U.S. patent application number 13/516173 was filed with the patent office on 2013-02-21 for multifunctional zwitterionic polymer conjugates.
This patent application is currently assigned to Oligasis. The applicant listed for this patent is Didier G. Benoit, Stephen A. Charles, Lane A. Clizbe, D. Victor Perlroth, Wayne To. Invention is credited to Didier G. Benoit, Stephen A. Charles, Lane A. Clizbe, D. Victor Perlroth, Wayne To.
Application Number | 20130045522 13/516173 |
Document ID | / |
Family ID | 44167643 |
Filed Date | 2013-02-21 |
United States Patent
Application |
20130045522 |
Kind Code |
A1 |
Charles; Stephen A. ; et
al. |
February 21, 2013 |
MULTIFUNCTIONAL ZWITTERIONIC POLYMER CONJUGATES
Abstract
The present invention provides random copolymers containing
zwitterions and one or more functional agents, and methods of
preparing such random copolymers.
Inventors: |
Charles; Stephen A.; (San
Jose, CA) ; Perlroth; D. Victor; (Palo Alto, CA)
; Clizbe; Lane A.; (Redwood City, CA) ; Benoit;
Didier G.; (San Jose, CA) ; To; Wayne;
(Fremont, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Charles; Stephen A.
Perlroth; D. Victor
Clizbe; Lane A.
Benoit; Didier G.
To; Wayne |
San Jose
Palo Alto
Redwood City
San Jose
Fremont |
CA
CA
CA
CA
CA |
US
US
US
US
US |
|
|
Assignee: |
Oligasis
Palo Alto
CA
|
Family ID: |
44167643 |
Appl. No.: |
13/516173 |
Filed: |
December 20, 2010 |
PCT Filed: |
December 20, 2010 |
PCT NO: |
PCT/US2010/061358 |
371 Date: |
August 27, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61288127 |
Dec 18, 2009 |
|
|
|
Current U.S.
Class: |
435/188 ;
525/326.5; 525/326.6; 525/375; 525/54.1 |
Current CPC
Class: |
A61K 38/1816 20130101;
C12N 9/96 20130101; A61K 49/0043 20130101; C08F 230/02 20130101;
A61K 38/00 20130101; C12Y 301/03001 20130101; A61K 49/0002
20130101; A61P 35/02 20180101; C12N 9/16 20130101; A61K 49/0054
20130101; A61P 35/00 20180101; A61K 47/6805 20170801; A61K 47/6811
20170801; A61P 29/00 20180101; C07K 16/00 20130101; A61P 43/00
20180101; C08F 2438/01 20130101; C07K 2317/55 20130101; A61K 47/544
20170801; A61K 39/395 20130101; C08F 8/32 20130101; A61K 38/465
20130101; C07K 2317/21 20130101; A61K 47/58 20170801; C08F 230/08
20130101; C08F 230/02 20130101 |
Class at
Publication: |
435/188 ;
525/326.5; 525/375; 525/326.6; 525/54.1 |
International
Class: |
C08F 230/02 20060101
C08F230/02; C12N 9/96 20060101 C12N009/96; C08F 8/32 20060101
C08F008/32 |
Claims
1. A random copolymer of Formula I: ##STR00179## wherein each
M.sup.1 and M.sup.2 are independently selected from the group
consisting of acrylate, methacrylate, acrylamide, methacrylamide,
styrene, vinyl-pyridine and vinyl-pyrrolidone; R.sup.1 is selected
from the group consisting of H, L.sup.1-A.sup.1, LG.sup.1 and
L.sup.1-LG.sup.1; each R.sup.2 is independently selected from the
group consisting of H, C.sub.1-20 alkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, C.sub.1-6 haloalkyl, C.sub.1-6 heteroalkyl,
C.sub.3-8 cycloalkyl, C.sub.3-8 heterocycloalkyl, aryl, heteroaryl,
A.sup.2, L.sup.2-A.sup.2, LG.sup.2, L.sup.2-LG.sup.2, I.sup.2 and
L.sup.2-I.sup.2; ZW is a zwitterionic moiety; I is an initiator
fragment and I' is a radical scavenger, such that the combination
of I-I' is an initiator, I.sup.1, for the polymerization of the
random copolymer of Formula I; alternatively, I' is selected from
the group consisting of H and C.sub.1-6 alkyl; I.sup.2 is an
initiator; each of L.sup.1 and L.sup.2 is a linker; each of A.sup.1
and A.sup.2 is a functional agent; each of LG.sup.1 and LG.sup.2 is
a linking group; subscripts x and y.sup.1 are each independently an
integer of from 1 to 1000; subscript z is an integer of from 1 to
10; subscript s is an integer of from 1 to 100; and subscript n is
an integer of from 1 to 20, wherein either R.sup.1 is
L.sup.1-A.sup.1 or one of R.sup.2 is L.sup.2-A.sup.2.
2. The random copolymer of claim 1, wherein the random copolymer
has the formula: ##STR00180##
3. The random copolymer of claim 1, wherein the random copolymer
has the formula: ##STR00181##
4. The random copolymer of claim 1, having the formula:
##STR00182##
5. The random copolymer of claim 1, having the formula:
##STR00183##
6. The random copolymer of claim 1, having the formula:
##STR00184##
7. The random copolymer of claim 1, wherein at least one R.sup.2 is
selected from the group consisting of H, C.sub.1-6 alkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 haloalkyl, C.sub.1-6
heteroalkyl, C.sub.3-8 cycloalkyl, C.sub.3-8 heterocycloalkyl,
aryl, and heteroaryl.
8. The random copolymer of claim 1, wherein the random copolymer
has the formula: ##STR00185##
9. The random copolymer of claim 8, wherein the random copolymer
has the formula: ##STR00186##
10. The random copolymer of claim 8, wherein the random copolymer
has the formula: ##STR00187##
11. The random copolymer of claim 10, having the following formula:
##STR00188##
12. The random copolymer of claim 1, wherein each of L.sup.1 and
L.sup.2 is a cleavable linker independently selected from the group
consisting of hydrolyzable linkers, enzymatically cleavable
linkers, pH sensitive linkers, disulfide linkers, photolabile
linkers, and self-immolative or double prodrug linkers.
13. The random copolymer of claim 1, wherein at least one of
L.sup.1 and L.sup.2 is a cleavable linker independently selected
from the group consisting of hydrolyzable linkers, enzymatically
cleavable linkers, pH sensitive linkers, disulfide linkers,
photolabile linkers, and self-immolative or double prodrug
linkers.
14. The random copolymer of claim 1, wherein the functional agent
is a bioactive agent selected from the group consisting of a drug,
a therapeutic protein and a targeting agent.
15. The random copolymer of claim 1, wherein the radical scavenger
I' is a halogen.
16. The random copolymer of claim 1, wherein the initiator I.sup.1
is selected from the group consisting of: ##STR00189## ##STR00190##
##STR00191## ##STR00192## ##STR00193## ##STR00194##
17. The random copolymer of claim 1, wherein the random copolymer
is selected from the group consisting of: ##STR00195## ##STR00196##
##STR00197## ##STR00198##
18. The random copolymer of claim 1, wherein the random copolymer
has the formula: ##STR00199## ##STR00200## ##STR00201## wherein PC
is phosphorylcholine; HEMA is hydroxyethyl methacrylate; GMA is
glycidyl methacrylate; and Glu is glutamic acid.
19. The random copolymer of claim 1, wherein the random copolymer
has the formula: ##STR00202## wherein the block copolymer has the
formula: ##STR00203##
20. The random copolymer of claim 1, wherein the random copolymer
has the formula: ##STR00204## wherein subscripts x and y.sup.1 are
such that the Mn of the polymer portion is about 95,000 g/mol;
A.sup.1 is an antibody; and L-CTP has the formula: ##STR00205##
21. The random copolymer of claim 1, wherein the random copolymer
has the formula: ##STR00206## wherein subscripts x, y.sup.1a and
y.sup.1b are such that the Mn of the polymer portion is about
107,100 g/mol; A.sup.1 is an antibody; and L-CTP has the formula:
##STR00207##
22. The random copolymer of claim 1, wherein the random copolymer
has the formula: ##STR00208## wherein subscripts x and y.sup.1 are
such that the Mn of the polymer portion is about 95,000 g/mol;
A.sup.1 is an IgG; and L-CTP has the formula: ##STR00209##
23. The random copolymer of claim 1, wherein each of A.sup.1 and
A.sup.2 is independently selected from the group consisting of an
antibody, an antibody fragment, a Fab, IgG, a peptide, a protein,
an enzyme, an oligonucleotide, a polynucleotide, nucleic acids, and
an antibody drug conjugate (ADC).
24. The random copolymer of claim 1, wherein A.sup.1 is
independently selected from an antibody, an antibody fragment, a
Fab, a scFv, an immunoglobulin domain, an IgG, and A.sup.2 is
independently selected from an anti-cancer agent, a toxin, a small
molecule drug, a chemotherapy agent, a kinase inhibitor, an
anti-inflammatory agent, and an antifibrotic agent.
25. The random copolymer of claim 1, wherein R.sup.1 is LG.sup.1,
and L.sup.2-A.sup.2 is independently selected from an anti-cancer
agent, a toxin, a small molecule drug, a chemotherapy agent, a
kinase inhibitor, an anti-inflammatory agent, and an antifibrotic
agent.
26. A process for preparing a random copolymer, the process
comprising: contacting a mixture of a first monomer and a second
monomer with an initiator, I.sup.I, under conditions sufficient to
prepare a random copolymer via free radical polymerization, wherein
the first monomer comprises a phosphorylcholine, and each of the
second monomer and initiator independently comprise at least one of
a functional agent or a linking group for linking to the functional
agent.
27. The process of claim 26, wherein the mixture further comprises
a catalyst and a ligand.
28. A random copolymer comprising a first monomer comprising
phosphorylcholine; a second monomer comprising a functional agent
or a linking group; and an initiator moiety comprising a functional
agent or a linking group, wherein the functional agent is linked to
the second monomer or the initiator moiety via a linker.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 61/288,127, filed Dec. 18, 2009.
BACKGROUND OF THE INVENTION
[0002] An arms race of sorts is happening right now amongst the
pharmaceutical companies who are all trying to deliver `medically
differentiated products`. Current drug formats are inflexible, in
that they generally allow for a single activity. For example, a
recombinant monoclonal antibody generally is designed and optimized
to bind and inhibit a single target protein. For example, a small
molecule drug is generally designed and optimized to bind and
activate (or inhibit) a single target. In some cases, the drug is
not selective and there are multiple activities (for example, a
small molecule kinase inhibitor that is designed to bind the ATP
binding site of a single kinase but which shows a level of affinity
and bioactivity against adjacent kinase family members). But
generally drug developers optimize using today's drug formats for
single activities and non-selectivity is seen as something to
engineer away in the drug development process.
[0003] In today's drug development, then, the selection of the
single target is the key variable. Drugs, therefore, are developed
from a format-centric point of view. But drugs are developed to
treat disease. And diseases generally are composed of more than one
pathophysiologic mechanism happening in series or in parallel. A
mechanism being a pathway or set of intersecting pathways occurring
either in a localized cell or tissue or organ or systemically
throughout the organism. A pathway being a set of moieties that
interact with each other. A more ideal way to engage in drug
development is to be able to take a disease-centric or
biology-centric approach. For example, based on the sum of academic
and corporate and historical research and experience to date,
disease x involves pathways a, b, and c. Within pathway a, target
protein z is known to be upregulated (and could be bound and
inhibited by an antibody fragment). Within pathway b, cell-type y
is known to be proliferating inappropriately (and could be impacted
by a small molecule anti-proliferative agent). And the
pathophysiology of pathway a and b is occurring within tissue
subtype x (and which could be targeted or enriched with drug by
including on the drug several copies of a small tissue-targeting
peptide). It would be ideal to have a drug technology or format
that allowed these multiple functions and different types of
bioactive moieties (protein, oligonucleotide, small molecule,
lipid, etc.) to be integrated into a single, adaptable,
multi-functional drug that is a practical best-of-breed and
straightforward in its design, implementation, manufacturing, and
administration. In addition, the technology should allow for
certain of the bioactive moieties to be unstably attached such that
they can be released under the desired conditions (time, aqueous pH
environment, other). These drugs should demonstrate higher efficacy
and safety while providing a higher overall probability of
technical, regulatory, and commercial success from early in the
drug development process.
[0004] Most diseases are complex and multifactorial in origin.
Therefore, in applying this biology-centric or disease-centric
approach, one could imagine a future ten or fifteen years down the
road where a big disease such as rheumatoid arthritis is actually
divided through diagnostic (molecular, imaging, biomarker, genetic)
or other approaches into, say, ten major subtypes each of which is
driven by a particular set of pathophysiologies and which can be
targeted using one multi-functional drug such that ten
multi-functional drugs are developed in order to treat the ten
different disease types.
[0005] The present invention describes such a drug technology
format that can be the backbone of the next-generation of
multi-functional drug development. The technology delivers a
polymer backbone which (i) itself delivers fundamental
biocompatibility to the drug through the selection of hydrophilic
monomer and architecture, and (ii) also forms a core backbone or
scaffold for conjugation and/or adsorption to multiple agents of
different types (amino acid, small molecule, oligonucleotide,
lipid, other, diagnostic agent, imaging agent, therapy monitoring
agent), predefined stoichiometries and functions (biocompatibility,
spacer, bioactivity, targeting, diagnostic, imaging, other), and
(iii) can employ any stable or flexible (under predefined
conditions) conjugation linker and chemistries.
[0006] Hydrophilic polymers for drug conjugation have been well
described and the drug conjugates are generating in excess of $5
billion revenue per annum. What is important for these polymers is
the extent to which they bind water molecules and the physical
properties of those water binding interactions. This combination of
properties drives the fundamental biocompatibility of the polymer.
PEG is one example of a hydrophilic polymer, but there are other
examples of hydrophilic polymers that bind water to a different
extent and with different physical properties and therefore with
different fundamental biocompatibility. One such example is
phosphorylcholine-based polymers, specifically polymers derived
from 2-methacryloyloxyethyl phosphorylcholine, which polymers have
been commercialized in various forms in medical devices such as
coronary drug eluting stents and contact lenses. In recent years,
new methods of controlled radical polymerization have been
developed with the promise to enable the manufacture of large,
complex-architecture polymers with low cost and high quality.
[0007] The present invention integrates a drug technology and
format that allows for a new paradigm of drug development, starting
with a set of biologies driving disease pathophysiology;
integrating biocompatibility moieties, drug moieties of different
classes, extended architectures, flexible chemistries, all in a
practical package. More simply put, the present invention presents
a drug format that allows the user to create a nanoscale biomachine
with the goal of creating magic bullets for combating diseases to
the benefit of patients.
[0008] Efforts to formulate biologically active agents for delivery
must deal with a variety of variables including the route of
administration, the biological stability of the active agent and
the solubility of the active agents in physiologically compatible
media. Choices made in formulating biologically active agents and
the selected routes of administration can affect the
bioavailability of the active agents. For example, the choice of
parenteral administration into the systemic circulation for
biologically active proteins and polypeptides avoids the
proteolytic environment found in the gastrointestinal tract.
However, even where direct administration, such as by injection, of
biologically active agents is possible, formulations may be
unsatisfactory for a variety of reasons including the generation of
an immune response to the administered agent and responses to any
excipients including burning and stinging. Even if the active agent
is not immunogenic and satisfactory excipients can be employed,
biologically active agents can have a limited solubility and short
biological half-life that can require repeated administration or
continuous infusion, which can be painful and/or inconvenient.
[0009] For some biologically active agents a degree of success has
been achieved in developing suitable formulations of functional
agents by conjugating the agents to water soluble polymers. The
conjugation of biologically active agents to water soluble polymers
is generally viewed as providing a variety of benefits for the
delivery of biologically active agents, and in particular, proteins
and peptides. Among the water soluble polymers employed,
polyethylene glycol (PEG) has been most widely conjugated to a
variety of biologically active agents including biologically active
peptides. A reduction in immunogenicity or antigenicity, increased
half-life, increased solubility, decreased clearance by the kidney
and decreased enzymatic degradation have been attributed to
conjugates of a variety of water soluble polymers and functional
agents, including PEG conjugates. As a result of these attributes,
the polymer conjugates of biologically active agents require less
frequent dosing and may permit the use of less of the active agent
to achieve a therapeutic endpoint. Less frequent dosing reduces the
overall number of injections, which can be painful and which
require inconvenient visits to healthcare professionals.
Conjugation of PEG or other polymers can also modify the core
activity of the drug itself--the idea of "additional bioactivities
conferred to the drug by virtue of polymer conjugation (for
example, the large hydrodynamic radius broadens the scope of
inhibition from drug (antibody fragment) inhibits binding to
receptor A but polymer-drug conjugate inhibits binding to receptor
A plus receptor B as a function of any number of different
mechanisms but certainly steric hindrance.
[0010] Although some success has been achieved with PEG
conjugation, "PEGylation" of biologically active agents remains a
challenge. As drug developers progress beyond very potent agonistic
proteins such as erythropoietin and the various interferons, the
benefits of the PEG hydrophilic polymer are insufficient to drive
the increases in solubility, stability and the decreases in
viscosity and immunogenicity that are necessary for a commercially
successful product that is subcutaneously administered. PEG
conjugation may also result in the loss of biological activity. A
variety of theories have been advanced to account for loss of
biological activity upon conjugation with PEG. These include
blockage of necessary sites for the agent to interact with other
biological components, either by the conjugation linkage or by the
agent being buried within the PEG conjugate, particularly where the
polymer is long and may "wrap" itself around some of the active
agent, thereby blocking access to potential ligands required for
activity.
[0011] Branched forms of PEG for use in conjugate preparation have
been introduced to alleviate some of the difficulties encountered
with the use of long straight PEG polymer chains. While branched
polymers may overcome some of the problems associated with
conjugates formed with long linear PEG polymers, neither branched
nor linear PEG polymer conjugates completely resolve the issues
associated with the use of conjugated functional agents. Both
linear and branched PEG conjugates can, for example, suffer from
rates of degradation that are either too long or too short. A rapid
rate of degradation can result in a conjugate having too short of
an in vivo half-life, whereas, too slow of a rate of degradation
can result in an unacceptably long conjugate half-life in vivo.
[0012] In view of the recognized advantages of conjugating
functional agents to water soluble polymers, and the limitations of
water soluble polymers such as PEG in forming conjugates suitable
for therapeutic purposes, additional water soluble polymers for
forming conjugates with functional agents are desirable. Water
soluble polymers, particularly those which have many of the
advantages of PEG for use in conjugate formation, and which do not
suffer from the disadvantages observed with PEG as a conjugating
agent would be desirable for use in forming therapeutic and
diagnostic agents. To this end, polymers containing zwitterionic
monomers, in particular, 2-methacryloyloxyethyl-phosphorylcholine
are set forth for use in preparing conjugates of biologically
active agents.
BRIEF SUMMARY OF THE INVENTION
[0013] In one embodiment, the random copolymers of the present
invention have formula I:
##STR00001##
Each monomer M.sup.1 and M.sup.2 of formula I can independently be
an acrylate, methacrylate, acrylamide, methacrylamide, styrene,
vinyl-pyridine or a vinyl-pyrrolidone. Moreover, R.sup.1 of formula
I can independently be H, L.sup.1-A.sup.1, a linking group LG.sup.1
or L.sup.1-LG.sup.1, and each R.sup.2 of formula I is independently
H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, C.sub.1-6 heteroalkyl, C.sub.3-8 cycloalkyl, C.sub.3-8
heterocycloalkyl, aryl, heteroaryl, A.sup.2, L.sup.2-A.sup.2,
LG.sup.2, L.sup.2-LG.sup.2, I.sup.2 and L.sup.2-I.sup.2. The group
ZW of formula I is a zwitterionic moiety. The groups I is an
initiator fragment and I' is a radical scavenger, such that the
combination of I-I' is an initiator, I.sup.1, for the
polymerization of the random copolymer of Formula I. Alternatively,
I' can be H or C.sub.1-6 alkyl. The group I.sup.2 is an initiator.
In addition, each of the groups L.sup.1 and L.sup.2 is a linker,
each of the groups A.sup.1 and A.sup.2 is a functional agent, and
each of the groups LG.sup.1 and LG.sup.2 is a linking group. In
formula I above, subscripts x and y.sup.1 are each independently an
integer of from 1 to 1000, subscript z is an integer of from 1 to
10, subscript s is an integer of from 1 to 100, and subscript n is
an integer of from 1 to 20, wherein either R.sup.1 is
L.sup.1-A.sup.1 or one of R.sup.2 is L.sup.2-A.sup.2.
[0014] In other embodiments, the present invention provides a
process for preparing a random copolymer of the present invention,
the process including the step of contacting a mixture of a first
monomer and a second monomer with an initiator, I.sup.1, under
conditions sufficient to prepare a random copolymer via free
radical polymerization, wherein the first monomer comprises a
phosphorylcholine, and each of the second monomer and initiator
independently comprise at least one of a functional agent or a
linking group for linking to the functional agent.
[0015] In another embodiment, the random copolymers of the present
invention have a first monomer with a zwitterion such as
phosphorylcholine, at least one second monomer having a functional
agent or a linking group, and an initiator moiety having a
functional agent or a linking group, wherein the functional agent
is linked to the second monomer or the initiator moiety via a
linker.
[0016] In another embodiment, the random copolymers of the present
invention have a first monomer with a zwitterion such as
phosphorylcholine, at least one second monomer having a functional
agent or a linking group, said second monomer having a different
reactivity ratio than the first monomer allowing the final polymer
to be an alternating copolymer, a periodic copolymer, a gradient
copolymer, a block copolymer or a statistical copolymer.
[0017] In another embodiment, the random copolymers of the present
invention have a first monomer with a zwitterion such as
phosphorylcholine, at least one second monomer having a functional
agent and a tunable linking group, said second monomer having the
same reactivity ratio as the first monomer allowing the final
polymer to be an alternating copolymer, a periodic copolymer, a
gradient copolymer, a block copolymer or a statistical
copolymer.
[0018] In another embodiment, the random copolymers of the present
invention have a first monomer with a zwitterion such as
phosphorylcholine, at least one second monomer having a functional
agent or a linking group and other monomers that have differing
environment affinities allowing for the formation of new topologies
by non-covalent binding.
[0019] In another embodiment, the random copolymers of the present
invention have a first monomer with a zwitterion such as
phosphorylcholine, at least one second monomer having a functional
agent and a tunable linking group and other monomers that have
differing environment affinities allowing for the formation of new
topologies by non-covalent binding.
[0020] In another embodiment, the random copolymers of the present
invention have a first monomer with a zwitterion such as
phosphorylcholine, at least one second monomer having a functional
agent or a linking group and other monomers of similar environment
affinities allows for the formation of new topologies by
non-covalent binding (e.g. chelation between carboxylic groups in
aqueous environments, or pH sensitive groups).
[0021] In another embodiment, the random copolymers of the present
invention have a first monomer with a zwitterion such as
phosphorylcholine, at least one second monomer having a functional
agent and a tunable linking group allowing for the formation of new
topologies by non-covalent binding (e.g. chelation between
carboxylic groups in aqueous environments, or pH sensitive
groups).
[0022] In another embodiment, the random copolymers of the present
invention have a first monomer with a zwitterion such as
phosphorylcholine, at least one second monomer having a tunable
linking group allowing for the release of functional agents in
response to predefined triggers such as aqueous environments or low
pH environments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 shows a scheme for the preparation of the random
copolymers of the present invention. The initiator I-I' is cleaved
into initiator fragment I and radical scavenger I'. The initiator
fragment I then reacts with comonomers M.sup.1 and M.sup.2 to
initiate the polymerization process and generate species A. The
radical scavenger I' can then reversibly react with species A to
form species B. Alternatively, species A can react with additional
monomers to continue propagation of the polymer (species C).
Concomitantly, the growing polymer chain of species C reversibly
reacts with radical scavenger I' to form the random copolymer,
species D.
DETAILED DESCRIPTION OF THE INVENTION
I. General
[0024] The present invention provides random copolymers having a
zwitterion such as phosphorylcholine, and at least one functional
agent (as defined herein). A zwitterion such as phosphorylcholine
as a highly biocompatible molecule drives fundamental
biocompatibility. It also has chaperone type functions, in terms of
protecting proteins under temperature or other stress. It also can
allow other functions such as reversible cellular uptake. The
functional agent can be a bioactive agent such as a drug,
therapeutic protein or targeting agent, as well as a detection
agent, imaging agent, labeling agent or diagnostic agent. The
random copolymers are useful for the treatment of a variety of
conditions and disease states by selecting one or more appropriate
functional agents. Multiple bioactive agents can be linked to the
random copolymer, thus enabling treatment of not just a single
disease symptom or mechanism, but rather the whole disease.
Furthermore, the bioactive agents can be linked via non-cleavable
linkers in a stable manner, or via a variety of cleavable linkers
such that different predefined triggers release the respective
bioactive agents through the use of prodrug or double prodrug
linker and linking group strategies. In addition, the random
copolymers are useful for diagnostic and imaging purposes by
attachment of suitable targeting agents and imaging agents. The
random copolymers can include both therapeutic and diagnostic
agents in a single polymer, providing theranostic agents that treat
the disease as well as detect and diagnose.
[0025] The random polymers can be prepared via a conventional
free-radical polymerization or controlled/living radical
polymerization, such as atom transfer radical polymerization
(ATRP), using monomers that contain the zwitterion such as
phosphorylcholine and monomers that contain one or more bioactive
agents which may be the same or different, or linking groups that
are able to link to the bioactive agents. The initiators used for
preparation of the random copolymers can have multiple initiating
sites such that multi-arm polymers, such as stars, can be prepared.
The initiator can also contain either a bioactive agent, or linking
groups, or flexible chemistries that are able to link to bioactive
agents.
II. Definitions
[0026] For the purpose of the present invention the following
terminology will be used in accordance with the definitions set
forth below.
[0027] "Random copolymer" refers to a polymer having at least two
different monomer groups that are distributed randomly throughout
the polymer backbone. The monomers of the random copolymer are the
chemical moieties that are bonded together to form the polymer.
Each distinct chemical moiety is termed a monomer. The random
copolymers are prepared from monomers that include, but are not
limited to, acrylates, methacrylates, acrylamides, methacrylamides,
styrenes, vinyl-pyridine and vinyl-pyrrolidone. Additional monomers
are useful in the random copolymers of the present invention. When
two different monomers are used, such as in the random copolymers
of the present invention, the two monomers are called "comonomers,"
meaning that the different monomers are copolymerized to form a
single polymer.
[0028] "Zwitterionic moiety" refers to a compound having both a
positive and a negative charge. Zwitterionic moieties useful in the
random copolymers can include a quaternary nitrogen and a
negatively charged phosphate, such as phosphorylcholine:
RO--P(.dbd.O)(O.sup.-)--O--CH.sub.2CH.sub.2--N.sup.+(Me).sub.3.
Other zwitterionic moieties are useful in the random copolymers of
the present invention, and Patents WO 1994/016748 and WO
1994/016749 are incorporated in their entirety herein.
[0029] "Initiator" refers to a compound capable of initiating a
polymerization using the comonomers of the present invention. The
polymerization can be a conventional free radical polymerization or
a controlled/living radical polymerization, such as Atom Transfer
Radical Polymerization (ATRP), Reversible
Addition-Fragmentation-Termination (RAFT) polymerization or
nitroxide mediated polymerization (NMP). The polymerization can be
a "pseudo" controlled polymerization, such as degenerative
transfer. When the initiator is suitable for ATRP, it contains a
labile bond which can homolytically cleave to form an initiator
fragment, I, being a radical capable of initiating a radical
polymerization, and a radical scavenger, I', which reacts with the
radical of the growing polymer chain to reversibly terminate the
polymerization. The radical scavenger I' is typically a halogen,
but can also be an organic moiety, such as a nitrile.
[0030] "Linker" refers to a chemical moiety that links two groups
together. The linker can be cleavable or non-cleavable. Cleavable
linkers can be hydrolyzable, enzymatically cleavable, pH sensitive,
photolabile, or disulfide linkers, among others. Other linkers
include homobifunctional and heterobifunctional linkers. A "linking
group" is a functional group capable of forming a covalent linkage
consisting of one or more bonds to a bioactive agent. Nonlimiting
examples include those illustrated in Table 1.
[0031] "Hydrolyzable linker" refers to a chemical linkage or bond,
such as a covalent bond, that undergoes hydrolysis under
physiological conditions. The tendency of a bond to hydrolyze may
depend not only on the general type of linkage connecting two
central atoms between which the bond is severed, but also on the
substituents attached to these central atoms. Non-limiting examples
of hydrolytically susceptible linkages include esters of carboxylic
acids, phosphate esters, acetals, ketals, acyloxyalkyl ether,
imines, orthoesters, and some amide linkages.
[0032] "Enzymatically cleavable linker" refers to a linkage that is
subject to degradation by one or more enzymes. Some hydrolytically
susceptible linkages may also be enzymatically degradable. For
example esterases may act on esters of carboxylic acid or phosphate
esters, and proteases may act on peptide bonds and some amide
linkages.
[0033] "pH sensitive linker" refers to a linkage that is stable at
one pH and subject to degradation at another pH. For example, the
pH sensitive linker can be stable at neutral or basic conditions,
but labile at mildly acidic conditions.
[0034] "Photolabile linker" refers to a linkage, such as a covalent
bond, that cleaves upon exposure to light. The photolabile linker
includes an aromatic moiety in order to absorb the incoming light,
which then triggers a rearrangement of the bonds in order to cleave
the two groups linked by the photolabile linker.
[0035] "Self-immolative or double prodrug linker" refers to a
linkage in which the main function of the linker is to release a
functional agent only after selective trigger activation (for
example, a drop in pH or the presence of a tissue-specific enzyme)
followed by spontaneous chemical breakdown to release the
functional agent.
[0036] "Functional agent" is defined to include a bioactive agent
or a diagnostic agent. A "bioactive agent" is defined to include
any agent, drug, compound, or mixture thereof that targets a
specific biological location (targeting agent) and/or provides some
local or systemic physiological or pharmacologic effect that can be
demonstrated in vivo or in vitro. Non-limiting examples include
drugs, vaccines, antibodies, antibody fragments, vitamins and
cofactors, polysaccharides, carbohydrates, steroids, lipids, fats,
proteins, peptides, polypeptides, nucleotides, oligonucleotides,
polynucleotides, and nucleic acids (e.g., mRNA, tRNA, snRNA, RNAi,
DNA, cDNA, antisense constructs, ribozymes, etc). A "diagnostic
agent" is defined to include any agent that enables the detection
or imaging of a tissue or disease. Examples of diagnostic agents
include, but are not limited to, radiolabels, fluorophores and
dyes.
[0037] "Therapeutic protein" refers to peptides or proteins that
include an amino acid sequence which in whole or in part makes up a
drug and can be used in human or animal pharmaceutical
applications. Numerous therapeutic proteins are known to
practitioners of skill in the art including, without limitation,
those disclosed herein.
[0038] "Phosphorylcholine," also denoted as "PC," refers to the
following:
##STR00002##
where * denotes the point of attachment. The phosphorylcholine is a
zwitterionic group and includes salts (such as inner salts), and
protonated and deprotonated forms thereof.
[0039] "Phosphorylcholine containing polymer" is a polymer that
contains phosphorylcholine. It is specifically contemplated that in
each instance where a phosphorylcholine containing polymer is
specified in this application for a particular use, a single
phosphorylcholine can also be employed in such use. "Zwitterion
containing polymer" refers to a polymer that contains a
zwitterion.
[0040] "Poly(acryloyloxyethyl phosphorylcholine) containing
polymer" refers to a polymer of acrylic acid containing at least
one acryloyloxyethyl phosphorylcholine monomer such as
2-methacryloyloxyethyl phosphorylcholine (i.e.,
2-methacryloyl-2'-trimethylammonium ethyl phosphate).
[0041] "Contacting" refers to the process of bringing into contact
at least two distinct species such that they can react. It should
be appreciated, however, that the resulting reaction product can be
produced directly from a reaction between the added reagents or
from an intermediate from one or more of the added reagents which
can be produced in the reaction mixture.
[0042] "Water-soluble polymer" refers to a polymer that is soluble
in water. A solution of a water-soluble polymer may transmit at
least about 75%, more preferably at least about 95% of light,
transmitted by the same solution after filtering. On a weight
basis, a water-soluble polymer or segment thereof may be at least
about 35%, at least about 50%, about 70%, about 85%, about 95% or
100% (by weight of dry polymer) soluble in water.
[0043] "Molecular weight" in the context of the polymer can be
expressed as either a number average molecular weight, or a weight
average molecular weight or a peak molecular weight. Unless
otherwise indicated, all references to molecular weight herein
refer to the peak molecular weight. These molecular weight
determinations, number average, weight average and peak, can be
measured using gel permeation chromatography or other liquid
chromatography techniques. Other methods for measuring molecular
weight values can also be used, such as the use of end-group
analysis or the measurement of colligative properties (e.g.,
freezing-point depression, boiling-point elevation, or osmotic
pressure) to determine number average molecular weight, or the use
of light scattering techniques, ultracentrifugation or viscometry
to determine weight average molecular weight. The polymeric
reagents of the invention are typically polydisperse (i.e., number
average molecular weight and weight average molecular weight of the
polymers are not equal), possessing low polydispersity values of
preferably less than about 1.5, as judged by gel permeation
chromatography. In other embodiments the polydispersities may be in
the range of about 1.4 to about 1.2, more preferably less than
about 1.15, still more preferably less than about 1.10, yet still
more preferably less than about 1.05, and most preferably less than
about 1.03.
[0044] The phrase "a" or "an" entity as used herein refers to one
or more of that entity; for example, a compound refers to one or
more compounds or at least one compound. As such, the terms "a" (or
"an"), "one or more", and "at least one" can be used
interchangeably herein.
[0045] "About" as used herein means variation one might see in
measurements taken among different instruments, samples, and sample
preparations.
[0046] "Protected,", "protected form", "protecting group" and
"protective group" refer to the presence of a group (i.e., the
protecting group) that prevents or blocks reaction of a particular
chemically reactive functional group in a molecule under certain
reaction conditions. Protecting group will vary depending upon the
type of chemically reactive group being protected as well as the
reaction conditions to be employed and the presence of additional
reactive or protecting groups in the molecule, if any. The skilled
artisan will recognize protecting groups known in the art, such as
those found in the treatise by Greene et al., "Protective Groups In
Organic Synthesis," 3.sup.rd Edition, John Wiley and Sons, Inc.,
New York, 1999.
[0047] "Spacer," and "spacer group" are used interchangeably herein
to refer to an atom or a collection of atoms optionally used to
link interconnecting moieties such as a terminus of a water-soluble
polymer and a reactive group of a functional agent and a reactive
group. A spacer may be hydrolytically stable or may include a
hydrolytically susceptible or enzymatically degradable linkage.
[0048] "Alkyl" refers to a straight or branched, saturated,
aliphatic radical having the number of carbon atoms indicated. For
example, C.sub.1-C.sub.6 alkyl includes, but is not limited to,
methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,
tert-butyl, pentyl, isopentyl, hexyl, etc. Other alkyl groups
include, but are not limited to heptyl, octyl, nonyl, decyl, etc.
Alkyl can include any number of carbons, such as 1-2, 1-3, 1-4,
1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6,
4-5, 4-6 and 5-6. The alkyl group is typically monovalent, but can
be divalent, such as when the alkyl group links two moieties
together.
[0049] The term "lower" referred to above and hereinafter in
connection with organic radicals or compounds respectively defines
a compound or radical which can be branched or unbranched with up
to and including 7, preferably up to and including 4 and (as
unbranched) one or two carbon atoms.
[0050] "Alkylene" refers to an alkyl group, as defined above,
linking at least two other groups, i.e., a divalent hydrocarbon
radical. The two moieties linked to the alkylene can be linked to
the same atom or different atoms of the alkylene. For instance, a
straight chain alkylene can be the bivalent radical of
--(CH.sub.2).sub.n, where n is 1, 2, 3, 4, 5 or 6. Alkylene groups
include, but are not limited to, methylene, ethylene, propylene,
isopropylene, butylene, isobutylene, sec-butylene, pentylene and
hexylene.
[0051] Substituents for the alkyl and heteroalkyl radicals
(including those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be a
variety of groups selected from: --OR', .dbd.O, .dbd.NR',
.dbd.N--OR', --NR'R'', --SR', -halogen, --SiR'R''R''', --OC(O)R',
--C(O)R', --CO.sub.2R', --CONR'R'', --OC(O)NR'R'', --NR''C(O)R',
--NR'--C(O)NR''R''', --NR''C(O).sub.2R', --NH--C(NH.sub.2).dbd.NH,
--NR'C(NH.sub.2).dbd.NH, --NH--C(NH.sub.2).dbd.NR', --S(O)R',
--S(O).sub.2R', --S(O).sub.2NR'R'', --CN and --NO.sub.2 in a number
ranging from zero to (2 m'+1), where m' is the total number of
carbon atoms in such radical. R', R'' and R''' each independently
refer to hydrogen, unsubstituted (C.sub.1-C.sub.8)alkyl and
heteroalkyl, unsubstituted aryl, aryl substituted with 1-3
halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or
aryl-(C.sub.1-C.sub.4)alkyl groups. When R' and R'' are attached to
the same nitrogen atom, they can be combined with the nitrogen atom
to form a 5-, 6-, or 7-membered ring. For example, --NR'R'' is
meant to include 1-pyrrolidinyl and 4-morpholinyl. From the above
discussion of substituents, one of skill in the art will understand
that the term "alkyl" is meant to include groups such as haloalkyl
(e.g., --CF.sub.3 and --CH.sub.2CF.sub.3) and acyl (e.g.,
--C(O)CH.sub.3, --C(O)CF.sub.3, --C(O)CH.sub.2OCH.sub.3, and the
like). Preferably, the substituted alkyl and heteroalkyl groups
have from 1 to 4 substituents, more preferably 1, 2 or 3
substituents. Exceptions are those perhalo alkyl groups (e.g.,
pentafluoroethyl and the like) which are also preferred and
contemplated by the present invention.
[0052] Substituents for the alkyl and heteroalkyl radicals
(including those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one
or more of a variety of groups selected from, but not limited to:
--OR', .dbd.O, .dbd.NR', .dbd.N--OR', --NR'R'', --SR', -halogen,
--SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R', --CONR'R'',
--OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''',
--NR''C(O).sub.2R', --NR--C(NR'R''R''').dbd.NR'''',
--NR--C(NR'R'').dbd.NR''', --S(O)R', --S(O).sub.2R',
--S(O).sub.2NR'R'', --NRSO.sub.2R', --CN and --NO.sub.2 in a number
ranging from zero to (2 m'+1), where m' is the total number of
carbon atoms in such radical. R', R'', R''' and R'''' each
preferably independently refer to hydrogen, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g.,
aryl substituted with 1-3 halogens, substituted or unsubstituted
alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a
compound of the invention includes more than one R group, for
example, each of the R groups is independently selected as are each
R', R'', R''' and R'''' groups when more than one of these groups
is present. When R' and R'' are attached to the same nitrogen atom,
they can be combined with the nitrogen atom to form a 5-, 6-, or
7-membered ring. For example, --NR'R'' is meant to include, but not
be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above
discussion of substituents, one of skill in the art will understand
that the term "alkyl" is meant to include groups including carbon
atoms bound to groups other than hydrogen groups, such as haloalkyl
(e.g., --CF.sub.3 and --CH.sub.2CF.sub.3) and acyl (e.g.,
--C(O)CH.sub.3, --C(O)CF.sub.3, --C(O)CH.sub.2OCH.sub.3, and the
like).
[0053] "Alkoxy" refers to alkyl group having an oxygen atom that
either connects the alkoxy group to the point of attachment or is
linked to two carbons of the alkoxy group. Alkoxy groups include,
for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy,
2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy,
etc. The alkoxy groups can be further substituted with a variety of
substituents described within. For example, the alkoxy groups can
be substituted with halogens to form a "halo-alkoxy" group.
[0054] "Carboxyalkyl" means an alkyl group (as defined herein)
substituted with a carboxy group. The term "carboxycycloalkyl"
means an cycloalkyl group (as defined herein) substituted with a
carboxy group. The term alkoxyalkyl means an alkyl group (as
defined herein) substituted with an alkoxy group. The term
"carboxy" employed herein refers to carboxylic acids and their
esters.
[0055] "Haloalkyl" refers to alkyl as defined above where some or
all of the hydrogen atoms are substituted with halogen atoms.
Halogen (halo) preferably represents chloro or fluoro, but may also
be bromo or iodo. For example, haloalkyl includes trifluoromethyl,
fluoromethyl, 1,2,3,4,5-pentafluoro-phenyl, etc. The term
"perfluoro" defines a compound or radical which has all available
hydrogens that are replaced with fluorine. For example,
perfluorophenyl refers to 1,2,3,4,5-pentafluorophenyl,
perfluoromethyl refers to 1,1,1-trifluoromethyl, and
perfluoromethoxy refers to 1,1,1-trifluoromethoxy.
[0056] "Fluoro-substituted alkyl" refers to an alkyl group where
one, some, or all hydrogen atoms have been replaced by
fluorine.
[0057] "Cytokine" in the context of this invention is a member of a
group of protein signaling molecules that may participate in
cell-cell communication in immune and inflammatory responses.
Cytokines are typically small, water-soluble glycoproteins that
have a mass of about 8-35 kDa.
[0058] "Cycloalkyl" refers to a cyclic hydrocarbon group that
contains from about 3 to 12, from 3 to 10, or from 3 to 7
endocyclic carbon atoms. Cycloalkyl groups include fused, bridged
and spiro ring structures.
[0059] "Endocyclic" refers to an atom or group of atoms which
comprise part of a cyclic ring structure.
[0060] "Exocyclic" refers to an atom or group of atoms which are
attached but do not define the cyclic ring structure.
[0061] "Cyclic alkyl ether" refers to a 4 or 5 member cyclic alkyl
group having 3 or 4 endocyclic carbon atoms and 1 endocyclic oxygen
or sulfur atom (e.g., oxetane, thietane, tetrahydrofuran,
tetrahydrothiophene); or a 6 to 7 member cyclic alkyl group having
1 or 2 endocyclic oxygen or sulfur atoms (e.g., tetrahydropyran,
1,3-dioxane, 1,4-dioxane, tetrahydrothiopyran, 1,3-dithiane,
1,4-dithiane, 1,4-oxathiane).
[0062] "Alkenyl" refers to either a straight chain or branched
hydrocarbon of 2 to 6 carbon atoms, having at least one double
bond. Examples of alkenyl groups include, but are not limited to,
vinyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl,
butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl,
1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl,
1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or
1,3,5-hexatrienyl. Alkenyl groups can also have from 2 to 3, 2 to
4, 2 to 5, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6 and 5 to 6
carbons. The alkenyl group is typically monovalent, but can be
divalent, such as when the alkenyl group links two moieties
together.
[0063] "Alkenylene" refers to an alkenyl group, as defined above,
linking at least two other groups, i.e., a divalent hydrocarbon
radical. The two moieties linked to the alkenylene can be linked to
the same atom or different atoms of the alkenylene. Alkenylene
groups include, but are not limited to, ethenylene, propenylene,
isopropenylene, butenylene, isobutenylene, sec-butenylene,
pentenylene and hexenylene.
[0064] "Alkynyl" refers to either a straight chain or branched
hydrocarbon of 2 to 6 carbon atoms, having at least one triple
bond. Examples of alkynyl groups include, but are not limited to,
acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl,
sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl,
1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl,
1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or
1,3,5-hexatriynyl. Alkynyl groups can also have from 2 to 3, 2 to
4, 2 to 5, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6 and 5 to 6
carbons. The alkynyl group is typically monovalent, but can be
divalent, such as when the alkynyl group links two moieties
together.
[0065] "Alkynylene" refers to an alkynyl group, as defined above,
linking at least two other groups, i.e., a divalent hydrocarbon
radical. The two moieties linked to the alkynylene can be linked to
the same atom or different atoms of the alkynylene. Alkynylene
groups include, but are not limited to, ethynylene, propynylene,
butynylene, sec-butynylene, pentynylene and hexynylene.
[0066] "Cycloalkyl" refers to a saturated or partially unsaturated,
monocyclic, fused bicyclic or bridged polycyclic ring assembly
containing from 3 to 12 ring atoms, or the number of atoms
indicated. Monocyclic rings include, for example, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Bicyclic and
polycyclic rings include, for example, norbornane,
decahydronaphthalene and adamantane. For example,
C.sub.3-8cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, cyclooctyl, and norbornane.
[0067] "Cycloalkylene" refers to a cycloalkyl group, as defined
above, linking at least two other groups, i.e., a divalent
hydrocarbon radical. The two moieties linked to the cycloalkylene
can be linked to the same atom or different atoms of the
cycloalkylene. Cycloalkylene groups include, but are not limited
to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene,
and cyclooctylene.
[0068] "Heterocycloalkyl" refers to a ring system having from 3
ring members to about 20 ring members and from 1 to about 5
heteroatoms such as N, O and S. Additional heteroatoms can also be
useful, including, but not limited to, B, Al, Si and P. The
heteroatoms can also be oxidized, such as, but not limited to,
--S(O)-- and --S(O).sub.2--. For example, heterocycle includes, but
is not limited to, tetrahydrofuranyl, tetrahydrothiophenyl,
morpholino, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl,
pyrazolidinyl, pyrazolinyl, piperazinyl, piperidinyl, indolinyl,
quinuclidinyl and 1,4-dioxa-8-aza-spiro[4.5]dec-8-yl.
[0069] "Heterocycloalkylene" refers to a heterocyclalkyl group, as
defined above, linking at least two other groups. The two moieties
linked to the heterocycloalkylene can be linked to the same atom or
different atoms of the heterocycloalkylene.
[0070] "Aryl" refers to a monocyclic or fused bicyclic, tricyclic
or greater, aromatic ring assembly containing 6 to 16 ring carbon
atoms. For example, aryl may be phenyl, benzyl or naphthyl,
preferably phenyl. "Arylene" means a divalent radical derived from
an aryl group. Aryl groups can be mono-, di- or tri-substituted by
one, two or three radicals selected from alkyl, alkoxy, aryl,
hydroxy, halogen, cyano, amino, amino-alkyl, trifluoromethyl,
alkylenedioxy and oxy-C.sub.2-C.sub.3-alkylene; all of which are
optionally further substituted, for instance as hereinbefore
defined; or 1- or 2-naphthyl; or 1- or 2-phenanthrenyl.
Alkylenedioxy is a divalent substitute attached to two adjacent
carbon atoms of phenyl, e.g. methylenedioxy or ethylenedioxy.
Oxy-C.sub.2-C.sub.3-alkylene is also a divalent substituent
attached to two adjacent carbon atoms of phenyl, e.g. oxyethylene
or oxypropylene. An example for oxy-C.sub.2-C.sub.3-alkylene-phenyl
is 2,3-dihydrobenzofuran-5-yl.
[0071] Preferred as aryl is naphthyl, phenyl or phenyl mono- or
disubstituted by alkoxy, phenyl, halogen, alkyl or trifluoromethyl,
especially phenyl or phenyl-mono- or disubstituted by alkoxy,
halogen or trifluoromethyl, and in particular phenyl.
[0072] Examples of substituted phenyl groups as R are, e.g.
4-chlorophen-1-yl, 3,4-dichlorophen-1-yl, 4-methoxyphen-1-yl,
4-methylphen-1-yl, 4-aminomethylphen-1-yl,
4-methoxyethylaminomethylphen-1-yl,
4-hydroxyethylaminomethylphen-1-yl,
4-hydroxyethyl-(methyl)-aminomethylphen-1-yl,
3-aminomethylphen-1-yl, 4-N-acetylaminomethylphen-1-yl,
4-aminophen-1-yl, 3-aminophen-1-yl, 2-aminophen-1-yl,
4-phenyl-phen-1-yl, 4-(imidazol-1-yl)-phen-yl,
4-(imidazol-1-ylmethyl)-phen-1-yl, 4-(morpholin-1-yl)-phen-1-yl,
4-(morpholin-1-ylmethyl)-phen-1-yl,
4-(2-methoxyethylaminomethyl)-phen-1-yl and
4-(pyrrolidin-1-ylmethyl)-phen-1-yl, 4-(thiophenyl)-phen-1-yl,
4-(3-thiophenyl)-phen-1-yl, 4-(4-methylpiperazin-1-yl)-phen-1-yl,
and 4-(piperidinyl)-phenyl and 4-(pyridinyl)-phenyl optionally
substituted in the heterocyclic ring.
[0073] "Arylene" refers to an aryl group, as defined above, linking
at least two other groups. The two moieties linked to the arylene
are linked to different atoms of the arylene. Arylene groups
include, but are not limited to, phenylene.
[0074] "Arylene-oxy" refers to an arylene group, as defined above,
where one of the moieties linked to the arylene is linked through
an oxygen atom. Arylene-oxy groups include, but are not limited to,
phenylene-oxy.
[0075] Similarly, substituents for the aryl and heteroaryl groups
are varied and are selected from: -halogen, --OR', --OC(O)R',
--NR'R'', --SR', --R', --CN, --NO.sub.2, --CO.sub.2R', --CONR'R'',
--C(O)R', --OC(O)NR'R'', --NR''C(O)R', --NR''C(O).sub.2R',
--NR'--C(O)NR''R''', --NH--C(NH.sub.2).dbd.NH,
--NR'C(NH.sub.2).dbd.NH, --NH--C(NH.sub.2).dbd.NR', --S(O)R',
--S(O).sub.2R', --S(O).sub.2NR' R'', --N.sub.3, --CH(Ph).sub.2,
perfluoro(C.sub.1-C.sub.4)alkoxy, and
perfluoro(C.sub.1-C.sub.4)alkyl, in a number ranging from zero to
the total number of open valences on the aromatic ring system; and
where R', R'' and R''' are independently selected from hydrogen,
(C.sub.1-C.sub.8)alkyl and heteroalkyl, unsubstituted aryl and
heteroaryl, (unsubstituted aryl)-(C.sub.1-C.sub.4)alkyl, and
(unsubstituted aryl)oxy-(C.sub.1-C.sub.4)alkyl.
[0076] Two of the substituents on adjacent atoms of the aryl or
heteroaryl ring may optionally be replaced with a substituent of
the formula -T-C(O)--(CH.sub.2).sub.q--U--, wherein T and U are
independently --NH--, --O--, --CH.sub.2-- or a single bond, and q
is an integer of from 0 to 2. Alternatively, two of the
substituents on adjacent atoms of the aryl or heteroaryl ring may
optionally be replaced with a substituent of the formula
-A-(CH.sub.2).sub.r--B--, wherein A and B are independently
--CH.sub.2--, --O--, --NH--, --S--, --S(O)--, --S(O).sub.2NR'-- or
a single bond, and r is an integer of from 1 to 3. One of the
single bonds of the new ring so formed may optionally be replaced
with a double bond. Alternatively, two of the substituents on
adjacent atoms of the aryl or heteroaryl ring may optionally be
replaced with a substituent of the formula
--(CH.sub.2).sub.s--X--(CH.sub.2).sub.t--, where s and t are
independently integers of from 0 to 3, and X is --O--, --NR'--,
--S--, --S(O)--, --S(O).sub.2--, or --S(O).sub.2NR'--. The
substituent R' in --NR'-- and --S(O).sub.2NR'-- is selected from
hydrogen or unsubstituted (C.sub.1-C.sub.6)alkyl.
[0077] "Heteroaryl" refers to a monocyclic or fused bicyclic or
tricyclic aromatic ring assembly containing 5 to 16 ring atoms,
where from 1 to 4 of the ring atoms are a heteroatom each N, O or
S. For example, heteroaryl includes pyridyl, indolyl, indazolyl,
quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl,
benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl,
oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl,
thienyl, or any other radicals substituted, especially mono- or
di-substituted, by e.g. alkyl, nitro or halogen. Pyridyl represents
2-, 3- or 4-pyridyl, advantageously 2- or 3-pyridyl. Thienyl
represents 2- or 3-thienyl. Quinolinyl represents preferably 2-, 3-
or 4-quinolinyl. Isoquinolinyl represents preferably 1-, 3- or
4-isoquinolinyl. Benzopyranyl, benzothiopyranyl represents
preferably 3-benzopyranyl or 3-benzothiopyranyl, respectively.
Thiazolyl represents preferably 2- or 4-thiazolyl, and most
preferred, 4-thiazolyl. Triazolyl is preferably 1-, 2- or
5-(1,2,4-triazolyl). Tetrazolyl is preferably 5-tetrazolyl.
[0078] Preferably, heteroaryl is pyridyl, indolyl, quinolinyl,
pyrrolyl, thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl,
imidazolyl, thienyl, furanyl, benzothiazolyl, benzofuranyl,
isoquinolinyl, benzothienyl, oxazolyl, indazolyl, or any of the
radicals substituted, especially mono- or di-substituted.
[0079] As used herein, the term "heteroalkyl" refers to an alkyl
group having from 1 to 3 heteroatoms such as N, O and S. Additional
heteroatoms can also be useful, including, but not limited to, B,
Al, Si and P. The heteroatoms can also be oxidized, such as, but
not limited to, --S(O)-- and --S(O).sub.2--. For example,
heteroalkyl can include ethers, thioethers, alkyl-amines and
alkyl-thiols.
[0080] As used herein, the term "heteroalkylene" refers to a
heteroalkyl group, as defined above, linking at least two other
groups. The two moieties linked to the heteroalkylene can be linked
to the same atom or different atoms of the heteroalkylene.
[0081] "Electrophile" refers to an ion or atom or collection of
atoms, which may be ionic, having an electrophilic center, i.e., a
center that is electron seeking, capable of reacting with a
nucleophile. An electrophile (or electrophilic reagent) is a
reagent that forms a bond to its reaction partner (the nucleophile)
by accepting both bonding electrons from that reaction partner.
[0082] "Nucleophile" refers to an ion or atom or collection of
atoms, which may be ionic, having a nucleophilic center, i.e., a
center that is seeking an electrophilic center or capable of
reacting with an electrophile. A nucleophile (or nucleophilic
reagent) is a reagent that forms a bond to its reaction partner
(the electrophile) by donating both bonding electrons. A
"nucleophilic group" refers to a nucleophile after it has reacted
with a reactive group. Non limiting examples include amino,
hydroxyl, alkoxy, haloalkoxy and the like.
[0083] "Maleimido" refers to a pyrrole-2,5-dione-1-yl group having
the structure:
##STR00003##
which upon reaction with a sulfhydryl (e.g., a thio alkyl) forms an
--S-maleimido group having the structure
##STR00004##
where ".cndot." indicates the point of attachment for the maleimido
group and "" indicates the point of attachment of the sulfur atom
the thiol to the remainder of the original sulfhydryl bearing
group.
[0084] For the purpose of this disclosure, "naturally occurring
amino acids" found in proteins and polypeptides are L-alanine,
L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamine,
L-glutamic acid, L-glycine, L-histidine, L-isoleucine, L-leucine,
L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine,
L-threonine, L-tryptophan, L-tyrosine, and or L-valine.
"Non-naturally occurring amino acids" found in proteins are any
amino acid other than those recited as naturally occurring amino
acids. Non-naturally occurring amino acids include, without
limitation, the D isomers of the naturally occurring amino acids,
and mixtures of D and L isomers of the naturally occurring amino
acids. Other amino acids, such as 4-hydroxyproline, desmosine,
isodesmosine, 5-hydroxylysine, epsilon-N-methyllysine,
3-methylhistidine, although found in naturally occurring proteins,
are considered to be non-naturally occurring amino acids found in
proteins for the purpose of this disclosure as they are generally
introduced by means other than ribosomal translation of mRNA.
[0085] "Linear" in reference to the geometry, architecture or
overall structure of a polymer, refers to polymer having a single
monomer derived backbone.
[0086] "Branched," in reference to the geometry, architecture or
overall structure of a polymer, refers to polymer having 2 or more
polymer "arms" extending from a single group, such as an L group
that may be derived from an initiator employed in an atom transfer
radical polymerization reaction. A branched polymer may possess 2
polymer arms, 3 polymer arms, 4 polymer arms, 5 polymer arms, 6
polymer arms, 7 polymer arms, 8 polymer arms or more. For the
purpose of this disclosure, compounds having three or more polymer
arms extending from a single linear group are denoted as having a
"comb" structure or "comb" architecture. Branched can also be
achieved through "statistical" structures to create broader
dendrimer-like architectures.
[0087] "Pharmaceutically acceptable" composition or "pharmaceutical
composition" refers to a composition comprising a compound of the
invention and a pharmaceutically acceptable excipient or
pharmaceutically acceptable excipients.
[0088] "Pharmaceutically acceptable excipient" and
"pharmaceutically acceptable carrier" refer to an excipient that
can be included in the compositions of the invention and that
causes no significant adverse toxicological effect on the patient.
Non-limiting examples of pharmaceutically acceptable excipients
include water, NaCl, normal saline solutions, lactated Ringer's,
normal sucrose, normal glucose and the like.
[0089] "Patient" or "subject in need thereof" refers to a living
organism suffering from or prone to a condition that can be
prevented or treated by administration of a pharmaceutical
composition as provided herein. Non-limiting examples include
humans, other mammals and other non-mammalian animals.
[0090] "Therapeutically effective amount" refers to an amount of a
conjugated functional agent or of a pharmaceutical composition
useful for treating, ameliorating, or preventing an identified
disease or condition, or for exhibiting a detectable therapeutic or
inhibitory effect. The effect can be detected by any assay method
known in the art.
[0091] The "biological half-life" of a substance is a
pharmacokinetic parameter which specifies the time required for one
half of the substance to be removed from an organism following
introduction of the substance into the organism.
III. Zwitterion-Containing Random Copolymers
[0092] The present invention provides random copolymers having
zwitterionic groups, such as phosphorylcholine, and at least one
functional agent. In some embodiments, the random copolymers of the
present invention have a first monomer with phosphorylcholine, at
least one second monomer having a functional agent or a linking
group, and an initiator moiety having a functional agent or a
linking group, wherein the functional agent can be linked to the
second monomer or the initiator moiety via a linker.
[0093] In other embodiments, the random copolymers of the present
invention have formula I:
##STR00005##
In formula I, the monomer units M.sup.1 and M.sup.2 are any
monomers suitable for polymerization via controlled free radical
methods, such as atom-transfer radical polymerization (ATRP). Each
of monomers M.sup.1 and M.sup.2 can have any suitable number of
comonomers in the random copolymer, as defined by radicals x and
y.sup.1, respectively. The M.sup.1 monomer is linked to a
zwitterionic group ZW, such as phosphorylcholine, via an alkylene
chain (as defined by radical n). The random copolymers can include
a single comonomer M.sup.2 (radical z is 1), or can include several
comonomers M.sup.2 (z is greater than 1) wherein the different
comonomers M.sup.2 are the same or different. The comonomers
M.sup.2 are each linked to an R.sup.2 group that can be inert but
modifies the properties of the random copolymer (such as alkyl,
aryl, etc.), or the R.sup.2 groups can be functional such as when
the R.sup.2 group includes a functional agent A, a linking group LG
or an initiator I. When the R.sup.2 group includes one of these
functional groups, the functional group can optionally be linked to
the comonomer M.sup.2 via a linker L. The R.sup.2 groups can
include a variety of functional groups and inert groups to tune the
properties and functionality of the random copolymer. For example,
several different targeting agents can be included along with
several different drugs or therapeutic proteins as functional
agents A. The monomers M.sup.1 and M.sup.2 can be polymerized by an
initiator, I-I', that can be cleaved into initiator fragment I and
radical scavenger I'. The initiator fragment I can be any group
that initiates the polymerization. The radical scavenger I' can be
any group that will reversibly terminate the growing polymer chain.
The radical scavenger I' can be a halogen such as bromine, allowing
the end of the polymer to be functionalized after polymerization.
In addition, the initiator fragment I can be (but does not need to
be) functionalized with an R.sup.1 group that can include a variety
of functional groups to tune the functionality of the random
copolymer. For example, the R.sup.1 group can include a functional
agent A or a linking group LG, each optionally linked to initiator
fragment I via a linker L. Moreover, the initiator fragment I can
have multiple initiating sites such that the product polymer has
several polymer arms (radical s greater than 1).
[0094] In some embodiments, each monomer M.sup.1 and M.sup.2 of
formula I can independently be an acrylate, methacrylate,
acrylamide, methacrylamide, styrene, vinyl-pyridine or a
vinyl-pyrrolidone. Moreover, R.sup.1 of formula I can independently
be H, L.sup.1-A.sup.1, a linking group LG.sup.1 or
L.sup.1-LG.sup.1, and each R.sup.2 of formula I is independently H,
C.sub.1-20 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, C.sub.1-6 heteroalkyl, C.sub.3-8 cycloalkyl, C.sub.3-8
heterocycloalkyl, aryl, heteroaryl, A.sup.2, L.sup.2-A.sup.2,
LG.sup.2, L.sup.2-LG.sup.2, I.sup.2 and L.sup.2-I.sup.2. The group
ZW of formula I is a zwitterionic moiety. The groups I and I' of
formula I can each independently be an initiator fragment, such
that the combination of I-I' is an initiator, I', for the
polymerization of the random copolymer of formula I. Alternatively,
I' can be H or C.sub.1-6 alkyl. The group I.sup.2 is an initiator.
In addition, each of the groups L.sup.1 and L.sup.2 is a linker,
each of the groups A.sup.1 and A.sup.2 is a functional agent, and
each of the groups LG.sup.1 and LG.sup.2 is a linking group. In
formula I above, subscripts x and y.sup.1 are each independently an
integer of from 1 to 1000, subscript z is an integer of from 1 to
10, subscript s is an integer of from 1 to 100, and subscript n is
an integer of from 1 to 20, wherein either R.sup.1 is
L.sup.1-A.sup.1 or one of R.sup.2 is L.sup.2-A.sup.2.
[0095] The random copolymers of the present invention can have any
suitable number of repeat units for each of the monomers M.sup.1
and M.sup.2. Exemplary ranges of repeat units for each comonomer
include, but are not limited to, from about 1 to about 10,000, from
about 10 to about 5,000, from about 10 to about 2,000, from about
10 to about 1,500, from about 10 to about 1,000, from about 100 to
about 1,000, from about 100 to about 900, from about 100 to about
800, from about 100, to about 700, from about 100 to about 600, and
from about 100 to about 500. When multiple M.sup.2 monomers are
present, each M.sup.2 monomer can have a different number of repeat
units.
[0096] The random copolymers of the present invention can have any
suitable molecular weight. Exemplary molecular weights for the
random copolymers of the present invention can be from about 1000
to about 1,500,000 Daltons (Da). In some embodiments, the random
copolymers of the present invention can have a molecular weight of
about 5,000 Daltons, about 10,000 Daltons, about 25,000 Daltons,
about 50,000 Daltons, about 75,000 Daltons, about 100,000 Daltons,
about 150,000 Daltons, about 200,000 Daltons, about 250,000
Daltons, about 300,000 Daltons, about 350,000 Daltons, about
400,000 Daltons, about 450,000 Daltons, about 500,000 Daltons,
about 550,000 Daltons, about 600,000 Daltons, about 650,000
Daltons, about 700,000 Daltons, about 750,000 Daltons, about
800,000 Daltons, about 850,000 Daltons, about 900,000 Daltons,
about 950,000 Daltons, about 1,000,000 Daltons and about 1,250,000
Daltons.
[0097] The random copolymers of the present invention can also have
any suitable number of comonomers, M.sup.2. For example, the number
of comonomers, subscript z, can be from 1 to 10, such as 1, 2, 3,
4, 5, 6, 7, 8, 9 or 10. The number of comonomers, subscript z, can
also be from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some
embodiments, the random copolymer of the present invention can have
two different monomers where subscript z is 1, such as in formula
II:
##STR00006##
In other embodiments, the random copolymer can have 3 different
monomers where subscript z is 2, such as in formula III:
##STR00007##
Additional comonomers M.sup.2 can be present in the random
copolymers of the present invention, such as M.sup.2c, M.sup.2d,
M.sup.2e, M.sup.2f, M.sup.2g, M.sup.2h, etc., where each comonomer
is present in a same or different y.sup.1 value, and each comonomer
having a corresponding R.sup.2 group attached, R.sup.2c, R.sup.2d,
R.sup.2e, R.sup.2f, R.sup.2g, R.sup.2h, etc., respectively. Each
M.sup.2 group, such as M.sup.2a, M.sup.2b, M.sup.2c, etc., can be
as defined above for M.sup.2. Each R.sup.2 group, such as R.sup.2a,
R.sup.2b, R.sup.2c, etc., can be as defined above for R.sup.2.
Similarly, each y.sup.1 group, such as y.sup.1a, y.sup.1b,
y.sup.1c, etc., can be as defined above for y.sup.1.
[0098] In some embodiments, the random copolymer can be of formula
III, wherein R.sup.2a and R.sup.2b are each independently H,
C.sub.1-20 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, C.sub.1-6 heteroalkyl, C.sub.3-8 cycloalkyl, C.sub.3-8
heterocycloalkyl, aryl, heteroaryl, A.sup.2, L.sup.2-A.sup.2,
LG.sup.2, or L.sup.2-LG.sup.2; M.sup.2a and M.sup.2b are each
independently acrylate, methacrylate, acrylamide, methacrylamide,
styrene, vinyl-pyridine or vinyl-pyrrolidone; and subscripts
y.sup.1a and y.sup.1b are each independently an integer of from 1
to 1000.
[0099] The different monomers of the random copolymers can also be
present in any suitable ratio. For example, the M.sup.2 monomers,
collectively or individually, can be present relative to the
M.sup.1 monomer in a ratio of 100:1, 50:1, 40:1, 30:1, 20:1, 10:1,
9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5,
1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:30, 1:40, 1:50 and 1:100. In
addition, each M.sup.2 monomer can be present in any suitable ratio
relative to the M.sup.1 or any other M.sup.2 monomer, such as
100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1,
3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20,
1:30, 1:40, 1:50 and 1:100.
[0100] The random copolymers of the present invention can have any
suitable architecture. For example, the random copolymers can be
linear or branched. When the random copolymers are branched, they
can have any suitable number of copolymer arms, as defined by
subscript s of formula I, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,
30, 40, 50, 60, 70, 80, 90 and up to 100 arms. In some embodiments,
subscript s can be from 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8,
1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2. The random
copolymers of the present invention can adopt any suitable
architecture. For example, the random copolymers can be linear,
branched, stars, dendrimers, dendrigrafts, combs, etc.
[0101] A functional agent of the random copolymers can be linked to
either one of the comonomers M.sup.2, or to the initiator fragment
I, or both. When multiple functional agents are present, a
functional agent can be linked to both the comonomer M.sup.2 and
the initiator fragment I. In some embodiments, the random copolymer
has formula IIa:
##STR00008##
In formula IIa, functional agent A.sup.1 can be a drug or
therapeutic protein and functional agent A.sup.2 can be a targeting
agent. Alternatively, functional agent A.sup.1 can be a targeting
agent and functional agent A.sup.2 can be a drug or therapeutic
protein. Furthermore, functional agents A.sup.1 and A.sup.2 can
both be therapeutic agents. Functional agents can be chosen to
inhibit (or activate) distinct targets in the same molecular
pathway, provide inhibition (or activation) of both a primary and
compensatory pathway, or inhibit (or activate) the same target at
different binding sites to decrease resistance or allow use of
lower doses to minimize toxicity. Moreover, the linkers L.sup.1 and
L.sup.2 can be the same or different. For example, linker L.sup.1
can be a cleavable linker, such as when attached to a drug or
therapeutic protein to facilitate release of the drug or
therapeutic protein, while linker L.sup.2 can be a non-cleavable
linker, such as when attached to a targeting agent. Furthermore,
linker L.sup.1 can be a non-cleavable linker, while linker L.sup.2
can be a cleavable linker. Alternatively, both linkers L.sup.1 and
L.sup.2 can be cleavable linkers or non-cleavable linkers. In
addition, the linker attached to the targeting agent can also be a
cleavable linker. Alternatively, one or both of L.sup.1 and L.sup.2
can be self-immolative or double prodrug linkers.
[0102] When multiple comonomers M.sup.2 are present, each comonomer
M.sup.2 can have a different functional agent attached. For
example, the random copolymer can have formula IIIa:
##STR00009##
[0103] When multiple comonomers M.sup.2 are present, each comonomer
M.sup.2 can have a different functional agent attached. For
example, the random copolymer can have formula IIIa:
##STR00010##
In formula IIIa, M.sup.2a and M.sup.2b can be as defined above for
M.sup.2; A.sup.2a and A.sup.2b can be as defined above for A.sup.2;
L.sup.2a and L.sup.2b can be as defined above for L.sup.2; and
y.sup.1a and y.sup.1b can be as defined above for y.sup.1. In some
embodiments, each of L.sup.2a and L.sup.2b is a linker; and each of
A.sup.2a and A.sup.2b is a functional agent.
[0104] Functional agents A.sup.2a and A.sup.2b can be the same or
different in formula IIIa. Functional agent A.sup.2a can be a drug
or therapeutic protein and functional agent A.sup.2b can be a
targeting agent. Alternatively, functional agents A.sup.2a and
A.sup.2b can both be targeting agents, and functional agent A.sup.1
can be the drug or therapeutic agent. The functional agents
A.sup.2a and A.sup.2b can also both be a drug or therapeutic agent,
while functional agent A.sup.1 is the targeting agent. When
functional agents A.sup.2a and A.sup.2b are both a drug or
therapeutic agent, each functional agent A.sup.2a and A.sup.2b can
be a different drug or therapeutic agent. In addition, one of
functional agents A.sup.2a and A.sup.2b can be a drug or
therapeutic agent and the other can be a targeting agent, where
functional agent A.sup.1 can be any functional agent.
[0105] As described above for formula IIa, the linkers L.sup.1,
L.sup.2a and L.sup.2b of formula IIIa can be the same or different.
For example, linker L.sup.1 can be a cleavable linker when attached
to a drug or therapeutic agent to facilitate release of the drug or
therapeutic agent, while linkers L.sup.2a and L.sup.2b can be
non-cleavable linkers when attached to targeting agents.
Alternatively, linker L.sup.1 can also be a non-cleavable linker
and linkers L.sup.2a and L.sup.2b can be cleavable linkers.
Furthermore, linkers L.sup.2a and L.sup.2b can be the same or
different, such as where one is a cleavable linker and the other is
a non-cleavable linker. Linkers L.sup.2a and L.sup.2b can also be
different cleavable linkers, such as when each is attached to a
drug, to provide different release rates for the different
drugs.
[0106] In some embodiments, there is no functional agent linked to
the initiator fragment I, such as in formula IIIb:
##STR00011##
In formula IIIb, M.sup.2a and M.sup.2b can be as defined above for
M.sup.2; A.sup.2a and A.sup.1b can be as defined above for A.sup.2;
L.sup.2a and L.sup.2b can be as defined above for L.sup.2; and
y.sup.1a and y.sup.1b can be as defined above for y.sup.1. In some
embodiments, each of L.sup.2a and L.sup.2b is a linker; and each of
A.sup.2a and A.sup.2b is a functional agent.
[0107] In formula IIIb, functional agents A.sup.2a and A.sup.2b can
be the same or different, as described above, and linkers L.sup.2a
and L.sup.2b can be the same or different. In other embodiments,
one of the comonomers M.sup.2 can have no functional agent or
linking group, such as in formula IIIc:
##STR00012##
In formula IIIc, M.sup.2a and M.sup.2b can be as defined above for
M.sup.2; A.sup.2a can be as defined above for A.sup.2; L.sup.2a can
be as defined above for L.sup.2. Similarly, y.sup.1a and y.sup.1b
can be as defined above for y.sup.1.
[0108] When additional comonomers, M.sup.2 are present in the
random copolymers of the present invention, the corresponding
linkers L.sup.2 can be the same or different as linkers L.sup.1,
L.sup.2a and L.sup.2b, as described above. Moreover, the
corresponding functional agents A.sup.2 can be the same or
different as functional agents A.sup.1, A.sup.2a and A.sup.2b, as
described above.
[0109] In some embodiments, the random copolymers have linking
groups LG linked to either or both of the initiator fragment I and
the comonomers M.sup.2, such as shown in the structures below:
##STR00013##
The linking groups LG.sup.2 facilitate the "clicking" on or
covalent chemical attachment of functional agents and initiator
groups following polymerization.
[0110] When a plurality of comonomers M.sup.2 is present, the
comonomers can be linked to either a functional agent or a linking
group, for example as shown in the following formula:
##STR00014##
wherein M.sup.2a and M.sup.2b can be as defined above for M.sup.2;
LG.sup.2a can be as defined above for LG.sup.2; L.sup.2b can be as
defined above for L.sup.2; A.sup.2b can be as defined above for
A.sup.2; and y.sup.1a and y.sup.1b can be as defined above for
y.sup.1. In addition, the linking group can be present on the
initiator fragment I while functional agents A.sup.2 are linked to
the comonomers M.sup.2. Alternatively, when the linking group LG is
linked to the initiator fragment I, a second linking group LG can
be linked to one of the comonomers M.sup.2:
##STR00015##
Moreover, a functional agent A.sup.1 can be linked to the initiator
fragment I while linking groups LG are linked to the comonomers
M.sup.2, where the linking groups can be the same or different:
##STR00016##
wherein M.sup.2a and M.sup.2b can be as defined above for M.sup.2;
L.sup.2a and L.sup.2b can be as defined above for L.sup.2;
LG.sup.2a and LG.sup.2b can be as defined above for LG.sup.2; and
y.sup.1a and y.sup.1b can be as defined above for y.sup.1.
[0111] In some embodiments when there are multiple comonomers
M.sup.2, one of the comonomers M.sup.2 can be linked to a group
other than a linking group LG, a functional agent A or an initiator
I. In other embodiments, at least one R.sup.2 group is H,
C.sub.1-20 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, C.sub.1-6 heteroalkyl, C.sub.3-8 cycloalkyl, C.sub.3-8
heterocycloalkyl, aryl, or heteroaryl. For example, such structures
include the following:
##STR00017##
wherein R.sup.2a can be H, C.sub.1-20 alkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, C.sub.1-6 haloalkyl, C.sub.1-6 heteroalkyl,
C.sub.3-8 cycloalkyl, C.sub.3-8 heterocycloalkyl, aryl, or
heteroaryl. In other embodiments, R.sup.2a can be a species having
one or more positive or negative charges, such as aspartic acid,
glutamic acid, lysine, histidine, arginine, choline or hyaluronic
acid. Other radicals M.sup.2a, M.sup.2b, y.sup.1a, y.sup.1b,
R.sup.2a and LG.sup.2b can be as defined above.
[0112] When R.sup.2 of some comonomers M.sup.2 is the initiator
I.sup.2, more complex architectures can be prepared of the random
copolymers. For example, comb polymers, hyperbranched polymers,
dendrimers, and dendrigrafts can be prepared. When initiator
I.sup.2 is present on a comonomer M.sup.2, polymerization using
initiator I.sup.2 typically occurs following polymerization using
initiator I-I'. In some embodiments, polymerization via I-I' and
I.sup.2 can be simultaneous. Moreover, the initiator I.sup.2 can be
linked to the comonomer M.sup.2 via a cleavable or non-cleavable
linker L.sup.2.
[0113] In some embodiments, the random copolymers of the present
invention can be modified via a subsequent polymerization with one
or more additional monomers. For example, in formula III above,
monomers M.sup.1 and M.sup.2a can be copolymerized in a first
polymerization, and monomer M.sup.2b can be polymerized in a second
polymerization. A block copolymer would be formed having two
blocks, the first block being a random copolymer of M.sup.1 and
M.sup.2a, and the second block a homopolymer of M.sup.2b.
Alternatively, following polymerization of monomers M.sup.1 and
M.sup.2a, monomer M.sup.2b can be copolymerized with monomer
M.sup.2c, thus forming a block copolymer where the first block is a
random copolymer of M.sup.1 and M.sup.2a, and the second block is a
random copolymer of M.sup.2b and M.sup.2c. Additional polymer
structures can be prepared by copolymerizing monomers M.sup.1,
M.sup.2a and M.sup.2b in a first polymerization, followed by
copolymerization of monomers M.sup.2c, M.sup.2d, and others, in a
second copolymerization. Additional blocks can be prepared by yet a
third polymerization using additional monomers. Such polymers
provide blocks of copolymers that can have different properties,
drugs and functional agents.
[0114] In other embodiments, the random copolymer has the
formula:
##STR00018## ##STR00019## ##STR00020## ##STR00021##
wherein L-CTP has the formula:
##STR00022##
[0115] In some other embodiments, the random copolymer has the
formula:
##STR00023## ##STR00024## ##STR00025##
wherein PC is phosphorylcholine; HEMA is hydroxyethyl methacrylate;
GMA is glycidyl methacrylate; and Glu is glutamic acid.
[0116] In still other embodiments, the random copolymer has the
formula:
##STR00026##
wherein the block copolymer has the formula:
##STR00027##
[0117] A. Initiators
[0118] The random copolymers of the present invention are
polymerized using any suitable initiator. Initiators useful in the
present invention can be described by the formula: I-(I').sub.m,
where subscript m is an integer from 1 to 20. The initiator
fragment I can be any group that initiates the polymerization. The
radical scavenger I' can be any group that will reversibly
terminate the growing polymer chain. The radical scavenger I' can
be a halogen such as bromine, allowing the end of the polymer to be
functionalized after polymerization. In addition, the initiator
fragment I can optionally be functionalized with an R.sup.1 group
that can include a variety of functional groups to tune the
functionality of the random copolymer.
[0119] Initiators useful in the present invention can have a single
radical scavenger I', or any suitable number of branches such that
there are multiple radical scavengers I' each capable of reversibly
terminating a growing polymer chain. When the initiator fragment I
is branched and is capable of initiating multiple polymer chains,
subscript m is greater than one such that there are as many radical
scavengers I' as there are growing polymer chains.
[0120] The bond between initiator fragment I and radical scavenger
I' is labile, such that during the polymerization process monomers
M.sup.1 and comonomers M.sup.2 are inserted between initiator
fragment I and radical scavenger I'. For example, during a free
radical polymerization, such as ATRP, initiator fragment I and
radical scavenger I' dissociate, as shown in FIG. 1, to form
radicals of I and I'. The radical of initiator fragment I then
reacts with the monomers in solution to grow the polymer and forms
a propagating polymer radical (species A and species C of FIG. 1).
During the polymerization process, the radical of the radical
scavenger I' will reversibly react with the propagating polymer
radical to temporarily stop polymer growth. The bond between the
monomer and the radical savenger I' is also labile, such that the
bond can cleave and allow the propagating polymer radical to react
with additional monomer to grow the polymer. The end result of the
polymerization process is that initiator fragment I is at one end
of the polymer chain and radical scavenger I' is at the opposite
end of the polymer chain.
[0121] The radical of initiator fragment I is typically on a
secondary or tertiary carbon, and can be stabilized by an adjacent
carbonyl carbon. The radical scavenger I' is typically a halogen,
such as bromine, chlorine or iodine. Together, initiator fragment I
and radical scavenger I' form the initiators I.sup.1 and I.sup.2
useful in the preparation of the random copolymers of the present
invention.
[0122] A broad variety of initiators can be used to prepare the
random copolymers of the invention, including a number of
initiators set forth in U.S. Pat. No. 6,852,816 (incorporated
herein by reference). In some embodiments, the initiators employed
for ATRP reactions to prepare random copolymers of the invention
are selected from alkanes, cycloalkanes, alkyl carboxylic acids or
esters thereof, cycloalkylcarboxylic acids or esters thereof,
ethers and cyclic alkyl ethers, alkyl aryl groups, alkyl amides,
alkyl-aryl carboxylic acids and esters thereof, and also bearing
one radical scavenger I' where unbranched random copolymers are
prepared, and more than one radical scavenger I' where branched
molecules are prepared.
[0123] Radical scavengers I' useful in the present invention
include, but are not limited to, halogens, such as Br, Cl and I,
thiocyanate (--SCN) and isothiocyanate (--N.dbd.C.dbd.S). Other
groups are useful for the radical scavenger I' of the present
invention. In some embodiments, the radical scavenger I' is
bromine.
[0124] Initiators employed for ATRP reactions can be hydroxylated.
In some embodiments, the initiators employed for ATRP reactions to
prepare random copolymers of the invention are selected from
alkanes, cycloalkanes, alkyl carboxylic acids or esters thereof,
cycloalkylcarboxylic acids or esters thereof, ethers, cyclic alkyl
ethers, alkyl aryl groups, alkyl amides, alkyl-aryl carboxylic
acids and esters thereof, bearing a hydroxyl group, and also
bearing one radical scavenger I' where unbranched random copolymers
are to be prepared, or alternatively, more than one radical
scavenger I' where branched molecules are to be prepared.
[0125] Initiators employed for ATRP reactions can bear one or more
amine groups. In some embodiments, the initiators employed for ATRP
reactions to prepare random copolymers of the invention are
alkanes, cycloalkanes, alkyl carboxylic acids or esters thereof,
cycloalkylcarboxylic acids or esters thereof, ethers, cyclic alkyl
ethers, alkyl-aryl groups, alkyl amides, alkyl-aryl carboxylic
acids and esters thereof, bearing an amine group and also bearing
one radical scavenger I' where unbranched random copolymers are to
be prepared, or alternatively, more than one radical scavenger I'
where branched molecules are to be prepared.
[0126] Alkylcarboxylic acids, including alkyl dicarboxylic acids,
having at least one radical scavenger I', and substituted with
amino or hydroxy groups can also be employed as initiators. In some
embodiments of the invention where ATRP is employed to prepare
random copolymers of the present invention, the initiators can be
alkylcarboxylic acids bearing one or more halogens selected from
chlorine and bromine.
[0127] Alkanes substituted with two or more groups selected from
--COOH, --OH and --NH.sub.2, and at least one radical scavenger I',
can also be employed as initiators for the preparation of random
copolymers where ATRP is employed to prepare random copolymers of
the present invention.
[0128] Initiators can also contain one or more groups including,
but not limited to, --OH, amino, monoalkylamino, dialkylamino,
--O-alkyl, --COOH, --COO-alkyl, or phosphate groups (or protected
forms thereof).
[0129] A broad variety of initiators are commercially available,
for example bromoacetic acid N-hydroxysuccinimide ester available
from Sigma-Aldrich (St. Louis, Mo.). Suitably protected forms of
those initiators can be prepared using standard methods in the art
as necessary.
[0130] Other initiators include thermal, redox or photo initiators,
including, for example, alkyl peroxide, substituted alkyl
peroxides, aryl peroxides, substituted aryl peroxides, acyl
peroxides, alkyl hydroperoxides, substituted aryl hydroperoxides,
aryl hydroperoxides, substituted aryl hydroperoxides, heteroalkyl
peroxides, substituted heteroalkyl peroxides, heteroalkyl
hydroperoxides, substituted heteroalkyl hydroperoxides, heteroaryl
peroxides, substituted heteroaryl peroxides, heteroaryl
hydroperoxides, substituted heteroaryl hydroperoxides, alkyl
peresters, substituted alkyl peresters, aryl peresters, substituted
aryl peresters, azo compounds and halide compounds. Specific
initiators include cumene hydroperoxide (CHP), tert-butyl
hydroperoxide (TBHP), tert-butyl perbenzoate, (TBPB), sodium
carbonateperoxide, benzoyl peroxide (BPO), lauroyl peroxide (LPO),
methylethyl ketone 45%, potassium persulfate, ammonium persulfate,
2,2-azobis(2,4-dimethyl-valeronitrile),
1,1-azobis(cyclo-hexanecarbonitrile),
2,2-azobis(N,N-dimethyleneisobutyramidine) dihydrochloride, and
2,2-azobis (2-amido-propane) dihydrochloride. Redox pairs such as
persulfate/sulfite and Fe (2+) peroxide or ammonium persulfate and
N,N,N'N'-tetramethylethylenediamine (TEMED).
[0131] Still other initiators useful for preparing the random
copolymers of the present invention, are branched. Suitable
initiators having a single branch point include the following:
##STR00028##
where radical R can be any of the following:
##STR00029##
[0132] In some embodiments, the initiator can be:
##STR00030##
which is a protected maleimide that can be deprotected after
polymerization to form the maleimide for reaction with additional
functional groups.
[0133] Additional branched initiators include, but are not limited
to, the following, where radical R is as defined above:
##STR00031##
[0134] In some embodiments, the branched initiators include, but
are not limited to, the following:
##STR00032##
[0135] Other branched initiators useful for preparing the random
copolymers of the present invention include the following:
##STR00033##
where radical R is as defined above, and radical X can be CHO,
SO.sub.2Cl, SO.sub.2CH.dbd.CH.sub.2, NHCOCH.sub.2I, N.dbd.C.dbd.O
and N.dbd.C.dbd.S, among others. Additional X groups can include
the following:
##STR00034##
Still other initiators include, but are not limited to, the
following:
##STR00035##
[0136] In other embodiments, the initiator can have several branch
points to afford a plurality of polymer arms, such as:
##STR00036##
where radical R is as defined above. In some other embodiments, the
initiator can have the following structure:
##STR00037##
[0137] In some other embodiments, the initiator can have the
following structures:
##STR00038## ##STR00039## ##STR00040## ##STR00041## ##STR00042##
##STR00043##
[0138] As described above, the initiator can be added to the
polymerization mixture separately, or can be incorporated into
another molecule, such as a monomer (hyperbranched structure) or a
polymer fragment (such as graft copolymers). Initiation of the
polymerization can be accomplished by heat, UV light, or other
methods known to one of skill in the art.
[0139] In some embodiments, the initiator I-I' of the present
invention has the formula:
(F).sub.r-Sp.sup.1-C-Sp.sup.2-I'
where the initiator fragment I corresponds to
F-Sp.sup.1-C-Sp.sup.2. Each radical F is a functional group for
reaction with a functional agent or linking group of the present
invention. Radical r is from 1 to 10. Radicals Sp.sup.1 and
Sp.sup.2 are spacers and can be any suitable group for forming a
covalent bond, such as C.sub.1-6 alkyl, aryl or heteroaryl. Radical
C can be any core providing one or a plurality of points for
linking to one or more spacers, Sp.sup.2 (which can be the same or
different), and one or more radical scavengers, I', and providing
one or a plurality of points for linking to one or more spacers,
Sp.sup.1 (which can be the same or different), and one or more
functional groups, F (which can be the same or different). Core C
can be any suitable structure, such as a branched structure, a
crosslinked structure including heteroatoms, such as
silsesquiloxanes, and a linear, short polymer with multiple pendant
functional groups. In addition, core C can be attached to the one
or more Sp.sup.1 and Sp.sup.2 spacers by any suitable group for
forming a covalent bond including, but not limited to, esters,
amides, ethers, and ketones. Radical scavenger I' is a radically
transferable atom or group such as, but not limited to, a halogen,
Cl, Br, I, OR.sup.10, SR.sup.11, SeR.sup.11, OC(.dbd.O)R.sup.11,
OP(.dbd.O)R.sup.11, OP(.dbd.O)(OR.sup.11).sub.2,
O--(R.sup.11).sub.2, S--C(.dbd.S)N(R.sup.11).sub.2, CN, NC, SCN,
CNS, OCN, CNO, N.sub.3, OH, O, C1-C6-alkoxy, (SO.sub.4), PO.sub.4,
HPO.sub.4, H.sub.2 PO.sub.4, triflate, hexafluorophosphate,
methanesulfonate, arylsulfonate, carboxylic acid halide. R.sup.10
is an alkyl of from 1 to 20 carbon atoms or an alkyl of from 1 to
20 carbon atoms in which each of the hydrogen atoms may be replaced
by a halide, alkenyl of from 2 to 20 carbon atoms, alkynyl of from
2 to 10 carbon atoms, phenyl, phenyl substituted with from 1 to 5
halogen atoms or alkyl groups with from 1 to 4 carbon atoms,
aralkyl, aryl, aryl substituted alkyl, in which the aryl group is
phenyl or substituted phenyl and the alkyl group is from 1 to 6
carbon atoms, and R.sup.11 is aryl or a straight or branched
C.sub.1-C.sub.20 alkyl group or where an N(R.sup.11).sub.2 group is
present, the two R.sup.11 groups may be joined to form a 5-, 6- or
7-member heterocyclic ring. Spacer Sp.sup.1 covalently links
functional group F and core C while spacer Sp.sup.2 covalently
links core C and radical scavenger I'.
[0140] In other embodiments, the initiator of the present invention
has the formula:
LG.sup.2-L.sup.5-C L.sup.4-I'].sub.p
wherein each I' is independently selected from halogen, --SCN, or
--NCS. L.sup.4 and L.sup.5 are each independently a bond or a
linker, such that one of L.sup.4 and L.sup.5 is a linker. C is a
bond or a core group. LG.sup.2 is a linking group. And subscript p
is from 1 to 32, wherein when subscript p is 1, C is a bond, and
when subscript p is from 2 to 32, C is a core group. In some other
embodiments, the initiator has the formula:
##STR00044##
wherein each R.sup.3 and R.sup.4 is independently selected H, CN or
C.sub.1-6 alkyl.
[0141] B. Monomers
[0142] Monomers useful for preparing the random copolymers of the
present invention include any monomer capable of radical
polymerization. Typically, such monomers have a vinyl group.
Suitable monomers include, but are not limited to, acrylate,
methacrylate, acrylamide, methacrylamide, styrene, vinyl-pyridine
and vinyl-pyrrolidone monomers. Monomers, M.sup.1, containing the
zwitterionic moiety, ZW, include, but are not limited to, the
following:
##STR00045##
Monomers, M.sup.2, containing the linking group or functional agent
include, but are not limited to, the following structures:
##STR00046##
Other monomers are well-known to one of skill in the art, and
include vinyl acetate and derivatives thereof.
[0143] In some embodiments, the monomers are acrylate or
methacrylate monomers. In other embodiments, the random copolymer
has the formula:
##STR00047##
wherein each of R.sup.3 and R.sup.4 are independently H or
C.sub.1-6 alkyl; and PC is phosphatidylcholine.
[0144] In some other embodiments, the random copolymer has the
formula:
##STR00048##
[0145] In other embodiments, the random copolymer has the
formula:
##STR00049##
wherein A.sup.2 is camptothecin.
[0146] In still other embodiments, the random copolymer has the
formula:
##STR00050##
wherein R.sup.4a and R.sup.4b can be as defined above for R.sup.4;
R.sup.2a and R.sup.2b can be as defined above for A.sup.2; and
y.sup.1a and y.sup.1b can be as defined above for y'. In some
embodiments, R.sup.2a and R.sup.2b are each independently H,
C.sub.1-20 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, C.sub.1-6 heteroalkyl, C.sub.3-8 cycloalkyl, C.sub.3-8
heterocycloalkyl, aryl, heteroaryl, A.sup.2, L.sup.2-A.sup.2,
LG.sup.2, L.sup.2-LG.sup.2; each of R.sup.3, R.sup.4a and R.sup.4b
are independently H or C.sub.1-6 alkyl; subscripts y.sup.1a and
y.sup.1b are each independently an integer of from 1 to 1000; and
PC is phosphatidylcholine.
[0147] In still yet other embodiments, the random copolymer can
have any of the following formulas:
##STR00051##
wherein R.sup.4a and R.sup.4b can be as defined above for R.sup.4;
L.sup.2a and L.sup.2b can be as defined above for L.sup.2; A.sup.2a
and A.sup.2b can be as defined above for A.sup.2; and y.sup.1a and
y.sup.1b can be as defined above for y.sup.1. In some embodiments,
each of L.sup.2a and L.sup.2b is a linker; and each of A.sup.2a and
A.sup.2b is a functional agent.
[0148] C. Zwitterions
[0149] The zwitterions of the present invention include any
compound having both a negative charge and a positive charge.
Groups having a negative charge and suitable for use in the
zwitterions of the present invention include, but are not limited
to, phosphate, sulfate, other oxoanions, etc. Groups having a
positive charge and suitable for use in the zwitterions of the
present invention include, but are not limited to, ammonium ions.
In some embodiments, the zwitterion can be phosphorylcholine.
[0150] D. Linkers
[0151] The random copolymers of the present invention can also
incorporate any suitable linker L. The linkers provide for
attachment of the functional agents to the initiator fragment I and
the comonomers M.sup.2. The linkers can be cleavable or
non-cleavable, homobifunctional or heterobifunctional. Other
linkers can be both heterobifunctional and cleavable, or
homobifunctional and cleavable.
[0152] Cleavable linkers include those that are hydrolyzable
linkers, enzymatically cleavable linkers, pH sensitive linkers,
disulfide linkers and photolabile linkers, among others.
Hydrolyzable linkers include those that have an ester, carbonate or
carbamate functional group in the linker such that reaction with
water cleaves the linker. Enzymatically cleavable linkers include
those that are cleaved by enzymes and can include an ester, amide,
or carbamate functional group in the linker. pH sensitive linkers
include those that are stable at one pH but are labile at another
pH. For pH sensitive linkers, the change in pH can be from acidic
to basic conditions, from basic to acidic conditions, from mildly
acidic to strongly acidic conditions, or from mildly basic to
strongly basic conditions. Suitable pH sensitive linkers are known
to one of skill in the art and include, but are not limited to,
ketals, acetals, imines or imminiums, siloxanes, silazanes,
silanes, maleamate-amide bonds, ortho esters, hydrazones, activated
carboxylic acid derivatives and vinyl ethers. Disulfide linkers are
characterized by having a disulfide bond in the linker and are
cleaved under reducing conditions. Photolabile linkers include
those that are cleaved upon exposure to light, such as visible,
infrared, ultraviolet, or electromagnetic radiation at other
wavelengths.
[0153] Other linkers useful in the present invention include those
described in U.S. Patent Application Nos. 2008/0241102 (assigned to
Ascendis/Complex Biosystems) and 2008/0152661 (assigned to Mirus),
and International Patent Application Nos. WO 2004/010957 and
2009/117531 (assigned to Seattle Genetics) and 01/24763,
2009/134977 and 2010/126552 (assigned to Immunogen) (incorporated
in their entirety herein). Mirus linkers useful in the present
invention include, but are not limited to, the following:
##STR00052##
Other linkers include those described in Bioconjugate Techniques,
Greg T. Hermanson, Academic Press, 2d ed., 2008 (incorporated in
its entirety herein), and those described in Angew. Chem. Int. Ed.
2009, 48, 6974-6998 (Bertozzi, C. R. and Sletten, E. M)
(incorporated in its entirety herein).
[0154] In some embodiments, linkers L.sup.1 and L.sup.2 can have a
length of up to 30 atoms, each atom independently C, N, O, S, and
P. In other embodiments, the linkers L.sup.1 and L.sup.2 can be any
of the following: --C.sub.1-12 alkyl-, --C.sub.3-12 cycloalkyl-,
--(C.sub.1-8 alkyl)-(C.sub.3-12 cycloalkyl)-(C.sub.0-8 alkyl)-,
--(CH.sub.2).sub.1-12O--,
(--(CH.sub.2).sub.1-6--O--(CH.sub.2).sub.1-6--).sub.1-12--,
(--(CH.sub.2).sub.1-4--NH--(CH.sub.2).sub.1-4).sub.1-12--,
(--(CH.sub.2).sub.1-4--O--(CH.sub.2).sub.1-4).sub.1-12--O--,
(--(CH.sub.2).sub.1-4--O--(CH.sub.2).sub.1-4--).sub.1-12--O--(CH.sub.2).s-
ub.1-12--, --(CH.sub.2).sub.1-12--(C.dbd.O)--O--,
--(CH.sub.2).sub.1-12--O--(C.dbd.O)--,
-(phenyl)-(CH.sub.2).sub.1-3--(C.dbd.O)--O--,
-(phenyl)-(CH.sub.2).sub.1-3--(C.dbd.O)--NH--, --(C.sub.1-6
alkyl)-(C.dbd.O)--O--(C.sub.0-6 alkyl)-,
--(CH.sub.2).sub.1-12--(C.dbd.O)--O--(CH.sub.2).sub.1-12--,
--CH(OH)--CH(OH)--(C.dbd.O)--O--CH(OH)--CH(OH)--(C.dbd.O)--NH--,
--S-maleimido-(CH.sub.2).sub.1-6--, --S-maleimido-(C.sub.1-3
alkyl)-(C.dbd.O)--NH--, --S-maleimido-(C.sub.1-3alkyl)-(C.sub.5-6
cycloalkyl)-(C.sub.0-3alkyl)-, --(C.sub.1-3 alkyl)-(C.sub.5-6
cycloalkyl)-(C.sub.0-3alkyl)-(C.dbd.O)--O--, --(C.sub.1-3
alkyl)-(C.sub.5-6 cycloalkyl)-(C.sub.0-3 alkyl)-(C.dbd.O)--NH--,
--S-maleimido-(C.sub.0-3alkyl)-phenyl-(C.sub.0-3alkyl)-,
--(C.sub.0-3 alkyl)-phenyl-(C.dbd.O)--NH--,
--(CH.sub.2).sub.1-12--NH--(C.dbd.O)--,
--(CH.sub.2).sub.1-12--(C.dbd.O)--NH--,
-(phenyl)-(CH.sub.2).sub.1-3--(C.dbd.O)--NH--,
--S--(CH.sub.2)--(C.dbd.O)--NH-(phenyl)-,
--(CH.sub.2).sub.1-12--(C.dbd.O)--NH--(CH.sub.2).sub.1-12--,
--(CH.sub.2).sub.2--(C.dbd.O)--O--(CH.sub.2).sub.2--O--(C.dbd.O)--(CH.sub-
.2).sub.2--(C.dbd.O)--NH--, --(C.sub.1-6
alkyl)-(C.dbd.O)--N--(C.sub.1-6 alkyl)-, acetal, ketal,
acyloxyalkyl ether, --N.dbd.CH--, --(C.sub.1-6
alkyl)-S--S--(C.sub.0-6, --(C.sub.1-6 alkyl)-S--S--(C.sub.1-6
alkyl)-(C.dbd.O)--O--, --(C.sub.1-6 alkyl)-S--S--(C.sub.1-6
alkyl)-(C.dbd.O)--NH--,
--S--S--(CH.sub.2).sub.1-3--(C.dbd.O)--NH--(CH.sub.2).sub.1-4--NH--(C.dbd-
.O)--(CH.sub.2).sub.1-3--, --S--S--(C.sub.0-3 alkyl)-(phenyl)-,
--S--S--(C.sub.1-3-alkyl)-(phenyl)-(C.dbd.O)--NH--(CH.sub.2).sub.1-5--,
--(C.sub.1-3
alkyl)-(phenyl)-(C.dbd.O)--NH--(CH.sub.2).sub.1-5--(C.dbd.O)--NH--,
--S--S--(C.sub.1-3-alkyl)-,
--(C.sub.1-3-alkyl)-(phenyl)-(C.dbd.O)--NH--, --O--(C.sub.1-C.sub.6
alkyl)-S(O.sub.2)--(C.sub.1-6 alkyl)-O--(C.dbd.O)--NH--,
--S--S--(CH.sub.2).sub.1-3--(C.dbd.O)--,
--(CH.sub.2).sub.1-3--(C.dbd.O)--NH--N.dbd.C--S--S--(CH.sub.2).sub.1-3--(-
C.dbd.O)--NH--(CH.sub.2).sub.1-5--,
--(CH.sub.2).sub.1-3--(C.dbd.O)--NH--(CH.sub.2).sub.1-5--(C.dbd.O)--NH--,
--(CH.sub.2).sub.0-3--(heteroaryl)--(CH.sub.2).sub.0-3--,
--(CH.sub.2).sub.0-3-phenyl-(CH.sub.2).sub.0-3--, --N.dbd.C(R)--,
--(C.sub.1-6 alkyl)-C(R).dbd.N--(C.sub.1-6 alkyl)-, --(C.sub.1-6
alkyl)-(aryl)-C(R).dbd.N--(C.sub.1-6 alkyl)-, --(C.sub.1-6
alkyl)-C(R).dbd.N-(aryl)-(C.sub.1-6 alkyl)-, and --(C.sub.1-6
alkyl)-O--P(O)(OH)--O--(C.sub.0-6 alkyl)-, wherein R is H,
C.sub.1-6 alkyl, C.sub.3-6 cycloalkyl, or an aryl group having 5-8
endocyclic atoms.
[0155] In some other embodiments, linkers L.sup.1 and L.sup.2 can
be any of the following: --C.sub.1-C.sub.12 alkyl-,
--C.sub.3-C.sub.12 cycloalkyl-,
(--(CH.sub.2).sub.1-6--O--(CH.sub.2).sub.1-6--).sub.1-12--,
(--(CH.sub.2).sub.1-4--NH--(CH.sub.2).sub.1-4).sub.1-12--,
--(CH.sub.2).sub.1-12--O--,
(--(CH.sub.2).sub.1-4--O--(CH.sub.2).sub.1-4).sub.1-12--O--,
--(CH.sub.2).sub.1-12--(CO)--O--,
--(CH.sub.2).sub.1-12--(CO)--NH--,
--(CH.sub.2).sub.1-12--O--(CO)--,
--(CH.sub.2).sub.1-12--NH--(CO)--,
(--(CH.sub.2).sub.1-4--O--(CH.sub.2).sub.1-4).sub.1-12--O--(CH.sub.2).sub-
.1-12--, --(CH.sub.2).sub.1-12--(CO)--O--(CH.sub.2).sub.1-12--,
--(CH.sub.2).sub.1-12--(CO)--NH--(CH.sub.2).sub.1-12--,
--(CH.sub.2).sub.1-12--O--(CO)--(CH.sub.2).sub.1-12--,
--(CH.sub.2).sub.1-12--NH--(CO)--(CH.sub.2).sub.1-12--,
--(C.sub.3-C.sub.12 cycloalkyl)-,
--(C.sub.1-C.sub.8alkyl)-(C.sub.3-C.sub.12 cycloalkyl)-,
--(C.sub.3-C.sub.12 cycloalkyl)-(C.sub.1-8alkyl)-,
--(C.sub.1-8alkyl)-(C.sub.3-C.sub.12 cycloalkyl)-(C.sub.1-8alkyl)-,
and --(CH.sub.2).sub.0-3-aryl-(CH.sub.2).sub.0-3--.
[0156] In still other embodiments, each of linkers L.sup.1 and
L.sup.2 is a cleavable linker independently selected from
hydrolyzable linkers, enzymatically cleavable linkers, pH sensitive
linkers, disulfide linkers and photolabile linkers.
[0157] Other linkers useful in the present invention include
self-immolative linkers. Useful self-immolative linkers are known
to one of skill in the art, such as those useful for antibody drug
conjugates. Exemplary self-immolative linkers are described in U.S.
Pat. No. 7,754,681.
[0158] E. Linking Groups LG
[0159] The linkers and functional agents of the present invention
can react with a linking group on the initiator fragment I or the
comonomers M.sup.2 to form a bond. The linking groups LG of the
present invention can be any suitable functional group capable of
forming a bond to another functional group, thereby linking the two
groups together. For example, linking groups LG useful in the
present invention include those used in click chemistry, maleimide
chemistry, and NHS-esters, among others. Linking groups involved in
click chemistry include, but are not limited to, azides and alkynes
that form a triazole ring via the Huisgen cycloaddition process
(see U.S. Pat. No. 7,375,234, incorporated herein in its entirety).
The maleimide chemistry involves reaction of the maleimide olefin
with a nucleophile, such as --OH, --SH or --NH.sub.2, to form a
stable bond. Other linking groups include those described in
Bioconjugate Techniques, Greg T. Hermanson, Academic Press, 2d ed.,
2008 (incorporated in its entirety herein).
[0160] Some non-limiting examples of the reaction of the linking
groups and some groups typically found or introduced into
functional agents are set forth in Table I.
TABLE-US-00001 TABLE I Illustrative Groups Exemplary Reactive that
may react with Linking Groups a linking group (LG) (shown as
appended to --X) Product Y--X Y--COOH HO--X Y--C(.dbd.O)O--X
(hydroxyl or activated forms thereof (e.g., tresylate, mesylate
etc.)) Y--COOH HS--X Y--C(.dbd.O)S--X Y--SH (thiol) Y--S--S--X
Y--SH R'--S--S--X Y--S--S--X (disulfide) Y--SH (pyridyl)-S--S--X
Y--S--S--X (dithiopyridyl) Y--NH.sub.2 H(O.dbd.)C--X Y--N.dbd.CH--X
aldehyde or Y--NH--CH.sub.2--X following reduction Y--NH.sub.2
(HO).sub.2HC--X Y--N.dbd.CH--X aldehyde hydrate or
Y--NH--CH.sub.2--X following reduction Y--NH.sub.2 ##STR00053##
Y--N.dbd.CH--X or Y--NH--CH--X following reduction Y--NH.sub.2
R'OCH(OH)--X or Y--N.dbd.CH--X hemiacetal or Y--NH--CH--X following
reduction Y--NH.sub.2 R'(O.dbd.)C--X Y--N.dbd.CR'--X ketone or
Y--NH--C(R')H--X following reduction Y--NH.sub.2 ##STR00054##
Y--N.dbd.C(R')--X or Y--NH--C(R')H--X following reduction
Y--NH.sub.2 R'OC(R')(OH)--X Y--N.dbd.C(R')--X hemiketal or
Y--NH--C(R')H--X following reduction Y--NH.sub.2 R'(S.dbd.)C--X
Y--N.dbd.C(R')--X ketone or thione (thioketone) Y--NH--C(R')H--X
following reduction Y--NH.sub.2 ##STR00055## Y--N.dbd.C(R')--X or
Y--NH--C(R')H--X following reduction Y--NH.sub.2 R'SC(R')(SH)--X or
Y--N.dbd.C(R')--X dithiohemiketal or Y--NH--C(R')H--X following
reduction Y--NH.sub.2 ##STR00056## Y--N.dbd.C(R')--X or
Y--NH--C(R')H--X following reduction Y--SH Y--OH Y--COOH (anion)
Y--NHR'' ##STR00057## Y--S--CH.sub.2--C(OH)(R'')--X--
Y--O--CH.sub.2--C(OH)(R'')--X--
Y--C(.dbd.O)O--CH.sub.2--C(OH)(R'')--X--
Y--NR''--CH.sub.2--C(OH)(R'')--X-- Y--SH Y--OH Y--COOH (anion)
Y--NHR'' ##STR00058## Y--S--CH.sub.2--C(OH)(R'')--X--
Y--O--CH.sub.2--C(OH)(R'')--X--
Y--C(.dbd.O)O--CH.sub.2--C(OH)(R'')--X--
Y--NR''--CH.sub.2--C(OH)(R'')--X-- Y--SH HO--(C.dbd.O)--X
Y--S--(C.dbd.O)--X Y--OH carboxyl Y--O--(C.dbd.O)--X Y--NHR''
Y--N(R'')--(C.dbd.O)--X Y--SH (alcohol)-(C.dbd.O)--X
Y--S--(C.dbd.O)--X Y--OH carboxylic acid ester Y--O--(C.dbd.O)--X
Y--NHR'' (alcohol indicates an esterified Y--N(R'')--(C.dbd.O)--X
suitable alcohol leaving group e.g., p-nitrophenyl) Y--NH.sub.2
##STR00059## Y--NH--R'''--X Y--SH ##STR00060## ##STR00061##
Y--NH.sub.2 ##STR00062## Y--NH--R'''--X Y--NH.sub.2
CH.sub.3((CH.sub.2).sub.1-3)--O(C.dbd.NH)--X Y--NH--(C.dbd.NH)--X
(imidoester) (amidine)
Y--(C.dbd.NH)--O--((CH.sub.2).sub.1-3)--CH.sub.3 H.sub.2N--X
Y--(C.dbd.NH)--HN--X (imidoester) (amidine) Y--COOH H.sub.2N--X
Y--(C.dbd.O)--NH--X Y--(C.dbd.O)--R'' amine Y--(R'')C.dbd.N--X or
Y--(R'')CH--NH--X following reduction Y--COOH
H.sub.2N--(C.dbd.O)--NH--X Y--(C.dbd.O)--NH--(C.dbd.O)--NH--X
Y--(C.dbd.O)--R'' urea Y--(R'')C.dbd.N--(C.dbd.O)--NH--X or
Y--(R'')CH--NH--(C.dbd.O)--NH--X following reduction Y--COOH
H.sub.2N--(C.dbd.O)--O--X Y--(C.dbd.O)--NH--(C.dbd.O)--O--X
Y--(C.dbd.O)--R'' carbamate Y--(R'')C.dbd.N--(C.dbd.O)--O--X or
Y--(R'')CH--NH--(C.dbd.O)--O--X following reduction Y--COOH
H.sub.2N--(C.dbd.S)--NH--X Y--(C.dbd.O)--NH--(C.dbd.S)--NH--X
Y--(C.dbd.O)--R'' thiourea Y--(R'')C.dbd.N--(C.dbd.S)--NH--X or
Y--(R'')CH--NH--(C.dbd.S)--NH--X following reduction Y--COOH
H.sub.2N--(C.dbd.S)--O--X Y--(C.dbd.O)--NH--(C.dbd.S)--O--X
Y--(C.dbd.O)--R'' thiocarbamate Y--(R'')C.dbd.N--(C.dbd.S)--O--X or
Y--(R'')CH--NH--(C.dbd.S)--O--X following reduction
Y--(C.dbd.O)--R'' H.sub.2N--HN--X Y--(R'')C.dbd.N--HN--X hydrazone
Y--NH--NH.sub.2 R''--(O.dbd.C)--X Y--NH--N.dbd.C(R'')--X hydrazone
Y--NH.sub.2 O.dbd.C.dbd.N--X Y--NH--(C.dbd.O)--NH--X Y--OH
isocyanate Y--O--(C.dbd.O)--NH--X Y--NH.sub.2 S.dbd.C.dbd.N--X
Y--NH--(C.dbd.S)--NH--X Y--OH isothiocyanate Y--O--(C.dbd.S)--NH--X
Y--SH H.sub.2C.dbd.CH--(C.dbd.O)--X
Y--S--CH.sub.2CH.sub.2--(C.dbd.O)--X or
Y--S--CH.sub.2--CH(CH.sub.3)--(C.dbd.O)--X
H.sub.2C.dbd.C(CH.sub.3)--(C.dbd.O)--X alpha-beta unsubstituted
carbonyls Y--SH H.sub.2C.dbd.CH--(C.dbd.O)O--X
Y--S--CH.sub.2CH.sub.2--(C.dbd.O)O--X alpha-beta unsubstituted
carboxyl Y--SH H.sub.2C.dbd.C(CH.sub.3)--(C.dbd.O)--O--X
Y--S--CH.sub.2CH(CH.sub.3)--(C.dbd.O)O--X alpha-beta unsubstituted
carboxyls (methacrylates) Y--SH H.sub.2C.dbd.CH--(C.dbd.O)NH--X
Y--S--CH.sub.2CH.sub.2--(C.dbd.O)NH--X alpha-beta unsubstituted
amides (acrylamides) Y--SH vinylpyridine-X
Y--S--CH.sub.2--CH.sub.2-(pyridyl)-X (2- or 4-vinylpyridine) Y--SH
H.sub.2C.dbd.CH--SO.sub.2--X Y--S--H.sub.2C--CH.sub.2--SO.sub.2--X
(vinyl sulfone) Y--SH ClH.sub.2C--CH.sub.2--SO.sub.2--L
Y--S--H.sub.2C--CH.sub.2--SO.sub.2--X (chloroethyl sulfone) Y--SH
(halogen)-CH.sub.2--(C.dbd.O)--O--X Y--S--CH.sub.2--(C.dbd.O)--O--X
(halogen)-CH.sub.2--(C.dbd.O)--NH--X
Y--S--CH.sub.2--(C.dbd.O)--NH--X (halogen)-CH.sub.2--(C.dbd.O)--X
Y--S--CH.sub.2--(C.dbd.O)--X (halogen is preferably I or Br)
Y--O(C.dbd.O)--CH.sub.2-(halogen) HS--X
Y--O(C.dbd.O)--CH.sub.2--S--X Y--NH(C.dbd.O)--CH.sub.2-(halogen)
Y--NH(C.dbd.O)--CH.sub.2--S--X Y--(C.dbd.O)--CH.sub.2-(halogen)
Y--(C.dbd.O)--CH.sub.2--S--X (halogen is preferably I or Br) Y--SH
(halogen)-CH.sub.2(C.dbd.O)O--X Y--S--CH.sub.2(C.dbd.O)O--X
(halogen)-CH.sub.2(C.dbd.O)NH--X Y--S--CH.sub.2(C.dbd.O)NH--X
(halogen)-CH.sub.2(C.dbd.O)--X Y--S--CH.sub.2(C.dbd.O)--X (halogen
is preferably I or Br) Y--N.sub.3 HC.ident.C--X ##STR00063##
Y--N.sub.3 ##STR00064## ##STR00065## Y--N.sub.3 ##STR00066##
##STR00067## Y--SH ##STR00068## ##STR00069## Y--NH.sub.2
(F.sub.5--Ph)--OC(O)--X Y--NH--C(O)--X
R' is C.sub.1-6 alkyl, C.sub.3-6 cycloalkyl, or an aryl group
having 5-8 endocyclic atoms; R'' is H, C.sub.1-6 alkyl, C.sub.3-6
cycloalkyl, or an aryl group having 5-8 endocyclic atoms; R''' is a
carbonyl derivative *--(C.dbd.O)--,
*--(C.dbd.O)--(CH.sub.2).sub.1-8--S--S--,
*--(C.dbd.O)--(CH.sub.2).sub.1-8--(C.dbd.O)--O--,
*--(C.dbd.O)--(CH.sub.2).sub.1-8--O--(C.dbd.O)--,
*--(C.dbd.O)--(CH.sub.2).sub.1-8--(C.dbd.O)--NH--, or
*--(C.dbd.O)--(CH.sub.2).sub.1-8--NH--(C.dbd.O)--, or
alternatively, R'' is carbonyl derivative of the form
*--(C.dbd.O)--O--(CH.sub.2).sub.1-8--S--S--,
*--(C.dbd.O)--O--(CH.sub.2).sub.1-8--(C.dbd.O)--O--,
*-(C.dbd.O)--O--(CH.sub.2).sub.1-8--O--(C.dbd.O)--,
*--(C.dbd.O)--O--(CH.sub.2).sub.1-8--(C.dbd.O)--NH--, or
*--(C.dbd.O)--O--(CH.sub.2).sub.1-8--NH--(C.dbd.O)--, where "*"
indicates the point of attachment to succinimidyl or benzotriazolyl
groups; X and Y are each the active agent, linker, monomer or
initiator fragment I. --C(O)NR.sup.1aR.sup.1b, --NR.sup.1aR.sup.1b,
C.sub.1-6 alkyl-NR.sup.1aR.sup.1b, --N(R.sup.1a)C(O)R.sup.1b,
--N(R.sup.1a)C(O)OR.sup.1b, --N(R.sup.1a)C(O)NR.sup.1aR.sup.1b,
--OP(O)(OR.sup.1a).sub.2, --S(O).sub.2OR.sup.1a,
--S(O).sub.2NR.sup.1aR.sup.1b, --CN, --NO.sub.2, cycloalkyl,
heterocycloalkyl, aryl and heteroaryl.
[0161] F. Functional Agents
[0162] Functional agents useful in the random copolymers of the
present invention include any biological agent or synthetic
compound capable of targeting a particular ligand, receptor,
complex, organelle, cell, tissue, epithelial sheet, or organ, or of
treating a particular condition or disease state. Of particular
interest, is a combination of bioactive agents that together target
mechanisms common to a particular disease. For example, a first
bioactive agent (stably attached) that is a biopharmaceutical agent
that binds to a protein upregulated in a disease; a second
bioactive agent (stably attached) that is a peptide that binds to
an extracellular matrix tissue constituent such as heparin sulfate;
a third bioactive agent (unstably attached) that is a small
molecule drug that releases over time and exerts a local,
intracellular effect, for example, an anti-proliferative effect. In
some embodiments, the bioactive agent is a drug, a therapeutic
protein, a small molecule, a peptide, a peptoid, an oligonucleotide
(aptamer, siRNA, microRNA), a nanoparticle, a carbohydrate, a
lipid, a glycolipid, a phospholipid, or a targeting agent. The
ratio of comonomers is chosen based on predefined stoichiometry
(for example, to match a biological avidity; to match a biological
stoichiometry; to impart a `gearing` effect). Other functional
agents useful in the random copolymers of the present invention
include, but are not limited to, radiolabels, fluorophores and
dyes.
[0163] The functional agents can be linked to the initiator
fragment I or the comonomers M.sup.2, or both, of the random
copolymers. The functional agents can be linked to the initiator
fragment I or the comonomers M.sup.2 either before or after
polymerization via cleavable, non-cleavable, or self-immolative
linkers described above. The functional agent can also be
physisorbed or ionically absorbed to the random copolymer instead
of covalently attached.
[0164] The preparation of the random copolymers of the present
invention linked to a functional agent can be conducted by first
linking the functional agent to a linking group attached to a
monomer and subjecting the coupled functional agent to conditions
suitable for synthesis of the inventive random copolymers. In those
cases, a suitable linking group can be an initiator (e.g.,
iodinated, brominated or chlorinated compound/group) for use in
ATRP reactions. Such a reaction scheme is possible where the
functional agent is compatible with the polymer polymerization
reactions and any subsequent workup required. However, coupling of
functional agents to preformed random copolymers can be used where
the functional agent is not compatible with conditions suitable for
polymerization. In addition, where cost makes the loss of an agent
to imperfect synthetic yields, oftentimes encountered particularly
in multistep synthetic reactions, coupling of functional agent to
preformed random copolymers of the present invention can be
employed.
[0165] Where a functional agent is not compatible with the
conditions employed for polymerization reactions, it can be
desirable to introduce the functional agent subsequent to the
polymerization reaction.
[0166] Bioactive agents, A, can be broadly selected. In some
embodiments the bioactive agents can be selected from one or more
drugs, vaccines, aptamers, avimer scaffolds based on human A domain
scaffolds, diabodies, camelids, shark IgNAR antibodies, fibronectin
type III scaffolds with modified specificities, antibodies,
antibody fragments, vitamins and cofactors, polysaccharides,
carbohydrates, steroids, lipids, fats, proteins, peptides,
polypeptides, nucleotides, oligonucleotides, polynucleotides, and
nucleic acids (e.g., mRNA, tRNA, snRNA, RNAi, microRNA, DNA, cDNA,
antisense constructs, ribozymes, etc., and combinations thereof).
In one embodiment, the bioactive agents can be selected from
proteins, peptides, polypeptides, soluble or cell-bound,
extracellular or intracellular, kinesins, molecular motors,
enzymes, extracellular matrix materials and combinations thereof.
In another embodiment, bioactive agents can be selected from
nucleotides, oligonucleotides, polynucleotides, and nucleic acids
(e.g., mRNA, tRNA, snRNA, RNAi, DNA, cDNA, antisense constructs,
ribozymes, etc., and combinations thereof). In another embodiment,
bioactive agents can be selected from steroids, lipids, fats and
combinations thereof. For example, the bioactive agent can bind to
the extracellular matrix, such as when the extracellular matrix is
hyaluronic acid or heparin sulfate proteoglycan and the bioactive
agent is a positively charged moiety such as choline for
non-specific, electrostatic, Velcro type binding interactions. In
another embodiment, the bioactive agent can be a peptide sequence
that binds non-specifically or specifically.
[0167] Bioactive agents can be designed and/or selected to have a
full activity (such as a high level of agonism or antagonism).
Alternatively, a multifunctional bioactive agent can be selected to
modulate one target protein's activity while impacting fully
another.
[0168] Just as mosaic proteins contain extracellular binding
domains or sub-domains (example, VEGF and Heparin Binding Epidermal
Growth Factor), sequences from these binding sites can be
replicated as a bioactive agent for polymer attachment. More
broadly, mosaic proteins represent strings of domains of many
functions (target binding, extracellular matrix binding, spacers,
avidity increases, enzymatic). The set of bioactives chosen for a
particular application can be assembled in similar fashion to
replicate a set of desired functional activities.
[0169] Other functional agents, A, include charged species such as
choline, lysine, aspartic acid, glutamic acid, and hyaluronic acid,
among others. The charged species are useful for facilitating ionic
attachment, to vitreous for example.
Therapeutic Proteins and Antibodies
[0170] In one particularly useful embodiment, the functional agent
is a therapeutic protein. Numerous therapeutic proteins are
disclosed throughout the application such as, and without
limitation, erythropoietin, granulocyte colony stimulating factor
(G-CSF), GM-CSF, interferon alpha, interferon beta, human growth
hormone, and imiglucerase.
[0171] In one embodiment, the functional agents can be selected
from specifically identified polysaccharide, protein or peptide
bioactive agents, including, but not limited to: A.beta.,
agalsidase, alefacept, alkaline phosphatase, aspariginase,
amdoxovir (DAPD), antide, becaplermin, botulinum toxin including
types A and B and lower molecular weight compounds with botulinum
toxin activity, calcitonins, cyanovirin, denileukin diftitox,
erythropoietin (EPO), EPO agonists, dornase alpha, erythropoiesis
stimulating protein (NESP), coagulation factors such as Factor V,
Factor VII, Factor VIIa, Factor VIII, Factor IX, Factor X, Factor
XII, Factor XIII, von Willebrand factor; ceredase, cerezyme,
alpha-glucosidase, N-Acetylgalactosamine-6-sulfate sulfatase,
collagen, cyclosporin, alpha defensins, beta defensins,
desmopressin, exendin-4, cytokines, cytokine receptors, granulocyte
colony stimulating factor (G-CSF), thrombopoietin (TPO), alpha-1
proteinase inhibitor, elcatonin, granulocyte macrophage colony
stimulating factor (GM-CSF), fibrinogen, filgrastim, growth
hormones human growth hormone (hGH), somatropin, growth hormone
releasing hormone (GHRH), GRO-beta, GRO-beta antibody, bone
morphogenic proteins such as bone morphogenic protein-2, bone
morphogenic protein-6, parathyroid hormone, parathyroid hormone
related peptide, OP-1; acidic fibroblast growth factor, basic
fibroblast growth factor, Fibroblast Growth Factor 21, CD-40
ligand, heparin, human serum albumin, low molecular weight heparin
(LMWH), interferon alpha, interferon beta, interferon gamma,
interferon omega, interferon tau, consensus interferon, human lysyl
oxidase-like-2 (LOXL2); interleukins and interleukin receptors such
as interleukin-1 receptor, interleukin-2, interleukin-2 fusion
proteins, interleukin-1 receptor antagonist, interleukin-3,
interleukin-4, interleukin-4 receptor, interleukin-6,
interleukin-8, interleukin-12, interleukin-17, interleukin-21,
interleukin-23, p40, interleukin-13 receptor, interleukin-17
receptor; lactoferrin and lactoferrin fragments, luteinizing
hormone releasing hormone (LHRH), insulin, pro-insulin, insulin
analogues, leptin, ghrelin, amylin, C-peptide, somatostatin,
somatostatin analogs including octreotide, vasopressin, follicle
stimulating hormone (FSH), imiglucerase, influenza vaccine,
insulin-like growth factor (IGF), insulintropin, macrophage colony
stimulating factor (M-CSF), plasminogen activators such as
alteplase, urokinase, reteplase, streptokinase, pamiteplase,
lanoteplase, and teneteplase; nerve growth factor (NGF),
osteoprotegerin, platelet-derived growth factor, tissue growth
factors, transforming growth factor-1, vascular endothelial growth
factor, leukemia inhibiting factor, keratinocyte growth factor
(KGF), glial growth factor (GGF), T Cell receptors, CD
molecules/antigens, tumor necrosis factor (TNF) (e.g., TNF-.alpha.
and TNF-.beta.), TNF receptors (e.g., TNF-.alpha. receptor and
TNF-.beta. receptor), CTLA4, CTLA4 receptor, monocyte
chemoattractant protein-1, endothelial growth factors, parathyroid
hormone (PTH), glucagon-like peptide, somatotropin, thymosin alpha
1, rasburicase, thymosin alpha 1 IIb/IIIa inhibitor, thymosin beta
10, thymosin beta 9, thymosin beta 4, alpha-1 antitrypsin,
phosphodiesterase (PDE) compounds, VLA-4 (very late antigen-4),
VLA-4 inhibitors, bisphosphonates, respiratory syncytial virus
antibody, cystic fibrosis transmembrane regulator (CFTR) gene,
deoxyribonuclease (Dnase), bactericidal/permeability increasing
protein (BPI), and anti-CMV antibody. Exemplary monoclonal
antibodies include etanercept (a dimeric fusion protein consisting
of the extracellular ligand-binding portion of the human 75 kD TNF
receptor linked to the Fc portion of IgG1), abciximab, adalimumab,
afelimomab, alemtuzumab, antibody to B-lymphocyte, atlizumab,
basiliximab, bevacizumab, biciromab, bertilimumab, CDP-484,
CDP-571, CDP-791, CDP-860, CDP-870, cetuximab, clenoliximab,
daclizumab, eculizumab, edrecolomab, efalizumab, epratuzumab,
fontolizumab, gavilimomab, gemtuzumab ozogamicin, ibritumomab
tiuxetan, infliximab, inolimomab, keliximab, labetuzumab,
lerdelimumab, olizumab, radiolabeled lym-1, metelimumab,
mepolizumab, mitumomab, muromonad-CD3, nebacumab, natalizumab,
odulimomab, omalizumab, oregovomab, palivizumab, pemtumomab,
pexelizumab, rhuMAb-VEGF, rituximab, satumomab pendetide,
sevirumab, siplizumab, tositumomab, I.sup.131tositumomab,
trastuzumab, tuvirumab, visilizumab, and fragments and mimetics
thereof. Functional agents also include agents which bind to these
specifically identified polysaccharide, protein or peptide
bioactive agents.
[0172] In one embodiment, the bioactive agent is a fusion protein.
For example, and without limitation, the bioactive component can be
an immunoglobulin or portion of an immunoglobulin fused to one or
more certain useful peptide sequences. For example, the bioactive
agent may contain an antibody Fc fragment. In one embodiment, the
bioactive agent is a CTLA4 fusion protein. For example, the
bioactive agent can be an Fc-CTLA4 fusion protein. In another
embodiment, the bioactive agent is a Factor VIII fusion protein.
For example, the bioactive agent can be an Fc-Factor VIII fusion
protein.
[0173] In one particularly useful embodiment, the bioactive agent
is a human protein or human polypeptide, for example, a
heterologously produced human protein or human polypeptide.
Numerous proteins and polypeptides are disclosed herein for which
there is a corresponding human form (i.e., the protein or peptide
is normally produced in human cells in the human body). Therefore,
in one embodiment, the bioactive agent is the human form of each of
the proteins and polypeptides disclosed herein for which there is a
human form. Examples of such human proteins include, without
limitation, human antibodies, human enzymes, human hormones and
human cytokines such as granulocyte colony stimulation factor,
granulocyte macrophage colony stimulation factor, interferons
(e.g., alpha interferons and beta interferons), human growth
hormone and erythropoietin.
[0174] Other examples of therapeutic proteins which may serve as
bioactive agents include, without limitation, factor VIII, b-domain
deleted factor VIII, factor VIIa, factor IX, anticoagulants;
hirudin, alteplase, tpa, reteplase, tpa, tpa--3 of 5 domains
deleted, insulin, insulin lispro, insulin aspart, insulin glargine,
long-acting insulin analogs, hgh, glucagons, tsh, follitropin-beta,
fsh, gm-csf, pdgh, ifn alpha2, ifn alpha2a, ifn alpha2b, inf-apha1,
consensus ifn, ifn-beta, ifn-beta 1b, ifn-beta 1a, ifn-gamma (e.g.,
1 and 2), ifn-lambda, ifn-delta, il-2, il-11, hbsag, ospa, murine
mab directed against t-lymphocyte antigen, murine mab directed
against tag-72, tumor-associated glycoprotein, fab fragments
derived from chimeric mab directed against platelet surface
receptor gpII(b)/III(a), murine mab fragment directed against
tumor-associated antigen cal 25, murine mab fragment directed
against human carcinoembryonic antigen, cea, murine mab fragment
directed against human cardiac myosin, murine mab fragment directed
against tumor surface antigen psma, murine mab fragments (fab/fab2
mix) directed against hmw-maa, murine mab fragment (fab) directed
against carcinoma-associated antigen, mab fragments (fab) directed
against nca 90, a surface granulocyte nonspecific cross reacting
antigen, chimeric mab directed against cd20 antigen found on
surface of b lymphocytes, humanized mab directed against the alpha
chain of the il2 receptor, chimeric mab directed against the alpha
chain of the il2 receptor, chimeric mab directed against tnf-alpha,
humanized mab directed against an epitope on the surface of
respiratory synctial virus, humanized mab directed against her 2,
human epidermal growth factor receptor 2, human mab directed
against cytokeratin tumor-associated antigen anti-ctla4, chimeric
mab directed against cd 20 surface antigen of b lymphocytes
dornase-alpha dnase, beta glucocerebrosidase, tnf-alpha,
il-2-diptheria toxin fusion protein, tnfr-lgg fragment fusion
protein laronidase, dnaases, alefacept, darbepoetin alpha (colony
stimulating factor), tositumomab, murine mab, alemtuzumab,
rasburicase, agalsidase beta, teriparatide, parathyroid hormone
derivatives, adalimumab (lgg1), anakinra, biological modifier,
nesiritide, human b-type natriuretic peptide (hbnp), colony
stimulating factors, pegvisomant, human growth hormone receptor
antagonist, recombinant activated protein c, omalizumab,
immunoglobulin e (lge) blocker, lbritumomab tiuxetan, ACTH,
glucagon, somatostatin, somatotropin, thymosin, parathyroid
hormone, pigmentary hormones, somatomedin, erythropoietin,
luteinizing hormone, chorionic gonadotropin, hypothalmic releasing
factors, etanercept, antidiuretic hormones, prolactin and thyroid
stimulating hormone. And any of these can be modified to have a
site-specific conjugation point (a N-terminus, or C-terminus, or
other location) using natural (for example, a serine to cysteine
substitution) (for example, formylaldehyde per method of Redwood
Biosciences) or non-natural amino acid.
[0175] Examples of therapeutic antibodies (or their respective scFv
or Fab fragments) that may serve as bioactive agents include, but
are not limited, to HERCEPTIN.TM. (Trastuzumab) (Genentech, CA)
which is a humanized anti-HER2 monoclonal antibody for the
treatment of patients with metastatic breast cancer; REOPRO.TM.
(abciximab) (Centocor) which is an anti-glycoprotein IIb/IIIc
receptor on the platelets for the prevention of clot formation;
ZENAPAX.TM. (daclizumab) (Roche Pharmaceuticals, Switzerland) which
is an immunosuppressive, humanized anti-CD25 monoclonal antibody
for the prevention of acute renal allograft rejection; PANOREX.TM.
which is a murine anti-17-IA cell surface antigen IgG2a antibody
(Glaxo Wellcome/Centocor); BEC2 which is a murine anti-idiotype
(GD3 epitope) IgG antibody (ImClone System); IMC-C225 which is a
chimeric anti-EGFR IgG antibody (ImClone System); VITAXIN.TM. which
is a humanized anti-.alpha.V.beta.3 integrin antibody (Applied
Molecular Evolution/MedImmune); Campath; Campath 1H/LDP-03 which is
a humanized anti CD52 IgG1 antibody (Leukosite); Smart M195 which
is a humanized anti-CD33 IgG antibody (Protein Design Lab/Kanebo);
RITUXAN.TM. which is a chimeric anti-CD2O IgG1 antibody (IDEC
Pharm/Genentech, Roche/Zettyaku); LYMPHOCIDE.TM. which is a
humanized anti-CD22 IgG antibody (Immunomedics); ICM3 is a
humanized anti-ICAM3 antibody (ICOS Pharm); IDEC-114 is a primate
anti-CD80 antibody (IDEC Pharm/Mitsubishi); ZEVALIN.TM. is a
radiolabelled murine anti-CD20 antibody (IDEC/Schering AG);
IDEC-131 is a humanized anti-CD40L antibody (IDEC/Eisai); IDEC-151
is a primatized anti-CD4 antibody (IDEC); IDEC-152 is a primatized
anti-CD23 antibody (IDEC/Seikagaku); SMART anti-CD3 is a humanized
anti-CD3 IgG (Protein Design Lab); 5G1.1 is a humanized
anti-complement factor 5 (CS) antibody (Alexion Pharm); D2E7 is a
humanized anti-TNF-.alpha. antibody (CATIBASF); CDP870 is a
humanized anti-TNF-.alpha. Fab fragment (Celltech); IDEC-151 is a
primatized anti-CD4 IgG1 antibody (IDEC Pharm/SmithKline Beecham);
MDX-CD4 is a human anti-CD4 IgG antibody (Medarex/Eisai/Genmab);
CDP571 is a humanized anti-TNF-.alpha. IgG4 antibody (Celltech);
LDP-02 is a humanized anti-.alpha.4.beta.7 antibody
(LeukoSite/Genentech); OrthoClone OKT4A is a humanized anti-CD4 IgG
antibody (Ortho Biotech); ANTOVA.TM. is a humanized anti-CD40L IgG
antibody (Biogen); ANTEGREN.TM. is a humanized anti-VLA-4 IgG
antibody (Elan); CAT-152, a human anti-TGF-.beta..sub.2 antibody
(Cambridge Ab Tech); Cetuximab (BMS) is a monoclonal anti-EGF
receptor (EGFr) antibody; Bevacizuma (Genentech) is an anti-VEGF
human monoclonal antibody; Infliximab (Centocore, JJ) is a chimeric
(mouse and human) monoclonal antibody used to treat autoimmune
disorders; Gemtuzumab ozogamicin (Wyeth) is a monoclonal antibody
used for chemotherapy; and Ranibizumab (Genentech) is a chimeric
(mouse and human) monoclonal antibody used to treat macular
degeneration.
[0176] The spectrum of existing approaches to creating antibody
drug conjugates depends on conjugation of single toxin-like
molecules together with a linker to an antibody generally at one to
eight sites. The approach outlined in this invention involves
attachment of a random copolymer to the immunoglobulin via a
cleavable or non-cleavable linkage chemistry. The copolymer is
designed to have multiple copies of the small molecule bioactive
moiety attached stoichiometrically via cleavable (including
self-immolative) or non-cleavable linkage chemistry. Because of the
additional flexibility inherent in the invention, more than one
type of bioactive moiety and many more copies of each bioactive
moiety can be included via conjugation or attachment to polymer
comonomers, for example 10, 20, 50, 100, 250, or 500. The ability
to include many more allows one to broaden the perspective of
antibody drug conjugates beyond toxins to include other small
molecule drugs with synergistic biologies (non-limiting examples
include panitumumab and has inhibitors; adalimumab and p38 or JAK
inhibitors; bevacizumab and cMet inhibitors). The result is
targeted distribution, focal delivery, tailored drug release
kinetics which decrease off-target effects. Furthermore, the
attachment of small molecule bioactives to the polymer backbone
comonomers rescues bioactives with poor oral absorption,
distribution, metabolism and/or elimination or other liabilities.
All in, the result is a step change in efficacy due to
multifunctionality, lower Cmax, increased drug loading, plus other
benefits. Importantly, this approach to create combination
therapeutics with the different bioactive agents attached to a
common polymer core or scaffold results in local tissue therapeutic
effects that can be synergistic and that can substantially increase
efficacy while decreasing toxicity.
[0177] The antibodies of the present invention can also be linked
to a therapeutic agent described within or known in the art to form
an antibody drug conjugate (ADC). Targeted therapeutics provide
several advantages over existing technologies, including reducing
nonspecific toxicities and increasing efficacy. The targeting
properties of antibodies, such as monoclonal antibodies (mABs) and
mAB fragments (scFv and FAb'), enable delivery of a potent
therapeutic agent that is coupled to the mAB. The therapeutic agent
can be any useful drug, protein, peptide, or radionuclide. Antibody
drug conjugates useful in combination with the random copolymers of
the present invention are described in, for example, U.S. Pat. Nos.
7,745,394 (Seattle Genetics), 7,695,716 (Seattle Genetics),
7,662,387 (Seattle Genetics), 7,514,080 (ImmunoGen), 7,491,390
(Seattle Genetics), 7,501,120 (ImmunoGen), 7,494,649 (ImmunoGen),
and 7,374,762 (ImmunoGen).
Proteins, Peptides and Amino Acids
[0178] Proteins and peptides for use as bioactive agents as
disclosed herein can be produced by any useful method including
production by in vitro synthesis and by production in biological
systems. Typical examples of in vitro synthesis methods which are
well known in the art include solid-phase synthesis ("SPPS") and
solid-phase fragment condensation ("SPFC"). Biological systems used
for the production of proteins are also well known in the art.
Bacteria (e.g., E. coli and Bacillus sp.) and yeast (e.g.,
Saccharomyces cerevisiae and Pichia pastoris) are widely used for
the production of heterologous proteins. In addition, heterologous
gene expression for the production of bioactive agents for use as
disclosed herein can be accomplished using animal cell lines such
as mammalian cell lines (e.g., CHO cells). In one particularly
useful embodiment, the bioactive agents are produced in transgenic
or cloned animals such as cows, sheep, goats and birds (e.g.,
chicken, quail, ducks and turkey), each as is understood in the
art. See, for example, U.S. Pat. No. 6,781,030, issued Aug. 24,
2004, the disclosure of which is incorporated in its entirety
herein by reference.
[0179] Bioactive agents such as proteins produced in domesticated
birds such as chickens can be referred to as "avian derived"
bioactive agents (e.g., avian derived therapeutic proteins).
Production of avian derived therapeutic proteins is known in the
art and is described in, for example, U.S. Pat. No. 6,730,822,
issued May 4, 2004, the disclosure of which is incorporated in its
entirety herein by reference.
[0180] In embodiments where the bioactive agent is a protein or
polypeptide, functional groups present in the amino acids of the
protein polypeptide sequence can be used to link the agent to the
random copolymer. Linkages to protein or polypeptide bioactive
agents can be made to naturally occurring amino acids in their
sequence or to naturally occurring amino acids that have either
been added to the sequence or inserted in place of another amino
acid, for example the replacement of a serine by a cysteine.
[0181] Protein or polypeptide bioactive agents may also comprise
non-naturally occurring amino acids in addition to the common
naturally occurring amino acids found in proteins and polypeptides.
In addition to being present for the purpose of altering the
properties of a polypeptide or protein, non-naturally occurring
amino acids can be introduced to provide a functional group that
can be used to link the protein or polypeptide directly to the
random copolymer. Furthermore, naturally occurring amino acids,
e.g., cysteine, tyrosine, tryptophan can be used in this way.
[0182] Non-naturally occurring amino acids can be introduced into
proteins and peptides by a variety of means. Some of the techniques
for the introduction of non-natural amino acids are discussed in
U.S. Pat. No. 5,162,218, the disclosure of which is incorporated in
its entirety herein by reference. First, non-naturally occurring
amino acids can be introduced by chemical modification of a
polypeptide or protein on the amino acid side chain or at either
the amino terminus or the carboxyl terminus. Non-limiting examples
of chemical modification of a protein or peptide might be
methylation by agents such as diazomethane, or the introduction of
acetylation at an amino group present in lysine's side chain or at
the amino terminus of a peptide or protein. Another example of the
protein/polypeptide amino group modification to prepare a
non-natural amino acid is the use of methyl
3-mercaptopropionimidate ester or 2-iminothiolane to introduce a
thiol (sulfhydryl, --SH) bearing functionality linked to positions
in a protein or polypeptide bearing a primary amine. Once
introduced, such groups can be employed to form a covalent linkage
to the protein or polypeptide.
[0183] Second, non-naturally occurring amino acids can be
introduced into proteins and polypeptides during chemical
synthesis. Synthetic methods are typically utilized for preparing
polypeptides having fewer than about 200 amino acids, usually
having fewer than about 150 amino acids, and more usually having
100 or fewer amino acids. Shorter proteins or polypeptides having
less than about 75 or less than about 50 amino acids can be
prepared by chemical synthesis.
[0184] The synthetic preparation methods that are particularly
convenient for allowing the insertion of non-natural amino acids at
a desired location are known in the art. Suitable synthetic
polypeptide preparation methods can be based on Merrifield
solid-phase synthesis methods where amino acids are sequentially
added to a growing chain (Merrifield (1963) J. Am. Chem. Soc.
85:2149-2156). Automated systems for synthesizing polypeptides by
such techniques are now commercially available from suppliers such
as Applied Biosystems, Inc., Foster City, Calif. 94404; New
Brunswick Scientific, Edison, N.J. 08818; and Pharmacia, Inc.,
Biotechnology Group, Piscataway, N.J. 08854.
[0185] Examples of non-naturally occurring amino acids that can be
introduced during chemical synthesis of polypeptides include, but
are not limited to: D-amino acids and mixtures of D and L-forms of
the 20 naturally occurring amino acids, N-formyl glycine,
ornithine, norleucine, hydroxyproline, beta-alanine, hydroxyvaline,
norvaline, phenylglycine, cyclohexylalanine, t-butylglycine
(t-leucine, 2-amino-3,3-dimethylbutanoic acid),
hydroxy-t-butylglycine, amino butyric acid, cycloleucine,
4-hydroxyproline, pyroglutamic acid (5-oxoproline), azetidine
carboxylic acid, pipecolinic acid, indoline-2-carboxylic acid,
tetrahydro-3-isoquinoline carboxylic acid, 2,4-diaminobutyricacid,
2,6-diaminopimelic acid, 2,4-diaminobutyricacid,
2,6-diaminopimelicacid, 2,3-diaminopropionicacid, 5-hydroxylysine,
neuraminic acid, and 3,5-diiodotyrosine.
[0186] Third, non-naturally occurring amino acids can be introduced
through biological synthesis in vivo or in vitro by insertion of a
non-sense codon (e.g., an amber or ocher codon) in a DNA sequence
(e.g., the gene) encoding the polypeptide at the codon
corresponding to the position where the non-natural amino acid is
to be inserted. Such techniques are discussed for example in U.S.
Pat. Nos. 5,162,218 and 6,964,859, the disclosures of which are
incorporated in their entirety herein by reference. A variety of
methods can be used to insert the mutant codon including
oligonucleotide-directed mutagenesis. The altered sequence is
subsequently transcribed and translated, in vivo or in vitro in a
system which provides a suppressor tRNA, directed against the
nonsense codon that has been chemically or enzymatically acylated
with the desired non-naturally occurring amino acid. The synthetic
amino acid will be inserted at the location corresponding to the
nonsense codon. For the preparation of larger and/or glycosylated
polypeptides, recombinant preparation techniques of this type are
usually preferred. Among the amino acids that can be introduced in
this fashion are: formyl glycine, fluoroalanine,
2-Amino-3-mercapto-3-methylbutanoic acid, homocysteine,
homoarginine and the like. Other similar approaches to obtain
non-natural amino acids in a protein include methionine
substitution methods.
[0187] Where non-naturally occurring amino acids have a
functionality that is susceptible to selective modification, they
are particularly useful for forming a covalent linkage to the
protein or polypeptide. Circumstances where a functionality is
susceptible to selective modification include those where the
functionality is unique or where other functionalities that might
react under the conditions of interest are hindered either
stereochemically or otherwise.
[0188] Other antibodies, such as single domain antibodies are
useful in the present invention. A single domain antibody (sdAb,
called Nanobody by Ablynx) is an antibody fragment consisting of a
single monomeric variable antibody domain. Like a whole antibody,
the sdAb is able to bind selectively to a specific antigen. With a
molecular weight of only 12-15 kDa, single domain antibodies are
much smaller than common whole antibodies (150-160 kDa). A single
domain antibody is a peptide chain of about 110 amino acids in
length, comprising one variable domain (VH) of a heavy chain
antibody, or of a common IgG.
[0189] Unlike whole antibodies, sdAbs do not show complement system
triggered cytotoxicity because they lack an Fc region. Camelid and
fish derived sdAbs are able to bind to hidden antigens that are not
accessible to whole antibodies, for example to the active sites of
enzymes.
[0190] A single domain antibody (sdAb) can be obtained by
immunization of dromedaries, camels, llamas, alpacas or sharks with
the desired antigen and subsequent isolation of the mRNA coding for
heavy chain antibodies. Alternatively they can be made by screening
synthetic libraries. Camelids are members of the biological family
Camelidae, the only living family in the suborder Tylopoda. Camels,
dromedaries, Bactrian Camels, llamas, alpacas, vicunas, and
guanacos are in this group.
[0191] Peptides useful in the present invention also include, but
are not limited to, a macrocyclic peptide, a cyclotide, an LDL
receptor A-domain, a protein scaffold (as discussed in U.S. Patent
No. 60/514,391, incorporated in its entirety herein), a soluble
receptor, an enzyme, a peptide multimer, a domain multimer, an
antibody fragment multimer, and a fusion protein.
Drugs
[0192] In another embodiment, the bioactive agents can also be
selected from specifically identified drug or therapeutic agents,
including but not limited to: tacrine, memantine, rivastigmine,
galantamine, donepezil, levetiracetam, repaglinide, atorvastatin,
alefacept, tadalafil, vardenafil, sildenafil, fosamprenavir,
oseltamivir, valacyclovir and valganciclovir, abarelix, adefovir,
alfuzosin, alosetron, amifostine, amiodarone, aminocaproic acid,
aminohippurate sodium, aminoglutethimide, aminolevulinic acid,
aminosalicylic acid, amlodipine, amsacrine, anagrelide,
anastrozole, aprepitant, aripiprazole, asparaginase, atazanavir,
atomoxetine, anthracyclines, bexarotene, bicalutamide, bleomycin,
bortezomib, buserelin, busulfan, cabergoline, capecitabine,
carboplatin, carmustine, chlorambucin, cilastatin sodium,
cisplatin, cladribine, clodronate, cyclophosphamide, cyproterone,
cytarabine, camptothecins, 13-cis retinoic acid, all trans retinoic
acid; dacarbazine, dactinomycin, daptomycin, daunorubicin,
deferoxamine, dexamethasone, diclofenac, diethylstilbestrol,
docetaxel, doxorubicin, dutasteride, eletriptan, emtricitabine,
enfuvirtide, eplerenone, epirubicin, estramustine, ethinyl
estradiol, etoposide, exemestane, ezetimibe, fentanyl,
fexofenadine, fludarabine, fludrocortisone, fluorouracil,
fluoxymesterone, flutamide, fluticazone, fondaparinux, fulvestrant,
gamma-hydroxybutyrate, gefitinib, gemcitabine, epinephrine, L-Dopa,
hydroxyurea, icodextrin, idarubicin, ifosfamide, imatinib,
irinotecan, itraconazole, goserelin, laronidase, lansoprazole,
letrozole, leucovorin, levamisole, lisinopril, lovothyroxine
sodium, lomustine, mechlorethamine, medroxyprogesterone, megestrol,
melphalan, memantine, mercaptopurine, mequinol, metaraminol
bitartrate, methotrexate, metoclopramide, mexiletine, miglustat,
mitomycin, mitotane, mitoxantrone, modafinil, naloxone, naproxen,
nevirapine, nicotine, nilutamide, nitazoxanide, nitisinone,
norethindrone, octreotide, oxaliplatin, palonosetron, pamidronate,
pemetrexed, pergolide, pentostatin, pilcamycin, porfimer,
prednisone, procarbazine, prochlorperazine, ondansetron,
palonosetron, oxaliplatin, raltitrexed, rosuvastatin, sirolimus,
streptozocin, pimecrolimus, sertaconazole, tacrolimus, tamoxifen,
tegaserod, temozolomide, teniposide, testosterone,
tetrahydrocannabinol, thalidomide, thioguanine, thiotepa,
tiotropium, topiramate, topotecan, treprostinil, tretinoin,
valdecoxib, celecoxib, rofecoxib, valrubicin, vinblastine,
vincristine, vindesine, vinorelbine, voriconazole, dolasetron,
granisetron, formoterol, fluticazone, leuprolide, midazolam,
alprazolam, amphotericin B, podophylotoxins, nucleoside antivirals,
aroyl hydrazones, sumatriptan, eletriptan; macrolides such as
erythromycin, oleandomycin, troleandomycin, roxithromycin,
clarithromycin, davercin, azithromycin, flurithromycin,
dirithromycin, josamycin, spiromycin, midecamycin, loratadine,
desloratadine, leucomycin, miocamycin, rokitamycin,
andazithromycin, and swinolide A; fluoroquinolones such as
ciprofloxacin, ofloxacin, levofloxacin, trovafloxacin,
alatrofloxacin, moxifloxicin, norfloxacin, enoxacin, gatifloxacin,
gemifloxacin, grepafloxacin, lomefloxacin, sparfloxacin,
temafloxacin, pefloxacin, amifloxacin, fleroxacin, tosufloxacin,
prulifloxacin, irloxacin, pazufloxacin, clinafloxacin, and
sitafloxacin; aminoglycosides such as gentamicin, netilmicin,
paramecin, tobramycin, amikacin, kanamycin, neomycin, and
streptomycin, vancomycin, teicoplanin, rampolanin, mideplanin,
colistin, daptomycin, gramicidin, colistimethate; polymixins such
as polymixin B, capreomycin, bacitracin, penems; penicillins
including penicllinase-sensitive agents like penicillin G,
penicillin V; penicillinase-resistant agents like methicillin,
oxacillin, cloxacillin, dicloxacillin, floxacillin, nafcillin; gram
negative microorganism active agents like ampicillin, amoxicillin,
and hetacillin, cillin, and galampicillin; antipseudomonal
penicillins like carbenicillin, ticarcillin, azlocillin,
mezlocillin, and piperacillin; cephalosporins like cefpodoxime,
cefprozil, ceftbuten, ceftizoxime, ceftriaxone, cephalothin,
cephapirin, cephalexin, cephradrine, cefoxitin, cefamandole,
cefazolin, cephaloridine, cefaclor, cefadroxil, cephaloglycin,
cefuroxime, ceforanide, cefotaxime, cefatrizine, cephacetrile,
cefepime, cefixime, cefonicid, cefoperazone, cefotetan,
cefinetazole, ceftazidime, loracarbef, and moxalactam, monobactams
like aztreonam; and carbapenems such as imipenem, meropenem, and
ertapenem, pentamidine isetionate, albuterol sulfate, lidocaine,
metaproterenol sulfate, beclomethasone diprepionate, triamcinolone
acetamide, budesonide acetonide, salmeterol, ipratropium bromide,
flunisolide, cromolyn sodium, and ergotamine tartrate; taxanes such
as paclitaxel; SN-38, and tyrphostines. Bioactive agents may also
be selected from the group consisting of aminohippurate sodium,
amphotericin B, doxorubicin, aminocaproic acid, aminolevulinic
acid, aminosalicylic acid, metaraminol bitartrate, pamidronate
disodium, daunorubicin, levothyroxine sodium, lisinopril,
cilastatin sodium, mexiletine, cephalexin, deferoxamine, and
amifostine in another embodiment.
[0193] Other bioactive agents useful in the present invention
include extracellular matrix targeting agents, functional transport
moieties and labeling agents. Extracellular matrix targeting agents
include, but are not limited to, heparin binding moieties, matrix
metalloproteinase binding moieties, lysyl oxidase binding domains,
negatively charged moieties or positively charged moieties and
hyaluronic acid. Functional transport moieties include, but are not
limited to, blood brain barrier transport moieties, intracellular
transport moieties, organelle transport moieties, epithelial
transport domains and tumor targeting moieties (folate, other). In
some embodiments, the targeting agents useful in the present
invention target anti-TrkA, anti A-beta (peptide 1-40, peptide
1-42, monomeric form, oligomeric form), anti-IGF1-4, agonist
RANK-L, anti-ApoE4 or anti-ApoA1, among others.
Diagnostic Agents
[0194] Diagnostic agents useful in the random copolymers of the
present invention include imaging agents and detection agents such
as radiolabels, fluorophores, dyes and contrast agents.
[0195] Imaging agent refers to a label that is attached to the
random copolymer of the present invention for imaging a tumor,
organ, or tissue in a subject. The imaging moiety can be covalently
or non-covalently attached to the random copolymer. Examples of
imaging moieties suitable for use in the present invention include,
without limitation, radionuclides, fluorophores such as
fluorescein, rhodamine, Texas Red, Cy2, Cy3, Cy5, Cy5.5, and the
AlexaFluor (Invitrogen, Carlsbad, Calif.) range of fluorophores,
antibodies, gadolinium, gold, nanomaterials, horseradish
peroxidase, alkaline phosphatase, derivatives thereof, and mixtures
thereof.
[0196] Radiolabel refers to a nuclide that exhibits radioactivity.
A "nuclide" refers to a type of atom specified by its atomic
number, atomic mass, and energy state, such as carbon 14
(.sup.14C). "Radioactivity" refers to the radiation, including
alpha particles, beta particles, nucleons, electrons, positrons,
neutrinos, and gamma rays, emitted by a radioactive substance.
Radionuclides suitable for use in the present invention include,
but are not limited to, fluorine 18 (.sup.18F), phosphorus 32
(.sup.32P), scandium 47 (.sup.47Sc), cobalt 55 (.sup.55Co), copper
60 (.sup.60Cu), copper 61 (.sup.61Cu), copper 62 (.sup.62Cu),
copper 64 (.sup.64Cu), gallium 66 (.sup.66Ga), copper 67
(.sup.67Cu), gallium 67 (.sup.67Ga), gallium 68 (.sup.68Ga),
rubidium 82 (.sup.82Rb), yttrium 86 (.sup.86Y), yttrium 87
(.sup.87Y), strontium 89 (.sup.89Sr), yttrium 90 (.sup.90Y),
rhodium 105 (.sup.105Rh), silver 111 (.sup.111Ag), indium 111
(.sup.111In), iodine 124 (.sup.124I), iodine 125 (.sup.125I),
iodine 131 (.sup.131I), tin 117m (.sup.117mSn), technetium 99m
(.sup.99mTc), promethium 149 (.sup.149Pm), samarium 153
(.sup.153Sm), holmium 166 (.sup.166Ho)lutetium 177 (.sup.177Lu),
rhenium 186 (.sup.186Re), rhenium 188 (.sup.188Re), thallium 201
(.sup.201Tl), astatine 211 (.sup.211At), and bismuth 212
(.sup.212Bi). As used herein, the "m" in .sup.117mSn and .sup.99mTc
stands for meta state. Additionally, naturally occurring
radioactive elements such as uranium, radium, and thorium, which
typically represent mixtures of radioisotopes, are suitable
examples of radionuclides. .sup.67Cu, .sup.131I, .sup.177Lu, and
.sup.186Re are beta- and gamma-emitting radionuclides. .sup.212Bi
is an alpha- and beta-emitting radionuclide. .sup.211At is an
alpha-emitting radionuclide. .sup.32P, .sup.47Sc, .sup.89Sr,
.sup.90Y, .sup.105Rh, .sup.111Ag, .sup.117mSn, .sup.149Pm,
.sup.153Sm, .sup.166Ho, and .sup.188Re are examples of
beta-emitting radionuclides. .sup.67Ga, .sup.111In, .sup.99mTc, and
.sup.201Tl are examples of gamma-emitting radionuclides. .sup.55Co,
.sup.60Cu, .sup.61Cu, .sup.62Cu, .sup.66Ga, .sup.68Ga, .sup.82Rb,
and .sup.86Y are examples of positron-emitting radionuclides.
.sup.64Cu is a beta- and positron-emitting radionuclide. Imaging
and detection agents can also be designed into the random
copolymers of the invention through the addition of naturally
occurring isotopes such as deuterium, .sup.13C, or .sup.15N during
the synthesis of the initiator, linkers, linking groups,
comonomers.
Nanoparticles
[0197] The functional agents can also include nanoparticles.
Nanoparticles useful in the present invention include particles
having a size ranging from 1 to 1000 nm. Nanoparticles can be
beads, metallic particles or can in some cases be micelles and in
some other be liposomes. Other nanoparticles include carbon
nanotubes, quantum dots and colloidal gold. Nanoparticles can be
packed with diagnostic and/or therapeutic agents.
[0198] Those skilled in the art will also recognize that the
invention can be used to enable coincident detection of more than
one agent of the same or different type. Also, the use of flexible
linker chemistries can also be used to witness the loss of one
fluorescent label, for example as the molecule is taken up into the
cell and into a low pH environment.
[0199] In some embodiments, the random copolymer has the following
formula:
##STR00070##
wherein subscripts x and y.sup.1 are such that the Mn of the
polymer portion is about 95,000 g/mol; A.sup.1 is an antibody; and
L-CTP has the formula:
##STR00071##
[0200] In other embodiments, the random copolymer has the
formula:
##STR00072##
wherein subscripts x, y.sup.1a and y.sup.1b are such that the Mn of
the polymer portion is about 107,100 g/mol; A.sup.1 is an antibody;
and L-CTP is as defined above.
[0201] In still other embodiments, the random copolymer has the
formula:
##STR00073##
wherein subscripts x and y.sup.1 are such that the Mn of the
polymer portion is about 95,000 g/mol; A.sup.1 is an IgG; and L-CTP
is as defined above.
[0202] In some embodiments, each of A.sup.1 and A.sup.2 is
independently an antibody, an antibody fragment, a Fab, IgG, a
peptide, a protein, an enzyme, an oligonucleotide, a
polynucleotide, nucleic acids, or an antibody drug conjugate
(ADC).
[0203] In some embodiments, A.sup.1 is independently selected from
an antibody, an antibody fragment, a Fab, a scFv, an immunoglobulin
domain, an IgG, and A.sup.2 is independently selected from an
anti-cancer agent, a toxin, a small molecule drug, a chemotherapy
agent, a kinase inhibitor, an anti-inflammatory agent, and an
antifibrotic agent.
[0204] In some embodiments, R.sup.1 is LG.sup.1, and
L.sup.2-A.sup.2 is independently selected from an anti-cancer
agent, a toxin, a small molecule drug, a chemotherapy agent, a
kinase inhibitor, an anti-inflammatory agent, and an antifibrotic
agent.
IV. Preparation of Zwitterion-Containing Random Copolymers
[0205] The random copolymers of the present invention can be
prepared by any means known in the art. In some embodiments, the
present invention provides a process for preparing a random
copolymer of the present invention, the process including the step
of contacting a mixture of a first monomer and a second monomer
with an initiator, I.sup.I, under conditions sufficient to prepare
a random copolymer via free radical polymerization, wherein the
first monomer comprises a phosphorylcholine, and each of the second
monomer and initiator independently comprise at least one of a
functional agent or a linking group for linking to the functional
agent.
[0206] The mixture for preparing the random copolymers of the
present invention can include a variety of other components. For
example, the mixture can also include catalyst, ligand, solvent,
and other additives. In some embodiments, the mixture also includes
a catalyst and a ligand. Suitable catalysts and ligands are
described in more detail below.
[0207] The mixture for preparing the random copolymers of the
present invention can be prepared using a semi-continuous process
to control the structure of the polymer when the reactivity ratio
of the monomers are different in order to allow the final polymer
to be an alternating copolymer, a periodic copolymer, a gradient
copolymer, a block copolymer or a statistical copolymer.
[0208] Any suitable monomer can be used in the process of the
present invention, such as those described above.
[0209] The random copolymers of the present invention can be
prepared by any suitable polymerization method, such as by living
radical polymerization. Living radical polymerization, discussed by
Odian, G. in Principles of Polymerization, 4.sup.th
Wiley-Interscience John Wiley & Sons: New York, 2004, and
applied to zwitterionic polymers for example in U.S. Pat. No.
6,852,816. Several different living radical polymerization
methodologies can be employed, including Stable Free Radical
Polymerization (SFRP), Radical Addition-Fragmentation Transfer
(RAFT) and Nitroxide-Mediated Polymerization (NMP). In addition,
Atom Transfer Radical Polymerization (ATRP), provides a convenient
method for the preparation of the random copolymers of the
invention.
[0210] The preparation of polymers via ATRP involves the radical
polymerization of monomers beginning with an initiator bearing one
or more halogens. The halogenated initiator is activated by a
catalyst (or a mixture of catalysts when CuBr) is employed) such as
a transition metal salt (CuBr) that can be solubilized by a ligand
(e.g., bipyridine or PMDETA). RAFT polymerization uses
thiocarbonylthio compounds, such as dithioesters, dithiocarbamates,
trithiocarbonates, and xanthates, to mediate the polymerization
process via a reversible chain-transfer process. Other "living" or
controlled radical processes useful in the preparation of the
inventive random copolymers include NMP.
Initiators
[0211] Initiators useful for the preparation of the random
copolymers of the present invention include any initiator suitable
for polymerization via atom transfer radical polymerization (ATRP),
such as those described above. Other useful initiators include
those for nitroxide-mediated radical polymerization (NMP), or
reversible addition-fragmentation-termination (RAFT or MADIX)
polymerization. Still other techniques to control a free-radical
polymerization process can be used, such as the use of iniferters,
degenerative transfer or telomerization process. Moreover, the
initiators useful in the present invention include those having at
least one branch point, such as those described above.
[0212] Random copolymers of the present invention having complex
architectures including branched compounds having multiple polymer
arms including, but not limited to, comb and star structures. Comb
architectures can be achieved employing linear initiators bearing
three or more halogen atoms, preferably the halogens are chlorine,
bromine, or iodine atoms, more preferably the halogens are chlorine
or bromine atoms. Star architectures can also be prepared employing
compounds bearing multiple halogens on a single carbon atom or
cyclic molecules bearing multiple halogens. In some embodiments
compounds having star architectures have 3 polymer arms and in
other embodiments they have 4 polymer arms. See initiators
described above.
Catalyst and Ligands
[0213] Catalyst for use in ATRP or group radical transfer
polymerizations may include suitable salts of Cu.sup.1+, Cu.sup.2+,
Fe.sup.2+, Fe.sup.3+, Ru.sup.2+, Ru.sup.3+, Cr.sup.2+, Cr.sup.3+,
Mo.sup.2+, Mo.sup.3+, W.sup.2+, W.sup.3+, Mn.sup.2+, Mn.sup.2+,
Mn.sup.4+, Rh.sup.3+, R.sup.4+, Re.sup.2+, Re.sup.3+, Co.sup.1+,
Co..sup.2+, Co.sup.3+, V.sup.2+, V.sup.3+, Zn..sup.1+, Zn.sup.2+,
Ni.sup.2+, Ni.sup.3+, Au.sup.1+, Au.sup.2+, Ag.sup.1+ and
Ag.sup.2+. Suitable salts include, but are not limited to: halogen,
C.sub.1-C.sub.6-alkoxy, sulfates, phosphate, triflate,
hexafluorophosphate, methanesulphonate, arylsulphonate salts. In
some embodiments the catalyst is a Chloride, bromide salts of the
above-recited metal ions. In other embodiments the catalyst is
CuBr, CuCl or RuCl.sub.2.
[0214] In some embodiments, the use of one or more ligands to
solubilize transition metal catalysts is desirable. Suitable
ligands are usefully used in combination with a variety of
transition metal catalysts including where copper chloride or
bromide, or ruthenium chloride transition metal salts are part of
the catalyst. The choice of a ligand affects the function of the
catalyst as ligands not only aid in solubilizing transition metal
catalysts in organic reaction media, but also adjust their redox
potential. Selection of a ligand is also based upon the solubility
and separability of the catalyst from the product mixture. Where
polymerization is to be carried out in a liquid phase soluble
ligands/catalyst are generally desirable although immobilized
catalysts can be employed. Suitable ligands include those pyridyl
groups (including alkyl pyridines e.g., 4.4. dialkyl-2,2'
bipyridines) and pyridyl groups bearing an alkyl substituted imino
group, where present, longer alkyl groups provide solubility in
less polar monomer mixtures and solvent media. Triphenyl phosphines
and other phosphorus ligands, in addition to indanyl, or
cyclopentadienyl ligands, can also be employed with transition
metal catalysts (e.g., Ru.sup.+2-halide or Fe.sup.+2-halide
complexes with triphenylphosphine, indanyl or cyclopentadienyl
ligands).
[0215] An approximately stoichiometric amount of metal compound and
ligand in the catalyst, based on the molar ratios of the components
when the metal ion is fully complexed, is employed in some
embodiments. In other embodiments the ratio between metal compound
and ligand is in the range 1:(0.5 to 2) or in the range 1:(0.8 to
1.25).
[0216] Generally, where the catalyst is copper, bidentate or
multidentate nitrogen ligands produce more active catalysts. In
addition, bridged or cyclic ligands and branched aliphatic
polyamines provide more active catalysts than simple linear
ligands. Where bromine is the counter ion, bidentate or one-half
tetradentate ligands are needed per Cu.sup.+1. Where more complex
counter ions are employed, such as triflate or hexafluorophosphate,
two bidentate or one tetradentate ligand can be employed. The
addition of metallic copper can be advantageous in some embodiments
particularly where faster polymerization is desired as metallic
copper and Cu.sup.+2 may undergo redox reaction to form Cu.sup.+1.
The addition of some Cu.sup.+2 at the beginning of some ATRP
reactions can be employed to decrease the amount of normal
termination.
[0217] In some embodiments, the amount of catalyst employed in the
polymerization reactions is the molar equivalent of the initiator
that is present. Since catalyst is not consumed in the reaction,
however, it is not essential to include a quantity of catalyst as
high as of initiator. The ratio of catalyst to each halogen
contained in the initiator, based on transition metal compound in
some embodiments is from about 1:(1 to 50), in other embodiments
from about 1:(1 to 10), in other embodiments from about 1:(1 to 5),
and in other embodiments from 1:1.
Polymerization Conditions
[0218] In some embodiments, the "living" or controlled radical
polymerization process of the invention is preferably carried out
to achieve a degree of polymerization in the range of 3 to about
2000, and in other embodiments from about 5 to about 500. The
degree of polymerization in other embodiments is in the range 10 to
100, or alternatively in the range of about 10 to about 50. The
degree of polymerization in group or atom transfer radical
polymerization techniques, is directly related to the initial ratio
of initiator to monomer. Therefore, in some embodiments the initial
ratios of initiator to monomer are in the range of 1:(3 to about
2,000) or about 1:(5 to 500), or about 1:(10 to 100), or about
1:(10 to 50).
[0219] Polymerization reactions are typically carried out in the
liquid phase, employing a single homogeneous solution. The reaction
may, however, be heterogeneous comprising a solid and a liquid
phase (e.g., a suspension or aqueous emulsion). The reaction may
proceed in the solid state where the polymer is attached to a
planar surface (wafer) or a non-planar surface (beads). In those
embodiments where a non-polymerizable solvent is employed, the
solvent employed is selected taking into consideration the nature
of the zwitterionic monomer, the initiator, the catalyst and its
ligand; and in addition, any comonomer that can be employed.
[0220] The solvent may comprise a single compound or a mixture of
compounds. In some embodiments the solvent is water, and in other
embodiments water is present in an amount from about 10% to about
100% by weight, based on the weight of the monomers present in the
reaction. In those embodiments where a water insoluble comonomer is
to be polymerized with a zwitterionic monomer, it can be desirable
to employ a solvent or co-solvent (in conjunction with water) that
permits solubilization of all the monomers present. Suitable
organic solvents include, without limitation, formamides (e.g.,
N,N'-dimethylformamide), ethers (e.g., tetrahydrofuran), esters
(ethyl acetate) and, most preferably, alcohols. In some embodiments
where a mixture of water and organic solvent is to be employed,
C.sub.1-C.sub.4 water miscible alkyl alcohols (methanol, ethanol,
propanol, isopropanol, butanol, isobutanol, and tertbutanol) are
useful organic solvents. In other embodiments, water and methanol
combinations are suitable for conducting polymerization reactions.
The reaction may also be conducted in supercritical solvents such
as CO.sub.2.
[0221] As noted above, in some embodiments it is desirable to
include water in the polymerization mixture in an amount from about
10% to about 100% by weight based on the weight of monomers to be
polymerized. In other embodiments the total non-polymerizable
solvent is from about 1% to about 500% by weight, based on the
weight of the monomers present in the reaction mixture. In other
embodiments, the total non-polymerizable solvent is from about 10%
to about 500% by weight or alternatively from 20% to 400%, based on
the weight of the monomers present in the reaction mixture. It is
also desirable in some cases to manipulate the solubility of an
input reagent, such as initiator or monomer, for example by
modifying temperature or solvent or other method so as to modify
the reaction conditions in a dynamic fashion.
[0222] In some embodiments, contact time of the zwitterionic
monomer and water prior to contact with the initiator and catalyst
are minimized by forming a premix comprising all components other
than the zwitterionic monomer and for the zwitterionic monomer to
be added to the premix last.
[0223] The polymerization reactions can be carried out at any
suitable temperature. In some embodiments the temperature can be
from about ambient (room temperature) to about 120.degree. C. In
other embodiments the polymerizations can be carried out at a
temperature elevated from ambient temperature in the range of about
60.degree. to 80.degree. C. In other embodiments the reaction is
carried out at ambient (room temperature).
[0224] In some embodiments, the compounds of the invention have a
polydispersity (of molecular weight) of less than 1.5, as judged by
gel permeation chromatography. In other embodiments the
polydispersities can be in the range of 1.2 to 1.4.
[0225] A number of workup procedures can be used to purify the
polymer of interest such as precipitation, fractionation,
reprecipitation, membrane separation and freeze-drying of the
polymers.
Non-Halogenated Polymer Terminus
[0226] In some embodiments, it can be desirable to replace the
halogen, or other radical scavenger I', with another functionality.
A variety of reactions can be employed for the conversion of the
aliphatic halogen. In some embodiments, the conversion of the
aliphatic halogen can include reaction to prepare an alkyl, alkoxy,
cycloalkyl, aryl, heteroaryl or hydroxy group. Halogens can also be
subject to an elimination reaction to give rise to an alkene
(double bond). Other methods of modifying the halogenated terminus
are described in Matyjaszewski et al. Prog. Polym. Sci. 2001, 26,
337, incorporated by reference in its entirety herein.
Attachment of Functional Agents
[0227] The coupling of functional agents to the random copolymers
of the present invention can be conducted employing chemical
conditions and reagents applicable to the reactions being
conducted. Exemplary methods are described in Bioconjugate
Techniques, Greg T. Hermanson, Academic Press, 2d ed., 2008
(incorporated in its entirety herein). Other bioconjugation
techniques are described in Bertozzi et al. Angewandte Chemie 2009,
48, 6974, and Gauthier et al. Chem. Commun. 2008, 2591, each
incorporated by reference in its entirety herein.
[0228] Where, for example, the coupling requires the formation of
an ester or an amide, dehydration reactions between a carboxylic
acid and an alcohol or amine may employ a dehydrating agent (e.g.,
a carbodiimide such as dicyclohexylcarbodimide, DCC, or the water
soluble agent 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide
hydrochloride, EDC). Alternatively, N-hydroxysuccinimide esters
(NHS) can be employed to prepare amides. Reaction to prepare amides
employing NHS esters are typically conducted near neutral pH in
phosphate, bicarbonate, borate, HEPES or other non-amine containing
buffers at 4.degree. to 25.degree. C. In some embodiments,
reactions employing EDC as a dehydrating agent, a pH of 4.5-7.5 can
be employed; in other embodiments, a pH of 4.5 to 5 can be
employed. Morpholinoethanesulfonic acid, MES, is an effective
carbodiimide reaction buffer.
[0229] Thiol groups can be reacted under a variety of conditions to
prepare different products. Where a thiol is reacted with a
maleimide to form a thioether bond, the reaction is typically
carried out at a pH of 6.5-7.5. Excess maleimide groups can be
quenched by adding free thiol reagents such as mercaptoethanol.
Where disulfide bonds are present as a linkage, they can be
prepared by thiol-disulfide interchange between a sulfhydryl
present in the bioactive group and an X functionality which is a
disulfide such as a pyridyl disulfide. Reactions involving pyridyl
disulfides can be conducted at pH 4-pH 5 and the reaction can be
monitored at 343 nm to detect the released pyridine-2-thione. Thiol
groups may also be reacted with epoxides in aqueous solution to
yield hydroxy thioethers. A thiol may also be reacted at slightly
alkaline pH with a haloacetate such as iodoacetae to form a
thioether bond.
[0230] The reaction of guanido groups (e.g., those of an arginine
in a protein or polypeptide of interest) with a glyoxal can be
carried out at pH 7.0-8.0. The reaction typically proceeds at
25.degree. C. The derivative, which contains two phenylglyoxal
moieties per guanido group, is more stable under mildly acidic
conditions (below pH 4) than at neutral or alkaline pHs, and
permits isolation of the linked materials. At neutral or alkaline
pH values, the linkage decomposes slowly. Where an arginine residue
of a protein or polypeptide is reacted with a phenylglyoxal
reagent, about 80% of the linkage will hydrolyze to regenerate the
original arginine residue (in the absence of excess reagent) in
approximately 48 hours at 37.degree. C. at about pH 7.
[0231] Imidoester reactions with amines are typically conducted at
pH of 8-10, and preferably at about pH 10. The amidine linkage
formed from the reaction of an imidoester with an amine is
reversible, particularly at high pH.
[0232] Haloacetals can be reacted with sulfhydryl groups over a
broad pH range. To avoid side reactions between histidine residues
that can be present, particularly where the sulfhydryl group is
present on a protein or polypeptide, the reaction can be conducted
at about pH 8.3.
[0233] Aldehydes can be reacted with amines under a variety of
conditions to form imines. Where either the aldehyde or the amine
is immediately adjacent to an aryl group the product is a Schiff
base that tends to be more stable than where no aryl group is
present. Conditions for the reaction of amines with aldehydes to
form an imine bond include the use of a basic pH from about pH 9 to
about pH 11 and a temperature from about 0.degree. C. to room
temperature, over 1 to 24 hours. Alternatively, where preferential
coupling to the N-terminal amine of a protein is desired, lower pHs
from about 4-7 can be employed. Buffers including borohydride and
tertiary amine containing buffers are often employed for the
preparation of imines. Where it is desired imine conjugates, which
are hydrolytically susceptible, can be reduced to form an amine
bond which is not hydrolytically susceptible. Reduction can be
conducted with a variety of suitable reducing agents including
sodium borohydride or sodium cyanoborohydride.
[0234] The reaction conditions provided above are intended to
provide general guidance to the artisan. The skilled artisan will
recognize that reaction conditions can be varied as necessary to
promote the attachment of the functional agent to the random
copolymers of the present invention and that guidance for
modification of the reactions can be obtained from standard texts
in organic chemistry. Additional guidance can be obtained from
texts such as Wong, S. S., "Chemistry of Protein Conjugation and
Cross-Linking," (CRC Press 1991), which discuss related chemical
reactions.
V. Compositions
[0235] The present invention includes and provides for
pharmaceutical compositions comprising one or more compounds of the
invention and one or more pharmaceutically acceptable excipients.
The compounds of the invention may be present as a pharmaceutically
acceptable salt, prodrug, metabolite, analog or derivative thereof,
in the pharmaceutical compositions of the invention. As used
herein, "pharmaceutically acceptable excipient" or
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration.
[0236] Pharmaceutically acceptable carriers for use in formulating
the random copolymers of the present invention include, but are not
limited to: solid carriers such as lactose, terra alba, sucrose,
talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic
acid and the like; and liquid carriers such as syrups, saline,
phosphate buffered saline, water and the like. Carriers may include
any time-delay material known in the art, such as glyceryl
monostearate or glyceryl distearate, alone or with a wax,
ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate or
the like.
[0237] Other fillers, excipients, flavorants, and other additives
such as are known in the art may also be included in a
pharmaceutical composition according to this invention. The use of
such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the
compositions of the invention is contemplated. Supplementary active
compounds can also be incorporated into the compositions of the
present invention.
[0238] The pharmaceutical preparations encompass all types of
formulations. In some embodiments they are parenteral (including
subcutaneous, intramuscular, intravenous, intradermal,
intraperitoneal, intrathecal, intraventricular, intracranial,
intraspinal, intracapsular, and intraosseous) formulations suited
for injection or infusion (e.g., powders or concentrated solutions
that can be reconstituted or diluted as well as suspensions and
solutions). Where the composition is a solid that requires
reconstitution or a concentrate that requires dilution with liquid
media, any suitable liquid media may be employed. Preferred
examples of liquid media include, but are not limited to, water,
saline, phosphate buffered saline, Ringer's solution, Hank's
solution, dextrose solution, and 5% human serum albumin.
[0239] Where a compound or pharmaceutical composition comprising a
random copolymer of the present invention is suitable for the
treatment of cell proliferative disorders, including but not
limited to cancers, the compound or pharmaceutical composition can
be administered to a subject through a variety of routes including
injection directly into tumors, the blood stream, or body
cavities.
[0240] While the pharmaceutical compositions may be liquid
solutions, suspensions, or powders that can be reconstituted
immediately prior to administration, they may also take other
forms. In some embodiments, the pharmaceutical compositions may be
prepared as syrups, drenches, boluses, granules, pastes,
suspensions, creams, ointments, tablets, capsules (hard or soft)
sprays, emulsions, microemulsions, patches, suppositories, powders,
and the like. The compositions may also be prepared for routes of
administration other than parenteral administration including, but
not limited to, topical (including buccal and sublingual),
pulmonary, rectal, transdermal, transmucosal, oral, ocular, and so
forth.
[0241] In some embodiments, the pharmaceutical compositions of the
present invention comprise one or more random copolymers of the
present invention.
[0242] Other pharmaceutical compositions of the present invention
may comprise one or more random copolymers of the present invention
that function as biological ligands that are specific to an antigen
or target molecule. Such compositions may comprise a random
copolymer of the present invention, where the bioactive agent is a
polypeptide that comprises the amino acid sequence of an antibody,
or an antibody fragment such as a FAb.sub.2 or FAb' fragment or an
antibody variable region. Alternatively, the compound may be a
random copolymer and the polypeptide may comprise the antigen
binding sequence of a single chain antibody. Where a bioactive
agent present in a random copolymer of the present invention
functions as a ligand specific to an antigen or target molecule,
those compounds may also be employed as diagnostic and/or imaging
reagents and/or in diagnostic assays.
[0243] The amount of a compound in a pharmaceutical composition
will vary depending on a number of factors. In one embodiment, it
may be a therapeutically effective dose that is suitable for a
single dose container (e.g., a vial). In one embodiment, the amount
of the compound is an amount suitable for a single use syringe. In
yet another embodiment, the amount is suitable for multi-use
dispensers (e.g., containers suitable for delivery of drops of
formulations when used to deliver topical formulations). A skilled
artisan will be able to determine the amount a compound that
produces a therapeutically effective dose experimentally by
repeated administration of increasing amounts of a pharmaceutical
composition to achieve a clinically desired endpoint.
[0244] Generally, a pharmaceutically acceptable excipient will be
present in the composition in an amount of about 0.01% to about
99.999% by weight, or about 1% to about 99% by weight.
Pharmaceutical compositions may contain from about 5% to about 10%,
or from about 10% to about 20%, or from about 20% to about 30%, or
from about 30% to about 40%, or from about 40% to about 50%, or
from about 50% to about 60%, or from about 60% to about 70%, or
from about 70% to about 80%, or from about 80% to about 90%
excipient by weight. Other suitable ranges of excipients include
from about 5% to about 98%, from about from about 15 to about 95%,
or from about 20% to about 80% by weight.
[0245] Pharmaceutically acceptable excipients are described in a
variety of well known sources, including but not limited to
"Remington: The Science & Practice of Pharmacy", 19.sup.th ed.,
Williams & Williams, (1995) and Kibbe, A. H., Handbook of
Pharmaceutical Excipients, 3.sup.rd Edition, American
Pharmaceutical Association, Washington, D.C., 2000.
VI. Methods
[0246] The random copolymers of the present invention are useful
for treating any disease state or condition. By combining
appropriate targeting agents, drugs and therapeutic proteins, along
with a zwitterion such as phosphorylcholine, the random copolymers
of the present invention can be used to address the panoply of
mechanisms provided by any one disease state or condition. For
example, the disease state or condition can be acute or
chronic.
[0247] Disease states and conditions that can be treated using the
random copolymers of the present invention include, but are not
limited to, cancer, autoimmune disorders, genetic disorders,
infections, inflammation, fibrotic disorders, and metabolic
disorders.
[0248] Cancers that can be treated using the random copolymers of
the present invention include, but are not limited to, ovarian
cancer, breast cancer, lung cancer, bladder cancer, thyroid cancer,
liver cancer, pleural cancer, pancreatic cancer, cervical cancer,
testicular cancer, colon cancer, anal cancer, bile duct cancer,
gastrointestinal carcinoid tumors, esophageal cancer, gall bladder
cancer, rectal cancer, appendix cancer, small intestine cancer,
stomach (gastric) cancer, renal cancer, cancer of the central
nervous system, skin cancer, choriocarcinomas; head and neck
cancers, osteogenic sarcomas, fibrosarcoma, neuroblastoma, glioma,
melanoma, leukemia, and lymphoma.
[0249] Autoimmune diseases that can be treated using the random
copolymers of the present invention include, but are not limited
to, multiple sclerosis, myasthenia gravis, Crohn's disease,
ulcerative colitis, primary biliary cirrhosis, type 1 diabetes
mellitus (insulin dependent diabetes mellitus or IDDM), Grave's
disease, autoimmune hemolytic anemia, pernicious anemia, autoimmune
thrombocytopenia, vasculitides such as Wegener's granulomatosis,
Behcet's disease, rheumatoid arthritis, systemic lupus
erythematosus (lupus), scleroderma, systemic sclerosis,
Guillain-Barre syndromes, fibrosis, hepatic fibrosis,
post-transplant fibrosis, idiopathic pulmonary fibrosis,
Hashimoto's thyroiditis spondyloarthropathies such as ankylosing
spondylitis, psoriasis, dermatitis herpetiformis, inflammatory
bowel diseases, pemphigus vulgaris and vitiligo.
[0250] Some metabolic disorders treatable by the random copolymers
of the present invention include lysosomal storage disorders, such
as mucopolysaccharidosis IV or Morquio Syndrome, Activator
Deficiency/GM2 Gangliosidosis, Alpha-mannosidosis,
Aspartylglucosaminuria, Cholesteryl ester storage disease, Chronic
Hexosaminidase A Deficiency, Cystinosis, Danon disease, Fabry
disease, Farber disease, Fucosidosis, Galactosialidosis, Gaucher
Disease, GM1 gangliosidosis, hypophosphatasia, I-Cell
disease/Mucolipidosis II, Infantile Free Sialic Acid Storage
Disease/ISSD, Juvenile Hexosaminidase A Deficiency, Krabbe disease,
Metachromatic Leukodystrophy, Mucopolysaccharidoses disorders such
as Pseudo-Hurler polydystrophy/Mucolipidosis IIIA, Hurler Syndrome,
Scheie Syndrome, Hurler-Scheie Syndrome, Hunter syndrome,
Sanfilippo syndrome, Hyaluronidase Deficiency, Maroteaux-Lamy, Sly
Syndrome, Mucolipidosis I/Sialidosis, Mucolipidosis, and
Mucolipidosis, Multiple sulfatase deficiency, Niemann-Pick Disease,
Neuronal Ceroid Lipofuscinoses, Pompe disease/Glycogen storage
disease type II, Pycnodysostosis, Sandhoff disease, Schindler
disease, Salla disease/Sialic Acid Storage Disease, Tay-Sachs/GM2
gangliosidosis and Wolman disease.
[0251] Conjugates of the invention and compositions (e.g.,
pharmaceutical compositions) containing conjugates of the invention
can be used to treat a variety of conditions. For example, there
are many conditions for which treatment therapies are known to
practitioners of skill in the art in which functional agents, as
disclosed herein, are employed. The invention contemplates that the
conjugates of the invention (e.g., phosphorylcholine containing
polymers conjugated to a variety of functional agents) and
compositions containing the conjugates of the invention can be
employed to treat such conditions and that such conjugates provide
for an enhanced treatment therapy relative to the same functional
agent not coupled to a phosphorylcholine containing polymer.
[0252] Therefore, the invention contemplates the treatment of a
condition known to be treatable by a certain bioactive agent by
treating the condition using the same certain bioactive agent
conjugated to a phosphorylcholine containing polymer.
[0253] Another aspect of the present invention relates to methods
of treating a condition responsive to a biological agent comprising
administering to a subject in need thereof a therapeutically
effective amount of a compound of the invention or of a
pharmaceutically acceptable composition of the invention as
described above. Dosage and administration are adjusted to provide
sufficient levels of the bioactive agent(s) to maintain the desired
effect. The appropriate dosage and/or administration protocol for
any given subject may vary depending on various factors including
the severity of the disease state, general health of the subject,
age, weight, and gender of the subject, diet, time and frequency of
administration, drug combination(s), reaction sensitivities, and
tolerance/response to therapy. Therapeutically effective amounts
for a given situation can be determined by routine experimentation
that is within the skill and judgment of the clinician.
[0254] The pharmaceutical compositions described herein may be
administered singly. Alternatively, two or more pharmaceutical
compositions may be administered sequentially, or in a cocktail or
combination containing two random copolymers of the present
invention or one random copolymer of the present invention and
another bioactive agent. Other uses of bioactive agents set forth
herein may be found in standard reference texts such as the Merck
Manual of Diagnosis and Therapy, Merck & Co., Inc., Whitehouse
Station, N.J. and Goodman and Gilman's The Pharmacological. Basis
of Therapeutics, Pergamon Press, Inc., Elmsford, N.Y., (1990).
[0255] The random copolymers of the present invention are useful
for treating, detecting and imaging a variety of disease states and
conditions. The random copolymers can be used as a chemotherapy
agent in the treatment of cancer where the initiator fragment I is
not functionalized and R.sup.2 includes a cancer chemotherapeutic
agent A.sup.2 that is loaded onto the random copolymer via click
chemistry or any suitable conjugation chemistry:
##STR00074##
[0256] Additional cancer treatment agents using the random
copolymers can include a targeting agent of an anti-angiogenic
protein such as an anti-VEGF scFv fragment A.sup.1 conjugated via a
C-terminal cysteine to a maleimide initiator I. The random
copolymer can also include a cancer chemotherapeutic agent A.sup.2a
that is linked to the polymer backbone via a cleavable or
self-immolative linker. Moreover, the cancer chemotherapeutic is
loaded onto the random copolymer via click chemistry or any
suitable conjugation chemistry. For example, in Ewing's sarcoma:
the targeting agent can be an anti-cancer antibody fragment such as
a Fab' or scFv fragment that binds to an angiogenic growth factor
such as VEGF. In addition, bone targeting comonomer A.sup.2b can
include an aspartate or glutamate rich peptide or a bisphosphonate.
Other comonomers A.sup.2c can include Vincristine, Doxorubicin,
and/or cyclophosphamide attached via cleavable or self-immolative
linkers:
##STR00075##
[0257] Random copolymers for more efficacious and longer residence
time therapy for wet or dry macular degeneration can include an
anti-inflammatory or anti-angiogenic protein such as anti-VEGF or
anti-IL-6 scFv fragment A.sup.1 conjugated via a C-terminal
cysteine to a maleimide initiator I. The random copolymer prepared
can be either a homopolymer of phosphorylcholine or a copolymer of
phosphorylcholine stably attached to the polymer backbone, in
combination with an anti-inflammatory small molecule or an
anti-angiogenic small molecule A.sup.2 linked to the polymer
backbone via a cleavable linker L.sup.2. Alternatively, the random
copolymer can include another comonomer having a vitreous
extracellular matrix (hyaluronic acid) binding moiety A.sup.2
attached via a non-cleavable linker L.sup.2 such as choline or a
positively charged amino acid:
##STR00076##
[0258] Random copolymers for real-time diagnostic estimate of tumor
burden and imaging for oncology can include an
anti-tumor-associated protein such as an anti-Carcino Embryonic
Antigen (CEA) scFv fragment A.sup.1 conjugated via a C-terminal
cysteine to a maleimide initiator I. The random copolymer can
include phosphorylcholine stably attached and an imaging reagent
A.sup.2a such as a fluorescent dye (fluorescent probe detection) or
gadolinium (for whole body imaging detection). Additional
comonomers can be added having small molecule chemotherapy agents
A.sup.1b attached via a cleavable linker L.sup.2b to add a
therapeutic element. These structures provide both therapeutic and
diagnostic functions, and are commonly referred to as
theranostics:
##STR00077##
[0259] Random copolymers for use as a targeted platform for bone
enzyme replacement therapies, specifically hypophosphatasia, can
include recombinant alkaline phosphatase enzyme A.sup.1 conjugated
via aldehyde-modified initiator I through a stable linkage L.sup.1.
The random copolymer can include phosphorylcholine stably attached
to the polymer, and a comonomer useful for targeting via a stably
attached bone targeting moiety A.sup.2 such as an aspartate or
glutamate rich peptide sequence or a bisphosphonate such that more
than five targeting moieties are present (y.sup.1 is greater than
5). A1, of course, can be any protein such as a growth factor, for
example human growth hormone, and the targeting peptide can be any
peptide suitable for locating the conjugate in any tissue. These
copolymers are useful for subcutaneous delivery:
##STR00078##
[0260] Other random copolymers are useful as a targeted platform
for bone enzyme replacement therapies, specifically Morquio
Syndrome (MPS type IVa). These types of random copolymers include a
recombinant N-Acetylgalactosamine-6-sulfate sulfatase enzyme
A.sup.1 conjugated via site specific chemistry initiator I through
a cleavable linker L.sup.1. The random copolymer can include
phosphorylcholine stably attached to the polymer, and a targeting
comonomer containing a bone targeting moiety A.sup.2 such as an
aspartate or glutamate rich peptide sequence or a bisphosphonate
linked via a non-cleavable linker L.sup.2, such that more than five
targeting moieties are present. These copolymers are useful for
subcutaneous delivery:
##STR00079##
[0261] Random copolymers for targeted platforms for safer, more
efficacious treatment of Rheumatoid Arthritis can include several
different drugs, including an anti-TNF.alpha. biopharmaceutical
such as an antibody fragment A.sup.1 that is linked to the
initiator I via a non-cleavable linker L.sup.1, or an anti-VEGFR2,
a small molecule A.sup.2a, as a kinase inhibitor, and methotrexate
A.sup.2b, an antineoplastic antimetabolite with immunosuppressant
properties both linked via cleavable linkers L.sup.2a and L.sup.2b
respectively:
##STR00080##
[0262] Similar random copolymers to those above can be prepared by
replacing the anti-TNF.alpha. biopharmaceutical of A.sup.1 with a
small protein dual domain inhibitor such as an avimer or a scFv
dimer that inhibits two proteins, for example TNF.alpha. and also
VEGF, but without the small molecule inhibitor. In addition, the
methotrexate A.sup.2 can be substituted for cyclophosphamide:
##STR00081##
[0263] Finally, a random copolymer for targeted and protected RNAi
can be prepared without a functionalized initiator I. The random
copolymer can include phosphorylcholine stably attached to the
polymer, and a comonomer having an siRNA A.sup.2a linked to the
polymer via a cleavable bond L.sup.2a, and another comonomer having
a cell- or tissue-targeting group A.sup.2b attached via a
non-cleavable linker L.sup.2b. The siRNA containing comonomer can
be prepared using a monomer having a linking group suitable for
click chemistry or any suitable conjugation chemistry wherein the
siRNA is linked to the linking group following polymerization. The
comonomer having the targeting moiety can either already contain
the targeting moiety, or link to the targeting moiety via a
comonomer having a linking group suitable for click chemistry or
any suitable conjugation chemistry via a different chemistry than
for attachment of the siRNA. The cleavable linker is preferably a
pH sensitive linker. The random copolymer can be prepared with a
target stoichiometry of approximately five oligonucleotide moieties
per drug A.sup.2c and five targeting moieties per drug (such that
the ratio of y.sup.1a:y.sup.1b:y.sup.1c is about 5:5:1). Moreover,
the phosphorylcholine polymer backbone can be optimized not for
half-life, but to protect the siRNA in its journey from injection
site to the targeted tissues. The siRNA can be replaced with
microRNA:
##STR00082##
In addition, the initiator I can optionally be linked to a
bioactive moiety A.sup.1 such as an antibody fragment for targeting
and therapy:
##STR00083##
[0264] In some other embodiments, the engineering of novel
multifunctional therapeutic systems can combine phosphorylcholine
polymers with drug or gene targeting agents with imaging and/or
sensing capabilities. Systems can have at least 3 components: (1) a
targeting moiety or molecular signatures that can target delivery
to specific sites, (2) the appropriate imaging agent/probe/tags for
visualization or monitoring of the systems, and (3) one or more
therapeutic agents to effectively treat a particular disease or
disorder.
[0265] The following are examples of multifunctional systems that
contain targeting, imaging, and drug/gene moieties. This list is
not intended to be exclusive of a phosphorylcholine containing
polymer system. Targeted systems that can be activated by internal
processes such as pH, enzyme cleavage or external stimuli such as
near IR light, ultrasound, heat, or magnetic field for therapeutic
delivery and imaging are also suitable. First, our approach
conceptually can be combined with all of the following: [0266]
Synthetic biodegradable polymer-based nanoparticles encapsulating a
therapeutic gene, a gadolinium contrast agent for MRI analysis, and
functionalized with antibodies to target specific disease sites.
[0267] Liposomes encapsulating small or large drug molecules,
labeled with .sup.18-Fluorine for PET analysis, and functionalized
with antibodies to target specific disease sites. [0268] Polyplexes
containing a siRNA molecule, an iron-oxide contrast agent for MRI
analysis, and modified with cell binding ligands and
cell-penetrating peptides for targeted cellular and intracellular
delivery respectively. [0269] Fluorescent quantum dots intercalated
with a drug molecule for optical imaging and sensing of the
delivery and functionalized with an RNA aptamer to target specific
diseases. [0270] Inorganic or organic nanoparticles containing an
antisense oligonucleotide for gene therapy, a gadolinium contrast
agent for MRI analysis, a fluorophore for optical imaging, and
surface modified to target specific diseases. [0271] pH sensitive
polymeric nanocomposites with a drug molecule that is released as a
function of pH, an iron oxide contrast agent for MRI imaging, CdTe
quantum dots for optical imaging, and functionalized with
antibodies to target specific diseases. [0272] Nanoparticle-DNA
aptamer conjugates containing a drug and a radiotracer such as
.sup.111In for SPECT imaging and functionalized with
disease-specific membrane antibodies.
[0273] Second, the polymers of the present invention can be
specifically combined with the above: [0274] Phosphorylcholine
polymer-based construct containing a therapeutic gene (bioactive
1), a gadolinium contrast agent for MRI analysis (functional 1),
and a small protein (such as an antibody fragment) to target
specific disease sites. [0275] Imaging agent .sup.18-Fluorine for
PET analysis, and functionalized with small protein (such as an
antibody fragment) to target specific disease sites. [0276]
Phosphorylcholine polymers containing one or more siRNA molecules,
an iron-oxide contrast agent for MRI analysis, and modified with
cell binding ligands and cell-penetrating peptides for targeted
cellular and intracellular delivery respectively. [0277]
Phosphorylcholine polymers containing fluorescent quantum dots
(functional agent) intercalated with a drug molecule (functional
agent) for optical imaging and sensing of the delivery and
functionalized with an RNA aptamer or a small protein (such as an
antibody fragment or scaffold derived protein) to target specific
diseases. [0278] Phosphorylcholine containing polymers containing
an antisense oligonucleotide for gene therapy, a gadolinium
contrast agent for MRI analysis, a fluorophore for optical imaging,
and an additional functional agent for targeting specific diseases
such as folate for tumor or choline for electrostatic interactions
for targeting extracellular matrix. [0279] pH sensitive
phosphorylcholine polymer with a drug molecule that is released as
a function of pH, an iron oxide contrast agent for MRI imaging,
CdTe quantum dots for optical imaging, and functionalized with
antibodies or other protein or aptamer to target and treat specific
diseases. [0280] Phosphorylcholine polymer with aptamer functional
agent conjugates containing a drug and a radiotracer such as
.sup.111In for SPECT imaging and further functionalized with
disease-specific membrane antibodies.
VII. Examples
Example 1
Preparation of
N-(2-hydroxyethyl)-exo-3,6-epoxy-1,2,3,6-tetrahydrophthalimide
##STR00084##
[0282] A 100-ml round-bottom flask equipped with a stir bar was
charged with 50 ml ethanol and 2.0 grams of
exo-3,6-epoxy-1,2,3,6-tetrahydrophthalic anhydride. The stirring
mixture was cooled with an ice water bath, and a solution of 0.73
grams of ethanolamine in 20 ml of ethanol was added drop wise over
10 minutes. The reaction was heated at reflux for 4 hours, then
refrigerated overnight. Filtration and rinsing with ethanol yielded
0.73 grams of the desired product as a white crystalline solid. The
filtrate was concentrated and chilled again to obtain a second
crystal crop. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.=2.90 (s,
2H, CH), 3.71 (m, 2H, OCH.sub.2), 3.77 (t, J=5.0 Hz, NCH.sub.2),
5.29 (t, J=1.0 Hz, 2H, OCH), 6.53 (t, J=1.0 Hz, 2H, CH.dbd.CH).
Example 2
Preparation of isopropylidene-2,2-bis(hydroxymethyl)propionic
acid
##STR00085##
[0284] A 100 ml round-bottom flask equipped with a stir bar was
charged with 50 ml of acetone, 13.8 ml of 2,2-dimethoxypropane, 10
grams of 2,2-bis(hydroxymethyl)propionic acid, and 0.71 grams
p-toluenesulfonic acid monohydrate. The mixture was stirred for two
hours at ambient temperature, then neutralized with 1 ml of 2M
ammonia in methanol. The solvent was evaporated and the mixture
dissolved in dichloromethane, then extracted twice with 20 ml of
water. The organic phase was dried over magnesium sulfate and
evaporated to give 10.8 grams of the product as a white crystalline
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.=1.20 (s, 3H,
CH.sub.3CC.dbd.O), 1.43 (s, 3H, CH.sub.3), 1.46 (s, 3H, CH.sub.3),
3.70 (d, J=12.4 Hz, 2H, OCH.sub.2), 4.17 (d, J=12.4 Hz, 2H,
OCH.sub.2).
Example 3
Preparation of N,N-dimethylpyridinium p-toluenesulfonate (DPTS)
##STR00086##
[0286] A solution of 1.9 grams of p-toluenesulfonic acid
monohydrate in 10 ml benzene was dried by azeotropic distillation
using a Dean-Stark trap, then 3.42 grams of 4-dimethylaminopyridine
were added. Much solid formed, and an additional 25 ml of benzene
were required to mobilize the reaction, which stirred slowly as it
cooled to room temperature. The resulting solid was isolated by
filtration, washed with 10 ml of benzene, and dried to yield 7.88
grams of the product as a white solid.
Example 4
Preparation of Protected Maleimide Bromopropionate Initiator
##STR00087##
[0288] A 100-ml round-bottom flask equipped with a stir bar was
charged with 50 ml tetrahydrofuran, 2 grams of
N-(2-hydroxyethyl)-exo-3,6-epoxy-1,2,3,6-tetrahydrophthalimide, and
2.0 ml triethylamine. The stirring mixture was cooled to 0 degrees,
and a solution of 1.18 ml of 2-bromoisobutyryl bromide in 5 ml
tetrahydrofuran was added drop wise over 30 minutes. The reaction
was allowed to stir on ice for 3 hours followed by room temperature
overnight. Concentration of the reaction mixture gave an oily
residue, which was purified by silica gel flash chromatography with
30-50% ethyl acetate in hexane, giving 1.96 grams of the desired
product as a white powder. .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta.=1.89 (s, 6H, CH.sub.3), 2.87 (s, 2H, CH), 3.82 (t, J=5.4
Hz, 2H, NCH.sub.2), 4.33 (t, J=5.4 Hz, 2H, OCH.sub.2), 5.27 (t,
J=1.0 Hz, 2H, OCH), 6.51 (t, J=1.0 Hz, 2H, CH.sub.vinyl).
Example 5
Preparation of Protected Maleimide Bis(Bromopropionate)
Initiator
[0289] Protected Maleimide Isopropylidene Acid
##STR00088##
[0290] A solution of 2.00 grams of
N-(2-hydroxyethyl)-exo-3,6-epoxy-1,2,3,6-tetrahydrophthalimide and
1.67 grams of isopropylidene-2,2-bis(hydroxymethyl)propionic acid
in 30 ml of dry dichloromethane, together with 563 mg of DPTS was
treated drop wise with a solution of 2.37 grams of
N,N'-dicyclohexylcarbodiimide in 10 ml of dry dichloromethane. Much
solid began to form as the reaction mixture was stirred at ambient
temperature overnight. The reaction was filtered, and the
precipitate was washed with a small amount of dichloromethane. The
combined organic layers were concentrated to give a clear oil
containing a small amount of solid. This oil was subjected to flash
column chromatography on silica gel, using first 20-100% ethyl
acetate in hexane. The fractions containing the desired product
were combined and concentrated to give 3.17 grams of the final
product as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta.=1.19 (s, 3H, CH.sub.3CC.dbd.OO), 1.37 (s, 3H, CH.sub.3),
1.41 (s, 3H, CH.sub.3), 1.55 (s, 6H, (CH.sub.3).sub.2C), 2.86 (s,
2H, C.dbd.OCHCHC.dbd.O), 3.58 (d, J=12 Hz, CH.sub.2O), 3.78 (t,
J=5.4 Hz, CH.sub.2CH.sub.2O), 4.14 (d, J=12H, CH.sub.2O), 4.30 (t,
J=5.4 Hz, CH.sub.2CH.sub.2O), 5.27 (t, 2H, CHOCH), 6.51 (s, 2H,
CH.dbd.CH).
[0291] Protected Maleimide Diol
##STR00089##
[0292] A solution of the isopropylidene compound from above in 50
ml of methanol was treated with 1.0 grams of Dowex 50Wx8-100 ion
exchange resin (H.sup.+ form) and the reaction was stirred at room
temperature overnight, at which time the reaction appeared complete
by tlc (silica gel, ethyl acetate). The mixture was filtered, and
the solid resin was washed with a small amount of methanol. The
combined organics were concentrated and placed under high vacuum to
give 1.55 grams of a slightly cloudy oil, which was used in the
next reaction without further purification.
[0293] Protected Maleimide Bis(Bromopropionate) Initiator
##STR00090##
[0294] A solution of the crude product from above in 40 ml of
anhydrous tetrahydrofuran (THF), together with 1.45 ml of
triethylamine was cooled in an ice water bath, and a solution of
1.23 ml of 2-bromoisobutyryl bromide in 20 ml of anhydrous THF was
added drop wise over a few minutes. The reaction was stirred in the
cold for 30 minutes, then allowed to warm to room temperature over
6 hours. Another 600 .mu.l of triethylamine were added, followed by
another 0.5 ml of 2-bromoisobutyryl bromide. The reaction was
acidic by pH paper, so another 200 .mu.l of triethylamine were
added to bring the pH of the solution to 9. The reaction was
stirred overnight, concentrated, and the residue was partitioned
between 50 ml of dichloromethane and 50 ml of water. The organic
layer was dried over sodium sulfate, filtered and concentrated to
give an oil. This was subjected to flash column chromatography on
silica gel, first with 20%, then 30% and finally 40% ethyl acetate
in hexane. The fractions containing product were combined and
concentrated to give 1.63 g of an oil which solidified to a white
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.=1.32 (s, 3H,
CH.sub.3CC.dbd.O), 1.91 [s, 12H, (CH.sub.3).sub.2CBr], 2.90 (s, 2H,
CHC.dbd.O), 3.78 (t, 2H, NCH.sub.2CH.sub.2O), 4.28 (t, 2H,
NCH.sub.2CH.sub.2O), 4.31 (app q, 4H, CH.sub.2OC.dbd.O), 5.30 (s,
2H, CHOCH), 6.52 (s, 2H, CH.dbd.CH).
Example 6
Preparation of
N-[2-(2-hydroxyethoxy)ethyl]-exo-3,6-epoxy-1,2,3,6-tetrahydrophthalimide
##STR00091##
[0296] A 250 ml round-bottom flask equipped with a stir bar was
charged with 100 ml methanol and 20 grams of
exo-3,6-epoxy-1,2,3,6-tetrahydrophthalic anhydride. The stirring
mixture was cooled to 0 degrees, and a solution of 0.73 grams
2-(2-aminoethoxy)ethanol in 40 ml of methanol was added drop wise
over 45 minutes. The reaction was stirred at room temperature for 2
hours, then heated at gentle reflux overnight. The solution was
concentrated and the product was dissolved in 100 ml of
dichloromethane, then washed with 100 ml brine. The organic layer
was dried over sodium sulfate, concentrated, and purified by
passage through a silica gel plug with 100 ml dichloromethane and
100 ml ethyl acetate. .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta.=2.90 (s, 2H, CH), 3.49 (m, 2H, OCH.sub.2), 3.59 (m, 4H,
OCH.sub.2), 3.65 (m, 2H, NCH2), 5.15 (t, J=0.8 Hz, 2H, OCH), 6.55
(t, J=0.8 Hz, 2H, CH.dbd.CH).
Example 7
Preparation of his 2,2-[(2-bromoisobutyryl)hydroxymethyl]propionic
acid
##STR00092##
[0298] To a solution of 17.5 ml of 2-bromoisobutyryl bromide in 100
ml of dichloromethane, cooled in an ice-water bath, was added
dropwise over 30 minutes a solution of 10.0 grams of
2,2-bis(hydroxymethyl)propionic acid and 41 ml of triethylamine in
100 ml of dichloromethane. The reaction was allowed to stir in the
cold for 1 hour, then allowed to warm to room temperature. The
reaction mixture was then washed with 200 ml of 1N HCl, then with
100 ml of 0.5N HCl, and finally with 50 ml of saturated NaCl. The
organic layer was dried over anhydrous sodium sulfate, filtered and
concentrated to give a yellow oil. This oil was taken up in 100 ml
of 15% ethyl acetate in hexane using a heat gun to effect solution
if necessary. The solution was then allowed to cool over 1 hour,
adding a seed crystal as the solution neared room temperature.
Crystallization was allowed to proceed for 2 hours, cooling first
in an ice-water bath, then in the refrigerator overnight. The
resulting solution had nearly solidified, so 25 ml of 10% ethyl
acetate in hexane were added, the mixture was stirred, and the
crystalline solid was recovered by filtration. It was washed with a
minimum amount of hexane and dried under vacuum to give 14.55 grams
of the desired product as a white solid. Additional product can be
obtained from the mother liquors if desired. .sup.1H NMR (400 MHz,
CD.sub.3OD): .delta.=1.33 (s, 3H, CCH.sub.3), 1.90 (s, 12H,
(CH.sub.3).sub.2CBr), 4.30 (d, J=5.4 Hz, 2H, NCH.sub.2), 4.39 (d,
J=5.4 Hz, 2H, OCH.sub.2).
Example 8
Preparation of Protected Maleimide Extended Bis(Bromopropionate)
Initiator
##STR00093##
[0300] A 250 ml round-bottom flask equipped with a stir bar was
charged with 100 ml dichloromethane, 1.0 grams of
N-[2-(2-hydroxyethoxy)ethyl]-exo-3,6-epoxy-1,2,3,6-tetrahydrophthalimide,
2.5 grams of the dibromo acid from Example 7, 0.5 grams of
dimethylaminopyridine, and 0.35 grams DPTS. Nitrogen was bubbled
through the solution briefly, and 1.6 grams DCC was added slowly.
The reaction was allowed to stir at room temperature overnight.
Filtration and evaporation gave a pink oily residue, which was
purified by silica gel flash chromatography. .sup.1H NMR (400 MHz,
CD.sub.3OD): .delta.=1.34 (s, 3H, CH.sub.3), 1.90 (s, 6H,
CH.sub.3), 2.94 (s, 2H, CH), 3.64 (m, 6H, OCH.sub.2), 4.22 (t,
J=5.4 Hz, 2H, NCH.sub.2), 4.35 (app q, 4H, OCH.sub.2), 5.15 (t,
J=1.0 Hz, 2H, OCH), 6.54 (t, J=1.0 Hz, 2H, CH.dbd.CH).
Example 9
Preparation of
N-[2-(2-hydroxyethoxy)ethyl]-exo-3,6-epoxy-1,2,3,6-tetrahydrophthalimide,
isopropylidene-2,2-bis(hydroxymethyl)propionate
##STR00094##
[0302] A solution of 11.0 grams of
N-[2-(2-hydroxyethoxy)ethyl]-exo-3,6-epoxy-1,2,3,6-tetrahydrophthalimide
and 8.22 grams of isopropylidene-2,2-bis(hydroxymethyl)propionic
acid in 250 ml of dichloromethane, together with 1.3 grams of DPTS
and 5.24 grams of DMAP was treated with 12.9 grams of DCC, and the
reaction was stirred overnight. The reaction was filtered and
concentrated to give a residue, which was subjected to flash column
chromatography in two portions on silica gel with 40-50% ethyl
acetate in hexane to give the desired product as a clear oil.
Example 10
Preparation of
N-[2-(2-hydroxyethoxy)ethyl]-exo-3,6-epoxy-1,2,3,6-tetrahydrophthalimide,
2,2-bis(hydroxymethyl)propionate
##STR00095##
[0304] The product from above was dissolved in 100 ml of methanol
and treated with 2.0 grams of Dowex 50Wx8-100 ion exchange resin
(H.sup.+ form) and the reaction was stirred at room temperature
overnight. The reaction was filtered and concentrated to give the
desired product as an oil which was used without further
purification. NMR (CD.sub.3OD): .delta. 6.546 (t, 2H, CH.dbd.CH,
J=0.8 Hz), 5.158 (t, 2H, CH--O, J=0.8 Hz), 4.180 (m, 2H,
CH.sub.2--CH.sub.2--O--C.dbd.O, 4.9 Hz), 3.63 (m, 10H, N--CH.sub.2
and N--CH.sub.2--CH.sub.2 and CH.sub.2--CH.sub.2--O--C.dbd.O and
CH.sub.2--OH), 2.936 (s, 2H, CH--CH), 1.147 (s, 3H, CH.sub.3).
Example 11
Preparation of
N-[2-(2-hydroxyethoxy)ethyl]-exo-3,6-epoxy-1,2,3,6-tetrahydrophthalimide,
2,2-bis-[2,2-bis(2-bromoisobutyryloxymethyl) propionyloxymethyl]
propionate initiator
##STR00096##
[0306] To a solution of 1.5 grams of the diol from the previous
step and 3.72 grams of
2,2-bis[(2-bromoisobutyryloxy)methyl]propionic acid in 50 ml of
dichloromethane, together with 500 mg of DPTS and 810 mg of DMAP,
was treated with 1.40 grams of diisopropylcarbodiimide, and the
reaction was stirred at room temperature overnight. The reaction
was concentrated and the residue was chromatographed several times
on silica gel with 40% ethyl acetate in hexane. The appropriate
fractions in each case were combined and concentrated to give the
desired product as an oil. NMR (CD.sub.3OD): .delta. 6.55 (t, 2H,
CH.dbd.CH, J=0.8 Hz), 5.17 (t, 2H, CH--O, J=0.8 Hz), 3.34 (m, 1214,
CCH.sub.2), 4.23 (m, 2H, CH.sub.2--CH.sub.2--O--C.dbd.O, J=4.7 Hz),
3.68 (m, 2H, N--CH.sub.2, J=4.7 Hz), 3.64 (app q, 4H,
N--CH.sub.2--CH.sub.2 and CH.sub.2--CH.sub.2--O--C.dbd.O), 2.95 (s,
2H, CH--CH), 1.907 (s, 24H, Br--C--CH.sub.3), 1.34 (s, 6H,
CH.sub.3), 1.308 (s, 3H, CH.sub.3).
Example 12
Preparation of N-(3-propionic
acid)-exo-3,6-epoxy-3,6-dimethyl-1,2,3,6-tetrahydrophthalimide,
ester with 2,2-bis[(2-bromoisobutyryloxy)methyl]propionic acid,
3-hydroxypropyl ester initiator
##STR00097##
[0308] A solution of 738 mg of
2,2-bis[(2-bromoisobutyryloxy)methyl]propionic acid,
3-hydroxypropyl ester and 399 mg of N-(3-propionic
acid)-exo-3,6-epoxy-3,6-dimethyl-1,2,3,6-tetrahydrophthalimide in
20 ml of dry acetonitrile, together with 50 mg of DPTS and 100 mg
of DMAP, was treated with 375 mg of DCC and the reaction was
stirred at room temperature overnight. The reaction was filtered to
give a residue, which was subjected to flash column chromatography
on silica gel with 30-40% ethyl acetate in hexane. The appropriate
fractions were combined and concentrated to give 1.02 grams of the
desired product as a clear oil. By .sup.1H NMR, it appeared that
about 10% of the product had already undergone retro Diels-Alder
reaction. NMR (CDCl.sub.3): .delta. 6.19 (s, 2H, CH.dbd.CH), 4.37
(app q, 4H, CCH.sub.2O, J=10.9, 29.7 Hz), 4.23 (t, 2H,
CH.sub.2CH.sub.2O, J=6.3 Hz), 4.15 (t, 2H, CH.sub.2CH.sub.2O, J=6.3
Hz), 3.62 (t, 2H, NCH.sub.2, J=7.4 Hz), 3.22 (s, 2H, CHC.dbd.O),
2.48 (t, 2H, CH.sub.2C.dbd.O, J=7.4 Hz), 2.00 (m, 2H,
CH.sub.2CH.sub.2CH.sub.2, J=6.3 Hz), 1.92 (s, 12H,
Br--C(CH.sub.3).sub.2), 1.78 (s, 6H, CH.sub.3), 1.35 (s, 3H,
CH.sub.3).
Example 13
Preparation of Acetal Bis(Bromopropionate) Initiator
##STR00098##
[0310] To a solution of 1.03 grams of 3,3-diethoxy-1-propanol and
3.0 grams of 2,2-bis(2-bromoisobutyryloxymethyl)propionic acid in
50 ml of dichloromethane, together with 817 mg of
N,N-dimethylpyridinium p-toluenesulfonate, was treated with 1.58
grams of N,N'-dicyclohexylcarbodiimide, and the reaction was
stirred at ambient temperature overnight. The reaction was
filtered, and the precipitate was washed with a small amount of
dichloromethane. The combined organics were concentrated, and the
residue was subjected to flash column chromatography on silica gel
with 10-20% ethyl acetate in hexane. The fractions containing the
desired product were combined and concentrated to give 2.87 grams
of a clear, colorless oil. This material was still not pure by
.sup.1H NMR, so it was again subjected to flash column
chromatography on silica gel using dichloromethane. The appropriate
fractions were combined and concentrated to give 2.00 grams of the
desired product as a viscous, clear oil. .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta.=1.20 (t, 6H, CH.sub.3CH.sub.2O), 1.34 (s, 3H,
CH.sub.3CC.dbd.O), 1.92 [s, 12H, (CH.sub.3).sub.2CBr], 1.98 (app q,
2H, CHCH.sub.2CH.sub.2), 3.50 (m, 2H, OCH.sub.2CH.sub.3), 3.66 (m,
2H, OCH.sub.2CH.sub.3), 4.24 (t, 2H, CH.sub.2CH.sub.2OC.dbd.O),
4.37 (app q, 4H, CH.sub.2OC.dbd.OCBr), 4.60 (t, 1H, O--CH--O).
Example 14
Preparation of Vinyl Bis(Bromopropionate) Initiator 1
##STR00099##
[0312] A 100 ml round-bottom flask equipped with a stir bar was
charged with 30 ml of dichloromethane, 86 milligrams of
4-penten-1-ol, 432 milligrams of the dibromo acid from Example 7,
and 88 milligrams of DPTS. Nitrogen was bubbled through the
solution briefly, and 169 .mu.l of N,N'-diisopropylcarbodiimide was
added slowly. The reaction was allowed to stir at room temperature
overnight, then another 0.1 grams DPTS was added and the reaction
was again stirred overnight. Filtration and evaporation gave an
oily residue, which was purified by flash chromatography on silica
gel using 20-40% ethyl acetate in hexane. The solvent was removed
from the first product to come off the column, yielding 0.13 grams
of the desired product as a colorless oil. .sup.1H NMR (400 MHz,
CD.sub.3OD): .delta.=1.34 (s, 3H, CH.sub.3), 1.77 (m, 2H,
CH.sub.2CH.sub.2CH.sub.2), 1.90 (s, 12H, CH.sub.3), 2.15 (q, J=7.2
Hz, 2H, CHCH.sub.2CH.sub.2), 4.16 (t, J=6.4 Hz, 2H, OCH.sub.2),
4.36 (app q, 4H, CCH.sub.2O), 5.02 (m, 2H, CH.sub.2.dbd.CH), 5.82
(m, 1H, CH.sub.2.dbd.CH).
Example 15
Preparation of Vinyl Bis(Bromopropionate) Initiator 2
##STR00100##
[0314] A 100 ml round-bottom flask equipped with a stir bar was
charged with 25 ml dichloromethane, 370 milligrams of ethylene
glycol monovinyl ether, 432 milligrams of the dibromo acid from
Example 7, and 590 grams of DPTS. The flask was flushed with
nitrogen, and 681 .mu.l of N,N'-diisopropylcarbodiimide was added
slowly. The reaction was allowed to stir at room temperature
overnight. The mixture was filtered and then dried onto silica gel
for flash chromatography using 5-10% ethyl acetate in hexane,
yielding the product as a colorless oil. .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta.=1.36 (s, 3H, CH.sub.3), 1.92 (s, 12H,
CH.sub.3), 3.90 (app q, J=5.4 Hz, 2H, NCH.sub.2CH.sub.2O), 4.05
(dd, 1H, J=2.4, 6.8 Hz, .dbd.CH), 4.19 (dd, J=2.4, 14.4 Hz, 1H,
.dbd.CH), 4.39 (m, 2H, NCH.sub.2CH.sub.2O), 4.40 (app q, 4H,
OCH.sub.2), 6.45 (dd, 1H, J=6.8, 14.4 Hz, .dbd.CHO).
Example 16
Preparation of Boc-Amino Bis(Maleimide) Initiator
##STR00101##
[0316] A solution of 2.19 grams of N-Boc-3-amino-1-propanol and
5.20 grams of 2,2-bis(2-bromoisobutyryloxymethyl)propionic acid in
50 ml of dichloromethane, together with 350 mg of DPTS, was treated
with 3.0 grams of N,N'-dicyclohexylcarbodiimide and the reaction
was stirred at ambient temperature overnight. The reaction mixture
was filtered, and the precipitate was washed with a small amount of
dichloromethane. Concentration gave a residue, which was subjected
to flash column chromatography on silica gel with 5-20% ethyl
acetate in hexane. The appropriate fractions were combined and
concentrated to give an oil containing a little solid residue. This
material was taken up in ethyl acetate and filtered. Concentration
again gave an oil still containing a little solid, so the material
was again taken up in ethyl acetate, filtered, and concentrated to
give the desired product as a clear oil. .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta.=4.8 (br s, 1H, NH), 4.37 (app q, 4H,
CH.sub.2OC.dbd.OCBr), 4.22 (t, 2H, CH.sub.2CH.sub.2OC.dbd.O), 3.20
(app q, 2H, NHCH.sub.2), 1.92 [s, 12H, (CH.sub.3).sub.2CBr], 1.85
(t, 2H, CH.sub.2CH.sub.2CH.sub.2), 1.43 (s, 9H, (CH.sub.3).sub.3O),
1.35 (s, CH.sub.3CC.dbd.O).
Example 17
Preparation of N-(3-Propionic acid, t-butyl
ester)-2,2-Bis[(2-bromoisobutyryloxy)methyl]propionamide
##STR00102##
[0318] A solution of 1.00 grams of b-alanine t-butyl ester
hydrochloride in 50 ml of dichloromethane was treated with 25 ml of
saturated aqueous sodium bicarbonate, and the mixture was stirred
for 15 minutes. The layers were separated, and the organics were
dried over sodium sulfate. To this solution was added 2.38 grams of
2,2-bis[(2-bromoisobutyryloxy]methyl)propionic acid, followed by
1.92 ml of diisopropylethylamine and 2.1 grams of HBTU, and the
reaction was stirred at room temperature overnight. The reaction
mixture was then diluted with another 50 ml of dichloromethane,
washed with 2.times.50 ml of water, and dried over sodium sulfate.
Filtration and concentration gave an oil, which was subjected to
flash column chromatography with 20-25% ethyl acetate in hexane.
The appropriate fractions were combined and concentrated to give
730 mg of a white solid. NMR (CDCl.sub.3): .delta. 6.70 (t, 1H, NH,
J=5.4 Hz), 4.33 (app q, 4H, CH.sub.2O, J=16.3, 11.4 Hz), 3.51 (q,
2H, NCH.sub.2, J=6.0 Hz), 2.46 (t, 2H, CH.sub.2CO, J=6.0 Hz), 1.93
(s, 12H, Br--C(CH.sub.3).sub.2), 1.45 (s, 9H, C(CH.sub.3).sub.3),
1.33 (s, 3H, CH.sub.3).
Example 18
Preparation of Protected Maleimide 4-Ol
##STR00103##
[0320] A 100 ml round-bottom flask equipped with a stir bar was
charged with 30 ml of dichloromethane, 1.6 grams of the diol from
Example 7, 1.71 grams of
isopropylidene-2,2-bis(hydroxymethyl)propionic acid, and 0.5 grams
of DPTS. Nitrogen was bubbled through the solution briefly, 1.70 ml
of N,N'-diisopropylcarbodiimide was added slowly, and the reaction
was allowed to stir at room temperature overnight. Filtration and
evaporation gave an oily residue, which was purified by flash
chromatography on silica gel using 10-40% ethyl acetate in hexane.
A second purification by flash chromatography on silica gel using
2% methanol in dichloromethane yielded about 2 grams of colorless
oil. This oil was dissolved in 25 ml of methanol and stirred for 60
hours at room temperature with Dowex 50WX8-100 resin (H.sup.+
form). The reaction was filtered, concentrated, then passed through
a silica gel plug with 150 ml of 15% methanol in dichloromethane.
Evaporation yielded 1.3 grams of a nearly colorless hard foam.
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta.=1.13 (s, 6H, CH.sub.3),
1.25 (s, 3H, CH.sub.3), 2.96 (s, 2H, CHC.dbd.ON), 3.57-3.65 (m, 8H,
CH.sub.2OH), 3.64 (t, J=2.8 Hz, 2H, CH.sub.2CH.sub.2OC.dbd.O), 4.22
(app q, 4H, C(CH.sub.3)CH.sub.2OC.dbd.O.sub.1), 4.22 (t, J=2.8 Hz,
CH.sub.2CH.sub.2OC.dbd.O), 5.21 (t, J=0.8 Hz, CHOCH), 6.55 (t,
J=0.8 Hz, CH.dbd.CH).
Example 19
Preparation of Protected Maleimide Tetra(Bromopropionate)
Initiator
##STR00104##
[0322] A 100 ml round-bottom flask equipped with a stir bar was
charged with 20 ml of dichloromethane, 0.55 grams of the tetraol
from Example 13, and 1.69 ml of triethyl amine. The stirring
mixture was cooled to 0 degrees, and a solution of 0.99 ml of
2-bromoisobutyryl bromide in 10 ml dichloromethane was added drop
wise. The reaction was allowed to stir at room temperature
overnight, then washed with 50 ml of half-saturated sodium
bicarbonate. Concentration of the reaction mixture gave an oily
brown residue, which was purified by flash chromatography on silica
gel with 40% ethyl acetate in hexane. The brown residue was
dissolved in methanol and treated with charcoal to remove color,
yielding 0.68 grams of the desired product as a light brown oil.
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta.=1.26 (s, 3H,
CH.sub.3CC.dbd.O), 1.34 (s, 6H, CH.sub.3CC.dbd.O), 1.90 (s, 24H,
(CH.sub.3).sub.2CBr), 2.95 (s, 2H, CH), 3.78 (t, J=5 Hz, 2H,
NCH.sub.2), 4.25 (m, 6H, OCH.sub.2C (4H) and OCH.sub.2CH.sub.2N
(2H)), 4.35 (app q, 8H, OCH.sub.2), 5.23 (t, J=1 Hz, 2H, CHOCH),
6.55 (t, J=1 Hz, 2H, CH.dbd.CH).
Example 20
Preparation of 2,2-Bis[(2-bromoisobutyryloxy)methyl]propionic acid,
2-hydroxyethyl ester initiator
##STR00105##
[0324] A solution of 4.32 grams of
2,2-bis[(2-bromoisobutyryloxy]methyl)propionic acid and 12.41 grams
of ethylene glycol in 50 ml of dichloromethane, together with 883
mg of DPTS was treated with 1.39 grams of diisopropylcarbodiimide,
and the reaction was stirred at room temperature overnight. The
reaction mixture was concentrated, then partitioned between 150 ml
of ethyl acetate and 70 ml of water. The organic layer was
concentrated, and the residue was subjected to flash column
chromatography on silica gel with 20%-40% ethyl acetate in hexane.
The appropriate fractions were combined and concentrated to give
2.7 grams of the desired product as a clear oil. NMR (CD.sub.3OD):
.delta. 4.38 (app q, 4H, CCH.sub.2, J=11.2, 30.2 Hz), 4.20 (t, 2H,
CH.sub.2OH, J=5.0 Hz), 3.75 (t, 2H, CH.sub.2CH.sub.2OH, J=5.0 Hz),
1.90 (s, 12H, Br--CCH.sub.3), 1.36 (s, 3H, CH.sub.3).
Example 21
Preparation of 2,2-Bis[(2-bromoisobutyryloxy)methyl]propionic acid,
3-hydroxypropyl ester initiator
##STR00106##
[0326] A solution of 5.31 grams of
2,2-bis[(2-bromoisobutyryloxy)methyl]propionic acid and 4.68 grams
of 1,3-propanediol in 80 ml of dichloromethane and 20 ml of
acetonitrile was treated with 1.0 grams of DPTS, followed by 3.0
grams of DCC, and the reaction was stirred at room temperature for
2 hours. The reaction was then filtered, concentrated and the
residue was subjected to flash column chromatography on silica gel
with 30% ethyl acetate in hexane. The appropriate fractions were
combined and concentrated to give a clear oil, which was not quite
pure. Rechromatography on silica gel with 10-15% acetone in hexane
gave the desired product as a clear, colorless oil. NMR
(CDCl.sub.3): .delta. 4.3.8 (app q, 4H, CCH.sub.2O, J=11.2 Hz),
4.31 (t, 2H, CH.sub.2CH.sub.2O, J=6.3 Hz), 3.71 (q, 2H, CH.sub.2OH,
J=5.9 Hz), 1.92 (s, 12H, Br--C(CH.sub.3).sub.2), 1.9 (m, 2H,
CH.sub.2CH.sub.2CH.sub.2), 1.35 (s, 3H, CH.sub.3).
Example 22
2,2-Bis[(2-bromoisobutyryloxy)methyl]propionic acid,
11-hydroxy-3,6,9-trioxaundecanoate initiator
##STR00107##
[0328] A solution of 1.86 grams of
2,2-bis[(2-bromoisobutyryloxy)methyl]propionic acid and 4.18 grams
of tetraethylene glycol in 50 ml of dichloromethane, together with
250 mg of DPTS, was treated with 1.15 grams of DCC and the reaction
was stirred at room temperature overnight. The reaction was
filtered and the filtrate was diluted with 50 ml of dichloromethane
and washed with 20 ml of water. The organics were dried over sodium
sulfate, filtered and concentrated to give a residue, which was
subjected to flash column chromatography on silica gel first with
50-70% ethyl acetate in hexane. The appropriate fractions were
combined, filtered and concentrated to give 1.19 grams of the
desired product as a clear, colorless oil. NMR (CDCl.sub.3):
.delta. 4.38 (app q, 4H, CCH.sub.2O, J=31.8, 11.2 Hz), 4.31 (t, 2H,
CH.sub.2CH.sub.2OC.dbd.O, J=5.0 Hz), 3.6-3.73 (m, 14H, CH.sub.2O),
2.46 (t, 1H, OH, J=6.3 Hz), 1.92 (s, 12H, Br--C(CH.sub.3).sub.2),
1.35 (s, 3H, CH.sub.3).
Example 23
Preparation of 2,2-Bis[(2-bromoisobutyryloxy)methyl]propionic acid,
11-hydroxy-3,6,9-trioxaundecanoate, NHS carbonate initiator
##STR00108##
[0330] A solution of 630 grams of the above hydroxyl compound and
1.28 grams of disuccinimidyl carbonate in 3 ml of dry acetonitrile
was treated with 610 mg of DMAP and the reaction was stirred at
room temperature. The reaction was still heterogeneous, so 4 ml of
dry THF were added, and after 2 hours the reaction turned yellow
and became homogeneous, but contained several spots on tlc (silica
gel, 50% ethyl acetate in hexane). The reaction was concentrated to
give a residue which was subjected to flash column chromatography
on silica gel with 50-60% ethyl acetate in hexane. Two fractions
were isolated, and the fraction with a lower rf was concentrated to
give 260 mg of the desired product as a clear oil. NMR
(CDCl.sub.3): .delta. 4.47 (m, 2H, CH.sub.2O(C.dbd.O)O), 4.37 (app
q, 4H, CCH.sub.2O, J=11.2, 31.6 Hz), 4.30 (m, 2H,
CH.sub.2CH.sub.2O(C.dbd.O)C), 3.79 (m, 2H,
CH.sub.2CH.sub.2O(C.dbd.O)C), 3.71 (t, 2H,
CH.sub.2CH.sub.2O(C.dbd.O)O, J=5.0 Hz), 3.67 (s, 4H, CH.sub.2O),
3.65 (s, 4H, CH.sub.2O), 2.84 (s, 4H, CH.sub.2C.dbd.O), 1.92 (s,
12H, Br--C (CH.sub.3).sub.2), 1.35 (s, 3H, CH.sub.3).
Example 24
Preparation of 2,2-Bis[(2-bromoisobutyryloxy)methyl]propionic acid,
solketal ester initiator
##STR00109##
[0332] A solution of 918 mg of solketal and 3.0 grams of
2,2-bis[(2-bromoisobutyryloxy)methyl]propionic acid, together with
200 mg of DPTS was treated with 2.15 grams of DCC and the reaction
was stirred at room temperature overnight. The reaction was
filtered to give a residue, which was subjected to flash column
chromatography on silica gel with 10% ethyl acetate in hexane. The
appropriate fractions were combined and concentrated to give 1.85
grams of the desired product as a clear, colorless oil. NMR
(CDCl.sub.3): .delta. 4.38 (app q, 4H, CCH.sub.2O), 4.32 (m, 1H,
OCH), 4.19 (m, 2H, CHCH.sub.2OC.dbd.O), 4.07 (d of d, 1H,
OCH.sub.2CH, J=6.7, 8.6 Hz), 3.76 (d of d, 1H, OCH.sub.2CH, J=5.7,
8.6 Hz), 1.92 (s, 12H, Br--C(CH.sub.3).sub.2), 1.43 (s, 3H,
(CH.sub.3).sub.2CO), 1.36 (s, 3H, CH.sub.3), 1.35 (s, 3H,
(CH.sub.3).sub.2CO).
Example 25
Preparation of 2,2-Bis[(2-bromoisobutyryloxy)methyl]propionic acid,
2,3-dihydroxypropyl ester initiator
##STR00110##
[0334] A solution of 1.0 grams of the previous ketal in 50 ml of
methanol was treated with 750 mg of Dowex 50Wx8-100 and the
reaction was stirred overnight. The reaction was then filtered,
concentrated, and the residue was subjected to flash column
chromatography on silica gel with 20-40% ethyl acetate in hexane.
The appropriate fractions were combined and concentrated to give
630 mg of the desired product as a clear, colorless oil. NMR
(CDCl.sub.3+D.sub.2O): .delta. 4.40 (app q of d, 4H, CCH.sub.2O,
J=2.8, 11.5, 30.2 Hz), 4.24 (app q of d, 2H, CHCH.sub.2OC.dbd.O,
J=4.5, 6.6, 11.5 Hz), 3.96 (m, 1H, CH), 3.66 (app q of d, 2H,
HOCH.sub.2CH, J=3.8, 5.6, 11.5, 37.9 Hz), 1.92 (s, 12H,
Br--C(CH.sub.3).sub.2), 1.37 (s, 3H, CH.sub.3).
Example 26
Preparation of 2,2-Bis[(2-bromoisobutyryloxy)methyl]propionic acid,
2-(2,3-dihydroxypropoxy)ethyl ester initiator
##STR00111##
[0336] To a solution of 1.5 grams of
2-[(2-bromoisobutyryloxy)methyl]-2-hydroxymethylpropionic acid,
2-(allyloxy)ethyl ester in 15 ml of water and 15 ml of t-butanol
was added 2.86 grams (3 eq) of potassium ferricyanide, 1.20 grams
(3 eq) of potassium carbonate, 7.5 mg of potassium osmate
dehydrate, 11 mg of quinuclidine, and 276 mg (1 eq) of
methanesulfonamide, and the reaction mixture was stirred at room
temperature overnight. The reaction appeared to be complete by TLC
(silica gel, 50% ethyl acetate in hexane), so the reaction was
poured into 100 ml of water, then extracted with 100 ml of
dichloromethane. The combined organics were dried over sodium
sulfate, filtered and concentrated to give an oily residue, which
was subjected to flash column chromatography on silica gel with
30-40% ethyl acetate in hexane. The appropriate fractions were
combined, treated with decolorizing carbon, filtered and
concentrated to give 850 mg of the desired product as a nearly
colorless oil. NMR (CDCl.sub.3): .delta.4.39 (app q of d, 4H,
CCH.sub.2O, J=4.1, 11.1, 3.0, 37.6 Hz), 4.31 (t, 2H,
OCH.sub.2CH.sub.2OC.dbd.O, J=4.7 Hz), 3.87 (m, 1H, CH--OH),
3.54-3.77 (m, 2H, CH.sub.2--OH), 3.72 (m, 2H, OCH.sub.2CH), 3.58
(app t, 2H, OCH.sub.2CH.sub.2OC.dbd.O), 2.68 (d, 1H, CH--OH, J=5.1
Hz), 2.15 (app t, 1H, CH.sub.2--OH, J=6.1 Hz), 1.92 (s, 12H,
Br--C(CH.sub.3).sub.2), 1.36 (s, 3H, CH.sub.3).
Example 27
2,2-Bis[(2-bromoisobutyryloxy)methyl]propionic acid,
12-(allyloxy)-3,6,9,12-tetraoxadodecanoate initiator
##STR00112##
[0338] To a solution of 1.60 g of
2,2-bis[(2-bromoisobutyryloxy)methyl]propionic acid and 870 mg of
12-(allyloxy)-3,6,9,12-tetraoxadodecane in 30 ml of dry
acetonitrile, together with 218 mg of DPTS and 362 mg of DMAP, was
added 917 mg of DCC and the reaction was stirred at room
temperature overnight. The mixture was then filtered and
concentrated, and the residue was subjected to flash column
chromatography on silica gel first with 50-60% ethyl acetate in
hexanes, and the product containing fractions were combined and
concentrated to give 1.35 grams of the desired product as a clear,
colorless oil. NMR (CDCl.sub.3): .delta. 5.87-5.97 (m, 1H,
CH.sub.2CH.dbd.CH.sub.2), 5.28 (dq, 1H, H--CH.dbd.CH), 5.18 (dq,
1H, H--CH.dbd.CH), 4.37 (app q, CH.sub.2OC.dbd.O), 4.30 (dd, 2H,
CH.sub.2CH.sub.2OC.dbd.O), 4.02 (d, 2H, CH.sub.2.dbd.CHCH.sub.2),
3.60-3.72 (m, 14H, CH.sub.2CH.sub.2OCH.sub.2), 1.92 (s, 12H,
Br--C(CH.sub.3).sub.2), 1.35 (s, 3H, CH.sub.3).
Example 28
Preparation of 2,2-Bis[(2-bromoisobutyryloxy)methyl]propionic acid,
12-(2,3-dihydroxypropoxy)-3,6,9,12-tetraoxadodecyl ester
initiator
##STR00113##
[0340] To a mixture of 1.29 grams of
2,2-bis[(2-bromoisobutyryloxy)methyl]propionic acid,
12-(allyloxy)-3,6,9,12-tetraoxadodecyl ester in 15 ml of water and
15 ml of t-butanol was added 1.98 grams (3 eq) of potassium
ferricyanide, 829 mg (3 eq) of potassium carbonate, 8 mg of
potassium osmate dehydrate, 11 mg of quinuclidine, and 190 mg (1
eq) of methanesulfonamide, and the reaction mixture was stirred at
room temperature overnight. The reaction appeared to be complete by
TLC (silica gel, 50% ethyl acetate in hexane), so the reaction was
poured into 50 ml of water, then extracted with 100 ml of
dichloromethane. The combined organics were dried over sodium
sulfate, filtered and concentrated to give an oily residue, which
was subjected to flash column chromatography on silica gel with 5%
methanol in dichloromethane. The product containing fractions were
combined and treated twice with two small spatulafuls of activated
carbon, filtering between treatments. Filtration and concentration
gave a light gray oil containing a small amount of solid, so it was
taken up in ethyl acetate and filtered, then concentrated to give
1.06 grams of the desired product as a light gray oil, still
containing a tiny amount of solid. NMR (CDCl.sub.3): .delta. 4.38
(app q, 4H, CCH.sub.2OC.dbd.O), 4.30 (t, 2H,
CH.sub.2CH.sub.2OC.dbd.O, J=5.0 Hz), 3.85 (p, 1H, CHOH, J=5 Hz),
3.71 (t, 2H, OCH.sub.2CHOH, J=4.8 Hz), 3.72-3.55 (m, 16H,
OCH.sub.2CH.sub.2O and CH.sub.2OH), 3.12 (s, 1H, CHOH), 2.37 (s,
1H, CH.sub.2OH), 1.92 (s, 12H, Br--C(CH.sub.3).sub.2), 1.35 (s, 3H,
CH.sub.3).
Example 29
Preparation of 2,2,5-Trimethyl-1,3-dioxane-5-carboxylic acid,
2-(allyloxy)ethyl ester
##STR00114##
[0342] A solution of 1.4 grams of ethylene glycol monoallyl ether
and 2.35 grams of 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid in
25 ml of anhydrous THF was treated with 500 mg of
4-dimethylaminopyridinium p-toluenesulfonate (DPTS) and 1.44 grams
of dimethylaminopyridine (DMAP), followed by the addition of 3.38
grams of dicyclohexylcarbodiimide, and the reaction was stirred at
room temperature for 3 days. The reaction mixture was filtered and
concentrated to give a semisolid residue, which was subjected to
flash column chromatography on silica gel with 20% ethyl acetate in
hexane. The product containing fractions were combined,
concentrated and filtered to give 2.83 grams (81%) of a clear oil
containing a small amount of solid. .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta.=1.23 (s, 3H, C.dbd.OCCH.sub.3), 1.39 (s, 3H,
CH.sub.3), 1.43 (s, 3H, CH.sub.3), 3.66 (m, 4H), 4.02 (dd, 2H,
CH.sub.2.dbd.CHCH.sub.2), 4.20 (d, 2H), 4.31 (t, 2H,
C.dbd.OOCH.sub.2), 5.18 (dd, 1H, .dbd.CH), 5.28 (dd, 1H, .dbd.CH),
5.89 (m, .dbd.CHCH.sub.2).
Example 30
2,2-Bis(hydroxymethyl)propionic acid, 2-(allyloxy)ethyl ester
##STR00115##
[0344] A solution of 2.72 grams of
2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid, 2-(allyloxy)ethyl
ester in 50 ml of methanol was treated with 1.0 gram of Dowex
50W-X8 resin (H+ form) and the reaction was stirred at room
temperature overnight. The reaction was filtered, and the filtrate
was concentrated to give an oil, which was subjected to flash
column chromatography on silica gel with 5% methanol in
dichloromethane. The product containing fractions were combined and
concentrated to give 2.23 grams of the product as a clear, light
yellow oil. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.=5.84-5.94
(ddt, 1H, H.sub.2C.dbd.CHCH.sub.2), 5.28 (dq, 1H,
HHC.dbd.CHCH.sub.2), 5.22 (dq, 1H, HHC.dbd.CHCH.sub.2), 4.36 (app
t, 2H, OCH.sub.2CH.sub.2), 4.02 (dt, 2H, H.sub.2C.dbd.CHCH.sub.2),
3.86 (dd, 2H, CH.sub.2OH), 3.74 (dd, 2H, CH.sub.2OH), 3.68 (app t,
2H, OCH.sub.2CH.sub.2), 2.90 (br d, 2H, OH), 1.11 (s,
CH.sub.3).
Example 31
Preparation of 2,2-Bis[(2-bromoisobutyryloxy)methyl]propionic acid,
2-(allyloxy)ethyl ester initiator
##STR00116##
[0346] A solution of 1.2 grams of allyloxyethanol, 5.0 grams of
2,2-bis(2-bromoisobutyryloxymethyl) propionic acid and 690 mg of
DPTS in 100 ml of dichloromethane was stirred at room temperature
as 2.86 grams of DCC were added as a solution in a small amount of
dichloromethane. The reaction was stirred at room temperature
overnight, then filtered and concentrated to give an oil. This was
subjected to flash chromatography on silica gel with 10% ethyl
acetate in hexane. The appropriate fractions were combined and
concentrated to give a clear oil, which was not sufficiently pure.
This oil was again subjected to flash chromatography on silica gel
with 3-4% ethyl acetate in hexane. The product containing fractions
were combined and concentrated to give 2.78 grams of the desired
product as a clear, colorless oil. NMR (CDCl.sub.3): .delta. 5.89
(m, 1H, CH.sub.2CH.dbd.CH.sub.2), 5.28 (d of q, 1H, H--CH.dbd.CH,
J=17.2, 1.7 Hz), 5.20 (d of q, 1H, H--CH.dbd.CH, J=10.5, 1.5 Hz),
4.38 (app q, 4H, CH.sub.2OC.dbd.O), 4.31 (t, 2H, OCH.sub.2, J=4.7
Hz), 4.01 (d of t, 2H, OCH.sub.2, J=5.6, 1.5 Hz), 3.65 (t, 2H,
OCH.sub.2, J=4.7 Hz), 1.91 (s, 12H, Br--C(CH.sub.3).sub.2), 1.35
(s, 3H, CH.sub.3).
Example 32
2,2-Bis-[2,2-bis(2-bromoisobutyryloxymethyl)propionyloxymethyl]propionic
acid, 2-(allyloxy)ethyl ester initiator
##STR00117##
[0348] A solution of 2.42 grams of
2-[(2-bromoisobutyryloxy)methyl]-2-hydroxymethylpropionic acid,
2-(allyloxy)ethyl ester and 1.73 grams of
2,2-[bis-(2-bromoisobutyryloxy)methyl]propionic acid in 25 ml of
dry acetonitrile, together with 200 mg of DPTS and 580 mg of DMAP,
was treated with 1.03 grams of DCC, and the reaction was stirred at
room temperature overnight. By TLC (silica gel, 30% ethyl acetate
in hexane) it appeared that the reaction was incomplete, so another
812 mg of 2,2-[bis-(2-bromoisobutyryloxy)methyl]propionic acid and
400 mg of DCC were added, and the reaction was again stirred at
room temperature overnight. The reaction mixture was filtered and
concentrated, and the residue was subjected to flash column
chromatography on silica gel first with 20%, and then with 30%
ethyl acetate in hexanes. The product containing fractions were
combined and concentrated to give 1.27 grams of the desired
compound as a clear, colorless oil. NMR (CDCl.sub.3): .delta. 5.88
(m, 1H, CH.sub.2CH.dbd.CH.sub.2), 5.28 (d of q, 1H, H--CH.dbd.CH,
J=17.4, 1.6 Hz), 5.20 (d of q, 1H, H--CH.dbd.CH, J=10.3, 1.3 Hz),
4.24-4.44 (m, 14H, CH.sub.2OC.dbd.O), 4.01 (d, 2H,
CH.sub.2.dbd.CHCH.sub.2, J=5.6), 3.65 (t, 2H,
CH.sub.2CH.sub.2OCH.sub.2, J=4.7 Hz), 1.91 (s, 24H,
Br--C(CH.sub.3).sub.2), 1.33 (s, 6H, CH.sub.3), 1.30 (s, 3H,
CH.sub.3).
Example 33
Preparation of 2,2-Bis-[2,2-Bis[(2-Bromoisobutyryloxy)
propionyloxymethyl]propionic acid], 2-[(2,3-dihydroxy)propoxy]ethyl
ester initiator
##STR00118##
[0350] To a mixture of 1.21 grams of
2,2-bis[(2-bromoisobutyryloxy)methyl]propionic acid,
2-(allyloxy)ethyl ester in 15 ml of water and 15 ml of t-butanol
was added 1.14 grams (3 eq) of potassium ferricyanide, 480 mg (3
eq) of potassium carbonate, 7.5 mg of potassium osmate dehydrate,
11 mg of quinuclidine, and 110 mg (1 eq) of methanesulfonamide, and
the reaction mixture was stirred at room temperature overnight. The
reaction appeared to be complete by tlc (silica gel, 50% ethyl
acetate in hexane), so the reaction was poured into 50 ml of water,
then extracted with 100 ml of dichloromethane, followed by another
50 ml of dichloromethane. The combined organics were dried over
sodium sulfate, filtered and concentrated to give an oily residue,
which was subjected to flash column chromatography on silica gel
with 50% ethyl acetate in hexane, and the product containing
fractions were combined and concentrated to give 620 mg of the
desired product as a clear, colorless oil. NMR (CDCl.sub.3):
.delta. 4.28-4.41 (m, 14H, CCH.sub.2OC.dbd.O), 3.86 (m, 1H,
CH.sub.2CHOHCH.sub.2), 3.69-3.75 (m, 3H), 3.56-3.65 (m, 3H), 2.78
(dd, 1H, OH), 2.23 (app t, 1H, OH), 1.92 (s, 24H, CH.sub.3CBr),
1.34 (s, 6H, CH.sub.3), 1.31 (s, 3H, CH.sub.3).
Example 34
Preparation of 2,2-bis[(2-bromoisobutyryloxy)methyl]propionic acid,
(2-azidoethoxy)ethyl ester initiator
##STR00119##
[0352] To a solution of 3.30 grams of
2,2-bis[(2-bromoisobutyryloxy)methyl]propionic acid and 1.0 gram of
2-(2-azidoethoxy)ethanol in 20 mL of dry acetonitrile, together
with 225 mg of DPTS, was added 1.89 grams of DCC and the reaction
was stirred at room temperature overnight. The reaction was
filtered and concentrated to give a residue, which was subjected to
flash column chromatography on silica gel with 10-15% ethyl acetate
in hexane. The appropriate fractions were combined and concentrated
to give 2.06 grams of the desired product as a clear, colorless
oil. NMR (CDCl.sub.3): .delta. 4.39 (app q, 4H, CCH.sub.2O, J=11.1,
33.8 Hz), 4.31 (t, 2H, OCH.sub.2CH.sub.2OC.dbd.O, J=5 Hz), 3.72 (t,
2H, CH.sub.2N.sub.3, J=5 Hz), 3.67 (t, 2H, CH.sub.2CH.sub.2N.sub.3,
J=5 Hz), 3.38 (t, 2H, OCH.sub.2CH.sub.2OC.dbd.O, J=5 Hz), 1.92 (s,
12H, Br--C(CH.sub.3).sub.2), 1.36 (s, 3H, CH.sub.3).
Example 35
Preparation of 3,5-bis-(2-bromoisobutyryloxy)benzaldehyde
##STR00120##
[0354] A solution of 1.0 gram of 3,5-dihydroxybenzaldehyde and 4.0
ml (4 eq) of triethylamine in 20 ml of dichloromethane was cooled
with an ice-water bath, and a solution of 3.35 grams of
2-bromoisobutyryl bromide in 5 ml of dichloromethane was added
dropwise over a few minutes as much solid formed. The reaction was
stirred at room temperature for 1.5 hr, at which time the reaction
appeared to be complete by TLC (silica gel, 30% ethyl acetate in
hexane). The reaction was washed with 25 ml of water, then
concentrated to give a residue, which was subjected to flash column
chromatography on silica gel with 10% ethyl acetate in hexane. The
appropriate fractions were combined, treated with a small amount of
decolorizing carbon, filtered and concentrated to give 2.2 grams of
an oil, which crystallized in the refrigerator to give a white
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.=2.08 (s, 12H,
CH.sub.3), 7.29 (t, 1H, J=2.4 Hz, ArH), 7.61 (d, J=2.4 Hz, 211,
ArH), 10.0 (s, 1H, CHO).
Example 36
Preparation of
7-(13-allyloxy-2,5,8,11-tetraoxamidecyl)-2,4,9-triphenyl-1,3,5-triazatric-
yclo[3.3.1.13,7]decane
##STR00121##
[0356] A solution of 870 mg of 11-allyloxy-3,6,9-trioxaundecan-1-ol
methanesulfonate and 1.01 grams of
2,4,9-triphenyl-1,3,5-triazatricyclo[3.3.1.13,7]decane-7-methanol
(WO2000/037658) in 10 ml of dry THF was treated with 410 mg of
sodium hydride (60% in oil) and the reaction was heated at
80.degree. C. for 20 hours. The reaction was then quenched
carefully by the addition of a few ml of water, poured into 20 ml
of sat NaCl, then extracted with 3.times.10 ml of dichloromethane.
The organics were dried over sodium sulfate, filtered and
concentrated to give a residue, which was subjected to flash
chromatography on silica gel with 25-35% ethyl acetate in hexane.
The appropriate fractions were combined and concentrated to give
920 mg of the desired product as a colorless oil. NMR
(DMSO-d.sub.6): .delta. 7.70-7.82 (m, 6H, PhH), 7.26-7.51 (m, 9H,
PhH), 3.69-3.75 (m, 3H), 3.56-3.65 (m, 3H), 2.78 (dd, 1H, OH), 2.23
(app t, 1H, OH), 1.92 (s, 24H, CH.sub.3CBr), 1.34 (s, 6H,
CH.sub.3), 1.31 (s, 3H, CH.sub.3).
Example 37
Preparation of
1-Amino-15-allyloxy-2,2-bis(aminomethyl)-4,7,10,13-tetraoxapentadecane
trihydrochloride
##STR00122##
[0358] The triazaadamantane compound from the previous reaction was
taken up in 20 ml of ethanol and 4 ml of ether, then treated with 2
ml of concentrated hydrochloric acid. The reaction was mixed and
then left to stand at 4.degree. C. for 1.5 hours. Then 30 ml of
ether were added and the mixture was cooled again for another 30
minutes. Then added 100 ml of ether and the solid product was
recovered by filtration, washed with ether and dried under vacuum
to give 564 mg of the product as a white solid. NMR (DMSO-d.sub.6):
.delta. 7.75 (m, 6H, CCH), 7.44 (m, 6H, CCHCH), 7.30 (m, 3H,
CCHCHCH), 5.86 (m, 1H, CH.sub.2.dbd.CH), 5.70 (s, 1H, NCH
(equatorial)), 5.250 (s, 2H, NCH(axial)), 5.23 (d of q, 1H,
CH.sub.2.dbd.CH), 5.11 (d of q, 1H, CH.sub.2.dbd.CH), 3.93 (d of t,
2H, CH--CH.sub.2--O), 3.55-3.25 (m, 16H, OCH.sub.2CH.sub.2O), 3.26
(m, 2H, NCH.sub.2), 3.19 (d, 2H, NCH.sub.2), 2.88 (s, 2H,
NCH.sub.2), 2.719 (s, 2H, CCH.sub.2O).
Example 38
Preparation of
N-(2-Bromo-2-methylpropionyl)-1-Amino-15-allyloxy-2,2-bis[N-(2-bromo-2-me-
thylpropionyl)aminomethyl]-4,7,10,13-tetraoxapentadecane
initiator
##STR00123##
[0360] The triamine hydrochloride from the previous procedure was
taken up in 25 ml of dichloromethane, the solution was cooled with
and ice water bath, and treated with 1.35 ml of triethylamine,
followed by the addition of 0.46 ml of 2-bromoisobutyryl bromide.
The reaction was then stirred as it was allowed to warm to room
temperature over 2 hours. The reaction mixture was then washed with
3.times.10 ml of 1N HCl, 2.times.10 mL of sat NaHCO.sub.3, 10 ml of
sat NaCl, and dried over magnesium sulfate. The solution was
filtered and concentrated to give a residue, which was flushed
through a plug of silica gel with ethyl acetate. Concentration gave
989 mg of the desired product as a viscous oil. NMR (DMSO-d.sub.6):
.delta. 8.004 (t, 3H, NH), 5.87 (m, 1H, CH), 5.23 (d of q, 1H,
CH.sub.2.dbd.CH), 5.12 (d of q, 1H, CH.sub.2.dbd.CH), 3.93 (d of t,
2H, CH.sub.2--CH), 3.6-3.45 (m, 16H, OCH.sub.2CH.sub.2O), 3.289 (s,
2H, CCH.sub.2O), 3.12 (d, 6H, CCH.sub.2N), 1.907 (s, 18H,
CH.sub.3).
Example 39
Preparation of
N-(2-Bromo-2-methylpropionyl)-1-Amino-15-(2,3-dihydroxypropyl)-2,2-bis[N--
(2-bromo-2-methylpropionyl)aminomethyl]-4,7,10,13-tetraoxapentadecane
initiator
##STR00124##
[0362] To a mixture of 350 mg of the alkene from the previous
procedure in 5 ml of t-butanol and 5 ml of water was added 433 mg
(3 eq) of potassium ferricyanide, 182 mg (3 eq) of potassium
carbonate, 42 mg (1 eq) of methanesulfonamide, 7.5 mg of
quinuclidine, and 4 mg of potassium osmate dihydrate, and the
solution was stirred at room temperature overnight. The reaction
appeared to be complete by TLC (silica gel, 5% methanol in
dichloromethane), so 50 ml of water were added and the solution was
extracted with 50 ml of dichloromethane, followed by another
2.times.25 ml of dichloromethane. The combined extracts were dried
over sodium sulfate, concentrated, and the dark gray residue was
subjected to flash column chromatography on silica gel with 2-5%
methanol in dichloromethane. The appropriate fractions were
combined and concentrated to give 310 mg of the desired dihydroxy
compound as a light gray oil. NMR (CDCl.sub.3): .delta. 7.91 (t,
3H, NH), 3.88 (m, 1H, HOCH.sub.2CHOHCH.sub.2), 3.55-3.72 (complex
m, 21H), 3.35 (s, 1H, OCH.sub.2C(CH.sub.2).sub.3), 3.19 (d, 6H,
J=6.4 Hz, CH.sub.2NH), 1.99 (s, 18H, CH.sub.3).
Example 40
Preparation of
7-(7-Azido-2,5-dioxaheptyl)-2,4,9-triphenyl-1,3,5-triazatricyclo[3.3.1.13-
,7]decane
##STR00125##
[0364] To a solution of 1.1 grams of
2,4,9-triphenyl-1,3,5-triazatricyclo[3.3.1.13,7]decane-7-methanol
(WO2000/037658) and 585 mg of 2-(2-azidoethoxy)ethyl
methanesulfonate in 15 ml of anhydrous THF was added 224 mg of NaH
(60% in oil), and the solution was heated at 70.degree. C.
overnight. Another 245 mg of NaH and 600 mg of
2-(2-azidoethoxy)ethyl methanesulfonate were added, and heating was
again continued overnight. The reaction mixture was cooled, diluted
with 25 ml of water, and extracted with 50 ml of dichloromethane.
The organic layer was washed with saturated NaCl, dried over sodium
sulfate, filtered and concentrated to give a residue. This material
was subjected to flash column chromatography on silica gel with
10-25% ethyl acetate in hexane. The appropriate fractions were
combined and concentrated to give 1.15 grams of the desired product
as an oil, which was not completely pure, but used in the next
reaction without further purification. NMR (DMSO) extremely
complex.
Example 41
Preparation of 1-Amino-9-azido-2,2-bis(aminomethyl)-4,7-dioxanonane
trihydrochloride
##STR00126##
[0366] A solution of 1.15 grams of the triazaadamantane compound
from the previous procedure in 20 ml of ethanol and 4 ml of ether
was cooled with an ice water bath, and 3 ml of concentrated HCl
were added. Solid product began to form immediately, and the
reaction was allowed to stand in the cold for 10 minutes. Another
30 ml of ether were added, and the reaction was refrigerated
overnight. The reaction mixture was diluted with another 100 ml of
ether, and the solid product was isolated by filtration, washed
with more ether and dried under vacuum to give 800 mg of the
product as a white solid.
Example 42
Preparation of
N-(2-Bromo-2-methylpropionyl)-1-Amino-9-azido-2,2-bis[N-(2-bromo-2-methyl-
propionyl)aminomethyl]-4,7-dioxanonane initiator
##STR00127##
[0368] A solution of 800 mg of the trihydrochloride salt from the
previous procedure in 25 ml of dichloromethane was cooled with an
ice water bath, then treated with 3.5 ml of triethylamine. To this
mixture was added dropwise 1.07 ml of 2-bromoisobutyryl bromide,
and the reaction was stirred while warming to room temperature over
2 hours. The mixture was then washed with 3.times.10 ml of 1N HCl,
2.times.10 ml of saturated NaHCO3, and with 10 ml of saturated
NaCl, then dried over magnesium sulfate. Filtration and
concentration gave a residue, which was subjected to flash column
chromatography on silica gel with 20-30% ethyl acetate in hexane.
The appropriate fractions were combined and concentrated to give
630 mg of the desired product as an oil. NMR (CDCl.sub.3): .delta.
7.76 (t, 3H, NH, J=6.3 Hz), 3.68 (m, 4H, OCH.sub.2CH.sub.2O), 3.63
(m, 2H, N.sub.3CH.sub.2CH.sub.2O), 3.40 (t, 2H, N.sub.3CH.sub.2,
J=5.0 Hz), 3.37 (s, 2H, CCH.sub.2O), 3.19 (d, 6H, CCH.sub.2N, J=6.8
Hz), 1.99 (s, 18H, CH.sub.3).
Example 43
13-Allyloxy-2,5,8,11-tetraoxamidecyl 6-arm initiator
##STR00128##
[0370] To a solution of 0.9 grams of
1-amino-15-allyloxy-2,2-bis(aminomethyl)-4,7,10,13-tetraoxapentadecane
trihydrochloride and 3.89 grams of
2,2-bis[(2-bromoisobutyryloxy]methyl)propionic acid in 25 ml of
dichloromethane, together with 530 mg of DPTS and 890 mg of DMAP,
was added 2.7 grams of DCC and the reaction was stirred at room
temperature overnight. The reaction was filtered and concentrated,
and the residue was subjected to flash column chromatography on
silica gel with 50-70% ethyl acetate in hexane. The appropriate
fractions were combined and concentrated to give 1.9 grams of the
desired product as a viscous oil. NMR (CDCl.sub.3): .delta. 7.78
(t, 3H, NH, J=6.5 Hz), 5.91 (m, 1H, CH), 5.27 (d of q, 1H,
CH.sub.2.dbd.CH, J=17.4, 1.6 Hz), 5.18 (d of q, 1H,
CH.sub.2.dbd.CH, J=10.4, 1.4 Hz), 4.38 (app q, 12H,
CH.sub.2OC.dbd.O), 4.01 (d of t, 2H, CH--CH.sub.2, J=5.7, 1.4 Hz),
3.61 (two m, 16H, OCH.sub.2CH.sub.2O), 3.30 (s, 2H, CCH.sub.2O),
3.14 (d, 6H, CH.sub.2N, J=6.1 Hz), 1.92 (d, 36H,
BrC(CH.sub.3).sub.2, J=1.2 Hz), 1.38 (s, 9H, CH.sub.3).
Example 44
13-(2,3-Dihydroxypropyl)-2,5,8,11-tetraoxamidecyl 6-arm
initiator
##STR00129##
[0372] To a mixture of 1.0 gram of the alkene from the previous
procedure in 10 ml of water and 10 ml of t-butanol was added 638 mg
(3 eq) of potassium ferricyanide, 268 mg (3 eq) of potassium
carbonate, 10 mg of potassium osmate dehydrate, 12 mg of
quinuclidine, and 61 mg (1 eq) of methanesulfonamide, and the
reaction mixture was stirred at room temperature overnight. The
reaction was poured into 50 ml of water, then extracted with 50 ml
of dichloromethane, followed by another 25 ml of dichloromethane.
The combined organics were dried over sodium sulfate, filtered and
concentrated to give an oily residue, which was subjected to flash
column chromatography on silica gel with 2-4% methanol in
dichloromethane, and the product containing fractions were combined
and concentrated to give 417 mg of the desired product as a viscous
oil. NMR (CDCl.sub.3): .delta. 7.78 (t, 3H, NH, J=6.0 Hz), 4.39
(app q, 12H, CH.sub.2OC.dbd.O), 3.86 (broad s, 1H, OH--CH), 3.62
(m, 20H, OCH.sub.2CH.sub.2O and OHCHCH.sub.2O and OH--CH.sub.2),
3.27 (s, 2H, CCH.sub.2O), 3.13 (s, 6H, NCH.sub.2), 2.40 (s, 2H,
OH), 1.92 (s, 36H, BrC(CH.sub.3).sub.2), 1.38 (s, 9H,
CH.sub.3).
Example 45
Preparation of Hexaglutamic acid amide with
9-Azido-4,7-dioxanononanoic acid
Preparation of t-Butyl 9-hydroxy-4,7-dioxanonanoate methane
sulfonate
##STR00130##
[0374] A solution of 3.0 grams of t-Butyl
9-hydroxy-4,7-dioxanonanoate (Bioconjugate Chem, 2004, 15, 1349) in
50 ml of dichloromethane was cooled with an ice water bath, treated
with 2.5 ml of triethylamine followed by the addition of 1.60 grams
of methanesulfonyl chloride. The reaction was stirred in the cold
for 10 minutes, then allowed to stir while warming to room
temperature over 1 hour. The reaction was diluted with 50 ml of
dichloromethane, washed with 50 ml of water and dried over sodium
sulfate. Filtration and concentration gave an oil, which was
subjected to flash column chromatography on silica gel with 50%
ethyl acetate in hexane. The appropriate fractions were combined
and concentrated to give 3.99 grams of the product as a clear,
colorless oil. NMR (CDCl.sub.3): .delta. 4.38 (m, 2H), 3.76 (m,
2H), 3.70 (t, 2H, J=6.4 Hz, C.dbd.OCH.sub.2), 3.61-3.66 (m, 4H),
3.08 (s, 3H, OSO.sub.2CH.sub.3), 2.49 (t, 2H, J=6.4 Hz,
C.dbd.OCH.sub.2CH.sub.2), 1.45 (s, 9H, CH.sub.3).
Preparation of t-Butyl 9-azido-4,7-dioxanonanoate
##STR00131##
[0376] A solution of 2.0 grams of the mesylate from the previous
procedure in 25 ml of DMF, together with 1.25 grams (3 eq) of
sodium azide, was heated at 85.degree. C. overnight. The reaction
mixture was poured into 100 ml of water, then extracted with
4.times.50 ml of ether. The combined organic layers were dried over
sodium sulfate, filtered and concentrated to give a clear oil. This
oil was flushed through a plug of silica gel with 200 ml of 50%
ethyl acetate in hexane, and the filtrate was concentrated to give
1.63 grams of the product as a clear, colorless oil. NMR
(CDCl.sub.3): .delta. 3.73 (t, 2H, J=6.4 Hz, C.dbd.OCH.sub.2),
3.63-3.69 (m, 6H), 3.39 (app t, 2H, CH.sub.2N.sub.3), 2.51 (t, 2H,
J=6.4 Hz, C.dbd.OCH.sub.2CH.sub.2), 1.45 (s, 9H, CH.sub.3).
Preparation of 9-Azido-4,7-dioxanononanoic acid
##STR00132##
[0378] A solution of 1.63 grams of the azido ester from the
previous procedure in 5 ml of 88% formic acid was stirred at room
temperature overnight. The reaction mixture was diluted with 50 ml
of water, then extracted with 4.times.25 ml of ether. The combined
organics were dried over sodium sulfate, filtered and concentrated
to give 1.14 grams of the product as a clear oil. NMR (CDCl.sub.3):
.delta. 3.79 (t, 2H, J=6.4 Hz, C.dbd.OCH.sub.2), 3.68 (app t, 2H),
3.67 (s, 4H), 3.39 (app t, 2H, CH.sub.2N.sub.3), 2.66 (t, 2H, J=6.4
Hz, C.dbd.OCH.sub.2CH.sub.2).
Preparation of 9-Azido-4,7-dioxanononanoic acid,
N-hydroxysuccinimide ester
##STR00133##
[0380] A solution of 1.14 grams of the acid from the previous
procedure and 650 mg of N-hydroxysuccinimide in 15 ml of dry
acetonitrile, together with 150 mg of DMAP, was treated with 1.4
grams of DCC and the reaction was stirred at room temperature for 3
hours. The reaction was filtered and concentrated to give a
residue, which was subjected to flash column chromatography on
silica gel with 10-30% ethyl acetate in hexane. The appropriate
fractions were combined and concentrated to give 960 mg of the
product as a clear oil containing a small amount of solid. NMR
(CDCl.sub.3): .delta. 3.87 (t, 2H, J=6.4 Hz, C.dbd.OCH.sub.2), 3.68
(app t, 2H), 3.67 (s, 4H), 3.39 (app t, 2H, CH.sub.2N.sub.3), 2.91
(t, 2H, J=6.4 Hz, C.dbd.OCH.sub.2CH.sub.2), 2.84 (br s, 4H,
CH.sub.2CH.sub.2).
Preparation of Hexaglutamic acid amide with
9-Azido-4,7-dioxanononanoic acid
##STR00134##
[0382] A mixture of 17 mg of hexaglutamic acid in 1 ml of 25 mM
HEPES buffer pH 7 was prepared, adding 350 .mu.l of DMF to improve
solubility. Then was added 7 mg of the above NHS ester in DMF
solution, and checked the pH, which was about 5. A total of 240
.mu.L of 0.5 M NaOH were added to bring the pH back to about 7.5,
and added another 13 mg of the NHS ester. The reaction was followed
by reverse phase HPLC using a Waters HPLC system with a 2695
Alliance Solvent delivery system equipped with a Waters 2685 Dual
Wavelength Detector. Samples were chromatographed using a Jupitor
C18 HPLC column (8.times.260 mm) from Phenomenex at 1.2 ml/min with
pump A buffer as 0.08% TFA in water and pump B buffer as 0.1% TFA
in acetonitrile for 25 min. Following sample injection, the column
was washed for 1 min. with isocratic 100% A, then increased to 20%
B over 10 min. with a linear gradient followed by a linear increase
to 50% B over 6 min. The column was stripped with 95% B for 2 min.
before regeneration using an isocratic 100% A for 2 min. The
chromatogram was monitored at OD220 nm. The native peptide and the
azide modified peptide eluted as sharp peaks at 5.6 min. and 9.6
min., respectively. Following the overnight reaction, the peptide
peak was gone and the product peak was at its maximum. Product
purity was confirmed by anion exchange chromatography using a
Waters HPLC system with 2695 Alliance Solvent delivery system
equipped with a Waters 2685 Dual Wavelength Detector. Samples were
chromatographed using a weak anion exchange DEAE-825 HPLC column
(8.times.75 mm) from Shodex at 1 ml/min. with pump A buffer as 20
mM Tris pH 7.5 and pump B buffer as buffer A containing 0.5M NaCl
for 16 min. Following sample injection, the column was first washed
for 5 min. with isocratic 30% B, then increased to 100% B over 10
min. with a linear gradient and then maintained at 100% B for 2
min. The column was then regenerated using isocratic 30% B for 3
min. prior to the next injection. The chromatogram was monitored at
OD220 nm. The native peptide eluted as a single sharp peak at 10.1
min while the modified peptide eluted as a single sharp peak at
10.6 min. The reaction was concentrated using a vacuum pump on the
rotary evaporator to remove all solvent, and the residue was
triturated with 100 .mu.L of 2.5 M HCl, resulting in a white solid.
This mixture was vortexed and centrifuged for 5 min at 5000 rpm,
and the supernatant was decanted. The solid was washed again in a
similar manner with 2.times.100 .mu.L of 2.5 M HCl, then dried
under vacuum to give the desired product as a white solid.
Example 46
Preparation of Camptothecin PC-Copolymer
Synthesis of 2-(2-Azidoethoxy)ethanol
##STR00135##
[0384] A solution of 10.0 grams of 2-(2-chloroethoxy)ethanol in 50
ml of deionized water was treated with 10.4 grams (2 eq) of sodium
azide, and the reaction mixture was heated at 80.degree. C. for 48
hours. The solution was cooled to room temperature, saturated with
sodium chloride and extracted with 3.times.50 ml of ether. The
combined organics were dried over anhydrous sodium sulfate,
filtered and concentrated to give 7.25 grams (69%) of the desired
product as a clear, colorless oil. .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta.=2.05 (t, J=6.4 Hz, 1H, OH), 3.42 (t, J=5 Hz,
2H), 3.63 (dd, J=4.4, 5.6 Hz), 3.71 (dd, J=4.4, 4.8 Hz, 2H), 3.77
(dt, J=4.4, 6 Hz, 2H).
Synthesis of 5-[2-(2-Azidoethoxy)ethoxy]-4-oxopentanoic acid
##STR00136##
[0386] A solution of 3.0 grams of 2-(2-azidoethoxy)ethanol in 50 ml
of dichloromethane was treated with 280 mg of
4-(dimethylamino)pyridine and 64 ml (2 eq) of triethylamine, and
the solution was cooled with an ice bath. A solution of 2.61 grams
(1.0 eq) of glutaric anhydride in 5 ml of dichloromethane was then
added dropwise over a few minutes. The reaction was stirred, then
heated at gentle reflux overnight. The reaction was cooled to room
temperature, washed with 2.times.25 ml of 1N HCl and 25 ml of
H.sub.2O, then dried over sodium sulfate. Filtration and
concentration gave 4.66 grams (83%) of the desired product as a
clear, colorless oil. .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta.=1.97 (quintet, J=7.2 Hz, 2H), 2.45 (t, J=7.2 Hz, 4H), 3.39
(t, J=4.8 Hz, 2H), 3.66-3.72 (m, 4H), 4.26 (app t, J=4.6 Hz,
2H).
[0387] Synthesis of Camptothecin Azide Conjugate
##STR00137##
[0388] A solution of 70 mg of
5-[2-(2-azidoethoxy)ethoxy]-4-oxopentanoic acid in 10 ml of
dichloromethane was cooled in an ice-water bath, and treated with
55 mg of EDC, followed by 35 mg of DMAP and 50 mg of camptothecin.
The reaction was then allowed to warm to room temperature and
stirred overnight as the solution slowly became homogeneous. The
reaction mixture was then concentrated and applied to a silica gel
column, which was eluted first with 1-2% methanol in
dichloromethane. The appropriate fractions were then concentrated
to give the desired conjugate as a yellow solid. .sup.1H NMR (400
MHz, CDCl.sub.3): .delta.=0.98 (t, J=7.6H), 1.98 (quintet, J=7.2
Hz, 2H), 2.13-2.32 (complex m, 2H), 2.45 (t, J=7.6 Hz, 2H),
2.51-2.65 (complex m, 2H), 3.35 (t, J=5 Hz, 2H), 3.63-3.68 (m, 4H),
4.21-4.25 (m, 2H), 5.30 (br s, 2H), 5.41 (d, J=17.2 Hz, 1H), 5.68
(d, J=17.2 Hz, 1 H), 7.21 (s, 1H), 7.68 (t, J=6.8 Hz, 1H), 7.84
(app t, J=8.4 Hz, 1H), 7.95 (d, J=8 Hz, 1H), 8.23 (d, J=8 Hz, 1H),
8.40 (s, 1H).
[0389] Synthesis of Copolymer of Methacryloyloxyethyl
Phosphorylcholine and Trimethylsilyl (TMS)-Protected Propargyl
Methacrylate
[0390] Ethyl .alpha.-bromoisobutyrate (18.84 mg, 0.096 mmol),
bipyridine (30.1 mg, 0.192 mmol) and 450 mg of DMSO were initially
loaded into a Schlenk tube. The mixture was carefully degassed and
the tube filled with nitrogen. CuBr was then added to the tube
under inert conditions (13.8 mg, 0.096 mmol). The reaction mixture
was sealed and cooled at -78.degree. C. A mixture of
trimethylsilyl-protected propargyl methacrylate (TMS-PgMA) (66 mg,
0.336 mmol) and methacryloyloxyethyl phosphoryl choline (0.9, 3.04
mmol) were dissolved in 4 mL of degassed 200 proof ethanol. The
solution was added drop wise under inert conditions to the cooled
reaction vessel. The mixture was thoroughly degassed under vacuum
for 15 min at 0.degree. C. and filled with inert gas.
Polymerization was allowed to proceed for 15 hours.
##STR00138##
[0391] After 15 hours, the reaction mixture was found to be very
homogeneous with no apparent crosslinking. The reaction was
quenched by exposure to air and the mixture turned from dark brown
to green. GPC analysis of a crude sample before purification
performed on a Shodex column (OH806) calibrated with polyethylene
oxide standards indicated the formation of a polymer as a single
peak of narrow distribution (molecular weight at peak Mp was found
to be 13200 g/mol). Analysis by light scattering showed a Mn of
22900 g/mol, Mp of 25000 g/mol and PDi of 1.14. The crude reaction
was passed through silica gel, concentrated and precipitated
carefully into diethyl ether. The solid was isolated by filtration
and washed several times with diethyl ether. Copolymer was dried
inside an oven at 50.degree. C. overnight, yielding 0.9 g of
copolymer. Analysis by .sup.1H NMR spectroscopy showed no TMS
group. As a precautionary step, 0.5 g of the copolymer was further
treated by 100 mg of tetrabutyl ammonium fluoride trihydate and
purified by precipitation.
[0392] Grafting of Camptothecin Azide Conjugate onto the
Alkyne-Functionalized Copolymer
[0393] CuBr (13 mg) was loaded inside a degassed Schlenk tube
followed by the addition of 15 mg of N,N,N',N'',N''-pentamethyl
diethylenetriamine. 240 mg of copolymer was dissolved into 2 g of
200proof degassed ethanol and 50 mg of camptothecin azide conjugate
(CPT-L-N3) were dissolved into 1.5 g of DMF. The solution of
CPT-L-N3 was added dropwise under inert conditions to the Schlenk
tube while stirring, followed by the addition of the solution of
alkyne-functionalized copolymer. The mixture was degassed by three
cycles of vacuum-nitrogen and was allowed to react at room
temperature for 3 hours.
##STR00139##
[0394] After 3 hours, an aliquot was taken from the crude mixture
and analyzed by GPC at 370 nm which showed the disappearance of the
free camptothecin peak and a high molecular weight peak which
corresponded to the camptothecin copolymer conjugate.
[0395] The reaction mixture was exposed to air, concentrated to
half its volume, passed through silica gel to remove the copper
catalyst and then precipitated carefully into diethyl ether. The
polymer was washed with an excess of diethyl ether. The solid was
isolated by filtration and washed several times with diethyl ether.
The polymer was dried in an oven at 50.degree. C. overnight and was
isolated as a light-brown powder. .sup.1HNMR spectroscopy analysis
performed on the camptothecin grafted copolymers (CD.sub.3OD)
showed weak and broad aromatic signals in the 7-9 ppm area,
characteristic of protons from the incorporated camptothecin.
Example 47
Camptothecin Release Study from Camptothecin Grafted Copolymer
[0396] Samples of camptothecin grafted copolymer were prepared at
approximately 10 mg/ml in Tris Buffer, pH=8.0. Liver esterase from
rabbit liver (Sigma-Aldrich E0887-IKU, Lot #061K74451) was added to
the sample and the sample was incubated at 37.degree. C. for up to
65 hours.
[0397] GPC analysis of the samples was made using an HPLC system
consisting of a Waters Alliance 2995 with Waters 2410 Refractive
Index Detector, Waters 2996 Photodiode Array Detector, and a Shodex
Protein KW-803 column. The mobile phase used for the elution was
phosphate buffered saline containing 10% absolute ethanol. The flow
rate was set to 1 ml/min and the presence of camptothecin monitored
at 370 nm. Ten microliter injections of the samples were made at
each time point.
TABLE-US-00002 Camptothecin Released Time (h) (mg/ml) 0 0.059 1
0.079 2 0.132 3 0.130 4 0.128 17 0.208 26 0.251 41 0.335 65
0.427
Example 48
Preparation of Maleimide-Functionalized PC-Copolymer Containing
Camptothecin
[0398] Polymerization
##STR00140##
[0399] The polymerization protocol followed was essentially the
same as that described in Example 46 except that the protected
maleimide functionalized initiator described in Example 5 was used
in lieu of ethyl .alpha.-bromoisobutyrate. The amounts of reagents
utilized were as described in the following table:
TABLE-US-00003 Initiator HEMA- TMS-PgMA CuBr Bipyridyl Ethanol DMF
(mol) PC (g) (mg) (mg) (mg) (ml) (mg) 2.214 .times. 10.sup.-5 1.116
40.1 6.38 13.8 4 42.5
[0400] The polymerization reaction mixture was thoroughly degassed
at -78.degree. C. and the reaction allowed to proceed at room
temperature for 17 hours. The polymerization was quenched upon
exposure to air. A solution of 100 mg of tetrabutyl ammonium
fluoride dissolved in 1 ml of methanol was added to the reaction
mixture. The crude reaction was passed through silica gel,
concentrated and precipitated carefully into diethyl ether. The
solid was isolated by filtration and washed several times with
diethyl ether. Polymer was dried inside an oven at 40.degree. C.
overnight. Analysis by light scattering showed a Mn of 73000 g/mol,
Mp of 74000 g/mol and PDi of 1.15. Analysis by .sup.1H NMR
spectroscopy showed no TMS group.
[0401] Deprotection of the Protected Maleimide Functional Group
##STR00141##
[0402] The polymer from the previous step was sprayed as a thin
layer of powder on the bottom of a wide crystallizing dish. The
dish was placed in a vacuum oven preheated at 125.degree. C. and
vacuum applied. Heating at 125.degree. C. was carried out for 1
hour and vacuum was gradually discontinued once the temperature
reached room temperature. The resulting solid/powder was collected
on a frit/filtration device, washed several times with diethyl
ether and dried in a vacuum oven at room temperature.
##STR00142##
[0403] .sup.1H NMR analysis showed disappearance of signals at 5.2
and 6.6 ppm (representing the furan group) and the appearance of a
new signal at 6.95 ppm (representing the CH from maleimide).
Analysis by light scattering showed a Mn of 77000 g/mol, Mp of
69000 g/mol and PDi of 1.1.
[0404] Preparation of Maleimide-Functionalized PC-Copolymer
Containing Camptothecin
##STR00143##
[0405] The attachment of camptothecin to the maleimide
functionalized polymer from the previous step was essentially as
described in Example 43. 170 mg of the polymer from the previous
step was dissolved in 0.5 ml of 200proof ethanol in a Schlenk tube.
To the solution was added 50 .mu.L of a PMDETA solution in dry DMF
(5 mg in 50 .mu.L), followed by the addition of a 200 .mu.L of a
solution of camptothecin azide conjugate dissolved in DMF (125 mg
of CPT-L-N3 per ml of DMF). To the mixture was added an additional
210 mg of dry DMF to ensure the homogeneity of the reaction
mixture. The mixture was briefly degassed and 4 mg of CuBr were
added under inert conditions. The mixture was degassed and the
reaction allowed to proceed at room temperature overnight. The
crude mixture was dissolved in methanol and passed through a short
column of silica gel and purified by precipitation and washing in
THF. The solid was finally washed with diethyl ether and dried
overnight at 35-40.degree. C. Analysis by light scattering showed a
20% increase in molecular weight (Mp), with Mn of 95000 g/mol, Mp
of 84000 g/mol and PDi of 1.14. .sup.1H NMR analysis of the
resulting polymer in CD.sub.3OD showed weak aromatic signals in the
7-9 ppm range. A rough estimate based on CH from camptothecin at
8.4 ppm and methylene groups from HEMA-PC in the 4-4.5 ppm region
gave a camptothecin incorporation of 1.5-2%.
Example 49
Deprotection of Protected Maleimide Functionalized PC-Copolymer
Following Attachment of Camptothecin Azide Conjugate
##STR00144##
[0407] To 100 mg of the protected maleimide functionalized
copolymer from Example 46 in 3004 of ethanol was added 29.4 .mu.L
of a stock solution of PMDETA dissolved in DMF (10 mg/ml), followed
by the addition of 117 .mu.L of a stock solution of camtothecin
azide conjugate in DMF (30 mg in 2404 of DMF), 85 .mu.L of DMF and
2.1 mg of CuBr. The reaction mixture was thoroughly degassed and
stirred overnight. Deprotection of the maleimide functionality was
performed as described in Example 48.
Example 50
Preparation of Maleimide-Functionalized PC-Copolymer Containing
Camptothecin and Fluorescein
[0408] Polymerization
##STR00145##
[0409] The polymerization protocol followed was essentially the
same as that described in Example 46 except that a third comonomer,
fluorescein methacrylate (FLMA), was added:
##STR00146##
[0410] The amounts of reagents utilized were as described in the
following table:
TABLE-US-00004 HEMA- TMS- Initiator PC PgMA FLMA CuBr Bipyridyl
Ethanol DMF (mol) (g) (mg) (mg) (mg) (mg) (ml) (mg) 2.133 .times.
1.005 35.5 14.46 6.12 13.33 4 42.5 10.sup.-5
[0411] The polymerization reaction mixture was thoroughly degassed
at -78.degree. C. and the reaction allowed to proceed at room
temperature for 17 hours. The polymerization was quenched upon
exposure to air. A solution of 100 mg of tetrabutyl ammonium
fluoride dissolved in 1 ml of methanol was added to the reaction
mixture. The crude reaction was passed through silica gel,
concentrated and precipitated carefully into diethyl ether. The
solid was isolated by filtration and washed several times with
diethyl ether. The copolymer was dried inside an oven at 40.degree.
C. overnight. Analysis by light scattering showed a Mn of 69000
g/mol, Mp of 70000 g/mol and PDi of 1.15. .sup.1H NMR spectroscopy
of the dry polymer showed no TMS group.
[0412] Deprotection of the Protected Maleimide Functional Group
##STR00147##
[0413] The protected maleimide functional group of the polymer from
the previous step was deprotected using the protocol detailed in
Example 46. .sup.1H NMR analysis showed disappearance of signals at
5.2 and 6.6 ppm (representing the furan group) and the appearance
of a new signal at 6.95 ppm (representing the CH from maleimide).
Analysis by light scattering showed a Mn of 72,200 g/mol, Mp of
63,700 g/mol and PDi of 1.1.
[0414] Preparation of Maleimide-Functionalized PC-Copolymer
Containing Camptothecin and Fluorescein
##STR00148##
[0415] The attachment of camptothecin to the maleimide
functionalized polymer from the previous step was essentially as
described in Example 46. 170 mg of the polymer from the previous
step was dissolved in 0.5 ml of 200proof ethanol in a Schlenk tube.
To the solution was added 50 .mu.L of a PMDETA solution in dry DMF
(5 mg in 50 .mu.L), followed by the addition of a 200 .mu.L of a
solution of camptothecin azide conjugate dissolved in DMF (125 mg
of CPT-L-N3 per ml of DMF). To the mixture was added an additional
210 mg of dry DMF to ensure the homogeneity of the reaction
mixture. The mixture was briefly degassed and 4 mg of CuBr were
added under inert conditions. The mixture was degassed and the
reaction allowed to proceed at room temperature overnight. The
crude mixture was dissolved in methanol and passed through a short
column of silica gel and purified by precipitation and washing in
THF. The solid was finally washed with diethyl ether and dried
overnight at 35-40.degree. C. Analysis by light scattering showed a
20% increase in molecular weight (Mp), with Mn of 107,100 g/mol, Mp
of 98100 g/mol and PDi of 1.14. .sup.1H NMR analysis of the
resulting polymer in CD.sub.3OD showed weak aromatic signals in the
7-9 ppm range. A rough estimate based on CH from camptothecin at
8.4 ppm and methylene groups from HEMA-PC in the 4-4.5 ppm region
gave a camptothecin incorporation of 2.5-5%.
Example 51
Deprotection of Protected Maleimide Functionalized Fluorescein
PC-Copolymer Following Attachment of Camptothecin Azide
Conjugate
##STR00149##
[0417] To 100 mg of the protected maleimide functionalized
copolymer from Example 50 in 3004 of ethanol was added 29.44 of a
stock solution of PMDETA dissolved in DMF (10 mg/ml), followed by
the addition of 117 .mu.L of a stock solution of camtothecin azide
conjugate in DMF (30 mg in 240 .mu.L of DMF), 85 .mu.L of DMF and
2.1 mg of CuBr. The reaction mixture was thoroughly degassed and
stirred overnight. Deprotection of the maleimide functionality was
performed as described in Example 43.
Example 52
Preparation of 4-Arm Maleimide Functionalized HEMA-PC Choline Block
Copolymer
[0418] Preparation of 4-Arm Protected Maleimide Functionalized
PC-Polymer
##STR00150##
[0419] The 4-arm protected maleimide functionalized initiator from
Example 11 and the ligand 2,2'-bipyridyl were introduced into a
Schenk tube. Dimethyl formamide was introduced drop wise so that
the weight percent of initiator and ligand was approximately 20%.
The resultant solution was cooled to -78.degree. C. using a dry
ice/acetone mixture, and was degassed under vacuum for 10 min. The
tube was refilled under nitrogen and the catalyst CuBr, kept under
nitrogen, was introduced into the Schlenck tube (the Molar ratio of
bromine/catalyst/ligand was kept at 1/1/2). The solution became
dark brown immediately. The Schlenk tube was sealed and kept at
-78.degree. C. The solution was purged by applying a
vacuum/nitrogen cycle three times. A solution of HEMA-PC was
prepared by mixing a defined quantity of monomer, kept under
nitrogen, with 200proof degassed ethanol. The monomer solution was
added drop wise into the Schlenk tube and homogenized by light
stirring. The temperature was maintained at -78.degree. C. A
thorough vacuum was applied to the reaction mixture for at least 10
to 15 min. until bubbling from the solution ceased. The tube was
then refilled with nitrogen and warmed to room temperature. The
solution was stirred, and as the polymerization proceeded, the
solution became viscous. After 38 hours, the reaction was quenched
by direct exposure to air in order to oxidize Cu (I) to Cu (II),
the mixture became blue-green in color, and was passed through
silica gel, concentrated and precipitated carefully into diethyl
ether. The solid was isolated by filtration and washed several
times with diethyl ether. Polymer was dried inside an oven at
40.degree. C. overnight. The amounts of reagents utilized were as
described in the following table:
TABLE-US-00005 Initiator HEMA-PC CuBr Bipyridyl Ethanol DMF (mol)
(g) (mg) (mg) (ml) (mg) 3.66 .times. 10.sup.-5 2.203 2.1 4.58 5
42.5
[0420] Analysis by light scattering showed a Mn of 550,000 g/mol,
Mp of 640,000 g/mol and PDi of 1.18.
Preparation of 4-Arm Maleimide Functionalized HEMA-PC Choline Block
Copolymer
##STR00151##
[0421] wherein the block copolymer has the formula:
##STR00152##
[0422] To a mixture of 300 mg of the polymer from the previous step
in 0.7 ml of ethanol was added, under inert conditions, 1 mg of
PMDETA dissolved in 42 mg of DMF followed by 1 mg of CuBr. The
reaction mixture was immediately cooled to -78.degree. C. and
degassed thoroughly. 2(Methacryloyloxy)ethyltrimethyl ammonium
chloride (MC) as an aqueous solution (72% w/w) was preliminary
passed through a short column to remove the stabilizer. 177 mg of
the solution was added to the reaction mixture, and the mixture was
thoroughly degassed at -78.degree. C. for 30 min. until no bubbling
was seen. The reaction mixture was replenished with nitrogen and
the reaction allowed to proceed at room temperature for 44 hours.
Conversion estimated by .sup.1H NMR indicated that 15% of MC was
converted into a polymer. Crude mixture was purified by dialysis to
remove any low molecular weight impurities (MWCO 15 kDa) followed
by lyophilization. .sup.1H NMR analysis indicated a new peak in the
4.5 ppm region (CH.sub.2O) from choline group next to the three
peaks from phosphorylcholine (from 4 ppm to 4.5 ppm). NMR analysis
of the final polymer in CD.sub.3OD showed a Molar ratio of 5-10% of
MC versus HEMA-PC. The maleimide functional group was generated by
deprotection as described in Example 43. Similar chain extensions
have been observed in a one step process where the MC was added at
the end of the HEMA-PC polymerization.
Example 53
Preparation of 3-Arm Diol Functionalized HEMA-PC Fluorescein Block
Copolymer
[0423] Polymerization
##STR00153##
[0424] 4.66 mg of 2,2' bipyridyl were added to a Schlenk tube
followed by 41.3 .mu.L of a stock solution of the initiator from
Example 37 in DMF (10 mg/100 mL of DMF) and by 83.4 .mu.L of a
stock solution of CuBr.sub.2 in DMF (10 mg/ml of DMF). The mixture
was degassed under vacuum at -78.degree. C. To the reaction mixture
was added 1.6 mg of CuBr under inert conditions, followed by an
addition of 2 g of HEMA-PC dissolved in 3.75 ml of 200proof ethanol
dropwise. The vessel was sealed and degassed at -78.degree. C.
under vacuum until no bubbling was seen. The reaction mixture was
placed under inert conditions and the reaction allowed to proceed
at room temperature for 48 hours. Conversion was estimated by
.sup.1H NMR to be above 98%. Analysis by light scattering showed a
Mp of 457 kDa, Mn of 407 kDa and PDi of 1.13. The crude mixture was
passed through a plug of silica gel and purified by precipitation
into THF followed by washing with THF and then a final washing with
diethyl ether.
[0425] Preparation of 3-Arm Diol Functionalized HEMA-PC Fluorescein
Block Copolymer
##STR00154##
[0426] 1.409 g of the polymer from the previous step were dissolved
in 4 ml of 200proof ethanol. To the reaction mixture was added a
solution of 14 mg of FLMA dissolved in 182 mg of DMF, 510 mg of DMF
and 3 mg of 2,2' bipyridyl. The reaction mixture was thoroughly
degassed before the addition of 1.34 mg of CuBr and I mg of Cu(0).
The reaction mixture was thoroughly degassed and allowed to proceed
at room temperature for 8 hours. The crude mixture was passed
through a plug of silica gel and purified by precipitation in THF,
followed by a washing with THF and another washing with diethyl
ether. Final polymer was isolated as a yellow powder. The presence
of fluorescein was demonstrated by .sup.1H NMR in methanol and
absorbance at 370 nm. Molecular weight analysis performed on the
polymer by light scattering indicated an increase in molecular
weight to Mp of 501 kDa and a PDi of 1.28.
Example 54
Preparation of Aldehyde Functionalized PC Copolymer Containing
Alkyne Groups
[0427] Polymerization
##STR00155##
[0428] The amounts of reagents utilized were as described in the
following table:
TABLE-US-00006 TMS- Initiator HEMA- PgMA CuBr.sub.2 CuBr Bipyridyl
Ethanol DMF (mol) PC (g) (mg) (mg) (mg) (mg) (ml) (.mu.l) 2.133
.times. 2.005 30 1.7 3.27 9.53 3.8 555 10.sup.-5
The initiator from Example 39 was utilized. The polymerization
reaction mixture was thoroughly degassed at -78.degree. C. and
allowed to proceed at room temperature for 64 hours. The reaction
was quenched upon exposure to air. A solution of 100 mg of
tetrabutyl ammonium fluoride dissolved in 1 ml of methanol was
added to the reaction mixture. The crude reaction mixture was
passed through silica gel, concentrated and precipitated carefully
into diethyl ether. The solid was isolated by filtration and washed
several times with diethyl ether. The polymer was dried in an oven
at 40.degree. C. overnight. Analysis by light scattering showed a
Mn of 71,000 g/mol, Mp of 64000 g/mol and PDi of 1.15. .sup.1H NMR
spectroscopy of the dry polymer showed no TMS group.
[0429] Generation of Aldehyde Functional Group by Periodate
Oxidation
##STR00156##
[0430] To a solution of diol functionalized polymer in distilled
water (10 wt. %) was introduced a large excess of sodium periodate
dissolved in distilled water. The reaction was allowed to proceed
at room temperature for 90 min. in the dark. The reaction was
quenched with an aqueous solution of glycerol (1.5.times. vs.
NaIO.sub.4) to remove any unreacted sodium periodate. The mixture
was stirred at room temperature for 15 min. and placed in a
dialysis bag (MWCO 14 to 25 kDa) for purification at room
temperature for one day. Water was removed by lyophilization and
the polymer was collected as a dry powder.
##STR00157##
Example 55
Preparation of Diol Functionalized PC Copolymer Containing Epoxide
Groups
##STR00158##
[0432] 9.13 mg of 2,2' bipyridyl were added to a Schlenk tube
followed by 80 .mu.l of a stock solution of the initiator from
Example 26 in DMF (10 mg/100 ml of DMF). The mixture was degassed
under vacuum at -78.degree. C. To the reaction mixture was added
4.2 mg of CuBr under inert conditions, followed by an addition of a
mixture of 1 g of HEMA-PC and 23 .mu.L of purified glycidyl
methacrylate (GMA) (passed through a stabilizer remover, to remove
the MEHQ stabilizer) which was dissolved in 2 ml of 200proof
ethanol was added by drop wise addition. The vessel was sealed and
degassed at -78.degree. C. under vacuum until no bubbling was seen.
The reaction mixture was placed under inert conditions and allowed
to proceed at room temperature for 3 hours. The crude mixture was
passed through a plug of silica gel and purified by precipitation
into THF followed by a washing with THF and then a final washing
with diethyl ether. Analysis by light scattering showed a Mp of 92
kDa, Mn of 83 kDa and PDi of 1.1.
Example 56
Preparation of Protected Maleimide Functionalized PC Copolymer
Containing Epoxide Groups
##STR00159##
[0434] 13.55 mg of 2,2' bipyridyl were added to a Schlenk tube
followed by 13.52 mg of the initiator from Example 26. The solids
were dissolved in 142 mg of DMSO. The mixture was degassed under
vacuum at -78.degree. C. To the reaction mixture was added 6.22 mg
of CuBr under inert conditions, followed by an addition of a
mixture of 1 g of HEMA-PC and 78 .mu.L of purified GMA (passed
through a stabilizer remover, to remove the MEHQ stabilizer) which
was dissolved in 2 ml of 200proof ethanol was added by drop wise
addition. The vessel was sealed and degassed at -78.degree. C.
under vacuum until no bubbling was seen. The reaction mixture was
placed under inert conditions and allowed to proceed at room
temperature for 3 hours. The crude mixture was passed through a
plug of silica gel and purified by precipitation into THF followed
by a washing with THF and then a final washing with diethyl ether.
Analysis by light scattering showed a Mp of 71 kDa, Mn of 65 kDa
and PDi of 1.13.
Example 57
Preparation of Protected Maleimide Functionalized PC Copolymer
Containing Acetoacetate Groups
##STR00160##
[0436] 13.55 mg of 2,2' bipyridyl were added to a Schlenk tube
followed by 13.52 mg of the initiator from Example 26. The solids
were dissolved in 142 mg of DMSO. The mixture was degassed under
vacuum at -78.degree. C. To the reaction mixture was added 6.22 mg
of CuBr under inert conditions, followed by an addition of a
mixture of 1 g of HEMA-PC and 1104 of purified
2-(acetoacetyloxy)ethyl 2-methacrylate (MEA) (passed through a
stabilizer remover, to remove the MEHQ stabilizer) which was
dissolved in 2 ml of 200proof ethanol was added by drop wise
addition. The vessel was sealed and degassed at -78.degree. C.
under vacuum until no bubbling was seen. The reaction mixture was
placed under inert conditions and allowed to proceed at room
temperature for 3 hours. The crude mixture was passed through a
plug of silica gel and purified by precipitation into THF followed
by a washing with THF and then a final washing with diethyl ether.
Analysis by light scattering showed a Mp of 85 kDa, Mn of 79 kDa
and PDi of 1.15.
Example 58
Preparation of Protected Maleimide Functionalized PC Copolymer
Containing Alkyne and Acetoacetate Groups
##STR00161##
[0438] 13.55 mg of 2,2' bipyridyl were added to a Schlenk tube
followed by 13.52 mg of the initiator from Example 26. The solids
were dissolved in 142 mg of DMSO. The mixture was degassed under
vacuum at -78.degree. C. To the reaction mixture was added 6.22 mg
of CuBr under inert conditions, followed by an addition of a
mixture of 1 g of HEMA-PC, 56.3 mg of TMS-PgMA and 55 .mu.L of
purified MEA (passed through a stabilizer remover, to remove the
MEHQ stabilizer) which was dissolved in 2 ml of 200proof ethanol
was added by drop wise addition. The vessel was sealed and degassed
at -78.degree. C. under vacuum until no bubbling was seen. The
reaction mixture was placed under inert conditions and allowed to
proceed at room temperature for 3 hours. The crude mixture was
passed through a plug of silica gel and purified by precipitation
into THF followed by a washing with THF and then a final washing
with diethyl ether. Analysis by light scattering showed a Mp of 78
kDa, Mn of 72 kDa and PDi of 1.13.
Example 59
Preparation of Diol Functionalized PC Copolymer Containing Alkyne
Groups
##STR00162##
[0440] The amounts of reagents utilized were as described in the
following table:
TABLE-US-00007 HEMA- Initiator PC TMS-PgMA CuBr Bipyridyl Ethanol
DMF (mol) (g) (mg) (mg) (mg) (ml) (.mu.l) 2.37 .times. 10.sup.-5
1.983 69.2 6.76 14.71 8 35.2
[0441] The initiator from Example 26 was utilized. The
polymerization reaction mixture was thoroughly degassed at
-78.degree. C. and allowed to proceed at room temperature for 14
hours. The reaction was quenched upon exposure to air. A solution
of 100 mg of tetrabutyl ammonium fluoride dissolved in 1 ml of
methanol was added to the reaction mixture. The crude reaction
mixture was passed through silica gel, concentrated and
precipitated carefully into diethyl ether. The solid was isolated
by filtration and washed several times with diethyl ether. The
polymer was dried in an oven at 40.degree. C. overnight. Analysis
by light scattering showed a Mn of 222,000 g/mol, Mp of 277,000
g/mol and PDi of 1.2. .sup.1H NMR spectroscopy of the dry polymer
showed no TMS group.
Example 60
Attachment of Hexaglutamic Acid Amide with
9-Azido-4,7-Dioxanononanoic Acid to Diol Functionalized PC
Copolymer Containing Alkyne Groups and Subsequent Generation of
Aldehyde Functional Groups from Diol Precursors
##STR00163##
[0443] 45 mg of the diol functionalized PC copolymer with alkyne
groups from [0368] 13.55 mg of 2,2' bipyridyl were added to a
Schlenk tube followed by 13.52 mg of the initiator from Example 26.
The solids were dissolved in 142 mg of DMSO. The mixture was
degassed under vacuum at -78.degree. C. To the reaction mixture was
added 6.22 mg of CuBr under inert conditions, followed by an
addition of a mixture of 1 g of HEMA-PC, 56.3 mg of TMS-PgMA and 55
.mu.L of purified MEA (passed through a stabilizer remover, to
remove the MEHQ stabilizer) which was dissolved in 2 ml of 200proof
ethanol was added by drop wise addition. The vessel was sealed and
degassed at -78.degree. C. under vacuum until no bubbling was seen.
The reaction mixture was placed under inert conditions and allowed
to proceed at room temperature for 3 hours. The crude mixture was
passed through a plug of silica gel and purified by precipitation
into THF followed by a washing with THF and then a final washing
with diethyl ether. Analysis by light scattering showed a Mp of 78
kDa, Mn of 72 kDa and PDi of 1.13.
Example 59 were dissolved in 800-900 mg of de-ionized water. 13.5
.mu.l of PDMETA (from stock solution of 10 mg in 100 .mu.l of DMF)
were added to the reaction mixture in a round bottom flask. 7 mg of
hexaglutamic acid amide with 9-azido-4,7-dioxanononanoic acid from
Example 45 were dissolved in 70 .mu.l of DMF and added to the
reaction mixture, along with 2.1 mg of CuBr. The mixture was
degassed thoroughly, placed under inert conditions and stirred
overnight at room temperature.
[0444] Reaction efficiency was monitored by anion exchange
chromatography at OD220 nm as described in Example 45. Injection at
time zero showed the presence of unreacted polymer in the
flow-through, and the unreacted peptide at 10.6 min. Following the
overnight reaction, the polymer peak disappeared, and a new peak,
corresponding to the polymer modified peptide, appeared at 11.6
min. This peak was broad indicating the presence of multiple
polymer-peptide species due to the fact that each polymer has
multiple alkyne groups for potential attachment of the azide
modified peptide.
[0445] Purification by Anion Exchange Chromatography
[0446] Based upon the analytical anion exchange chromatography
experience, the polymer modified peptide was purified using anion
exchange chromatography on an Akta Prime Plus system using a Hitrap
DEAE FF column (5 ml) from GE Healthcare. Buffer A was 20 mM Tris
pH7.5, and Buffer B was Buffer A containing 0.5M NaCl. The column
was equilibrated with Buffer A, followed by three column volumes of
Buffer B, and then sufficient Buffer A to return the column eluate
to the same conductivity as Buffer A. 700 .mu.g of the crude
polymer modified peptide was loaded onto the column in Buffer A,
and the column was washed with sufficient Buffer A to return the
column eluate to the same conductivity as Buffer A. Elution was
performed in a step-wise fashion using 20% B, 30% B, 50% B, 70% B,
and 100% B. Fractions of 10 ml were collected and fractions 17 and
18 were pooled (20 ml) to form a 40% B pool, and fractions 19 and
20 were pooled to form a 70% B pool (20 ml). Both pools were
concentrated to a volume of 0.5-1 ml using an Amicon Ultra 30 kDa
MWCO concentrator. Analysis was performed using the analytical
anion exchange method from Example 45 which indicated that the 70%
B pool contained a single broad peak as described previously. The
40% B pool also contained a single broad peak which eluted slightly
earlier than the 70% B peak indicating the presence of polymer
modified peptide with fewer peptides per polymer. Further analysis
was performed using size exclusion chromatography on a Waters HPLC
system with a 2695 Alliance Solvent Delivery system equipped with a
Waters 2685 Dual Wavelength Detector. Samples were chromatographed
using a Superdex 200 column (10.times.300 mm) from GE Healthcare at
1 ml/min. with 1.times.PBS pH 7.4 for 25 min. The chromatogram was
monitored at OD220 nm and OD280 nm. Both the 40% B and 70% B pools
from anion exchange purification eluted with peak retention times
at around 9 min. equating to a molecular weight in the 500 kDa-600
kDa range. The peaks were visible at 220 nm and 280 nm indicating
the presence of polymer and peptide. Unreacted polymer eluted in a
similar position, but was only visible at 220 nm, while unreacted
peptide eluted with a retention time of 18 min, but was only
visible at 280 nm.
[0447] Conversion of Terminal Diol Functional Groups into Aldehyde
Functional Groups by Periodate Oxidation on the Hexaglutamic Acid
Modified PC Copolymer
[0448] The terminal diol functional groups on the anion exchange
purified and concentrated 70% B elution pool of the polymer
modified peptide from the previous step were converted into
aldehyde functional groups using periodate oxidation as described
in Example 54.
Example 61
Conjugation of Alkaline Phosphatase to Aldehyde Functionalized PC
Polymer Containing Hexaglutamic Acid
[0449] Alkaline phosphatase (Sigma-Aldrich) was buffer exchanged
into 25 mM Hepes pH 7 (conjugation buffer) and concentrated to 5-8
mg/ml. Conjugation reactions were carried out at 3-5.times. molar
excess of the aldehyde functionalized PC copolymer containing
hexaglutamic acid from Example 60 to protein in the presence of 40
mM sodium cyanoborohydride with a final protein concentration of
.about.1 mg/ml. All the reactions were carried out in crimp sealed
glass vials overnight at room temperature. The diol form of the
polymer was used as a negative control. 40 .mu.l of each reaction
were fractionated on a Superdex 200 column (10/300 mm) at 1 ml/min.
in 1.times.PBS pH 7.4. Fractions of 1 ml were collected and tested
for alkaline phosphatase activity as follows. 5 .mu.l of the SEC
fractions were diluted 5.times. with 20 mM Tris pH 7.5, and 100
.mu.l of the PNPP substrate was added and the samples were
incubated at 37.degree. C. for 20 min. OD405 nm was measured using
a SpectraMax Plus 384 plate reader from Molecular Devices. As
expected, no conjugation was observed when the diol functionalized
polymer was used. However, in the case of the aldehyde
functionalized polymer, alkaline phosphatase activity was
determined in the 8-10 min. retention time range, corresponding
with free polymer and higher molecular weight species, as well as
in the 12-13 min. range corresponding with free alkaline
phosphatase.
Example 62
Conjugation of Human Fab to Maleimide Functionalized PC Copolymer
Containing Camptothecin
[0450] Human Fab was prepared by pepsin digestion of whole human
IgG (Innovative Research) to yield Fab.sub.2, followed by
subsequent reduction with TCEP to yield Fab. Pepsin digestion of
IgG was performed in 0.1M sodium acetate pH 4.5 at 4.degree. C.
overnight to obtain over 90% digestion efficiency. The Fab.sub.2
fraction was then further purified using cation exchange
chromatography with a MacroCap SP column. The pure Fab.sub.2
fraction was eluted with 100-200 mM NaCl at pH 5 while the free
pepsin and all other contaminants eluted in the unbound fraction.
The purified Fab.sub.2 was then reduced with a 2.times. molar ratio
of TCEP at 37.degree. C. for 30 min., and gel filtration
chromatography was used to purify the Fab from unreduced Fab.sub.2
and free TCEP. The Fab fraction was then pooled and buffer
exchanged into the conjugation buffer. The conjugation experiment
described below is for conjugation of 1 mg of Fab to a 13.times.
molar excess of the 84 kDa maleimide functionalized PC copolymer
containing camptothecin from Example 48. The conjugation reaction
was performed in 10 mM sodium acetate pH 5 with 2 mM EDTA. The
final Fab concentration was 2.7 mg/ml in the presence of a
13.times. molar excess of polymer dissolved in the conjugation
buffer and 3.times. molar excess of TCEP as reducing agent. The
polymer was dissolved in conjugation buffer at a concentration of
100-300 mg/ml followed by addition of the TCEP and Fab. The
reaction mixture was gently mixed, and the conjugation carried out
in the dark at room temperature overnight.
[0451] The conjugation status can be monitored with SDS-PAGE where
under non-reducing conditions, the accumulation of high MW species
larger than the free Fab is a good indication of the conjugation
event. Such high MW conjugate species are characterized to be: (1)
fluorescent under UV illumination due to the presence of
camptothecin; (2) the conjugate bands should be stainable by
Coomassie Blue due to the presence of protein (the polymer does not
stain); (3) the high MW species does not shift under reducing
conditions which is a good indication that they are not due to
disulfide mediated aggregates.
[0452] Alternatively, the conjugation event can be monitored with
analytical SEC using a Superdex 200 (10/300 mm) column from GE
Healthcare at 1 ml/min in 1.times.PBS pH 7.4. Under such running
conditions, the free Fab elutes at 15.3 min. and the free polymer
elutes at 10.6 min.
[0453] To further characterize the presence of
Fab-polymer-camptothecin conjugate as described above, the reaction
mixture was further fractionated using a 1 ml cation exchange
chromatography (CEX) or MacroCap SP column from GE Healthcare at pH
5. The column was connected to an AKTA Prime Plus chromatography
system equipped with an OD280 nm detector, conductivity meter and
fraction collector. Buffer A was 10 mM sodium acetate pH 5 and
buffer B was Buffer A containing 0.5M NaCl. The eluted fractions
were further analyzed using SDS-PAGE.
[0454] As the Fab is protonated at pH 5, together with the low
ionic strength at 10 mM NaCl, Fab-conjugate and free Fab bind to
the cation exchange column while the unconjugated polymer should
not interact with the CEX and therefore should remain in the flow
through fraction. The unbound fraction was collected for analysis.
After washing with at least 15 column volumes (CV) of buffer A, the
column was eluted stepwise with 8%, 12%, 20%, 40% and 100% buffer B
which are equivalent to buffer A containing 40 mM, 60 mM, 100 mM,
200 mM and 500 mM NaCl, respectively. In each elution step, at
least 10 CV of each elution buffer was passed through the column
and 1.5 ml fractions were collected and the OD280 nm trace was
monitored continuously until the baseline dropped to at least 5% of
the initial buffer background before the higher salt elution
gradient was initiated.
[0455] The peak fractions of each step elution were collected and
concentrated with an Amicon Ultrafree concentrator with 10 kDa MW
cutoff (MWCO) membrane. The concentrate was analyzed with SDS-PAGE
under non-reducing and reducing conditions using a 1 mm NuPAGE
Novex 4-12% gradient gel, and electrophoresis was performed
according to the manufacturer's specifications (Invitrogen Corp).
Samples for SDS-PAGE analysis include the initial reaction mixture,
MacroCap SP column unbound fraction, column wash fractions, and
concentrated fractions of 8%, 12%, 20% and 40% elution pools. Once
the electrophoresis was completed, the PAGE was disassembled from
the cassette and placed on the UV illuminator to review the
fluorescence which is due to the camptothecin containing polymer
and conjugate. A picture was taken immediately before the gel was
subjected to Coomassie Blue stain using the SimplyBlue stain system
from Invitrogen to review the protein containing bands.
[0456] The results based on the SDS-PAGE analysis indicate the
following:
[0457] The bulk of the unbound MacroCap SP fraction contained no
protein based on Coomassie Blue staining but exhibited an extensive
fluorescent signal at the high MW range of the well (.gtoreq.160
kDa). In addition, when the fraction was analyzed by Bradford
protein assay, it showed no protein signal at all compared to the
MacroCap SP column-load; this is additional evidence to confirm
that the unbound fraction is devoid of any protein including Fab
and Fab-polymer conjugate. However, it contained mostly free
polymer but no free camptothecin as the camptothecin is too small
to migrate at this MW range.
[0458] Fractions at 8% and 12% B contain two major species that
were stained by Coomassie Blue, one was the free Fab and the other
a higher MW diffused band with MW spanning between 110-260 kDa,
only the latter band showed fluorescence but not free Fab. Based on
the previous evidence that the polymer cannot be stained by
Coomassie Blue and the fact that the unbound fraction contains
mostly polymer and showed no Coomassie Blue stain, we can conclude
that the high MW species is the conjugate that contains both Fab
and polymer with camptothecin.
[0459] No fluorescence was observed in the 20% and 40% eluted
fractions as these corresponded to the free Fab and Fab.sub.2
fraction. These two fractions constitute the majority of the eluted
protein (>80%) which is a good indication that the conjugate was
enriched in the low salt eluted fractions as expected (due to the
expected shielding effect of the polymer).
[0460] The eluted fraction pools were also subjected to reducing
conditions using DTT, and the Fab band was shifted down to the 25
kDa position which is a good indication of light chain and
half-heavy chain dissociation due to reduction of inter-chain
disulfide linkages. Under such conditions, the fluorescent signal
at high MW as described above was not shifted and was also stained
by Coomassie Blue. This observation confirms that the high MW
species is covalently attached to the polymer rather than
non-covalent association or connection through disulfide
linkages.
[0461] In addition to the SDS-PAGE analysis, the MacroCap SP eluted
fractions were subjected to analytical SEC using a Superdex 200
(10.times.300 mm) column from GE Healthcare and a Waters HPLC
system with 2695 Alliance Solvent delivery system with a Diode
Array detector 2669. The analysis was performed in 1.times.PBS pH
7.4 at a flow rate of 1 ml/min. The chromatogram was monitored
using OD220 nm, OD280 nm and OD355 nm where OD220 nm detects
protein, polymer and camptothecin, OD280 nm detects only protein
and camptothecin, and OD355 nm detects only the camptothecin. The
results are as follows:
TABLE-US-00008 Fraction contains Fraction Fab-polymer OD peak
signal (nm) elution % conjugate Fab 220 280 355 8 Yes Yes Yes Yes
Yes 12 Yes Yes Yes Yes Yes 20 No Yes Yes Yes No 40 No Yes Yes Yes
No
Example 63
Conjugation of Human Fab to Maleimide Functionalized PC Copolymer
Containing Camptothecin and Fluorescein
[0462] The conjugation reaction conditions, purification, analyses
and conclusions were essentially the same as for Example 62, except
for the following:
[0463] The 98 kDa maleimide functionalized PC copolymer containing
camptothecin and fluorescein from Example 50 was used.
[0464] For MacroCap SP elution, an additional 4% B elution preceded
the 8% B elution. Therefore, the 4% B elution pool was included in
both the SDS-PAGE and SEC analysis. The 4% B pool also showed
fluorescence, a good indication that this fraction contains
conjugate also.
[0465] SEC/MALS analysis of the unbound MacroCap SP fraction
confirmed that this fraction was composed of free polymer only.
Example 64
Conjugation of Traut's Reagent Modified Human Whole IgG to
Maleimide Functionalized PC Copolymer Containing Camptothecin and
Fluorescein
[0466] In this example, whole human IgG (Innovative Research) was
first modified with Traut's reagent at a 3 fold molar excess ratio
in 1.times.PBS pH 7.4. The reaction setup included 10 mg/ml IgG and
3 mg/ml Traut's reagent in 1.times.PBS pH 7.4, the reaction volume
was 300 .mu.l and the reaction was carried out for 1 hour at room
temperature in the dark with mixing. Upon completion of the
reaction, the reaction mixture was buffer exchanged into 10 mM
sodium acetate pH 5 with 2 mM EDTA using a 10 ml BioGel P30
desalting column. At pH 5 and in the presence of EDTA, oxidation of
sulfhydryl groups to form disulfide linkages was prevented. The
column was connected to an AKTA Prime Plus equipped with an OD280
nm detector, conductivity meter, and fraction collector. Protein
fractions were collected and concentrated to 4.45 mg/ml. The sample
was now ready for conjugation to the polymer. SDS-PAGE analysis
showed that modification of IgG with Traut's reagent under these
conditions did not result in protein aggregation.
[0467] Conjugation of the Traut's modified IgG to polymer was
performed in 10 mM sodium acetate pH 5 with 20 fold polymer molar
excess. The final concentration of IgG and polymer was 3.8 mg/ml
and 44 mg/ml, respectively. The reaction was carried out at room
temperature overnight. Upon completion of the reaction, the
conjugation reaction was subjected to cation exchanger
chromatography as described in Example 62 without modification. The
eluted fraction pools at 8% B, 12% B, 20% B and 40% B were analyzed
by SDS-PAGE and subjected to UV illumination. The results indicate
that only the 8% B eluted fraction contained high MW species larger
than the IgG monomer and the free polymer. In addition, the band
stained with Coomassie blue and exhibited fluorescence, indicating
presence of conjugate. Under reducing conditions, the band did not
shift down again indicating the presence IgG-polymer conjugate as
described in Examples 62 and 63.
Example 65
Preparation of 2-(Acryloyloxyethyl-2'-(trimethylammonium)ethyl
phosphate, inner salt
[0468] 1.sup.st Intermediate
##STR00164##
[0469] A solution of 11.6 grams of 2-hydroxyethylacrylate and 14.0
ml of triethylamine in 100 ml of dry acetonitrile, under a nitrogen
atmosphere, was cooled to -20.degree. C., and a solution of 14.2
grams of 2-chloro-2-oxo-1,3,2-dioxaphospholane in 10 ml of dry
acetonitrile was added dropwise over about 30 minutes. The reaction
was stirred in the cold for 30 minutes, then filtered under a
nitrogen atmosphere. The precipitate was washed with 10 ml of cold
acetonitrile, and the filtrate was used directly in the next
reaction.
2-(Acryloyloxyethyl-2'-(trimethylammonium)ethyl phosphate, inner
salt
##STR00165##
[0471] To the solution from the previous procedure was added 14.0
ml of trimethylamine (condensed using a dry ice-acetone condenser
under nitrogen), the reaction mixture was sealed into a pressure
vessel, and stirred at 65.degree. C. for 4 hours. The reaction
mixture was allowed to stir while cooling to room temperature, and
as it reached about 30.degree. C., a solid began to form. The
vessel was then placed in a 4.degree. C. refrigerator overnight.
Strictly under a nitrogen atmosphere, the solid was recovered by
filtration, washed with 20 ml of cold dry acetonitrile, then dried
under a stream of nitrogen for 15 minutes. The solid was then dried
under high vacuum overnight to give 12.4 grams of product as a
white solid. NMR (CDCl.sub.3): .delta. 6.41 (dd, 1H, J=1.6, 17.2
Hz, vinyl CH), 6.18 (dd, 1H, J=10.6, 17.2 Hz, vinyl CH), 5.90 (dd,
1H, J=1.6, 10.4 Hz, vinyl CH), 4.35 (m, 2H), 4.27 (m, 2H), 4.11 (m,
2H), 3.63 (m, 2H), 3.22 (s, 9H, N(CH.sub.3).sub.3).
Example 66
Preparation of 3-Trimethylsilylpropargyl methacrylate
##STR00166##
[0473] A solution of 3.0 grams of 3-(trimethylsilyl)propargyl
alcohol and 4.2 ml of triethylamine in 50 ml of ether was cooled to
-10.degree. C. with a dry ice/acetonitrile/ethylene glycol bath,
and a solution of 2.9 grams of methacryloyl chloride in 25 mL of
ether were added dropwise over 30 minutes. The reaction mixture was
stirred while warming to room temperature over 4 hours, then
filtered and concentrated to give an oily residue, which subjected
to flash column chromatography on silica gel with 1% ether in
hexane. The product containing fractions were combined,
concentrated, and subjected to a second chromatography as before to
give 2.46 g of the product as a clear, colorless oil. NMR
(CDCl.sub.3): .delta. 6.18 (t, 1H, CCH.sub.2, J=1.2 Hz), 5.62 (p,
1H, CCH.sub.2, J=1.6 Hz), 4.76 (s, 2H, CH.sub.2), 1.97 (d of d, 3H,
CCH.sub.3, J=1.0, 1.6 Hz), 0.187 (s, 9H, Si(CH.sub.3).sub.3).
Example 67
Preparation of N-Iodoacetylpropargylamine
##STR00167##
[0475] A solution of 1.05 grams of propargylamine hydrochloride in
20 ml of dry acetonitrile was treated with 4.0 ml of
diisopropylethylamine, followed by the addition of 4.29 grams of
iodoacetic anhydride in 20 ml of dry acetonitrile. The reaction was
stirred at room temperature for 1.5 hours, then concentrated to
give a residue, which was partitioned between 100 ml of ethyl
acetate and 100 ml of water. The organic phase was washed with 50
ml of saturated sodium chloride, then dried over sodium sulfate.
Concentration gave a dark solid, which was subjected to flash
column chromatography on silica gel with 30-40% ethyl acetate in
hexane. The product containing fractions which were clean were
combined and concentrated to give a solid, which was triturated
with a small amount of hexane and air-dried to give 940 mg of the
product as a very light yellow solid. NMR (CDCl.sub.3): .delta.
6.25 (s, 1H, CH), 4.08 (app d of d, 2H, NCH.sub.2, J=2.6, 5.3),
3.72 (s, 2H, ICH.sub.2), 2.28 (t, 1H, NH, J=2.6 Hz).
Example 68
Preparation of 4-Pentyn-1-ol, NHS ester
##STR00168##
[0477] A solution of 1.02 grams of 4-pentynoic acid and 1.20 grams
of N-hydroxysuccinimide in 20 ml of dry acetonitrile was treated
with 300 mg of DPTS, followed by 2.8 grams of DCC, and the reaction
was stirred at room temperature overnight. The reaction was
filtered and concentrated to give a residue, which was subjected to
flash column chromatography on silica gel with 30% ethyl acetate in
hexane. The product containing fractions were combined and
concentrated to give a 1.62 grams of the desired product as a white
solid. NMR (CDCl.sub.3): .delta. 2.89 (d of d, 2H, CH.sub.2C.dbd.O,
J=7.9, 6.4 Hz), 2.85 (s, 4H, O.dbd.CCH.sub.2CH.sub.2C.dbd.O), 2.62
(app d of d of d, 2H, CHCCH.sub.2, J=8.6, 6.9, 2.7 Hz), 2.06 (t,
1H, CH, J=2.7 Hz).
Example 69
Preparation of N-Propargylmaleimide
##STR00169##
[0479] A solution of 1.08 grams of propargylamine hydrochloride in
50 ml of saturated sodium bicarbonate was cooled with an ice water
bath, and 2.0 grams of N-carboethoxymaleimide were added
portionwise over a few minutes. The reaction was stirred in the
cold for 30 min., then while warming to room temperature over 25
min. The reaction was then extracted with 3.times.25 ml of
dichloromethane, which was dried over sodium sulfate, filtered and
concentrated. The residue was taken up in 10 ml of ethyl acetate
and heated at 50.degree. C. for two hours to complete the
cyclization. The reaction was concentrated and the residue was
which was subjected to flash column chromatography on silica gel
with 30% ethyl acetate in hexane. A second chromatography as before
gave 1.24 g of the product as a very light yellow oil. NMR
(CDCl.sub.3): .delta. 6.77 (s, 2H, CHC.dbd.O), 4.30 (d, 2H,
NCH.sub.2, J=2.4 Hz), 2.22 (t, 1H, CCH, J=2.5 Hz).
Example 70
Preparation of 5-Hexyn-1-al
##STR00170##
[0481] A solution of 694 mg of 5-hexyn-1-ol in 20 ml of
dichoromethane was treated at room temperature with 3.0 grams of
Dess-Martin periodinane, and the solution was stirred at room
temperature for 2 hr. The reaction was filtered and the filtrate
was concentrated to give a residue, which was subjected to flash
column chromatography on silica gel with ethyl acetate in hexane.
Concentration of the appropriate fractions gave the product as a
very light yellow oil. NMR (CDCl.sub.3): .delta. 9.81 (t, 1H,
CH.dbd.O, J=2.6 Hz), 2.61 (t of d, 2H, CH.sub.2CH.dbd.O, J=7.1, 1.2
Hz), 2.28 (t of d, 2H, CCH.sub.2, J=7.1, 2.6 Hz), 1.99 (t, 1H, CCH,
J=2.6 Hz), 1.86 (p, 2H, CCH.sub.2CH.sub.2, J=7.0 Hz).
Example 71
Conjugation of Recombinant Human Erythropoietin to Aldehyde
Functionalized PC Polymer Containing Hexaglutamic Acid
[0482] Recombinant human erythropoietin (R&D Systems) was
buffer exchanged into 25 mM Hepes pH 7 (conjugation buffer) and
concentrated to 5 mg/ml. Conjugation reactions were carried out at
3-5.times. molar excess of the aldehyde functionalized PC copolymer
containing hexaglutamic acid from Example 60 to protein in the
presence of 40 mM sodium cyanoborohydride with a final protein
concentration of .about.1 mg/ml. All the reactions were carried out
in crimp sealed glass vials overnight at room temperature. The diol
form of the polymer was used as a negative control. 400 of each
reaction were fractionated on a Superdex 200 column (10/300 mm) at
1 ml/min. in 1.times.PBS pH 7.4. Fractions of 1 ml were collected
and analyzed at OD220 nm and OD280 nm. As expected, no conjugation
was observed when the diol functionalized polymer was used.
However, in the case of the aldehyde functionalized polymer, the
presence of erythropoietin-polymer conjugate was observed because
the OD280 nm:OD220 nm ratio was much higher than for the free
polymer alone in the 8-10 min. retention time range, where free
polymer and higher molecular weight species elute. Free
erythropoietin eluted in the 14-15 min. range.
Example 72
Preparation of 9-(Methacryloyloxy)-4,7-dioxanonanoic acid,
4-sulfo-2,3,5,6-tetrafluorophenyl ester, sodium salt
Preparation of 9-(Methacryloyloxy)-4,7-dioxanonanoic acid, t-butyl
ester
##STR00171##
[0484] A solution of 5.0 grams of t-butyl
4,7-dioxa-9-hydroxynonanoate in 100 ml of ether, together with 5.9
ml (2 eq) of triethylamine, was cooled with an ice water bath, and
a solution of 2.3 grams of methacryloyl chloride in 5 ml of ether
was added dropwise over a few minutes. The reaction was stirred in
the cold for 30 minutes, then allowed to warm to room temperature.
By TLC (silica gel, 50% ethyl acetate in hexane) the reaction
appeared to be incomplete, so another 1.0 g of methacryloyl
chloride was added dropwise. After another 2 hours, the reaction
appeared complete, so the reaction mixture was washed with 50 ml of
water, then dried over sodium sulfate. Filtration and concentration
gave an oily residue, which was subjected to flash column
chromatography on silica gel with 20-30% ethyl acetate in hexane.
The appropriate fractions were combined and concentrated to give
4.07 grams of the desired product as a clear, nearly colorless oil.
NMR (CDCl.sub.3): .delta. 6.13 (br m, 1H, C.dbd.CHH), 5.57 (br app
t, 1H, J=1.6 Hz, C.dbd.CHH), 4.29 (app t, 2H, J=4.8 Hz,
C.dbd.OOCH.sub.2), 3.70-3.76 (m, 4H), 3.61-3.67 (m, 4H), 2.50 (t,
2H, J=6.4 Hz, C.dbd.OCH.sub.2), 1.95 (app t, 3H, CH2=CCH.sub.3),
1.45 (s, 9H, C(CH.sub.3).sub.3).
Preparation of 9-(Methacryloyloxy)-4,7-dioxanonanoic acid
##STR00172##
[0486] A solution of 3.70 grams of t-butyl
9-(methacryloyloxy)-4,7-dioxanonanoate in 15 ml of 88% formic acid
was stirred at room temperature for 5 hours, at which time the
reaction was complete by TLC (silica gel, 50% ethyl acetate in
hexane). Concentration gave an oil, which was partitioned between
100 ml of dichloromethane and 50 ml of water, and the organic layer
was dried over sodium sulfate. Filtration and concentration gave an
oil, which was subjected to flash column chromatography on silica
gel with 40% ethyl acetate in hexane. The appropriate fractions
were combined and concentrated to give 2.01 grams of the desired
product as a clear, colorless oil. NMR (CDCl.sub.3): .delta. 6.14
(br m, 1H, C.dbd.CHH), 5.58 (br app t, 1H, J=1.6 Hz, C.dbd.CHH),
4.31 (app t, 2H, J=4.8 Hz, C.dbd.OOCH.sub.2), 3.73-3.80
(overlapping tm, 4H, J=6 Hz), 3.66 (m, 4H), 2.65 (t, 2H, J=6 Hz,
C.dbd.OCH.sub.2), 1.95 (app t, 3H, CH2=CCH.sub.3).
Preparation of 9-(Methacryloyloxy)-4,7-dioxanonanoic acid,
4-sulfo-2,3,5,6-tetrafluorophenyl ester, sodium salt
##STR00173##
[0488] A mixture of 970 mg of 9-(methacryloyloxy)-4,7-dioxanonanoic
acid and 1.06 grams of 4-sulfo-2,3,5,6-tetrafluorophenyl, sodium
salt in 20 ml of dry acetonitrile was treated with 1.06 grams of
DCC, and the reaction was stirred at room temperature for 1.5
hours. Filtration and concentration nearly to dryness gave a
residue, which was subjected to flash column chromatography on
silica gel with 5% methanol in dichloromethane. The appropriate
fractions were combined and concentrated to give a solid, which was
placed under high vacuum overnight to afford 783 mg of the desired
product as a slightly sticky solid. NMR (.sup.1H, CD.sub.3OD):
.delta. 6.10 (h, 1H, CH.sub.2, J=0.9 Hz), 5.61 (p, 1H, CH.sub.2,
J=1.6 Hz), 4.27 (m, 2H, CH.sub.2OC.dbd.O), 3.87 (t, 2H,
CH.sub.2CH.sub.2C.dbd.O, J=6.0 Hz), 3.75 (m, 2H,
CH.sub.2CH.sub.2OC.dbd.O), 3.67 (s, 4H, OCH.sub.2CH.sub.2O), 2.98
(t, 2H, CH.sub.2C.dbd.O, J=6.0 Hz), 1.92 (d of d, 3H, CH.sub.3,
J=1.6, 0.9 Hz). NMR (.sup.19F, CD.sub.3OD): .delta. -140.92 (m, 2F,
SCCF), 155.00 (m, 2F, OCCF).
Example 73
Preparation of (2-Mercaptoethyl)methacrylate, S-sulfate
Preparation of (2-Bromoethyl)methacrylate
##STR00174##
[0490] A solution of 6.25 grams of bromoethanol and 8.36 ml of
triethylamine in 50 ml of dichloromethane was cooled with an ice
water bath, and a solution of 5.0 grams of methacryloyl chloride in
5 ml of dichloromethane was added dropwise. The reaction was
stirred at room temperature for 4 hours, then another 50 ml of
dichloromethane were added and the reaction was washed with
2.times.25 mL of water, then with 25 ml of saturated sodium
chloride. The organics were dried over sodium sulfate, filtered and
concentrated to give an orange residue, which was subjected to
flash column chromatography on silica gel with 10% ethyl acetate in
hexane. The appropriate fractions were combined and concentrated to
give 3.15 grams of the product as a clear oil, which was pure
enough to use in the next reaction. NMR (CDCl.sub.3): .delta. 6.18
(app p, 1H, J=1.1 Hz, C.dbd.CHH), 5.62 (p, 1H, C.dbd.CHH, J=1.6
Hz), 4.46 (t, 2H, J=6.0 Hz, CH.sub.2OC.dbd.O), 3.56 (t, 2H,
CH.sub.2Br, J=6.0 Hz), 1.97 (dd, 3H, J=1.4, 1.1 Hz,
CH.sub.3C.dbd.C).
Preparation of (2-Mercaptoethyl)methacrylate, S-sulfate
##STR00175##
[0492] To a solution of 5.25 grams of sodium thiosulfate
pentahydrate and 10 mg of hydroquinone in 45 ml of water and 30 ml
of isopropanol was added 3.0 grams of (2-bromoethyl)methacrylate
and the reaction was stirred at room temperature overnight.
Concentration gave a residue, which was taken up in 20 ml of
ethanol and 20 ml of methanol. Filtration and concentration gave a
white solid, which was slurried with 45 ml of isopropanol. After
stirring vigorously for 4 hours, the solid was recovered by
filtration washed with a small amount of isopropanol and dried
under high vacuum to give 940 mg of the desired product as a white
solid. NMR (CD.sub.3OD): .delta. 6.11 (h, 1H, CH.sub.2, J=0.9 Hz),
5.62 (p, 1H, CH.sub.2, J=1.6 Hz), 4.47 (t, 2H, OCH.sub.2, J=6.9
Hz), 3.31 (t, 2H, SCH.sub.2, J=6.9 Hz), 1.93 (d of d, 3H, CH.sub.3,
J=1.5, 1.0 Hz).
Example 74
Preparation of PC Copolymer Containing Trimethoxysilane Functional
Groups
##STR00176##
[0494] 32.18 mg of 2,2' bipyridyl were placed in a Schlenk tube
followed by 20.1 mg of the initiator ethyl .alpha.-bromo
isobutyrate and dissolved in 160 mg of DMSO. The mixture was
degassed under vacuum for 10 min. 14.78 mg of CuBr were added under
inert conditions, and the reaction mixture was cooled to
-78.degree. C., degassed and refilled with inert gas. 1.033 g of
HEMA-PC and 46 mg of 3-(trimethoxysilyl) propyl methacrylate were
dissolved in 4 ml of 200proof ethanol and added to the reaction
mixture dropwise. The vessel was sealed and thoroughly degassed at
-78.degree. C. under vacuum until no bubbling was seen. The
reaction mixture was placed under inert conditions and allowed to
proceed at room temperature for 2 hours. Analysis by light
scattering showed a Mp of 24 kDa, Mn of 22 kDa and PDi of 1.05.
Example 75
Preparation of PC Copolymer Containing Protected Thiol Functional
Groups
##STR00177##
[0496] 74.8 mg of 2,2' bipyridyl were placed in a Schlenk tube
followed by 46.58 mg of the initiator ethyl .alpha.-bromo
isobutyrate and dissolved in 520 mg of DMSO. The mixture was
degassed under vacuum for 10 min. 34.26 mg of CuBr were added under
inert conditions, and the reaction mixture was cooled to
-78.degree. C., degassed and refilled with inert gas. 1.601 g of
HEMA-PC and 70.8 mg of S-Sulfo-(2-thioethyl)methacrylate, sodium
salt (from Example 73) were dissolved in 6.2 ml of 200proof ethanol
and added to the reaction mixture dropwise. The vessel was sealed
and thoroughly degassed at -78.degree. C. under vacuum until no
bubbling was seen. The reaction mixture was placed under inert
conditions and allowed to proceed at room temperature for 3 hours.
Analysis by light scattering showed a Mp of 17 kDa, Mn of 16 kDa
and PDi of 1.05.
Example 76
Preparation of PC Copolymer Containing Protected Thiol Functional
Groups and Tetrafluorophenol Ester Functional Groups
##STR00178##
[0498] 64 mg of 2,2' bipyridyl were placed in a Schlenk tube
followed by 40 mg of the initiator ethyl .alpha.-bromo isobutyrate
and dissolved in 300 mg of DMSO. The mixture was degassed under
vacuum for 10 min. 29.4 mg of CuBr were added under inert
conditions, and the reaction mixture was cooled to -78.degree. C.,
degassed and refilled with inert gas. 1.8 g of HEMA-PC,
6.77.times.10-4 mol of S-Sulfo-(2-thioethyl)methacrylate, sodium
salt (from Example 73), and 6.77.times.10-4 mol of
4,7-Dioxa-9-(methacryloyloxy)nonanoic acid,
4-sulfo,2,3,5,6-tetrafluorophenyl ester, sodium salt (from Example
72) were dissolved in 7.5 ml of 200proof ethanol and added to the
reaction mixture dropwise. The vessel was sealed and thoroughly
degassed at -78.degree. C. under vacuum until no bubbling was seen.
The reaction mixture was placed under inert conditions and allowed
to proceed at room temperature for 2 hours. Analysis by light
scattering showed a Mp of 24 kDa, Mn of 25 kDa and PDi of 1.05.
[0499] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of 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.
Sequence CWU 1
1
216PRTArtificial Sequencesynthetic hexaglutamic acid amide with
9-azido-4,7-dioxanononanoic acid 1Glu Glu Glu Glu Glu Glu1 5
26PRTArtificial Sequencesynthetic hexaglutamic acid, [Glu]-6 2Glu
Glu Glu Glu Glu Glu1 5
* * * * *