U.S. patent application number 10/119546 was filed with the patent office on 2003-06-19 for multi-armed, monofunctional, and hydrolytically stable derivatives of poly (ethylene glycol) and related polymers for modification of surfaces and molecules.
This patent application is currently assigned to Shearwater Corporation. Invention is credited to Caliceti, Paolo, Harris, J. Milton, Schiavon, Oddone, Veronese, Francesco Maria.
Application Number | 20030114647 10/119546 |
Document ID | / |
Family ID | 27005224 |
Filed Date | 2003-06-19 |
United States Patent
Application |
20030114647 |
Kind Code |
A1 |
Harris, J. Milton ; et
al. |
June 19, 2003 |
Multi-armed, monofunctional, and hydrolytically stable derivatives
of poly (ethylene glycol) and related polymers for modification of
surfaces and molecules
Abstract
Multi-armed, monofunctional, and hydrolytically stable polymers
are described having the structure 1 wherein Z is a moiety that can
be activated for attachment to biologically active molecules such
as proteins and wherein P and Q represent linkage fragments that
join polymer arms poly.sub.a and poly.sub.b, respectively, to
central carbon atom, C, by hydrolytically stable linkages in the
absence of aromatic rings in the linkage fragments. R typically is
hydrogen or methyl, but can be a linkage fragment that includes
another polymer arm. A specific example is an mPEG disubstituted
lysine having the structure 2 where mPEG.sub.a and mPEG.sub.b have
the structure CH.sub.3O--(CH.sub.2CH.-
sub.2O).sub.nCH.sub.2CH.sub.2-- wherein n may be the same or
different for poly.sub.a- and poly.sub.b- and can be from 1 to
about 1,150 to provide molecular weights of from about 100 to
100,000.
Inventors: |
Harris, J. Milton;
(Huntsville, AL) ; Veronese, Francesco Maria;
(Padova, IT) ; Caliceti, Paolo; (Padova, IT)
; Schiavon, Oddone; (Padova, IT) |
Correspondence
Address: |
ALSTON & BIRD LLP
BANK OF AMERICA PLAZA
101 SOUTH TRYON STREET, SUITE 4000
CHARLOTTE
NC
28280-4000
US
|
Assignee: |
Shearwater Corporation
|
Family ID: |
27005224 |
Appl. No.: |
10/119546 |
Filed: |
April 10, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10119546 |
Apr 10, 2002 |
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09939867 |
Aug 27, 2001 |
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09939867 |
Aug 27, 2001 |
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09140907 |
Aug 27, 1998 |
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09140907 |
Aug 27, 1998 |
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08443383 |
May 17, 1995 |
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5932462 |
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08443383 |
May 17, 1995 |
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08371065 |
Jan 10, 1995 |
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Current U.S.
Class: |
530/402 ;
435/192; 435/199; 435/213 |
Current CPC
Class: |
Y10S 530/816 20130101;
A61K 47/60 20170801; C08G 65/48 20130101; C08G 65/329 20130101;
Y10S 530/815 20130101; C12N 9/96 20130101 |
Class at
Publication: |
530/402 ;
435/192; 435/213; 435/199 |
International
Class: |
C08H 001/00; C12N
009/08; C12N 009/22; C12N 009/76 |
Claims
What is claimed is:
1. A polymeric derivative represented by the structure 38wherein
poly.sub.a and poly.sub.b are nonpeptidic and substantially
nonreactive water soluble polymeric arms that may be the same or
different, wherein C is carbon, wherein P and Q comprise linkage
fragments that may be the same or different and join polymeric arms
poly.sub.a and poly.sub.b, respectively, to C by hydrolytically
stable linkages in the absence of aromatic rings in said linkage
fragments, wherein R is a moiety selected from the group consisting
of H, substantially nonreactive moieties, and linkage fragments
having attached thereto by a hydrolytically stable linkage in the
absence of aromatic rings one or more nonpeptidic and substantially
nonreactive water soluble polymeric arms, and wherein Z comprises a
moiety selected from the group consisting of moieties having a
single site reactive toward nucleophilic moieties, sites that can
be converted to sites reactive toward nucleophilic moieties, and
the reaction product of a nucleophilic moiety and moieties having a
single site reactive toward nucleophilic moieties.
2. The polymeric derivative of claim 1 wherein said hydrolytically
stable linkages are selected from the group consisting of amide,
amine, ether, carbamate, thiourea, urea, thiocarbamate,
thiocarbonate, thioether, thioester, and dithiocarbamate
linkages.
3. The polymeric derivative of claim 1 wherein said nucleophilic
moieties are selected from the group consisting of amino, thiol,
and hydroxyl moieties.
4. The polymeric derivative of claim 1 wherein said nucleophilic
moiety is a biologically active molecule.
5. The polymeric derivative of claim 4 wherein said biologically
active molecule is selected from the group consisting of
polypeptides, polynucleotides, and lipids.
6. The polymeric derivative of claim 1 wherein said nucleophilic
moiety is a solid surface or a particle.
7. The polymeric derivative of claim 6 wherein said solid particle
is a liposome.
8. The polymeric derivative of claim 1 wherein Z is selected from
the group consisting of carboxyl, hydroxyl, activated carboxyl,
activated hydroxyl, and conjugates of activated carboxyl or
hydroxyl sites and molecules having at least one reactive
nucleophilic moiety.
9. The polymeric derivative of claim 1 wherein Z comprises a moiety
selected from the group consisting of trifluoroethylsulfonate,
isocyanate, isothiocyanate, active esters, active carbonates,
aldehyde, vinylsulfone, maleimide, iodoacetamide, and
iminoesters.
10. The polymeric derivative of claim 9 wherein said active ester
is N-hydroxylsuccinimidyl ester and said active carbonates are
selected from the group consisting of N-hydroxylsuccinimidyl
carbonate, p-nitrophenylcarbonate, and
trichlorophenylcarbonate.
11. The polymeric derivative of claim 1 wherein said nonpeptidic
polymeric arms are selected from the group consisting of
poly(alkylene oxides), poly(oxyethylated polyols), and
poly(oxyethylated glucose).
12. The polymeric derivative of claim 1 wherein said nonpeptidic
polymeric arms are selected from the group consisting of
poly(ethylene glycol), poly(vinyl alcohol), poly(propylene glycol),
poly(oxyethylated glycerol), poly(oxyethylated sorbitol),
poly(oxyethylated glucose), poly(oxazoline),
poly(acryloylmorpholine), and poly(vinylpyrrolidone).
13. The polymeric derivative of claim 1 wherein said nonpeptidic
polymeric arms are linear mPEGs of molecular weight of from about
50 to 50,000.
14. The polymeric derivative of claim 1 wherein said linkage
fragments P and Q comprise hydrolytically stable linkages in the
absence of aromatic rings to one or more nonpeptidic and water
soluble polymeric arms.
15. The polymeric derivative of claim 1 wherein R comprises a
linkage fragment attached by a hydrolytically stable linkage in the
absence of aromatic rings to a nonpeptidic and substantially
nonreactive water soluble polymeric arm.
16. The polymeric derivative of claim 15 wherein R is represented
by the general structure --M-poly.sub.d, wherein poly.sub.d is said
polymeric arm and M is said linkage fragment.
17. The polymeric derivative of claim 1 wherein Z further comprises
a linkage fragment attached by a hydrolytically stable linkage in
the absence of aromatic rings to a nonpeptidic and substantially
nonreactive water soluble polymeric arm.
18. A polymeric derivative represented by the structure 39wherein
poly.sub.a and poly.sub.b may be the same or different and are
selected from the group consisting of linear poly(ethylene glycol),
poly(vinyl alcohol), poly(propylene glycol), poly(oxyethylated
glycerol), poly(oxyethylated sorbitol), poly(oxyethylated glucose),
poly(oxazoline), poly(acryloylmorpholine), and
poly(vinylpyrrolidone); wherein C is carbon; wherein P and Q
comprise linkage fragments that may be the same or different and
join polymeric arms poly.sub.a and poly.sub.b, respectively, to C
by hydrolytically stable linkages selected from the group
consisting of amide, amine, ether, carbamate, thiourea, urea,
thiocarbamate, thiocarbonate, thioether, thioester, and
dithiocarbamate linkages; wherein R is a moiety selected from the
group consisting of H, substantially nonreactive moieties, and
linkage fragments having attached thereto by a hydrolytically
stable linkage in the absence of aromatic rings one or more
nonpeptidic and substantially nonreactive water soluble polymeric
arms; and wherein Z comprises a moiety selected from the group
consisting of carboxyl, hydroxyl, trifluoroethylsulfonate,
isocyanate, isothiocyanate, N-hydroxylsuccinimidyl ester,
N-hydroxylsuccinimidyl carbonate, p-nitrophenylcarbonate,
trichlorophenylcarbonate, aldehyde, vinylsulfone, maleimide,
iodoacetamide, and iminoesters.
19. A multi-armed monofunctional polymeric derivative that is the
reaction product of at least one monofunctional nonpeptidic polymer
derivative and a linker moiety having two or more active sites that
form linkages with said monofunctional nonpeptidic polymer
derivatives in the absence of aromatic moieties, wherein said
linkages between said linker moiety and said monofunctional
nonpeptidic polymer derivatives are hydrolytically stable.
20. The multi-armed monofunctional polymeric derivative of claim 19
wherein said linker moiety is selected from the group consisting of
monohydroxy alcohols and monocarboxylic acids.
21. The multi-armed monofunctional polymer derivative of claim 19
wherein said active sites on said linker moiety are nucleophilic
moieties.
22. The multi-armed monofunctional polymer derivative of claim 21
wherein said nucleophilic moieties are selected from the group
consisting of amino, thiol, and hydroxyl moieties.
23. The multi-armed monofunctional polymer derivative of claim 19
wherein said active sites on said linker moiety are electrophilic
moieties.
24. The multi-armed monofunctional polymer derivative of claim 23
wherein said electrophilic moieties are selected from the group
consisting of trifluoroethylsulfonate, isocyanate, isothiocyanate,
active esters, active carbonates, aldehyde, vinylsulfone,
maleimide, iodoacetamide, and iminoesters.
25. The multi-armed monofunctional polymeric derivative of claim 24
wherein said active esters are N-hydroxylsuccinimidyl ester and
said active carbonates are selected from the group consisting of
N-hydroxylsuccinimidyl carbonates, p-nitrophenylcarbonates, and
trichlorophenylcarbonates.
26. The multi-armed monofunctional polymeric derivative of claim 19
wherein said hydrolytically stable linkages in the absence of
aromatic rings are selected from the group consisting of amide,
amine, ether, carbamate, thiourea, urea, thiocarbamate,
thiocarbonate, thioether, thioester, and dithiocarbamate
linkages.
27. The multi-armed monofunctional polymeric derivative of claim 19
wherein said monofunctionality is selected from the group
consisting of carboxyl, hydroxyl, activated carboxyl, activated
hydroxyl, and conjugates of activated carboxyl or hydroxyl sites
and molecules having at least one reactive nucleophilic moiety.
28. The multi-armed monofunctional polymeric derivative of claim 19
wherein said monofunctionality is selected from the group
consisting of trifluoroethylsulfonate, isocyanate, isothiocyanate,
active esters, active carbonates, aldehyde, vinylsulfone,
maleimide, iodoacetamide, and iminoesters.
29. The multi-armed monofunctional polymeric derivative of claim 28
wherein said active ester is N-hydroxylsuccinimide and said active
carbonates are selected from the group consisting of
N-hydroxylsuccinimide carbonates, p-nitrophenylcarbonates, and
trichlorophenylcarbonates.
30. The multi-armed monofunctional polymeric derivative of claim 19
wherein said nonpeptidic polymeric derivative is selected from the
group consisting of poly(alkylene oxides), poly(oxyethylated
polyols), and poly(oxyethylated glucose).
31. The multi-armed monofunctional polymeric derivative of claim 19
wherein said nonpeptidic polymer derivative is selected from the
group consisting of activated poly(ethylene glycol), poly(vinyl
alcohol), poly(propylene glycol), poly(oxyethylated glycerol),
poly(oxyethylated sorbitol), poly(oxyethylated glucose),
poly(oxazoline), poly(acryloylmorpholine), and
poly(vinylpyrrolidone).
32. The multi-armed monofunctional polymeric derivative of claim 19
wherein said nonpeptidic polymer derivative is a linear mPEG of
molecular weight of from about 50 to 50,000 and the multi-armed
monofunctional polymeric derivative has two arms of said linear
mPEG.
33. A material comprising a solid surface or particle having
attached thereto compounds of the structure claimed in claim
19.
34. The material of claim 33 wherein said solid surface or particle
is a liposome.
35. A biologically active structure comprising a biologically
active molecule having attached thereto one or more compounds of
the structure claimed in claim 19.
36. The biologically active structure of claim 35 wherein said
biologically active molecule is selected from the group consisting
of polypeptides, polynucleotides, and lipids.
37. The biologically active structure of claim 36 wherein said
polypeptide is selected from the group consisting of asparaginase,
catalase, ribonuclease, subtilisine, trypsin, and uricase.
38. A two-armed polymeric derivative having a structure selected
from the group consisting of: 40wherein poly.sub.a and poly.sub.b
may be the same or different and comprise moieties selected from
the group consisting of poly(ethylene glycol), poly(vinyl alcohol),
poly(propylene glycol), poly(oxyethylated glycerol),
poly(oxyethylated sorbitol), poly(oxyethylated glucose),
poly(oxazoline), poly(acryloylmorpholine), and
poly(vinylpyrrolidone) moieties; and wherein Z comprises a moiety
selected from the group consisting of moieties having a single site
reactive toward nucleophilic moieties, sites that can be converted
to sites reactive toward nucleophilic moieties, and the reaction
product of a nucleophilic moiety and moieties having a single site
reactive toward nucleophilic moieties.
39. The two-armed polymeric derivative of claim 38 wherein said
reactive site is selected from the group consisting of carboxyl,
activated carboxyl, hydroxyl, activated hydroxyl, and conjugates of
activated carboxyl or hydroxyl sites and molecules having at least
one reactive nucleophilic moiety.
40. The polymeric derivative of claim 38 wherein Z comprises a
moiety selected from the group consisting of
trifluoroethylsulfonate, isocyanate, isothiocyanate, active esters,
active carbonates, aldehyde, vinylsulfone, maleimide,
iodoacetamide, and iminoesters.
41. The polymeric derivative of claim 40 wherein said active ester
is N-hydroxylsuccinimidyl ester and said active carbonates are
selected from the group consisting of N-hydroxylsuccinimidyl
carbonate, p-nitrophenylcarbonate, and
trichlorophenylcarbonate.
42. A molecule having the structure 41wherein mPEG.sub.a and
mPEG.sub.b have the structure
CH.sub.3--(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2--, wherein n
equals from 1 to about 1,150, and wherein n may be the same or
different for mPEG.sub.a and mPEG.sub.b.
43. The molecule of claim 42 wherein n equals from 1 to about
570.
44. A method of synthesizing a multi-armed, water soluble,
monofunctional polymeric molecule comprising reacting one or more
nonpeptidic monofunctional polymers of the structure poly-W,
wherein W is an active moiety providing the monofunctionality for
the polymer, with a linker moiety having two or more active sites
with which W is reactive, and forming hydrolytically stable
linkages in the absence of aromatic rings between the
monofunctional polymer and the linker moiety at the linker moiety
active sites, the linker moiety having a reactive site for which
said active moiety --W is not reactive to provide the
monofunctionality for the multi-armed molecule.
45. The method of claim 44 wherein the method further comprises
activating the reactive site after the multi-armed polymeric
compound is formed with an electrophilic moiety.
46. The method of claim 45 wherein the electrophilic moiety is
reactive with nucleophilic moieties selected from the group
consisting of amino, thiol, and hydroxyl moieties.
47. The method of claim 44 wherein the active moiety W is an
electrophilic moiety selected from the group consisting of
trifluoroethylsulfonate, isocyanate, isothiocyanate, active esters,
active carbonates, aldehyde, vinylsulfone, maleimide,
iodoacetamide, and iminoesters.
48. The method of claim 47 wherein the active ester is
N-hydroxylsuccinimidyl ester and the active carbonates are selected
from the group consisting of N-hydroxylsuccinimidyl carbonate,
p-nitrophenylcarbonate, and trichlorophenylcarbonate.
49. The method of claim 44 wherein the active moiety W is a
nucleophilic moiety selected from the group consisting of amino,
thiol, and hydroxyl moieties.
50. The method of claim 44 wherein the active sites on the linker
moiety are nucleophilic moieties selected from the group consisting
of amino, thiol, and hydroxyl moieties.
51. The method of claim 44 wherein the active sites on the linker
moiety are electrophilic moieties selected from the group
consisting of trifluoroethylsulfonate, isocyanate, isothiocyanate,
active esters, active carbonates, aldehyde, vinylsulfone,
maleimide, iodoacetamide, and iminoesters.
52. The method of claim 51 wherein the active ester is
N-hydroxylsuccinimidyl ester and the active carbonates are selected
from the group consisting of N-hydroxylsuccinimidyl carbonate,
p-nitrophenylcarbonate, and trichlorophenylcarbonate.
53. The method of claim 44 wherein the hydrolytically stable
linkages are selected from the group consisting of amide, amine,
ether, carbamate, thiourea, urea, thiocarbamate, thiocarbonate,
thioether, thioester, and dithiocarbamate linkages.
54. A method for preparing a polymeric derivative represented by
the structure 42comprising the steps of: a) reacting nonpeptidic,
water soluble, monofunctional polymers of the structure
poly.sub.a-W and poly.sub.b-W with a linker moiety having at least
two active sites for which W is selective, a reactive site Z for
which W is not selective, and a moiety R which is substantially
nonreactive, wherein W is an active electrophilic moiety selected
from the group consisting of trifluoroethylsulfonate, isocyanate,
isothiocyanate, active esters, active carbonates, aldehyde,
vinylsulfone, maleimide, iodoacetamide, and iminoesters, and may be
the same or different on poly.sub.a and poly.sub.b, wherein
poly.sub.a and poly.sub.b are polymer moieties selected from the
group consisting of poly(ethylene glycol), poly(vinyl alcohol),
poly(propylene glycol), poly(oxyethylated glycerol),
poly(oxyethylated sorbitol), poly(oxyethylated glucose),
poly(oxazoline), poly(acryloylmorpholine), and
poly(vinylpyrrolidone) and may be the same or different, and
wherein the active sites of the linker moiety are nucleophilic
sites selected from the group consisting of amino, thiol, and
hydroxyl; and b) forming hydrolytically stable linkages P and Q,
which may be the same or different, in the absence of aromatic
rings between the polymer and the linker moiety that are selected
from the group consisting of amide, amine, ether, carbamate,
thiourea, urea, thiocarbamate, thiocarbonate, thioether, thioester,
and dithiocarbamate linkages.
55. The method of claim 54 wherein the linker moiety is substituted
with polymer at each active site in one step.
56. The method of claim 55 wherein the linker moiety is substituted
with polymer at each active site in more than one step.
57. The multi-armed polymeric derivative of claim 54 wherein said
linker moiety is selected from the group consisting of monohydroxy
alcohols and monocarboxilic acids having two or more active
moieties selected from the group consisting of thiol, amino, and
hydroxyl moieties.
58. The multi-armed polymeric derivative of claim 1 wherein Z is
selected from the group consisting of carboxyl, hydroxyl, activated
carboxyl, activated hydroxyl, and conjugates of precursor activated
carboxyl or hydroxyl sites and molecules having sites for which
said precursor activated sites are active.
59. A method for forming monofunctional monomethoxy-poly(ethylene
glycol) disubstituted lysene comprising the following step: 43
60. The method of claim 59 wherein the reaction takes place in
water at a pH of about 8.0.
61. The method of claim 59 further comprising the steps of 44
62. The method of claim 61 wherein steps a) and b) take place in
methylene chloride.
63. The method of claim 59 further comprising the steps of
activating the carboxyl moiety and reacting the activated carboxyl
moiety with an active moiety to join the disubstituted lysine to
the active moiety.
64. A method for forming a monofunctional monomethoxy-poly(ethylene
glycol) disubstituted compound comprising the following steps:
45
65. The method of claim 64 further comprising the steps of
activating the carboxyl moiety and reacting the activated carboxyl
moiety with an active moiety to join the disubstituted lysine to
the active moiety.
66. The method of claim 64 wherein step a) takes place in aqueous
buffer.
67. The method of claim 64 wherein step b) takes place in methylene
chloride.
Description
[0001] This application is related to and claims the benefit of the
filing date of U.S. Ser. No. 08/371,065, which was filed on Jan.
10, 1995 and is entitled MULTI-ARMED, MONOFUNCTIONAL, AND
HYDROLYTICALLY STABLE DERIVATIVES OF POLY(ETHYLENE GLYCOL) AND
RELATED POLYMERS FOR MODIFICATION OF SURFACES AND MOLECULES.
FIELD OF THE INVENTION
[0002] This invention relates to monofunctional derivatives of
poly(ethylene glycol) and related polymers and to methods for their
synthesis and activation for use in modifying the characteristics
of surfaces and molecules.
BACKGROUND OF THE INVENTION
[0003] Improved chemical and genetic methods have made many
enzymes, proteins, and other peptides and polypeptides available
for use as drugs or biocatalysts having specific catalytic
activity. However, limitations exist to use of these compounds.
[0004] For example, enzymes that exhibit specific biocatalytic
activity sometimes are less useful than they otherwise might be
because of problems of low stability and solubility in organic
solvents. During in vivo use, many proteins are cleared from
circulation too rapidly. Some proteins have less water solubility
than is optimal for a therapeutic agent that circulates through the
bloodstream. Some proteins give rise to immunological problems when
used as therapeutic agents. Immunological problems have been
reported from manufactured proteins even where the compound
apparently has the same basic structure as the homologous natural
product. Numerous impediments to the successful use of enzymes and
proteins as drugs and biocatalysts have been encountered.
[0005] One approach to the problems that have arisen in the use of
polypeptides as drugs or biocatalysts has been to link suitable
hydrophilic or amphiphilic polymer derivatives to the polypeptide
to create a polymer cloud surrounding the polypeptide. If the
polymer derivative is soluble and stable in organic solvents, then
enzyme conjugates with the polymer may acquire that solubility and
stability. Biocatalysts can be extended to organic media with
enzyme and polymer combinations that are soluble and stable in
organic solvents.
[0006] For in vivo use, the polymer cloud can help to protect the
compound from chemical attack, to limit adverse side effects of the
compound when injected into the body, and to increase the size of
the compound, potentially to render useful compounds that have some
medicinal benefit, but otherwise are not useful or are even harmful
to an organism. For example, the polymer cloud surrounding a
protein can reduce the rate of renal excretion and immunological
complications and can increase resistance of the protein to
proteolytic breakdown into simpler, inactive substances.
[0007] However, despite the benefits of modifying polypeptides with
polymer derivatives, additional problems have arisen. These
problems typically arise in the linkage of the polymer to the
polypeptide. The linkage may be difficult to form. Bifunctional or
multifunctional polymer derivatives tend to cross link proteins,
which can result in a loss of solubility in water, making a
polymer-modified protein unsuitable for circulating through the
blood stream of a living organism. Other polymer derivatives form
hydrolytically unstable linkages that are quickly destroyed on
injection into the blood stream. Some linking moieties are toxic.
Some linkages reduce the activity of the protein or enzyme, thereby
rendering the protein or enzyme less effective.
[0008] The structure of the protein or enzyme dictates the location
of reactive sites that form the loci for linkage with polymers.
Proteins are built of various sequences of alpha-amino acids, which
have the general structure 3
[0009] The alpha amino moiety (H.sub.2N--) of one amino acid joins
to the carboxyl moiety (--COOH) of an adjacent amino acid to form
amide linkages, which can be represented as 4
[0010] where n can be hundreds or thousands. The terminal amino
acid of a protein molecule contains a free alpha amino moiety that
is reactive and to which a polymer can be attached. The fragment
represented by R can contain reactive sites for protein biological
activity and for attachment of polymer.
[0011] For example, in lysine, which is an amino acid forming part
of the backbone of most proteins, a reactive amino (--NH.sub.2)
moiety is present in the epsilon position as well as in the alpha
position. The epsilon --NH.sub.2 is free for reaction under
conditions of basic pH. Much of the art has been directed to
developing polymer derivatives having active moieties for
attachment to the epsilon --NH.sub.2 moiety of the lysine fraction
of a protein. These polymer derivatives all have in common that the
lysine amino acid fraction of the protein typically is modified by
polymer attachment, which can be a drawback where lysine is
important to protein activity.
[0012] Poly(ethylene glycol), which is commonly referred to simply
as "PEG," has been the nonpeptidic polymer most used so far for
attachment to proteins. The PEG molecule typically is linear and
can be represented structurally as
HO--(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2--OH
[0013] or, more simply, as HO--PEG--OH. As shown, the PEG molecule
is difunctional, and is sometimes referred to as "PEG diol." The
terminal portions of the PEG molecule are relatively nonreactive
hydroxyl moieties, --OH, that can be activated, or converted to
functional moieties, for attachment of the PEG to other compounds
at reactive sites on the compound.
[0014] For example, the terminal moieties of PEG diol have been
functionalized as active carbonate ester for selective reaction
with amino moieties by substitution of the relatively nonreactive
hydroxyl moieties, --OH, with succinimidyl active ester moieties
from N-hydroxy succinimide. The succinimidyl ester moiety can be
represented structurally as 5
[0015] Difunctional PEG, functionalized as the succinimidyl
carbonate, has a structure that can be represented as 6
[0016] Difunctional succinimidyl carbonate PEG has been reacted
with free lysine monomer to make high molecular weight polymers.
Free lysine monomer, which is also known as alpha, epsilon
diaminocaproic acid, has a structure with reactive alpha and
epsilon amino moieties that can be represented as 7
[0017] These high molecular weight polymers from difunctional PEG
and free lysine monomer have multiple, pendant reactive carboxyl
groups extending as branches from the polymer backbone that can be
represented structurally as 8
[0018] The pendant carboxyl groups typically have been used to
couple nonprotein pharmaceutical agents to the polymer. Protein
pharmaceutical agents would tend to be cross linked by the
multifunctional polymer with loss of protein activity.
[0019] Multiarmed PEGs having a reactive terminal moiety on each
branch have been prepared by the polymerization of ethylene oxide
onto multiple hydroxyl groups of polyols including glycerol.
Coupling of this type of multi-functional, branched PEG to a
protein normally produces a cross-linked product with considerable
loss of protein activity.
[0020] It is desirable for many applications to cap the PEG
molecule on one end with an essentially nonreactive end moiety so
that the PEG molecule is monofunctional. Monofunctional PEGs are
usually preferred for protein modification to avoid cross linking
and loss of activity. One hydroxyl moiety on the terminus of the
PEG diol molecule typically is substituted with a nonreactive
methyl end moiety, CH.sub.3--. The opposite terminus typically is
converted to a reactive end moiety that can be activated for
attachment at a reactive site on a surface or a molecule such as a
protein.
[0021] PEG molecules having a methyl end moiety are sometimes
referred to as monomethoxy-poly(ethylene glycol) and are sometimes
referred to simply as "mPEG." The mPEG polymer derivatives can be
represented structurally as
H.sub.3C--O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--Z
[0022] where n typically equals from about 45 to 115 and --Z is a
functional moiety that is active for selective attachment to a
reactive site on a molecule or surface or is a reactive moiety that
can be converted to a functional moiety.
[0023] Typically, mPEG polymers are linear polymers of molecular
weight in the range of from about 1,000 to 5,000. Higher molecular
weights have also been examined, up to a molecular weight of about
25,000, but these mPEGs typically are not of high purity and have
not normally been useful in PEG and protein chemistry. In
particular, these high molecular weight mPEGs typically contain
significant percentages of PEG diol.
[0024] Proteins and other molecules typically have a limited number
and distinct type of reactive sites available for coupling, such as
the epsilon --NH.sub.2 moiety of the lysine fraction of a protein.
Some of these reactive sites may be responsible for a protein's
biological activity. A PEG derivative that attached to a sufficient
number of such sites to impart the desired characteristics can
adversely affect the activity of the protein, which offsets many of
the advantages otherwise to be gained.
[0025] Attempts have been made to increase the polymer cloud volume
surrounding a protein molecule without further deactivating the
protein. Some PEG derivatives have been developed that have a
single functional moiety located along the polymer backbone for
attachment to another molecule or surface, rather than at the
terminus of the polymer. Although these compounds can be considered
linear, they are often referred to as "branched" and are
distinguished from conventional, linear PEG derivatives since these
molecules typically comprise a pair of mPEG-- molecules that have
been joined by their reactive end moieties to another moiety, which
can be represented structurally as --T--, and that includes a
reactive moiety, --Z, extending from the polymer backbone. These
compounds have a general structure that can be represented as 9
[0026] These monofunctional mPEG polymer derivatives show a
branched structure when linked to another compound. One such
branched form of mPEG with a single active binding site, --Z, has
been prepared by substitution of two of the chloride atoms of
trichloro-s-triazine with mPEG to make mPEG-disubstituted
chlorotriazine. The third chloride is used to bind to protein. An
mPEG disubstituted chlorotriazine and its synthesis are disclosed
in Wada, H., Imamura, l., Sako, M., Katagiri, S., Tarui, S.,
Nishimura, H., and Inada, Y. (1990) Antitumor enzymes: polyethylene
glycol-modified asparaginase. Ann. N.Y. Acad. Sci. 613, 95-108.
Synthesis of mPEG disubstituted chlorotriazine is represented
structurally below. 10
[0027] However, mPEG-disubstituted chlorotriazine and the procedure
used to prepare it present severe limitations because coupling to
protein is highly nonselective. Several types of amino acids other
than lysine are attacked and many proteins are inactivated. The
intermediate is toxic. Also, the mPEG-disubstituted chlorotriazine
molecule reacts with water, thus substantially precluding
purification of the branched mPEG structure by commonly used
chromatographic techniques in water.
[0028] A branched mPEG with a single activation site based on
coupling of mPEG to a substituted benzene ring is disclosed in
European Patent Application Publication No. 473 084 A2. However,
this structure contains a benzene ring that could have toxic
effects if the structure is destroyed in a living organism.
[0029] Another branched mPEG with a single activation site has been
prepared through a complex synthesis in which an active succinate
moiety is attached to the mPEG through a weak ester linkage that is
susceptible to hydrolysis. An mPEG--OH is reacted with succinic
anhydride to make the succinate. The reactive succinate is then
activated as the succinimide. The synthesis, starting with the
active succinimide, includes the following steps, represented
structurally below. 11
[0030] The mPEG activated as the succinimide, mPEG succinimidyl
succinate, is reacted in the first step as shown above with
norleucine. The symbol --R in the synthesis represents the n-butyl
moiety of norleucine. The mPEG and norleucine conjugate (A) is
activated as the succinimide in the second step by reaction with
N-hydroxy succinimide. As represented in the third step, the mPEG
and norleucine conjugate activated as the succinimide (B) is
coupled to the alpha and epsilon amino moieties of lysine to create
an mPEG disubstituted lysine (C) having a reactive carboxyl moiety.
In the fourth step, the mPEG disubstituted lysine is activated as
the succinimide.
[0031] The ester linkage formed from the reaction of the mPEG--OH
and succinic anhydride molecules is a weak linkage that is
hydrolytically unstable. In vivo application is therefore limited.
Also, purification of the branched mPEG is precluded by commonly
used chromatographic techniques in water, which normally would
destroy the molecule.
[0032] The molecule also has relatively large molecular fragments
between the carboxyl group activated as the succinimide and the
mPEG moieties due to the number of steps in the synthesis and to
the number of compounds used to create the fragments. These
molecular fragments are sometimes referred to as "linkers" or
"spacer arms," and have the potential to act as antigenic sites
promoting the formation of antibodies upon injection and initiating
an undesirable immunological response in a living organism.
SUMMARY OF THE INVENTION
[0033] The invention provides a branched or "multi-armed"
amphiphilic polymer derivative that is monofunctional,
hydrolytically stable, can be prepared in a simple, one-step
reaction, and possesses no aromatic moieties in the linker
fragments forming the linkages with the polymer moieties. The
derivative can be prepared without any toxic linkage or potentially
toxic fragments. Relatively pure polymer molecules of high
molecular weight can be created. The polymer can be purified by
chromatography in water. A multi-step method can be used if it is
desired to have polymer arms that differ in molecular weight. The
polymer arms are capped with relatively nonreactive end groups. The
derivative can include a single reactive site that is located along
the polymer backbone rather than on the terminal portions of the
polymer moieties. The reactive site can be activated for selective
reactions.
[0034] The multi-armed polymer derivative of the invention having a
single reactive site can be used for, among other things, protein
modification with a high retention of protein activity. Protein and
enzyme activity can be preserved and in some cases is enhanced. The
single reactive site can be converted to a functional group for
highly selective coupling to proteins, enzymes, and surfaces. A
larger, more dense polymer cloud can be created surrounding a
biomolecule with fewer attachment points to the biomolecule as
compared to conventional polymer derivatives having terminal
functional groups. Hydrolytically weak ester linkages can be
avoided. Potentially harmful or toxic products of hydrolysis can be
avoided. Large linker fragments can be avoided so as to avoid an
antigenic response in living organisms. Cross linking is
avoided.
[0035] The molecules of the invention can be represented
structurally as poly.sub.a-P--CR(--Q-poly.sub.b)-Z or: 12
[0036] Poly.sub.a and poly.sub.brepresent nonpeptidic and
substantially nonreactive water soluble polymeric arms that may be
the same or different. C represents carbon. P and Q represent
linkage fragments that may be the same or different and that join
polymer arms poly.sub.aand poly.sub.b, respectively, to C by
hydrolytically stable linkages in the absence of aromatic rings in
the linkage fragments. R is a moiety selected from the group
consisting of H, substantially nonreactive, usually alkyl,
moieties, and linkage fragments attached by a hydrolytically stable
linkage in the absence of aromatic rings to a nonpeptidic and
substantially nonreactive water soluble polymeric arm. The moiety
--Z comprises a moiety selected from the group consisting of
moieties having a single site reactive toward nucleophilic
moieties, sites that can be converted to sites reactive toward
nucleophilic moieties, and the reaction product of a nucleophilic
moiety and moieties having a single site reactive toward
nucleophilic moieties.
[0037] Typically, the moiety --P--CR(--Q--)--Z is the reaction
product of a linker moiety and the reactive site of monofunctional,
nonpeptidic polymer derivatives, poly.sub.a-W and poly.sub.b-W, in
which W is the reactive site. Polymer arms poly.sub.a and
poly.sub.b are nonpeptidic polymers and can be selected from
polymers that have a single reactive moiety that can be activated
for hydrolytically stable coupling to a suitable linker moiety. The
linker has the general structure X--CR--(Y)--Z, in which X and Y
represent fragments that contain reactive sites for coupling to the
polymer reactive site W to form linkage fragments P and Q,
respectively.
[0038] In one embodiment, at least one of the polymer arms is a
poly(ethylene glycol) moiety capped with an essentially nonreactive
end group, such as a monomethoxy-poly(ethylene glycol) moiety
("mPEG--"), which is capped with a methyl end group, CH.sub.3--.
The other branch can also be an mPEG moiety of the same or
different molecular weight, another poly(ethylene glycol) moiety
that is capped with an essentially nonreactive end group other than
methyl, or a different nonpeptidic polymer moiety that is capped
with a nonreactive end group such as a capped poly(alkylene oxide),
a poly(oxyethylated polyol), a poly(olefinic alcohol), or
others.
[0039] For example, in one embodiment poly.sub.a and poly.sub.b are
each monomethoxy-poly(ethylene glycol) ("mPEG") of the same or
different molecular weight. The mPEG-disubstituted derivative has
the general structure mPEG.sub.a--P--CH(--Q-mPEG.sub.b)--Z. The
moieties mPEG.sub.a-- and mPEG.sub.b-- have the structure
CH.sub.3--(CH.sub.2CH.sub.2O).sub.nCH- .sub.2CH.sub.2-- and n may
be the same or different for mPEG.sub.a and mPEG.sub.b. Molecules
having values of n of from 1 to about 1,150 are contemplated.
[0040] The linker fragments P and Q contain hydrolytically stable
linkages that may be the same or different depending upon the
functional moiety on the mPEG molecules and the molecular structure
of the linker moiety used to join the mPEG moieties in the method
for synthesizing the multi-armed structure. The linker fragments
typically are alkyl fragments containing amino or thiol residues
forming a linkage with the residue of the functional moiety of the
polymer. Depending on the degree of substitution desired, linker
fragments P and Q can include reactive sites for joining additional
monofunctional nonpeptidic polymers to the multi-armed
structure.
[0041] The moiety --R can be a hydrogen atom, H, a nonreactive
fragment, or, depending on the degree of substitution desired, R
can include reactive sites for joining additional monofunctional
nonpeptidic polymers to the multi-armed structure.
[0042] The moiety --Z can include a reactive moiety for which the
activated nonpeptidic polymers are not selective and that can be
subsequently activated for attachment of the derivative to enzymes,
other proteins, nucleotides, lipids, liposomes, other molecules,
solids, particles, or surfaces. The moiety --Z can include a
linkage fragment --R.sub.z. Depending on the degree of substitution
desired, the R.sub.z fragment can include reactive sites for
joining additional monofunctional nonpeptidic polymers to the
multi-armed structure.
[0043] Typically, the --Z moiety includes terminal functional
moieties for providing linkages to reactive sites on proteins,
enzymes, nucleotides, lipids, liposomes, and other materials. The
moiety --Z is intended to have a broad interpretation and to
include the reactive moiety of monofunctional polymer derivatives
of the invention, activated derivatives, and conjugates of the
derivatives with polypeptides and other substances. The invention
includes biologically active conjugates comprising a biomolecule,
which is a biologically active molecule, such as a protein or
enzyme, linked through an activated moiety to the branched polymer
derivative of the invention. The invention includes biomaterials
comprising a solid such as a surface or particle linked through an
activated moiety to the polymer derivatives of the invention.
[0044] In one embodiment, the polymer moiety is an mPEG moiety and
the polymer derivative is a two-armed mPEG derivative based upon
hydrolytically stable coupling of mPEG to lysine. The mPEG moieties
are represented structurally as
CH.sub.3O--(CH.sub.2CH.sub.2O).sub.nCH.sub.2C- H.sub.2-- wherein n
may be the same or different for poly.sub.a- and poly.sub.b- and
can be from 1 to about 1,150 to provide molecular weights of from
about 100 to 100,000. The --R moiety is hydrogen. The --Z moiety is
a reactive carboxyl moiety. The molecule is represented
structurally as follows: 13
[0045] The reactive carboxyl moiety of hydrolytically stable
mPEG-disubstituted lysine, which can also be called alpha,
epsilon-mPEG lysine, provides a site for interacting with ion
exchange chromatography media and thus provides a mechanism for
purifying the product. These purifiable, high molecular weight,
monofunctional compounds have many uses. For example,
mPEG-disubstituted lysine, activated as succinimidyl ester, reacts
with amino groups in enzymes under mild aqueous conditions that are
compatible with the stability of most enzymes. The
mPEG-disubstituted lysine of the invention, activated as the
succinimidyl ester, is represented as follows: 14
[0046] The invention includes methods of synthesizing the polymers
of the invention. The methods comprise reacting an active suitable
polymer having the structure poly-W with a linker moiety having the
structure X--CR--(Y)Z to form poly.sub.a-P--CR(--Q-poly.sub.b)-Z.
The poly moiety in the structure poly-W can be either poly.sub.a or
poly.sub.b and is a polymer having a single reactive moiety W. The
W moiety is an active moiety that is linked to the polymer moiety
directly or through a hydrolytically stable linkage. The moieties X
and Y in the structure X--CR--(Y)Z are reactive with W to form the
linkage fragments Q and P, respectively. If the moiety R includes
reactive sites similar to those of X and Y, then R can also be
modified with a poly-W, in which the poly can be the same as or
different from poly.sub.a or poly.sub.b. The moiety Z normally does
not include a site that is reactive with W. However, X, Y, R, and Z
can each include one or more such reactive sites for preparing
monofunctional polymer derivatives having more than two
branches.
[0047] The method of the invention typically can be accomplished in
one or two steps. The method can include additional steps for
preparing the compound poly-W and for converting a reactive Z
moiety to a functional group for highly selective reactions.
[0048] The active Z moiety includes a reactive moiety that is not
reactive with W and can be activated subsequent to formation of
poly.sub.a-P--CR(--Q-poly.sub.b)-Z for highly selective coupling to
selected reactive moieties of enzymes and other proteins or
surfaces or any molecule having a reactive nucleophilic moiety for
which it is desired to modify the characteristics of the
molecule.
[0049] In additional embodiments, the invention provides a
multi-armed mPEG derivative for which preparation is simple and
straightforward. Intermediates are water stable and thus can be
carefully purified by standard aqueous chromatographic techniques.
Chlorotriazine activated groups are avoided and more highly
selective functional groups are used for enhanced selectivity of
attachment and much less loss of activity upon coupling of the mPEG
derivatives of the invention to proteins, enzymes, and other
peptides. Large spacer arms between the coupled polymer and protein
are avoided to avoid introducing possible antigenic sites. Toxic
groups, including triazine, are avoided. The polymer backbone
contains no hydrolytically weak ester linkages that could break
down during in vivo applications. Monofunctional polymers of double
the molecular weight as compared to the individual mPEG moieties
can be provided, with mPEG dimer structures having molecular
weights of up to at least about 50,000, thus avoiding the common
problem of difunctional impurities present in conventional, linear
mPEGs.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] FIGS. 1(a), 1(b), and 1(c) illustrate the time course of
digestion of ribonuclease (.circle-solid.), conventional, linear
mPEG-modified ribonuclease (.largecircle.), and ribonuclease
modified with a multi-armed mPEG of the invention (.box-solid.) as
assessed by enzyme activity upon incubation with pronase (FIG.
1(a)), elastase (FIG. 1(b)), and subtilisin (FIG. 1(c)).
[0051] FIGS. 2(a) and 2(b) illustrate stability toward heat (FIG.
2(a)) and pH (FIG. 2(b)) of ribonuclease (.circle-solid.), linear
mPEG-modified ribonuclease (.largecircle.), and ribonuclease
modified with a multi-armed mPEG of the invention (.quadrature.).
FIG. 2(a) is based on data taken after a 15 minute incubation
period at the indicated temperatures. FIG. 2(b) is based on data
taken over a 20 hour period at different pH values.
[0052] FIGS. 3(a) and 3(b) illustrate the time course of digestion
for catalase (.circle-solid.), linear mPEG-modified catalase
(.largecircle.), and catalase modified with a multi-armed mPEG of
the invention (.box-solid.) as assessed by enzyme activity upon
incubation with pronase (FIG. 3(a)) and trypsin (FIG. 3(b)).
[0053] FIG. 4 illustrates the stability of catalase
(.circle-solid.), linear mPEG-modified catalase (.quadrature.), and
catalase modified with a multi-armed mPEG of the invention
(.largecircle.) for 20 hours incubation at the indicated pH
values.
[0054] FIG. 5 illustrates the time course of digestion of
asparaginase (.circle-solid.), linear mPEG-modified asparaginase
(.largecircle.), and asparaginase modified with a multi-armed mPEG
of the invention (.box-solid.) as assessed by enzyme activity assay
upon trypsin incubation.
[0055] FIG. 6 illustrates the time course of autolysis of trypsin
(.circle-solid.), linear mPEG-modified trypsin (.box-solid.), and
trypsin modified with a multi-armed mPEG of the invention
(.tangle-solidup.) evaluated as residual activity towards TAME
(alpha N-p-tosyl-arginine methyl ester).
DETAILED DESCRIPTION
[0056] I. Preparation of a Hydrolytically Stable mPEG-Disubstituted
Lysine
[0057] Two procedures are described for the preparation of a
hydrolytically stable, two-armed, mPEG-disubstituted lysine. The
first procedure is a two step procedure, meaning that the lysine is
substituted with each of the two mPEG moieties in separate reaction
steps. Monomethoxy-poly(ethylene glycol) arms of different lengths
or of the same length can be substituted onto the lysine molecule,
if desired, using the two step procedure. The second procedure is a
one step procedure in which the lysine molecule is substituted with
each of the two mPEG moieties in a single reaction step. The one
step procedure is suitable for preparing mPEG-disubstituted lysine
having mPEG moieties of the same length.
[0058] Unlike prior multisubstituted structures, no aromatic ring
is present in the linkage joining the nonpeptidic polymer arms
produced by either the one or two step methods described below that
could result in toxicity if the molecule breaks down in vivo. No
hydrolytically weak ester linkages are present in the linkage.
Lengthy linkage chains that could promote an antigenic response are
avoided.
[0059] The terms "group," "functional group," "moiety," "active
moiety," "reactive site," "radical," and similar terms are 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 or
fragment of a molecule. "Reactive site," "functional group," and
"active moiety" refer to units that perform some function or have a
chemical activity and are reactive with other molecules or portions
of molecules. In this sense a protein or a protein residue can be
considered as a molecule and as a functional moiety when coupled to
a polymer. A polymer, such as mPEG--COOH has a reactive site, the
carboxyl moiety, --COOH, that can be converted to a functional
group for selective reactions and attachment to proteins and linker
moieties. The converted polymer is said to be activated and to have
an active moiety, while the --COOH group is relatively nonreactive
in comparison to an active moiety.
[0060] The term "nonreactive" is used herein primarily to refer to
a moiety that does not readily react chemically with other
moieties, such as the methyl alkyl moiety. However, the term
"nonreactive" should be understood to exclude carboxyl and hydroxyl
moieties, which, although relatively nonreactive, can be converted
to functional groups that are of selective reactivity.
[0061] The term "biologically active" means a substance, such as a
protein, lipid, or nucleotide that has some activity or function in
a living organism or in a substance taken from a living organism.
For example, an enzyme can catalyze chemical reactions. The term
"biomaterial" is somewhat imprecise, and is used herein to refer to
a solid material or particle or surface that is compatible with
living organisms or tissue or fluids. For example, surfaces that
contact blood, whether in vitro or in vivo, can be made nonfouling
by attachment of the polymer derivatives of the invention so that
proteins do not become attached to the surface.
[0062] A. Two Step Procedure
[0063] For the two step procedure, an activated mPEG is prepared
for coupling to free lysine monomer and then the lysine monomer is
disubstituted with the activated mPEG in two steps. The first step
occurs in aqueous buffer. The second step occurs in dry methylene
chloride. The active moiety of the mPEG for coupling to the lysine
monomer can be selected from a number of activating moieties having
leaving moieties that are reactive with the amino moieties of
lysine monomer. A commercially available activated mPEG,
mPEG-p-nitrophenylcarbonate, the preparation of which is discussed
below, was used to exemplify the two step procedure.
[0064] The two step procedure can be represented structurally as
follows: 15
[0065] Step 1. Preparation of mPEG-monosubstituted lysine.
Modification of a single lysine amino group was accomplished with
mPEG-p-nitrophenylcarbo- nate in aqueous solution where both lysine
and mPEG-p-nitrophenylcarbonate are soluble. The
mPEG-p-nitrophenylcarbonate has only limited stability in aqueous
solution. However, lysine is not soluble in organic solvents in
which the activated mPEG is stable. Consequently, only one lysine
amino group is modified by this procedure. NMR confirms that the
epsilon amino group is modified. Nevertheless, the procedure allows
ready chloroform extraction of mPEG-monosubstituted lysine from
unreacted lysine and other water soluble by-products, and so the
procedure provides a desirable monosubstituted product for
disubstitution.
[0066] To prepare the mPEG-monosubstituted lysine, 353 milligrams
of lysine, which is about 2.5 millimoles, was dissolved in 20
milliliters of water at a pH of about 8.0 to 8.3. Five grams of
mPEG-p-nitrophenylcarbon- ate of molecular weight 5,000, which is
about 1 millimole, was added in portions over 3 hours. The pH was
maintained at 8.3 with 0.2 N NaOH. The reaction mixture was stirred
overnight at room temperature. Thereafter, the reaction mixture was
cooled to 0.degree. C. and brought to a pH of about 3 with 2 N HCl.
Impurities were extracted with diethyl ether. The mPEG
monosubstituted lysine, having the mPEG substituted at the epsilon
amino group of lysine as confirmed by NMR analysis, was extracted
three times with chloroform. The solution was dried. After
concentration, the solution was added drop by drop to diethyl ether
to form a precipitate. The precipitate was collected and then
crystallized from absolute ethanol. The percentage of modified
amino groups was 53%, calculated by calorimetric analysis.
[0067] Step 2. Preparation of mPEG-Disubstituted Lysine. The
mPEG-monosubstituted lysine product from step 1 above is soluble in
organic solvents and so modification of the second lysine amino
moiety can be achieved by reaction in dry methylene chloride.
Activated mPEG, mPEG-p-nitrophenylcarbonate, is soluble and stable
in organic solvents and can be used to modify the second lysine
amino moiety.
[0068] Triethylamine ("TEA") was added to 4.5 grams of
mPEG-monosubstituted lysine, which is about 0.86 millimoles. The
mixture of TEA and mPEG-monosubstituted lysine was dissolved in 10
milliliters of anhydrous methylene chloride to reach a pH of 8.0.
Four and nine tenths grams of mPEG-p-nitrophenycarbonate of
molecular weight 5,000, which is 1.056 millimoles, was added over 3
hours to the solution. If it is desirable to make an mPEG
disubstituted compound having mPEG arms of different lengths, then
a different molecular weight mPEG could have been used. The pH was
maintained at 8.0 with TEA. The reaction mixture was refluxed for
72 hours, brought to room temperature, concentrated, filtered,
precipitated with diethyl ether and then crystallized in a minimum
amount of hot ethanol. The excess of activated mPEG,
mPEG-p-nitrophenycarbonate, was deactivated by hydrolysis in an
alkaline aqueous medium by stirring overnight at room temperature.
The solution was cooled to 0.degree. C. and brought to a pH of
about 3 with 2 N HCl.
[0069] p-Nitrophenol was removed by extraction with diethyl ether.
Monomethyl-poly(ethylene glycol)-disubstituted lysine and remaining
traces of mPEG were extracted from the mixture three times with
chloroform, dried, concentrated, precipitated with diethyl ether
and crystallized from ethanol. No unreacted lysine amino groups
remained in the polymer mixture as assessed by colorimetric
analysis.
[0070] Purification of mPEG-disubstituted lysine and removal of
mPEG were accomplished by gel filtration chromatography using a Bio
Gel P100 (Bio-Rad) column. The column measured 5 centimeters by 50
centimeters. The eluent was water. Fractions of 10 milliliters were
collected. Up to 200 milligrams of material could be purified for
each run. The fractions corresponding to mPEG-disubstituted lysine
were revealed by iodine reaction. These fractions were pooled,
concentrated, and then dissolved in ethanol and concentrated. The
mPEG-disubstituted lysine product was dissolved in methylene
chloride, precipitated with diethyl ether, and crystallized from
ethanol.
[0071] The mPEG-disubstituted lysine was also separated from
unmodified mPEG--OH and purified by an alternative method. Ion
exchange chromatography was performed on a QAE Sephadex A50 column
(Pharmacia) that measured 5 centimeters by 80 centimeters. An 8.3
mM borate buffer of pH 8.9 was used. This alternative procedure
permitted fractionation of a greater amount of material per run
than the other method above described (up to four grams for each
run).
[0072] For both methods of purification, purified
mPEG-disubstituted lysine of molecular weight 10,000, titrated with
NaOH, showed that 100% of the carboxyl groups were free carboxyl
groups. These results indicate that the reaction was complete and
the product pure.
[0073] The purified mPEG-disubstituted lysine was also
characterized by .sup.1H-NMR on a 200 MHz Bruker instrument in
dimethyl sulfoxide, d6, at a 5% weight to volume concentration. The
data confirmed the expected molecular weight of 10,000 for the
polymer. The chemical shifts and assignments of the protons in the
mPEG-disubstituted lysine are as follows: 1.2-1.4 ppm (multiplet,
6H, methylenes 3,4,5 of lysine); 1.6 ppm (multiplet, 2H, methylene
6 of lysine); 3.14 ppm (s, 3H, terminal mPEG methoxy); 3.49 ppm (s,
mPEG backbone methylene); 4.05 ppm (t, 2H, --CH.sub.2, --OCO--);
7.18 ppm (t, 1H, --NH-- lysine); and 7.49 ppm (d, 1H, --NH--
lysine).
[0074] The above signals are consistent with the reported structure
since two different carbamate NH protons are present. The first
carbamate NH proton (at 7.18 ppm) shows a triplet for coupling with
the adjacent methylene group. The second carbamate NH proton (at
7.49 ppm) shows a doublet because of coupling with the .alpha.-CH
of lysine. The intensity of these signals relative to the mPEG
methylene peak is consistent with the 1:1 ratio between the two
amide groups and the expected molecular weight of 10,000 for the
polymer.
[0075] The two step procedure described above allows polymers of
different types and different lengths to be linked with a single
reactive site between them. The polymer can be designed to provide
a polymer cloud of custom shape for a particular application.
[0076] The commercially available activated mPEG,
mPEG-p-nitrophenylcarbon- ate, is available from Shearwater
Polymers, Inc. in Huntsville, Ala. This compound was prepared by
the following procedure, which can be represented structurally as
follows: 16
[0077] Five grams of mPEG--OH of molecular weight 5,000, or 1
millimole, were dissolved in 120 milliliters of toluene and dried
azeotropically for 3 hours. The solution was cooled to room
temperature and concentrated under vacuum. Reactants added to the
concentrated solution under stirring at 0.degree. C. were 20
milliliters of anhydrous methylene chloride and 0.4 g of
p-nitrophenylchloroformate, which is 2 millimoles. The pH of the
reaction mixture was maintained at 8 by adding 0.28 milliliters of
triethylamine ("TEA"), which is 2 millimoles. The reaction mixture
was allowed to stand overnight at room temperature. Thereafter, the
reaction mixture was concentrated under vacuum to about 10
milliliters, filtered, and dropped into 100 milliliters of stirred
diethyl ether. A precipitate was collected from the diethyl ether
by filtration and crystallized twice from ethyl acetate. Activation
of mPEG was determined to be 98%. Activation was calculated
spectrophotometrically on the basis of the absorption at 400 nm in
alkaline media after 15 minutes of released 4-nitrophenol
(.epsilon. of p-nitrophenol at 400 nm equals 17,000).
[0078] B. One Step Procedure
[0079] In the one step procedure, mPEG disubstituted lysine is
prepared from lysine and an activated mPEG in a single step as
represented structurally below: 17
[0080] Except for molecular weight attributable to a longer PEG
backbone in the activated mPEG used in the steps below, the mPEG
disubstituted lysine of the one step procedure does not differ
structurally from the mPEG disubstituted lysine of the two step
procedure. It should be recognized that the identical compound,
having the same molecular weight, can be prepared by either
method.
[0081] Preparation of mPEG disubstituted lysine by the one step
procedure proceeded as follows: Succinimidylcarbonate mPEG of
molecular weight about 20,000 was added in an amount of 10.8 grams,
which is 5.4.times.10.sup.-4 moles, to 40 milliliters of lysine HCl
solution. The lysine HCL solution was in a borate buffer of pH 8.0.
The concentration was 0.826 milligrams succinimidylcarbonate mPEG
per milliliter of lysine HCL solution, which is
1.76.times.10.sup.-4 moles. Twenty milliliters of the same buffer
was added. The solution pH was maintained at 8.0 with aqueous NaOH
solution for the following 8 hours. The reaction mixture was
stirred at room temperature for 24 hours.
[0082] Thereafter, the solution was diluted with 300 milliliters of
deionized water. The pH of the solution was adjusted to 3.0 by the
addition of oxalic acid. The solution was then extracted three
times with dichloromethane. The combined dichloromethane extracts
were dried with anhydrous sodium sulphate and filtered. The
filtrate was concentrated to about 30 milliliters. The product, an
impure mPEG disubstituted lysine, was precipitated with about 200
milliliters of cold ethyl ether. The yield was 90%.
[0083] Nine grams of the above impure mPEG-disubstituted lysine
reaction product was dissolved in 4 liters of distilled water and
then loaded onto a column of DEAE Sepharose FF, which is 500
milliliters of gel equilibrated with 1500 milliliters of boric acid
in a 0.5% sodium hydroxide buffer at a pH of 7.0. The loaded system
was then washed with water. Impurities of succinimidylcarbonate
mPEG and mPEG-monosubstituted lysine, both of molecular weight
about 20,000, were washed off the column. However, the desired mPEG
disubstituted lysine of molecular weight 20,000 was eluted with 10
mM NaCl. The pH of the eluate was adjusted to 3.0 with oxalic acid
and then mPEG disubstituted lysine was extracted with
dichloromethane, dried with sodium sulphate, concentrated, and
precipitated with ethyl ether. Five and one tenth grams of purified
mPEG disubstituted lysine were obtained. The molecular weight was
determined to be 38,000 by gel filtration chromatography and 36,700
by potentiometric titration.
[0084] The one step procedure is simple in application and is
useful for producing high molecular weight dimers that have
polymers of the same type and length linked with a single reactive
site between them.
[0085] Additional steps are represented below for preparing
succinimidylcarbonate mPEG for disubstitution of lysine. 18
[0086] Succinimidylcarbonate mPEG was prepared by dissolving 30
grams of mPEG--OH of molecular weight 20,000, which is about 1.5
millimoles, in 120 milliliters of toluene. The solution was dried
azeotropically for 3 hours. The dried solution was cooled to room
temperature. Added to the cooled and dried solution were 20
milliliters of anhydrous dichloromethane and 2.33 milliliters of a
20% solution of phosgene in toluene. The solution was stirred
continuously for a minimum of 16 hours under a hood due to the
highly toxic fumes.
[0087] After distillation of excess phosgene and solvent, the
remaining syrup, which contained mPEG chlorocarbonate, was
dissolved in 100 milliliters of anhydrous dichloromethane, as
represented above. To this solution was added 3 millimoles of
triethylamine and 3 millimoles of N-hydroxysuccinimide. The
reaction mixture remained standing at room temperature for 24
hours. Thereafter, the solution was filtered through a silica gel
bed of pore size 60 Angstroms that had been wetted with
dichloromethane. The filtrate was concentrated to 70 milliliters.
Succinimidylcarbonate mPEG of molecular weight about 20,000 was
precipitated in ethyl ether and dried in vacuum for a minimum of 8
hours. The yield was 90%. Succinimidylcarbonate-mPEG is available
commercially from Shearwater Polymers in Huntsville, Ala.
[0088] The mPEG disubstituted lysine of the invention can be
represented structurally more generally as
poly.sub.a-P--CR(--Q-poly.sub.b)-Z or: 19
[0089] For the mPEG disubstituted lysines described above,
--P--CR(--Q--)--Z is the reaction product of a precursor linker
moiety having two reactive amino groups and active monofunctional
precursors of poly.sub.a and poly.sub.b that have been joined to
the linker moiety at the reactive amino sites. Linker fragments Q
and P contain carbamate linkages formed by joining the amino
containing portions of the lysine molecule with the functional
group with which the mPEG was substituted. The linker fragments are
selected from --O--C(O)NH(CH.sub.2).sub.4-- and --O--C(O)NH-- and
are different in the exemplified polymer derivative. However, it
should be recognized that P and Q could both be
--O--C(O)NH(CH.sub.2).sub.4-- or --O--C(O)NH-- or some other
linkage fragment, as discussed below. The moiety represented by R
is hydrogen, H. The moiety represented by Z is the carboxyl group,
--COOH. The moieties P, R, Q, and Z are all joined to a central
carbon atom.
[0090] The nonpeptidic polymer arms, poly.sub.a and poly.sub.b, are
mPEG moieties mPEG.sub.a and mPEG.sub.b, respectively, and are the
same on each of the linker fragments Q and P for the examples
above. The mPEG moieties have a structure represented as
CH.sub.3O--(CH.sub.2CH.sub.2O).s- ub.nCH.sub.2CH.sub.2--. For the
mPEG disubstituted lysine made by the one step method, n is about
454 to provide a molecular weight for each mPEG moiety of 20,000
and a dimer molecular weight of 40,000. For the mPEG disubstituted
lysine made by the two step method, n is about 114 to provide a
molecular weight for each mPEG moiety of 5,000 and a dimer
molecular weight of 10,000.
[0091] Lysine disubstituted with mPEG and having as dimer molecular
weights of 10,000 and 40,000 and procedures for preparation of
mPEG-disubstituted lysine have been shown. However, it should be
recognized that mPEG disubstituted lysine and other multi-armed
compounds of the invention can be made in a variety of molecular
weights, including ultra high molecular weights. High molecular
weight monofunctional PEGs are otherwise difficult to obtain.
[0092] Polymerization of ethylene oxide to yield mPEGs usually
produces molecular weights of up to about 20,000 to 25,000 g/mol.
Accordingly, two-armed mPEG disubstituted lysines of molecular
weight of about 40,000 to 50,000 can be made according to the
invention. Higher molecular weight lysine disubstituted PEGs can be
made if the chain length of the linear mPEGs is increased, up to
about 100,000. Higher molecular weights can also be obtained by
adding additional monofunctional nonpeptidic polymer arms to
additional reactive sites on a linker moiety, within practical
limits of steric hindrance. However, no unreacted active sites on
the linker should remain that could interfere with the
monofunctionality of the multi-armed derivative. Lower molecular
weight disubstituted mPEGs can also be made, if desired, down to a
molecular weight of about 100 to 200.
[0093] It should be recognized that a wide variety of linker
fragments P and Q are available, although not necessarily with
equivalent results, depending on the precursor linker moiety and
the functional moiety with which the activated mPEG or other
nonpeptidic monofunctional polymer is substituted and from which
the linker fragments result. Typically, the linker fragments will
contain the reaction products of portions of linker moieties that
have reactive amino and/or thiol moieties and suitably activated
nonpeptidic, monofunctional, water soluble polymers.
[0094] For example, a wide variety of activated mPEGs are available
that form a wide variety of hydrolytically stable linkages with
reactive amino moieties. Linkages can be selected from the group
consisting of amide, amine, ether, carbamate, which are also called
urethane linkages, urea, thiourea, thiocarbamate, thiocarbonate,
thioether, thioester, dithiocarbonate linkages, and others.
However, hydrolytically weak ester linkages and potentially toxic
aromatic moieties are to be avoided.
[0095] Hydrolytic stability of the linkages means that the linkages
between the polymer arms and the linker moiety are stable in water
and that the linkages do not react with water at useful pHs for an
extended period of time of at least several days, and potentially
indefinitely. Most proteins could be expected to lose their
activity at a caustic pH of 11 or higher, so the derivatives should
be stable at a pH of less than about 11.
[0096] Examples of the above linkages and their formation from
activated mPEG and lysine are represented structurally below.
[0097] a) Formation of Amide Linkage 20
[0098] b) Formation of Carbamate Linkage 21
[0099] c) Formation of Urea Linkage 22
[0100] d) Formation of Thiourea Linkage 23
[0101] e) Formation of Amine Linkage 24
[0102] One or both of the reactive amino moieties, --NH.sub.2, of
lysine or another linker moiety can be replaced with thiol
moieties, --SH. Where the linker moiety has a reactive thiol moiety
instead of an amino moiety, then the linkages can be selected from
the group consisting of thioester, thiocarbonate, thiocarbamate,
dithiocarbamate, thioether linkages, and others. The above linkages
and their formation from activated mPEG and lysine in which both
amino moieties have been replaced with thiol moieties are
represented structurally below.
[0103] a) Formation of Thioester Linkage 25
[0104] b) Formation of Thiocarbonate Linkage 26
[0105] c) Formation of Thiocarbamate Linkage 27
[0106] d) Formation of Dithiocarbamate Linkage 28
[0107] e) Formation of Thioether Linkage 29
[0108] It should be apparent that the mPEG or other monofunctional
polymer reactants can be prepared with a reactive amino moiety and
then linked to a suitable linker moiety having reactive groups such
as those shown above on the mPEG molecule to form hydrolytically
stable linkages as discussed above. For example, the amine linkage
could be formed as follows: 30
[0109] Examples of various active electrophilic moieties useful for
activating polymers or linking moieties for biological and
biotechnical applications in which the active moiety is reacted to
form hydrolytically stable linkages in the absence of aromatic
moieties include trifluoroethylsulfonate, isocyanate,
isosthiocyanate, active esters, active carbonates, various
aldehydes, various sulfones, including chloroethylsulfone and
vinylsulfone, maleimide, iodoacetamide, and iminoesters. Active
esters include N-hydroxylsuccinimidyl ester. Active carbonates
include N-hydroxylsuccinimidyl carbonate, p-nitrophenylcarbonate,
and trichlorophenylcarbonate. These electrophilic moieties are
examples of those that are suitable as Ws in the structure poly-W
and as Xs and Ys in the linker structure X--CR(--Y)--Z.
[0110] Nucleophilic moieties for forming the linkages can be amino,
thiol, and hydroxyl. Hydroxyl moieties form hydrolytically stable
linkages with isocyanate electrophilic moieties. Also, it should be
recognized that the linker can be substituted with different
nucleophilic or electrophilic moieties or both electrophilic and
nucleophilic moieties depending on the active moieties on the
monofunctional polymers with which the linker moiety is to be
substituted.
[0111] Linker moieties other than lysine are available for
activation and for disubstitution or multisubstitution with mPEG
and related polymers for creating multi-armed structures in the
absence of aromatic moieties in the structure and that are
hydrolytically stable. Examples of such linker moieties include
those having more than one reactive site for attachment of various
monofunctional polymers.
[0112] Linker moieties can be synthesized to include multiple
reactive sites such as amino, thiol, or hydroxyl groups for joining
multiple suitably activated mPEGs or other nonpeptidic polymers to
the molecule by hydrolytically stable linkages, if it is desired to
design a molecule having multiple nonpeptidic polymer branches
extending from one or more of the linker arm fragments. The linker
moieties should also include a reactive site, such as a carboxyl or
alcohol moiety, represented as --Z in the general structure above,
for which the activated polymers are not selective and that can be
subsequently activated for selective reactions for joining to
enzymes, other proteins, surfaces, and the like.
[0113] For example, one suitable linker moiety is a diamino alcohol
having the structure 31
[0114] The diamino alcohol can be disubstituted with activated mPEG
or other suitable activated polymers similar to disubstitution of
lysine and then the hydroxyl moiety can be activated as follows:
32
[0115] Other diamino alcohols and alcohols having more than two
amino or other reactive groups for polymer attachment are useful. A
suitably activated mPEG or other monofunctional, nonpeptidic, water
soluble polymer can be attached to the amino groups on such a
diamino alcohol similar to the method by which the same polymers
are attached to lysine as shown above. Similarly, the amino groups
can be replaced with thiol or other active groups as discussed
above. However, only one hydroxyl group, which is relatively
nonreactive, should be present in the --Z moiety, and can be
activated subsequent to polymer substitution.
[0116] The moiety --Z can include a reactive moiety or functional
group, which normally is a carboxyl moiety, hydroxyl moiety, or
activated carboxyl or hydroxyl moiety. The carboxyl and hydroxyl
moieties are somewhat nonreactive as compared to the thiol, amino,
and other moieties discussed above. The carboxyl and hydroxyl
moieties typically remain intact when the polymer arms are attached
to the linker moiety and can be subsequently activated. The
carboxyl and hydroxyl moieties also provide a mechanism for
purification of the multisubstituted linker moiety. The carboxyl
and hydroxyl moieties provide a site for interacting with ion
exchange chromatography media.
[0117] The moiety --Z may also include a linkage fragment,
represented as R.sub.z in the moiety, which can be substituted or
unsubstituted, branched or linear, and joins the reactive moiety to
the central carbon. Where a reactive group of the --Z moiety is
carboxyl, for activation after substitution with nonpeptidic
polymers, then the --Z moiety has the structure --R.sub.z--COOH if
the R.sub.z fragment is present. For hydroxyl, the structure is
--R.sub.z--OH. For example, in the diamino alcohol structure
discussed above, R.sub.z is CH.sub.2. It should be understood that
the carboxyl and hydroxyl moieties normally will extend from the
R.sub.z terminus, but need not necessarily do so.
[0118] R.sub.z can also include the reaction product of one or more
reactive moieties including reactive amino, thiol, or other
moieties, and a suitably activated mPEG arm or related nonpeptidic
polymer arm. In the latter event, R.sub.z can have the structure
(--L-poly.sub.c)-COOH or (--L-poly.sub.c)-OH in which --L-- is the
reaction product of a portion of the linker moiety and a suitably
activated nonpeptidic polymer, poly.sub.c-W, which is selected from
the same group as poly.sub.a-W and poly.sub.b-W but can be the same
or different from poly.sub.a-W and poly.sub.b-W.
[0119] It is intended that --Z have a broad definition. The moiety
--Z is intended to represent not only the reactive site of the
multisubstituted polymeric derivative that subsequently can be
converted to an active form and its attachment to the central
carbon, but the activated reactive site and also the conjugation of
the precursor activated site with another molecule, whether that
molecule be an enzyme, other protein or polypeptide, a
phospholipid, a preformed liposome, or on a surface to which the
polymer derivative is attached.
[0120] The skilled artisan should recognize that Z encompasses the
currently known activating moieties in PEG chemistry and their
conjugates. It should also be recognized that, although the linker
fragments represented by Q and P and R.sub.z should not contain
aromatic rings or hydrolytically weak linkages such as ester
linkages, such rings and such hydrolytically weak linkages may be
present in the active site moiety of --Z or in a molecule joined to
such active site. It may be desirable in some instances to provide
a linkage between, for example, a protein or enzyme and a
multisubstituted polymer derivative that has limited stability in
water. Some amino acids contain aromatic moieties, and it is
intended that the structure Z include conjugates of
multisubstituted monofunctional polymer derivatives with such
molecules or portions of molecules. Activated Zs and Zs including
attached proteins and other moieties are discussed below.
[0121] When lysine, the diamino alcohol shown above, or many other
compounds are linkers, then the central carbon has a nonreactive
hydrogen, H, attached thereto. In the general structure
poly.sub.a-P--CR(--Q-poly.sub.b)-Z, R is H. It should be recognized
that the moiety R can be designed to have another substantially
nonreactive moiety, such as a nonreactive methyl or other alkyl
group, or can be the reaction product of one or more reactive
moieties including reactive amino, thiol, or other moieties, and a
suitably activated mPEG arm or related nonpeptidic polymer arm. In
the latter event, R can have the structure --M-poly.sub.d, in which
--M-- is the reaction product of a portion of the linker moiety and
a suitably activated nonpeptidic polymer, poly.sub.d-W, which is
selected from the same group as poly.sub.a-W and poly.sub.b-W but
can be the same or different from poly.sub.a-W and
poly.sub.b-W.
[0122] For example, multi-armed structures can be made having one
or more mPEGs or other nonpeptidic polymer arms extending from each
portion P, Q, R, and R.sub.z, all of which portions extend from a
central carbon atom, C, which multi-armed structures have a single
reactive site for subsequent activation included in the structure
represented by Z. Upon at least the linker fragments P and Q are
located at least one active site for which the monofunctional,
nonpeptidic polymers are selective. These active sites include
amino moieties, thiol moieties, and other moieties as described
above.
[0123] The nonpeptidic polymer arms tend to mask antigenic
properties of the linker fragment, if any. A linker fragment length
of from 1 to 10 carbon atoms or the equivalent has been determined
to be useful to avoid a length that could provide an antigenic
site. Also, for all the linker fragments P, Q, R, and R.sub.z,
there should be an absence of aromatic moieties in the structure
and the linkages should be hydrolytically stable.
[0124] Poly(ethylene glycol) is useful in the practice of the
invention for the nonpeptidic polymer arms attached to the linker
fragments. PEG is used in biological applications because it has
properties that are highly desirable and is generally approved for
biological or biotechnical applications. PEG typically is clear,
colorless, odorless, soluble in water, stable to heat, inert to
many chemical agents, does not hydrolyze or deteriorate, and is
nontoxic. Poly(ethylene glycol) is considered to be biocompatible,
which is to say that PEG is capable of coexistence with living
tissues or organisms without causing harm. More specifically, PEG
is not immunogenic, which is to say that PEG does not tend to
produce an immune response in the body. When attached to a moiety
having some desirable function in the body, the PEG tends to mask
the moiety and can reduce or eliminate any immune response so that
an organism can tolerate the presence of the moiety. Accordingly,
the activated PEGs of the invention should be substantially
non-toxic and should not tend substantially to produce an immune
response or cause clotting or other undesirable effects.
[0125] The term "PEG" is used in the art and herein to describe any
of several condensation polymers of ethylene glycol having the
general formula represented by the structure
HO--(CH.sub.2CH.sub.2O).sub.nCH.sub.2 CH.sub.2--OH
[0126] or, more simply, as HO--PEG--OH. PEG is also known as
polyoxyethylene, polyethylene oxide, polyglycol, and polyether
glycol. PEG can be prepared as copolymers of ethylene oxide and
many other monomers.
[0127] Other water soluble polymers than PEG are suitable for
similar modification to create multi-armed structures that can be
activated for selective reactions. These other polymers include
poly(vinyl alcohol) ("PVA"); other poly(alkylene oxides) such as
poly(propylene glycol) ("PPG") and the like; and poly(oxyethylated
polyols) such as poly(oxyethylated glycerol), poly(oxyethylated
sorbitol), and poly(oxyethylated glucose), and the like. The
polymers can be homopolymers or random or block copolymers and
terpolymers based on the monomers of the above polymers, straight
chain or branched, or substituted or unsubstituted similar to mPEG
and other capped, monofunctional PEGs having a single active site
available for attachment to a linker.
[0128] Specific examples of suitable additional polymers include
poly(oxazoline), poly(acryloylmorpholine) ("PAcM"), and
poly(vinylpyrrolidone) ("PVP"). PVP and poly(oxazoline) are well
known polymers in the art and their preparation and use in the
syntheses described above for mPEG should be readily apparent to
the skilled artisan.
[0129] An example of the synthesis of a PVP disubstituted lysine
having a single carboxyl moiety available for activation is shown
below. The disubstituted compound can be purified, activated, and
used in various reactions for modification of molecules and
surfaces similarly to the mPEG-disubstituted lysine described
above. 33
[0130] Poly(acryloylmorpholine) "(PAcM)" functionalized at one end
is a new polymer, the structure, preparation, and characteristics
of which are described in Italian Patent Application No. MI 92 A
0002616, which was published May 17, 1994 and is entitled, in
English, "Polymers Of N-Acryloylmorpholine Functionalized At One
End And Conjugates With Bioactive Materials And Surfaces." Dimer
polymers of molecular weight up to at least about 80,000 can be
prepared using this polymer. The contents of the Italian patent
application are incorporated herein by reference.
[0131] PAcM can be used similarly to mPEG or PVP to create
multi-armed structures and ultra-high molecular weight polymers. An
example of a PAcM-disubstituted lysine having a single carboxyl
moiety available for activation is shown below. The disubstituted
compound can be purified, activated, and used in various reactions
for modification of molecules and surfaces similarly to the mPEG--
and PVP-disubstituted lysines described above. 34
[0132] It should also be recognized that the multi-armed
monofunctional polymers of the invention can be used for attachment
to a linker moiety to create a highly branched monofunctional
structure, within the practical limits of steric hindrance.
[0133] II. Activation of mPEG-Disubstituted Lysine and Modification
of Protein Amino Groups
[0134] Schemes are represented below for activating the
mPEG-disubstituted lysine product made by either the one step or
two step procedures and for linking the activated
mPEG-disubstituted lysine through a stable carbamate linkage to
protein amino groups to prepare polymer and protein conjugates.
Various other multisubstituted polymer derivatives as discussed
above can be activated similarly.
[0135] A. Activation of mPEG Disubstituted Lysine
[0136] Purified mPEG-disubstituted lysine produced in accordance
with the two step procedure discussed above was activated with
N-hydroxysuccinimide to produce mPEG-disubstituted lysine activated
as the succinimidyl ester. The reaction is represented structurally
below: 35
[0137] Six and two tenths grams of mPEG-disubstituted lysine of
molecular weight 10,000, which is about 0.6 millimoles, was
dissolved in 10 milliliters of anhydrous methylene chloride and
cooled to 0.degree. C. N-hydroxysuccinimide and
N,N-dicyclohexylcarbodiimide ("DCC") were added under stirring in
the amounts, respectively, of 0.138 milligrams, which is about 1.2
millimoles, and 0.48 milligrams, which is about 1.2 millimoles. The
reaction mixture was stirred overnight at room temperature.
Precipitated dicyclohexylurea was removed by filtration and the
solution was concentrated and precipitated with diethyl ether. The
product, mPEG disubstituted lysine activated as the succinimidyal
ester, was crystallized from ethyl acetate. The yield of
esterification, calculated on the basis of hydroxysuccinimide
absorption at 260 nm (produced by hydrolysis), was over 97%
(.epsilon. of hydroxysuccinimide at 260 nm=8,000
m.sup.-1cm.sup.-1). The NMR spectrum was identical to that of the
unactivated carboxylic acid except for the new succinimide singlet
at 2.80 ppm (2 Hs)
[0138] The procedure previously described for the activation of the
mPEG-disubstituted lysine of molecular weight 10,000 was also
followed for the activation of the higher molecular weight polymer
of molecular weight approximately 40,000 that was produced in
accordance with the one step procedure discussed above. The yield
was over 95% of high molecular weight mPEG-disubstituted lysine
activated as the succinimidyal ester.
[0139] It should be recognized that a number of activating groups
can be used to activate the multisubstituted polymer derivatives
for attachment to surfaces and molecules. Any of the activating
groups of the known derivatives of PEG can be applied to the
multisubstituted structure. For example, the mPEG-disubstituted
lysine of the invention was functionalized by activation as the
succinimidyl ester, which can be attached to protein amino groups.
However, there are a wide variety of functional moieties available
for activation of carboxilic acid polymer moieties for attachment
to various surfaces and molecules. Examples of active moieties used
for biological and biotechnical applications include
trifluoroethylsulfonate, isocyanate, isosthiocyanate, active
esters, active carbonates, various aldehydes, various sulfones,
including chloroethylsulfone and vinylsulfone, maleimide,
iodoacetamide, and iminoesters. Active esters include
N-hydroxylsuccinimidyl ester. Active carbonates include
N-hydroxylsuccinimidyl carbonate, p-nitrophenylcarbonate, and
trichlorophenylcarbonate.
[0140] A highly useful, new activating group that can be used for
highly selective coupling with thiol moieties instead of amino
moieties on molecules and surfaces is the vinyl sulfone moiety
described in co-pending U.S. patent application Ser. No.
08/151,481, which was filed on Nov. 12, 1993, the contents of which
are incorporated herein by reference. Various sulfone moieties can
be used to activate a multi-armed structure in accordance with the
invention for thiol selective coupling.
[0141] Various examples of activation of --Z reactive moieties to
created --Z activated moieties are presented as follows: 36
[0142] It should also be recognized that, although the linker
fragments represented by Q and P should not contain aromatic rings
or hydrolytically weak linkages such as ester linkages, such rings
and such hydrolytically weak linkages may be present in the moiety
represented by --Z. It may be desirable in some instances to
provide a linkage between, for example, a protein or enzyme and a
multisubstituted polymer derivative that has limited stability in
water. Some amino acids contain aromatic moieties, and it is
intended that the structure --Z include conjugates of
multisubstituted monofunctional polymer derivatives with such
molecules or portions of molecules.
[0143] B. Enzyme Modification
[0144] Enzymes were modified with activated, two-armed,
mPEG-disubstituted lysine of the invention of molecular weight
about 10,000 that had been prepared according to the two step
procedure and activated as the succinimidyl ester as discussed
above. The reaction is represented structurally below: 37
[0145] For comparison, enzymes were also modified with activated,
conventional, linear mPEG of molecular weight 5,000, which was mPEG
with a norleucine amino acid spacer arm activated as the
succinimide. In the discussion of enzyme modification below,
conventional, linear mPEG derivatives with which enzymes are
modified are referred to as "linear mPEG." The activated,
two-armed, mPEG-disubstituted lysine of the invention is referred
to as "two-armed mPEG." Different procedures were used for enzyme
modification depending upon the type of enzyme and the polymer used
so that a similar extent of amino group modification or attachment
for each enzyme could be obtained. Generally, higher molar ratios
of the two-armed mPEG were used. However, in all examples the
enzymes were dissolved in a 0.2 M borate buffer of pH 8.5 to
dissolve proteins. The polymers were added in small portions for
about 10 minutes and stirred for over 1 hour. The amount of polymer
used for modification was calculated on the basis of available
amino groups in the enzyme.
[0146] Ribonuclease in a concentration of 1.5 milligrams per
milliliter of buffer was modified at room temperature. Linear and
two-armed mPEGs as described were added at a molar ratio of polymer
to protein amino groups of 2.5:1 and 5:1, respectively.
Ribonuclease has a molecular weight of 13,700 D and 11 available
amino groups. Catalase has a molecular weight of 250,000 D with 112
available amino groups. Trypsin has a molecular weight of 23,000 D
with 16 available amino groups. Erwinia Caratimora asparaginase has
a molecular weight of 141,000 D and 92 free amino groups.
[0147] Catalase in a concentration of 2.5 milligrams per milliliter
of buffer was modified at room temperature. Linear and two-armed
mPEGs as described were added at a molar ratio of polymer to
protein amino groups of 5:1 and 10:1, respectively.
[0148] Trypsin in a concentration of 4 milligrams per milliliter of
buffer was modified at 0.degree. C. Linear and two-armed mPEGs as
described were added at a molar ratio of polymer to protein amino
groups of 2.5:1.
[0149] Asparaginase in a concentration of 6 milligrams per
milliliter of buffer was modified with linear mPEG at room
temperature. Linear mPEG as described was added at a molar ratio of
polymer to protein amino groups of 3:1. Asparaginase in a
concentration of 6 milligrams per milliliter of buffer was modified
with two-armed mPEG at 37.degree. C. Two-armed mPEG of the
invention as described was added at a molar ratio of polymer to
protein amino groups of 3.3:1.
[0150] The polymer and enzyme conjugates were purified by
ultrafiltration and concentrated in an Amicon system with a PM 10
membrane (cut off 10,000) to eliminate N-hydroxysuccinimide and
reduce polymer concentration. The conjugates were further purified
from the excess of unreacted polymer by gel filtration
chromatography on a Pharmacia Superose 12 column, operated by an
FPLC instrument, using 10 mM phosphate buffer of pH 7.2, 0.15 M in
NaCl, as eluent.
[0151] Protein concentration for the native forms of ribonuclease,
catalase, and trypsin was evaluated spectrophotometrically using
molar extinction coefficients of 945.times.10.sup.3 M.sup.-1
cm.sup.-1, 1.67.times.10.sup.5 M.sup.-1 cm.sup.-1 and
3.7.times.10.sup.4 M.sup.-1 cm.sup.-1 at 280 nm, respectively. The
concentration of native asparaginase was evaluated by biuret assay.
Biuret assay was also used to evaluate concentrations of the
protein modified forms.
[0152] The extent of protein modification was evaluated by one of
three methods. The first is a calorimetric method described in
Habeeb, A. F. S. A. (1966) Determination of free amino groups in
protein by trinitrobenzensulphonic acid. Anal. Biochem. 14,
328-336. The second method is amino acid analysis after acid
hydrolysis. This method was accomplished by two procedures: 1) the
post-column procedure of Benson, J. V., Gordon, M. J., and
Patterson, J. A. (1967) Accelerated chromatographic analysis of
amino acid in physiological fluids containing vitamin and
asparagine. Anal. Biol. Chem. 18, 288-333, and 2) pre-column
derivatization by phenylisothiocyanate (PITC) according to
Bidlingmeyer, B. A., Cohen, S. A., and Tarvin, T. L. (1984) Rapid
analysis of amino acids using pre-column derivatization. J.
Chromatography 336, 93-104.
[0153] The amount of bound linear mPEG was evaluated from
norleucine content with respect to other protein amino acids. The
amount of two-armed, mPEG-disubstituted lysine was determined from
the increase in lysine content. One additional lysine is present in
the hydrolysate for each bound polymer.
[0154] III. Analysis of Polymer and Enzyme Conjugates
[0155] Five different model enzymes, ribonuclease, catalase,
asparaginase, trypsin and uricase, were modified with linear,
conventional mPEG of molecular weight 5000 having a norleucine
amino acid spacer arm activated as succinimidl ester and with a
two-armed, mPEG-disubstituted lysine of the invention prepared from
the same linear, conventional mPEG as described above in connection
with the two step procedure. The molecular weight of the two-armed
mPEG disubstituted lysine of the invention was approximately
10,000.
[0156] A. Comparison of Enzyme Activity. The catalytic properties
of the modified enzymes were determined and compared and the
results are presented in Table 1 below. To facilitate comparison,
each enzyme was modified with the two polymers to a similar extent
by a careful choice of polymer to enzyme ratios and reaction
temperature.
[0157] Ribonuclease with 50% and 55% of the amino groups modified
with linear mPEG and two-armed mPEG, respectively, presented 86%
and 94% residual activity with respect to the native enzyme.
Catalase was modified with linear mPEG and with two-armed mPEG to
obtain 43% and 38% modification of protein amino groups,
respectively. Enzyme activity was not significantly changed after
modification. Trypsin modification was at the level of 50% and 57%
of amino groups with linear mPEG and with two-armed mPEG,
respectively. Esterolytic activity for enzyme modified with linear
mPEG and two-armed mPEG, assayed on the small substrate TAME, was
increased by the modification to 120% and 125%, respectively.
Asparaginase with 53% and 40% modified protein amino groups was
obtained by coupling with linear mPEG and two-armed mPEG,
respectively. Enzymatic activity was increased, relative to the
free enzyme, to 110% for the linear mPEG conjugate and to 133% for
the two-armed mPEG conjugate.
[0158] While not wishing to be bound by theory, it is possible that
in the case of trypsin and asparaginase, that modification produces
a more active form of the enzyme. The K.sub.m values of the
modified and unmodified forms are similar.
[0159] For the enzyme uricase a particularly dramatic result was
obtained. Modification of uricase with linear mPEG resulted in
total loss of activity. While not wishing to be bound by theory, it
is believed that the linear mPEG attached to an amino acid such as
lysine that is critical for activity. In direct contrast,
modification of 40% of the lysines of uricase with two-armed mPEG
gave a conjugate retaining 70% activity.
[0160] It is apparent that modification of enzymes with two-armed
mPEG gives conjugates of equal or greater activity than those
produced by conventional linear mPEG modification with
monosubstituted structures, despite the fact that two-armed mPEG
modification attaches twice as much polymer to the enzyme.
[0161] Coupling two-armed mPEG to asparaginase with chlorotriazine
activation as described in the background of the invention gave
major loss of activity. Presumably the greater activity of enzymes
modified with a two-armed mPEG of the invention results because the
bulky two-armed mPEG structure is less likely than monosubstituted
linear mPEG structures to penetrate into active sites of the
proteins.
1TABLE 1 Properties of enzymes modified by linear mPEG and
two-armed mPEG. NH.sub.2:POLYMER % % ENZYME.sup.a MOLAR RATIO
MODIFICATION ACTIVITY Km (M) Kcas (min.sup.-1) Ribonuclease RN 1:0
0 100 RP1 1:2.5 50 86 RP2 1:5 55 94 Catalase CN 1:0 0 100 CP1 1:5
43 100 CP2 1:10 38 90 Trypsin.sup.b TN 1:0 0 100 8.2 .times.
10.sup.-5 830 TP1 1:2.5 50 120 7.6 .times. 10.sup.-5 1790 TP2 1:2.5
57 125 8.0 .times. 10.sup.-5 2310 Asparaginase AN 1:0 0 100 3.31
.times. 10.sup.-6 523 AP1 1:3 53 110 3.33 .times. 10.sup.-6 710 AP2
1:3.3 40 133 3.30 .times. 10.sup.-6 780 Uricase UP 1:0 0 100 UP1
1:5 45 0 UP2 1:10 40 70 .sup.aN = native enzyme, P1 = enzyme
modified with linear mPEG, P2 = enzyme modified with two-armed
mPEG. .sup.bFor trypsin only the esterolytic activity is
reported.
[0162] Enzymatic activity of native and modified enzyme was
evaluated by the following methods. For ribonuclease, the method
was used of Crook, E. M., Mathias, A. P., and Rabin, B. R. (1960)
Spectrophotometric assay of bovine pancreatic ribonuclease by the
use of cytidine 2':3' phosphate. Biochem. J. 74, 234-238. Catalase
activity was determined by the method of Beers, R. F. and Sizer, I.
W. (1952) A spectrophotometric method for measuring the breakdown
of hydrogen peroxide by catalase. J. Biol. Chem. 195,133-140. The
esterolytic activity of trypsin and its derivatives was determined
by the method of Laskowski, M. (1955) Trypsinogen and trypsin.
Methods Enzymol. 2, 26-36. Native and modified asparaginase were
assayed according to a method reported by Cooney, D. A., Capizzi,
R. L. and Handschumacher, R. E. (1970) Evaluation of L-asparagine
metabolism in animals and man. Cancer Res. 30, 929-935. In this
method, 1.1 ml containing 120 .mu.g of .alpha.-ketoglutaric acid,
20 Ul of glutamic-oxalacetic transaminase, 30 Ul of malate
dehydrogenase, 100 .mu.g of NADH, 0.5 .mu.g of asparaginase and 10
.mu.moles of asparagine were incubated in 0.122 M Tris buffer, pH
8.35, while the NADH absorbance decrease at 340 nm was
followed.
[0163] B. Proteolytic Digestion of Free Enzyme and Conjugates. The
rates at which proteolytic enzymes digest and destroy proteins was
determined and compared for free enzyme, enzyme modified by
attachment of linear activated mPEG, and enzyme modified by
attachment of an activated two-armed mPEG of the invention. The
proteolytic activities of the conjugates were assayed according to
the method of Zwilling, R., and Neurath, H. (1981) Invertrebate
protease. Methods Enzymol. 80, 633-664. Four enzymes were used:
ribonuclease, catalase, trypsin, and asparaginase. From each enzyme
solution, aliquots were taken at various time intervals and enzyme
activity was assayed spectrophotometrically.
[0164] Proteolytic digestion was performed in 0.05 M phosphate
buffer of pH 7.0. The free enzyme, linear mPEG and protein
conjugate, and two-armed mPEG-protein conjugates were exposed to
the known proteolytic enzymes trypsin, pronase, elastase or
subtilisin under conditions as follows.
[0165] For native ribonuclease and its linear and two-armed mPEG
conjugates, 0.57 mg protein was digested at room temperature with
2.85 mg of pronase, or 5.7 mg of elastase, or with 0.57 mg of
subtilisin in a total volume of 1 ml. Ribonuclease with 50% and 55%
of the amino groups modified with linear mPEG and two-armed mPEG,
respectively, was studied for stability to proteolytic digestion by
pronase (FIG. 1(a)), elastase (FIG. 1(b)) and subtilisin (FIG.
1(c)). Polymer modification greatly increases the stability to
digestion by all three proteolytic enzymes, but the protection
offered by two-armed mPEG is much more effective as compared to
linear mPEG.
[0166] For native and linear and two-armed mPEG-modified catalase,
0.58 mg of protein were digested at room temperature with 0.58 mg
of trypsin or 3.48 mg of pronase in a total volume of 1 ml.
Catalase was modified with linear mPEG and two-armed mPEG to obtain
43% and 38% modification of protein amino groups, respectively.
Proteolytic stability was much greater for the two-armed mPEG
derivative than for the monosubstituted mPEG derivative,
particularly toward pronase (FIG. 3(a)) and trypsin (FIG. 3(b)),
where no digestion took place.
[0167] Autolysis of trypsin and its linear and two-armed mPEG
derivatives at 37.degree. C. was evaluated by esterolytic activity
of protein solutions at 25 mg/ml of TAME. Trypsin modification was
at the level of 50% and 57% of amino groups with linear mPEG and
two-armed mPEG, respectively. Modification with linear mPEG and
two-armed mPEG reduced proteolytic activity of trypsin towards
casein, a high molecular weight substrate: activity relative to the
native enzyme was found, after 20 minutes incubation, to be 64% for
the linear mPEG and protein conjugate and only 35% for the
two-armed mPEG conjugate. In agreement with these results, the
trypsin autolysis rate (i.e., the rate at which trypsin digests
trypsin), evaluated by enzyme esterolytic activity, was totally
prevented in two-armed mPEG-trypsin but only reduced in the linear
mPEG-trypsin conjugate. To prevent autolysis with linear mPEG,
modification of 78% of the available protein amino groups was
required.
[0168] For native and linear mPEG-- and two-armed mPEG-modified
asparaginase, 2.5 .mu.g were digested at 37.degree. C. with 0.75 mg
of trypsin in a total volume of 1 ml. Asparaginase with 53% and 40%
modified protein amino groups was obtained by coupling with linear
mPEG and two-armed mPEG, respectively. Modification with two-armed
mPEG had an impressive influence on stability towards proteolytic
enzyme. Increased protection was achieved at a lower extent of
modification with respect to the derivative obtained with the
two-armed polymer (FIG. 5).
[0169] These data clearly show that two-armed mPEG coupling is much
more effective than conventional linear mPEG coupling in providing
a protein with protection against proteolysis. While not wishing to
be bound by theory, it is believed that the two-armed mPEG, having
two polymer chains bound to the same site, presents increased
hindrance to approaching macromolecules in comparison to linear
mPEG.
[0170] C. Reduction of Protein Antigenicity. Protein can provoke an
immune response when injected into the bloodstream. Reduction of
protein immunogenicity by modification with linear and two-armed
mPEG was determined and compared for the enzyme superoxidedismutase
("SOD").
[0171] Anti-SOD antibodies were obtained from rabbit and purified
by affinity chromatography. The antigens (SOD, linear mPEG-SOD, and
two-armed mPEG-SOD) were labelled with tritiated succinimidyl
propionate to facilitate tracing. Reaction of antigen and antibody
were evaluated by radioactive counting. In a 500 .mu.L sample, the
antigen (in the range of 0-3 .mu.g) was incubated with 2.5 .mu.g of
antibody. The results show the practical disappearance of antibody
recognition for two-armed mPEG-SOD, while an appreciable
antibody-antigen complex was formed for linear mPEG-SOD and native
SOD.
[0172] D. Blood Clearance Times. Increased blood circulation half
lives are of enormous pharmaceutical importance. The degree to
which mPEG conjugation of proteins reduces kidney clearance of
proteins from the blood was determined and compared for free
protein, protein modified by attachment of conventional, linear
activated mPEG, and protein modified by attachment of the activated
two-armed mPEG of the invention. Two proteins were used. These
experiments were conducted by assaying blood of mice for the
presence of the protein.
[0173] For linear mPEG-uricase and two-armed mPEG-uricase, with 40%
modification of lysine groups, the half life for blood clearance
was 200 and 350 minutes, respectively. For unmodified uricase the
result was 50 minutes.
[0174] For asparaginase, with 53% modification with mPEG and 40%
modification with two armed mPEG, the half lives for blood
clearance were 1300 and 2600 minutes, respectively. For unmodified
asparaginase the result was 27 minutes.
[0175] E. Thermal Stability of Free and Conjugated Enzymes. Thermal
stability of native ribonuclease, catalase and asparaginase and
their linear mPEG and two-armed mPEG conjugates was evaluated in
0.5 M phosphate buffer pH 7.0 at 1 mg/ml, 9 .mu.g/ml and 0.2 mg/ml
respectively. The samples were incubated at the specified
temperatures for 15 min., 10 min., and 15 min, respectively, cooled
to room temperature and assayed spectrophotometrically for
activity.
[0176] Increased thermostability was found for the modified forms
of ribonuclease, as shown in FIG. 2, at pH 7.0, after 15 min.
incubation at different temperatures, but no significant difference
between the two polymers was observed. Data for catalase, not
reported here, showed that modification did not influence catalase
thermostability. A limited increase in thermal stability of linear
and two-armed mPEG-modified asparaginase was also noted, but is not
reported.
[0177] F. pH Stability of the Free and Conjugated Enzymes.
Unmodified and polymer-modified enzymes were incubated for 20 hrs
in the following buffers: sodium acetate 0.05 M at a pH of from 4.0
to 6.0, sodium phosphate 0.05 M at pH 7.0 and sodium borate 0.05 M
at a pH of from 8.0 to 11. The enzyme concentrations were 1 mg/ml,
9 .mu.g/ml, 5 .mu.g/ml for ribonuclease, catalase, and asparaginase
respectively. The stability to incubation at various pH was
evaluated on the basis of enzyme activity.
[0178] As shown in FIG. 2b, a decrease in pH stability at acid and
alkline pH values was found for the linear and two-armed
mPEG-modified ribonuclease forms as compared to the native enzyme.
As shown in FIG. 4, stability of the linear mPEG and two-armed mPEG
conjugates with catalase was improved for incubation at low pH as
compared to native catalase. However, the two-armed mPEG and linear
mPEG conjugates showed equivalent pH stability. A limited increase
in pH stability at acid and alkaline pH values was noted for linear
and two-armed mPEG-modified asparaginase as compared to the native
enzyme.
[0179] It should be recognized that there are thousands of proteins
and enzymes that can be usefully modified by attachment to the
polymer derivatives of the invention. Proteins and enzymes can be
derived from animal sources, humans, microorganisms, and plants and
can be produced by genetic engineering or synthesis.
Representatives include: cytokines such as various interferons
(e.g. interferon-.alpha., interferon-.beta., interferon-.gamma.),
interleukin-2 and interleukin-3), hormones such as insulin, growth
hormone-releasing factor (GRF), calcitonin, calcitonin gene related
peptide (CGRP), atrial natriuretic peptide (ANP), vasopressin,
corticortropin-releasing factor (CRF), vasoactive intestinal
peptide (VIP), secretin, .alpha.-melanocyte-stimulating hormone
(.alpha.-MSH), adrenocorticotropic hormone (ACTH), cholecystokinin
(CCK), glucagon, parathyroid hormone (PTH), somatostatin,
endothelin, substance P, dynorphin, oxytocin and growth
hormone-releasing peptide, tumor necrosis factor binding protein,
growth factors such as growth hormone (GH), insulin-like growth
factor (IGF-I, IGF-II), .beta.-nerve growth factor (.beta.-NGF),
basic fibroblast growth factor (bFGF), transforming growth factor,
erythropoietin, granulocyte colony-stimulating factor (G-CSF),
granulocyte macrophage colony-stimulating factor (GM-CSF),
platelet-derived growth factor (PDGF) and epidermal growth factor
(EGF), enzymes such as tissue plasminogen activator (t-PA),
elastase, superoxide dismutase (SOD), bilirubin oxydase, catalase,
uricase and asparaginase, other proteins such as ubiquitin, islet
activating protein (IAP), serum thymic factor (STF), peptide-T and
trypsin inhibitor, and derivatives thereof. In addition to protein
modification, the two-armed polymer derivative of the invention has
a variety of related applications. Small molecules attached to
two-armed activated mPEG derivatives of the invention can be
expected to show enhanced solubility in either aqueous or organic
solvents. Lipids and liposomes attached to the derivative of the
invention can be expected to show long blood circulation lifetimes.
Other particles than lipids and surfaces having the derivative of
the invention attached can be expected to show nonfouling
characteristics and to be useful as biomaterials having increased
blood compatibility and avoidance of protein adsorption.
Polymer-ligand conjugates can be prepared that are useful in two
phase affinity partitioning. The polymers of the invention could be
attached to various forms of drugs to produce prodrugs. Small drugs
having the multisubstituted derivative attached can be expected to
show altered solubility, clearance time, targeting, and other
properties.
[0180] The invention claimed herein has been described with respect
to 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 made within the scope and spirit of the
invention as described in the foregoing specification. 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.
* * * * *