U.S. patent application number 12/576153 was filed with the patent office on 2010-10-07 for stereocomplex hydrogels.
Invention is credited to Silvia Johanna De Jong, Wilhelmus Everhardus HENNINK, Cornelis Franciscus Van Nostrum.
Application Number | 20100255098 12/576153 |
Document ID | / |
Family ID | 8239899 |
Filed Date | 2010-10-07 |
United States Patent
Application |
20100255098 |
Kind Code |
A1 |
HENNINK; Wilhelmus Everhardus ;
et al. |
October 7, 2010 |
STEREOCOMPLEX HYDROGELS
Abstract
The invention relates to hydrogel compositions, which can be
applied as biodegradable materials and to the processes to prepare
such hydrogels. The hydrogel of the present invention comprises a
stereocomplex gel structure which is the result of the interaction
of oligomerized monomers of one chirality with that of oligomerized
monomers of the opposite chirality, both grafted to hydrophilic
polymers. The grafts form an interaction which is different from a
covalent chemical interaction and thus provide the gel with the
required coherence.
Inventors: |
HENNINK; Wilhelmus Everhardus;
(Waddinxveen, NL) ; Van Nostrum; Cornelis Franciscus;
(Vlijmen, NL) ; De Jong; Silvia Johanna; (De
Meern, NL) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
12531 HIGH BLUFF DRIVE, SUITE 100
SAN DIEGO
CA
92130-2040
US
|
Family ID: |
8239899 |
Appl. No.: |
12/576153 |
Filed: |
October 8, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09913967 |
Dec 31, 2001 |
7776359 |
|
|
PCT/NL00/00108 |
Feb 21, 2000 |
|
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12576153 |
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Current U.S.
Class: |
424/486 ;
514/1.1 |
Current CPC
Class: |
C08B 37/0021 20130101;
A61K 47/34 20130101; C08J 3/075 20130101; A61K 9/1652 20130101;
A61K 9/0019 20130101; C08L 101/14 20130101; A61K 47/36 20130101;
A61K 9/1647 20130101 |
Class at
Publication: |
424/486 ;
514/1.1 |
International
Class: |
A61K 9/00 20060101
A61K009/00; A61K 38/02 20060101 A61K038/02 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 19, 1999 |
EP |
99200472.1 |
Claims
1. A hydrogel composition comprised of a mixture of (A) a water
soluble or water dispersible hydrophilic polymer grafted with
oligomers or co-oligomers, wherein the oligomers or co-oligomers
comprise homo-oligomers of L-lactic acid, and (B) a water soluble
or water dispersible hydrophilic polymer grafted with oligomers or
co-oligomers, wherein the oligomers or co-oligomers comprise
homo-oligomers of D-lactic acid, in an aqueous system, wherein said
homo-oligomers of L-lactic acid and said homo-oligomers of D-lactic
acid are comprised of 7-25 lactic acid monomers on average, and
wherein said homo-oligomers of L-lactic acid and said
homo-oligomers of D-lactic acid interact noncovalently.
2. The hydrogel composition of claim 1, wherein the water soluble
or water dispersible hydrophilic polymer (A) is grafted with
homo-oligomers of L-lactic acid, and the water soluble or water
dispersible hydrophilic polymer (B) is grafted with homo-oligomers
of D-lactic acid.
3. The hydrogel composition according to claim 1, wherein the water
soluble or water dispersible hydrophilic polymer is selected from
the group consisting of dextran, starch, cellulose derivatives,
albumin, lysozyme, poly(aminoacids), poly(lysine) and related
copolymers, poly(glutamic acid) and related copolymers, poly
(meth)acrylates, poly(meth)acrylamides, poly(vinylalcohol),
poly(ethylene glycol), water soluble polyphosphazenes, and mixtures
thereof.
4. The hydrogel composition of claim 1, wherein the water soluble
or water dispersible hydrophilic polymer is dextran.
5. The hydrogel composition of claim 1, wherein the average degree
of substitution is from 3-25.
6. The hydrogel composition of claim 1, wherein the hydrogel is
formed in microspheres.
7. The hydrogel composition according to claim 1, further
comprising an active ingredient.
8. The hydrogel composition according to claim 7, wherein the
active ingredient is a drug to be released.
9. A process for the preparation of a hydrogel comprising: a)
polymerizing L-lactic acid, optionally in the presence of a
suitable initiator; b) polymerizing D-lactic acid, optionally in
the presence of a suitable initiator; c) reacting the product of
step a) with a suitable coupling compound and a water soluble or
water dispersible hydrophilic polymer to form a water soluble or
water dispersible hydrophilic polymer grafted with oligomers or
co-oligomers, wherein the oligomers or co-oligomers comprise
homo-oligomers of L-lactic acid; d) reacting the product of step b)
with a suitable coupling compound and a water soluble or water
dispersible hydrophilic polymer to form a water soluble or water
dispersible hydrophilic polymer grafted with oligomers or
co-oligomers, wherein the oligomers or co-oligomers comprise
homo-oligomers of D-lactic acid; and e) mixing the product of step
c) and the product of step d) in an aqueous system such that the
homo-oligomers of L-lactic acid and the homo-oligomers of D-lactic
acid interact noncovalently.
10. The process of claim 9, wherein said suitable initiator
comprises a primary or secondary hydroxyl group.
11. The process of claim 9, wherein an active ingredient is added
prior to or during step e).
12. The process of claim 11, wherein the active ingredient is a
drug to be released.
13. The process of claim 12, wherein the drug to be released is
selected from proteins and proteinaceous products.
14. A method for drug delivery comprising administering the
hydrogel composition of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
09/913,967 filed Dec. 31, 2001, which is a national phase of
PCT/NL00/00108 filed Feb. 21, 2000, which claims priority to EP
99200472.1 filed Feb. 19, 1999. The contents of each of these
applications are incorporated herein by reference in their
entirety.
TECHNICAL FIELD
[0002] The present invention relates to hydrogel compositions
comprised of mixtures of water soluble or water dispersible
polymers in an aqueous system, at least part of which polymers
contain at least two groups, which groups can give an interaction.
Furthermore, the present invention relates to processes for
preparing such hydrogels, and the use of oligomeric or
co-oligomeric groups on polymers for gel formation.
BACKGROUND
[0003] The fast developments in the field of molecular biology and
biotechnology have made it possible to produce a large number of
pharmaceutically interesting products in large quantities. For
instance, pharmaceutically active peptides and proteins can
suitably be used as drugs in the treatment of life-threatening
diseases, e.g., cancer, and of several types of viral, bacterial
and parasital diseases; in the treatment of, e.g., diabetes; in
vaccines, e.g., for prophylactic aims, and for anticonception
purposes.
[0004] Especially the specialized biological activities of these
types of drugs provide tremendous advantages over other types of
pharmaceutics.
[0005] To illustrate the fast developments, it has been reported
(see, e.g., Soeterboek and Verheggen, Pharm. Weekblad 130, pp.
670-675 (1995); R. P. Evens and R. D. Sindelar, `Biotechnology
products in the pipeline`, In: Pharmaceutical Biotechnology (D. J.
A. Crommelin and R. D. Sindelar, eds.), Harword Academic
Publishers, pp. 337-355 (1997)) that in the United States of
America, about 275 biotechnological products are in phase IV
studies, while more than 500 products are under investigation.
[0006] Examples of (recombinant) proteins, which are considered
very interesting from a pharmacological point of view, are
cytokines, such as interleukines, interferons, tumor necrosis
factor (TNF), insulin, proteins for use in vaccines, and growth
hormones.
[0007] Due to their nature, proteins and proteinaceous products,
including peptides, which group of products will be referred to as
protein drugs herein-below, cannot be administered orally, at least
not effectively. These products tend to degrade rapidly in the
gastro-intestinal tract, in particular because of the acidic
environment and the presence of proteolytic enzymes therein.
[0008] Moreover, to a high extent protein drugs are not able to
pass endothelial and epithelial barriers, due to their size and,
generally, polar character.
[0009] For these reasons, protein drugs have to be brought in the
system parenterally, i.e., by injection. The pharmacokinetical
profile of these products is, however, such that injection of the
product per se requires a frequent administration. For, it is a
known fact that proteinaceous material is eliminated from the blood
circulation within minutes.
[0010] In other words, since protein drugs are chemically and/or
physically unstable and generally have a short half-life in the
human or animal body, multiple daily injections or continuous
infusions are required for the protein drug to have a desired
therapeutic effect. It will be evident that this is inconvenient
for patients requiring these protein drugs. Furthermore, this type
of application often requires hospitalization and has logistic
drawbacks.
[0011] In addition, it appears that at least for certain classes of
pharmaceutical proteins, such as cytokines which are presently used
in, e.g., cancer treatments, the therapeutic efficacy is strongly
dependent on effective delivery, e.g., intra-or peritumoral. In
such cases, the protein drugs should be directed to the sites where
their activity is needed during a prolonged period of time.
[0012] Hence, there is a need for delivery systems which have the
capacity for controlled release. In the art, delivery systems
consisting of a matrix of biodegradable polymers, (e.g., poly
(DL-lactide-co-glycolide), see for example: J. L. Cleland, `Protein
delivery from biodegradable microspheres`, in: Protein Delivery,
Physical Systems, Pharmaceutical Biotechnology, Vol. 10, (L. M.
Sanders and R. W. Hendren, eds.), Plenum Press, pp. 1-39 (1997) or
L. Brannon-Peppas, `Recent advances on the use of biodegradable
microparticles and nanoparticles in controlled drug delivery`, Int.
J. Pharm., 116, pp. 1-9 (1995)) in which matrix proteins are
present and from which they are gradually released, have been
propose.
[0013] Delivery systems can be obtained by using such biodegradable
polymers for example in microspheres. However, in vitro or in vivo
application of such systems based on poly (lactic acid) or poly
(lactic-co-glycolic acid) have some inherent drawbacks. First,
organic solvents have to be used to encapsulate proteins in the
microspheres. Second, acidic products are formed during
degradation, which might result in a lowering of the pH. Both a low
pH and organic solvents can affect protein stability. Furthermore,
it appears to be difficult to control the protein release from
these systems, which can lead to a burst release.
[0014] A hydrogel system consisting of biodegradable polymers would
overcome such objections. Hydrogels can be obtained by crosslinking
of hydrophilic polymers. Crosslinking can be established by using
aggressive and toxic cross-linking agents which are not compatible
with proteins, such as bisfunctional agents, e.g., glutaraldehyde,
diisocyanates and epichlorohydrin (See: Biodegradable hydrogels for
drug delivery (K. Park, W. S. W. Shalaby and H. Park, eds.)
Technomic Publishing Co. Inc., p. 75 (1993)). These are toxic
compounds which have to be extracted from the gels before these
gels can be therapeutically applied. Moreover, these compounds can
also react with, e.g., epsilon amine residues or lysine side chains
of the protein that is present in the hydrogel matrix. This is
highly unwanted because these reactions might result in loss or
reduction of biological activity of the protein.
[0015] PCT/NL97/00374 describes a delivery system that provides one
solution for this problem that improves the degradation properties.
The hydrogel described in said PCT application is based on
biodegradable polymers. More in particular, chains of these
biodegradable polymers are intermolecularly crosslinked through
linking groups, and these linking groups that keep the hydrogel
together, are hydrolysable under physiological conditions, which
effects the degradation of the hydrogel.
[0016] Although these hydrolysable hydrogels provide a gel with
enhanced release properties, some disadvantages remain. The
polymerization of dextran (-derivatives), for example, requires
peroxydisulfate and TEMED as initiator/accelerator, which compounds
are essentially incompatible with in vivo application. Furthermore,
the use of an initiator may give rise to oxidation of proteins to
sulfoxides. Therefore, it is required that these compounds are
carefully removed.
BRIEF SUMMARY
[0017] It is the object of the present invention to provide a
release delivery system that consists of a biodegradable hydrogel
that can be applied in vitro and in vivo as a release system,
wherein the crosslinks are not of a chemical nature, in the sense
that the crosslinks are not covalent bonds. Consequently, such
crosslinks do not require cleavage of chemical bonds.
[0018] To obtain a hydrogel composition that meets the demands set
out in the above, several ways to modify polymeric chains have been
investigated, and the present inventors have now found that when a
hydrophilic polymer is substituted with grafts that contain
elements with chiral differences, in particular elements that are
enantiomers, a gel, i.e., a crosslinked structure, is obtained that
has excellent properties, for the application as a release system,
and in particular a controlled release system, for example for
protein drugs.
[0019] Without wishing to be limited to a certain theory, the
formation of this gelled structure is believed to be caused by the
interaction of the grafts, which is believed to be similar to the
interaction between the constituents of so called stereo-complexes.
These stereocomplexes are racemic crystallites that are known to be
formed from racemic mixtures of certain polymers. For example, it
has been found (see, e.g., De Jong, et al., Macromolecules, 31, pp.
6397-6402, (1998)) that the melting temperature of stereocomplexes
of poly (lactic acid) is considerably higher than the melting
temperature of both the enantiomer crystallites.
[0020] According to the present invention, the stereocomplex gel
structure is the result of the interaction of oligomerized monomers
of one chirality with that of oligomerized monomers of the opposite
chirality, which oligomerized monomers are both present on
hydrophilic polymers. The groups of oligomerized monomers can be
present anywhere on the polymer chains, viz. also on the head or
tail positions of the polymer chains. Preferably however, the
groups of oligomerized monomers are present as branches or grafts
on different polymer chains that constitute the gel structure. For
clarity's sake it is noted that in the present description and
claims, the term "graft" does not encompass oligomerized comonomers
at the head and tail of a polymer backbone nor blocks of such
oligomerized comonomers incorporated in a polymer backbone.
[0021] The gel is formed by mixing at least two different systems
(A) and (B), each system being a solution or a dispersion of water
soluble or water dispersible polymers, which polymers have
oligomeric grafts. The grafts are obtained by polymerization of
preferably one type of chiral monomer (e.g., D or L lactic
acid).
[0022] The hydrogel composition according to the present invention
is thus comprised of a mixture of (A) a water soluble or water
dispersible polymer in an aqueous system at least part of which
polymer contains at least two groups, which groups are oligomers or
co-oligomers at least partly formed from chiral monomers, and (B) a
water soluble or water dispersible polymer in an aqueous system at
least part of which polymer contains at least two groups, which
groups are oligomers or co-oligomers which are at least partly
formed from chiral monomers with a chirality that is opposite to
that of said monomers in mixture (A), such that the chiral part of
the oligomers or co-oligomers in mixture (B) are in essence
complementary to that of said groups of mixture (A); in which
hydrogel composition the groups on the polymers from mixture (A)
give a physical interaction with the groups from mixture (B).
[0023] System (A) comprises water soluble or water dispersible
polymers with grafts formed from monomers of a certain chirality.
System (B) comprises water soluble or water dispersible polymers
with groups formed from monomers of an opposite chirality, viz. the
enantiomers of the monomers used in system (A).
[0024] Optionally, a small mole percentage of another monomer
(e.g., glycolic acid) randomly placed in the group of oligomerized
monomers is present. The presence of this comonomer should not
prevent the ability of the groups, e.g., graft to give an
interaction to form an associated complex with groups, e.g., grafts
of the opposite chirality.
[0025] Alternatively, the graft is a co-oligomer that comprises a
block-like structure, such as X-Y, in which X is formed from
non-chiral monomers (e.g., a polyethylene glycol block) and Y is
formed from chiral monomers (e.g., a poly (D or L lactic acid)
block).
BRIEF DESCRIPTION OF FIGURES
[0026] FIG. 1 shows the reaction scheme for the synthesis of
dex-lactate (8).
[0027] FIGS. 2 and 3 show the development of the storage moduli in
time of dex-lactate products.
[0028] FIG. 4 shows the rheological characteristics as a function
of time of dex-(L)lactate solution and a mixture of dex-(L)lactate
and dex-(D)lactate solutions.
[0029] FIG. 5 shows the strain as a function of time in creep
experiments for (A) dex-(L)lactate, and (B) dex-lactate
mixture.
[0030] FIG. 6 shows the storage modulus (G') of dex-lactate
hydrogel and chemically cross-linked methacrylated-dextran gel as a
function of temperature.
[0031] FIG. 7 shows the storage modulus (G') of dex-lactate
hydrogel and chemically cross-linked methacrylated-dextran gel as a
function of frequency.
[0032] FIG. 8 shows the storage modulus (G')(A) and strain (B) of
dex-(L)lactate and dex-lactate mixture as a function of time.
[0033] FIG. 9 shows the influence of (A) the degree of
polymerization, and (B) the water content and the degree of
substitution on the storage modulus (G') of dex-lactate mixture and
of dex-(L)lactate.
[0034] FIG. 10 shows the FTIR-transmission spectrum of the free
monodisperse lactic acid oligomers (DP 8) and its corresponding
blend.
[0035] FIG. 11 shows the IR spectra of the dried
dex-(monodisperse)lactate products at wavenumbers between (A)
2000-1000 cm.sup.-1, and (B) 1500-1100 cm.sup.-1.
[0036] FIG. 12 shows the cumulative amount of (A) protein IgG, and
(B) lysozyme, released from dextran gels as a function of time.
DETAILED DESCRIPTION
[0037] According to the present invention, a hydrogel can be
obtained by mixing two or more water soluble or water dispersible
polymers of the invention, which hydrogel has excellent release
characteristics, is easily loaded with, e.g., protein material, and
that can be prepared without the use of organic solvents.
[0038] Preferably the oligomerized monomers, which are referred to
as "grafts" herein-below are biodegradable. The grafts are
preferably coupled to the polymer through hydrolysable bindings.
The entire release system can be biodegradable or
biocompatible.
[0039] It is also possible to obtain the desired effect, viz. the
stereocomplex interaction, when grafts formed of monomers of one
chirality and grafts formed of monomers of an opposite chirality
are present on the same polymer chain. When such grafted polymers
are present in the mixture, they are also able to display the
stereocomplex interaction. Preferably, however, each polymeric
chain is grafted with oligomers formed from monomers of the same
chirality. More preferably, the grafts have a monodispersed
distribution of the chain length, i.e., all grafts have essentially
the same length.
[0040] It will be understood from the above that the
characteristics of the formed gel will be determined to a great
extent by the type, the number and the length of the grafts
applied.
[0041] In the hydrogel according to the present invention, the
average chain length of the oligomeric or co-oligomeric groups is
preferably sufficiently low to render the polymer soluble or
dispersible in water.
[0042] The hydrogel according the present invention comprises water
soluble or water dispersible polymers in which the average chain
length of the graft groups is preferably sufficiently high to
obtain physical interaction between the grafts formed from monomers
of opposite chirality, which physical interaction is different from
the physical interaction that occurs when the grafts were formed
from a racemic mixture of the same monomers.
[0043] Different amounts of enantiomers can be applied in one
graft. It will be understood that the interaction between the
grafts will be highest when the grafts on the polymer in one system
is formed by monomers of one chirality, while the other portion of
the grafts has an opposite chirality. Alternatively, it is also
possible to use grafts that are enriched in monomers having one
chirality instead of being exclusively formed by monomers of one
chirality, as long as the grafts of the polymer in the other system
is enriched in a fashion that it is in essence complementary to
that of the first. This can be realized by, e.g., oligomers or
co-oligomers in the form of blocks of monomers with the same
chirality.
[0044] In order to form the hydrogel of the present invention it is
required that for a polymer chain, in order to contribute to the
coherency of the gel structure, that it is grafted with at least
two grafts. In this way a network can be formed.
[0045] The number of grafts is represented by the degree of
substitution (DS) of the water soluble or water dispersible
polymer. A DS of 100 refers to a polymer in which each monomeric
unit of the polymer is substituted by a graft, whereas a DS of 0
refers to an ungrafted polymer. A minimum DS is required for a gel
to be formed. DS can be varied by changing the synthesis
conditions, in particular the conditions during the coupling of the
grafts to the polymer.
[0046] This can be carried out for example, by changing the ratio
of oligomeric grafts/polymer in the synthesis mixture. Other
parameters which may be used to control DS comprise reaction
temperature and reaction time. As explained in the previous
hereinabove, the minimum DS, for the portion of the polymer chains
that contribute to the gel structure, should correspond to two
grafts per polymeric chain.
[0047] On the other hand, in order for the constituent to be water
soluble or water dispersible, it is required that DS is not too
large. The value of DS required for the desired solubility or
dispersibility can be experimentally obtained in an experimental
procedure which procedure per se is standard for the person skilled
in the art. Preferably the number of grafts per polymer chain is
larger than 2, more preferably it is between 2 and a number that
corresponds to a DS of about 25. The corresponding value of DS will
depend on the molecular weight of the polymer.
[0048] In a preferred embodiment of the hydrogel composition
according to the present invention, the average degree of
substitution of the water dispersible polymer with oligomeric or
co-oligomeric groups is sufficiently high to obtain a network in
which the crosslinks are formed by physical interaction of the
water soluble or water dispersible polymers.
[0049] Preferably, the average degree of substitution is
sufficiently low to render the polymeric structure water soluble or
water dispersible.
[0050] The graft length is represented by the degree of
polymerization (DP), which is the amount of monomers that
constitute a graft. DP can be varied by changing the synthesis
conditions, e.g., by changing the initiator/graft monomers
ratio.
[0051] The DP should be sufficiently high in order for the grafts
to interact so that a gel can be formed. On the other hand, when
the grafts are too long, the water solubility or water
dispersibility becomes insufficient. Typically, for a poly (lactic
acid) graft the average DP is greater than 6, more preferably it is
between 7 and 25, but of course this value will depend on the type
of oligomer used as graft as well as on the type of polymer.
[0052] For a hydrogel that comprises or is comprised of water
soluble polymers, such as dextran polymers, part of which are
grafted with poly (D-lactic acid) and the other part with poly
(L-lactic acid), the lower value of DS is preferably a value that
corresponds to 2 grafts per polymer chain. The upper value is
preferably about 25, and average DP is between 6 and 25. Preferably
both said parts are equal.
[0053] It will be understood that the preferable values for DS and
DP depend essentially on the type of grafts and the required
properties of the resulting gel, although also the type of polymer
used and other conditions can be of influence. For example, when
the oligomeric or co-oligomeric groups of one mixture comprise poly
(D-lactic acid) and the oligomeric or co-oligomeric groups of the
other mixture of the hydrogel composition according to the
invention comprises poly (L-lactic acid), it is preferred that both
oligomeric or co-oligomeric groups have an average chain length of
7-25 monomers.
[0054] In fact, the release properties and other properties of the
resulting gel, when applied in controlled release, can be
fine-tuned and adopted to a specific application by varying DP and
DS, which is a tremendous advantage of the present invention.
[0055] It is also possible that the grafts are co-oligomers of
different monomers, as long as at least one of the monomers used
has a chirality that is opposite from at least one of the monomers
that is used in the grafts of the polymer of the other system.
[0056] In one embodiment of the present invention, the grafts of
the polymer in system (A) are in essence formed from the
L-enantiomers of a monomer and the grafts of the polymer in system
(B) are in essence formed from D-enantiomers of the same
monomer.
[0057] In a preferred embodiment of the hydrogel according to the
present invention, the said oligomers or co-oligomers of mixtures
(A) or (B) are chosen from the group comprising homo-oligomers of
D-lactic acid, random co-oligomers of D-lactide/s-caprolactone, di-
and triblock blends of D-rich poly (lactic acid),
poly(D-lactide-co-glycolide), di-and triblock co-oligomers of
poly(ethylene glycol)/poly(D-lactic acid), poly(methyl
methacrylate),
poly(.alpha.-methyl-.alpha.-ethyl-.beta.-propiolactone),
poly(tert-butylethylene oxide), poly(tert-butylethylene sulfide),
poly[.beta.,-(1,1-dichloropropyl)-.beta.-propiolactone],
poly(.alpha.-benzyl glutamate), poly(methylbenzyl methacrylate),
poly(vinyl-N-butylpyridium bromide), poly(sodium styrenesulfonate),
poly(tert-butylthiirane), poly(.alpha.-methylbenzyl methacrylate),
poly[.beta.-(1,1-dichloroethyl)-.beta.-propiolactone], and mixtures
thereof; and said monomers of the other mixture are formed by the
enantiomers of said monomers of the first mixture.
[0058] In another preferred embodiment the water soluble or water
dispersible polymer of the hydrogel of the present invention is
chosen from the group consisting of dextran, starch, cellulose
derivates, albumin, lysozym, poly(aminoacids), poly(lysine) and
related copolymers, poly(glutamic acid) and related copolymers,
poly((meth) acrylates)/((meth)acrylamides), poly(vinylalcohol),
poly(ethylene glycol), water soluble polyphosphazenes, or mixtures
thereof.
[0059] When applied as a controlled release system, the drug to be
released is incorporated after the formation of the gel, viz. after
the addition of mixtures (A) and (B).
[0060] Alternatively, because the gel of the present invention is
prepared in the absence of organic solvents, the drug to be
released can be added to the composition prior to the formation of
the gel, viz. prior to the addition of system (A) and system (B).
The drug is thus mixed with system (A) and/or system (B), which
systems are subsequently mixed, upon which the gel will be formed
under suitable reaction conditions.
[0061] The graft groups can be linked directly to the polymers or
by means of a linking group, depending on the reactivity of the
groups and the polymer. An example of such a linking group is
carbonyldiimidazole (CDI). Such linking groups are converted
further when the grafts are linked to the polymer. The linking
group could also be applied to enhance the biodegradability of the
product. According to a preferred embodiment of the present
invention there is a linking group between the water soluble or
water dispersible polymer and the oligomeric or co-oligomeric
group, which linking group comprises a hydrolysable group.
[0062] The grafts can be formed by oligomerization of the monomers,
which is preferably carried out by using an initiator. The
initiator is usually incorporated in the oligomeric graft. Such
initiators are compounds with a primary or secondary hydroxyl
group, e.g.: ethyl lactate or other aliphatic or aromatic lactate
esters, benzyl alcohol, lauryl alcohol, 1,4-butanediol, adipic
acid, (monomethoxy)PEG, 2-(2-methoxyethoxy)ethanol, or mixtures
thereof. Care should be taken that the use of these initiators does
not give rise to toxic levels of (reaction products of) these
initiators in the resulting gel when applied in vivo. For this
reason it is preferred to use endogenous compounds or compounds
derived from endogenous compounds as an initiator. The use of such
compounds as initiator prevents unacceptable (i.e., toxic) levels
of these compounds or the reaction products thereof. An example of
a suitable initiator is ethyl lactate, which is easily hydrolyzed
to the relatively harmless compounds ethanol and lactate in, e.g.,
mammals.
[0063] When an initiator is applied, the grafts in the resulting
product may carry (part of) the initiator as an end group. The
amount of initiator relative to the amount of graft monomers can be
used to tailor the value of DP.
[0064] The oligomerization is carried out in the presence of a
suitable catalyst. Such a catalyst can be chosen from the group
comprising: stannous octoate, aluminum alkoxides (e.g., aluminum
tris (2-propanolate), zinc powder, CaH.sub.2,
Sn(IV)tris-2-ethylhexanoate, tetraphenylporphinatoaluminum,
aluminum triisopropoxide, chiral Schiff's base/aluminum alkoxides,
Al (Acac), SALEN-Al--OCH.sub.3, t-BuOLi, Bu.sub.3SnOCH.sub.3, PbO,
zinc oxide, diethyl zinc, zinc chloride, stannous chloride,
magnesium salt, Zn(Acac).sub.2, ZnEt.sub.2-Al (OiPr).sub.3,
(ZnEt.sub.2+AlEt.sub.3+nH.sub.2O), yttrium oxide, or mixtures
thereof.
[0065] The grafting of the polymers can be effected by mixing the
grafts with or without the linking groups and the polymers in a
suitable solvent. Preferably the grafts are mixed with the linking
groups. Such solvents can be chosen from aprotic solvents,
depending on the polymer used, e.g., dimethyl sulfoxide for, e.g.,
polydextrans, after which the grafting reaction is carried out
under suitable conditions, which conditions can be easily
determined by a skilled person. After this the solvent is
removed.
[0066] The degree of substitution can be controlled by changing the
amount of (co-)oligomeric graft and water soluble polymer, e.g.,
the ratio of lactides to dextran.
[0067] Another parameter that can be used in the hydrogels
according to the present invention is the presence of
(co-)oligomers in the hydrogel which are not grafted to the
polymer. Such non-bonded (co-)oligomers form physical interaction,
which interaction is similar as described in the above. The
non-bonded (co-)oligomers interact with the grafts present on the
polymer, thus `occupying` the grafts and consequently reduce the
interaction between the different polymer chains through the
grafts, rendering a gel which is softer and/or has a lower shear
modulus.
[0068] The oligomeric groups can be coupled to the polymer by a
coupling reaction, e.g., by forming a carbonate or ester bond
between hydroxyl moieties present on the polymer with hydroxyl
groups (carbonate bond) or carboxylic acid groups (ester bond) of
the oligomeric groups.
[0069] Some oligomeric groups which find particular use in the
present invention are bifunctional, i.e., have two functional
groups with which the coupling to the polymer can be carried out.
Lactic acid oligomers, for example, bear a hydroxyl group on one
end and a carboxylic acid group on the other end of the chain. This
carboxylic acid end group may be in its free form or may be
blocked, e.g., in the form of an ester with a group derived, e.g.,
from the initiator.
[0070] Stereocomplexes may be formed from a mixture of such
oligomers (i.e., the non-bonded oligomeric groups per se), i.e., a
racemic mixture of oligomers formed from D-monomers and oligomers
formed from L-monomers (which will be referred to as "D-oligomers"
and "L-oligomers", respectively). It is found that the
stereocomplex formation takes place in either the so-called
parallel or anti-parallel orientation of the chirally different
oligomers.
[0071] In stereocomplexes with the parallel orientation each
D-oligomer is paired with an L-oligomer, such that the beginning
(.alpha.-position) of each D-oligomer is next to the
.alpha.-position of an L-oligomer with which it forms the
stereocomplex Likewise, the end position .omega.-position) of the
chirally different oligomers are paired .omega.-.omega..
[0072] In anti-parallel stereocomplexes, however, the D-oligomers
show a preference for pairing such that their .alpha.-position is
next to the .omega.-position (end position) of the L-oligomers.
[0073] Whether parallel or anti-parallel stereocomplexes are
formed, depends mainly on the type of oligomers, viz. the monomers
used to make these oligomers. For example, in a mixture of
poly(D-lactide) and poly(D-lactide), a poly(L-lactide) segment and
poly(D-lactide) segment are packed in parallel fashion (Okihara,
T.; Tsuji, M.; Kawaguchi, A.; Katayama, K. I.; Tsuji, H.; Hyon, S.
H.; Ikada, Y. J. Macromol. Sci. Phys. 1991, B30 (1&2),
119-140).
[0074] It was found that this preference for parallel or
anti-parallel orientation of the oligomers can be employed to
obtain a hydrogel which has particular favorable rheological
behavior.
[0075] For example, a hydrogel can be prepared from a mixture (A)
and (B) as described above of polymers grafted with groups derived
from bifunctional oligomers that show preference to parallel
orientation. When the majority of the oligomeric groups on the
polymers in mixture (A) are linked to the polymer through a moiety
(for example the moiety at the .alpha.-position), while the
oligomers in mixture (B) are linked to the polymer through the
moiety at the opposite position of the bifunctional oligomer (i.e.,
the moiety at the .omega.-position), a mixture is obtained which
shows enhanced gel formation, viz. stronger gels (higher value of
G') compared with polymers grafted with oligomeric groups which are
linked to the polymer by the same functional group in both mixtures
(A) and (B), having the same DP and DS.
[0076] By choosing the synthesis conditions for grafting the
polymers in mixture (A) differently from the synthesis conditions
for grafting the polymers in mixture (B), a gel can be obtained in
which the parallel orientation of the oligomeric groups is
facilitated. When the parallel orientation of the corresponding
oligomers is preferred, this will result in a stronger gel than
when the same oligomeric groups are in anti-parallel orientation
(which orientation is facilitated by employing the same synthesis
conditions for grafting in both mixtures).
[0077] Conversely, when grafts are used derived from oligomers that
form anti-parallel stereocomplexes, the stronger gel is obtained
when synthesis conditions for grafting are the same in both
mixtures.
[0078] Therefore, according to a further embodiment of the
invention, there is provided a hydrogel composition as described
above, in which the oligomeric groups are derived from bifunctional
oligomers that form parallel stereocomplexes, in which a
substantial part of the oligomeric groups are coupled to the
polymer in mixture (A) through a chemical moiety that is different
from the corresponding moiety through which a substantial part of
the oligomeric groups are coupled to the polymer in mixture
(B).
[0079] Conversely, when anti-parallel oligomeric groups (i.e.,
groups derived from oligomers that form anti-parallel
stereocomplexes) are employed, stronger gels are obtained when the
groups are coupled to the polymer in mixtures (A) and (B) through
the same group. Coupling through different groups, as described
above, could in this case be employed to lower the strength (value
of G') of the resulting gels.
[0080] Facilitating the parallel or anti-parallel orientation of
the oligomeric groups can be obtained easily by choosing proper
synthesis conditions. For example, when polymers having hydroxyl
groups are used in mixtures (A) and (B) and parallel complexing
oligomers are used as oligomeric groups, which oligomers are
bifunctional in that they have an hydroxyl group on one end and a
carboxylic acid group on the other end, the skilled person can
choose synthesis conditions in mixture (A) which result in the
formation of a carbonate bond. The carbonate bond is formed between
the hydroxyl group of the oligomeric group and the hydroxyl group
of the polymer. The synthesis conditions in mixture (B) can be
chosen such that an ester bond is formed between the carboxylic
acid group of the oligomeric group and the hydroxyl group of the
polymer. When the two mixtures (A) and (B) are subsequently mixed
together, gel formation is enhanced by the anti-parallel
orientation of the oligomeric groups.
[0081] Prior to mixing (A) and (B) the individual mixtures can be
subjected to solvatation, which comprises mixing for a certain
period of time.
[0082] An interesting possible use of the invention is the use of
mixture (A) and (B) for the in vivo formation of the hydrogel. When
mixtures (A) and (B) are mixed, the resulting mixture will
initially be liquid, since the formation of a hydrogel will take
some time. When this mixture is injected before the formation of a
hydrogel is completed, the hydrogel will be formed in vivo. This is
carried out by mixing ex vivo the mixtures (A) and (B) and
injecting this mixture in liquid form after which said hydrogel
forms in vivo.
[0083] The stereocomplex hydrogels according to the present
invention, are macroscopic gels. The gels can be used in their
macroscopic form, applied, e.g., in implants. The stereocomplex
hydrogels may also be used for topical application in which an
active ingredient can be administered, by applying the drug loaded
gel to skin This can be used, e.g., in the treatment of bums and
scalds.
[0084] Alternatively, the gels can be formed in microspheres, for
example by spray drying. Another possibility for forming
microspheres is described in PCT/NL97/00625 which discloses a two
phase method in which a two phase system is formed from two
incompatible water soluble polymers and at least one releasable
compound. According to the present invention, a process for the
preparation of a hydrogel in the form of microspheres comprises the
formation of a two phase system, optionally in the presence of a
releasable compound, by choosing two of said water soluble or water
dispersible polymers such that they are incompatible; from which
two phase system the hydrogel is formed.
[0085] Injectable microspheres, suitable for controlled release of
an active ingredient, which is incorporated preferably before the
gel formation takes place, can thus be obtained.
[0086] Another interesting application of the gels of the present
invention is to encapsulate cell material, in particular living
cells. This is of particular interest for tissue engineering. This
encapsulation can be effected by mixing the cells and/or another
active ingredients, such as growth factors, in one of the mixtures
(A) or (B), after which the other mixture is added, by which the
gel is formed. Instead of cells, other biological or non-biological
compounds can be used as active ingredient. Examples are plasmid
DNA, viral vectors, and colloidal carriers such as liposomes,
iscoms, polyplexes (i.e., combinations of (cationic) polymers--such
as organic polyphosphazenes or polyacrylates--and DNA), lipoplexes
(i.e., combinations of (cationic) lipids and DNA), nanoparticles,
(i.e., polymer based spheres in the nanometer size range), solid
lipid particles in the colloidal size range, emulsions, such as
intralipid-like systems, and combinations thereof. Also low
molecular weight compounds may be encapsulated.
[0087] The present invention also provides a process for the
preparation of a hydrogel comprises the steps of preparing two
mixtures of a substituted water soluble or water dispersible
polymer, the preparation of each mixture comprising:
[0088] 1) polymerization, optionally in the presence of a suitable
initiator, of a monomer, where the monomer of one mixture is the
enantiomer of the monomer of the other mixture,
[0089] 2) reaction of the product of the previous step with a
suitable coupling compound,
[0090] 3) reaction of the product of the previous step with said
water soluble or water dispersible polymer, and
[0091] 4) mixing two said mixtures.
[0092] Preferably, the suitable initiator in step 1) contains a
primary or secondary hydroxyl group.
[0093] The invention further relates to the use of two opposite
enantiomeric forms of a monomer in an oligomer or co-oligomer which
oligomer or co-oligomer are attached to polymeric chains to
physically link these polymeric chains.
[0094] Whether a specific mixture of grafted polymers will give
rise to the formation of a gel can effectively be assessed by
measuring the rheologic behavior of the presence of physical
interactions (stereocomplexes) will give rise to (more) elastic
behavior, as is reflected for example in the storage modulus (G'),
the loss modulus (G''), tan .delta.(=G''/G') and/or the creep,
which can be determined experimentally.
[0095] The present invention will now be illustrated in the
following Examples, which are not intended to limit the scope of
the invention.
Example 1
Synthesis of Dex-lactate ((DP).sub.av=15, (DS).sub.av=10)
Hydrogels
[0096] Ring-opening polymerization of lactide (L-lactide for system
(A) and D-lactide for system B, each 5 g) was carried out using
2-(2-methoxyethoxy) ethanol (MEE, 0.556 g) as an initiator, and
stannous octoate (0.093 g) as the catalyst. The polymerization was
carried out in the melt at 130.degree. C. for four hours to yield
MEE-L-lactic acid oligomer (A) and MEE-D-lactic acid oligomer (B).
FIG. 1 shows the reaction scheme for the synthesis of dex-lactate
(8), in which the DP is the degree of polymerization and DS is the
degree of substitution (number of lactic acid oligomers per 100
glucopyranose units of dextran.
[0097] Subsequently, carbonyldiimidazole (CDI, 1.122 g, 2 eq., 6.92
mmol) was dissolved in tetrahydrofuran (THF) in a nitrogen
atmosphere. The MEE-lactates (A) and (B) (each 4 g, 3.46 mmol) from
the previous step were added in THF to form separate CDI mixtures.
Each reaction mixture was stirred for four hours at room
temperature under nitrogen atmosphere. The products were purified
by precipitation in water to inactivate residual CDI. After
centrifugation the product was dissolved in acetonitrile and dried
over MgSO.sub.4. After filtration, the organic solvent was removed
under reduced pressure, yielding a viscous oil for each mixture (A)
and (B). Both products (A) and (B) were dissolved in dried
DMSO.
[0098] After this, two mixtures were made in which dextran (5.6 g)
was dissolved in dry dimethyl sulfoxide (DMSO) before
4-(N,N-dimethylamino)pyridine (DMAP, 1.06 g, 0.25 eq. to dextran,
8.65 mmol) was added. After dissolution of DMAP, the
MEE-lactate-carbonylimidazole mixtures (A) and (B) (each 4.48 g,
3.46 mmol) of the previous step were each added to one portion to
yield two new mixtures (A) and (B). These mixtures were stirred at
room temperature for 4 days under nitrogen atmosphere, after which
the reaction was stopped by adding concentrated HCl to neutralize
DMAP and imidazole. The reaction mixtures were dialyzed against
demineralized water at 4.degree. C. The crude dex-lactate products
were lyophilized and the uncoupled lactate oligomers were removed
by extraction with dichloromethane. After filtration and
evaporation the pure product was obtained.
[0099] The products had grafts with an average degree of <BR>
<BR> <BR> polymerization (DP).sub.av of 15 and the
average degree of substitution of the polymer (DS).sub.av was
10.
[0100] 80% Hydrogels were prepared by blending a mixture of 200 mg
of product (A) from the previous step in 800 .mu.l acetate buffer,
pH=about 4 (in order to minimize hydrolysis), with product (B)
(also 200 mg/800 .mu.l acetate buffer, pH=about 4) in equal
amounts. Before blending the individual products were dispersed
during three days at room temperature.
Example 2
Synthesis of Dex-lactate ((DP).sub.av=9, (DS).sub.av=10)
Hydrogels
[0101] The procedure of Example 1 was repeated, but different
amounts of reactants were used instead: L (or D)-lactide (10 g),
MEE (1.85 g, 0.015 mol), stannous octoate (0.3 g, 0.74 mmol, 5 mol%
to MEE).
[0102] From the product of this first step 4.23 g (5.5 mmol) was
used with CDI (1.79 g, 2 eq., 11 mmol).
[0103] The dex-lactate-MEE was obtained using the product of the
previous step (5.32 g, 6.17 mmol) and DMAP (1.88 g, 0.25 eq., 15
mmol).
[0104] The products had grafts with an average degree of
polymerization (DP).sub.av of 9 and the average degree of
substitution of the polymer (DS).sub.av was 10.
[0105] The remainder of the procedure was similar to that of
Example 1.
Example 3
Rheology Behavior
[0106] The rheology of samples prepared in the previous Examples
was studied. The mixtures (A) and (B) from the previous Examples
were either solvatated before they were mixed, in which they were
stirred for a certain period of time, or mixed directly.
[0107] Upon mixing (A) and B, the gel formation was followed with a
rheometer (TA Instruments AR 1000-N) using a 2 cm, 1 degree steel
cone plate and a solvent trap filled with silicon oil of 100 mPas,
at a frequency of 1 Hz. The measurements were performed in
controlled strain mode, in which the force to obtain a deformation
of 1% is measured. The results are expressed as the storage modulus
G', which represents the elastic storage of energy in the sample.
For a real Newtonian fluid, G' will be zero. A higher value of G'
corresponds to a gelled structure. By definition, tan .delta.=1
defines the sol-gel transition. A value of tan .delta. smaller than
1 also indicates gel formation. Generally, the gels which are
obtained in accordance with the present invention have a value of
tan .delta. which is smaller than 0.3.
[0108] Typical rheograms for a 20 wt % dextran-L-lactate (average
DP=15 (FIG. 2) or 9 (FIG. 3)) in water as well as a mixture of 10
wt % dextran-L-lactate (average DP=15 (FIG. 2) or 9 (FIG. 3)) and
10 wt % dextran-D-lactate (average DP=15 (FIG. 2) or 9 (FIG. 3)) in
water are shown.
[0109] The mixture of the L and D forms (mixture (A) and (B))
showed an increase of G' as a function of time, whereas one
enantiomeric form of dex-lactate product did not show such an
increase. The increase of G' in case of the mixture of (A) and (B)
shows that the product becomes more and more elastic. This
phenomenon was not observed with one enantiomeric form. This is
explained by the formation of stereocomplexes between two
enantiomeric forms.
TABLE-US-00001 TABLE 1 Storage Moduli of Dex-Lactate Products of
Example 1 and 2. Enantiomeric G' (Pa) after G' (Pa)
(DP).sub.av.sup.1) (DS).sub.av.sup.2) form 2-3 minutes after 15 h 9
5 L and D (blend) 1323 3680 9 5 L and D (blend) 2059 3184 9 5 L or
D.sup.3) 629 683 15 4 L and D (blend) 4049 8384 15 4 L and D
(blend) 3453 7934 15 4 L or D.sup.3) 1972 2306 .sup.1)average
degree of polymerization .sup.2)average degree of substitution
.sup.3)reference
TABLE-US-00002 TABLE 2 Development of storage moduli in time of
dex-lactate products of Example 1 with (DP).sub.av = 15. a.sup.1)
b.sup.1) c.sup.1) d.sup.1) Solvatation G'.sup.2) G'.sup.3)
G'.sup.2) G'.sup.3) G'.sup.2) G'.sup.3) G'.sup.2) G'.sup.3) time
(h) (Pa) (Pa) (Pa) (Pa) (Pa) (Pa) (Pa) (Pa) 0 1972 2306 16 2402
1823 6039 40 3453 7934 48 2359 4911 232 302 60 982 2383 62 1259
4064 68 1253 1476 72 1721 4432 80 1164 6717 95 1705 1715 96 3140
6084 100 1553 5026 120 2947 6639 144 1515 1903 240 2926 8459 264
1323 1783 .sup.1)a: Non-purified dex-lactate product containing non
coupled oligomer, 1:1 Dex-(L)lactate and Dex-(D)lactate mixture (10
+ 10 wt % gel). b: Reference; Non-purified dex-lactate product
containing non coupled oligomer, Dex-(L)lactate or Dex-(D)lactate
(20 wt % gel). c: Purified dex-lactate product, 1:1 Dex-(L)lactate
and Dex-(D)lactate mixture (10 + 10 wt % gel). d: Reference;
Purified dex-lactate product, Dex-(L)lactate or Dex-(D)lactate (20
wt % gel). .sup.2)Storage moduli measured 2-3 minutes after mixing.
.sup.3)Storage moduli measured after gelation, viz. after 24-72
h.
TABLE-US-00003 TABLE 3 Development of storage moduli in time of
dex-lactate products of Example 2 with (DP).sub.av = 9. a.sup.1)
b.sup.1) c.sup.1) d.sup.1) Solvatation G'.sup.2) G'.sup.3)
G'.sup.2) G'.sup.3) G'.sup.2) G'.sup.3) G'.sup.2) G'.sup.3) time
(h) (Pa) (Pa) (Pa) (Pa) (Pa) (Pa) (Pa) (Pa) 0 550 727 48 1323 3680
60 2059 5067 529 683 156 275 1353 180 242 1276 252 176 1354 276 130
1917 .sup.1)a: Non-purified dex-lactate product containing non
coupled oligomer, 1:1 Dex-(L)lactate and Dex-(D)lactate mixture (10
+ 10 wt % gel). b: Reference; Non-purified dex-lactate product
containing non coupled oligomer, Dex-(L)lactate or Dex-(D)lactate
(20 wt % gel). c: Purified dex-lactate product, 1:1 Dex-(L)lactate
and Dex-(D)lactate mixture (10 + 10 wt % gel). d: Reference;
Purified dex-lactate product, Dex-(L)lactate or Dex-(D)lactate (20
wt % gel). .sup.2)Storage moduli measured 2-3 minutes after mixing.
.sup.3)Storage moduli measured after gelation, viz. after 24-72
h.
[0110] The experimental data in Tables 1-3 and FIGS. 2 and 3
clearly demonstrate the different rheology behavior of the
hydrogels of the present invention when compared with the reference
examples (`L or D` in Table 1 and experiments `b` and `d` in Tables
2 and 3).
[0111] Moreover, the purified examples according to the present
invention (`c` in Tables 2 and 3) show higher storage moduli when
compared with the unpurified samples according to the present
invention (`a` in Tables 2 and 3).
[0112] Finally, a longer solvatation time gives rise to formation
of a gel with a higher final value of the modulus.
Example 4
Synthesis, Characterization and Properties of
dex-(L)lactate/dex-(D)lactate Gels
[0113] Materials. L-Lactide
((3S-cis)-3,6-dimethyl-1,4-dioxane-2,5-dione, >99.5%) and
D-lactide ((3R-cis)-3,6-dimethyl-1,4-dioxane-2,5-dione, >99.5%)
were obtained from Purac Biochem BV (Gorinchem, The Netherlands)
and used without further treatment. Stannous octoate (tin(II)
bis(2-ethylhexanoate), SnOct.sub.2, 95%) (Sigma Chemical Co., St.
Louis, Mo., USA), dichloromethane, potassium peroxydisulfate (KPS)
(Merck, Darmstadt, Germany), and 2(2-methoxyethoxy)ethanol
(Aldrich-Chemie, Steinheim, Germany) were used as received.
Tetrahydrofuran (THF) and acetonitrile (HPLC-S, gradient grade)
were purchased from Biosolve LTD (Valkenswaard, The Netherlands).
THF was distilled from LiAlH4 immediately before use. Dextran (from
Leuconostoc mesenteroides, Mn=15,000 Da, and MW=32,500 Da, as
determined by GPC analysis), dimethyl sulfoxide (DMSO, <0.01%
water), glycidyl methacrylate (GMA, (.+-.)-2,3-epoxypropyl
methylpropenoate, 95% by GPC), N,N,N',N'-tetramethylethylenediamine
(TEMED) and silicon oil (DC 200, 110 mPa. s), were obtained from
Fluka Chemie AG (Buchs, Switzerland). 4-(N,N-dimethylamino)pyridine
(DMAP, 99%) and N,N'-carbonyldiimidazole (CDI, 98%) were from Acros
Chimica (Geel, Belgium). Dialysis tubes (cellulose, molecular
weight cut off 12,000-14,000 (based on proteins)) were purchased
from Medicell International Ltd. (London, UK). Methacrylated
dextran (dex-MA) with a degree of substitution (DS, the number of
methacryl residues per 100 glucopyranose units of dextran) of 4 was
synthesized according to the procedure described in detail in Van
Dijk-Wolthuis, W. N. E.; Franssen, O.; Talsma, H.; Van Steenbergen,
M. J.; Kettenes-van den Bosch, J. J.: Hennink, W. E. Macromolecules
1995, 28, 6317-6322. Van Dijk-Wolthuis, W. N. E.; Kettenes-van den
Bosch, J. J.; Van der Kerk-van Hoof, A.; Hennink, W. E.
Macromolecules 1997, 30, 3411-3413.
[0114] Synthesis of Polydisperse Lactic Acid Oligomers.
[0115] Lactic acid oligomers with varying DP were synthesized by a
ring opening polymerization reaction of lactide with
2(2-methoxyethoxy) ethanol (MEE) and stannous octoate as initiator
and catalyst, respectively, according to De Jong, et al. (De Jong,
S. J.; Van Dijk-Wolthuis, W. N. E.; Kettenes-van den Bosch, J. J.;
Schuyl, P. J. W.; Hennink, W. E. Macromolecules 1998, 31,
6397-6402). The average degree of polymerization (DP.sub.aV) of the
formed MEE-lactate was controlled by the MEE/lactide ratio.
[0116] Preparation of Monodisperse Lactic Acid Oligomers.
[0117] Monodisperse lactic acid oligomers were prepared by
fractionation of polydisperse MEE-lactate with preparative HPLC
(column: Econosphere C8, 10 micron, 250.times.22 mm; Alltech, Ill.,
USA) with an AKTA purifier (Pharmacia Biotech AB, Sweden).
Polydispers oligomer (1 g) was dissolved in 1 ml water/acetonitrile
(50 w/w %) and 500 yl of this solution was injected onto the column
A gradient was run from 100% A (water/actonitrile 95:5) to 100% B
(acetonitrile/water 95:5) in 50 minutes. The flow rate was 5.0
ml/min; detection by UV (.lamda.=195 nm). The chromatograms were
analyzed with Unicorn Analysis module (version 2.30) software. The
individual oligomers were collected and fractions with
corresponding DP were pooled. The solvent was removed under reduced
pressure. The oligomers were characterized by HPLC, NMR and MS.
[0118] Synthesis of Activated Lactic Acid Oligomer.
[0119] To couple the oligomers to dextran, the hydroxyl group of
the oligomer was activated using N,N'-carbonyldiimidazole
(CDI).
[0120] Essentially the same procedure was used as for the synthesis
of hydroxyethyl methacrylate (HEMA)-lactate-CI. In brief, CDI (3.6
g, 22 mmol, 2 eq.) was dissolved in dried tertrahydrofuran (THF,
100 ml) in a nitrogen atmosphere. MEE- lactate (e.g., DP.sub.av 9;
8.46 g, 11 mmol, 1 eq.) was dissolved in THF (10 ml) and added to
the CDI solution. The reaction mixture was stirred for four hours
at room temperature in a nitrogen atmosphere. Thereafter,
dichloromethane (DCM, 200 ml) was added and the reaction mixture
was washed with water (100 ml), to decompose the excess of CDI and
to remove the imidazole. Next, the water layer was extracted with
DCM (50 ml) for two times. The organic layers were combined and
dried over magnesium sulfate. After filtration, the organic solvent
was removed under reduced pressure to yield the MEE-lactate-CI
DP.sub.av, 9.
[0121] .sup.1H NMR (CDCl.sub.3): .delta. 8.16 (m, 1H,
C(O)--N--CH.dbd.N), 7.44 (m, 1H, C(O)--N--N--CH.dbd.CH), 7.07 (m,
1H, C(O)--N--CH.dbd.CH), 5.35 (q, 1H, CH--O--C(O)--N), 5.23-5.12
(overlapping q, CH), 4.28 (m, 2H, CH.sub.2--O--C(O)), 3.66 (m, 2H,
CH.sub.3--O--CH.sub.2), 3.60 (m, 2H, CH.sub.2-O), 3.51 (m, 2H,
CH.sub.2--O), 3.37 (s, 3H, CH.sub.3--O), 1.72 (d,
CH.sub.3--CH--O--C(O)N), 1.63-1.50 (overlapping d,
CH--CH.sub.3)
[0122] .sup.13C NMR (CDCl.sub.3): .delta. 169.5 C.dbd.O, 168.8
C(O)--N, 137.1 N--CH--N, 121.6+117.1 N--C.dbd.C--N, 71.7 CH.sub.2,
71.5 (CH.sub.3)CH--O--C(O)--N, 70.3 CH.sub.2, 69.3-68.7
CH--CH.sub.3+CH.sub.2--OCH.sub.3, 64.3 CH.sub.2--O--C(O), 58.9
CH.sub.2O, 25.5 CH.sub.3--CH--O--C(O)--N, 16.6/16.5 CH.sub.3
[0123] Synthesis of Dex-Lactate.
[0124] Dextran-lactate (dex- lactate) was synthesized using the
same procedure as for the synthesis of dex-lactate-HEMA. In brief,
dextran 40 000 (10 g) and DMAP (2 g, 16 3 mmol, 0.25 eq. to
glycopyranose units of dextran) were dissolved in dried DMSO (90
ml). Next, MEE-lactate-CI (e.g., DP.sub.av 9, 5.7 g, 6.17 mmol)
dissolved in dry DMSO (5 ml) was added. The solution was stirred at
room temperature for 4 days in a nitrogen atmosphere, after which
the reaction was stopped by addition of concentrated HCl (2 ml, 1
eq.) to neutralize DMAP and imidazol. The reaction mixture was
extensively dialyzed against water (reversed osmosis) at 4.degree.
C. The dex-lactate product was collected by lyophilization. To
remove traces of uncoupled lactic acid oligomers, the dex-lactate
product (10 g) was extracted with dichloromethane (400 ml). The
product was dried in a vacuum oven at 40.degree. C., to yield
dex-lactate with DP.sub.av 9 and degree of substitution (DS, the
number of oligomers per 100 glucopyranose units of dextran) of 3.
The DS was calculated by .sup.1H NMR as (x-100)/y, in which x is
the integral of the CH.sub.3 groups of the lactic acid oligomer at
1.41 ppm divided by (3-DP), with DP is the degree of
polymerization, and y is the integral of the anomeric proton of
dextran at 5.14-4.95 ppm.
[0125] .sup.1H NMR (12.5% .sup.2H.sub.2O/DMSO-d.sub.5) : .delta.
5.14-4.95 (broad m, residual OH, CH, CH--O--C(O)--N), 4.65 (broad
s, anomeric proton dextran), 4.16 (m, 2H, CH.sub.2--O--C(O)),
3.84-3.18 (m, (6H) dextran, (2H) CH.sub.3--O--CH.sub.2, (4H)
CH.sub.2--O, (3H) CH.sub.3--O), 1.41 (overlapping d,
CHH.sub.2--CH--O--C(O)N, CH--CH.sub.3)
[0126] .sup.13C NMR (12.5% .sup.1H.sub.2O/DMSO-d.sub.6) : .delta.
170.4 C(O)--O--CH.sub.2, 169.8 C.dbd.O, 98.5 C.sub.anomeric, 73.7
C.sub.3, 72.1 C.sub.2, 71.6 CH.sub.2, 70. 7 C.sub.5, 70.3 C.sub.4,
69.9 CH.sub.2, 69.8 (CH.sub.3)CH--O--C(O)--O-dex, 69.3 CH, 68.5
CH.sub.2, 66.2 CH.sub.2(dex), 64.8 CH.sub.2, 58.5 CH, 20.6
CH.sub.3--CH--O--C(O)--O-dex, 16.9 CH.sub.3
[0127] Rheological Experiments.
[0128] For the rheological experiments two types of polymer
solutions were prepared: one contains dex-(L)lactate and the other
dex-(D)lactate. The dissolution time was at least one day at room
temperature. Acetate buffer pH 4 was selected as solvent to prevent
hydrolysis of the dex-lactate.
[0129] Equal amounts of the dex-(L)lactate and the dex-(D)lactate
solutions were mixed, homogenized and quickly applied on the
rheometer (AR 100 instrument of TA Instruments, Gent, Belgium). For
most of the experiments a flat-plate measuring geometry (acrylic, 4
cm diameter; gap 1 mm) was used. A cone-plate measuring geometry
(steel, 2 cm diameter with an angle of 1 degree; gap 31 .mu.m) was
used when only a small amount of material was available. Gelation
of the dex-lactate solutions took place between the cone and plate
of the measuring geometry. A solvent trap was used to prevent
evaporation of the solvent. In addition, a thin layer of silicon
oil (110 mPas) was applied to surround the dex-lactate sample
thereby preventing evaporation.
[0130] Gelation of the mixture of dex-lactate solutions was
monitored by measuring the shear storage modulus (G'), as well as
the loss modulus (G'') at 20.degree. C. for 6 to 18 hours. A
frequency of 1 Hz and a controlled strain of 1% were applied. The
strain used in these experiments was as low as possible to minimize
the influence of deformation on the formation of the dex-lactate
hydrogels. At the end of gelation, two types of rheological
measurements were done. Firstly, creep experiments were carried out
to establish the visco-elastic properties of the samples.
Therefore, a constant force (equal to the force applied at the end
of the gelation measurement to obtain 1% deformation, which is in
the linear visco-elastic range) was applied during 60 seconds while
the strain was monitored. Secondly, the temperature was raised from
20.degree. C. to 80.degree. C. in 30 minutes while several
rheological parameters were monitored (frequency 1 Hz, 1% strain).
At 80.degree. C., a creep experiment was carried out again. A
constant force, equal to the force applied at 80.degree. C. to
obtain 1% deformation, was applied. Thereafter, the sample was
cooled to 20.degree. C. in 30 minutes while G' and G'' were
monitored (frequency 1 Hz, 1% strain), again followed by a creep
experiment.
[0131] As a control, the same rheological experiments were
performed with a solution of dex-(L)lactate. To compare the
rheological behavior of physically cross-linked dex-lactate
hydrogels with chemically cross-linked gels, measurements were
performed on a hydrogel based on methacrylated-dextran (dex-MA).
Dex-MA hydrogels were prepared by radical reaction of aqueous
dex-MA solution (DS 4,10 w/w % dex-MA in 0.01M phosphate buffer pH
7). Solution A was obtained by adding 50 .mu.l TEMED (500 .mu.l/ml,
pH adjusted to 7) to 450 .mu.l of a dex-MA solution, while solution
B was a mixture of 410 .mu.l dex-MA solution and 90 .mu.l KPS
solution (50 mg/ml in 0.01M phosphate buffer pH 7). 100 .mu.l of
solutions A and B were mixed and directly applied on the measuring
geometry of the rheometer.
[0132] Rheology experiments were carried out in the same way as for
the dex-lactate products.
[0133] NMR Spectrometry. NMR spectra were recorded with a Gemini
300 MHz spectrometer (Varian Associates Inc. NMR Instruments, Palo
Alto, Calif., USA). Approximately 30 mg of lactic acid oligomer was
dissolved in 0.8 ml deuterochloroform and 30 mg of dex-lactate was
dissolved in a mixture of 0.8 ml dimethylsulfoxide-d6 and 0.1 ml
.sup.2H.sub.2O (all solvents were obtained from Cambridge Isotope
Laboratories, Andover, USA). For H NMR, chloroform (at 7.26 ppm)
was used as the reference line, whereas for DMSO-d.sub.6 the
central DMSO line was set at 2.50 ppm. A pulse length of 4.5 ms
(PW.sub.90.apprxeq.12 .mu.s) was used with a relaxation delay 15 s.
For .sup.13C NMR spectra, the pulse length was set at 4.5 ms
(PW.sub.90.apprxeq.12 .mu.s), and the relaxation delay at 2 s. The
central line in the chloroform triplet at 76.9 ppm was used as the
reference line, whereas for DMSO-d.sub.6 (99.9% .sup.2H, Cambridge
Isotope Laboratories, Andover, USA) the central DMSO line was set
at 39.5 ppm.
[0134] FTIR-Transmission and Photoacoustic Spectroscopy (PAS).
[0135] IR-transmission spectra of the lactic acid oligomers were
recorded with a Biorad FTS-25 interferometer/spectrometer. Thin
films of L-lactic acid oligomer, as well as the blend of L and D
form, were casted from dichloromethane solutions onto a KBr tablet.
The interferograms were recorded at a spectral resolution of 2
cm.sup.-1 in the rapid-scan mode (5 kHz) with a deuterated
triglycine sulfate (DTGS) detector. 32 scans were averaged to gain
a good signal-to-noise ratio. Fourier transformation yields the
IR-transmission spectra, which were normalized on background
spectra obtained from the pure KBr tablet at an identical parameter
set-up. IR-Photoacoustic (PA) spectra of dex-lactate were obtained
with a BioRad FTS-6000 step-scan interferometer/spectrometer. After
the rheology experiment, selected dex-lactate products were dried
overnight at room temperature. The IR spectra of these products
were measured with the PA detection method. The general benefit of
this method is that no further preparation of the sample (i.e., the
dex-lactate products) is needed. Therefore, IR spectra of solid
materials can be obtained directly and rather quickly. The
interferograms were recorded at a spectral resolution of 8
cm.sup.-1 using an MTEC-200 photoacoustic detector. They were
obtained in the step-scan mode of the interferometer, at 800 Hz
stepping frequency of the moving mirror. Scanning in the step-scan
mode of the interferometer resulted in a far better signal to noise
ratio. 32 Scans were averaged, and Fourier transformed to yield the
IR-PA-spectra of dex-lactate. All IR-PA spectra were normalized
using the PA-reference-sample carbon black.
[0136] Results
[0137] Synthesis of the Lactate Grafted Dextrans.
[0138] In the synthesis of dex-lactate (FIG. 1) essentially the
same strategy was used as for the synthesis of dex-lactate-HEMA.
First, the lactide acid oligomer (3) was synthesized via a ring
opening polymerization of lactide. After activation of the hydroxyl
end-group with N,N'-carbonyldiimidazole (CDI, 4), the resulting
lactate-CI (5) was coupled to dextran (7) to yield dex-lactate (8).
The incorporation of the lactic acid oligomers under the standard
reaction circumstances, 4 days reaction time at room temperature,
was about 30%. Higher degrees of incorporation, up to 60%, could be
achieved by longer reaction times (18-24 days) or higher reaction
temperatures (80.degree. C.). HPLC analysis demonstrated that under
the selected reaction conditions no transesterification
occurred.
[0139] Gelation of Dex-Lactate Solutions.
[0140] FIG. 4 shows the rheological characteristics as a function
of time of dex-(L)lactate solution and a mixture of dex-(L)lactate
and dex-(D)lactate solutions. G' and tan .delta. of dex-(L)lactate
did not change in time. In contrast, the dex-(L)lactate and
dex-(D)lactate mixture showed an increase in G' (even after 18
hours no real plateau value was reached) and a dramatic decrease of
tan .delta. (from 1.2 to 0.1) in time. The network formation can be
attributed to association of the enantiomeric lactic acid chains
(stereocomplex formation). The fact that no real plateau value of
G' was reached is often observed in physically cross-linked
networks, such as gelatin, see for example Bot, A.; Van Amerongen,
I. A.; Groot, R. D.; Hoekstra, N. L.; Agterof, W. G. M. Polymer
Gels and Networks 1996, 4, 189-227.
[0141] FIG. 5 shows the results of creep experiments on the samples
of FIG. 4 after about 18 hours. The retardation profile shows full
viscous behavior for dex-(L)lactate (FIG. 5A), which is in
agreement with the high value of tan .delta. observed (FIG. 4). For
the dex-lactate mixture elastic behavior was observed in the creep
experiment (FIG. 5B), in agreement with the low value of tan
.delta. (FIG. 4).
[0142] Thermoreversibility.
[0143] The rheological characteristics of a chemically cross-linked
methacrylated-dextran gel were monitored as a function of
temperature. FIG. 6 shows that G' increased proportionally with
temperature, which is in agreement with the rubber elasticity
theory of Flory. At 80.degree. C. the dex-MA hydrogel remained
fully elastic as observed from a creep experiment (result not
shown). During cooling the reverse G' profile was observed, and G'
at 20.degree. C. equaled G' before heating.
[0144] In contrast, the dex-lactate gels showed a completely
different temperature dependency. Upon heating the dex-lactate
hydrogels G' dropped (FIG. 6). The loss modulus (G'') of the
mixture at 80.degree. C. was almost equal to the G'' of a single
isomer of dex-lactate (not shown), indicating that no cross-links
were left in the mixture. Creep experiments of both systems at this
temperature showed viscous behavior as in FIG. 5A. FIG. 6 shows
that upon cooling G' increased and finally reached the original
value of G' at 20.degree. C., indicated by the vertical arrow. A
creep experiment showed the same pattern as FIG. 5B, which proves
the full thermo- reversible properties of the mixture of both
isomers and the physical nature of the cross-links
[0145] Elastic properties as function of the frequency. FIG. 7
compares a G' profile of a dex-lactate hydrogel and a chemically
cross-linked methacrylated-dextran hydrogel, respectively, as a
function of the frequency. G' of the dex-methacrylate hydrogel was
independent of the applied frequency again indicating the existence
of a real rubbery network, which is expected in hydrogels with
permanent (chemical) cross-links. (See for example De Smedt, S. C.;
Lauwers, A.; Demeester, J.; Van Steenbergen, M. J.; Hennink, W. E.;
Roefs, S. P. F. M. Micromolecules 1995, 28, 5082-5088). However, G'
of the dex-lactate gel decreased considerably with decreasing
frequency. This again demonstrates the physical, i.e., reversible
(transient) nature of the cross-links (FIG. 7). At low frequencies
the cross-links break and re-form at long time scales (the network
relaxation process), whereas at high frequencies the time scale
becomes smaller and the cross-links act as if permanent, resulting
in increasing G'.
[0146] Influence of DP, DS and Water Content on Gel Formation.
[0147] The G' profiles shown in FIG. 4 were obtained using a
dex-lactate sample with DP.sub.av 9 and DS 3. When dex-lactates
with a higher DP and higher DS were used, the dex-(L)lactate (or
dex-(D)lactate) solutions showed visco-elastic behavior. This is
probably caused by the association of the longer lactic acid chains
with oligomers of the same chirality, which also results in a
poorer water solubility. However, in this sample gelation as
function of the time was not observed. In contrast, the dex-lactate
mixture clearly gelled as observed from an increase in G' (FIG.
8A). The formation of a gel was confirmed by creep experiments: the
dex-lactate mixture showed an almost elastic behavior, whereas the
dex-(L)lactate system is typical visco-elastic material (FIG.
8B).
[0148] FIG. 9 shows the influence of the degree of polymerization
(A), the water content and the degree of substitution (B) on G', 18
hours after mixing the dex-(L)lactate with the dex-(D)lactate
solution. Mixing dex-(L)lactate DP.sub.av 5 with dex-(D)lactate
DP.sub.av 5 solution resulted in a slight increase in G' compared
with the G' value of the corresponding dex-(L)lactate system. This
indicates that a weak hydrogel was formed, as a result of the fact
that the oligomers did not have sufficient length to associate with
each other. Mixing of dex-lactates with higher degrees of
polymerization resulted in formation of a gel as reflected by a
substantially greater value for G' of the mixture compared to the
G' for one of the isomers (compare open and closed symbols, FIG.
9A). As FIG. 9B shows, stronger gels were obtained by increasing
the degree of substitution and by decreasing the water content of
the system.
[0149] Monodisperse Lactic Acid Oligomers Grafted to Dextran
Rheology.
[0150] Monodisperse lactic acid oligomers, with a degree of
polymerization ranging from 8 to 12, were coupled to dextran to
determine the effect of the lactic acid chain length on the gel
formation. Mixtures of dex-(L)lactate and dex-(D)lactate with the
same degree of polymerization of the lactic acid oligomers were
investigated for their ability to form a gel (Table 4). From these
data it can be seen that mixtures of dex-(L)lactate and
dex-(D)lactate with a DP lower than 11 are mainly viscous, even
when a high degree of substitution (DS 17) or low water content
(70%) was used. On the other hand, for dex-lactate DP 11 or 12, an
increase in G' was observed after mixing the dex-(L)lactate and
dex-(D)lactate product, and a hydrogel was formed. Gel formation
was confirmed by a creep experiment, which showed almost elastic
behavior of the mixture. A gel was also obtained with
dex-(L)lactate DP 12 and DS 17.
TABLE-US-00004 TABLE 4 Rheology and PAS data of the mixtures of
monodisperse dex-(L)lactate and dex-(D)lactate. Rheology PAS DP DS
% water Creep Gelation Stereocomplex 8 17 70 V - - 8 17 80 V - - 10
6 70 V - +/- 10 6 80 V - - 10 15 70 V - + 10 15 80 V + - 11 17 80 E
+ ++ 12 8 80 E + ++ V = mainly viscous, E = mainly elastic
[0151] FTIR Transmission and Photoacoustic Spectrometry.
[0152] Infrared spectroscopy was applied to investigate the
possible stereocomplex formation in the monodisperse dex-lactate
gels.
[0153] It was shown by Vert, et al. (Kister, G.; Cassanas, G.;
Vert, M. Polymer 1995, 39, 267-273) that IR spectroscopy can be
used to distinguish between 103 and 31 helical conformations of the
crystalline homopolymer of poly (lactide) (PLA) and the PLA
stereocomplex, respectively. Since sharp IR-band shapes were
observed for the monodisperse products, they were used for
interpretation purposes. FTIR-transmission spectra of the free
monodisperse lactic acid oligomer (DP 8) and its corresponding
blend, which form stereocomplexes upon mixing, were recorded (FIG.
10). In the stereocomplex the most pronounced difference was the
disappearance of the absorption peak at 1270 cm.sup.-1
(.delta.CH.sub.3, vCOC, indicated by the dotted line).
[0154] After rheology measurement, the dex-(monodisperse) lactate
products were dried at ambient temperature. Next, IR spectra of
these dried products were recorded with the PA-detection method,
which turned out to be qualitatively comparable to
FTIR-transmission method. FIG. 11 shows that similar IR spectra
were obtained for the dex-(L)lactate with DP 8 and DP 11, as well
as for the dex- lactate mixture with DP 8, in which less or no
gelation occurred (Table 4). Interestingly, for the dex-lactate
mixture with DP 11 (rheology experiments demonstrated the formation
of a gel, Table 4) some clear IR-frequency shifts were observed in
the IR spectrum. The observed shift from 1270 to 1260 cm.sup.-1
(FIG. 11B) can likely be ascribed to the formation of
stereocomplexes in the dex-lactate mixture. In Table 4 the presence
of stereocomplexes, as detected by IR-PA, in different dex-lactate
mixtures is given. For systems with DP 11 and 12, gelation (based
on rheology) was clearly associated with the presence of
stereocomplexes.
[0155] The dex-lactate mixtures with DP 10 (DS6 and DS15) and a
water content of 70% showed the same IR-frequency shift (from 1270
to 1260 cm.sup.-1) as well, although for these mixtures the
rheology data indicated mainly viscous behavior. This indicates
that stereocomplexes are formed in the dex-lactate mixture with DP
10 and a water content of 70%. However, only a few physical
interactions (stereocomplexes) may be present, which are not
sufficient to form an completely elastic network. For the free
lactic acid oligomers a minimum DP of 7 is required for the
stereocomplexation. For dex-lactates longer lactic acid chains are
required to obtain the parallel orientation of at least 7 lactic
acid units of opposite chirality needed for the formation of
stereocomplexes.
Example 5
Protein Release from Dextran Gels Crosslinked by Stereocomplex
Formation.
[0156] Gels with an initial water content of 80% were prepared as
follows. Around 400 mg (accurately weighed) of dex-L-lactate
(DP.sub.av 9, DS 12) was added to 1.6 ml of an aqueous solution of
lysozyme or IgG (30 mg/ml in 100 mM phosphate buffer pH 7.2). The
lysozyme and IgG were used as model active ingredients. In another
vial, 400 mg of dex-D-lactate (DP.sub.av 9, DS 14) was added to 1.6
ml of an aqueous solution of lysozyme or IgG (30 mg/ml in 100 mM
phosphate buffer pH 7.2). The modified dextran were allowed to
solubilize for 48 hours at 4.degree. C. Thereafter 900 mg of both
solutions were mixed well and allowed to gelatinize in a plastic
tube (diameter 1.0 cm, length 2.0 cm) for 72 hours at 4.degree.
C.
[0157] Gels with an initial water content of 60% were prepared by
adjusting the amount of dex-lactate MEE added to the protein
solution. The gels were removed from the plastic tubes, weighed,
added to 20 ml of an aqueous solution of phosphate buffer (100 mM,
pH 7.2) and incubated at 37.degree. C. At regular time points,
sample of 2 ml were withdrawn and replaced by fresh buffer.
[0158] The protein concentration in the different samples was
determined by the BCA-assay (K. Smith, R. I. Krohn, G. T.
Hermanson, A. K. Mallia, F. H. Provenza, E. K. Fujimoto, N. M.
Goeke, B. J. Olson, D. C. Klenk Anal. Biochem., vol. 150, p.
76-85,1985) and used to calculate the cumulative amount of protein
released. FIGS. 12A and 12B show the results.
[0159] From these results it follows that the release is dependent
on the hydrodynamic size of the protein and the initial water
content of the gel.
Example 6
Synthesis of Lactate Oligomer with Terminal Free Carboxylic Acid
Groups
[0160] In the stereocomplex of poly(L-lactide) and poly(D-lactide),
a poly(L-lactide) segment and poly(D-lactide) segment are packed in
parallel fashion (Okihara, T.; Tsuji, M.; Kawaguchi, A.; Katayama,
K. I.; Tsuji, H.; Hyon, S. H.; Ikada, Y. J. Macromol. Sci. Phys.
1991, B30 (1&2), 119-140). It was studied whether stereocomplex
formation between of oligomers grafted to dextrans was enhance once
one of the oligomers is coupled via its terminal hydroxyl group to
dextran and the other via carboxylic acid. To this end one oligomer
was coupled via its terminal hydroxyl group to dextran and the
oligomer of opposite chirality via its carboxylic acid group.
[0161] A lactate acid oligomer with a terminal carboxylic acid
group was synthesized as follows. A mixture of L-lactide (5 g, 35
mmol) and benzyl alcohol (0.85 g, 7 8 mmol) was heated to
120.degree. C. Stannous octoate (tin(II) bis(2-ethylhexanoate),
SnOct.sub.2, 70 mg, 0.17 mmol) was added and the mixture was
stirred at 120.degree. C. for 2 hr. To protect the terminal
hydroxyl group, acetic anhydride (0.875 g, 8.6 mmol) was added and
the heating was continued for 3 hr. The formed acetic acid and the
excess of acetic anhydride was removed by applying vacuum before
cooling down the reaction mixture. A viscous clear paste was
obtained in a quantitative yield. .sup.1H NMR (CDCl.sub.3): .delta.
(ppm) 7.34 (m, 5H, ArH), 5.02-5.23 (m, 11H, CH--OC.dbd.O) and
ArCH.sub.2O), 2.12 (s, 3H, CH.sub.3.dbd.O), 1.47-1.62 (m, 27 H,
CH.sub.3 lactate).
[0162] The obtained benzyl-lactate.sub.9acetate was dissolved in 30
ml ethyl acetate. Nitrogen was bubbled through the solution and 30
mg of Pd/C was added. The mixture was connected a balloon filled
with hydrogen and stirred for 24 hr at room temperature. The
mixture was filtered over celite and the solvent was removed by
rotary evaporation. The last traces of ethyl acetate were removed
under high vacuum. A viscous clear liquid was obtained in
quantitative yield (.alpha.-carboxyl lactate.sub.9acetate) .sup.1H
NMR (CDCl.sub.3): .delta. (ppm) 7.34 (m, 5H, ArH), 5.02-5.23 (m,
11H, CH--OC.dbd.O) and ArCH.sub.2O), 2.12 (s, 3H, CH.sub.3.dbd.O),
1.47-1.68 (m, 27 H, CH.sub.3 lactate).
[0163] Synthesis of dextran-(lactate.sub.9) acetate. Glassware was
dried in an oven at 150.degree. C. for at least one hour. Dextran
was dried in a vacuum oven at 40.degree. C. A hot 10 ml flask was
loaded with 100 mg LiCl and evacuated to dry the salt. After
cooling to room temperature, 535 mg dry dextran 40.000 was added
and the flask was evacuated three times and filled with nitrogen.
The flask was then closed with a septum and 5 ml dry DMF was added
with a dry syringe. The mixture was heated to 100.degree. C. to
dissolve the dextran and then cooled to room temperature.
[0164] Another dry 10 ml flask was loaded with .alpha.-carboxyl
lactate.sub.9 acetate (235 mg, 0.33 mmol),
4(dimethylamino)-pyridinium-4-toluene sulfonate (16 mg, 0.05 mmol)
and dicyclohexyl carbodiimide (100 mg, 0.50 mmol). The flask was
evacuated three times and filled with nitrogen, while slightly
warming the flask with a hairdryer. The flask was then closed with
a septum and the dextran solution was transferred into the flask
with the help of a needle. The mixture was stirred at room
temperature for 24 h.
[0165] The reaction mixture was diluted with 20 ml water and
dialysed against water (5 L) at 4.degree. C. during one night. The
product was freeze dried, and then stirred with 50 ml
CH.sub.2Cl.sub.2 for one hour, filtered and dried in vacuum.
[0166] Yield: 82% of a white powder. The DS was 9.7 (NMR-analysis).
Products with other degree of substitutions were synthesized by
adjusting the ratio dextran/oligomer.
[0167] Rheological Evaluation.
[0168] For the rheological experiments two types of polymer
solutions were prepared: one contains dex-(L)lactate and the other
dex-(D)lactate. The dissolution time was at least one day at room
temperature. Acetate buffer pH 4 was selected as solvent to prevent
hydrolysis of the dex-lactate.
[0169] Equal amounts of the dex-(L)lactate and the dex-(D)lactate
solutions were mixed, homogenized and quickly applied on the
rheometer (AR 100 instrument of TA Instruments, Gent, Belgium). A
cone-plate measuring geometry (steel, 2 cm diameter with an angle
of 1 degree; gap 31 .mu.m) was used when only a small amount of
material was available. Gelation of the dex-lactate solutions took
place between the cone and plate of the measuring geometry. A
solvent trap was used to prevent evaporation of the solvent. In
addition, a thin layer of silicon oil (110 mPas) was applied to
surround the dex-lactate sample thereby preventing evaporation.
[0170] Gelation of the mixture of dex-lactate solutions was
monitored by measuring the shear storage modulus (G'), as well as
the loss modulus (G'') at 20.degree. C. for 18 hours. A frequency
of 1 Hz and a controlled strain of 1% were applied. The strain used
in these experiments was as low as possible to minimize the
influence of deformation on the formation of the dex-lactate
hydrogels. The rheological characteristics of two systems were
compared.
[0171] In system I both the L-lactic acid oligomer and the D-lactic
acid oligomer are coupled to dextran via their terminal hydroxyl
groups (dex-lactate-MEE) essentially as described in Examples 1-4.
In System II the L-lactic acid oligomer is coupled via its terminal
carboxylic group (dex-lactate-acetate) as described in the present
Example above. The D-oligomer was coupled via its terminal hydroxyl
group (dex-lactate-MEE) essentially as described in Examples 1-4.
Table 5 summarizes the results.
TABLE-US-00005 TABLE 5 L-Oligomer D-oligomer System, % H.sub.2O
graft* graft* G' (Pa)** tan .delta.** I, 70% L-lactate-MEE,
D-lactate-MEE, 5749 0.11 DS 3 DS 4 II, 70% L-lactate-Ac,
D-lactate-MEE, 7806 0.16 DS 4 DS 4 I, 80% L-lactate-MEE,
D-lactate-MEE, 5807 0.17 DS 12 DS 14 II, 80% L-lactate-Ac,
D-lactate-MEE, 17240 0.14 DS 10 DS 14 I, 70% L-lactate-MEE,
D-lactate-MEE, 20040 0.14 DS 12 DS 14 II, 70% L-lactate-Ac,
D-lactate-MEE, 30860 0.11 DS 10 DS 14 *DP.sub.av 9, **after 18
hours
[0172] Table 5 clearly shows that by coupling of oligomer via its
hydroxyl group and the other oligomer of opposite chirality via its
carboxylic acid group, stronger gels were obtained in comparison
with gels in which both oligomers were coupled via their hydroxyl
groups (compare G' values for system II with those of the
corresponding gels for system I).
[0173] It is therefore advantageous to couple one oligomer via its
terminal hydroxyl group to dextran and the oligomer of opposite
chirality via its carboxylic acid group.
* * * * *