U.S. patent application number 10/228398 was filed with the patent office on 2003-04-24 for degradable cross-linking agents and cross-linked network polymers formed therewith.
Invention is credited to Kiser, Patrick F., Thomas, Allen A..
Application Number | 20030078339 10/228398 |
Document ID | / |
Family ID | 23324692 |
Filed Date | 2003-04-24 |
United States Patent
Application |
20030078339 |
Kind Code |
A1 |
Kiser, Patrick F. ; et
al. |
April 24, 2003 |
Degradable cross-linking agents and cross-linked network polymers
formed therewith
Abstract
Degradable cross-linkers which are used to form polymer networks
which degrade under aqueous conditions are described. These
cross-linkers comprise a central polyacid, monomeric or oligomeric
degradable regions and an optional water soluble regions. These
monomers are preferably polymerized using free radical or
condensation polymerization. Degradation occurs at the ester
linkages after cross-linking polymer filaments, and results in
soluble polymer filaments which may be cleared from the body.
Preferred applications of these materials include, for example,
controlled release of drugs and cosmetics, tissue engineering,
wound healing, hazardous waste remediation, metal chelation,
swellable devices for absorbing liquids and the prevention of
surgical adhesions.
Inventors: |
Kiser, Patrick F.; (Salt
Lake, UT) ; Thomas, Allen A.; (Loveland, CO) |
Correspondence
Address: |
JACKSON WALKER, L.L.P.
SUITE 2100
112 EAST PECAN ST.
SAN ANTONIO
TX
78205
US
|
Family ID: |
23324692 |
Appl. No.: |
10/228398 |
Filed: |
August 27, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10228398 |
Aug 27, 2002 |
|
|
|
09338404 |
Jun 22, 1999 |
|
|
|
6521431 |
|
|
|
|
Current U.S.
Class: |
525/54.5 ;
442/50 |
Current CPC
Class: |
C07C 233/20 20130101;
C08F 8/00 20130101; A61L 31/148 20130101; C07C 243/28 20130101;
A61K 9/2027 20130101; C08F 2800/10 20130101; Y10S 530/813 20130101;
Y10S 530/812 20130101; C08G 63/00 20130101; C07C 69/675 20130101;
A61L 24/0042 20130101; Y10S 977/906 20130101; Y10T 442/184
20150401; A61L 27/58 20130101; A61K 9/5146 20130101; Y10S 530/815
20130101; C08F 2810/20 20130101; A61K 9/5138 20130101; C07D 207/46
20130101; A61K 9/1635 20130101; C08F 8/00 20130101; C08F 220/20
20130101 |
Class at
Publication: |
525/54.5 ;
442/50 |
International
Class: |
C08G 063/48; C08G
063/91; D04H 005/02 |
Claims
What is claimed:
1. A monomeric or oligomeric cross-linker comprising a polyacid
core with at least two acidic groups being connected to reactive
groups usable to cross-link polymer filaments wherein between at
least one reactive group and the polyacid core is a region
degradable under aqueous conditions; the cross-linker being usable
to form cross-linked polymer filaments.
2. A monomeric or oligomeric cross-linker comprising a polyacid
core with at least two acidic groups connected to a region
degradable under aqueous conditions and having a covalently
attached reactive group usable to form cross-linked polymer
filaments.
3. The cross-linker of claim 1 or 2 where the polyacid is a
polycarboxylic acid.
4. The cross-linker of claim 3 described further as containing a
water soluble region between at least one carboxyl group and its
associated reactive group.
5. The cross-linker of claim 1 or 2 wherein the polymer is a
hydrogel.
6. The cross-linker of claim 1 or 2 wherein the polymer is
hydrophobic.
7. The cross-linker of claim 1 or 2 wherein the polyacid core
comprises at least one acidic group attached to a water soluble
region that is linked to a biodegradable region having a reactive
group attached thereto.
8. The cross-linker of claim 1 or 2 where the polyacid core is a
diacid.
9. The cross-linker of claim 1 or 2 where the polyacid core is a
triacid.
10. The cross-linker of claim 1 or 2 where the polyacid is a
pentaacid or tetraacid.
11. The cross-linker of claim 1 where the polyacid is succinic
acid, adipic acid, fumaric acid, maleic acid, sebacic acid, malonic
acid, tartaric acid, or citric acid.
12. The cross-linker of claim 1 or 2 where the polyacid is citric
acid, ethylenediaminetetraacetic acid (EDTA) or
diethylenetriaminepentaacetic acid (DTPA).
13. The cross-linker of claim 1 or 2 wherein cross-linked polymer
filaments are separated by at least two degradable regions.
14. The cross-linker of claim 1 or 2 wherein cross-linked polymer
filaments are separated by one degradable region.
15. The cross-linker of claim 1 or 2 where the degradable region
comprises a hydroxyalkyl acid ester.
16. The cross-linker of claim 1 or 2 where the degradable region
comprises an alpha hydroxy acid ester.
17. The cross-linker of claim 1 or 2 wherein the degradable region
comprises a peptide.
18. The cross-linker of claim 1 or 2 wherein the degradable region
comprises an glycolic polyester, a DL lactic acid polyester, a L
lactic acid ester, or combinations thereof.
19. The cross-linker of claim 1 or 2 where the degradable region
comprises at least one anhydride, orthoester or phosphoester or
combinations thereof, optionally attached to one or more
hydroxyalkyl acid esters.
20. The cross-linker of claim 1 or 2 wherein the degradable region
comprises at least one amide functionality.
21. The cross-linker of claim 1 or 2 defined further as comprising
at least one of an ethylene glycol oligomer, poly(ethylene) glycol,
poly(ethylene) oxide, poly(vinylpyrolidone), poly(ethylene) oxide,
co-poly(propylene) oxide), and poly (ethyloxazoline).
22. The cross-linker of claim 1 or 2 where the reactive group
contains a carbon-carbon double bond.
23. The cross-linker of claim 1 or 2 where the reactive group is an
end group.
24. The cross-linker of claim 1 or 2 where the reactive group
contains a carbonate, carbamate, hydrazone, hydrazino, cyclic
ether, acid halide, acyl azide, succinimidyl ester, imidazolide or
amino functionality.
25. The cross-linker of claim 1 or 2 wherein networks of polymer
filaments are formed by thermal, catalytic or photochemical
initiation.
26. The cross-linker of claim 1 or 2 wherein networks of polymer
filaments are formed by pH change.
27. The cross-linker of claim 1 or 2 wherein networks of polymer
filaments are formed by free radical addition or Michael
addition.
28. The cross-linker of claim 1 or 2 where the aqueous conditions
are physiological conditions.
29. A network of polymer filaments formed by precipitation,
dispersion or emulsion polymerization and cross-linked by a
monomeric or oligomeric cross-linker having a polyacid core with at
least two acidic groups connected to a covalently attached reactive
group used to cross-link polymer filaments and at least one acidic
group having a region degradable under aqueous conditions between
the acidic group and the reactive group.
30. A network of polymer filaments formed via condensation reaction
of preformed polymer filaments of polynucleic acids, polypeptides,
proteins or carbohydrates and cross-linked by a monomeric or
oligomeric cross-linker comprising a polyacid core with at least
two acidic groups connected to at least one region degradable under
in vivo conditions, and having a covalently attached reactive group
cross-linking the polymer filaments.
31. The network of claim 29 or 30 further comprising biologically
active molecules.
32. A network of polymer filaments cross-linked by a monomeric or
oligomeric cross-linker comprising a polyacid core with at least
two acidic groups connected to at least one region degradable under
in vivo conditions, and both acidic groups connected to a
covalently attached reactive group and defined further as
comprising an organic molecule, inorganic molecule, protein,
carbohydrate, poly(nucleic acid), cell, tissue or tissue
aggregate.
33. A network of polymer filaments cross-linked by monomeric or
oligomeric cross-linker comprising a central polyacid core with at
least two acidic groups connected to at least one region degradable
under in vivo conditions, and terminated by a covalently attached
reactive end group usable to cross-link polymer filaments, the
network comprising an organic radioisotope, inorganic radioisotope
or nuclear magnetic resonance relaxation reagent.
34. The cross-linker monomer or macromer of claim 1 or network of
claim 26 wherein the polyacid core has a molecular weight between
60 and 400 Da; the degradable region has a molecular weight between
70 and 500 Da and the reactive group has a molecular weight between
10 and 300 Da.
35. A microparticle or nanoparticle polymer composition containing
a monomeric or oligomeric cross-linker comprising a polyacid core
with at least two acidic groups connected to at least one region
degradable under in vivo conditions, and having a covalently
attached reactive group usable to cross-link polymer filaments.
Description
[0001] This patent application is a Continuation of U.S. patent
application Ser. No. 09/338,404, filed Jun. 22, 1999.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to novel cross-linking agents,
more particularly to novel biodegradable cross-linking agents.
Earlier use of cross-linking agents in a variety of fields
involving proteins, carbohydrates or polymers is well established.
Even biodegradable cross-links have previously been prepared and
utilized. However, none before have utilized particular and
advantageous cross-linker designs of the present invention.
[0003] Within the pharmaceutical, agricultural, veterinary, and
environmental industries, much attention has been directed to the
applications of biodegradable polymers. The Oxford English
dictionary defines biodegradable as: "susceptible to the
decomposing action of living organisms especially bacteria or
broken down by biochemical processes in the body." However, due to
the advent of the widespread use of polyhydroxyacids as degradable
polymers, this definition should be extended to include
non-enzymatic chemical degradation which can progress at an
appreciable rate under biologically relevant conditions (the most
relevant condition being water at pH 7; 100 mM salt and 37.degree.
C.). Thus, the meaning of the term biodegradation can be broadened
to include the breakdown of high molecular weight structures into
less complicated, smaller, and soluble molecules by hydrolysis or
other biologically derived processes.
[0004] In the biomaterials/pharmaceutical area, there is great
interest in the use of biodegradable materials in vivo, due to
performance and regulatory requirements. However, most of the
reports on biodegradable materials have focused on linear
water-insoluble hydroxyacid polyesters. Much less work has been
done on biodegradable network polymers which are cross-linked.
Therefore, due to the unique properties of network polymers, it is
to be expected that biodegradable networks will find many new and
important applications
[0005] Biodegradable Polymers
[0006] Much work has been accomplished in the last 20 years in the
area of hydrophobic biodegradable polymers, wherein the
biodegradable moieties comprise esters, lactones, orthoesters,
carbonates, phosphazines, and anhydrides. Generally the polymers
made of these biodegradable linkages are not water soluble and
therefore in themselves are not amenable for use in systems where
water is required, such as in hydrogels.
[0007] Since the mechanism of biodegradation in these polymers is
generally through the hydrolytically-active components of water
(hydronium and hydroxide ions), the rate of hydrolytic scission of
the bonds holding a polymer network together is generally pH
sensitive, with these moieties being susceptible to both
specific-acid catalyzed hydrolysis and base hydrolysis. Other
factors affecting the degradation of materials made of these
polymers are the degree of polymer crystallinity, the polymer
volume fraction, the polymer molecular weight, the cross-link
density, and the steric and electronic effects at the site of
degradation.
[0008] Degradable Network Structures
[0009] Biodegradable network structures are prepared by placing
covalent or non-covalent bonds within the network structure that
are broken under biologically relevant conditions. This involves
the use of two separate structural motifs. The degradable structure
is either placed into (i) the polymer backbone or (ii) into the
cross-linker structure. The method described herein creates a
degradable structure through placing degradable regions in the
cross-linking domain of the network. One of the first occurrences
of degradable hydrogels was published in 1983 by Heller. This
system contains a water soluble linear copolymer containing PEG,
glycolylglycolic acid and fumaric acid linkages. The fumaric acid
allowed the linear polymer to be cross-linked through free radical
polymerization in a second network forming polymerization step,
thus creating a polymer network which could degrade through
hydrolysis of the glycolic ester linkages. This is an example of
creating degradable linkages in the polymer backbone.
[0010] Biodegradable Cross-linkers
[0011] The first truly degradable cross-linking agents were made
from aryl diazo compounds for delivery of drugs in the digestive
tract. The diazo moiety is cleaved by a bacterial azoreductase
which is present in the colon. This has been used to create colon
specific delivery systems (Brondsted et al. & Saffan et al.).
Another biodegradable cross-linking agent appears in the work of
Ulbrich and Duncan where a bis-vinylic compound based on hydroxyl
amine was synthesized. Hydrogels made from this degradable
cross-linker were shown to undergo hydroxide induced hydrolysis of
the nitrogen-oxygen bond.
[0012] Hubbell et al. have made hydrogels composed of macromonomers
composed of a central PEG diol which was used as a bifunctional
alcohol in the tin octanoate catalyzed transesterifying ring
opening polymerization of lactide to give a bis-oligolactate PEG.
This compound was then reacted with acryloyl chloride to give a
macromolecular cross-linker which could be formed into a
homo-polymer interpenetrating network of PEG and
oligolactylacrylate through free radical polymerization (Pathak et
al.). Hubbell mostly intended these compounds for use as
photopolymerizable homo-polymers useful to prevent surgical
adhesions.
[0013] A second solution to this problem has been recently reported
in the work of Van Dijk et al. which is the first report of a
biodegradable cross-linking macromonomer composed of alpha-hydroxy
esters (Van Dijk-Wolthius et al.). This work combines natural
polymers with synthetic polymers in an interpenetrating network.
This group functionalized dextran with oligo-alpha hydroxy acid
domains which were end capped with vinyl regions that were
polymerized into biodegradable networks via free radical
polymerization.
[0014] The most recent report of a biodegradable cross-linking
agent was one designed to undergo enzymatic degradation. This
cross-linker is composed of a centro-symmetric peptide terminated
by acrylamide moieties with a central diamine linking the two ends
(Kurisawa et al.). This report is related to the invention
described herein in that the property of biodegradability is built
into the polymer network by first synthesizing a small symmetrical
cross-linker which can undergo cleavage, then incorporating this in
a polymer network.
[0015] Properties of Degradable Gels: Swelling and Porosity
[0016] Since degradability is a kinetic effect, the properties of
degradable gel networks are the similar to those standard gel
networks, except they change with time. The two main properties
that are exhibited by degradable hydrogel networks are swelling and
network porosity that increase with time as the network
degrades.
[0017] The main feature observed with degradable cross-linked
polymer networks in solvents which cause them to swell is that the
polymer network swells as it degrades. This is because network
degradation results in a decrease in cross-link density. As the
cross-link density decreases there is more available volume for
solvent within the network. The solvent increasingly permeates the
network structure, driven by a favorable thermodynamic mixing of
solvent with the polymer network.
[0018] Important uses envisioned for degradable gels are as
controlled drug delivery devices and as degradable polymers for
other in vivo uses. These devices are able to change from a high
viscosity material (gel) to a lower viscosity soluble material
(sol). The resulting water soluble linear polymer can then be
readily transported and excreted or degraded further.
[0019] Degradable hydrogel networks offer the opportunities to
effect the diffusitivity of materials bound in the hydrogel
network, because as the network degrades the diffusion coefficient
of molecules in the network increases with time thus facilitating
the release of materials locked within the polymer network
(Park).
[0020] Moreover, because the hydrogel network structure itself is
of such a high molecular weight, transport of the hydrogel network
out of the body or environment is slow. This is especially true in
vivo where non-degradable implanted hydrogel networks can remain in
the body for many years (Torchilin et al.). Therefore, such devices
would be more useful if they could be made of a high molecular
weight polymer that would degrade into smaller molecular weight
components after the device has performed its task and then could
be excreted through normal routes of clearance.
[0021] Since excretion of polymers is molecular weight- dependent
(Drobnik et al.), with the preferred route being through the renal
endothelia (Taylor et al. & Tomlinson), the chains making up
the polymer backbone should be between 10 and 100 kDa. Because the
material is engineered to degrade into excretable parts,
biodegradable hydrogel networks offer increased
biocompatibility.
[0022] Biodegradable Network Polymers as Controlled Release
Depots
[0023] Biodegradable network polymers can be used as carriers for
biologically active substances. These include proteins, peptides,
hormones, anti-cancer agents antibiotics, herbicides, insecticides
and cell suspensions. The hydrophilic or hydrophobic polymer
network can act as a stabilizing agent for the encapsulated species
and as a means to effect a controlled release of the agent in to
the surrounding tissue or systemic circulation. By changing the
size of the depot, the degree of porosity, and the rate of
degradation (through modification of the degradable regions in the
polymer network) controlled release depots with a variety of
release characteristics can be fashioned for application in the
medical and diagnostic areas.
[0024] Biodegradable Network Polymers as Water Adsorbents
[0025] Owing to the ability of hydrophilic network polymers to
adsorb water, biodegradable versions of these networks may prove to
have many uses in items for example, sanitary napkins, wound
dressings, and diapers. When these materials are used in consort
with other degradable materials a completely biodegradable and
disposable product could be produced. Although a literature search
in the Chemical Abstracts database for biodegradable adsorbents
produced no citations, the use of degradable adsorbents in the
above mentioned products would be very desirable.
[0026] Biodegradable Network Polymers as Adhesives
[0027] There is a great need for biodegradable adhesives and
sealers in surgery and elsewhere. Synthetic polymers have been used
as adhesives in surgery with the cyano acrylate esters being the
most commonly cited. Recent reports using biodegradable networks as
sealants in dentistry and orthopedics have displayed the utility of
biodegradable polymers (Burkoth). Here the use of a biodegradable
cross-linking monomer (bis-methacrylated diacid anhydride) which
has been photopolymerized is envisioned for use in dentistry. Here
a hydrophobic network-forming monomer is photopolymerized in situ
to form a mechanically stable and non-swellable bonding material.
Degradability would be a desirable property for any short term
application and of course would be undesirable for long term
applications.
[0028] Use of Biodegradable Polymers in Drug Delivery
[0029] Since most biodegradable polymers are not soluble in water,
a hydrophilic drug is formulated in these polymers by a dispersion
method using a two phase system of water (containing drug) and
organic solvent (containing the polymer). The solvent is removed by
evaporation resulting in a solid polymer containing aqueous
droplets. This type of system suffers from the need to use organic
solvents which would be undesirable for protein delivery since the
solvent may denature the protein. Therefore it is envisioned that
hydrophilic biodegradable network polymers will improve the range
of drugs delivered from this general glass of polymers.
[0030] Biodegradable Nanoparticles
[0031] The use of nanoparticles for colloidal drug delivery has
been a goal of formulation scientists for the last 20 years.
Nanoparticles are defined as any solid particle between 10 and 1000
nm and are composed of natural or more commonly synthetic polymers.
The most useful method of production for the lower end of this size
range is emulsion polymerization, where micelles act as a reaction
template for the formation of a growing polymer particle. For
passive delivery of anticancer agents to tumors, nanometer size
particles (50-200 nm) are required. The small size is required for
extravasation of the nanoparticles through the permeable tumor
vasculature in a process termed the EPR effect (enhanced
permeability and retention) (Duncan).
[0032] Another important feature of any nanocarrier is the
biocompatibility of the particle. This requires that the polymer
particle degrades after some period so that it may be excreted.
These criteria require polymer compositions that are well
tolerated. To date there are no reports in the literature of
degradable nanogels composed of well-tolerated parenternal
polymers.
[0033] Hydrogel particles can be made in several sizes according to
the performance requirements of the drug delivery system being
engineered. Gel particles in the nanometer size range that are
capable of being retained in tumor tissue are preferred for
delivery of anticancer agents. Methods for the creation of
approximately 100 nm in diameter hydrogel particles involve the use
of surfactant-based emulsion polymerizations in water. To make
ionomeric nanogels by this method it is necessary to include a
hydrophobic component in the monomer mixture, thus allowing
partitioning of the monomers into the micellar phase followed by
particle nucleation and further monomer adsorption (normally
emulsion polymerizations are used to make hydrophobic latexes).
[0034] Another important consideration is the means by which the
carrier will load the drug substance to be delivered. The loading
capacity of non-ionic hydrogels is generally limited by the aqueous
solubility of the drug. However if the drug is charged, groups of
opposite charge to the drug can be incorporated into the polymer to
allow high drug loading through ion exchange. An interesting and
perhaps useful property resulting from inclusion of charged
monomers in the polymer network is a pH induced volume response of
the polymer.
[0035] Current State of the Art
[0036] To date most biodegradable polymers have been synthesized
using stepwise condensation of monomer resulting in a polydispersed
molecular architecture. Since the rate of degradation is in part
directly related to this architecture, this method results in the
undesirable property that the material will contain cross-links
with a variety of degradation rates. Secondly, since synthetic
biodegradable polymers are generally water insoluble, there is a
need for degradable moieties that are readily incorporated into
water soluble monomers or polymers. Biodegradable moieties based on
the non-soluble degradable units can be combined with water soluble
oligomeric regions or polymers, resulting in a biodegradable
structure.
[0037] Therefore as an object of the present invention the new
material would have the preferred characteristics that it was
easily synthesized, composed of biocompatible components, and have
a well defined molecular structure leading to defined
biodegradation rates.
[0038] It is a further object of the present invention that it be
easily incorporated in many different polymer processing options
such as polymer microparticles, nanoparticles and slab gels.
[0039] Therefore, the use of organic synthesis methodology to
incorporate monodispersed degradable sequences into the monomer
structure before polymer formation permits control of overall
degradation as well as the release rate of entrapped
substances.
[0040] Previous work in the area of creating biodegradable
cross-linkers by Hubbel teaches a method to create degradable
sequences using ring opening polymerization of lactide or
glycolide. This method creates a mixture of degradable units with
varying molecular weights or chain lengths in the end product. The
present invention described herein teaches a method of stepwise
synthesis of the degradable region which creates a pure compound at
the end of the synthesis. Therefore, since the length of the
degradable region will be the major structural determinant of the
degradation rate, the present invention provides for a more
controlled degradation rate than the Hubbel invention. Our
invention also provides compounds which will be easier to purify
than the Hubbel invention owing to stepwise syntheses of the
degradable region and the resulting purity of the reaction product.
Other advantages of our invention over Hubbel's invention are that
the invention described herein is applicable to hydrophobic
networks as well as hydrophilic networks whereas Hubbel is
restricted to hydrophilic networks, and the invention herein can
generate all useful properties such as rapid degradation rate and
water solubility through the syntheses of oligomeric cross-linking
compounds without resorting to polymeric cross-linking
compounds.
[0041] The present preferred embodiment of this invention is as
cross-linkers which are composed of a symmetrical diacid attached
to at least one biodegradable region. These regions may consist of
alpha hydroxy acids such as glycolic or lactic acid. These
degradable portions are then terminated directly or indirectly by a
functional group which may be polymerized. Moreover component
pieces of the degradable gel such as lactic, glycolic and succinic
acids are members of the Krebs cycle and therefore readily
metabolized in vivo, while the end groups become incorporated into
water-soluble polymer, which is eliminated by renal excretion.
SUMMARY OF THE INVENTION
[0042] In one important aspect the present invention concerns a
monomeric or oligomeric cross-linker comprising a polyacid core
with at least two acidic groups directly or indirectly connected to
a reactive group usuable to cross-link polymer filaments, with at
least one acidic group being connected to a region degradable under
aqueous conditions and the degradable regions or (in the case of a
single degradable region), the degradable region at at least one
other acidic group directly or indirectly having a covalently
attached reactive group usable to cross-link polymer filaments
interceding between the acidic group and a reactive group. Thus the
at least two reactive groups are always interspaced by at least one
degradable region. In many preferred applications, the cross-linker
is utilized to cross-link water soluble polymeric filaments. The
polyacid core may be attached to a water soluble region that is in
turn attached to a degradable region (or vice versa) having an
attached reactive group. A polycarboxylic acid is the preferred
polyacid. The polyacid core is preferably a diacid, triacid,
tetraacid or pentaacid. The most preferred polyacid core is a
diacid. Preferred polyacids or polycarboxylic acids. Alkyl-based
diacids such as malonic, succinic, adipic, fumaric, maleic, sebacic
and tartaric are preferred. Diacids such as succinic, adipic or
malonic acid are particularly preferred. A triacid such as citric
acid, for example, is usable. Tetra-and penta-acids such as
ethylenediamine tetraacidic acid (EDTA) or diethylenetramine
pentaacetic acid (DTPA) are usable, for example. When cross-linked
polymer filaments are formed according to the present invention,
they are cross-linked by a component having at least one degradable
region. Preferred degradable regions include poly (alpha-hydroxy
acids), although other hydroxy alkyl acids that may form polyesters
can be used to form biodegradable regions. Preferred polyesters
include those of glycolic acid, DL lactic acid, L lactic acid,
oligomers, monomers or combinations thereof. Cross-linkers of the
present invention may also include a degradable region containing
one or more groups such as anhydride, a orthoester and/or a
phosphoester. In certain cases the biodegradable region may contain
at least one amide functionality. The cross-linker of the present
cross-linker may also include an ethylene glycol oligomer,
oligo(ethylene glycol), poly(ethylene oxide), poly(vinyl
pyrolidone), poly(propylene oxide), poly(ethyloxazoline), or
combinations of these substances.
[0043] Preferred reactive groups are those that contain a
carbon-carbon double bond, a carbonate, a carbamate, a hydrazone, a
hydrazino, a cyclic ether, acid halide, a acylazide, succinimidyl
ester, imidazolide or amino functionality. Other reactive groups
may be used that are known to those skilled in the art to be
precursors to polymers.
[0044] Utilizing the cross-linkers of the present invention,
networks of polymer filaments may be formed by thermal, catalytic
or photochemical initiation. Networks of polymer filaments may
likewise be formed by pH changes. Networks of polymer filaments may
also be formed for example by free radical addition or Michael
addition.
[0045] The present invention comprises a network of polymer
filaments formed by precipitation or emulsion polymerization and
cross-linked by a monomeric or oligomeric cross-linker comprising a
poly acid core with at least one acidic group connected to a region
degradable under in vivo conditions and having at least two
covalently attached reactive groups usable to cross-link polymer
filaments. Polymeric filaments to be cross-linked include preformed
polymer filaments such as polynucleic acids, polypeptides, proteins
or carbohydrates. Such cross-linked polymeric filaments may be
utilized to contain biologically active molecules. The biologically
active molecules may be organic molecules, proteins, carbohydrates,
polynucleic acids, whole cells, tissues or tissue aggregates.
[0046] The preferred monomeric or oligomeric cross-linker of the
present invention has a polyacid core with a molecular weight
between about 60 and about 400 Daltons. The degradable region(s)
has a preferable molecular weight range of about 70 to about 500
Daltons. The reactive groups of the cross-linker of the present
invention may be end groups and have preferred molecular weights
between about 10 and 300 Daltons.
[0047] An important aspect of the present invention is a monomeric
or oligomeric cross-linker comprising a polyacid core with at least
two esterified groups being connected (directly or indirectly) to
reactive groups usable to cross-link polymer filaments. Between at
least one reactive group and polyacid core is a region degradable
under aqueous conditions. Thus the cross-linker is usable to form
cross-linked polymer filaments. In a preferred embodiment, the
polyacid core has two acidic groups connected to a region
degradable under aqueous conditions, each having a covalently
attached reactive group usable to form cross-linked polymer
filaments. In certain cases the cross-linkers of the present
invention may contain a water soluble region located between at
least one carboxyl group and its associated reactive group. A
preferred polymer filament for cross-linking is a hydrogel. In
certain cases the polymer filament being cross-linked may be
hydrophobic.
[0048] In many cases the polyacid core of the present inventive
cross-linker is a diacid, such as for example succinic acid, adipic
acid, fumaric acid, maleic acid, sebacic acid or malonic acid.
Triacids such as citric acid are also usable. Other triacids will
be apparent to those of skill in the art. Tetraacids and pentaacids
may also be used. A preferred tetraacid is ethylene diamine
tetraacetic acid (EDTA) and a preferred pentaacid is
diethylenetriamine pentaaceticic acid (DTPA).
[0049] Acids that may be used as a polyacid core include citric
acid, tartaric acid and the like. A preferred biodegradable region
for use in the cross-linkers of the present invention is one that
comprises a hydroxy alkyl acid ester. A preferred hydroxy acid
ester is an alpha hydroxy acid ester. Under some circumstances the
degradable region may be a peptide. Preferred degradable polyesters
include glycolic polyester, DL lactic acid polyester and L lactic
acid ester or combinations thereof. In certain cases the degradable
region of the cross-linker of the present invention may comprise an
anhydride, orthoester or phosphoester linkages. In certain cases
the reactive group of the present inventive cross-linker contains a
carbon-carbon double bond. In some cases the reactive group is an
end group, e.g. at the end of a degradable region. The reactive
group may also contain a carbonate, carbamate hydrazone, hydrazino,
cyclic ether, acid halide, acyl azide, succinimidyl ester,
imidazolide or amino functionality.
[0050] The cross-linker of the present invention may be utilized to
form networks of polymer films formed by thermal catalytic or
photochemical initiation. In certain cases networks of polymer
films may be formed as induced by a pH change and then
cross-linked. In other cases, networks of polymer films may be
formed through reactions involving free radical addition or Michael
addition. The aqueous conditions under which the cross-linkers of
the present invention are degradable are most frequently
physiological conditions.
[0051] In an important aspect, the present invention comprises a
network of polymer filaments formed by precipitation, dispersion or
emulsion polymerization and cross-linked by a monomeric or
oligomeric cross-linker having a polyacid core with at least two
esterified groups connected to a covalently attached reactive group
used to cross-ink polymer filaments and at least one acidic group
having a region degradable under aqueous conditions between the
acidic group and the reactive group.
[0052] Also included in the present invention are networks of
polymer filaments of polynucleic acids, polypeptides, proteins or
carbohydrates and cross-linked by a monomeric or oligomeric
cross-linker comprising a polyacid core with at least two
esterified groups connected to at least one region degradable under
in vivo conditions, and having a covalently attached reactive group
cross-linking the polymer filaments.
[0053] In both cases of networked polymer filaments, these networks
may contain biologically active molecules. Because the cross-links
are degradable, these biological molecules will be expected to be
released.
[0054] In one important aspect, the present invention comprises a
network of polymer filaments cross-linked by a monomeric or
oligomeric cross-linker comprising a polyacid core with at least
two acidic groups connected to at least one region degradable under
in vivo conditions, and both acidic groups connected to a
covalently attached reactive group and defined further as
comprising an organic molecule, inorganic molecule, protein,
carbohydrate, poly(nucleic acid), cell, tissue or tissue
aggregate.
[0055] Additionally, the invention includes a network of polymer
filaments cross-linked by monomeric or oligomeric cross-linker
comprising a central polyacid core with at least two acidic groups
connected to at least one region degradable under in vivo
conditions, and terminated by a covalently attached reactive end
group usable to cross-link polymer filaments, the network
comprising an organic radioisotope, inorganic radioisotope or
nuclear magnetic resonance relaxation reagent.
[0056] According to the present invention the polyacid core has a
preferred molecular weight between about 60 and about 400 daltons.
The degradable region of the cross-linker has a preferred molecular
weight between about 70 and about 500 daltons. The reactive groups
of the present invention generally have molecular weights between
about 10 and about 300 daltons.
BRIEF DESCRIPTION OF DRAWINGS
[0057] FIG. 1 schematically illustrates a representative
lactate-based cross-linking agent of the present invention.
[0058] FIG. 2 schematically displays a synthetic method for
symmetrical biodegradable cross-linkers such as HPMALacSuc 5a,
HPMAGlySuc 5b, HPMALacLacSuc 7a, and HPMAGlyGlySuc 7b. Conditions:
(a) CH2Cl2, pyridine 0.degree. C.; (b) Pd/C 50 psi H2, i-PrOH;
0.degree. C.; (c) carbonyldiimidazole CDI, DMF, 0.degree. C.; HPMA,
rt.; (e) (CDI), DMF, 0.degree. C.; benzyl lactate (6a); benzyl
glycolate (6b); (f) Pd/C 50 psi H2, i-PrOH.
[0059] FIG. 3 displays a photograph of biodegradable gels of the
same composition with 1.5 mole % cross-linker after incubation in
pH 7 phosphate buffer at 37.degree. C. for varying amounts of time.
(a) control gel made up of compound 2 after 15 days (b-d) compound
5b after 2, 5 and 15 days, respectively.
[0060] FIG. 4 displays the degradative swelling of HPMA-co-XL gels
made from 4 different cross-linkers in pH 7.3 buffer; 100 mM
phosphate buffer; I=200 mM at 37.degree. C. The cross-linker
labeled HPMASuc is non-degradable.
[0061] FIG. 5 displays a plot of the half-life to dissolution
versus pH for three different degradable cross-linkers studied at
37.degree. C.
[0062] FIG. 6 displays a photograph of p (HPMA) degradable gels
with 1.5 mole % cross-linker and containing a deep red fluorescent
dye--thus the dark color--after incubation in pH 7 phosphate buffer
at 37.degree. C. for varying amounts of time. (a) control gel made
from compound 2 after 15 days (b and c) compound 7b after 4, 8 days
respectively.
[0063] FIG. 7 displays a plot comparing the swelling response and
the release of tetramethyl rhodamine labeled albumin from the
degradable gel network for HPMAGlyGlySuc 7b at pH 7.3 at 37.degree.
C.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0064] This invention discloses a representative synthesis and
application of symmetrical biodegradable cross-linking agents for
use in cross-linked polymer matrices formed into particles or slabs
that may be used e.g., in drug delivery. The cross-linking agents
will be monomers or oligomers of biocompatible units in the
preferred biological applications. In the preferred practice of
this invention the cross-linker is composed of a central diacid
(such as succinic); to this diacid is attached one or more
biodegradable regions, which are then terminated by reactive
moieties which are used for incorporation into the polymer network.
This invention requires there be at least two reactive moieties
(two representative cross-linkers are depicted in FIG. 1). The
cross-linkers may be incorporated into matrices of various sizes
ranging from hundreds of cm's to 10 nm so as to control the
diffusion of substance such as drugs e.g., from the matrix by
biodegradation of the cross-linkers under physiological conditions.
Ultimately the cross-linkers described above may be included in all
variety of hydrophilic and hydrophobic polymer networks to which
the desirable property of degradation is required.
[0065] Design of Centro-Symmetric Degradable Cross-Linkers Based on
the Alpha-Hydroxy Acids
[0066] Of importance in hydrogel engineering is the control the
structural properties of a random polymeric network. In standard
stepwise growth of polymers there is heterogeneity in copolymer
composition and dispersity in the molecular weight of the polymer
filaments thus making it difficult to precisely control bulk
material properties of the polymer network such as crystallinity
and mesh size. By engineering homogeneous structures into the
polymer structure, usefully tuned macromolecular properties such as
biodegradability can be obtained.
[0067] Hydrogel networks in the form of colloidal particles which
are being explored for use in drug delivery (Kiser et al.) are not
biodegradable owing to their carbon-carbon bond containing backbone
and their methylene-bis-acrylamide cross-links. This fact initiated
the design of a new class of centro-symmetric cross-linking
monomers. One of the preferred characteristics of the new material
was that it must be easily synthesized. A second preferred
characteristic is that the cross-linkers be composed of
biocompatible components. The third characteristic which separates
this work from all other work in this area is that the
biodegradable cross-linker be synthesized to be a single pure
molecule and not a mixture. This characteristic should lead to
defined biodegradation rates versus the use of a cross-linker
mixture as in previous work (Pathak et al.).
[0068] Therefore by utilizing classical organic synthesis
methodology to synthesize monodispersed degradable sequences into
the monomer structure before polymer formation presents an
opportunity to carefully control the overall degradation as well as
possibly the release rate of entrapped substances. One of the
particularly preferred embodiments of these cross-linkers is that
they are composed of a symmetrical diacid each acid attached to a
biodegradable regions consisting of acids, such as the
alpha-hydroxy acids glycolic or lactic acid for example. These
portions are then preferably terminated by the monomer
methacrylate.
[0069] The Monomers
[0070] The monomers are composed of a central polyacid as in FIG. 1
and are attached to the degradable region through oxygen, nitrogen,
or phosphorous atoms. Structure A shows a monomer having a central
diacid region (--), and a degradable region
(/.backslash./.backslash./.backslash-
./.backslash./.backslash./.backslash./) which is then terminated by
a reactive polymerizable region (------------X). Structure B is
similar and uses the same symbols except that the central core is a
triacid symbolized by a T structure. FIG. 2 displays a more
specific embodiments of this invention. In structure C, a
symmetrical centerpiece (succinic acid) is attached to two
degradable regions containing alpha-hydroxy esters. These are then
attached to a moiety (R.sub.2) which may or may not impart water
solubility through the connecting portion labeled Y. Finally, the
cross-linker is terminated with vinyl groups. Structure D is again
similar to structure C except in this case the monomer is
terminated with two nucleophilic moieties which could be used to
cross-link preformed polymer chains. These structures are exemplary
only. Many more are conceivable by those skilled in the art.
[0071] In a preferred embodiment the network begins with a
cross-linker containing two equal degradable regions attached to a
central diacid and each containing a terminal reactive group. In a
particularly preferred embodiment, the core is made of succinic
acid, each degradable region is composed of either symmetrical
units of glycolic or lactic acid where n in FIG. 1 is between 1 and
5 and the terminal reactive group is a acrylate type moiety where
R2 in FIG. 1 is CH(CH3)CH2CO and Y is equal to oxygen.
[0072] Central Component
[0073] In preferred embodiments the central piece can consist of
esters of dicarboxylic acids such as malonic succinic, adipic,
sebacic, maleic fumaric acids or even possibly (alpha,
omega-(oligo(ethylene glycol)) dicarboxylic acid (alpha,
omega-(oligo(propylene glycol)) dicarboxylic acid. Other diacids
such as aromatic polycarboxylic acids may also be used. In another
embodiment tri-acids such as citric acid or tetra and penta acids
such as EDTA and DTPA (possibly as protected derivatives) could
also be utilized. Also protected versions of tartaric, citric,
aspartic or glutamic acid may be used in certain embodiments.
[0074] Biodegradable Component
[0075] The biodegradable region is preferred to be hydrolyzable
under environmental or in vivo conditions. In the most preferred
embodiment the degradable regions will be composed of glycolic or
lactic acid domains containing anywhere from one to six members in
each oligomeric region attached to the central piece. Other hydroxy
esters that may be embodied include: (3-hydroxy butyric acid,
2-hydroxy propanoic acid, and 5-hydroxy caproic acid. Other useful
biodegradable regions include amino acids, ortho-esters,
anhydrides, phosphazines, phosphoesters and their oligomers and
polymers.
[0076] Reactive Cross-linking Polymerizable Region
[0077] This region is necessary for the invention because it is the
chemical functionality terminating the two or more ends of the
cross-linker which will chemically bind polymer filaments together.
The preferred method of achieving this end is through an acrylate
moiety, with polymerization through free radical generation. Free
radical generation can be accomplished via thermal, photochemical
or redox catalysis initiation systems (Odian). The preferred
polymerizable regions for free radical generation are acrylates,
vinyl ethers, diacrylates, oligoacrylates, methacrylates,
dimethacrylates, and oligomethacrylates. Alternatively another
preferred method of cross-linking preformed chains in solution is
to attach two or more nucleophiles to the end of the chains which
would be reactive with an electrophile attached to the polymer
chain. The preferred chemical reactive moieties for this method are
carbonate, carbamate, hydrazone, hydrazino, cyclic ether, acid
halide, acyl azide, alkylazide, succinimidyl ester, imidazolide,
amino groups, alcohol, carbonyl, carboxylic acid, carboxylic ester,
alkyl halide, aziridino, nitrile, isocyanate, isothiocyanate,
phosphine, phosphonodihalide, sulfide, sulfonate, sulfonamide,
sulfate, silane, or silyloxy groups.
[0078] Initiators
[0079] Several initiation systems for the formation of polymer
networks are useful with these compounds, depending on the
application and the conditions used.
[0080] For generation of polymer slabs either irradiation of vinyl
groups with high energy light such as in the UV is a suitable
method for initiation. Other preferred methods include the use of
thermally activated initiators such as azobisisobutyronitrile or
benzoyl peroxide for initiation in water or mixed water/organic
solvents, other water soluble alkyl diazo compounds, ammonium
persulphate with or without N,N,N',N'-tetramethyethylene
diamine.
[0081] For generation of particles by emulsion polymerization
generation of radicals by thermal initiation is convenient.
Generally this is accomplished with water soluble initiators such
as ammonium persulphate. Other initiators include the water soluble
alkyl diazo compounds.
[0082] For generation of polymer networks in vivo the most useful
initiation system is photochemical. Photochemical initiation of
free radical polymerization involves light activation of a light
absorbing compound (a dye), radical abstraction of a hydrogen to
generate the initiation radical (usually an amine), and attack of
this radical on a vinylic moiety beginning the polymerization. This
system preferably requires free radicals to be generated locally
and within a short time period, preferably in seconds. Initiation
in this system begins with irradiation of light at the appropriate
wavelength. The wavelength is chosen to be as close to the
absorption maximum of the dye as possible. The preferred light
absorbing compounds which will begin the radical generation process
are eosin dyes, 2,2'-dimethoxy-2-phenyl acetophenone and other
acetophenone derivatives. Other photo redox active dyes include
acridine dyes, xanthene dyes and phenazine dyes, for example,
acriblarine, rose bengal and methylene blue, respectively. These
dyes when photoactivated assume a triplet excited state which can
abstract a proton from an amine and thus generate a radical which
begins the polymerization. Compounds which act as the initiating
radical are amines such as triethanolamine, sulfur containing
compounds such as ammonium persulphate, and nitrogen
containing-heterocycles such as imidazoles.
[0083] Applications for the Cross-Linkers
[0084] Nature of the Polymer
[0085] In the preferred embodiment of this invention, these
cross-linkers can be incorporated in biodegradable network polymers
that are either hydrophilic or are hydrophobic. Hydrophobic
networks will contain less than 5% of the total mass of the polymer
network as water. Whereas hydrophilic networks can contain as great
as 99% water as the total mass. Hydrophilic network polymers are
known as hydrogels to those skilled in the art. Those skilled in
the art will generally recognize the polymer structures which are
generally considered to be hydrophilic or hydrophobic.
[0086] In Vivo Drug Delivery
[0087] One preferred application of these materials is in the use
of controlled delivery of bioactive compounds. In this method the
cross-linker is homopolymerized or copolymerized with other monomer
or polymers which may be charged or uncharged. The drug is placed
in the polymer network by polymerizing the network around the drug
(i.e., by co-dissolving or dispersing the drug with the monomer
solution) or by incubating the resulting polymer with a solution of
the drug whereby it diffuses into the polymer network. In this
embodiment the drug may be anywhere from 1 to 90% by weight of the
device. The biologically active compounds can be (but are not
limited to) proteins, peptides, carbohydrates, polysaccharides,
antineoplastic agents, water soluble linear and branched polymeric
prodrugs, particles containing drug, antibiotics, antibodies,
neurotransmitters, psychoactive substances, local anesthetics,
anti-inflammatory agents, spermicidal agents, imaging agents,
phototherapeutic agents, DNA, oligonucleotides and anti-sense
oligonucleotides.
[0088] An alternative method of producing a biodegradable drug
delivery system is through the production of particles. The
preferred size range is between 10 nm and 10 .mu.m. These particles
can be produced by emulsion polymerization in water containing a
surfactant such as sodium dodecyl sulfate, an initiator such as
ammonium persulphate, and cross-linking monomer and co-monomer(s)
such as 2-hydroxypropyl methacrylamide, 2-hydroxyethylmethacrylate,
acrylic acid, methacrylic acid, methyl methacrylate, methyl
acrylate, or other suitable monomers by themselves or in mixtures.
Alternatively the particles can be synthesized by precipitation
polymerization in organic solvent containing organic soluble
initiator such as azobisisobutronitrile and co-monomer(s) such as
acrylamide, as 2-hydroxypropyl methacrylamide, 2-hydroxyethyl
methacrylate, acrylic acid, methacrylic acid, methyl methacrylate
or methyl acrylate by themselves or in mixtures. In this method the
preferred route of incorporating drug in the particles is by first
synthesizing the particle, followed by purification through
washing. The particle is then incubated with drug which is bound to
the polymer network by either hydrophilic or ionic forces or by
entrapment within the network.
[0089] Another method which is well known to those skilled in the
art of producing polymer particles includes dissolving the
cross-linking monomer, co-monomer, initiator with or with our drug
in water and then dispersing this solution in oil. The resulting
oil droplets then act as templates for the formation of the gel
network. Polymerization is initiated either thermally, chemically
or photochemically depending on the monomer system and initiator
system. Which combination of systems to use will be obvious to
those skilled in the art. The resulting particles can then be
sedimented and isolated and purified. This technique is
particularly useful for producing larger particles in the 5- to
1000 micron in diameter size range.
[0090] Another preferred method for the creation of a drug delivery
device is to create a homopolymer network of the cross-linker in
organic solvent in the presence of a organic soluble drug. The
network is then dried and contains drug dispersed within it. The
highly cross-linked network will begin to erode when hydrated and
release drug.
[0091] Water Absorbents
[0092] In this application an important consideration is to
copolymerize the biodegradable cross-linker with charged monomers
(either negative charges or positive charges or mixtures thereof).
Very high charge densities in the polymer network can be obtained
by copolymerization of charge monomers into networks (>5 M). The
presence of charges in the polymer network require counterions for
electroneutrality. These counterions bind water to a lesser or
greater extent, depending on their size and polarizabilities. Since
the volume of the hydrated gel is equal to the volume of polymer,
the volume of water bound to the polymer and the volume of the
hydrated ions bound to the polymer, the presence of a large amount
of hydrated ions can create a super-water adsorbent hydrogel. The
molar ratio of cross-linker to other monomers should be kept as low
as possible so as to not inhibit the swellability of the network,
preferably in the range of 5 mol % or less. The preferred
copolymers include methacrylic acid, acrylic acid, acrylic and
methacrylic monomers containing sulfate, alkyl carboxylate,
phosphate, amino, quaternary amino and other charged groups and
their salts. In this application large batches of the degradable
network will be synthesized either by dispersion polymerization or
in bulk. The material could be synthesized in the presence of a
suitable counterion such as sodium for negatively charged filaments
or chloride for positively charged filaments. Alternatively the
polymer may be formed in its neutral state and then incubated with
a suitable acid or base such as hydrochloride in the case of
nitrogen containing co-monomers, and soluble metal hydroxides in
the case of acidic co-monomers. The most preferred method is to
polymerize the cross-linker with the salt form of the
co-monomer.
[0093] Adhesives
[0094] Another use of the monomer is in temporarily binding two
surfaces together. The biodegradable cross-linking monomer and
co-monomer or just the biodegradable cross-linking monomer itself
are mixed together with a solvent and an initiator by itself or
with a co-catalyst. The mixture is then spread on the surfaces
which are to be adhered, then polymerization is initiated by
addition of heat or by light. In the case of light initiation at
least one of the surfaces to be adhered must be transparent to the
light beam in order for the polymer network to form. The initiation
systems described above can be used to this end. Such biodegradable
adhesives should have many uses.
[0095] Tissue Supports
[0096] There is a need for degradable polymers as cell scaffolds in
tissue engineering. In this application the tissue scaffold would
be synthesized under sterile conditions in a suitable biocompatible
buffer. The cross-linking density should be controlled so as to
obtain a pore size large enough to allow cell migration. Pore size
may be determined by scanning electron microscopy and by using
macromolecular probes. A cell suspension containing cells such as,
but not limited to, keratinocytes, chrondocytes and osteoblasts,
would be injected into the polymer network along with suitable
growth factors. The cells would then be allowed to grow within the
network. As the cells grow the network around them would degrade.
Bioadhesive moieties such as RGD peptide sequence (Arg-Gly-Asp)
could be connected to matrix and thereby provide adhesive domains
for the growing cells. The timing of the network degradation should
coincide with the cells forming their own network (artificial
tissue) through inter-cell contacts.
[0097] The following examples are presented to describe preferred
embodiments and utilities of this invention but are not intended to
limit the use or scope of the methods, compositions or compounds
claimed in this invention unless otherwise stated in the claims.
Taken together, these examples describe the best currently
understood mode of synthesizing and incorporating these materials
into polymer networks.
[0098] The synthesis of the four members of the preferred class of
molecules claimed herein are given in FIG. 2. This invention has
several advantages over related inventions in this area, including:
(1) the cross-linking agents are biodegradable to biocompatible
substances, (2) the syntheses are both general and flexible,
allowing for a variety of monomeric units to be incorporated, (3)
the end groups (e.g., acrylate or hydrazide) can be readily
modified to accommodate either condensation or radical-type
polymerizations.
EXAMPLE 1
Synthesis of Symmetrical Biodegradable Cross-Linker
(HPMALacSuc)
[0099] Preparation of di(S)-1-[benzyloxycarbonyl]ethyl
butane-1,4-dioate (BnLacSuc) (3a). 3a was prepared by reaction of
benzyl (S)-(-) lactate (27.0 g, 150 mmol) with pyridine (15.2 mL,
188 mmol), and succinyl chloride (8.21 mL, 75.0 mmol) in
dichloromethane (100 mL) at 0.degree. C. with subsequent stirring
for 16 hours at 25.degree. C. An additional aliquot of succinyl
chloride (1.6 mL, 15 mmol) was then added to ensure complete
consumption of benzyl lactate. The reaction was allowed to stir 4
additional hours. After filtering the suspension through activated
carbon, the dark solution was washed with 100 mL water, 2-50 mL
portions of 1M HCl, 2-50 mL portions of 2-100 mL sat. NaHCO3 and
100 mL brine. The organic phase was then dried over Na2SO4 and
concentrated in vacuo to a viscous brown oil. Yield of 3a: 32.3 g
(97%). [a]D=-43.2 (c=1.0, CHCl3). Elution through a short column
(8.5 cm i.d. by 4 cm) of silica gel (70-230 mesh) using 3:7 ethyl
acetate/hexane resulted in a yellow oil of high purity by NMR. 1H
NMR (CDCl3): 1.49 (d, 6H, J=7.1 Hz), 2.65-2.72 (m, 4H), 5.08-5.21
(m, 6H), 7.29-7.34 (m, 10H). 13C NMR (CDCl3): 16.63, 28.47, 66.76,
68.68, 76.49, 77.52, 127.91, 128.21, 128.40, 135.13, 170.30,
171.34. Anal. Calcd. for C24H26O8: C, 65.15; H, 5.92. found: C,
65.06; H, 6.02.
[0100] Preparation of
(2S)-2-{3-[((1S)-1-carboxyethyl)oxycarbonyl]propanoy-
loxy}propanoic acid (LacSuc) (4a). LacSuc was prepared by
hydrogenolysis of BnLacSuc (3a) (10.2 g, 23.1 mmol) over Pd/C (1.0
g, 10% wt. Pd, Degussa type) in 2-propanol (100 mL). The material
was placed on a Parr hydrogenator at 50 psi. at 25.degree. C. When
hydrogen uptake had ceased, the sample was removed from the
hydrogenator, and the Pd-C was then removed by filtration through
celite. The solvent was removed in vacuo at 40.degree. C. (16
hours). The crude product was purified by crystallization of its
dicyclohexylamine salt as follows: crude 5 (6.4 g, 23 mmol) was
dissolved in 50 mL of a toluene/ethyl acetate/ethanol (2:2:1)
solvent mixture. Dicyclohexylamine (9.2 mL, 46 mmol) was added to
the diacid solution at 0.degree. C. Crystallization was induced by
cooling to -10.degree. C. and scratching the sides of the flask.
The white solid was washed with 30 mL portions of ethyl ether.
Concentration of the mother liquor allowed isolation of a second
crop. The first and second crop were combined to give a total yield
of 30.2 g [a]D=-26.9, (c=1.0, CHCl3). The dicyclohexylamine salt
was dissolved in 5:1 water/ethanol (10 mL) and subjected to strong
cation exchange chromatography (BioRad AG 50W-X4, 200-400 mesh) to
regenerate the dicarboxylic acid form. The eluate was lyophilized
to remove water/ethanol. The light yellow oil which resulted was
taken up in 100 mL dichloromethane/ethyl acetate (5:1) and dried
over Na2SO4, to remove residual water. The organic solvents were
removed in vacuo, and heating the viscous residue to 65.degree. C.
under vacuum (0.5 mm Hg) was required to induce crystallization of
the diacid 4a. Yield of 4a: 3.75 g (63%): mp 59-61.degree. C.;
[a]D=-54.5, (c=1.0, CHCl3): 1H NMR (CDCl3): 1.54 (d, 6H, J=7.1 Hz),
2.72-2.77 (m, 4H), 5.13 (q, 2H, J=7.1 Hz), 10.97 (br, 2H). 13C NMR
(CDCl3): 16.56, 28.51, 68.40, 171.62, 176.28. Anal. Calcd. for
C10H14O8: C, 45.81; H, 5.38. found: C, 46.01; H, 5.55.
[0101] Preparation of
di(1S)-1-{[1-methyl-2-(2-methylprop-2-enoylamino)
ethyl]oxycarbonyl}ethyl butane-1,4-dioate (HPMALacSuc) (5a). LacSuc
(4a) (2.20 g, 8.3 mmol) was dissolved in dichloromethane (30 mL)
and cooled to 0.degree. C. under an argon atmosphere in a
three-necked flask equipped with a stir bar and a powder addition
funnel. The reaction vessel was then charged with CDI (2.75 g, 17.0
mmol) via the powder addition funnel. Upon addition of the CDI the
reaction frothed copiously. The reaction vessel was allowed to warm
to 25.degree. C., and then HPMA (2.57 g, 17.0 mmol) was added. The
reaction was stirred at 25.degree. C. for 2 hours, and then washed
with 1M NaH2PO4 (2-100 mL), sat. Na2CO3, (10 mL) and brine (10 mL).
The dichloromethane phase was then dried over Na2SO4 and
concentrated in vacuo to a light yellow, viscous oil. Yield of 5a:
4.08 g (95%). Although the purity was >90% by TLC and NMR, the
purity could be improved by flash chromatography. Elution on 300 mL
silica gel (230400 mesh) using 3% methanol/dichloromethane resulted
in 3.22 g (75%) of 5a: [a]D=-21.3, (c=1.0 CHCl3) 1H NMR (CDCl3):
1.24-1.29 (m, 6H), 1.47-1.51 (m, 6H), 1.96 (s, 6H), 2.70-2.74 (m,
4H), 3.20-3.38 (m, 2H), 3.57-3.72 (m, 2H), 4.87-5.00 (m, 2H),
5.03-5.16 (m, 2H), 5.33-5.36 (m, 2H), 5.71-5.75 (m, 2H), 6.25-6.55
(m, 2H). 13C NMR (62.9 MHz, DMSO-d6 several peaks exhibited duality
which maybe due to hindered rotation or diastereomers): 16.53,
17.21, 17.37, 18.55, 28.20, 42.99, 54.88, 68.74, 70.17, 70.22,
119.11, 139.83, 139.87, 167.68, 167.83, 169.72, 169.89, 171.27,
171.35. HRMS (FAB+) Calc for C24H27N2O10 (M+H) 513.2448, found
513.2418.
[0102] Materials and Characterization
[0103] All chemicals were reagent grade and were used without
purification unless otherwise noted. 1H NMR and 13C NMR spectra
were recorded at 400 and 100.4 MHz respectively on a Varian
INOVA-400 spectrometer equipped with a temperature-controlled
probe. Abbreviations for NMR data are as follows: s=singlet,
d=doublet, m-multiplet, dd=doublet of doublets, t=triplet. Melting
points are uncorrected. Coupling constants (J) are reported in
Hertz. Chemical shifts are reported in parts per million. 1H shifts
are referenced to CHCl3 (7.24) or to DMSO (2.54) and 13C spectra
are referenced to CHCl3 (77.14) or to DMSO (40.45). Solvent
mixtures are given in volume to volume ratios unless otherwise
stated. Flash chromatography was performed on SiO2 Kieselgel 60
(70-230 mesh E. Merck). Mass spectroscopy was performed at the Duke
University Mass Spectrometry Laboratory. Optical rotations were
obtained using the Na+589 nm line at in CHCl3 or acetone using a
Perkin-Elmer 241 polarimeter in a 1 dm cell.
[0104] THF was used freshly distilled from sodium benzophenone
ketyl under nitrogen. 2-propanol was dried by distilling from CaO
and storing over 4A molecular sieves. Dichloromethane was distilled
from P205 and stored over molecular sieves. All other solvents were
obtained in their anhydrous state or stored over molecular sieves
before use. Hydrogenations were performed on Parr hydrogenator at
30 to 50 psi of hydrogen gas.
EXAMPLE 2
Synthesis of Symmetrical Biodegradable Cross-Linker HPMAGlySuc
[0105] Preparation of di[benzyloxycarbonyl]methyl butane-1,4-dioate
(BnGlySuc) (3b). Compound 3b was synthesized by dissolving benzyl
glycolate (15.0 g, 90.3 mmol) and pyridine (7.9 mL, 97 mmol) in 150
mL of CH2Cl2 at 0.degree. C. and adding succinyl chloride (4.7 mL
43 mmol), via a syringe while stirring under an argon atmosphere.
The reaction was allowed to warm to room temperature and stir for 3
hours. After 3 hours, TLC (5:95 methanol/CHCl3 Rf=0.5) indicated
almost complete reaction, and 0.5 mL of succinyl chloride was
added. The reaction was allowed to stir for 12 more hours. The
reaction was washed with 2-50 mL of saturated NaHCO3 followed by
2-50 mL 1M NaH2PO4 and then 1-50 mL of brine. The organic layer was
dried over Na2SO4. The crude brown solid was concentrated in vacuo.
The compound was purified using flash chromatography on a 7 cm i.d.
by 40 cm bed of SiO2 eluting isocratically with CHCl3.
Alternatively, the solid could be purified by recrystallization
from (1:1 ethyl acetate/hexane). The pure fractions were combined
and concentrated in vacuo to yield 3b as a white solid. Yield of
3b: 14.6 g (82%). 1H NMR (CDCl3): 2.77 (s, 4H), 4.65 (s, 4H), 5.17
(s, 2H1), 7.29-7.34 (m, 10H); 13C NMR (CDCl3): 28.71, 61.01 67.21,
128.65, 135.13, 167.58, 171.51. Anal. Calcd. for C22H18O10: C,
59.73; H, 4.10 found: C, 59.64; H, 4.25.
[0106] Preparation of
2-{3-[(carboxymethyl)oxycarbonyl]propanoyloxy} acetic acid (GlySuc)
(4b). Compound 4b was prepared by dissolving 3b (5.0g, 11.3 mmol)
in 2:1 2-propanol/CH2Cl2 (150 mL) in the presence of 500 mg of Pd/C
(Degussa type). The reaction mixture was place on a Parr
hydrogenator at 50 PSI for 5 hours, at which time uptake of
hydrogen gas had stopped. The reaction was filtered through celite
to remove the catalyst and the reaction was concentrated in vacuo
resulting in a white solid. The solid was triturated with diethyl
ether and dried further. Attempts to further purify this material
through the dicyclohexylamine salt resulted in low yields due to
liability of this material in water. However, the NMR of the
titurated product displayed no extraneous NMR resonances. Yield of
4b 2.54 g (96%): 1H NMR (d6-DMSO): 2.62 (s, 4H), 4.44 (s, 4H), 5.74
(m, 4H); 13C NMR (d6-DMSO): 28.40, 60.61, 169.32, 171.30. HRMS
(FAB+) calcd. for C8H10O8 (M+H) 233.0376, found 233.0290.
[0107] Preparation of
di{[1-methyl-2-(2-methylprop-2-enoylamino)ethyl]oxyc-
arbonyl}methyl butane-1,4-dioate (HPMAGlySuc) (5b). The
cross-linker HPMAGlySuc was prepared by adding 4b (3.40 g, 14.5
mmol) to a 100 mL three necked round bottomed flask under an argon
atmosphere at 0.degree. C. The reaction vessel was evacuated three
times and dry DMF (25 mL) was added to the vessel under pressure.
CDI (4.71 g, 29.0 mmol) was added rapidly via a powder addition
funnel with vigorous stirring and was accompanied by copious
frothing and the formation of the partially soluble diimidazolide.
The slurry was allowed to warm to room temperature and HPMA (1)
(4.16 g 29.0 mmol), dissolved in 10 mL of DMF, was added to the
reaction through a syringe. The reaction was allowed to stir for 15
hours during which time the precipitate dissolved. TLC of the
reaction mixture indicated complete conversion of the HPMA (10:90
methanol/CHCl3 Rf 5b=0.55). The reaction was diluted with CH2Cl2
(300 mL) and was washed with 1M NaH2PO4, (2-75 mL), NaHCO3 (2-75
mL) and of brine (100 mL). The organic layer was dried over Na2SO4.
The solvent was removed in vacuo (T<35.degree. C.) to yield a
light yellow oil. The material was purified by flash chromatography
on a SiO2 column (6 cm i.d. by 20 cm) eluting with CH2Cl2 followed
by 2-propanol/CH2Cl2. Fractions containing pure product were
combined and the solvent removed in vacuo (T<35.degree. C.) to
yield a colorless oil. Yield of 5b: 5.83 g (83%). 1H NMR (CDCl3):
1.23 (d, J=6.4 Hz, 6H), 1.92 (s, 6H), 2.74 (s, 4H), 3.21-3.28 (m,
2H), 3.55-3.62 (m, 2H), 4.54 (dd, 4H J1=10 Hz J2=3.2 Hz), 5.01-5.12
(m, 2H ), 5.31 (d, J=1.0 Hz, 2H), 5.67 (d, 2H, J=1H2), 6.15-6.25
(m, 2H); 13C NMR (CDC1.sub.3): 17.64, 18.70, 28.51, 28.54, 43.95,
43.01, 61.45, 71.90, 119.92, 119.94, 139.86, 167.42, 167.46,
168.66, 172.026, 172.07. HRMS (FAB+) Calcd for (M+H) C22H33N2O10
485.2057 found, 485.2123.
EXAMPLE 3
Synthesis of Symmetrical Biodegradable Cross-Linker
HPMALacLacSuc
[0108] Preparation of (1S)-1-[benzyloxycarbonyl]ethyl
(2S)-2-hydroxy propionate (BnLacLacOH). BnLacLacOH was prepared by
the acid catalyzed ring opening of l-lactide. A 250 mL round
bottomed flask was charged with l-lactide (15.0 g, 104 mmol) benzyl
alcohol (12.4 g, 114 mmol) and camphor sulfonic acid (139 mg, 624
.mu.mol) along with dry benzene (100 mL). The reaction was refluxed
under argon for 36 hours. TLC indicated that the reaction had
consumed most of the I-lactide (THF/hexanes/EtOH 45:45:10 Rf
lactide =0.1 (phosphomolybdic acid stain)). The reaction was washed
with of 200 mM NaHCO3 (2-50 mL), dried over Na2SO4 and the solvent
was removed in vacuo. The resultant clear oil was fractionally
distilled under high vacuum (30 mtorr) using a vacuum-jacketed
Vigreux column. The product was collected in a fraction between 108
and 115.degree. C. Yield of BnLacLacOH: 19.8 g (69%) 1H NMR (CDCl3)
1.40 (d, 3H J=6.8 Hz), 1.49 (d, 3H J=3.8 Hz), 3.00 (br, 1H),
4.284.38 (q, 2H J=6.8 Hz), 5.10-5.23 (m, 2H), 7.30-7.4 (m 5H ) 13C
NMR (CDCl3): 16.79, 20.33, 66.68, 66.82, 67.19, 69.26, 128.20,
128.48, 128.59, 135.05, 170.09, 175.00. HRMS (FAB+) Calcd C13H16O5
(M+H) 253.0998 found 253.1066.
[0109] Preparation of (1S)-1-({(1S)-1[benzyloxycarbonyl]ethyl}oxy
carbonyl)ethyl(1S)-1-({(1S)-1-[benzyloxycarbonyl]ethyl}oxy
carbonyl)ethyl butane-1,4-dioate. BnLacLacOH (4.00 g, 15.9 mmol),
pyridine (1.32 mL, 16.4 mmol) was dissolved in dichloromethane (50
mL) and cooled to 0.degree. C. under a N2 atmosphere. To this
mixture was added succinyl chloride (0.90 mL, 8.2 mmol) over a
period of 20 minutes. The reaction vessel was allowed to warm to
25.degree. C., and was stirred for 3 hours. TLC indicated the
reaction had nearly reached completion and an additional aliquot of
succinyl chloride was added (0.5 mL, 4.5 mmol). The reaction was
stirred for 1 hour more. The reaction was diluted with of CH2Cl2,
(50 mL) and poured into water, and washed with 2N HCl (2-50 mL),
water (2-50 mL), 2 M NaHCO3, (100 mL) and brine (50 mL). The CH2Cl2
phase was then dried over Na2SO4,and concentrated in vacuo to a
bronze-colored oil. Yield of BnLacLacOH: 35.8 g (85%). An
analytically pure sample of 9 was prepared by flash chromatography
on silica gel (230-400 mesh) using 30:70 ethyl acetate hexane.
[a]D=-71.60, (c=1.0, CHCl3); 1H NMR (CDCl3): 1.48 (d, 6H, J=7.0
Hz), 1.50 (d, 6H, J=7.1 Hz), 2.60-2.70 (m, 4H), 5.06-5.24 (m, 8H),
7.28-7.36 (m, 1H). 13C NMR (CDCl3,): 16.38, 16.50, 28.36, 66.86,
68.36, 68.87, 127.98, 128.24, 128.37, 134.92, 169.74, 169.83,
171.29.
[0110] Preparation of (1S)-1-({(1S)-1[benzyloxycarbonyl]ethyl}oxy
carbonyl)
ethyl(1S)-1-({(1S)-1-[benzyloxycarbonyl]ethyl}oxycarbonyl) ethyl
butane-1,4-dioate (BnLacLacSuc). LacSuc (4a) (19.1 g, 73.0 mmol)
was dissolved in CH2Cl2 (75 mL) and cooled to 0.degree. C. under N2
atmosphere. CDI (26.0 g, 161 mmol) was then added to the reaction
vessel. Much bubbling of CO2 gas was observed. The reaction vessel
was allowed to warm to 25.degree. C., and then benzyl
(S)-(-)-lactate (25.7 g, 143 mmol) was added. The reaction was
stirred at 25.degree. C. for 1 hour, and then washed with 2N HCI
(2-100 mL), water (100 mL), 10% NaHCO3 (2-100 mL), and brine (100
mL). The CH2Cl2 phase was then dried over MgSO4, and concentrated
in vacuo to a bronze-colored oil. Yield of BnLacLacSuc: 35.8 g
(85%). An analytically pure sample of BnLacLacSuc was prepared by
flash chromatography on silica gel (230-400 mesh) using 30:70 ethyl
acetate/hexane. [a]D=-71.4, (c=1.0 CHCl3) 1H NMR (250 MHz, CDCl3):
1.48 (d, 6H, J=7.0 Hz), 1.50 (d, 6H, J=7.1 Hz), 2.60-2.70 (m, 4H),
5.06-5.24 (m, 8H), 7.28-7.36 (m, 1H). 13C NMR (62.9 MHz, CDCl3):
16.38, 16.50, 28.36, 66.86, 68.36, 68.87, 127.98, 128.24, 128.37,
134.92, 169.74, 169.83, 171.29. Anal. Calcd. for C30H36O12: C,
61.43; H, 5.84. found: C, 61.47; H, 6.01.
[0111] Preparation of LacLacSuc (9a): LacLacSuc was prepared by
hydrogenation of BnLacLacSuc (9a) (35.8 g, 60.8 mmol) over 12.9g
Pd-C (10% wt. Pd, Degussa type; 6.08 mmol Pd) in 100 mL
2-propanol/ethyl acetate (2:1). Positive hydrogen pressure was
maintained using a gas dispersion tube for 4 hours at 25.degree. C.
and then under a balloon of hydrogen for 2 days. The Pd-C was then
removed by filtration, and the solvent was removed in vacuo. The
crude product was purified by crystallization of its
dicyclohexylamine salt as follows: Dicyclohexylamine (24.2 mL, 122
mmol) was added to the crude diacid dissolved in 200 mL 50% ethyl
acetate/hexane at 25.degree. C. Crystallization was induced by
cooling to -78.degree. C. for 16 hours. The white solid was washed
with 30 mL portions of 50% ethyl acetate/hexane. Concentration of
the mother liquor allowed isolation of a second crop. The first and
second crop were combined to give a total yield of 21.4 g
([a]D=42.5, c=1.0, CHCl3). The dicyclohexylamine salt was dissolved
in 25 mL water/ethanol (4:1) and subjected to strong cation
exchange chromatography (Dowex 5OX4-400) to regenerate the
dicarboxylic acid form. The fractions containing the pure diacid
were saturated with NaCl and extracted with 3-100 mL portions of
ethyl acetate. The combined organic phases were dried over MgSO4,
and concentrated in vacuo. The light yellow, viscous oil was then
heated to 65.degree. C. under vacuum (0.5 mm Hg) to remove residual
solvent. Yield of 6a LacLacSuc: 9.31 g (38%). ([a]D=-86.2, c=1.0,
CHCl3) 1H NMR (250 MHz, CDCl3): 1.55 (d, 6H, J=7.1 Hz); 1.56 (d,
6H, J=7.1 Hz); 2.70-2.80 (m, 4H); 5.09-5.22 (m, 4H); 11.07 (b, 2H).
13C NMR (62.9 MHz, CDCl3): 16.44, 28.42, 68.50, 170.00, 171.61,
175.75.
[0112] Preparation of HPMALacLacSuc (7a). LacLacSuc (2.01 g, 4.92
mmol) was dissolved in 10 mL dichloromethane and cooled to
0.degree. C. under N2 atmosphere. The reaction vessel was then
charged with carbonyldiimidazole (1.78 g, 11.0 mmol). Much bubbling
of C02 gas was observed. The reaction vessel was allowed to warm to
25.degree. C., and then HPMA (1.43 g, 10.0 mmol) was added. The
reaction was stirred at 25.degree. C. for 2 hours, and then washed
with 3-10 mL portions of 5% citric acid solution, 10 mL water, 10
mL 10% NaHCO3, and 10 mL brine. The dichloromethane phase was then
dried over MgSO4, and concentrated in vacuo to a light yellow,
viscous oil. Yield of 7a: 2.55 g (79%). Although the purity was
>90% by TLC and NMR, the purity could be improved by flash
chromatography. Elution on 400 mL silica gel (230-400 mesh) using
3% methanol/dichloromethane resulted in 2.20g (68%) of
HPMALacLacSuc (10a). ([a]D=-24.9, c=1.0, CHCl3) 1H NMR (250 MHz,
CDCl3,): 1.23 (d, 6H, J=6.4 Hz); 1.39-1.54(m, 12H); 1.92-1.93 (m,
6H); 2.63-2.78 (m, 4H); 3.18-3.37 (m, 2H); 3.55-3.67 (m, 2H);
4.91-5.11 (m, 6H); 5.29-5.32 (m, 2H); 5.68 (d, 2H, J=9.5 Hz);
6.28-6.33 (m, 2H); 13C NMR (62.9 MHz, CDCl3; several peaks
exhibited duality which is due to diastereomers): 16.23, 16.44,
16.50, 17.11, 17.19, 18.31, 28.30, 43.41, 43.63, 68.34, 68-51,
69.58, 71.24, 71.36, 119.47, 119.60, 139.40, 139.49, 168.20,
168.38, 169.46, 169.89, 170.16, 170.45, 171.26.; HRMS(FAB+) Calcd
MH+C30H44N2O14 657.2839, found 657.2849.
EXAMPLE 4
Synthesis of Symmetrical Biodegradable Cross-Linker
HPMAGlyGlySuc
[0113] Preparation of
di({[benzyloxycarbonyl]methyl}oxycarbonyl)methyl butane-1,4-dioate
(BnGlyGlySuc). GlySuc (4b) (3.50g 14.95 mmol) was dissolved in
CH2Cl2 (30 mL) and anhydrous DMF (60 mL) and cooled to 0.degree. C.
under argon atmosphere in a three necked flask equipped with a stir
bar and a powder addition funnel. The reaction vessel was then
charged with CDI (4.85 g, 30.0 mmol) via a powder addition funnel.
Upon the addition of the CDI the reaction frothed copiously. The
insoluble diimidazolide formed a thick precipitate. The reaction
vessel was allowed to warm to 25.degree. C., and then benzyl
glycolate (3.82 mL, 30.0 mmol) was added via a syringe in anhydrous
DMF (10 mL). The reaction was allowed to run overnight at
25.degree. C. As the reaction proceeded, the reaction mixture
slowly became less viscous. The reaction was diluted with CH2Cl2
(500 mL) and was washed with 1M NaH2PO4 (2-100 mL), NaHC3O3 (2-100
mL), and brine (100 mL). The organic layer was dried over Na2SO4
and the solvent was removed in vacuo yield of 4b: 7.67 g 98% (a
light yellow crystalline solid). The compound was purified by flash
chromatography on a 4.5 cm i.d. by 12 cm column over silica gel.
The sample was loaded in 2:1 CH2Cl2/hexanes (100 mL) eluted with of
the same (200 mL), of CH2Cl2 (200 mL), of 1:99 THF/CH2Cl2 (200 mL),
and finally with THF/CH2Cl2 (3:97). The fractions containing pure
product were combined and the solvent removed in vacuo to yield 26
as a pure crystalline solid. Yield of 4b: 6.57 g (83%). 1H NMR
(MHz, CDCl3): 2.78 (s, 4H), 4.71 (s, 4H), 4.73 (s, 4H), 5.18 (s,
4H), 7.33-7.39 (m, 10H); 13C NMR CDCl3) 28.65, 60.66, 61.21, 67.42,
128.55, 128.74, 128.78, 135.00, 167.03, 167.22, 171.40; Anal.
Calcd. for C26H22O14 C, 55.92; H, 3.97; Found: C, 55.64; H,
4.01.
[0114] Preparation of 2-{2-[3-({[Carboxymethyl)oxycarbonyl]methyl}
oxy carbonyl)propanoyloxy]acetyloxy}acetic acid (6b). Compound 6b
was prepared by suspending 4b (4.58 g, 8.61 mmol) in 1:1
2-propanol/CH2Cl2 (250 mL) in the presence of Pd/C (2.0 g, Degussa
type). The reaction mixture was placed on a Parr hydrogenator at 50
PSI for 18 hours at which time uptake of hydrogen gas had stopped.
The reaction was filtered through celite to remove the catalyst,
and the solution was concentrated in vacuo, resulting in a white
solid. The solid was titurated with diethyl ether, and dried
further yielding a white solid. Attempts at purification by
recrystallization of the dicyclohexylamine salt resulted in complex
mixtures upon trying to remove the amine by semi-aqueous ion
exchange. This was likely due to the instability of this compound.
However the NMR of the titurated product was adequate with a purity
>95%. Yield of 6b: 2.87 g (93%): 1H NMR (d6-DMSO): 2.80 (s, 4H),
4.66 (s, 4H), 4.77 (s, 4H), 13C NMR (d6-DMSO): 29.31, 61.34, 61.91,
167.92, 169.57, 171.97. HRMS (FAB) Calcd for (M-H) C12H13O12,
349.0485 found 349.0403.
[0115] Preparation of di[({[1-methyl-2-(2-methylprop-2-enoylamino)
ethyl]oxycarbon-yl}methyl)oxycarbonyl]methylbutane-1,4-dioate
(HPMAGlyGlySuc) (7b). The cross-linker HPMAGlyGlySuc was prepared
by adding 6b (1.50 g, 4.28 mmol) to 100 mL three-necked round
bottomed flask under an argon atmosphere at 0.degree. C. The
reaction vessel was evacuated three times and dry 1:1 DMF/CH2Cl2
(35 mL) was added to the vessel under pressure. The CDI (1.39 g,
8.57 mmol) was added rapidly with vigorous stirring via a powder
addition funnel and was accompanied by frothing and the formation
of the insoluble diimidazolide. The slurry was allowed to warm to
room temperature and HPMA (1.23g, 8.57 mmol) dissolved in DMF (10
mL) was added to the reaction through a syringe. The reaction was
allowed to stir for 10 hours during which time the precipitate
dissolved. TLC of the reaction mixture indicated complete
conversion of the HPMA (10:90 methanol/CHCl3 Rf 5b=0.73). The
reaction was diluted with CH2Cl2 (200 mL) and was washed with 1M
NaH2PO4 (2-50 mL), NaHCO3 (2-50 mL) and brine (100 mL). The organic
layer was dried over Na2SO4. The solvent was removed in vacuo
(T<35.degree. C.) to yield a clear oil. The material was
purified by flash chromatography on a 6 cm i.d. by 20 cm silica gel
column eluting with CH2Cl2 followed by 3:97 2-propanol/CH2Cl2. Pure
fractions were combined and the solvent removed in vacuo
(T<35.degree. C.) to yield a colorless oil. Yield of 7: 2.08g
(81%): 1H NMR CDCl3): 1.23 (d, 6H J=6.3 Hz), 1.89 (s, 6H), 2.73 (s,
4H), 3.21-3.30 (m, 2H), 3.53-3.62 (m, 2H), 4.524.8 (m, 8H),
5.01-5.17 (m, 2H), 5.28 (s, 4H), 5.64 (s, 4H), 6.23-6.35 (b, 2H),
13C NMR .degree.CDCl3): 17.45, 18.62, 43.80, 60.64, 61.54, 72.12,
119.74, 139.84, 166.75, 167.49, 168.67, 171.50. HRMS (FAB+) Calcd
for C26H36N2O14 (M+H) 601.2167 found 601.2219.
EXAMPLE 5
Synthesis of Degradable Hydrogels With TMED Initiation
[0116] Biodegradable hydrogels are synthesized by free radical
polymerization of the biodegradable cross-linkers and other
monomers described herein using the APS/TMED couple.
[0117] The vinyl groups on the terminus of the cross-linking
structure can be used to form a gel network structure. Gels were
synthesized using the ammonium persulphate (APS)
N,N,N',N'-tetramethylethylenediamine (TMED) couple as the
free-radical initiator system. This system proved very useful in
the synthesis of clear isotropic gels, without having to degas the
polymerization reactions. The gels in this section were made at a
mole feed ratio of 1.5 mole % cross-linker, as a copolymer with
98.5 mole % HPMA. Before the gels were polymerized, three 1.0 mL
plastic syringes to be used as a slab gel template were silylanized
by briefly incubating them in a heptane solution containing
Sigmacote and oven drying at 90.degree. C.
[0118] Also, three 8 cm lengths of 25 gauge tungsten wire were
silylanized for use in the gel making process and each was threaded
through 7 mm Suba Seal rubber septa. The procedure to form gels was
as follows: A 7 mL test tube was charged with HPMA (2.115 g, 14.8
mmol [HPMA] final .about.5 M), the oily compound 5a (HPMAGlySuc)
was adsorbed to the end of a tarred spatula (109.0 mg, 0.225 mmol,
[XL]final =0.075 M). The end of the spatula was placed in the test
tube and 1.5 mL of DI water was added to the mixture. The
cross-linker was dissolved in the mixture by rapid rotation of the
spatula and gentle bath sonication.
[0119] The dissolution of the HPMA has a negative heat of solution
but the mixture should not be warmed above room temperature. To
this solution was added a solution of APS in water (99 mg, 0.438
mmol, 166 .mu.L of a 2.63 M solution, [APS] final=0.143 M). This
was again agitated until homogeneous. To this mixture was added
TMED to initiate the polymerization (49 mg, 0.429 mmol, 204 .mu.L
of a 2.10 M solution of TMED adjusted to pH 7 with HCl). In this
preferred embodiment the concentration of TMED must be
approximately 0.15 M or greater. It was important to control the pH
of the TMED because TMED solutions in water are basic enough to
cause significant degradation of the hydrolytically reactive
cross-linker. Immediately after the TMED was added the mixture of
monomers and APS was vigorously mixed on a vortexer for 15 seconds
and then drawn into the 1.0 mL plastic syringes by plunger
aspiration. The syringe acts as a mold for gel formation. The
syringes were inverted and the tungsten wires were inserted into
the gel through the opening so that it runs through the center of
the forming 1.0 mL gel cylinder.
[0120] The wire was held in place by a septa which was placed over
the tip of the syringe, as the solution polymerized. This formed a
hole in the center of the cylinder, which was later used as a place
to insert a wire hanger for the initially brittle and finally
fragile gel, in order to measure its swelling and degradation
kinetics as a change of mass with time. Gelation occurred within
one to five minutes and the syringe was allowed to sit for 4 hours
at room temperature. At this point the wire was removed from the
center of the solid and the end of the plastic syringe was removed
with a razor blade. The plunger was then used to extrude the gel
from the syringe in 100 .mu.L increments which were cut into small
cylinders as they hung out from the end of the syringe. The gels
were then placed on tarred wire holders and the initial mass of the
assembly was determined. The resulting clear isotropic gels had the
composition of poly(HPMA-co-HPMAGlySuc) 98.5:1.5. The gels were
then incubated in pH 5, 100 mM sodium acetate buffer for 24 hours.
They were then charged into vials of differing pH to study the
degradation kinetics.
[0121] The gels of the four different compositions contained the
following amounts of cross-linkers:
1 Compound MW Mole % XL moles Mass (mg) HPMASuc 366.38 0.015
2.25E-04 82.4 HPMALacSuc 512.5 0.015 2.25E-04 115.3 HPMAGlySuc
484.51 0.015 2.25E-04 109.0 HPMAGlyGlySuc 600.58 0.015 2.25E-04
135.1
EXAMPLE 6
Synthesis of Biodegradable Hydrogel Using AIBN Thermal
Initiation.
[0122] To a 10 mL round bottomed flask was charged HPMALacLacSuc
(60.75 mg, 125 mol) and azobisisobutryonitrile (free radical
initiator) (4.0 mg). To this was added 1.0 mL of a 1:1
methanol:water mixture. The contents were dissolved and degassed
under N2 for 0.5 hours followed by 5 minutes in a bath sonicator
under a stream of N2. The mixture was charged in 300 ml aliquots
into 36.times.50 mm glass tubes which have been evacuated and
capped with rubber septa. The tubes were placed in a 60.degree. C.
bath overnight. The next morning the polymer gel had formed. It was
removed from the glass tube under vacuum and cut into 2.times.5 mm
disks. The disks were incubated in water for two days. The water
was changed at 8 hour intervals to remove any water soluble monomer
or reaction byproducts.
EXAMPLE 7
Degradation of Biodegradable Hydrogels
[0123] Measuring the Degradation of the Gel Network
[0124] The gels were placed in 15 mL vials containing 10 mL of
buffers. The masses of wire holders were determined before the gels
were placed on them. The original mass of the gel in its relaxed
state was also known by subtraction from the total mass of the
assembly. The original dry mass of the gel was determined by drying
three gels in their relaxed state from each composition and
determining the dry mass of the gel. These values were then used to
calculate the inverse of the volume fraction of polymer in the gel
(Qv) respectively, using the densities of the polymer and water.
The incubation solutions were changed each time the gel was
weighed. The gels were incubated in a gyratory water-bath shaker
(New Brunswick Scientific, New Brunswick, N.J.). The temperature
was regulated to be 37.+-.2.degree. C. and the shaker was set to 30
rotations per minute.
[0125] Explanation of the Order of the Rates of Degradation for
Different Cross-Inkers
[0126] Hydrogels are cross-linked structures composed of elastic
networks of water-soluble polymers. The maximum degree of swelling
is limited by the network elasticity. So as the gel's network
structure degrades the cross-link density decreases and the network
becomes more elastic. This allows the network to swell further as
it imbibes more water. This swelling results in a increase in the
volume fraction of water and a corresponding decrease in the volume
fraction of polymer. The property of the change of volume of the
polymer network can be measured by weighing the gel at different
time points.
[0127] Since the swelling is related to network cross-link density
by weighing the macroscopic gels at various times throughout their
swelling one can obtain information about the change in cross-link
density and thus the rate of degradation.
[0128] The cross-linkers in the gel degrade hydrolytically by the
action of the two hydrolytically active components of water: the
hydronium and hydroxide ions. Therefore, the rate of degradation is
strongly dependent on pH.
[0129] HPMALacSuc is electronically similar to HPMAGlySuc 5b yet
the lactic ester shows slower degradation than the glycolic. This
is because the lactic ester has a methyl group to the carbonyl
where the first step of ester hydrolysis takes place, and is
sterically hindered in 5a than 5b. HPMAGlyGlySuc 7b shows the
fastest hydrolysis and swelling kinetics with complete degradation
after about 5 days (See FIGS. 4 through 6).
[0130] Moreover, since we are not measuring the rate of hydrolysis
of individual bonds but measuring the swelling which comes about as
a result of cleaving the connection between two polymer chains, the
concentration of cleavable sites comes into play. Therefore, when
comparing gels composed of 5b and 7b at the same cross-link
density, compound 5b has four potential sites of cleavage and 7b
has six. This increased concentration of cleavable sites may result
in a difference in swelling rate depending on the relative
microscopic rate constants for hydrolysis of the different bonds
making up the cross-linker.
[0131] Synthesis of the Control Cross-Linker HPMASuc
[0132] Preparation of the control non-degradable cross-linker
bis-1-methyl-2-(2-methylprop-2-enoylamino) ethyl -1,4-butanedioate
(HPMASuc) (2). To a solution of HPMA (4.00 g, 27.9 mmol), DMAP (340
mg, 2.8 mmol) and Na2CO3 (3.26 g, 30.7 mmol) in CH2Cl2 (100 mL) at
0.degree. C. was added succinyl chloride (1.54 mL, 13.97 mmol)
dropwise. The reaction was allowed to warm to 25.degree. C. and
stir for 8 hours at which time another aliquot of succinyl chloride
was added (0.61g, 4 mmol). The reaction was allowed to stir for
another 4 hours. The reaction mixture was poured into 50 mL of
water and filtered through activated carbon. The mixture was then
washed with 1M NaH2PO4 (50 mL), sat. NaHCO3 (50 mL) and brine (100
mL). The organic phase was then dried over Na2SO4, and concentrated
in vacuo to a tan residue. This was purified by flash
chromatography in 15:85 2-propanol/CHCl3 on a 3 i.d. by 20 cm
column. Yield of 2 3.51g (68%): mp 103-105.degree. C., 1H NMR
.degree.CDCl3): 1.24 (d, 6H, J=6.3 Hz); 1.93 (d, 6H, J=0.6 Hz),
2.54-2.65 (m, 4H), 3.31-3.38 (m, 2H), 3.52-3.60 (m, 2H), 4.98-5.05
(m, 2H), 5.31 (d, 2H, J=0.4 Hz), 5.66 (s, 2H), 6.23 (b, 2H). 13C
NMR .degree.CDCl3); (several peaks exhibited duality which is most
likely due to diastereomers).sub.--17.48, 18.51, 29.38, 43.81,
43.87, 70.48, 70.62, 119.50, 119.56, 139.78, 168.48, 172.24. Anal.
Calcd. for C18H28N2O6: C, 58.68; H, 7.65; N, 7.60. found: C, 58.71;
H, 7.72; N, 7.48.
EXAMPLE 8
Release of a Soluble Macromolecule from a Degrading Network and
Degradation of a Polymer Network Labeled with a Chromophoric
Agent.
[0133] Gels were formed by the same method as above, but in this
case other compounds were included during the preparation of the
gels to study the release of small molecules from the network. In
one case, the network itself was labeled with a polymerizable
derivative of tetramethyl rhodamine (TMRAHMAM) in order to show the
release (degradation) of the network itself (see FIG. 6). In the
other case fluorescent rhodamine labeled albumin (Molecular Probes,
Eugene Oreg.) was included in the uncharged network to show
diffusive release of a macromolecule from the network (see FIG.
7).
[0134] To a 3 mL test tube was charged TMRAHMAm (3.0 mg, 5.0
.mu.mol; 30 .mu.L of a 100 mg/mL solution in CHCl3) which was then
placed under a 7 mtorr vacuum for 3 hours. To another 3 mL test
tube, 4.5 mg of 5+(6) carboxytetramethylrhodamine labeled albumin
(Molecular probes) was added. To a third 7.0 mL test tube HPMA was
added (1.692 g, 11.8 mmol, [HPMA] final .about.5 M), the oily
compound 7b (HPMAGlyGlySuc) was adsorbed to the end of a tarred
spatula (108.0 mg, 180 .mu.mol, [XL]final=0.075 M). The end of the
spatula was placed in the test tube and 1.2 mL of DI water was
added to the mixture.
[0135] The cross-linker was dissolved in the mixture by rapid
rotation of the spatula and gentle bath sonication. HPMA has a
negative heat of solution but the mixture should not be warmed
above room temperature. To this solution was added a solution of
APS in water (80 mg, 0.350 mmol, 79 .mu.L of a 2.63 M,
[APS]final=0.143 M). This was again agitated until homogeneous.
This viscous mixture was separated into 3-890 .mu.L aliquots. One
was mixed with the polymerizable dye (TMRAHMAm) and the other with
fluorescent albumin. All resulting monomer mixtures were thoroughly
homogenized. To each of these three 890 .mu.L mixtures was added an
aliquot of TMED to initiate the polymerization (13.2 mg, 114
.mu.mol, 54 .mu.L of a 2.10 M solution of TMED adjusted to pH 7
with HCI, 244 mg TMED freebase/mL).
[0136] All mixtures were mixed for 15 seconds and then placed in
the syringe template, with each solution having a final solid
volume of about 850 .mu.L. The gels were then allowed to polymerize
for 4 hours after which time they were extruded and cut into
slices. The gels were weighed, attached to wires and were placed in
separate vials for the release studies. The dye labeled gels were
incubated in water for two days to allow any unreacted monomer to
diffuse out of the network. The gels were placed in 15 mL of buffer
solutions at pH 4, 7, and 9. All solutions were incubated at
37.degree. C. on a temperature-regulated orbital-shaking bath at 30
rpm.
[0137] Gels were suspended in buffers of different pH's. All
buffers were adjusted to the same ionic strength. The release of
the rhodamine labeled albumin and the rhodamine labeled HPMA was
monitored at 550 nm. 750 .mu.L of the sample was removed from the
vial and periodically measured on a spectrophotometer. Release
values were normalized to the maximum amount released.
[0138] Explanation of the Release Data
[0139] The examination of the release of macromolecules entrained
in the polymer network provides another way to study the
performance of these materials. In this section, the degradation of
the network is ascertained by analysis spectrophotometrically
through the release of the HPMA polymer backbone itself by labeling
it with the polymerizable dye TMRAHMAm. Moreover, the release of a
model macromolecular solute (TMRA-albumin, molecular weight of
.sup..about.66,000 Da.) from the network is measured
spectrophotometrically.
[0140] FIG. 6 displays a photograph of three different gels in pH
7.3 buffer made with HPMASuc, HPMAGlyGlySuc (4 days) and
HPMAGlyGlySuc (8 days), which were co-polymerized with the
chromophoric label and HPMA. FIG. 6 displays not only the different
degrees of swelling but also the release of rhodamine labeled HPMA
into the solution at a given time versus control. FIG. 7 shows the
release curve for rhodamine labeled HPMA polymer backbone as well
as the corresponding swelling data. The release of rhodamine
labeled HPMA largely occurs to the greatest extent at the onset of
complete degradation of the polymer. In contrast to the release of
the polymer backbone, the release of the globular macromolecule BS
albumin more closely follows the swelling of the network.
EXAMPLE 9
Synthesis of Anionic Slab Gels and Loading of DX (Doxorubicin).
[0141] Synthesis of Gels Containing Methacrylic Acid.
[0142] The gels in this section were made at a mole feed ratio of
1.45 mole percent cross-linker as a copolymer with HPMA 95.4 mole %
and methacrylic acid sodium salt 3.18 mol %. Before the gels were
polymerized, three 1.0 mL plastic syringes to be used as a slab gel
template were silylanized with Sigmacote by briefly incubating them
in the heptane solution and oven drying at 90.degree. C. (see 1-1).
Also, three 8 cm lengths of 25 gauge tungsten wire. The procedure
to form gels is as follows: to a 7 mL test tube is charged HPMA
(564.1 mg, 0.00394 mol, [HPMA] final .sup..about.5 M), the
cross-linker (6.00E-05 mol, [XL] final=.sup..about.0.075M) and the
sodium salt of methacrylic acid (42 mg 3.89E-04 mol) are charged
into the same vial with the HPMA and 0.4 mL of DI water is added to
the mixture. The components of the mixture are dissolved by
agitation and gentle bath sonication at 15.degree. C. The
dissolution of the HPMA is retarded by its negative heat of
solution but the mixture should not be warmed above room
temperature. To this solution is added 44.4 .mu.L of a 2.63 M
solution of (APS) in water (27 mg, 1.17E-04 mol,
[APS]final=0.143M). This is again agitated until homogeneous. To
this mixture is added 55 .mu.L of a 2.10 M solution of TMED to
initiate the polymerization (13.3 mg, 1.14E-04 mol, TMED solution
adjusted to pH 7). It is important to control the pH of the TMED
because TMED solutions in water are basic enough to cause
significant degradation of the hydrolytically reactive
cross-linker. Immediately after the TMED is added the mixture of
monomers and APS is vigorously mixed on a vortexer for 15 seconds
and then drawn into the 1.0 mL plastic syringes by plunger
aspiration which acts as a mold for the forming gel (see above for
a description of gel processing).
[0143] For gels of the three different compositions synthesized the
following amounts of cross-linkers were used in addition to the
materials described above.
2 Compound MW Mole fraction Moles Mass (mg) HPMASuc 366.38 0.0136
0.00006 22 HPMAGlySuc 484.51 0.0136 0.00006 29 HPMAGlyGlySuc 600.58
0.0136 0.00006 36
[0144] Loading with Doxorubicin
[0145] The gels were cut into approximately equal volumes (100
.mu.L, .about.100 mg) and the unloaded masses were determined for
the gels in the rubbery state. Each gel was placed in 1.9 mL of a
2.0 mg/ml solution of doxorubicin hydrochloride which was buffered
to pH 7.4 with 5 mM TRIS buffer. The gels were agitated with the
solution for 4 days at room temperature on a temperature-regulated
orbital-shaking bath at 30 rpm. As the red doxorubicin was taken up
into the gels the gels became red. The solution around the gels
became depleted of doxorubicin due to the ion exchange of
doxorubicin for the sodium counterions.
EXAMPLE 10
Method for Making a Biodegradable Water Absorbant Device.
[0146] As discussed earlier the preferred embodiment of a network
polymer for use as a degradable water absorbent will include
ionomeric monomers which bring ions and water into the gel network.
Below is a description of the method to make a highly charged gel
of these cross-linkers.
[0147] To a 5 mL test tube was charged acrylic acid (675 .mu.L,
9.85 mmol), water (1120 .mu.L) and HPMAGlySuc (73 mg, 0.15 mmol)
(5b). The cross-linker was weighed into the mixture as described
earlier. The mixture was homogenized and APS was added (66.6 .mu.L,
0.175 mmol) from a 2.43 M solution in water. This solution was
again mixed. To this solution was added TMED (137 .mu.L, 0.287
mmol) from a 2.10 M pH 7.0 solution in water. The mixture was
vortexed rapidly for 15 seconds and the polymerizing solution was
charged into two 1.0 mL syringes that acted as a mold for the
polymerization. The syringes were allowed to sit for four hours.
The gel was removed from the syringe and cut into pieces
(.about.100 .mu.L cylinders). The mass of the cylinder was recorded
and placed in a 20 mL vial containing 18 mL of PBS at pH 7.4. The
gels were incubated overnight with buffer. The next day the buffer
was changed twice in order to keep a constant external pH as the
gel was charged. After incubating in PBS for 3 days the gel has
swollen with water to approximately 20 times the total initial
polymer volume.
[0148] The salt form of the gel will be synthesized and the gel
material processed into smaller pieces either before or after
drying. The dry gel pieces would then be incorporated as one
component in an absorbable layer of the absorbent device. Generally
the pieces should to small so as to increase the surface area of
the gel and therefore to increase the rate at which water would be
absorbed by the gel material.
EXAMPLE 11
General Method for Synthesizing the Cross-Linker
[0149] Those skilled in the art of organic synthesis will be aware
of the general considerations in designing cross-linkers of this
class. Generally if any alcohol groups are present in the
poly-acids used they must be protected unless it desired that they
react with the activated acids to be used in the formation of the
oligo-ester. Generally the synthesis must be performed under
anhydrous condition except when performing acid or base washes of
water immiscible organic solvents where the cross-linker or
intermediate largely partitions into the organic phase. If the
materials are to be used in an aqueous environment it is generally
best to keep the acid in the anionic form only a few units away
from its pKa. This is due to the well-known effect of inhibition of
attack of hydroxide by negatively charged electrophiles. In the
most preferred cases the cross-linkers are constructed by adding a
protected degradable piece to a polyacid. In a preferred embodiment
the degradable piece contains a nucleophilic moiety and a protected
acidic moiety, e.g. benzyl lactate. The protecting groups are
removed under appropriate conditions known to those skilled in the
art. The activation and reaction with a protected bifunctional
degradable molecule can be repeated on the molecule as many times
as desired. Alternatively, the final step of the synthesis can be
accomplished by terminating the molecule with reactive groups that
are later used to cross-link polymer filaments. The preferred
embodiment of the protecting group are groups that can be removed
under neutral anhydrous conditions such as the benzyl protecting
group. The next preferred protecting groups are ones that can be
removed with anhydrous acids or bases such as the BOC or MEM
protecting groups.
EXAMPLE 12
Preparation of HydLacSuc (8)
[0150] Preparation of
di[N-carbobenzoxy-N'-hydrazidooxycarbonyl]ethyl butane-1,4-dioate
(BnHydLacSuc). To a 25 mL round-bottomed flask was charged 4a (262
mg, 1.00 mmol), THF (2.0 mL), and pyridine (162 .mu.L, 2.00 mmol).
The flask was placed on an ice bath and to the reaction was added
isobutyl chloroformate (260 .mu.L, 2.0 mmol). The reaction was
allowed to stir and carbobenzoxyhydrazide was added (380 mg, 2.3
mmol). The reaction was allowed to stir overnight. The white solid
was dissolved in ethyl acetate and washed with 1M HCl (2-5 mL),
water (5 mL) and saturated NaHCO3 (5 mL). The organic layer was
dried over MgSO4. The solvent was removed in vacuo resulting in a
white solid. Yield of BnHydLacSuc: 392 mg (62%).
[0151] Preparation of di[N-hydrazidooxycarbonyl]ethyl
butane-1,4-dioate (HydLacSuc) (8). To a 5 mL pressure tube was
BnHydLacSuc (279 mg, 0.5 mmol), Pd-C (Degussa Type, 10% Pd, 50%
H20) (600 mg) and cyclohexene (1.25 mL, 12.5 mmol) and MeOH/DMF
(1:1, 1.25 mL). The reaction was heated to 60.degree. C. for 3
hours. Evolution of CO2 was observed. The Pd-C was removed by
filtration and the solvent was removed in vacuo resulting in an oil
(8).
EXAMPLE 13
Preparation of HEMAGlyAdp
[0152] Preparation of di[benzyloxycarbonyl]methyloctane-1,8-dioate
(BnGlyAdp). Compound BnGlyAdp was synthesized by methods similar to
those described for BnGlySuc, compound 3b, by dissolving benzyl
glycolate (9.08 g. 54.6 mmoles) and pyridine (4.42 mL., 54.6
mmoles) in 150 mL CH2CH1 at 0.degree. C. and adding adipoyl
chloride (5.00 g, 27.3 mmoles) via a syringe while stirring under
nitrogen atmosphere. The reaction was allowed to warm to room
temperature and stir for 5 hours. After 5 hours, TLC (5:95
methanol/CH2Cl2 Rf=0.63) indicated almost complete conversion, and
0.1 mL of adipoyl chloride was added. The reaction was allowed 12
more hours. The medium was then cooled to 0.degree. C. in a freezer
for 2 hours to facilitate precipitation of pyridinium chloride salt
(PyCl). After 2 hours, the medium was filtered through a medium
porous frit funnel and the filtrate was washed with 3-100 mL water
washings. The organic layer was dried over Na2S04 for 2 hours. The
CH2Cl2 was stripped on a roto-evaporator to concentrate the
CnGlyAdp. The material was purified by recrystallization (from 1:1
ethyl acetate/hexane). Yield of BnGlyAdp: 7.72g (64.0%). .sub.1H
NMR (d.sub.7 DMF): .delta.1.67 (s, 4H), 2.46 (s, 4H), 4.80 (s, 4H),
5.23 (s, 4H), 7.44 (m, 10H).
[0153] Preparation of
2,3-[(carboxymethyl)oxycarbonyl]octanoyloxyacetic acid (HOGlyAdp).
Compound HOGlyAdp was synthesized by methods similar to those
described for HOGlySuc, compound 4b, by dissolving BnGlyAdp (5.01
g, 11.3 mmoles) in 250 mL 2-proponal at room temperature in the
presence of 1.51 g Pd/C (Degussa type). An air stone was immersed
in the medium through rubber septum at the top of the flask. The
medium was sparged with hydrogen gas at 1 atm. The system was
isolated from air using a closed system bubbler. The medium was
sparged with hydrogen gas for 12 hours. After 12 hours, the
reaction mixture was filtered through celite to remove the catalyst
and the reaction product was concentrated in vacuo resulting in a
white solid. The white product was triturated with 1:1 diethyl
ether/hexane. The white product was recovered by filtration through
a medium porous filter funnel and then dried under vacuum in a
desiccator. Yield of HOGlyAdp: 1.74 g (60.0%).
[0154] Preparation of
di{1-methyl-2-(2-ethyl)oyloxycarbonyl}methyloctane-1- ,8-dioate
(HEMAGlyAdp). Compound HEMAGlyAdp was synthesized by methods
similar to those described for HPMAGlySuc, compound 5b. The
cross-linker HEMAGlyAdp was prepared by adding HOGlyAdp (500 mg,
1.92 mmoles) and CDI (622 mg, 3.83 mmoles) to a 50 mL boiling
flask. The flask was evacuated 3 times while iteratively purging
with nitrogen. The temperature of the reaction vessel was reduced
from room temperature to 0.degree. C. with an ice bath and dry DMF
(5 mL) was rapidly added to the vessel under pressure with vigorous
stirring via a magnetic stir bar. Addition was accompanied with
frothing and the formation of a white slurry, the intermediate
precursor, the diimidazolide of GlyAdp. The slurry was allowed to
come to room temperature and hydroxyethyl methacrylate (HEMA, 466
.mu.L, 3.84 mmoles) was added via a syringe. The vessel was covered
with aluminum foil to shield it from light and the reaction mixture
was stirred under nitrogen atmosphere for 15 hours over which time
the slurry completely dissolved. TLC of the reaction mixture showed
the presence of both unreacted HEMA and HEMAGlyAdp (5:95
methanol/CH.sub.2Cl.sub.2 Rf=0.80). The reaction was diluted with
100 mL CH.sub.2Cl.sub.2 and washed with 1M NaH.sub.2PO.sub.4 (pH
4.5, 2-50 mL), 1M NaHCO.sub.3 (pH 8.3, 2-50 mL) and brine (2-50
mL). The organic layer was dried over Na2SO.sub.4. The organic
layer was recovered by filtration and solvent removed in vacuo
(T<30.degree. C.) yielding a yellow oil that was purified by
flash chromatography on a SiO2 column (5 cm id by 30 cm) eluting
with CH.sub.2Cl.sub.2. Fractions containing pure product were
combined and the solvent was removed in vacuo (T<30.degree. C.)
yielding a colorless oil.
[0155] The following citations are incorporated in pertinent part
by reference herein for the reasons cited in the above text.
REFERENCES
[0156] Allcock, H. R., Polyphasphazines as new biomedical
materials. Biodegradable polymers as drug delivery systems. M.
Chasin and R. Langer. New York, N.Y., Marcel Decker. 45, 163
(1990).
[0157] Alfrey, T., Gurnee, E. F. & Lloyd, W. G., Diffusion in
glassy polymers, J. Polymer. Sci. C, 12 (1966) 249.
[0158] Allcock, H. R., in Polyphasphazines as new biomedical
materials, eds. Chasin, M. & Langer, R. (Marcel Decker, New
York, N.Y.), Vol. 45, pp. 163 (1990).
[0159] Devices Tripartite Biocompatibility Guidance (FDA,
Washington D.C.) 1987.
[0160] Baker, R. W. in Biodegradable Systems (John Wiley &
Sons, New York, N.Y.), pp. 120 (1987).
[0161] Bender, M. L., Mechanisms of catalysis of nucleophilic
reactions of carboxylic acid derivatives, Chem. Rev., 60 (1960)
53.
[0162] Burkoth, A. K., Anseth, K. S. "MALDI-TOF Characterization of
Highly Cross-linked, Degradable Polymer Networks." Macromolecules,
32 (1999) 1438.
[0163] Brondsted, H. & Kopecek, J., Hydrogels for site-specific
oral drug delivery: Synthesis and characterization, Biomaterials,
12 (1991) 584.
[0164] Chasin, M. & Langer R., Biodegradable polymers as drug
delivery systems (Marcel Decker, New York, N.Y.) (1990).
[0165] Tripartite Subcommittee for Medical Devices, Tripartite
Biocompatibility Guidance. (Washington D.C., FDA) (1987).
[0166] Drobnik, J. & Rypacek, F., Soluble synthetic polymers in
biological systems, ed. Dusek, K. (Springer-Verlag, Berlin, UDR),
57 (1984) 30.
[0167] Duncan, R., Dimitrijevic, S. & Evagorou, E. G., Polymer
therapeutics for tumor specific delivery, STP Pharma. Sci., 4
(1996) 237.
[0168] Eichenbaum, G., P. Kiser, et al., pH and Ion-Triggered
Volume Response of Anionic Hydrogel Microspheres. Macromolecules 31
(1998) 5084.
[0169] Flory, P. J., Principles of Polymer Chemistry (Cornell
University PRESS, Ithaca) (1953).
[0170] Heller, J., in Bioerodible Hydrogels, ed. Peppas, N. (CRC
Press, Boca Raton), Vol. Volume III, pp. 137-148 (1986).
[0171] Heller, J., Sparer, R. V. & Zentner, G. M., in
Poly(ortho-esters), eds. Chasin, M. & Langer, R. (Marcel
Decker, New York, N.Y.), Vol. 45 (1990).
[0172] Heller, J., Controlled release of water soluble
macromolecules from bioerodable hydrogels, Biomaterials. (1983)
262.
[0173] Isrealachvilli, J. (1991). Intersurface Forces. London,
Academic Press.
[0174] Kenny, J. F. & Willcockson, G. W., Structure-property
relationships of PVA. III. Relationships between stereoregularity,
crystallinity, and water resistance in PVA, J. Polym. Sci, Al
(1966) 699.
[0175] Kiser, P. F., Needham, D. & Wilson, G., A synthetic
mimic of the secretory granule for drug delivery, Nature, 394
(1998) 459.
[0176] Kreuter, J., Ed. Colloidal Drug Delivery Systems. Drugs and
the pharmaceutical sciences. N.Y., Marcel Dekker (1994).
[0177] Kopecek, J. & Bazilova, H., Poly [N-(2-Hydroxypropyl)
Methacrylamide]-I Radical polymerization and copolymerization,
European Polymer Journal, 9 (1974) 7.
[0178] Kost, J. & Langer, R., in Equilibrium Swollen hydrogels
in controlled release applications, ed. Peppas, N. (CRC Press, Boca
Raton), Vol. 3, pp. 95-105 (1986).
[0179] Kurisawa, M., Matsuo, Y. & Yui, N., Modulated
degradation of hydrogels with thermo-responsive network in relation
to their swelling behavior, Macromol. Chem. Phys., 199 (1998)
707.
[0180] Lasic, D. D. and D. Needham, The Stealth liposome: a
prototypical biomaterial." Chemical Reviews 95 (1995) 2601.
[0181] Lee, P., Kinetic considerations of drug delivery from
swelling-controlled and erosion/diffusion-controlled systems,
Proceed. Intern. Symp. Control. Rel. Bioact. Mat., 18 (1991)
315.
[0182] Mark, J. E., The use of model polymer networks to elucidate
molecular aspects of rubberlike elasticity, Adv. Poly, Sci., 44
(1982) 1.
[0183] Munk, P. Introduction to macromolecular science (John Wiley
& Sons, New York, N.Y.) (1989).
[0184] Odian, G., Principles of polymerization (John Wiley and
Sons, N.Y.) (1991).
[0185] Park, K., Shalaby, W. S. W. & Park H., Biodegradable
hydrogels for drug delivery (Technomic Publishing Co., Lancaster,
Pa.) (1993).
[0186] Park, K., Biodegradable Hydrogels for Drug Delivery
(Technomic Publishing Company, Inc., Lancaster, Pa.) (1993).
[0187] Pathak, C. P., Barman, S. P., Coury, A. J., Sawhney, A. S.
& Hubbell, J. A., Biodegradable thermoresponsive hydrogels and
macromonomers, Proc. Int. Symp. Controlled Release Bioact. Mater.,
22 (1995) 85.
[0188] Peppas, N. A., Hydrogels in Medicine and Pharmacy, Volume
II, Polymers (CRC Press, Boca Raton, Fla.) (1986).
[0189] Peppas, N., In Characterization of the Cross-linked
Hydrogels, ed. Peppas, N. (CRC Press, Boca Raton), Vol. 1, pp. 27
(1986).
[0190] Peppas, N., In Dynamically swelling hydrogels in controlled
release applications, ed. Peppas, N. (CRC Press, Boca Raton), Vol.
3, pp. 109 (1986).
[0191] Rembaum, A., S. P. Yen, et al., Functional polymeric
microspheres based on 2-hydroxyethyl methacrylate for
immunochemical studies, Macromolecules, 9 (1976) 328.
[0192] Saffran, M., Kumar, G. S., Savarian, C., Burnham, J. C.,
Williams, F. & Necker, D. C., A New Approach to oral
administration of insulin and other peptide drugs, Science, 233
(1986) 1081.
[0193] St. Pierre, T. & Chiellini, E., Biodegradability of
medical polymers used in medical and pharmaceutical applications:
Part 1-Principles of hydrolysis mechanisms, J. Bioact. Compatible
Polym., 1 (1986) 467.
[0194] Subr, V., Duncan, R. & Kopecek, J., Release of
macromolecules and daunomycin from hydrophilic gels containing
enzymatically degradable bonds, J. Biomater, Sc. Polym. Ed., 1
(1990) 261.
[0195] Taft, R. W., Polar and steric substituent constants for
aliphatic and o-benzoate groups from rates of esterification and
hydrolysis of esters, J. Amer. Chem. Soc., 74 (1952) 3120.
[0196] Taft, R. W., Linera free energy relationships from the rates
of esterification and hydrolysis of aliphatic and ortho-substituted
benzoate esters, J. Amer. Chem. Soc., 74 (1952) 2729.
[0197] Taylor, A. E. & Grainger, D.N., Exchange of
macromolecules across the microcirculation, Handbook of Physiol., 6
(1984) 467-520.
[0198] Tomlinson, E., Theory and practice of site-specific drug
delivery, Advanced Drug Delivery Reviews, 1(2) (1987) 87-198.
[0199] Torchilin, V. P., Tischenko, E. G., Smirnov, V. N. &
Chazov, E. I., Immobilization of enzymes on slowly soluble
carriers, J. Biome. Mat. Res., 11 (1977) 223.
[0200] Ulbrich, K., Subr, V., Seymour, L. W. & Duncan, R.,
Novel Biodegradable hydrogels prepared using the divinylic
cross-linking agent N,0-dimethylacryloylhydroxylamine 1. Synthesis
and characterization of rates of gel degradation, and rate of
release of model drugs in vitro and in vivo., J. Controlled
Release, 24 (1993) 181.
[0201] Van Dijk-Wolthius, W., Hoogeboom, J. A. M., van Steenbergen,
M. J., Tsang, S. K. Y. & Hennink, W. E., Degradation and
release behavior of dextran-based hydrogels, Macromolecules, 30
(1997) 4639.
[0202] Van Dijk-Wolthius, W. N. K., Tsang, S. K. Y., Kettees-van
den Bosch, J. J. & Hennink, W. E., A new class of polymerizable
dextrans with hydrolyzable groups: hydroxyethyl methacrylated
dextran with and without oligolactate spacer, Polymer, 38 (1997)
6235. Wang, P. Y. & Arlitt, B. P., in Structural requirements
for the degradation of condensation polymers in vivo, ed. Gregor,
H. P. (Plennum Press, New York, N.Y.), Ch. 16 (1975).
* * * * *