U.S. patent application number 08/928049 was filed with the patent office on 2002-05-30 for degradable poly(ethylene glycol) hydrogels with controlled half-life and precursors therefor.
Invention is credited to HARRIS, J. MILTON.
Application Number | 20020064546 08/928049 |
Document ID | / |
Family ID | 25455637 |
Filed Date | 2002-05-30 |
United States Patent
Application |
20020064546 |
Kind Code |
A1 |
HARRIS, J. MILTON |
May 30, 2002 |
DEGRADABLE POLY(ETHYLENE GLYCOL) HYDROGELS WITH CONTROLLED
HALF-LIFE AND PRECURSORS THEREFOR
Abstract
This invention relates to hydrolytically degradable gels of
crosslinked poly(ethylene) glycol (PEG) structures. Addition of
water causes these crosslinked structures to swell and become
hydrogels. The hydrogels can be prepared by reacting two different
PEG derivatives containing functional moieties at the chain ends
that react with each other to form new covalent linkages between
polymer chains. The PEG derivatives are chosen to provide covalent
linkages within the crosslinked structure that are hydrolytically
degradable. Hydrolytic degradation can provide for dissolution of
the gel components and for controlled release of trapped molecules,
including drugs. Reagents other than PEG can be avoided. The
hydrolysis rates can be controlled by varying atoms adjacent to the
hydrolytically degradable functional groups to provide
substantially precise control for drug delivery in vivo.
Inventors: |
HARRIS, J. MILTON;
(HUNTSVILLE, AL) |
Correspondence
Address: |
BELL SELTZER INTELLECTUAL PROPERTY LAW
ALSTON & BIRD
POST OFFICE DRAWER 34009
CHRLOTTE
NC
28234
|
Family ID: |
25455637 |
Appl. No.: |
08/928049 |
Filed: |
September 12, 1997 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60026066 |
Sep 13, 1996 |
|
|
|
Current U.S.
Class: |
424/426 ;
424/428; 424/486; 424/78.3; 514/944; 525/408; 525/488 |
Current CPC
Class: |
A61K 47/34 20130101;
C08G 65/329 20130101; A61K 47/10 20130101 |
Class at
Publication: |
424/426 ;
424/428; 424/486; 424/78.3; 514/944; 525/488; 525/408 |
International
Class: |
A61K 031/765; A61K
031/77; A61K 031/785; A61K 031/795; A61K 031/80; C08G 065/32; C08L
067/00; C08L 071/02; C08L 077/00; A61F 002/00; A61K 009/14 |
Claims
What is claimed is:
1. A crosslinked polymeric structure comprising poly(ethylene
glycol) (PEG) polymers in the substantial absence of non-PEG
polymers and having linkages between said PEG polymers wherein at
least some of said linkages comprise hydrolytically unstable
linkages.
2. The crosslinked polymeric structure of claim 1 wherein said
hydrolytically unstable linkages are sufficient to cause said
crosslinked polymeric structure to degrade by hydrolysis in aqueous
solution.
3. The crosslinked polymeric structure of claim 1 wherein said
structure forms a PEG hydrogel in aqueous solution that is subject
to hydrolysis.
4. The crosslinked polymeric structure of claim 3 wherein the PEG
hydrogel formed therefrom has a rate of hydrolysis that is
determined at least in part by the structure of said linkages
between said PEG polymers.
5. The crosslinked polymeric structure of claim 4 wherein said
linkages comprise one or more methylene groups in proximity to said
hydrolytically unstable linkages sufficient to determine at least
in part said rate of hydrolysis of said hydrolytically unstable
linkages.
6. The crosslinked polymeric structure of claim 5 wherein said
hydrolysis rate is decreased as the number of said methylene groups
is increased.
7. The crosslinked polymeric structure of claim 1 wherein said
hydrolytically unstable linkages comprise linkages selected from
the group consisting of esters, imines, hydrazones, acetals,
orthoesters, peptides, and oligonucleotides.
8. The crosslinked polymeric structure of claim 7 wherein said
hydrolytically unstable ester linkages comprise linkages selected
from the group consisting of carboxylate esters and phosphate
esters.
9. The crosslinked polymeric structure of claim 8 wherein said
hydrolytically unstable carboxylate ester linkages are the reaction
product of a PEG alcohol and a PEG carboxylic acid and wherein said
hydrolytically unstable phosphate ester linkages are the reaction
product of a PEG alcohol and a PEG phosphate.
10. The crosslinked polymeric structure of claim 7 wherein said
imines are the reaction product of an amine and an aldehyde,
wherein said hydrazones are the reaction product of a hydrazide and
an aldehyde, wherein said acetals are the reaction product of an
aldehyde and an alcohol, wherein said orthoesters are the reaction
product of a formate and an alcohol, wherein said hydrolytically
unstable peptide linkages comprise linkages selected from the group
consisting of peptide linkages that are the reaction product of
amines and PEG-peptide conjugates terminated with carboxyl and
peptide linkages that are the reaction product of a carboxylic acid
and PEG-peptide conjugates terminated with amine, and wherein said
hydrolytically unstable oligonucleotide linkages are the reaction
product of a phosphoramidite with a 5'-hydroxyl-terminated PEG
oligonucleotide.
11. The crosslinked polymeric structure of claim 1 wherein said
structure also comprises hydrolytically stable linkages that do not
degrade in aqueous solution.
12. The crosslinked polymeric structure of claim 11 wherein said
hydrolytically stable linkages comprise linkages selected from the
group consisting of amides, urethanes, ureas, amines, and
sulfonamides.
13. The crosslinked polymeric structure of claim 12 wherein said
amide linkages are the reaction product of an ester and an amine,
wherein said urethane linkages are the reaction product of an
isocyanate and an alcohol, wherein said urea linkages are the
reaction product of an isocyanate and an amine, wherein said
hydrolytically stable amine linkages are selected from the group
consisting of the reaction product of an aldehyde and an amine in
the presence of a reducing agent and the reaction product of an
epoxide and an amine, and wherein said sulfonamide linkages are the
reaction product of an amine and a sulfonate ester.
14. The crosslinked polymeric structure of claim 13 wherein said
amide linkages are the reaction product of a carboxylate ester and
an amine.
15. A drug delivery system comprising a poly(ethylene glycol)
hydrogel made from the crosslinked polymeric structure of claim
1.
16. A poly(ethylene glycol) (PEG) hydrogel comprising PEG polymers
in the substantial absence of non-PEG polymers and having linkages
between said PEG polymers wherein at least some of said linkages
are hydrolyzable under hydrolysis conditions, said hydrolyzable
linkages comprising linkages selected from the group consisting of
esters, imines, hydrazones, acetals, orthoesters, peptides, and
oligonucleotides.
17. A drug delivery system comprising the PEG hydrogel of claim
15.
18. A crosslinked polymeric structure comprising poly(ethylene
glycol) (PEG) and having a formula selected from the group
consisting of: {R[CH.sub.2--O-PEG-W-PEG-W-].sub.p}.sub.m
{R[CH.sub.2--O-PEG-X-PEG-W-PEG-- X-].sub.p}.sub.m
{R[CH.sub.2--O-PEG-X-R'-W-PEG-W-R'-X-].sub.p}.sub.m wherein m means
"matrix" and indicates that the crosslinked structure is a solid
aggregate; p is from about 3 to 10 and indicates the number of arms
on the polymers forming said crosslinked structure; R is a central
branching moiety suitable for making multiarmed PEGs; R' is a
hydrocarbon fragment having from about 1 to 10 carbons; W is a
hydrolytically unstable linkage comprising linkages selected from
the group consisting of esters, imines, hydrazones, acetals,
orthoesters, peptides, and oligonucleotides; and X is a
hydrolytically stable linkage comprising linkages selected from the
group consisting of amides, urethanes, ureas, amines, and
sulfonamides.
19. The crosslinked polymeric structure of claim 18 wherein R is a
moiety selected from the group consisting of glycerol, glycerol
oligomers, pentaerythritol, sorbitol, trimethyolpropane, and
di(trimethylolpropane).
20. The crosslinked polymeric structure of claim 18 wherein said
hydrolytically unstable linkages W comprise carboxylate ester
linkages that are the reaction product of an alcohol and a
carboxylic acid; phosphate ester linkages that are the reaction
product of an alcohol and a phosphate, imine linkages that are the
reaction product of an amine and an aldehyde; hydrazones linkages
that are the reaction product of a hydrazide and an aldehyde;
acetal linkages that are the reaction product of an aldehyde and an
alcohol; orthoester linkages that are the reaction product of a
formate and an alcohol; peptide linkages that comprise linkages
selected from the group consisting of peptide linkages that are the
reaction product of amines and PEG-peptide conjugates terminated
with carboxyl and peptide linkages that are the reaction product of
a carboxylic acid and PEG-peptide conjugates terminated with amine;
and oligonucleotide linkages that are the reaction product of a
phosphoramidite with a 5'-hydroxyl-terminated PEG
oligonucleotide.
21. The crosslinked polymeric structure of claim 18 wherein said
hydrolytically stable linkages X comprise amide linkages that are
the reaction product of an ester and an amine; urethane linkages
that are the reaction product of an isocyanate and an alcohol; urea
linkages that are the reaction product of an isocyanate and an
amine; amine linkages that are selected from the group consisting
of the reaction product of an aldehyde and an amine in the presence
of a reducing agent and the reaction product of an epoxide and an
amine; and sulfonamide linkages that are the reaction product of an
amine and a sulfonate ester.
22. The crosslinked polymeric structure of claim 21 wherein said
amide linkages are the reaction product of a carboxylate ester and
an amine.
23. A drug delivery system comprising a poly(ethylene glycol)
hydrogel made from the crosslinked polymeric structure of claim
18.
24. A crosslinked polymeric structure comprising poly(ethylene
glycol) (PEG) and having the formula:
{R[CH.sub.2--O-PEG-O.sub.2C--(CH.sub.2).sub-
.n--O-PEG-O(CH.sub.2).sub.n--CO.sub.2--].sub.p}.sub.m wherein m
means "matrix" and indicates that the crosslinked structure is a
solid aggregate; p is from about 3 to 10 and indicates the number
of arms on the polymers forming said crosslinked structure; R is a
moiety selected from the group consisting of glycerol, glycerol
oligomers, pentaerythritol, sorbitol, trimethyolpropane, and
di(trimethylolpropane); and wherein n is from about 1 to 10.
25. A crosslinked polymeric structure comprising poly(ethylene
glycol) (PEG) and having the formula:
{CH.sub.3C[CH.sub.2--O-PEG-O.sub.2C--(CH.su-
b.2).sub.n--O-PEG-O(CH.sub.2).sub.n--CO.sub.2--].sub.p}.sub.m
wherein m means "matrix" and indicates that the crosslinked
structure is a solid aggregate, and wherein n is from about 1 to
10.
26. The crosslinked polymeric structure of claim 25 wherein when n
equals 2, then the ester linkages have a hydrolysis half life of
about 4 days at pH 7 and 37 degrees Centrigrade, and wherein when n
equals 3, then the ester linkages have a hydrolysis half life of
about 43 days at pH 7 and 37 degrees Centrigrade.
27. A method of making a crosslinked polymeric structure comprising
poly(ethylene glycol) (PEG) polymers in the substantial absence of
non-PEG polymers and having linkages between said PEG polymers
wherein at least some of said linkages comprise hydrolytically
unstable linkages, said method comprising reacting a linear
poly(ethylene glycol) (PEG) with a branched PEG to provide a
crosslinked structure having linkages between said PEG polymers
wherein at least some of said linkages comprise hydrolyzable
linkages.
28. The method of claim 27 wherein the step of reacting a linear
PEG with a branched PEG includes the steps of separately injecting
the linear PEG and the branched PEG into a living organism or into
a substance taken from a living organism in close proximity in time
and space and reacting the linear and branched PEGs in vivo to form
a hydrogel.
29. A method for delivering biologically active substances to a
living organism or to a substance taken from a living organism
comprising mixing at least one biologically active substance with a
linear PEG or a branched PEG as set forth in claim 28, separately
injecting the linear PEG and the branched PEG into a living
organism or into a substance taken from a living organism in close
proximity in time and space, reacting the linear and branched PEGs
in vivo to form a degradable hydrogel matrix in which the
biologically active substance is trapped, and subjecting the
hydrogel to hydrolysis to degrade the hydrogel and allow the
biologically active substances to be delivered.
30. A method for making a crosslinked polymeric structure
comprising reacting. a linear poly(ethylene glycol) (PEG) polymer
of the formula Z-PEG-Z with a branched PEG polymer of the formula
R(CH.sub.2--O-PEG-Y).sub.p to provide a crosslinked structure of
the formula {R[CH.sub.2--O-PEG-W-PEG-].sub.p}.sub.m, wherein m
means "matrix" and indicates that the crosslinked structure is a
solid aggreagte; p is from about 3 to 10 and indicates the number
of arms on the polymers forming said crosslinked structure; R is a
central branching moiety suitable for making multiarmed PEGs, and
wherein Z reacts with Y to form the hydrolytically unstable group
W, and Z and Y are selected from the group consisting of alcohols,
carboxylic acids, amines, aldehydes, hydrazides, aldehydes,
phosphate, formate, PEG-peptide terminated with carboxyl,
PEG-peptide terminated with amine, PEG phosphoramidite, and
5'-hydroxyl-terminated PEG oligonucleotide, and wherein W is
selected from the group consisting of esters, imines, hydrazones,
acetals, orthoesters, peptides, and oligonucleotides.
31. A method for making a crosslinked polymeric structure
comprising reacting a linear poly(ethylene glycol) (PEG) with a
branched PEG polymer according to the following equation:
U-PEG-W-PEG-U+R(CH.sub.2--O-PEG-V).s-
ub.p.fwdarw..fwdarw.{[CH.sub.2--O-PEG-X-PEG-W-PEG-X-].sub.p}.sub.m
wherein W is selected from the group consisting of esters, imines,
hydrazones, acetals, orthoesters, peptides, and oligonucleotides;
wherein U reacts with V to form X, and U and V are selected from
the group consisting of active esters, amine, isocyanate, aldehyde,
epoxide, and sulfonate ester; wherein X is selected from the group
consisting of amides, urethanes, ureas, amines, and sulfonamides;
and wherein m means "matrix" and indicates that the crosslinked
structure is a solid aggreagte; p is from about 3 to 10 and
indicates the number of arms on the polymers forming said
crosslinked structure; and R is a central branching moiety suitable
for making multiarmed PEGs.
32. A method for making a crosslinked polymeric structure
comprising reacting a linear poly(ethylene glycol) (PEG) with a
branched PEG polymer according to the following equation:
U-R'-W-PEG-W-R'-U+R(CH.sub.2--O-PEG--
V).sub.p.fwdarw.{R[CH.sub.2--O-PEG-X-R'-W-PEG-W-R'-X].sub.p}.sub.m
wherein R' is a hydrocarbon fragment having from about 1 to 10
carbons; wherein W is selected from the group consisting of esters,
imines, hydrazones, acetals, orthoesters, peptides, and
oligonucleotides; wherein U reacts with V to form X, and U and V
are selected from the group consisting of active esters, amine,
isocyanate, aldehyde, epoxide, and sulfonate ester; wherein X is
selected from the group consisting of amides, urethanes, ureas,
amines, and sulfonamides; and wherein m means "matrix" and
indicates that the crosslinked structure is a solid aggreagte; p is
from about 3 to 10 and indicates the number of arms on the polymers
forming said crosslinked structure; and R is a central branching
moiety suitable for making multiarmed PEGs.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is related to commonly owned copending
Provisional Application Ser. No. 60/026,066, filed Sep. 13, 1996,
and claims the benefit of its earlier filing date under 35 U.S.C.
119(e).
FIELD OF THE INVENTION
[0002] This invention relates to poly(ethylene glycol) hydrogels,
precursors therefor, methods for making the precursors and
hydrogels, and the use of the precursors and hydrogels.
BACKGROUND OF THE INVENTION
[0003] In its most common form, poly(ethylene glycol) (PEG) is a
linear polymer terminated at each end with hydroxyl groups:
HO--CH.sub.2CH.sub.2O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--OH
[0004] This polymer can be represented in brief form as HO-PEG-OH
where it is understood that -PEG- represents the following
structural unit:
--CH.sub.2CH.sub.2O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--
[0005] n typically ranges from approximately 10 to 2000.
[0006] PEG is of great utility in biotechnology and is useful in a
variety of applications for drug delivery and modification of
surfaces to promote nonfouling characteristics, including as
hydrogels and for covalent attachment to various drugs and
surfaces. PEG is not toxic, does not tend to promote an immune
response, and is soluble in water and in many organic solvents.
[0007] The PEG polymer can be covalently attached to insoluble
molecules to make the resulting PEG-molecule conjugate soluble. For
example, Greenwald, Pendri and Bolikal in J. Org. Chem., 60,
331-336 (1995) recite that the water-insoluble drug taxol, when
coupled to PEG, becomes water soluble.
[0008] Davis et al. in U.S. Pat. No. 4,179,337 recite that proteins
coupled to PEG have an enhanced blood circulation lifetime because
of a reduced rate of kidney clearance and reduced immunogenicity.
The lack of toxicity of the polymer and its rate of clearance from
the body are important considerations in pharmaceutical
applications. Pharmaceutical applications and many leading
references are described in the book by Harris (J. M. Harris, Ed.,
"Biomedical and Biotechnical Applications of Polyethylene Glycol
Chemistry," Plenum, New York, 1992).
[0009] PEG is commonly used as methoxy-PEG-OH, or mPEG in brief, in
which one terminus is the relatively inert methoxy group, while the
other terminus is an hydroxyl group that is subject to ready
chemical modification.
CH.sub.3O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--OH mPEG
[0010] PEG is also commonly used in branched forms that can be
prepared by addition of ethylene oxide to various polyols,
including glycerol, pentaerythritol and sorbitol. For example, the
four-armed branched PEG prepared from pentaerythritol is shown
below:
C(CH.sub.2--OH).sub.4+nC.sub.2H.sub.4O.fwdarw.C[CH.sub.2O--(CH.sub.2CH.sub-
.2O).sub.n--CH.sub.2CH.sub.2--OH].sub.4
[0011] The branched PEGs can be represented in general form as
R(-PEG-OH).sub.n, in which R represents the central core molecule,
which can include glycerol or pentaerythritol, and n represents the
number of arms.
[0012] It is necessary to use an "activated derivative" of PEG to
couple PEG to a molecule. The hydroxyl group located at the PEG
terminus or other group subject to ready chemical modification is
activated by modifying or replacing the group with a functional
group suitable for reacting with a group on another molecule,
including proteins, surfaces, enzymes, and others. For example, the
succinimidyl "active ester" of carboxymethylated PEG forms covalent
bonds with amino groups on proteins as described by K. Iwasaki and
Y. Iwashita in U.S. Pat. No. 4,670,417.
[0013] The synthesis described in U.S. Pat. No. 4,670,417 is
illustrated below with the active ester reacting with amino groups
of a protein in which the succinimidyl group is represented as NHS
and the protein is represented as PRO-NH.sub.2:
PEG-O--CH.sub.2--CO.sub.2--NHS+PRO-NH.sub.2.fwdarw.PEG-O--CH.sub.2--CO.sub-
.2--NH-PRO
[0014] Succinimidyl "active esters", such as
PEG-O--CH.sub.2--CO.sub.2--NH- S, are commonly used forms of
activated carboxylic acid PEGs, and they are prepared by reacting
carboxylic acid PEGs with N-hydroxylsuccinimide.
[0015] Problems have arisen in the art. Some of the functional
groups that have been used to activate PEG can result in toxic or
otherwise undesirable residues when used for in vivo drug delivery.
Some of the linkages that have been devised to attach functional
groups to PEG can result in an undesirable immune response. Some of
the functional groups do not have sufficient or otherwise
appropriate selectivity for reacting with particular groups on
proteins and can tend to deactivate the proteins.
[0016] PEG hydrogels, which are water-swollen gels, have been used
for wound covering and drug delivery. PEG hydrogels are prepared by
incorporating the soluble, hydrophilic polymer into a chemically
crosslinked network or matrix so that addition of water produces an
insoluble, swollen gel. Substances useful as drugs typically are
not covalently attached to the PEG hydrogel for in vivo delivery.
Instead, the substances are trapped within the crosslinked matrix
and pass through the interstices in the matrix. The insoluble
matrix can remain in the body indefinitely and control of the
release of the drug typically can be somewhat imprecise.
[0017] One approach to preparation of these hydrogels is described
by Embrey and Grant in U.S. Pat. No. 4,894,238. The ends of the
linear polymer are connected by various strong, nondegradable
chemical linkages. For example, linear PEG is incorporated into a
crosslinked network by reacting with a triol and a diisocyanate to
form hydrolytically stable urethane linkages that are nondegradable
in water.
[0018] A related approach for preparation of PEG hydrogels has been
described by Gayet and Fortier in J. Controlled Release, 38,
177-184 (1996) in which linear PEG was activated as the
p-nitrophenylcarbonate and crosslinked by reaction with a protein,
bovine serum albumin. The linkages formed are hydrolytically stable
urethane groups and the hydrogels are nondegradable in water.
[0019] In another approach, described by N. S. Chu in U.S. Pat. No.
3,963,805, nondegradable PEG networks have been prepared by random
entanglement of PEG chains with other polymers formed by use of
free radical initiators mixed with multifunctional monomers. P. A.
King described nondegradable PEG hydrogels in U.S. Pat. No.
3,149,006 that have been prepared by radiation-induced crosslinking
of high molecular weight PEG.
[0020] Nagaoka et al. in U.S. Pat. No. 4,424,311 have prepared PEG
hydrogels by copolymerization of PEG methacrylate with other
comonomers such as methyl methacrylate. Substantial non-PEG
polymeric elements are introduced by this method. Vinyl
polymerization produces a polyethylene backbone with PEG attached.
The methyl methacrylate comonomer is added to give the gel
additional physical strength.
[0021] Sawhney, Pathak and Hubbell in Macromolecules, 26, 581
(1993) describe the preparation of block copolymers of
polyglycolide or polylactide and PEG that are terminated with
acrylate groups, as shown below.
CH.sub.2.dbd.CH--CO--(O--CH.sub.2--CO).sub.n--O-PEG-O--(CO--CH.sub.2--O).s-
ub.n--CO--CH.dbd.CH.sub.2
[0022] In the above formula, the glycolide blocks are the
--O--CH.sub.2--CO-- units; addition of a methyl group to the
methylene gives a lactide block; n can be multiples of 2. Vinyl
polymerization of the acrylate groups produces an insoluble,
crosslinked gel with a polyethylene backbone.
[0023] Substantial non-PEG elements are introduced into the
hydrogel. The polylactide or polyglycolide segments of the polymer
backbone shown above, which are ester groups, are susceptible to
slow hydrolytic breakdown, with the result that the crosslinked gel
undergoes slow degradation and dissolution.
[0024] Non-PEG elements tend to introduce complexity into the
hydrogel and degradation and dissolution of the matrix can result
in undesirable or toxic components being released into the blood
stream when the hydrogels are used in vivo for drug delivery.
[0025] It would be desirable to provide alternative PEG hydrogels
that are suitable for drug delivery and that have unique properties
that could enhance drug delivery systems.
SUMMARY OF THE INVENTION
[0026] The invention provides chemically crosslinked degradable PEG
hydrogels capable of controlled degradability and methods for
making these PEG hydrogels in the absence of substantial non-PEG
elements. Weak chemical linkages are introduced into the hydrogel
that provide for hydrolytic breakdown of the crosslinks and release
of drug molecules that can be trapped within the matrix. The gels
break down to substantially nontoxic PEG fragments that typically
are cleared from the body. Variation of the atoms near the
hydrolytically unstable linkages can provide precise control of
hydrolytic breakdown rate and drug release.
[0027] Examples of hydrolytically unstable linkages include
carboxylate ester, phosphate ester, acetals, imines, orthoesters,
peptides and oligonucleotides. These weak links are formed by
reaction of two PEGs having different terminal groups as
illustrated below:
-PEG-Z+Y-PEG-.fwdarw.-PEG-W-PEG-
[0028] In the above illustration, -W- represents the hydrolytically
unstable weak link. Z- and Y- represent groups located at the
terminus of the PEG molecule that are capable of reacting with each
other to form weak links -W-.
[0029] For example, the following pairs of Z and Y groups can be
used to form some of the W groups described above:
1 -PEG-CO.sub.2H + HO-PEG- .fwdarw. -PEG-CO.sub.2-PEG- ester
-PEG-OPO.sub.3H.sub.2 + HO-PEG .fwdarw. -PEG-OPO.sub.3(H)-PEG-
phosphate ester -PEG-CHO + (HO-PEG).sub.2- .fwdarw.
-PEG-CH(O-PEG).sub.2- acetal -PEG-CHO + NH.sub.2-PEG- .fwdarw.
-PEG-CH.dbd.N-PEG- imine
[0030] The PEG hydrogels of the invention can be made by either a
two-step or a one-step method. In the one-step approach, two
different PEGs with the appropriate terminal groups are reacted in
a single step. A specific example of the one-step approach
according to the invention is shown in the following equation for
coupling of linear PEG acids with a three-armed PEG terminated with
hydroxyl groups. Weak ester linkages are formed.
HO.sub.2C--(CH.sub.2).sub.n--O-PEG-O--(CH.sub.2).sub.n--CO.sub.2H+CH.sub.3-
C(CH.sub.2--O-PEG-OH).sub.3.fwdarw.{CH.sub.3C[CH.sub.2--O-PEG-O.sub.2C--(C-
H.sub.2).sub.n--O-PEG-O(CH.sub.2).sub.n--Co.sub.2--].sub.3}.sub.m--H.sub.2-
O
[0031] The degree of polymerization is given by m, which refers to
"matrix" and is intended to indicate that a crosslinked polymer has
been formed as a solid aggregate. It should be understood that the
degree of polymerization by the formation of crosslinks is large
and indeterminate. The PEG hydrogel that is formed is a visible and
solid aggregate that swells in water in which, in theory, all
available crosslinks are formed. However, it is not usually
possible to determine the degree of crosslinking that has
occurred.
[0032] The rate of release of drug molecules trapped within the
matrix is controlled by controlling the hydrolytic breakdown rate
of the gel. The hydrolytic breakdown rate of the gel can be
adjusted by controlling the degree of bonding of the PEGs that form
the hydrogel matrix. A multiarmed PEG having 10 branches or arms
will break down and release drug molecules more slowly than a 3
armed PEG.
[0033] Substantially precise control of hydrolytic breakdown rate
and drug release can be provided by varying the atoms near the
hydrolytically unstable linkages. Typically, increasing the n value
(the number of methylene groups) in the above structure decreases
the hydrolysis rate of esters and increases the time required for
the gel to degrade. If n in the above example is 1, then the ester
linkages of the gel will hydrolyze with a half life of about 4 days
at pH 7 and 37.degree. C. If n is 2, then the half life of
hydrolytic degradation of the ester linkages is about 43 days at pH
7 and 37.degree. C.
[0034] Phosphate esters, acetals, imines, and other hydrolytically
unstable linkages can be similarly formed and the hydrolysis rate
can be similarly controlled by controlling the number of methylene
groups adjacent the hydrolytically unstable linkage and by
controlling the degree of branching of the PEG.
[0035] The degradable hydrogels of this invention can also be made
by a two-step process. In the first step, soluble, uncrosslinked
PEGs are prepared that have hydrolytically unstable linkages in
their backbones. In the second step, these PEGs with hydrolytically
unstable linkages in their backbones are coupled together with
other PEGs by hydrolytically stable linkages. For example, the
following PEG has two hydrolytically unstable ester linkages in its
backbone:
NHS--O.sub.2C--CH.sub.2--O-PEG-O--CH.sub.2--CO.sub.2-PEG-O.sub.2C--CH.sub.-
2--O-PEG-O--CH.sub.2--CO.sub.2--NHS
[0036] The above PEG is activated at each terminus with an
N-hydroxylsuccinimide moiety (NHS) in which the active succinimidyl
ester moiety is NHS--CO.sub.2-- and is reactive with amino groups.
When this PEG is coupled with a multiarmed PEG amine, a crosslinked
network is produced that is held together by stable amide linkages
that are formed from the reaction of the active esters with amine
and by the hydrolytically unstable ester linkages already present
in the backbone. As in the previous example, the degradation rate
of the gel is controlled by varying the number of methylene groups
adjacent to the ester linkage.
[0037] The two-step method described above for making the PEG
hydrogels can be used to form the gel and to trap substances in
situ, in living tissue, for injectable drug systems. A drug can be
combined with one reactive PEG component of the hydrogel and
injected along with another reactive PEG component that will form
the gel. The drug is trapped within the matrix that is formed
because of its proximity to the reactive system.
[0038] Thus, the invention provides, among other things, degradable
PEG hydrogels having hydrolytically unstable linkages in which the
rate of hydrolysis of the unstable linkages can be controlled. The
PEG hydrogels of the invention can physically trap drugs, including
proteins, enzymes, and a variety of other substances, in the
absence of covalent linkages, for precisely controlled release in
vivo. The degraded gel can be more readily cleared from the body
than can gels that do not significantly degrade.
[0039] The foregoing and other objects, advantages, and features of
the invention, and the manner in which the same are accomplished,
will be more readily apparent upon consideration of the following
detailed description of the invention taken in conjunction with the
accompanying drawing, which illustrates an exemplary
embodiment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 is a schematic representation of a PEG hydrogel in
which the PEGs have three branches or arms.
DETAILED DESCRIPTION
[0041] FIG. 1 illustrates a poly(ethylene glycol) (PEG) matrix held
together by hydrolytically unstable or weak linkages W. The PEGs
shown in FIG. 1 have three branches or arms. The degree of
branching can be varied in the hydrogels of the invention to
control the physical strength and compressibility of the gels; in
general the greater the degree of branching and the shorter the
branches, the greater the strength (resistance to compression or
stretching) of the gels. Similarly, greater degrees of branching
and shorter branches also give smaller pores and lower water
content.
[0042] Degradable PEG hydrogels having hydrolytically unstable PEGs
can be prepared in one step, as shown in the following general
equation:
Z-PEG-Z+R(CH.sub.2--O-PEG-Y).sub.p.fwdarw.{R[CH.sub.2--O-PEG-W-PEG-W-].sub-
.p}.sub.m
[0043] where m means "matrix" and indicates a degree of
polymerization such that a crosslinked polymer, which is a solid
aggregate is formed. m is large and indeterminate. p is 3 to 10 and
refers to the degree of branching, which is the number of arms, of
the reactant branched PEG, R(CH.sub.2--O-PEG-Y).sub.p. The rate of
hydrolysis of the PEG gel typically is lengthened by increasing p.
R is a central branching moiety suitable for making multiarmed PEGs
and includes moieties selected from the group consisting of
glycerol, glycerol oligomers, pentaerythritol, sorbitol,
trimethyolpropane, and di(trimethylolpropane). Z and Y are groups
that react to form hydrolytically unstable linkages W. Examples of
pairs of the groups Z and Y that can be reacted to form
hydrolytically unstable linkages W include pairs selected from the
group consisting of alcohol and carboxylic acid reacting to form
carboxylate esters, amine and aldehyde reacting to form imines,
hydrazide and aldehyde reacting to form hydrazones, alcohol and
phosphate reacting to form phosphate ester, aldehyde and alcohol
reacting to form acetals, alcohols and formate reacting to form
orthoesters, peptides formed by reaction of PEG amine with
PEG-peptide terminated with carboxyl to form a new peptide linkage,
peptides formed by reaction of PEG carboxylic acid with PEG-peptide
terminated with amine to form a new peptide linkage, and
oligonucleotides formed by reaction of PEG phosphoramidite with an
5'-hydroxyl-terminated PEG oligonucleotide.
[0044] It should be noted that the Z groups are shown on a linear
PEG and the Y groups are shown on a branched PEG. However, the
reaction will proceed and the gel will be formed with the Y groups
on the linear PEG and the Z groups on the branched PEG to form the
same weak linkages W.
[0045] A specific example of the one-step method for making a PEG
hydrogel having hydrolytically unstable carboxylate ester linkages
W formed by the reaction of PEG carboxylic acid and PEG hydroxyl
groups Z and Y, respectively, is shown by the following
equation:
HO.sub.2C--(CH.sub.2).sub.n--O-PEG-O--(CH.sub.2).sub.n--CO.sub.2H+R(CH.sub-
.2--O-PEG-OH).sub.p.fwdarw..fwdarw.{R[CH.sub.2--O-PEG-O.sub.2C--(CH.sub.2)-
.sub.n--O-PEG-O (CH.sub.2).sub.n--CO.sub.2--].sub.p}.sub.m
[0046] In the above equation, m, p, and R are as characterized
above. n is from about 1 to 10, and can be varied to control the
rate of hydrolysis of the gel. Increasing n typically decreases the
rate of hydrolysis.
[0047] Note that in this example the hydroxyl group is on the
branched PEG while the carboxylic acid groups are on the linear
PEG. Alternatively, the hydroxyl group could be on the linear PEG
while the carboxylic acid could be on the branched PEG.
[0048] Degradable PEG hydrogels can also be prepared in two steps.
In the first step a linear PEG is prepared having one or more
hydrolytically unstable linkages W in its backbone. The linear PEG
has the general formula U-PEG-W-PEG-U, in which U represents a
reactive terminal moiety and W is the hydrolytically unstable
linkage.
[0049] In the second step the PEG with the hydrolytically unstable
linkages in its backbone is reacted with a second PEG. The second
PEG is a branched PEG, as shown in the general formula
R(CH.sub.2--O-PEG-V).sub.- p, in which V represents a reactive
terminal moiety. P is 3 to 10 and refers to the degree of
branching, which is the number of arms, of the reactant branched
PEG, R(CH.sub.2--O-PEG-V).sub.p. The rate of hydrolysis of the PEG
gel typically is lengthened by increasing p. R is a central
branching moiety suitable for making multiarmed PEGs and includes
moieties selected from the group consisting of glycerol, glycerol
oligomers, pentaerythritol, sorbitol, trimethyolpropane, and
di(trimethylolpropane).
[0050] The functional groups U and V at the ends of the PEG polymer
chains in the first and second PEGs, respectively, react to form
hydrolytically stable crosslinks X, as shown by the following
equation.
U-PEG-W-PEG-U+R(CH.sub.2--O-PEG-V).sub.p.fwdarw..fwdarw.{R[CH.sub.2--O-PEG-
-X-PEG-W-PEG-X-].sub.p}.sub.m
[0051] Again, m means "matrix" and indicates a degree of
polymerization such that a crosslinked polymer, which is a solid
aggregate is formed. W is a hydrolytically unstable group including
carboxylate esters, imines, phosphate esters, acetals, orthoesters,
peptides, and oligonucleotides. U and V are groups reactive toward
each other, including active esters, which includes carbonate
esters, reacting with amines, isocyanates reacting with alcohols,
isocyanates reacting with amines, aldehydes reacting with amines
and a reducing agent, epoxide reacting with amines, and sulfonate
esters reacting with amines.
[0052] The hydrolytically stable linkages X that are formed by the
reaction of U and V include amide from the reaction of active
esters with amine, urethane from the reaction of isocyanate with
alcohol, urea from the reaction of isocyanate with amine, amine
from the reaction of aldehyde with amine and reducing agent, amine
from the reaction of epoxide with amine, and sulfonamide from the
reaction of sulfonate ester with amine.
[0053] A specific example of the two-step method is the preparation
of degradable PEG hydrogels having hydrolytically unstable
carboxylate ester linkages W and hydrolytically stable amide
linkages X that are formed by the reaction of active esters U and
amines V as shown in the following equation.
NHS--O.sub.2C--(CH.sub.2).sub.n--O-PEG-W-PEG-O--(CH.sub.2).sub.n--CO.sub.2-
--NHS+R(CH.sub.2--O-PEG-NH.sub.2).sub.p.fwdarw.{R[CH.sub.2--O-PEG-NHCO--(C-
H.sub.2).sub.n--O-PEG-W-PEG-O--(CH.sub.2).sub.n--CONH--].sub.p}.sub.m
[0054] The symbols n, m, p, and R are as previously described. W is
a hydrolytically unstable ester linkage according to the formula
--O--(CH.sub.2).sub.r--CO.sub.2-- in which r is from about 1 to
10.
[0055] The amino group V is on the branched PEG while the active
esters U are on the linear PEG. It should be recognized that the
two groups could be exchanged so that the amino group is presented
on the linear PEG while the active ester is presented on the
branched PEG.
[0056] In a second two-step method, a reactant linear PEG is
prepared in a first step having hydrolytically unstable linkages W
near the polymer chain terminal groups U-R'. In a second step the
PEG having hydrolytically unstable linkages W near the polymer
chain terminal groups is reacted with a branched PEG having a
reactive moiety V to form hydrolytically stable crosslinks X.
U-R'-W-PEG-W-R'-U+R(CH.sub.2--O-PEG-V).sub.p.fwdarw.{R[CH.sub.2--O-PEG-X-R-
'-W-PEG-W-R'-X].sub.p}.sub.m
[0057] The symbols m, p, and R are as previously defined. R' is a
small hydrocarbon fragment having from about 1 to 10 carbons. W is
a hydrolytically unstable group including carboxylate esters,
imines, phosphate esters, acetals, orthoesters, peptides, and
oligonucleotides, as previously defined. U and V are groups
reactive toward each other, including active esters, which includes
carbonate esters, reacting with amines, isocyanates reacting with
alcohols, isocyanates reacting with amines, aldehydes reacting with
amines and a reducing agent, epoxides reacting with amines, and
sulfonate esters reacting with amines.
[0058] The hydrolytically stable linkage formed by reaction of U
and V is X. X includes amide from the reaction of active ester with
amine, urethane from the reaction of carbonate ester with amine,
urethane from the reaction of isocyanate with alcohol, urea from
the reaction of isocyanate with amine, amine from the reaction of
aldehyde with amine and reducing agent, amine from the reaction of
epoxide with amine, and sulfonamide from the reaction of sulfonate
ester with amine.
[0059] A specific example, which is shown in the following
equation, is the formation of PEG hydrogels containing
hydrolytically unstable carboxylate ester groups W and
hydrolytically stable amides X formed by the reaction of active
esters U and amines V, and in which the hydrolytically unstable
carboxylate ester groups W have been separated from the U and or V
groups by a small hydrocarbon fragment in the precursor linear
PEG.
NHS--O.sub.2C--(CH.sub.2).sub.i--O.sub.2C--(CH.sub.2).sub.n--O-PEG-O--(CH.-
sub.2).sub.n--CO.sub.2--(CH.sub.2).sub.i--CO.sub.2--NHS+R(CH.sub.2--O-PEG--
NH.sub.2).sub.p.fwdarw.{R
[CH.sub.2--O-PEG-NHCO--(CH.sub.2).sub.i--O.sub.2-
C--(CH.sub.2).sub.n--O-PEG-O--(CH.sub.2)--CO.sub.2--(CH.sub.2).sub.n--CONH-
--].sub.p}.sub.m
[0060] In the above equation, i is from about 1 to 10 and defines
the length of the small hydrocarbon fragment R'. The symbols n, m,
p and R are as previously defined. An amino group is shown on the
branched PEG while the active esters are shown on the linear PEG.
It should be recognized that the two groups could be exchanged so
that the amino group is on the linear PEG and the active ester is
on the branched PEG.
[0061] The skilled artisan should recognize that when reference is
made to a Z moiety reacting with a Y moiety or to a U moiety
reacting with a V moiety, that additional reagents or steps may be
employed according to commonly accepted chemical procedures and
standards to achieve the desired linkage W or X as the case may be.
There are many possible routes, too numerous to mention here, that
could be taken and that should be readily apparent to the skilled
artisan. For example, one of skill in the art can be expected to
understand that when an alcohol and a carboxylic acid are reacted,
the acid typically is converted to another form, the acid chloride,
prior to reaction with alcohol. Several examples are demonstrated
in the Examples below.
[0062] Hydrogels made from the crosslinked PEG polymeric structures
of the invention can be used in drug delivery systems and for wound
dressings. Wound dressings could be used internally to provide
dressings that degrade within the body over time. The hydrogels of
the invention could be usefully applied in drug delivery systems to
burns to apply therapeutic agents to burns. Drug delivery systems
can be prepared in which the rate of hydrolysis of the hydrogel is
controlled to provide controlled release of drug components. By
"drug" is meant any substance intended for the diagnosis, cure,
mitigation, treatment, or prevention of disease in humans and other
animals, or to otherwise enhance physical or mental well being. The
invention could be used for delivery of biologically active
substances generally that have some activity or function in a
living organism or in a substance taken from a living organism.
[0063] The terms "group," "functional group," "moiety," "active
moiety," "reactive site," and "radical" are all somewhat synonymous
in the chemical arts and are used in the art and herein to refer to
distinct, definable portions or units of a molecule and to units
that perform some function or activity and are reactive with other
molecules or portions of molecules.
[0064] The term "linkage" is used to refer to groups that normally
are formed as the result of a chemical reaction and typically are
covalent linkages. Hydrolytically stable linkages means that the
linkages are stable in water and do not react with water at useful
pHs for an extended period of time, potentially indefinitely.
Hydrolytically unstable linkages are those that react with water,
typically causing degradation of a hydrogel and release of
substances trapped within the matrix. The linkage is said to be
subject to hydrolysis and to be hydrolyzable. The time it takes to
degrade the crosslinked polymeric structure is referred to as the
rate of hydrolysis and is usually measured in terms of its half
life.
[0065] The skilled artisan should recognize that when reference is
made to a Z moiety reacting with a Y moiety or to a U moiety
reacting with a V moiety, that additional reagents or steps may be
employed according to commonly accepted chemical procedures and
standards to achieve the desired linkage W or X as the case may be.
There are many possible routes, too numerous to mention here, that
could be taken and that should be readily apparent to the skilled
artisan. For example, one of skill in the art can be expected to
understand that when an alcohol and a carboxylic acid are reacted,
the acid typically is converted to another form, the acid chloride,
prior to reaction with alcohol. Several examples are demonstrated
in the Examples below.
[0066] The following examples show the synthesis of various
examples of the invention.
EXAMPLES
Example 1
[0067] Example 1 shows preparation of a degradable PEG hydrogel
having a hydrolytically unstable ester linkage. In an aluminum pan
of 1 inch diameter, difunctional PEG 2000 acid (600 mg, 0.6 mmole
end groups, available from Shearwater Polymers in Huntsville, Ala.)
and one equivalent of 8-arm PEG 10,000 (750 mg, Shearwater
Polymers) were mixed with 30 mg stannous 2-ethylhexanoate (Sigma
Chemical) and melted. PEG acids used included PEG carboxymethyl
acid (-PEG-OCH.sub.2COOH), PEG propionic acid
(-PEG-O--CH.sub.2CH.sub.2COOH), and PEG succinic acid
(-PEG-OOCCH.sub.2CH.sub.2COOH). After a thin film of the melt
covered the pan surface uniformly, the pan was heated under vacuum
at 130.degree. C. and 100 millitorr for 6-24 hours. A firm,
transparent gel formed. After cooling in a N.sub.2 stream, the gel
became translucent and was cut into thin disks and purified by the
following procedures.
[0068] The crude gels were swollen in glacial acetic acid and
washed three times with this solvent during a 2-3 days period. For
hydrogels with a low swelling degree, swelling was conducted in
dioxane before the wash with glacial acetic acid to avoid breaking
of highly crosslinked gels. After washing, the gels were dried
under vacuum. The tin content of the gel was determined by
inductively coupled plasma spectroscopy to be less than 60 ppm.
Example 2
[0069] Example 2 shows preparation of a degradable PEG hydrogel
having a hydrolytically unstable imine linkage. In a test tube,
difunctional PEG propionic aldehyde 3400 (100 mg, 58.8 .mu.mole,
Shearwater Polymers) and 8-arm PEG amine 10,000 (74 mg, 58.8
.mu.mole) were dissolved in 1,4-dioxane (Aldrich Chemical). The
test tube was heated on an oil bath at 70.degree. C. for about two
hours. The gel was then dried under reduced pressure at room
temperature.
[0070] The PEG aldehydes used included PEG propionaldehyde
(-PEG-OCH.sub.2CH.sub.2CHO), PEG acetaldehyde (-PEG-OCH.sub.2CHO),
and PEG benzaldehyde (-PEG-O--C.sub.6H.sub.4--CHO).
[0071] Examples 3 and 4, below, show preparation of PEG derivatives
having hydrolytically unstable linkages for use in preparing the
degradable hydrogel of the invention.
Example 3
[0072] Example 3 shows synthesis of PEG derivatives having
hydrolytically unstable backbone linkages and NHS active carbonates
at each terminus thereof. The PEG derivative can be represented as
NHS-OOCO-PEG-W-PEG-OCOO- --NHS where W represents the
hydrolytically unstable linkage. In a 100 ml round-bottom flask,
benzyloxy-PEG carboxymethyl acid 3400 (3.4 g, 1 mmol, Shearwater
Polymers) in toluene was azeotropically distilled for two hours and
then cooled to room temperature. A solution of thionyl chloride
(2M, 4 ml, 8 mmole, Aldrich) in methylene chloride was injected and
the mixture was stirred under N.sub.2 overnight. The solvent was
condensed by rotary evaporation and the syrup was dried in vacuo
for about four hours over P.sub.2O.sub.5 powder. To the residue was
added anhydrous methylene chloride (5 ml) and azeotropically dried
benzyloxy-PEG 3400 (2.55 g, 0.75 mmol) in toluene (20 ml). After
the benzyloxy-PEG acyl chloride was dissolved, freshly distilled
triethylamine (0.6 ml) was added. The mixture was stirred
overnight, the triethylamine salt filtered off, and the product
collected by precipitation with ethyl ether. It was further
purified by dissolving in water and extracting with methylene
chloride. The organic phase was dried over anhydrous sodium
sulfate, condensed under vacuum, and precipitated into ethyl ether.
The precipitate was dried in vacuo. HPLC (GPC) of the product
showed that 100% of benzyloxy-PEG had been converted into the PEG
ester and about 15% wt % benzyloxy-PEG acid remained.
[0073] The mixture was chromatographically purified on an
ion-exchange column (DEAE sepharose fast flow, Pharmacia) to remove
the benzyloxy-PEG acid. 100% pure .alpha.-benzyloxy-{overscore
(.omega.)}-benzyloxy PEG ester 6800 was obtained. Yield: 4.1 gram
(80%).
[0074] A solution of .alpha.-benzyloxy-{overscore
(.omega.)}-benzyloxy PEG ester 6800 (2 g, 0.59 mmole) in
1,4-dioxane (20 ml) was hydrogenolyzed with H.sub.2 (2 atm
pressure) and Pd/C (1 g, 10% Pd) overnight. The catalyst was
removed by filtration and the product precipitated into ethyl ether
after most of the solvent was removed on a rotary evaporator.
.alpha.-hydroxy-{overscore (.omega.)}-hydroxy PEG ester 6800 was
collected by filtration and dried in vacuo. Yield: 1.5 gram
(75%).
[0075] .alpha.-hydroxy-{overscore (.omega.)}-hydroxy PEG ester 6800
(1.5 g, 0.44 mmole end group) was azeotropically dried with 100 ml
acetonitrile and cooled to room temperature. To this solution was
added disuccimidyl carbonate (DSC) (0.88 mmole, Fluka) and pyridine
(0.1 ml), and the solution was stirred at room temperature
overnight. The solvent was removed under vacuum and the syrup was
dried in vacuo. The product was dissolved in 35 ml of dry methylene
chloride, the insoluble solid was removed by filtration, and the
filtrate washed with pH 4.5 sodium chloride saturated acetate
buffer. The organic phase was dried over anhydrous sodium sulfate,
condensed under vacuum, and precipitated into ethyl ether. The
precipitate was dried over P.sub.2O.sub.5 in vacuo. Yield: 1.4 g
(93%). NMR (DMSO-d.sub.6): (1) product from benzyloxy-PEG propionic
acid: .delta. 3.5 (br m, PEG), 2.55 (t, --OCH.sub.2CH.sub.2COOP-
EG-), 4.13 (t, -PEG-COOCH.sub.2CH.sub.2O--), 4.45 (t,
-PEGOCH.sub.2CH.sub.2OCO--NHS), 2.80 (s, NHS, 4H); (2) product from
benzyloxy-PEG carboxymethyl acid: .delta. 3.5 (br m, PEG), 4.14 (s,
--OCH.sub.2COOPEG-), 4.18 (t, --OCH.sub.2COOCH.sub.2CH.sub.2--),
4.45 (t, -PEGO--CH.sub.2CH.sub.2OCONHS), 2.81 [s, NHS, 4H].
Example 4
[0076] Example 4 shows synthesis of PEG derivatives having
hydrolytically unstable backbone linkages and terminal NHS active
esters. The PEG derivative can be represented by the formula
NHS--OOC--(CH.sub.2).sub.n---
O-PEG-W-PEG-O--(CH.sub.2).sub.n--COONHS where W is a hydrolytically
unstable linkage. In a 100 ml round-bottom flask,
.alpha.-hydroxy-PEG acid 2000 (4 g, 2 mmol, Shearwater Polymers)
and difunctional PEG propionic acid 2000 (4 g, 2 mmole, Shearwater
Polymers) were azeotropically distilled with 70 ml toluene under
N.sub.2. After two hours, the solution was cooled to room
temperature and stannous 2-ethylhexanoate (200 mg, Sigma Chemical)
was added. The solution was then refluxed under N.sub.2 for 24
hours. The solvent was then condensed under vacuum and the syrup
precipitated into 100 ml of ether. The product was collected by
filtration, dried under vacuum, and dissolved in a sodium acetate
buffer solution at pH 5.0. The slightly milky solution was
centrifuged and the upper clear solution was extracted three times
with methylene chloride. The organic phase was dried over anhydrous
sodium sulfate, filtered, condensed under vacuum, and precipitated
into ether. The product was collected by filtration and dried under
vacuum. Yield 7 g (88%). HPLC: 70% product, 15% di-acid reactant
and 15% monoacid. The mixture was further purified by ion exchange
chromatography and gel permeation chromatography. .sup.1H NMR
(DMSO-d.sub.6): (1) product from PEG carboxymethyl acid: .delta.
3.5 (br m, PEG), 4.15 (s, --OCH.sub.2COOCH.sub.2--), 4.18 (t,
--OCH.sub.2COOCH.sub.2CH.sub.2--); (2) product from PEG propionic
acid: .delta. 3.5 (br m, PEG), 2.58 (t,
--OCH.sub.2CH.sub.2COOCH.sub.2--), 4.13 (t,
--OCH.sub.2CH.sub.2COOCH.sub.- 2CH.sub.2--).
[0077] In a round-bottom flask, the difunctional acid having weak
linkages (obtained from previous step) (2 g. approx. 1 mmole end
group) and N-hydroxysuccinimide (NHS) (126 mg, 1.05 mmole) were
dissolved in 50 ml of dry methylene chloride. To this solution was
added dicyclohexylcarbodiimide (240 mg, 1.15 mmole) in 5 ml dry
methylene chloride. The mixture was stirred under N.sub.2
overnight. The solvent was condensed and the syrup was redissolved
in 15 ml of anhydrous toluene. The insoluble salt was removed by
filtration and the filtrate was precipitated into 200 ml of dry
ethyl ether. The precipitate was collected by filtration and dried
in vacuo. Yield 1.88 g (94%). .sup.1H NMR(DMSO-d.sub.6): .delta.
3.5 (br m, PEG), 2.8 (s, NHS, 4H), 4.6 (s, -PEG-O--CH.sub.2-COONHS)
or 2.85 (t, -PEG-O--CH.sub.2CH.sub.2--COONHS).
Example 5
[0078] Example 5 shows preparation of a degradable PEG hydrogel
from branched PEG amine and PEG derivatives made in accordance with
Example 3 in which the PEG derivatives have hydrolytically unstable
backbone linkages and terminal NHS active carbonates, which can be
represented as NHS--OOCO-PEG-W-PEG-OCOO--NHS. In a test tube, 100
mg (4.7 .mu.mole) of difunctional PEG active carbonate 6800
(NHS--OOCO-PEG-W-PEG-OCOONHS, prepared in Example 3) was dissolved
in 0.75 ml of water, and a buffered solution (0.1M phosphate, pH 7)
of 0.15 ml 8-arm-PEG-amine 10,000 (250 mg/ml) was added. After
rapid shaking, it was allowed to sit and a gel formed in a few
minutes. A suitable buffer pH range was found to be 5.5 to 8.
Example 6
[0079] Example 6 shows preparation of degradable PEG hydrogels from
branched PEG amine and PEG derivatives made in accordance with
Example 4 in which the PEG derivatives have hydrolytically unstable
backbone linkages and terminal NHS active carbonates that can be
represented as
NHS--OOC--(CH.sub.2).sub.n--O-PEG-W-PEG-O--(CH.sub.2).sub.n-COO--NHS.
100 mg (approx. 50 .mu.mole) difunctional PEG active ester
(NHS--OOC--(CH.sub.2).sub.n--O-PEG-W-PEG-O--(CH.sub.2).sub.n--COO--NHS,
prepared in Example 4) was dissolved in 0.75 ml of water, and a
buffered solution (0.1M phosphate, pH 7) of 0.25 ml 8-arm-PEG-amine
10,000 (250 mg/ml) was added. After rapid shaking, it was allowed
to sit and a gel formed in a few minutes. A suitable buffer pH
range was found to be 5.5 to 8.
Example 7
[0080] Example shows the synthesis of difunctional
PEG-hydroxybutyric acid (HBA), which can be represented as
HOOC--CH.sub.2--CH(CH.sub.3)--OOC--(CH-
.sub.2).sub.n--O-PEG-O--(CH.sub.2).sub.n--COOCH(CH.sub.3)CH.sub.2--COOH
for use in preparing the reactive PEGs of Example 8. PEG acid 2000
(2.0 g, 1 mmole, carboxymethyl acid (CM) or propionic acid (PA))
was azeotropically dried with 60 ml toluene under N.sub.2. After
two hours, the solution was cooled to room temperature and thionyl
chloride (3 ml, 6 mmole, in CH.sub.2Cl.sub.2) was added. The
mixture was then stirred at room temperature overnight and the
solution condensed by rotary evaporation. The residue was dried in
vacuo for about four hours with P.sub.2O.sub.5, powder.
3-hydroxybutyric acid (0.30 g, 2.7 mmole) was azeotropically dried
with 70 ml 1,4-dioxane until approximately 20 ml of solution
remained. The solution was then cooled to room temperature under
N.sub.2 and to it was added dried PEG acyl chloride from the above
step. After the PEG was dissolved, 0.6 ml dry triethylamine was
injected into the system and the reaction mixture was stirred
overnight. The salt was filtered from the solution, the solvent
condensed on a rotary evaporator, and the syrup was dried in vacuo.
The crude product was dissolved in 100 ml distilled water and the
pH adjusted to 3.0. The product was extracted three times with a
total of 80 ml of methylene chloride. The organic phase was dried
over anhydrous sodium sulfate, filtered, condensed under vacuum,
and precipitated into 100 ml of ethyl ether. The product was
collected by filtration and dried in vacuo. Yield 1.84 g (92%).
.sup.1H NMR (DMSO-d.sub.6): .delta. 3.5 (br m, PEG), 2.54 (d,
PEGCOOCH(CH.sub.3) CH.sub.2COOH), 5.1 (h, PEGCOOCH(CH.sub.3)
CH.sub.2COOH), 1.21 (d, PEG-COOCH(CH.sub.3)CH.sub.2COOH), 2.54 (t,
PEGOCH.sub.2CH.sub.2COO (PA)), 4.05 (s, PEGOCH.sub.2COO (CM)).
Example 8
[0081] Example 8 shows the synthesis of difunctional PEG-HBA-NHS
double ester, which can be represented as
NHS--OOC--CH.sub.2--CH(CH.sub.3)--OOC--
-(CH.sub.2).sub.n--O-PEG-O--(CH.sub.2).sub.n--COOCH(CH.sub.3)CH.sub.2--COO-
NHS, for use in preparing PEG hydrogels of the invention.
PEG-3-butyric acid (1 g, approx. 0.5 mmole, prepared in example 7)
and 64 mg N-hydroxysuccinimide (NHS) (0.53 mmole) were dissolved in
30 ml of dry methylene chloride, followed by addition of
dicyclohexylcarbodiimide (DCC, 126 mg, 0.6 mmole) in 5 ml dry
methylene chloride. The solution was stirred under nitrogen
overnight and the solvent removed by rotary evaporation. The
residue was stirred with 10 ml dry toluene at 45.degree. C. and the
insoluble solid was removed by filtration. The product was
precipitated into 100 ml of dry ethyl ether and the precipitate was
collected by filtration and dried in vacuo. Yield 0.94 g (94%).
.sup.1H NMR(DMSO-d.sub.6): .delta. 3.5 (br m, PEG), 3.0-3.2 (m,
--COOCH(CH.sub.3)CH.sub.2COONHS), 5.26 (h,
--COOCH(CH.sub.3)CH.sub.2COONH- S), 1.3 (d,
--CO--OCH(CH.sub.3)CH.sub.2COONHS), 2.54 (t,
-PEGOCH.sub.2CH.sub.2COO-(PA)), 4.1 (s, -PEGOCH.sub.2COO-(CM)).
Example 9
[0082] Example 9 shows the preparation of a degradable PEG hydrogel
from branched PEG amine and the PEG-HBA-NHS double ester of Example
8, which can be represented as
NHS--OOC--CH.sub.2--CH(CH.sub.3)--OOC--(CH.sub.2).s-
ub.n--O-PEG-O--(CH.sub.2).sub.n--COOCH(CH.sub.3)CH.sub.2--COONHS.
PEG-HBA-NHS double ester 2000 (100 mg, approx. 0.1 mmole, Example
8) was dissolved in 0.5 ml of water and a buffered solution of
8-arm-PEG-amine 10,000 (0.5 ml, 250 mg/ml) was added. After rapid
shaking, it was allowed to sit and a gel formed in a few minutes. A
suitable buffer pH range was found to be 5.5 to 8.
[0083] The invention has been described in particular exemplified
embodiments. However, the foregoing description is not intended to
limit the invention to the exemplified embodiments, and the skilled
artisan should recognize that variations can be mad within the
scope and spirit of the invention as described in the foregoing
specification. On the contrary, the invention includes all
alternatives, modifications, and equivalents that may be included
within the true spirit and scope of the invention as defined by the
appended claims.
* * * * *