery composition of the present invention.
[0102] In another embodiment, the methods of the present invention
include a method of determining the suitability of a subject for a
targeted therapeutic treatment, comprising administering to the
subject a targeted delivery composition that includes a
nanocarrier, wherein the nanocarrier comprises a diagnostic agent,
and imaging the subject to detect the diagnostic agent.
Administration
[0103] In some embodiments, the present invention can include a
targeted delivery composition and a physiologically (i.e.,
pharmaceutically) acceptable carrier. As used herein, the term
"carrier" refers to a typically inert substance used as a diluent
or vehicle for a drug such as a therapeutic agent. The term also
encompasses a typically inert substance that imparts cohesive
qualities to the composition. Typically, the physiologically
acceptable carriers are present in liquid form. Examples of liquid
carriers include physiological saline, phosphate buffer, normal
buffered saline (135-150 mM NaCl), water, buffered water, 0.4%
saline, 0.3% glycine, glycoproteins to provide enhanced stability
(e.g., albumin, lipoprotein, globulin, etc.), and the like. Since
physiologically acceptable carriers are determined in part by the
particular composition being administered as well as by the
particular method used to administer the composition, there are a
wide variety of suitable formulations of pharmaceutical
compositions of the present invention (See, e.g., Remington's
Pharmaceutical Sciences, 17.sup.th ed., 1989).
[0104] The compositions of the present invention may be sterilized
by conventional, well-known sterilization techniques or may be
produced under sterile conditions. Aqueous solutions can be
packaged for use or filtered under aseptic conditions and
lyophilized, the lyophilized preparation being combined with a
sterile aqueous solution prior to administration. The compositions
can contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions, such as pH
adjusting and buffering agents, tonicity adjusting agents, wetting
agents, and the like, e.g., sodium acetate, sodium lactate, sodium
chloride, potassium chloride, calcium chloride, sorbitan
monolaurate, and triethanolamine oleate. Sugars can also be
included for stabilizing the compositions, such as a stabilizer for
lyophilized targeted delivery compositions.
[0105] The targeted delivery composition of choice, alone or in
combination with other suitable components, can be made into
aerosol formulations (i.e., they can be "nebulized") to be
administered via inhalation. Aerosol formulations can be placed
into pressurized acceptable propellants, such as
dichlorodifluoromethane, propane, nitrogen, and the like.
[0106] Suitable formulations for rectal administration include, for
example, suppositories, which includes an effective amount of a
packaged targeted delivery composition with a suppository base.
Suitable suppository bases include natural or synthetic
triglycerides or paraffin hydrocarbons. In addition, it is also
possible to use gelatin rectal capsules which contain a combination
of the targeted delivery composition of choice with a base,
including, for example, liquid triglycerides, polyethylene glycols,
and paraffin hydrocarbons.
[0107] Formulations suitable for parenteral administration, such
as, for example, by intraarticular (in the joints), intravenous,
intramuscular, intratumoral, intradermal, intraperitoneal, and
subcutaneous routes, include aqueous and non-aqueous, isotonic
sterile injection solutions, which can contain antioxidants,
buffers, bacteriostats, and solutes that render the formulation
isotonic with the blood of the intended recipient, and aqueous and
non-aqueous sterile suspensions that can include suspending agents,
solubilizers, thickening agents, stabilizers, and preservatives.
Injection solutions and suspensions can also be prepared from
sterile powders, granules, and tablets. In the practice of the
present invention, compositions can be administered, for example,
by intravenous infusion, topically, intraperitoneally,
intravesically, or intrathecally. Parenteral administration and
intravenous administration are the preferred methods of
administration. The formulations of targeted delivery compositions
can be presented in unit-dose or multi-dose sealed containers, such
as ampoules and vials.
[0108] The pharmaceutical preparation is preferably in unit dosage
form. In such form the preparation is subdivided into unit doses
containing appropriate quantities of the active component, e.g., a
targeted delivery composition. The unit dosage form can be a
packaged preparation, the package containing discrete quantities of
preparation. The composition can, if desired, also contain other
compatible therapeutic agents.
[0109] In therapeutic use for the treatment of cancer, the targeted
delivery compositions including a therapeutic and/or diagnostic
agent utilized in the pharmaceutical compositions of the present
invention can be administered at the initial dosage of about 0.001
mg/kg to about 1000 mg/kg daily. A daily dose range of about 0.01
mg/kg to about 500 mg/kg, or about 0.1 mg/kg to about 200 mg/kg, or
about 1 mg/kg to about 100 mg/kg, or about 10 mg/kg to about 50
mg/kg, can be used. The dosages, however, may be varied depending
upon the requirements of the patient, the severity of the condition
being treated, and the targeted delivery composition being
employed. For example, dosages can be empirically determined
considering the type and stage of cancer diagnosed in a particular
patient. The dose administered to a patient, in the context of the
present invention, should be sufficient to affect a beneficial
therapeutic response in the patient over time. The size of the dose
will also be determined by the existence, nature, and extent of any
adverse side-effects that accompany the administration of a
particular targeted delivery composition in a particular patient.
Determination of the proper dosage for a particular situation is
within the skill of the practitioner. Generally, treatment is
initiated with smaller dosages which are less than the optimum dose
of the targeted delivery composition. Thereafter, the dosage is
increased by small increments until the optimum effect under
circumstances is reached. For convenience, the total daily dosage
may be divided and administered in portions during the day, if
desired.
[0110] In some embodiments, the targeted delivery compositions of
the present invention may be used to diagnose a disease, disorder,
and/or condition. In some embodiments, the targeted delivery
compositions can be used to diagnose a cancerous condition in a
subject, such as lung cancer, breast cancer, pancreatic cancer,
prostate cancer, cervical cancer, ovarian cancer, colon cancer,
liver cancer, esophageal cancer, and the like. In some embodiments,
methods of diagnosing a disease state may involve the use of the
targeted delivery compositions to physically detect and/or locate a
tumor within the body of a subject. For example, tumors can be
related to cancers that sufficiently express (e.g., on the cell
surface or in the vasculature) a receptor that is being targeted by
a targeting agent of a targeted delivery composition of the present
invention. In some embodiments, the targeted delivery compositions
can also be used to diagnose diseases other than cancer, such as
proliferative diseases, cardiovascular diseases, gastrointestinal
diseases, genitourinary disease, neurological diseases,
musculoskeletal diseases, hematological diseases, inflammatory
diseases, autoimmune diseases, rheumatoid arthritis and the
like.
[0111] As disclosed herein, the targeted delivery compositions of
the invention can include a diagnostic agent that has intrinsically
detectable properties. In detecting the diagnostic agent in a
subject, the targeted delivery compositions, or a population of
particles with a portion being targeted delivery compositions, can
be administered to a subject. The subject can then be imaged using
a technique for imaging the diagnostic agent, such as single photon
emission computed tomography (SPECT), magnetic resonance imaging
(MRI), optical imaging, positron emission tomography (PET),
computed tomography (CT), x-ray imaging, gamma ray imaging, and the
like. Any of the imaging techniques described herein may be used in
combination with other imaging techniques. In some embodiments, the
incorporation of a radioisotope for imaging in a particle allows in
vivo tracking of the targeted delivery compositions in a subject.
For example, the biodistribution and/or elimination of the targeted
delivery compositions can be measured and optionally be used to
alter the treatment of patient. For example, more or less of the
targeted delivery compositions may be needed to optimize treatment
and/or diagnosis of the patient.
Targeted Delivery
[0112] In certain embodiments, the targeted delivery compositions
of the present invention can be delivered to a subject to release a
therapeutic or diagnostic agent in a targeted manner. For example,
a targeted delivery composition can be delivered to a target in a
subject and then a therapeutic agent embedded in, encapsulated in,
or tethered to the targeted delivery composition, such as to the
nanocarrier, can be delivered based on solution conditions in
vicinity of the target. Solution conditions, such as pH, salt
concentration, and the like, may trigger release over a short or
long period of time of the therapeutic agent to the area in the
vicinity of the target. Alternatively, an enzyme can cleave the
therapeutic or diagnostic agent from the targeted delivery
composition to initiate release. In some embodiments, the targeted
delivery compositions can be delivered to the internal regions of a
cell by endocytosis and possibly later degraded in an internal
compartment of the cell, such as a lysosome. One of ordinary skill
will appreciate that targeted delivery of a therapeutic or
diagnostic agent can be carried out using a variety of methods
generally known in the art.
Kits
[0113] The present invention also provides kits for administering
the targeted delivery compositions to a subject for treating and/or
diagnosing a disease state. Such kits typically include two or more
components necessary for treating and/or diagnosing the disease
state, such as a cancerous condition. Components can include
targeted delivery compositions of the present invention, reagents,
containers and/or equipment. In some embodiments, a container
within a kit may contain a targeted delivery composition including
a radiopharmaceutical that is radiolabeled before use. The kits can
further include any of the reaction components or buffers necessary
for administering the targeted delivery compositions. Moreover, the
targeted delivery compositions can be in lyophilized form and then
reconstituted prior to administration.
[0114] In certain embodiments, the kits of the present invention
can include packaging assemblies that can include one or more
components used for treating and/or diagnosing the disease state of
a patient. For example, a packaging assembly may include a
container that houses at least one of the targeted delivery
compositions as described herein. A separate container may include
other excipients or agents that can be mixed with the targeted
delivery compositions prior to administration to a patient. In some
embodiments, a physician may select and match certain components
and/or packaging assemblies depending on the treatment or diagnosis
needed for a particular patient.
[0115] It is understood that the embodiments described herein are
for illustrative purposes only and that various modifications or
changes in light thereof will be suggested to persons skilled in
the art and are to be included within the spirit and purview of
this application and scope of the appended claims. All
publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
VI. Examples
[0116] Abbreviations: mL, milliliters; HOBT, hydroxybenzotriazole;
LCMS, liquid chromatography mass spectrum; DMF, dimethylformamide;
DMSO, dimethyl sulfoxide; EA, ethyl acetate; H, hexane; rt, ambient
temperature; h, hour(s); TLC, thin layer chromatography; TEA,
triethylamine; HRMS, high resolution mass spectrum; Boc,
tert-butyloxy carbonyl.
Example 1
Synthesis of MMP-Targeting Conjugate for Preparation of Targeted
Delivery Compositions
Step 1: Preparation of 1-tert-butyl 4-ethyl
4-(4-(pyridin-3-yloxy)phenylsulfonyl)piperidine-1,4-dicarboxylate
[0117] 1-tert-butyl 4-ethyl
4-(4-(pyridin-3-yloxy)phenylsulfonyl)piperidine-1,4-dicarboxylate
was prepared according to FIG. 2. A round bottom flask (100 mL)
equipped with magnetic stir bar (Teflon covered) and condenser was
charged with 1.5 g p-fluro-sulfone, 0.5 g (1.5 eq.) 3-hydroxy
pyridine and 1.76 g (1.5 eq.) cesium carbonate in DMF (50 mL). The
reaction was heated to 90.degree. C. for 18 hours. LCMS after 2
hours shows desired product at 4.4 min and starting material
sulfone at 4.8 minutes. Volatiles were removed and the residue
partitioned between ethyl acetate and saturated aqueous sodium
bicarbonate. The organic layer was washed with 10% aqueous citric
acid, aqueous sodium bicarbonate, brine, and dried with anhydrous
sodium sulfate. TLC (silica) shows one spot (40% ethyl
acetate:hexane). Ethyl acetate was removed and the resulting amber
semi solid was vacuum dried to obtain 1.72 g of product as an amber
foam. .sup.1H-NMR (DMSO-d6) is consistent with desired product.
This intermediate was used in the next step without further
purification.
Step 2. Preparation of
1-(tert-butoxycarbonyl)-4-(4-(pyridin-3-yloxy)phenylsulfonyl)piperidine-4-
-carboxylic acid
[0118]
1-(tert-butoxycarbonyl)-4-(4-(pyridin-3-yloxy)phenylsulfonyl)piperi-
dine-4-carboxylic acid was prepared according to FIG. 3. A 250-mL
round bottom flask was charged with 1.7 g ethyl ester (from step
1), 0.78 g (4 eq.) potassium hydroxide in 16 mL ethanol and 4 mL
water. The reaction mixture was heated to 90.degree. C. LCMS after
1 hour shows complete reaction. The mixture was partitioned between
ethyl acetate and 10% aqueous KHSO.sub.4-Brine. The yellow organic
phase was separated, dried, filtered and concentrated. The residue
was vacuum dried overnight to produce 1.32 g off-white foam that
was used without further purification.
Step 3: Preparation of tert-butyl
4-(benzyloxycarbamoyl)-4-(4-(pyridin-3-yloxy)phenylsulfonyl)piperidine-1--
carboxylate
[0119] tert-Butyl
4-(benzyloxycarbamoyl)-4-(4-(pyridin-3-yloxy)phenylsulfonyl)piperidine-1--
carboxylate was prepared according to FIG. 4. A 100 mL round bottom
flask equipped with magnetic stir bar was charged with 1.32 g acid,
0.66 g (1.2 eq.) EDC, 0.58 (1.5 eq.) HOBT, and 0.58 g (2 eq.) TEA
in 15 mL CH.sub.2Cl.sub.2. This was stirred 10 minutes when 0.55 g
(1.2 eq.) of the amine HCl salt was added. The reaction was stirred
at RT. LCMS after 2 hours shows little starting material and
.about.1:1 mixture of product and active ester. Another 1 eq. of
amine --HCl salt was added to the reaction mixture. LCMS shows a
trace of acid and mostly product. The HOBt ester is not observed.
The reaction was concentrated to a solid and partitioned between
ethyl acetate and aqueous sodium bicarbonate. The organics were
washed with 10% aqueous KHSO.sub.4, brine, and dried. TLC (silica,
1:1 ethyl acetate: hexane) showed one spot (R.sub.f=.about.0.4 and
some material at the origin). The resulting ethyl acetate solution
was concentrated 10 mL) and filtered through a plug of silica gel.
The silica was washed with ethyl acetate and combined organics were
concentrated and vacuum dried to yield 1.29 g (80%) of an off-white
foam.
Step 4. Preparation of
N-(benzyloxy)-4-(4-(pyridin-3-yloxy)phenylsulfonyl)piperidine-4-carboxami-
de
[0120]
N-(benzyloxy)-4-(4-(pyridin-3-yloxy)phenylsulfonyl)piperidine-4-car-
boxamide was prepared according to FIG. 5. A 100 mL round bottom
flask was charged with 1.3 g of the Boc protected amine prepared in
Step 3 and 4 N HCl-Dioxane (10 mL) and the mixture stirred for 20
minutes. LCMS after 20 minutes shows no starting material. The
reaction mixture was concentrated in vacuo and vacuum dried
overnight to afford
N-(benzyloxy)-4-(4-(pyridin-3-yloxy)phenylsulfonyl)piperidine-4-carboxami-
de (1.3 g bis HCl salt) that was used without further
purification.
Step 5: Preparation of PEG 1000 piperidine amido amine derivative
of
N-(benzyloxy)-4-(4-(pyridin-3-yloxy)phenylsulfonyl)piperidine-4-carboxami-
de
[0121] The PEG 1000 piperidine amido amine derivative of
N-(benzyloxy)-4-(4-(pyridin-3-yloxy)phenylsulfonyl)piperidine-4-carboxami-
de was prepared according to FIG. 6. A 100 mL round bottom flask
was charged with 1.16 g (1.0 eq.) PEG acid mono Boc amine, 0.25 g
(1.2 eq.) EDC, 0.2 g (1.5 eq.) HOBt, and 0.09 g (1.0 eq.) TEA in 5
mL dichloromethane. 0.5 g (1.0 eq.) of the amine and 0.28 g (3.0
eq.) additional TEA were added in 7 mL dichloromethane and the
reaction was stirred under argon for 10 minutes then stirred at RT
overnight. The reaction was diluted with 85 mL CHCl.sub.3 and
washed with 15 mL deionized water, 25 mL 5% aqueous citric acid,
and then a mixture of 25 mL aqueous sodium bicarbonate -25 mL
brine. The organic layer stayed pale yellow and was dried (sodium
sulfate, anhydrous) and concentrated in vacuo. TLC (20%
MeOH--CHCl.sub.3) shows R.sub.f=0.4 but, more importantly, clean
one spot with nothing UV visible at the origin. After concentration
the product was vacuum dried to afford 1.7 g. LC-HRMS.sub.(obs)
M+H=1695.8782 g/mol; M+NH.sub.4=1712.9046 g/mol.
HRMS.sub.(calculated) M+H=1695.8775 g/mol; M+NH.sub.4=1712.9040
g/mol. .sup.1H-NMR (CDCl.sub.3) was consistent with the desired
product.
Step 6:
[0122] Deprotection of the PEG 1000 piperidine amido amine
derivative of
N-(benzyloxy)-4-(4-(pyridin-3-yloxy)phenylsulfonyl)piperidine-4-carboxami-
de was conducted according to FIG. 7. A 100 mL round bottom flask
was charged with 1.66 g of the Boc compound in 15 mL 4 N
HCl-Dioxane and tumbled for 30 minutes on a rotary evaporator. The
reaction was concentrated to an oil that was subsequently vacuum
dried to a clear thick yellow syrup. The de-BOC amine was obtained
(1.68 g of the bis-HCl salt) and used without further purification.
Material was submitted for LC-HRMS and chloride content. Cl content
was determined to be 4%, consistent with 2 eq. HCl.
Step 7:
[0123] Conjugation of DSPE-PEG 5000 to the PEG 1000 piperidine
amido amine derivative of
N-(benzyloxy)-4-(4-(pyridin-3-yloxy)phenylsulfonyl)piperidine-4-carboxami-
de was conducted according to FIG. 8. A 50 mL round bottom flask
was charged with 60 mg (1.0 eq.) amine prepared in Step 6 and 15 mg
(4 eq.) triethylamine in 8 mL methylene chloride. NHS ester of
DSPE-PEG 5000 (220 mg, 1.0 eq) was added and the reaction was
stirred at RT for 21/2 hours and a sample was analyzed by LCMS. The
reaction was concentrated to a clear oil. This was redissolved in
acetonitrile: water, frozen and lyophilized overnight to afford
.about.260 mg of crude white foam that was used without further
purification. LC-HRMS: M/2-2H.sub.(obs)=3780.2141 LC-HRMS
M/2-2H.sub.(calc)=3780.2244.
Step 8:
[0124] Cleavage of the benzylgroup from the
N-benzyloxy-4-carboxamide moiety of the DSPE-PEG-MMPi intermediate
obtained in Step 8 was conducted according to FIG. 9. A 200 mL
round bottom flask equipped with magnetic stir bar containing 325
mg lyophilized benzyl ester prepared in Step 7 was charged with 15
mL methanol. Wet 10% Pd--C catalyst (75 mg, Degussa) was added and
the reaction mixture was purged with argon for 10 minutes then
hydrogen was slowly bubbled over the stirring solution. The
reaction proceeded for 1 hour 20 minutes and then analyzed by MS.
LCMS showed partial conversion to product. The reaction mixture was
filtered through Celite.RTM. and washed with warm methanol (40 mL).
The resulting solution was treated with fresh catalyst (75 mg) and
hydrogenated for an additional 90 min. LCMS showed no starting
material. The reaction mixture was purged with argon and filtered
through Celite then poured through a paper funnel to remove slight
cloudiness and the filtrate was concentrated in vacuo to afford a
clear oil. This was dissolved in water/acetonitrile, frozen and
lyophilized for 2 days. The desired DSPE-PEG-MMPi product was
obtained as a dry white powder (230 mg). See FIG. 1 for LC-HRMS
results.
Example 2
Procedure for Preparation of MMP.sup.i Targeted Liposomal TD-1
##STR00005##
[0126] A representative procedure for the formation of MMP.sup.i
targeted liposomes containing a cytotoxic pro-drug is provided in
the following example.
[0127] To a 3-neck, 500 mL round-bottom flask containing a solution
of 10 mM aqueous sodium acetate/300 mM sucrose at pH 5.5 (90 mL)
was added TD-1 (189 mg, 0.181 mmol). Upon complete dissolution the
homogeneous solution was adjusted to pH 5.50. Independently, the
volume of a previously prepared solution of pre-formed liposomes
(DSPC:Cholesterol (55:45)) was measured and diluted with 10 mM
aqueous sodium acetate/300 mM sucrose at pH 5.5 to 100 mL. The pH
of the heterogeneous solution was adjusted to pH 5.5. Both
solutions were gently heated to 65.degree. C. at which time the
TD-1 solution was rapidly added to the liposome solution. The
combined mixture was held at 65.degree. C. for 15 min., then cooled
to 55.degree. C. Meanwhile, DSPE-PEG(2000) obtained from Lipoid
(138 mg, 0.049 mmol) and Conjugate 1 (see previous example, 5 mg,
0.553 umol) was dissolved in solution of 10 mM acetate/300 mM
sucrose at pH 5.5 (5 mL). Once the TD-1/liposome mixture reached
55.degree. C., the pH was measured (pH 5.98), particle size
(intensity and volume) was obtained (Z.Ave.=109.4 nm), and the
homogeneous solution of Lipoid/Conjugate 1 target was added. The
resulting mixture was heated at 55.degree. C. for 30 min then
allowed to cool to room temperature. The pH was measured (pH 5.89),
particle size (intensity and volume) was obtained (Z.Ave.=117.0).
This liposome solution was concentrated to 60 mL, then diafiltered
against 1.8 L of 20 mM histadine in saline at pH 6.5 (minimally
1.5.times. concentrated volume). The resulting solution was
concentrated to 14 mL (collecting final 2 mL of permeate solution
for analysis). The pH was measured (pH 6.42), particle size
(intensity and volume) was obtained (Z.Ave.=118.9) and the presence
of targeting MMPi ligand confirmed by rphplc analysis as greater
than 100 MMPi ligand molecules per liposome particle.
[0128] A sample (1.0 mL) was submitted for TD-1 assay, MMPi
targeting assay, docetaxel area %, DSPC, Cholesterol,
DSPE-PEG(2000) and Lyso-DSPC assay.
TABLE-US-00002 10 mM Acetate in 300 mM Formulation Sucrose pH 5.5
pH 6.42 Particle Size 118.90 Volume (mL) 34.00 Area %, TD-1 98.03
%-BOC, RRT 0.41 0.00 %-BOC, RRT 0.43 0.00 %-ORO RRT 0.89 0.00 %-ORO
RRT 0.91 0.00 %-ORO RRT 0.95 0.00 % 7-epi M3528, RRT 1.11 1.03 %
Docetaxel, RRT 1.44 0.95 Prodrug, mg/ml 3.34 Target, ug/ml 262.00
Wt % Chol 26.9 Wt % DSPC 58.3 Wt % DSPE_PEG 2000 9.6 Wt % Lyso-DSPC
5.2 TD1/lipid 0.157
Example 3
Preparation of MMP.sup.i-Targeted Liposomal Oxaliplatin
Step 1.
[0129] 1-Benzyl 4-methyl
4-((4-phenoxyphenyl)sulfonyl)piperidine-1,4-dicarboxylate was
synthesized as shown in FIG. 10. A 100 ml RBF equipped with
magnetic stir bar was charged with 1.1 g (2.67 mmole) 4-methyl
4-((4-phenoxyphenyl)sulfonyl)piperidine-4-carboxylate 0.73 g (2.94
mmole) Cbz-OSu, 0.8 g (8.01 mmole) triethylamine. LCMS after 1 hour
showed complete reaction with M+H=510 g/mol. The reaction was
partitioned between EA and satd. aq. sodium bicarbonate. The
organics were washed with 10 aq. KHSO.sub.4, dried and concentrated
to thick syrup that turned into white dry foam upon high vacuum
drying. The product was dried overnight to afford 1.29 g (95%
yield) of the Cbz ester that was used in Step 2.
Step 2.
[0130]
1-((Benzyloxy)carbonyl)-4-((4-phenoxyphenyl)sulfonyl)piperidine-4-c-
arboxylic acid was synthesized as shown in FIG. 11. A 250 ml RBF
was charged with the 1.75 g of 1-benzyl 4-methyl
4-((4-phenoxyphenyl)sulfonyl)piperidine-1,4-dicarboxylate (3.39
mmole), 0.57 g (10.16 mmole) potassium hydroxide in 30 ml
ethanol/7.5 ml water. The reaction mixture was stirred at
50.degree. C. with LCMS monitoring. LCMS analysis indicated
.about.80% conversion after 1 hr and approximately 90% conversion
after 2 hr with a trace of impurities appearing. The solution was
concentrated to 1/4 volume and partitioned between EA and 10% aq.
Citric acid. The organics were washed with brine, dried, and
concentrated in vacuo. The product was vacuum dried overnight to
yield 1.6 g (95% yield) white solid that was used in Step 3.
Step 3.
[0131] Benzyl
4-((4-phenoxyphenyl)sulfonyl)-4-(((tetrahydro-2H-pyran-2-yl)oxy)carbamoyl-
)piperidine-1-carboxylate was synthesized as shown in FIG. 12. A
200 ml RBF equipped with magnetic stir bar was charged with 1.48 g
(2.99 mmole)
1-((benzyloxy)carbonyl)-4-((4-phenoxyphenyl)sulfonyl)piperidine-4-carboxy-
lic acid, 0.49 g (4.18 mmole) OTHP-hydroxylamine, 0.8 g (4.18
mmole) EDC, 0.64 g (4.18 mmole) HOBt, and 1.25 ml (8.96 mmole)
triethylamine in 30 ml DMF. The reaction mixture was stirred at
room temperature overnight. The reaction was concentrated in vacuo
and partitioned between EA and satd. aq. sodium bicarbonate. The
organics were washed with 10% aq. Citric acid, brine, dried, and
concentrated in vacuo. This material was vacuum dried overnight to
afford 1.50 g (85%) of dry white foam. The sample analyzed by
direct infusion MS, which indicated >90% product with some trace
impurities. HRMS.sub.(theoretical) M+H=595.2108 g/mol.
HRMS.sub.(observed) M+H=595.2109 g/mol.
Step 4.
[0132]
4-((4-Phenoxyphenyl)sulfonyl)-4-(((tetrahydro-2H-pyran-2-yl)oxy)car-
bamoyl)piperidine was synthesized as shown in FIG. 13. A 200 ml RBF
equipped with magnetic stir bar was charged with the 1.5 g of
benzyl
4-((4-phenoxyphenyl)sulfonyl)-4-(((tetrahydro-2H-pyran-2-yl)oxy)carbamoyl-
)piperidine-1-carboxylate compound and 100 mg wet Degussa 5%
Palladium on Carbon in 45 ml Methanol. The reaction mixture was
purged with Argon for 5 minutes. Hydrogen was then bubbled over the
solution for 1 hr. MS analysis (direct infusion) at this point
indicated that the reaction was complete. The crude was filtered
through Celite and the Celite was washed with 40 ml additional
methanol. The methanol solution was concentrated in vacuo to 1.2 g
white solid that was vacuum dried for 4 hours to yield 1.1 g
product that was used in Step 5. HRMS.sub.(observed) M+H=451.1737
g/mol.
Step 5.
[0133] Benzyl tert-butyl
((5S)-6-oxo-6-(4-((4-phenoxyphenyl)sulfonyl)-4-(((tetrahydro-2H-pyran-2-y-
l)oxy)carbamoyl)piperidin-1-yl)hexane-1,5-diyl)dicarbamate was
synthesized as shown in FIG. 14. A 50 ml RBF equipped with magnetic
stir bar was charged with 330 mg (0.72 mmole)
4-((4-phenoxyphenyl)sulfonyl)-4-(((tetrahydro-2H-pyran-2-yl)oxy)carbamoyl-
)piperidine, 286 mg (0.75 mmole) acid, 172 mg (0.9 mmole) EDC, 165
mg (1.1 mmole) HOBt, and 218 mg (2.15 mmole) triethylamine in 10 ml
dry DMF. The reaction mixture was stirred at room temperature
overnight. The DMF was removed and the reaction residue was
partitioned between EA and satd. aq. sodium bicarbonate. The
organics were washed with brine, dried, concentrated and vacuum
dried to afford 585 mg (97% yield) crude white foam that was used
in Step 7. FIRMS.sub.(theoretical)M+Na=845.3402 g/mol.
HRMS.sub.(observed)M+H=845.3406 g/mol.
Step 6.
[0134] Tert-butyl
((2S)-6-amino-1-oxo-1-(4-((4-phenoxyphenyl)sulfonyl)-4-(((tetrahydro-2H-p-
yran-2-yl)oxy)carbamoyl)piperidin-1-yl)hexan-2-yl)carbamate was
synthesized as shown in FIG. 15. A 100 ml RBF was charged with 585
mg crude from Step 6, 88 mg 5% wet Palladium on Carbon (Degussa) in
45 ml Methanol. The reaction mixture was purged with Argon for
.about.5 minutes then hydrogen was slowly bubbled over the
solution. LCMS analysis after 1 hour showed .about.50-60%
conversion to product with a M+H=689 g/mol. LCMS analysis after
indicated about 75% conversion after three hours and about 90%
conversion after four hours. The mixture was left to react for an
additional hour. After a 5 minute Argon purge the reaction mixture
was filtered through Celite. The reaction was concentrated and
volatiles were chased 2.times. with dichloromethane and the white
foam/solid was vacuum dried overnight to afford 492 mg (87%) white
solid.
Step 7.
[0135] A protected
4-((4-phenoxyphenyl)sulfonyl)-4-(((tetrahydro-2H-pyran-2-yl)oxy)carbamoyl-
)piperidine-PEG5000-DSPE conjugate was synthesized as shown in FIG.
16. A 25 ml RBF equipped with magnetic stir bar was charged with
200 mg DSPE-PEG5000-GS active ester (0.03 mmole), 19 mg (0.027
mmole) amine, and 12 mg (0.12 mmole) triethylamine. The reaction
was stirred overnight under Argon. The product was concentrated and
vacuum dried overnight. RPHPLC purification was done on a C8 column
using 30 ml/min gradient of 20-100% over 13 minutes. The solvents
were initially 20% 1:1 acetonitrile:isopropanol/80% 25 mM ammonium
acetate in water with 5% acetonitrile. The product containing
fractions were combined, concentrated to remove organic and the
remainder was lyophilized to afford 93.5 mg (46% yield) white
powder.
Step 8.
[0136] An
N-hydroxy-4-((4-phenoxyphenyl)sulfonyl)piperidine-4-carboxamide--
PEG5000-DSPE conjugate (Conjugate 2) was synthesized as shown in
FIG. 17. A 100 ml RBF was charged with 93 mg in 1 ml TFA and 0.1 ml
triethylsilane. The reaction was tumbled for 60 minutes. A sample
was analyzed by LCMS, showing only desired formation of product. 10
ml water was added and aq. NH.sub.4OH was used to bring the pH to
7.1. This was frozen and lyophilized overnight. To remove excess
NH.sub.4TFA, the crude was dissolved in 20 ml Millipore water and
the solution was put it in a 3500 MW Pierce Slide-A-Lyzer cassette
and stirred overnight in a 1 L beaker filled with Millipore water.
The water was changed out twice more during the next 12 hours. The
resulting aqueous dialyzed solution was frozen and lyophilized
overnight to afford .about.83 mgs of dry powder. A high resolution
mass spectra for the product is shown in FIG. 18.
Preparation of Liposomes.
[0137] Conjugate 2 was dissolved in DI H.sub.2O to generate either
a 1.0 mg/mL or 2.0 mg/mL solution. It was then added to a
phosphatidylcholine-based oxaliplatin-containing liposome
preparation (at 1.0 mg/mL of liposome), and the resulting mixture
was stirred at 37.degree. C. for 8 h. The crude material was
analyzed by SEC-HPLC. To remove free MMPi and free oxapliplatin,
the crude formulation was passed through Spectrum Filter Module
P/N: P-DI-500E-100-01N (prewashed with 1 L Millipore water) and
washed with 900 mL (10-fold volume) of buffer (300 mM Sucrose with
20 mM sodium acetate, freshly prepared). This was typically
accomplished over two days. Buffer and formulation were kept in the
refrigerator overnight. The Spectrum Filter Module was rinsed with
0.1 N NaOH before storing overnight. After purification, the
formulation was concentrated to a desired oxaliplatin
concentration. The final sample was analyzed by SEC-HPLC. (10 uL of
sample were diluted with 90 uL of PBS (1:9 dilution), and 5 uL were
injected.) Particle size was measured on the Malvern Zetasizer.
Lipids were analyzed via HPLC while Pt was quantified by ICP-MS.
"Free" Pt was determined by ICP-MS of the filtrate obtained from 30
KDa Amicon centrifuge filters (9000 rpm for 10 min. at ambient
temperature). The amount of targeting ligand inserted was
determined by an HPLC method employing a calibration curve.
Liposomes had an average particle size of 100 nm and included 54
ligands (Conjugate 2) per particle.
Example 4
Efficacy of MMPi-Liposomes in Nude Mice Bearing BxPC3 Xenograft
Tumors
[0138] The MMP.sup.i-targeted liposomal oxaliplatin prepared in
Example 3 was administered to mice bearing BxPC3 pancreatic
tumors.
[0139] FemaleHsd:Athymic Nude-Foxn1 nu/nu mice ((.apprxeq.20 g)
were implanted with (2.5.times.10.sup.6) BxPC3 cells subcutaneously
into the right flank. Ten mice were used per dose group. The eight
dose groups included saline, oxaliplatin, base
oxaliplatin-containing liposomes at 22 mg/kg, 44 mg/kg, and 66
mg/kg, and the corresponding MMPi-liposomes at 31 mg/kg, 62 mg/kg,
and 94 mg/kg.
[0140] Once tumors reached a median size of 150 mm.sup.3, animals
were randomized into groups, normalized by tumor volume among the
groups. Animals without tumors were not included in this study.
Test articles were dosed intravenously once.
[0141] Tumor length and width were measured with calipers 3 times
per week and volume was calculated from the formula: Tumor Volume
(mm.sup.3)=Length*Width2*0.5. Animals were weighed once per week.
Tumor volume was expressed as median and plotted as a function of
time. Any animal removed from the study due to excess size beyond
2000 mm.sup.3 had its value carried forward as 2000 mm.sup.3 in
subsequent plots. Tumor volume was also expressed as mean and
plotted as a function of time (groups with less than 50% animals
remaining were not be carried forward). Statistical significance of
observed differences between growth curves was evaluated by One-Way
ANOVA followed by posthoc test if significant.
[0142] Mean tumor volume and survival rates for mice treated with
the targeted liposomes were compared to mean tumor volume and
survival rates for control mice and mice that were administered
untargeted oxaliplatin liposomes and Eloxatin (non-liposomal
oxaliplatin). Administration of liposomal oxaliplatin led to lower
tumor volumes than for Eloxatin, and administration of
MMP.sup.i-targeted liposomal oxaliplatin led to lower tumor volumes
than for comparable doses of untargeted liposomal oxaliplatin (FIG.
19A. Mean tumor volume was measured after a single intravenous
injection of test article. All doses are given as oxaliplatin molar
equivalents. Values are mean.+-.SEM for 5-10 mice/group.). FIG. 19B
shows a Kaplan-Meier plot showing percent survival of mice bearing
BxPC-3 human pancreatic xenografts after a single intravenous
injection of MMP14 receptor targeting liposomes containing
oxaliplatin (Targeted Liposome), non-targeted liposomes containing
oxaliplatin, Eloxatin or saline. All doses are given as oxaliplatin
molar equivalents. Each group started with 10 female mice bearing
tumors. Significant differences in body weight change from the
control group and the Eloxatin group were not observed for targeted
and untargeted liposomes at most dosage levels (FIG. 20A; values
are mean.+-.SEM for 5-10 mice/group).
Example 5
Efficacy of MMPi-Liposomes in Nude Mice Bearing MMP14 Overexpressed
HT-1080 Xenograft Tumors
[0143] The MMP.sup.i-targeted liposomal oxaliplatin prepared in
Example 3 was administered to nude mice mice bearing humar
fibrosarcoma HT1080 tumors over-expressing MMP14.
[0144] FemaleHsd:Athymic Nude-Foxn1 nu/nu mice (25 g) were
implanted with (5.times.10.sup.6) HT1080/MMP14 tumor cells
subcutaneously into the side. Ten mice were used per dose group.
The six dose groups included saline, oxaliplatin, base
oxaliplatin-containing liposomes at 15 mg/kg and 30 mg/kg, and the
corresponding MMPi-liposomes at 15 mg/kg and 30 mg/kg.
[0145] Once tumors reached a median size of 150 mm.sup.3, animals
were randomized into groups, normalized by tumor volume among the
groups. Animals without tumors were not included in this study.
Test articles were dosed intravenously once.
[0146] Tumor length and width were measured with calipers 3 times
per week and volume was calculated from the formula: Tumor Volume
(mm.sup.3)=Length*Width2*0.5. Animals were monitored and weighed
twice per week. Tumor volume was expressed as median and plotted as
a function of time. Any animal removed from the study due to excess
size beyond 2000 mm.sup.3 had its value carried forward as 2000
mm.sup.3 in subsequent plots. Tumor volume was also expressed as
mean and plotted as a function of time (groups with less than 50%
animals remaining were not be carried forward). Statistical
significance of observed differences between growth curves was
evaluated by One-Way ANOVA followed by posthoc test if
significant.
[0147] Mean tumor volume and survival rates for mice treated with
the targeted liposomes were compared to mean tumor volume and
survival rates for control mice and mice that were administered
untargeted oxaliplatin liposomes and Eloxatin (non-liposomal
oxaliplatin). Administration of liposomal oxaliplatin led to lower
tumor volumes than for Eloxatin, and administration of MMP-targeted
liposomal oxaliplatin at a dose of 30 mg/kg led to lower tumor
volumes than for untargeted liposomal oxaliplatin at the same dose
(FIG. 21A). Administration of MMP-targeted liposomal oxaliplatin at
a dose of 30 mg/kg led to the highest percent survival of all test
groups (FIG. 21B).
Example 6
MMP Inhibition by an MMP.sup.i-Targeted Liposomal Formulation
[0148] The activity of MMP.sup.i-targeted liposomal oxaliplatin
sample prepared in Example 3 was tested against metalloproteinases
MMP2 and MMP14.
[0149] rhMMP-2 (100 .mu.g/mL) was activated by incubation with 1 mM
APMA (p-aminophenylmercuric acetate) in assay buffer (50 mM Tris,
10 mM CaCl.sub.2, 150 mM NaCl, 0.05% (v/v) Brij-35, pH 7.5) at
37.degree. C. for 1 hr. The activated rhMMP-2 was diluted to 248
ng/mL in assay buffer. TAMP substrate
(Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH.sub.2) was diluted to 25
.mu.M in assay buffer. 25 .mu.L of 5.times. test samples containing
the targeted liposomal formulation and 50 .mu.L of 248 ng/mL
activated rhMMP-2 were added to a 96 well black-sided plate. 50
.mu.L of 25 .mu.M substrate was added to start the enzymatic
reactions, and fluorescence measurements (.lamda..sub.ex320 nm;
.lamda..sub.em=405 nm) were recorded in kinetic mode for 5
minutes.
[0150] rhMMP-14 (40 .mu.g/mL) was activated by incubation with 0.86
.mu.g/mL rhFurin in activation buffer (50 mM Tris, 1 mM CaCl.sub.2,
0.05% (v/v) Brij-35, pH 9.0) at 37.degree. C. for 1 hr. The
activated rhMMP-14 was diluted to 1.24 .mu.g/mL in assay buffer (50
mM Tris, 3 mM CaCl.sub.2, 1 .mu.M ZnCl.sub.2, pH 8.5). MMP
substrate (Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH.sub.2) was
diluted to 20 .mu.M in assay buffer. 25 .mu.L of 5.times. test
samples containing the targeted liposomal formulation and 50 .mu.L
of 1.24 .mu.g/mL activated rhMMP-14 were added to a 96 well
black-sided plate. 50 .mu.L of 20 .mu.M substrate was added to
start the enzymatic reactions, and fluorescence measurements
(.lamda..sub.ex320 nm; .lamda..sub.em405 nm) were recorded in
kinetic mode for 5 minutes.
[0151] IC.sub.50 values of 12.7 nm and 3.9 nm were observed for
MMP2 and MMP14, respectively (FIG. 22A and FIG. 22B).
[0152] Although the foregoing has been described in some detail by
way of illustration and example for purposes of clarity and
understanding, one of skill in the art will appreciate that certain
changes and modifications can be practiced within the scope of the
appended claims. In addition, each reference provided herein is
incorporated by reference in its entirety to the same extent as if
each reference was individually incorporated by reference.
* * * * *