U.S. patent application number 10/982303 was filed with the patent office on 2005-09-29 for method of preparing carboxylic acid functionalized polymers.
Invention is credited to Guo, Lihong, Harris, J. Milton, Kozlowski, Antoni.
Application Number | 20050214250 10/982303 |
Document ID | / |
Family ID | 34590189 |
Filed Date | 2005-09-29 |
United States Patent
Application |
20050214250 |
Kind Code |
A1 |
Harris, J. Milton ; et
al. |
September 29, 2005 |
Method of preparing carboxylic acid functionalized polymers
Abstract
Methods for preparing water soluble, non-peptidic polymers
carrying carboxyl functional groups, particularly carboxylic acid
functionalized poly(ethylene glycol) (PEG) polymers, are disclosed,
as are the products of these methods. In general, an ester reagent
R(C.dbd.O)OR', where R' is a tertiary group and R comprises a
functional group X, is reacted with a water soluble, non-peptidic
polymer POLY-Y, where Y is a functional group which reacts with X
to form a covalent bond, to form a tertiary ester of the polymer,
which is then treated with a strong base in aqueous solution, to
form a carboxylate salt of the polymer. Typically, this carboxylate
salt is then treated with an inorganic acid in aqueous solution, to
convert the carboxylate salt to a carboxylic acid, thereby forming
a carboxylic acid functionalized polymer.
Inventors: |
Harris, J. Milton;
(Huntsville, AL) ; Kozlowski, Antoni; (Huntsville,
AL) ; Guo, Lihong; (Huntsville, AL) |
Correspondence
Address: |
Nektar Therapeutics
150 Industrial Road
San Carlos
CA
94070
US
|
Family ID: |
34590189 |
Appl. No.: |
10/982303 |
Filed: |
November 4, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60517794 |
Nov 6, 2003 |
|
|
|
Current U.S.
Class: |
424/78.38 ;
525/530 |
Current CPC
Class: |
C08G 65/332 20130101;
C08F 289/00 20130101; C08G 65/3346 20130101; A61K 31/765 20130101;
C08F 261/04 20130101; C08F 265/00 20130101; C08F 271/00 20130101;
C08G 65/30 20130101; C08G 65/3322 20130101; C08F 251/00 20130101;
C08F 291/00 20130101; C08F 283/06 20130101; C08G 63/91
20130101 |
Class at
Publication: |
424/078.38 ;
525/530 |
International
Class: |
A61K 031/765; C08F
283/10 |
Claims
We claim:
1. A method for preparing a water soluble, non-peptidic polymer
functionalized with a carboxyl group, the method comprising: i)
reacting an ester reagent R(C.dbd.O)OR', where R' is a tertiary
group and R comprises a functional group X, with a water soluble,
non-peptidic polymer POLY-Y, where Y is a functional group which
reacts with X to form a covalent bond, to form a tertiary ester of
the polymer; and ii) treating the tertiary ester of the polymer
with a strong base in aqueous solution, to form a carboxylate salt
of the polymer.
2. The method of claim 1, further comprising iii) treating the
carboxylate salt of the polymer with an inorganic acid in aqueous
solution, to convert the carboxylate salt to a carboxylic acid,
thereby forming a carboxylic acid functionalized polymer.
3. The method of claim 1, wherein X is a leaving group and Y is a
hydroxyl group.
4. The method of claim 1, wherein said strong base is an alkali
metal hydroxide.
5. The method of claim 1, wherein said treating with strong base is
effective to produce a reaction pH of about 11 to 13.
6. The method of claim 2, wherein said inorganic acid is an acid
that produces non-nucleophilic anions in aqueous solution.
7. The method of claim 6, wherein the acid is selected from the
group consisting of sulfuric acid, nitric acid, phosphoric acid,
and hydrochloric acid.
8. The method of claim 1, wherein the tertiary ester reagent has
the structure: 3wherein: X is a leaving group, each of R.sup.1 and
R.sup.2 is independently selected from hydrogen, alkyl, cycloalkyl,
alkoxy, aryl, aralkyl, and heterocycle; each of R.sup.3-R.sup.5 is
independently selected from lower alkyl, aryl, aralkyl, and
cycloalkyl, where any of R.sup.3-R.sup.5 may be linked to form a
ring or ring system; where any of R.sup.1 to R.sup.5, excepting
hydrogen, may be substituted with a group selected from lower
alkyl, lower alkoxy, C.sub.3-C.sub.6 cycloalkyl, halo, cyano,
oxo(keto), nitro, and phenyl; and n is 1 to about 24.
9. The method of claim 8, wherein n is 1 to 6.
10. The method of claim 9, wherein n is 1 or 2.
11. The method of claim 10, wherein each of R.sup.1 and R.sup.2 is
independently hydrogen or unsubstituted lower alkyl, and each of
R.sup.3 to R.sup.5 is independently unsubstituted lower alkyl or
phenyl.
12. The method of claim 11, wherein each of R.sup.1 and R.sup.2 is
independently hydrogen or methyl, and each of R.sup.3 to R.sup.5 is
independently methyl, ethyl, or phenyl.
13. The method of claim 12, wherein each of R.sup.1 and R.sup.2 is
H and n is 1.
14. The method of claim 13, wherein the tertiary ester reagent is a
t-butyl haloacetate.
15. The method of claim 1, wherein the polymer is selected from the
group consisting of poly(alkylene glycols), poly(olefinic alcohol),
poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),
poly(hydroxyalkylmethacrylate), poly(saccharides),
poly(.alpha.-hydroxyacetic acid), poly(acrylic acid), poly(vinyl
alcohol), polyphosphazene, polyoxazolines,
poly(N-acryloylmorpholine), and copolymers or terpolymers
thereof.
16. The method of claim 15, wherein the polymer is a poly(ethylene
glycol).
17. The method of claim 16, wherein the poly(ethylene glycol) is
linear and is terminated at one end with said functional group Y
and at the other end with another functional group Y' or a capping
group.
18. The method of claim 2, further comprising converting the
carboxylic acid to an activated carboxylic acid derivative.
19. The method of claim 18, wherein said derivative is an activated
ester.
20. The method of claim 18, further comprising conjugating said
polymer with a biologically active molecule, by reacting said
carboxylic acid derivative with a functional group on said
molecule.
21. The method of claim 20, wherein the carboxylic acid derivative
is an activated ester, and the functional group on said molecule is
a nucleophilic group.
22. The method of claim 21, wherein said nucleophilic group is an
amino group, a hydroxyl group, or a thiol.
23. A method for preparing a poly(ethylene glycol) (PEG)
functionalized with a carboxyl group, the method comprising: i)
reacting a tertiary ester reagent R(C.dbd.O)OR', where R' is a
tertiary alkyl group and R comprises a functional group X, with a
polymer PEG-Y, where Y is a functional group which reacts with X to
form a covalent bond, to form a PEG tertiary ester; and ii)
treating the PEG tertiary ester with a strong base in aqueous
solution, to form a PEG carboxylate salt.
24. The method of claim 23, further comprising iii) treating the
PEG carboxylate salt with an inorganic acid in aqueous solution, to
convert the carboxylate salt to a carboxylic acid, thereby forming
a PEG carboxylic acid.
25. The method of claim 23, wherein X is a leaving group and Y is a
hydroxyl group.
26. The method of claim 23, wherein said strong base is an alkali
metal hydroxide.
27. The method of claim 24, wherein the acid is selected from the
group consisting of sulfuric acid, nitric acid, phosphoric acid,
and hydrochloric acid.
28. The method of claim 23, wherein the tertiary ester reagent has
the structure: 4wherein: X is a leaving group; each of R.sup.1 and
R.sup.2 is independently selected from hydrogen, alkyl, cycloalkyl,
alkoxy, aryl, aralkyl, and heterocycle; each of R.sup.3-R.sup.5 is
independently selected from lower alkyl, aryl, aralkyl, and
cycloalkyl, where any of R.sup.3-R.sup.5 may be linked to form a
ring or ring system; where any of R.sup.1 to R.sup.5, excepting
hydrogen, may be substituted with a group selected from lower
alkyl, lower alkoxy, C3-C6 cycloalkyl, halo, cyano, oxo(keto),
nitro, and phenyl; and n is 1 to about 24.
29. The method of claim 28, wherein n is 1 to 6.
30. The method of claim 29, wherein n is 1 or 2.
31. The method of claim 28, wherein each of R.sup.1 and R.sup.2 is
independently hydrogen or unsubstituted lower alkyl, and each of
R.sup.3 to R.sup.5 is independently unsubstituted lower alkyl or
phenyl.
32. The method of claim 28, wherein each of R.sup.1 and R.sup.2 is
H and n is 1.
33. The method of claim 32, wherein the tertiary ester reagent is a
t-butyl haloacetate.
34. The method of claim 23, wherein the poly(ethylene glycol) is
linear and is terminated at one end with said functional group Y
and at the other end with another functional group Y' or a capping
group.
35. The method of claim 23, wherein the PEG has a molecular weight
of about 100 to about 100,000 Da.
36. The method of claim 35, wherein the PEG has a molecular weight
of about 300 to about 60,000 Da.
37. The method of claim 24, further comprising converting the
PEG-carboxylic acid to an activated carboxylic acid derivative.
38. The method of claim 37, wherein said derivative is an activated
ester.
39. The method of claim 37, further comprising conjugating said PEG
with a biologically active molecule, by reacting said carboxylic
acid derivative with a functional group on said molecule.
40. An isolated polymer product comprising a carboxylic acid
functionalized polymer, made by the method of claim 2, wherein the
product contains less than 5% by weight of said POLY-Y polymer,
with the balance consisting essentially of said carboxylic acid
functionalized polymer.
41. The polymer product of claim 40, containing less than 2% by
weight of said POLY-Y polymer.
42. The polymer product of claim 40, containing less than 0.5% by
weight of said POLY-Y polymer.
43. The polymer product of claim 40, containing substantially no
amount of low molecular weight organic acid.
44. The polymer product of claim 40, containing substantially no
amount of monomeric organic carboxylic acid.
45. The polymer product of claim 40, containing substantially no
amount of trifluoroacetic acid.
46. The polymer product of claim 40, wherein said carboxylic acid
functionalized polymer is a PEG carboxylic acid.
47. The polymer product of claim 46, wherein said carboxylic acid
functionalized polymer is mPEG-CH.sub.2--COOH, and said polymer
product contains less than 5% by weight of mPEG-OH.
48. The polymer product of claim 47, containing less than 2% by
weight of mPEG-OH.
49. The polymer product of claim 48, containing less than 0.5% by
weight of mPEG-OH.
50. The polymer product of claim 49, containing substantially no
amount of trifluoroacetic acid.
51. The polymer product of claim 46, wherein said carboxylic acid
functionalized polymer is HOOC--CH.sub.2--PEG-CH.sub.2--COOH, and
said product contains less than 5% by weight of HO-PEG-OH.
52. The polymer product of claim 51, containing less than 0.5% by
weight of HO-PEG-OH.
53. The polymer product of claim 52, containing substantially no
amount of trifluoroacetic acid.
54. The polymer product of claim 46, wherein said carboxylic acid
functionalized polymer is a multifunctional branched or multiarm
carboxylic acid functionalized PEG represented by
PEG-(CH.sub.2--COOH).su- b.x, where x is 3 to 8, and said product
contains less than 5% by weight of PEG-(OH).sub.x.
55. The polymer product of claim 54, containing substantially no
amount of trifluoroacetic acid.
56. In a method of preparing a poly(ethylene glycol) (PEG)polymer
functionalized with a carboxyl group, by reaction of a tertiary
ester reagent R(C.dbd.O)OR', where R' is a tertiary alkyl group and
R comprises a functional group X, with a polymer PEG-Y, where Y is
a functional group which reacts with X to form a covalent bond, to
form a PEG tertiary ester, an improvement comprising: treating the
PEG tertiary ester with a strong base in aqueous solution, to form
a PEG carboxylate salt.
57. The improvement of claim 56, wherein the method further
comprises treating the PEG carboxylate salt with an inorganic acid
in aqueous solution, to convert the carboxylate salt to a
carboxylic acid, thereby forming a PEG carboxylic acid.
58. The improvement of claim 56, wherein said strong base is an
alkali metal hydroxide.
59. The improvement of claim 56, wherein said treating with strong
base is effective to produce a reaction pH of about 11 to 13.
60. The improvement of claim 57, wherein the acid is selected from
the group consisting of sulfuric acid, nitric acid, phosphoric
acid, and hydrochloric acid.
Description
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 60/517,794, filed Nov. 6, 2003, which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to methods for preparing water
soluble, non-peptidic polymers carrying carboxyl functional groups,
particularly carboxylic acid functionalized poly(ethylene glycol)
(PEG)polymers.
BACKGROUND OF THE INVENTION
[0003] Poly(ethylene glycol) (PEG) derivatives activated with
electrophilic groups are useful for coupling to nucleophilic
groups, such as amino groups, of biologically active molecules. In
particular, active esters and other carboxylic acid derivatives of
PEG have been used to attach PEG to proteins bearing amino
groups.
[0004] PEG molecules having terminal carboxymethyl groups have been
described, for example, by Martinez et al., U.S. Pat. No.
5,681,567, Veronese et al., Journal of Controlled Release
10:145-154 (1989), and Buckmann et al., Makromol. Chem. 182(5):
1379-1384 (1981). U.S. Pat. No. 5,672,662 (Harris et al.) discloses
PEG derivatives having a terminal propionic or butanoic acid
moiety. Such carboxyl-terminated PEGs are used to prepare active
esters suitable for conjugation to proteins or other molecules
bearing amino groups.
[0005] However, a persistent problem associated with preparation of
carboxyl-functionalized polymers has been the difficulty in
obtaining the desired polymer product at a sufficiently high purity
level. For example, Veronese et al. and Buckmann et al., cited
above, employ a method of synthesizing mPEG carboxylic acids which
comprises converting mPEG-OH to an ethyl ester of mPEG carboxylic
acid, by base-catalyzed reaction of mPEG-OH with an .alpha.-halo
ethyl ester, followed by base-promoted hydrolysis of the ester.
However, this approach provides mPEG acids of only about 85%
purity, with the main contaminant being mPEG-OH, which cannot be
separated from the mPEG carboxylic acid using typical purification
methods such as precipitation, crystallization or extraction.
Removal of mPEG-OH requires the use of preparative ion exchange
column chromatography, which is time consuming and expensive. PEG
carboxylic acids obtained commercially frequently contain residual
amounts of PEG-OH, which complicates the preparation of derivatives
or bioconjugates based on these materials.
[0006] U.S. Pat. Nos. 5,278,303, 5,605,976 and 5,681,567 report the
preparation of PEG carboxylic acids containing little or no
starting material (PEG alcohol) by employing a tertiary alkyl
haloacetate to prepare a tertiary alkyl ester-functionalized PEG,
which is then hydrolyzed with acid, preferably trifluoroacetic acid
(TFA).
[0007] Various treatises on the use of protecting groups note that
tertiary alkyl esters, such as t-butyl esters, are stable to mild
base hydrolysis typically used to hydrolyze primary alkyl esters,
such as ethyl esters. Strong base hydrolysis could cause cleavage
of carboxylic acid groups. See, for example, T. W. Greene,
Protective Groups in Organic Synthesis, 3.sup.rd edition, 1999, p.
406; or P. J. Kocienski, Protecting Groups, 1994, p. 125.
Accordingly, these tertiary alkyl esters are conventionally cleaved
with acid, typically with TFA.
[0008] However, use of trifluoroacetic acid can result in
purification and product stability problems. Trifluoroacetic acid
is difficult to completely remove from the final
carboxyl-functionalized polymer, particularly the amount of TFA
suggested in the above-referenced patents. The presence of residual
trifluoroacetic acid results in poor product stability, due to
degradation of the polymer caused by acid-promoted autoxidation.
See, for example, M. Donbrow, "Stability of the Polyoxyethylene
Chain", in Nonionic Surfactants: Physical Chemistry, M. J. Schick,
ed., Marcel Dekker, 1987, pp. 1011 ff This article reports that
acids catalyze the formation of hydroperoxides and hydroperoxide
rupture, leading to cleavage of polyoxyethylene chains.
[0009] Although U.S. Pat. No. 5,605,976 suggests distillation as a
means for separating organic materials from the polymer product,
even compounds with very low boiling points are difficult to remove
from high molecular weight polymers using a distillation process,
and the difficulty increases as the molecular weight of the polymer
increases.
[0010] There is a need in the art for alternative methods for
preparing carboxylic acid functionalized polymers in high yield and
free from significant amounts of polymer contaminants, particularly
the polymer starting material. There is also a need in the art for
alternative synthesis methods that do not utilize reagents that are
either difficult to remove from the final polymer product or cause
product stability problems.
SUMMARY OF THE INVENTION
[0011] In one aspect, the invention provides a method for preparing
a water soluble, non-peptidic polymer functionalized with a
carboxyl group, the method comprising:
[0012] (i) reacting an ester reagent R(C.dbd.O)OR', where R' is a
tertiary group and R comprises a functional group X, with a water
soluble, non-peptidic polymer POLY-Y, where Y is a functional group
which reacts with X to form a covalent bond, to form a tertiary
ester of the polymer; and
[0013] (ii) treating the tertiary ester of the polymer with a
strong base, such as an alkali metal hydroxide, in aqueous
solution, to form a carboxylate salt of the polymer. The method may
further comprise the step of (iii) treating the carboxylate salt of
the polymer with an inorganic acid in aqueous solution, to convert
the carboxylate salt to a carboxylic acid, thereby forming a
carboxylic acid functionalized polymer. The carboxylic acid
functionalized polymer can then be extracted from the aqueous
solution with a suitable solvent, preferably a chlorinated
solvent.
[0014] In one embodiment, X is a leaving group, such as a halide or
a sulfonate ester, and Y is a hydroxyl group. When Y is a hydroxyl
group, the reaction (i) is preferably carried out in the presence
of a base, e.g. a base of the form R'O.sup.-M.sup.+, where M.sup.+
is a cation.
[0015] The treatment with strong base in reaction (ii) is
preferably effective to produce a reaction pH of about 11 to 13.
The inorganic acid, e.g. a mineral acid, in step (iii) is
preferably an acid that produces non-nucleophilic anions in aqueous
solution. Preferred acids include sulfuric acid, nitric acid,
phosphoric acid, and hydrochloric acid. The acid treatment of (iii)
is preferably effective to produce a reaction pH of about 1 to
3.
[0016] The tertiary ester reagent employed in reaction (i)
preferably has the structure (I): 1
[0017] In structure (I), X is a leaving group; and each of R.sup.1
and R.sup.2 is independently selected from hydrogen, alkyl,
cycloalkyl, alkoxy, aryl, aralkyl, and heterocycle. Preferably, the
group (CR.sup.1R.sup.2).sub.n does not include two heteroatoms
attached to the same carbon atom; for example, R.sup.1 and R.sup.2
on the same carbon atom are preferably not both alkoxy. Each of
R.sup.3-R.sup.5 is independently selected from lower alkyl, aryl,
aralkyl, and cycloalkyl, where any of R.sup.3-R.sup.5 may be linked
to form a ring or ring system, such as adamantyl. Any of R' to
R.sup.5, excepting hydrogen, may be substituted with a group
selected from lower alkyl, lower alkoxy, C3-C6 cycloalkyl, halo,
cyano, oxo(keto), nitro, and phenyl. The variable n is 1 to about
24, preferably 1 to 6, more preferably 1 to 4, and most preferably
1 or 2. In one embodiment, n is 1.
[0018] In selected embodiments of structure (I), each of R.sup.1
and R.sup.2 is independently hydrogen or unsubstituted lower alkyl,
preferably hydrogen or methyl, and each of R.sup.3 to R.sup.5 is
independently unsubstituted lower alkyl or phenyl, preferably
methyl, ethyl, or phenyl. In one embodiment, each of R.sup.1 and
R.sup.2 is H and n is 1.
[0019] The leaving group X in structure (I) is preferably a halide
or a sulfonate ester. In one embodiment, the tertiary ester reagent
is a tertiary alkyl haloacetate, such as a t-butyl haloacetate.
[0020] The water soluble, non-peptidic polymer is preferably
selected from the group consisting of poly(alkylene glycols),
poly(olefinic alcohol), poly(vinylpyrrolidone),
poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),
poly(saccharides), poly(.alpha.-hydroxyacetic acid), poly(acrylic
acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines,
poly(N-acryloylmorpholine), and copolymers or terpolymers thereof.
In a preferred embodiment, the polymer is a poly(ethylene glycol).
The poly(ethylene glycol) may be linear and terminated at one end
with the functional group Y and at the other end with another
functional group Y' or a capping group, such as a methoxy group.
Alternatively, the poly(ethylene glycol) may be branched, forked,
or multiarmed.
[0021] The method may further comprise converting the carboxylic
acid of the carboxylic acid functionalized polymer to an activated
carboxylic acid derivative, e.g. an activated ester, such as, for
example, an N-succinimidyl ester, o-, m-, or p-nitrophenyl ester,
1-benzotriazolyl ester, imidazolyl ester, or N-sulfosuccinimidyl
ester. The polymer can then be conjugated with a biologically
active molecule, by reacting the carboxylic acid derivative with a
functional group, preferably a nucleophilic group such as a
hydroxyl, thiol, or amino group, on the biologically active
molecule. Preferably, the nucleophilic group is an amino group.
[0022] In a preferred embodiment of the method, as noted above, the
polymer is a PEG polymer. In this aspect, the invention provides a
method for preparing a poly(ethylene glycol) (PEG) functionalized
with a carboxyl group, the method comprising:
[0023] (i) reacting a tertiary ester reagent R(C.dbd.O)OR', where
R' is a tertiary alkyl group and R comprises a functional group X,
with a polymer PEG-Y, where Y is a functional group which reacts
with X to form a covalent bond, to form a PEG tertiary ester;
and
[0024] (ii) treating the PEG tertiary ester with a strong base,
such as an alkali metal hydroxide, in aqueous solution, to form a
PEG carboxylate salt. The method may further comprise (iii)
treating the PEG carboxylate salt with an inorganic acid in aqueous
solution, to convert the carboxylate salt to a carboxylic acid,
thereby forming a PEG carboxylic acid. Preferred embodiments of the
method correspond to those described above. The method may further
comprise converting the PEG-carboxylic acid to an activated
carboxylic acid derivative, such as an activated ester, and
conjugating the polymer to a biologically active molecule, by
reacting the carboxylic acid derivative with a functional group on
the molecule, as described above.
[0025] In one embodiment, the poly(ethylene glycol) is linear and
is terminated at one end with the functional group Y and at the
other end with another functional group Y' or with a capping group,
such as a methoxy group. The molecular weight of the PEG is
preferably in the range of about 100 Da to about 100 kDa, more
preferably in the range of about 300 Da to about 40, 50, or 60 kDa.
In other embodiments, the PEG is branched, forked, or multiarmed,
as described further below.
[0026] In a related aspect, the invention provides an isolated
polymer product comprising a carboxylic acid functionalized
polymer, made by the method disclosed herein, wherein the product
contains less than 5% by weight of the starting material; that is,
the POLY-Y or PEG-Y polymer, with the balance consisting
essentially of the carboxylic acid functionalized polymer.
Preferably, the isolated polymer product contains less than 2%,
more preferably less than 1%, and most preferably less than 0.5% by
weight of POLY-Y or PEG-Y polymer. In further preferred
embodiments, the isolated polymer product contains less than 0.4%,
more preferably less than 0.3%, and most preferably less than 0.2%
by weight of POLY-Y or PEG-Y polymer.
[0027] In a further preferred aspect, the isolated polymer product
contains substantially no amount of low molecular weight organic
acid. In one embodiment, the isolated polymer product contains
substantially no amount of monomeric organic carboxylic acid, such
as trifluoroacetic acid.
[0028] In one embodiment of the polymer product of the invention,
the carboxylic acid functionalized polymer is a PEG carboxylic
acid. For example, the carboxylic acid functionalized polymer may
be mPEG-CH.sub.2--COOH, and contains less than 5%, preferably less
than 2%, more preferably less than 0.5%, and most preferably less
than 0.2% by weight of mPEG-OH. Preferably, the product contains
substantially no amount of trifluoroacetic acid.
[0029] In another embodiment of the product, the carboxylic acid
functionalized polymer is HOOC--CH.sub.2--PEG-CH.sub.2--COOH, and
contains less than 5%, preferably less than 2%, more preferably
less than 0.5%, and most preferably less than 0.2% by weight of
HO-PEG-OH. Preferably, the product contains substantially no amount
of trifluoroacetic acid.
[0030] In a further embodiment of the product, the carboxylic acid
functionalized polymer is a multifunctional branched or multiarm
carboxylic acid functionalized PEG represented by
PEG-(CH.sub.2--COOH).su- b.x, where x is 3 to 8, and contains less
than 5%, preferably less than 2%, more preferably less than 0.5%,
and most preferably less than 0.2% by weight of PEG-(OH).sub.x.
Preferably, the product contains substantially no amount of
trifluoroacetic acid.
[0031] The invention further provides an improvement in a method of
preparing a poly(ethylene glycol) (PEG)polymer functionalized with
a carboxyl group, by reaction of a tertiary ester reagent
R(C.dbd.O)OR', where R' is a tertiary alkyl group and R comprises a
functional group X, with a polymer PEG-Y, where Y is a functional
group which reacts with X to form a covalent bond, to form a PEG
tertiary ester. The improvement comprises treating the PEG tertiary
ester with a strong base, preferably an alkali metal hydroxide, in
aqueous solution, to form a PEG carboxylate salt. The strong base
is preferably one that is strong base is effective to produce a
reaction pH of about 11 to 13 in the aqueous solution.
[0032] The improved method may further comprise treating the PEG
carboxylate salt with an inorganic acid in aqueous solution, to
convert the carboxylate salt to a carboxylic acid, thereby forming
a PEG carboxylic acid. The inorganic acid is preferably a mineral
acid selected from the group consisting of sulfuric acid, nitric
acid, phosphoric acid, and hydrochloric acid.
[0033] These and other objects and features of the invention will
become more fully apparent when the following detailed description
of the invention is read in conjunction with the included
drawings.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The present invention now will be described more fully. This
invention may, however, be embodied in many different forms and
should not be construed as limited to the embodiments set forth
herein; rather, these embodiments are provided so that this
disclosure will be thorough and complete, and will fully convey the
scope of the invention to those skilled in the art. The invention
is not limited to the particular polymers, synthetic techniques,
active agents, and the like set forth in this description, as such
may vary within the scope of the invention as embodied by the
appended claims. The terminology used herein is for describing
particular embodiments only, and is not intended to be
limiting.
[0035] I. Definitions
[0036] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions described below.
[0037] As used in this specification, the singular forms "a,""an,"
and "the" include plural referents unless the context clearly
dictates otherwise.
[0038] As used herein, "non-peptidic" refers to a polymer
substantially free of peptide linkages. However, the polymer may
include a minor number of peptide linkages spaced along the length
of the backbone, such as, for example, no more than about 1 peptide
linkage per about 50 monomer units.
[0039] "PEG" or "polyethylene glycol," as used herein, is meant to
encompass any water-soluble poly(ethylene oxide). Typically, PEGs
for use in the present invention will comprise one of the two
following structures: --O(CH.sub.2CH.sub.2O).sub.m-- or
--CH.sub.2CH.sub.2O(CH.sub.- 2CH.sub.2O).sub.m--CH.sub.2CH.sub.2--,
where m is generally from 3 to about 3000. In a broader sense,
"PEG" can refer to a polymer that contains a majority, i.e. greater
than 50%, of subunits that are --CH.sub.2CH.sub.2O--.
[0040] The terminal groups and architecture of the overall PEG may
vary. The PEG may contain an end-capping group on a terminal oxygen
which is generally a carbon-containing group typically comprised of
1-20 carbons and is preferably selected from alkyl, alkenyl,
alkynyl, aryl, aralkyl, cycloalkyl, heterocyclo, and substituted
forms of any of the foregoing. The end-capping group can also be a
silane. Most preferred are alkyl(alkoxy) or aralkyl(aralkoxy)
capping groups, such as methyl, ethyl or benzyl.
[0041] The end-capping group can also advantageously comprise a
detectable label. Such labels include, without limitation,
fluorescers, chemiluminescers, moieties used in enzyme labeling,
colorimetric (e.g., dyes), metal ions, radioactive moieties, and
the like.
[0042] The other ("non-end-capped") terminus is a typically
hydroxyl, amine or an activated group that can be subjected to
further chemical modification.
[0043] Specific PEG forms for use in the invention include PEGs
having a variety of molecular weights, structures or geometries
(e.g., branched, linear, forked, multiarmed).
[0044] A "multifunctional" polymer has 3 or more functional groups,
which may be the same or different. Multifunctional polymers will
typically contain from about 3-100 functional groups, or from 3-50
functional groups, or from 3-25 functional groups, or from 3-15
functional groups, or from 3 to 10 functional groups, or will
contain 3, 4, 5, 6, 7, 8, 9 or 10 functional groups.
[0045] A "difunctional" polymer has two functional groups contained
therein, which may be the same (i.e., homodifunctional) or
different (i.e., heterodifunctional).
[0046] "Molecular mass" or "molecular weight" refers to the average
molecular mass of a polymer, typically determined by size exclusion
chromatography, light scattering techniques, or intrinsic velocity
determination in 1,2,4-trichlorobenzene. Unless otherwise noted,
molecular weight is expressed herein as number average molecular
weight (M.sub.n), which is defined as .SIGMA.NiMi/.SIGMA.Ni,
wherein Ni is the number of polymer molecules (or the number of
moles of those molecules) having molecular weight Mi.
[0047] The polymers of the invention, or employed in the invention,
are typically polydisperse; i.e., the number average molecular
weight and weight average molecular weight of the polymers are not
equal. The polydispersity values, expressed as a ratio of weight
average molecular weight (Mw) to number average molecular weight
(Mn), (Mw/Mn), are generally low; that is, less than about 1.2,
preferably less than about 1.15, more preferably less than about
1.10, still more preferably less than about 1.05, yet still most
preferably less than about 1.03, and most preferably less than
about 1.025.
[0048] An "activated carboxylic acid" refers to a functional
derivative of a carboxylic acid that is more reactive than the
parent carboxylic acid, particularly with respect to nucleophilic
attack. Activated carboxylic acids include but are not limited to
acid halides (such as acid chlorides), anhydrides, and esters.
[0049] More generally, the term "activated" or "reactive", when
used in conjunction with a particular functional group, refers to a
functional group that reacts readily with an electrophile or a
nucleophile on another molecule, in contrast to groups that require
strong catalysts or impractical reaction conditions in order to
react (i.e., "nonreactive" or "inert" groups).
[0050] The term "protecting group" or "protective group" refers to
a moiety that prevents or blocks reaction of a particular
chemically reactive functional group in a molecule under certain
reaction conditions. The protecting group will vary depending upon
the type of chemically reactive group being protected as well as
the reaction conditions to be employed and the presence of
additional reactive or protecting groups in the molecule, if any.
Protecting groups known in the art can be found in Greene, T. W.,
et al., Protective Groups in Organic Synthesis, 3rd ed., John Wiley
& Sons, New York, N.Y. (1999). As used herein, the term
"functional group" or any synonym thereof is meant to encompass
protected forms thereof.
[0051] The term "spacer" or "spacer moiety" refers to an atom or a
collection of atoms used to link interconnecting moieties, such as
a terminus of a water-soluble polymer portion and an electrophile.
A typical spacer includes bonds selected from alkylene
(carbon-carbon), ether, amino, amide, ester, carbamate, urea, and
keto, and combinations thereof. A spacer may include short alkylene
moieties alternating with, or flanked by, one or more types of
heteroatom-containing linkages listed above. Various examples
include --CH.sub.2OCH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2C(O)NHCH.sub.2--, --C(O)OCH.sub.2--,
--OC(O)NHCH.sub.2CH.sub.2-- -, --CH.sub.2CH.sub.2NHCH.sub.2,
--CH.sub.2CH.sub.2C(O)CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2C(O)NHCH.sub.2CH.sub.2NH--, and
--CH.sub.2CH.sub.2CH.sub.2C(O)NHCH.sub.2CH.sub.2NHC(O)CH.sub.2CH.sub.2--.
The spacer moieties of the invention may be hydrolytically stable
or may include a physiologically hydrolyzable or enzymatically
degradable linkage (e.g. an ester linkage).
[0052] "Alkyl" refers to a hydrocarbon chain, typically ranging
from about 1 to 20 atoms in length. Such hydrocarbon chains are
preferably but not necessarily saturated and may be branched or,
preferably, linear (unbranched). Exemplary alkyl groups include
ethyl, propyl, butyl, pentyl, 2-methylbutyl,
2-methylpropyl(isobutyl), 3-methylpentyl, and the like. As used
herein, "alkyl" includes cycloalkyl when three or more carbon atoms
are referenced.
[0053] "Lower alkyl" refers to an alkyl group containing from 1 to
6 carbon atoms, as exemplified by methyl, ethyl, n-butyl, i-butyl,
t-butyl.
[0054] "Cycloalkyl" refers to a saturated or unsaturated cyclic
hydrocarbon chain, including bridged, fused, or spiro cyclic
compounds, preferably made up of 3 to about 12 carbon atoms, more
preferably 3 to about 8.
[0055] As used herein, "alkenyl" refers to a branched or unbranched
hydrocarbon group having 2 to 15 carbon atoms and containing at
least one double bond, such as ethenyl, n-propenyl, isopropenyl,
n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, and the
like.
[0056] The term "alkynyl" as used herein refers to a branched or
unbranched hydrocarbon group having 2 to 15 atoms and containing at
least one triple bond, such as ethynyl, n-propynyl, isopentynyl,
n-butynyl, octynyl, decynyl, and so forth.
[0057] "Alkoxy" refers to an --OR group, wherein R is alkyl or
substituted alkyl, preferably C1-C20 alkyl (e.g., methoxy, ethoxy,
propyloxy, etc.), more preferably lower alkyl (i.e. C1-C6).
[0058] "Aryl" refers to a substituted or unsubstituted monovalent
aromatic radical having a single ring (e.g., phenyl) or two
condensed or fused rings (e.g., naphthyl). Multiple aryl rings may
also be unfused (e.g. biphenyl). The term includes heteroaryl
groups, which are aromatic ring groups having one or more nitrogen,
oxygen, or sulfur atoms in the ring, such as furyl, pyrrole,
pyridyl, and indole.
[0059] "Aralkyl" refers to an alkyl, preferably lower
(C.sub.1-C.sub.4, more preferably C.sub.1-C.sub.2)alkyl,
substituent which is further substituted with an aryl group;
examples are benzyl and phenethyl. "Aralkoxy" refers to a group of
the form --OR where R is aralkyl; one example is benzyloxy.
[0060] A "heterocycle" refers to a ring, preferably a 5- to
7-membered ring, whose ring atoms are selected from the group
consisting of carbon, nitrogen, oxygen and sulfur. Preferably, the
ring atoms include 3 to 6 carbon atoms. Examples of aromatic
heterocycles (heteroaryl) are given above; non-aromatic
heterocycles include, for example, pyrrolidine, piperidine,
piperazine, and morpholine.
[0061] A "substituted" group or moiety is one in which a hydrogen
atom has been replaced with a non-hydrogen atom or group, which is
preferably a non-interfering substituent.
[0062] "Non-interfering substituents" are those groups that, when
present in a molecule, are typically non-reactive with other
functional groups contained within the molecule. These include, but
are not limited to, lower alkyl, alkenyl, or alkynyl; lower alkoxy;
C3-C6 cycloalkyl; halo, e.g., fluoro, chloro, bromo, or iodo;
cyano; oxo(keto); nitro; and phenyl.
[0063] A "tertiary group" is a group of the form --CR.sub.3, where
each R is an organic moiety linked to C via a carbon atom. Each R
may be, for example, alkyl, cycloalkyl, aryl, or aralkyl,
substituted or unsubstituted. Examples of tertiary groups include
t-butyl, where each R is methyl; triphenylmethyl(trityl), where
each R is phenyl; and dimethoxytrityl (DMT), where two R's are
p-methoxyphenyl and one is phenyl. Also included are groups where
one or more R's form a ring or ring system, such as adamantyl.
[0064] A "tertiary ester" is an ester having a tertiary group as
its alcohol portion; i.e. R'--(C.dbd.O)--OCR.sub.3, where CR.sub.3
is a "tertiary group" as defined above, and R' is the acid portion
of the ester.
[0065] A "carboxyl group" as used herein refers to the group
--C(.dbd.O)OH (carboxylic acid) or --C(.dbd.O)O.sup.-M.sup.+, where
M+is a positively charged ion, such as an alkali metal ion
(carboxylate group).
[0066] A "low molecular weight" organic acid refers to an acidic
organic compound having a molecular weight less than about 400,
preferably less than about 300, and more preferably less than about
200. The term typically refers to a non-polymeric and
non-oligomeric acid, and generally refers to an acid used as a
reagent. Examples include formic acid, acetic acid, trifluoroacetic
acid (TFA), and p-toluenesulfonic acid.
[0067] An "electrophile" is an atom or collection of atoms having
an electrophilic center, i.e., a center that is electron-seeking or
capable of reacting with a nucleophile.
[0068] A "nucleophile" refers to an atom or a collection of atoms
having a nucleophilic center, i.e., a center that is seeking an
electrophilic center or capable of reacting with an
electrophile.
[0069] A "physiologically cleavable" or "hydrolyzable" or
"degradable" bond is a relatively weak bond that reacts with water
(i.e., is hydrolyzed) under physiological conditions. The tendency
of a bond to hydrolyze in water will depend not only on the general
type of linkage connecting two central atoms but also on the
substituents attached to these central atoms. Appropriate
hydrolytically unstable or weak linkages include, but are not
limited to, carboxylate ester, phosphate ester, anhydrides,
acetals, ketals, acyloxyalkyl ether, imines, and orthoesters.
[0070] An "enzymatically degradable linkage" is a linkage that is
subject to degradation by one or more enzymes.
[0071] A "hydrolytically stable" linkage or bond refers to a
chemical bond, typically a covalent bond, that is substantially
stable in water; i.e. it does not undergo hydrolysis under
physiological conditions to any appreciable extent over an extended
period of time. Generally, a hydrolytically stable linkage is one
that exhibits a rate of hydrolysis of less than about 1-2% per day
under physiological conditions. Examples of hydrolytically stable
linkages include carbon-carbon bonds, ethers, amines, and amides.
Hydrolysis rates of representative chemical bonds can be found in
most standard chemistry textbooks.
[0072] A product containing "substantially no amount" of a
specified component either contains no amount of the specified
component, or contains an amount which is undetectable by
conventional methods of analysis of the product, and/or has no
detectable effect on the properties or stability of the product.
For example, a product which has never knowingly or deliberately
been exposed to or contacted with a particular substance would be
considered to contain substantially no amount of the substance.
[0073] Each of the terms "drug," "biologically active molecule,"
"biologically active moiety," and "biologically active agent", when
used herein, means any substance which can affect any physical or
biochemical property of a biological organism, where the organism
may be selected from viruses, bacteria, fungi, plants, animals, and
humans. In particular, as used herein, biologically active
molecules include any substance intended for diagnosis, cure
mitigation, treatment, or prevention of disease in humans or other
animals, or to otherwise enhance physical or mental well-being of
humans or animals. Examples of biologically active molecules
include, but are not limited to, peptides, proteins, enzymes, small
molecule drugs, dyes, lipids, nucleosides, oligonucleotides,
polynucleotides, nucleic acids, cells, viruses, liposomes,
microparticles and micelles. Classes of biologically active agents
that are suitable for use with the invention include, but are not
limited to, antibiotics, fungicides, anti-viral agents,
anti-inflammatory agents, anti-tumor agents, cardiovascular agents,
anti-anxiety agents, hormones, growth factors, steroidal agents,
and the like. Also included are foods, food supplements, nutrients,
nutriceuticals, drugs, vaccines, antibodies, vitamins, and other
beneficial agents.
[0074] The term "conjugate" refers to an entity formed as a result
of covalent attachment of a molecule, e.g., a biologically active
molecule, to a reactive polymer molecule, preferably a
poly(ethylene glycol).
[0075] "Pharmaceutically acceptable excipient" or "pharmaceutically
acceptable carrier" refers to an excipient that can be included in
the compositions of the invention and that causes no significant
adverse toxicological effects to a patient.
[0076] "Pharmacologically effective amount," "physiologically
effective amount," and therapeutically effective amount" are used
herein to refer to mean the amount of a polymer-active agent
conjugate present in a pharmaceutical preparation that is needed to
provide a desired level of active agent and/or conjugate in the
bloodstream or in the target tissue. The precise amount will depend
upon numerous factors, e.g., the particular active agent, the
components and physical characteristics of pharmaceutical
preparation, intended patient population, patient considerations,
and the like, and can readily be determined by one skilled in the
art, based upon the information provided herein and available in
the relevant literature.
[0077] The term "patient" refers to a living organism suffering
from or prone to a condition that can be prevented or treated by
administration of a biologically active agent or conjugate thereof,
and includes both humans and animals.
[0078] II. Method of Preparing Carboxylic Acid Functionalized
Polymers
[0079] A. Overview
[0080] The present invention provides, in one aspect, a method of
preparing a water soluble, non-peptidic polymer functionalized with
a carboxyl group, i.e. a carboxylate salt or carboxylic acid. The
method involves reacting a tertiary ester reagent R(C.dbd.O)OR',
where R' is a tertiary group, as defined above, and R includes a
functional group X, with a water soluble, non-peptidic polymer
POLY-Y, where Y is a functional group which reacts with X to form a
covalent bond, to form a tertiary ester of the polymer, which may
be represented as POLY-R--(C.dbd.O)OR'. The nature of the linkage
between POLY and R depends on the functional groups Y and X.
[0081] The starting material of the reaction, represented by
POLY-Y, or by PEG-Y when the polymer is a polyethylene glycol, may
include more than one functional group Y, in various
configurations. Examples include linear, branched, and multiarmed
PEGs containing multiple hydroxyl groups, as discussed further
below. The product of the reaction, i.e. the
carboxyl-functionalized polymer, contains a number of carboxyl
groups which is equal to the number of functional groups Y in the
starting material (or greater than Y, if the starting material has
existing carboxyl groups).
[0082] Preferably, the functional group Y of the polymer is a
hydroxyl group, or other nucleophilic group, and the functional
group X of the tertiary ester reagent is a leaving group capable of
being displaced by Y. Other possible functional group combinations
are described below.
[0083] Once the tertiary ester group is attached to the polymer, it
is converted to a carboxylate by base hydrolysis in aqueous
solution, which is preferably followed by acidification to produce
the carboxylic acid. Surprisingly, it has been found that the
tertiary ester, while stable in the presence of the base used in
the initial nucleophilic substitution reaction, can be removed by
base-promoted hydrolysis. As noted above, tertiary alkyl esters,
such as t-butyl esters, are conventionally thought to be resistant
to base hydrolysis.
[0084] The general reaction scheme below depicts a preferred
embodiment of the method of the invention, where Y is hydroxyl and
X is a leaving group, and the ester reagent has the structure shown
as (I). 2
[0085] B. Reaction Components
[0086] In the preferred ester reagent (I), each of R.sup.1 and
R.sup.2 is independently selected from H, lower alkyl, cycloalkyl,
alkoxy, aryl, aralkyl, and heterocycle; and each of R.sup.3-R.sup.5
is independently selected from lower alkyl, aryl, and aralkyl, each
as defined above. Preferably, the group (CR.sup.1R.sup.2).sub.n
does not include two heteroatoms attached to the same carbon atom;
for example, R.sup.1 and R.sup.2 on the same carbon atom are
preferably not both alkoxy. Any of R.sup.1 to R.sup.5, excepting
hydrogen, may be substituted with a non-interfering substituent, as
defined above.
[0087] Preferably, each of R.sup.1 and R.sup.2 is independently
hydrogen or unsubstituted lower alkyl, and each of R.sup.3 to
R.sup.5 is independently unsubstituted lower alkyl or phenyl. In
selected embodiments, each of R.sup.1 and R.sup.2 is independently
hydrogen or methyl, more preferably hydrogen, and each of R.sub.3
to R.sup.5 is independently methyl, ethyl, or phenyl.
[0088] The variable n is 1 to about 24, preferably 1 to about 12.
In selected embodiments, n is 1 or 2, 1 to 3, 1 to 4, 1 to 5, 1 to
6, 1 to 7, 1 to 8, 1 to 9, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to
15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22,
1 to 23, or 1 to 24. In further selected embodiments, n is 1 to 6;
preferably, n is 1 to 4; and more preferably, n is 1 or 2. When n
is greater than 1, the moiety --(CR.sup.1R.sub.2).sub.n--
preferably includes at most two, and more preferably at most one,
non-hydrogen embodiment of R.sup.1 or R.sup.2.
[0089] In further embodiments, n is 1, and R.sup.1 and R.sup.2 are
independently hydrogen or methyl. In one such embodiment, when both
R.sup.1 and R.sup.2 are hydrogen, the product (IV) contains a
carboxymethyl group.
[0090] Preferably, the functional group X on the ester reagent (II)
is a leaving group, such as halo, e.g. chloro or bromo, or
sulfonate ester, such as p-toluenesulfonyl(tosyl),
methanesulfonyl(mesyl), trifluorosulfonyl, or
trifluoroethylsulfonyl(tresyl). However, other functional groups
capable of reacting with a functional group on the polymer, to form
a covalent linkage, could also be used. Preferably, the functional
group on the polymer is a nucleophilic group, such as amine,
hydrazide (--C(.dbd.O)NHNH.sub.2), or thiol, and the functional
group X on the ester reagent is an electrophilic group. In addition
to leaving groups such as those described above, electrophilic
groups include carboxylic ester, including imide ester, orthoester,
carbonate, isocyanate, isothiocyanate, aldehyde, ketone, thione,
alkenyl, acrylate, methacrylate, acrylamide, sulfone, maleimide,
disulfide, iodo, epoxy, thiosulfonate, silane, alkoxysilane,
halosilane, and phosphoramidate. More specific examples of these
groups include succinimidyl ester or carbonate, imidazolyl ester or
carbonate, benzotriazole ester or carbonate, p-nitrophenyl
carbonate, vinyl sulfone, chloroethylsulfone, vinylpyridine,
pyridyl disulfide, iodoacetamide, glyoxal, and dione. Also included
are other activated carboxylic acid derivatives, as well as
hydrates or protected derivatives of any of the above moieties
(e.g. aldehyde hydrate, hemiacetal, acetal, ketone hydrate,
hemiketal, ketal, thioketal, thioacetal). Preferred electrophilic
groups include succinimidyl carbonate, succinimidyl ester,
maleimide, benzotriazole carbonate, glycidyl ether, imidazoyl
ester, p-nitrophenyl carbonate, acrylate, aldehyde, and
orthopyridyl disulfide.
[0091] In general, the functional group X on the reagent is chosen
such that it reacts with the functional group Y on the polymer much
more readily than the functional group Y would react with the
t-butyl ester portion of the reagent. When the polymeric functional
group Y is a nucleophile, such as hydroxyl, X is most suitably a
good leaving group such as halo or sulfonate ester.
[0092] Particularly preferred ester reagents include t-butyl
haloacetates, such as t-butyl bromoacetate, t-butyl chloroacetate,
and t-butyl iodoacetate. Such t-butyl haloacetates are available,
for example, from Sigma Chemical Co., St. Louis, Mo.
[0093] In Scheme I, POLY-OH is a water soluble, non-peptidic
polymer, such as, for example, mPEG-OH. In general, the polymer can
be any water soluble, non-peptidic polymer, having any available
geometric configuration (e.g., linear, branched, forked, etc.), as
discussed further below. For the sake of simplicity, the reaction
scheme given above utilizes a polymer with a single hydroxyl group.
However, as would be appreciated by one of ordinary skill in the
art, the polymer may comprise more than one hydroxyl group, such as
1 to about 25 hydroxyl groups (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
or more hydroxyl groups). Also, the hydroxyl group could be
replaced with any nucleophilic functional group reactive with the
functional group X on the tertiary ester reagent. Such nucleophilic
functional groups include thiols, amines, and stabilized
carbanions.
[0094] C. Reaction Process
[0095] For the first stage of the process, shown in the top line of
exemplary Scheme I above, the components are preferably dissolved
in a suitable organic solvent, such as t-butanol, benzene, toluene,
xylenes, tetrahydrofuran (THF), dimethylformamide (DMF),
dimethylsulfoxide (DMSO) and the like.
[0096] As shown in the embodiment of the invention represented by
Scheme I, reaction of a polymeric hydroxyl group with the tertiary
ester reagent is typically carried out in the presence of a base.
Exemplary bases include potassium t-butoxide, butyl lithium, sodium
amide, and sodium hydride. Other strong bases could also be
used.
[0097] The reaction is typically carried out at a temperature of
about 0-120.degree. C., preferably about 20-80.degree. C., more
preferably about 25-50.degree. C., although reaction conditions
will vary based on the polymer and the functional groups reacting.
As shown in the Examples provided below, reactions of
hydroxy-containing PEGs with t-butyl bromoacetate were effectively
carried out at temperatures between room temperature and about
45.degree. C.
[0098] The reaction time is typically about 0.5 hours to about 24
hours; e.g. about 1 to 20, 3 to 18, 4 to 12, or 6 to 8 hours.
Typical reaction times for reaction of a hydroxylated polymer with
t-butyl bromoacetate, as shown in the Examples below, are in the
range of 12 to 20 hours. The reaction may be monitored for
completion according to standard methods. Preferably, the reaction
is carried out under an inert atmosphere such as nitrogen or
argon.
[0099] The reaction preferably employs a molar excess of the ester
reagent (e.g., a twofold, threefold, 6 fold, 10 fold, or 20 fold,
up to about 30 fold molar excess), in order to ensure that complete
conversion of the polymer starting material is achieved. Following
this stage of the reaction, the organic solvent is removed,
typically by evaporation or distillation.
[0100] The ester-containing product (III) is dissolved in water,
preferably distilled or deionized water, for the second stage of
the process, in which the ester-containing polymer is subjected to
base-promoted hydrolysis by treatment with a strong base, such as
hydroxide, in aqueous solution. The base hydrolysis is typically
carried out at a pH to about 9 or above, preferably about 10 or
above, and more preferably about 11 or above (e.g., about 11 to
about 13). Accordingly, the base is one that is strong enough to
produce a pH in this range in aqueous solution. In one embodiment,
the pH is adjusted to fall in the range from about 12 to about
12.5. Preferably, base is added as necessary throughout the
reaction to maintain the pH in this range. The base is also
effective to hydrolyze any remaining ester reagent.
[0101] The base should produce a highly water soluble salt when
neutralized with acid in the step subsequent to hydrolysis.
Preferred are alkali metal hydroxides, such as sodium hydroxide
(NaOH) or potassium hydroxide (KOH).
[0102] The use of distilled or deionized water, or water having no
detectable levels of divalent cations such as calcium and magnesium
ions, is also preferred. The base hydrolysis step is typically
conducted at a temperature of about 0-50.degree. C., preferably
about 10-30.degree. C. The reaction time is typically about 12 to
36 hours; e.g. about 18 to 24 hours.
[0103] The polymer carboxylate salt produced by the base hydrolysis
can be isolated and stored as the salt, or, preferably, it is
directly converted to the carboxylic acid by treatment with acid,
as described below. Generally, the carboxylic acid is more suitable
for further derivatization than the carboxylate salt.
[0104] The carboxylate-containing polymer is treated with aqueous
acid to convert the salt to the free acid form. The acid is
preferably one that produces a non-nucleophilic anion in aqueous
solution. Mineral acids (i.e., inorganic acids) are preferred, such
as sulfuric acid, nitric acid, phosphoric acid, hydrochloric acid,
and the like. Typically, sufficient acid is added to adjust the pH
of the solution to about 1-3, more preferably about 2-3, which is
effective to convert the polymer carboxylate salt to a free acid
form, as well as to neutralize (and convert to a freely water
soluble salt) any base remaining in solution. The acidification
step is typically conducted at a temperature of about 0.degree. to
about 50.degree. C., preferably about 10.degree. to about
30.degree. C.
[0105] The carboxylic acid-containing polymer is then separated
using a conventional organic extraction step, preferably employing
a halogenated solvent such as dichloromethane or chloroform. The
polymer product is extracted into the organic phase, while any
hydrolyzed reagent and excess mineral acid or its salt remains in
the aqueous phase. Thus, separation of the mineral acid from the
polymer product is relatively simple.
[0106] The organic extract is dried and concentrated, and the
polymeric product is then purified using standard methods. For
example, the polymer may be isolated by precipitation, followed by
filtration and drying. The choice of precipitating solvent will
depend on the nature of the polymer, for PEG polymers, as described
in the Examples below, ethyl ether is a suitable precipitating
solvent. Recrystallization from solvents such as ethyl acetate or
ethanol can also be used for purification.
[0107] D. Reaction Products
[0108] Using the method of the invention, carboxyl-functionalized
polymers are produced with high purity, typically with a purity of
at least about 95%, preferably at least about 96%, 97%, or 98%,
more preferably at least about 99%, and most preferably at least
about 99.5% by weight. In selected embodiments, the polymer product
contains at least about 99.6%, 99.7%, 99.8%, or 99.9% by weight of
the desired carboxyl-functionalized polymer. Accordingly, the
product of the synthetic method disclosed herein contains less than
5%, preferably less than 4%, 3%, or 2%, more preferably less than
1%, and most preferably less than 0.5% by weight of starting
polymer (e.g., mPEG-OH, PEG diol, or multifunctional PEG polyol) or
other polymeric impurities. In selected embodiments, the product
contains less than 0.4%, 0.3%, 0.2% or 0.1% by weight of polymeric
starting material (e.g., mPEG-OH) or other polymeric
impurities.
[0109] By "product" or "polymer product" is meant the material
obtained by carrying out the synthetic process disclosed above,
including routine workup procedures such as extraction,
precipitation and removal of solvent. As shown in the Examples
below, reaction mixtures containing the products of the methods
disclosed herein were worked up by extraction with a chlorinated
solvent, followed by precipitation of the product from ethyl ether.
Ion exchange chromatographic analysis of these products showed
essentially 100% of the desired PEG-carboxylic acid product, with
no detectable amount of starting material or other polymeric
impurity present. Accordingly, polymer products having the
above-disclosed purities are obtained without the need for removal
of polymeric impurities, such as starting material. These products
can often be used directly for further derivatization and/or
conjugation, as described below. A further advantage of this
process is that it provides high purity polymeric carboxylic acids,
such as mPEG carboxylic acids, starting from inexpensive starting
materials such as mPEG-OH, in contrast to the use of commercially
available polymeric carboxylic acids, which tend to be expensive
and frequently contain residual amounts of polymeric hydroxyl
compound as well.
[0110] As described above, the reagents employed in the synthetic
process disclosed herein are readily removed from the polymeric
product. In particular, no low molecular weight organic acids, such
as TFA, are used in the process. Accordingly, the
carboxyl-containing polymer products of this invention contain no
trace amounts of low molecular weight organic acids, such as TFA,
as would commonly occur in polymeric carboxylic acids made using a
hydrolysis process which employs such a reagent. The present
products therefore do not suffer the disadvantage of reduced
stability associated with the presence of residual acids, as
described above. For example, the polymer described in Example 4
below showed no sign of degradation (by GPC analysis) after 8
months of storage at -20.degree. C.
[0111] III. Suitable Water-Soluble Non-Peptidic Polymers
[0112] Any of a variety of non-peptidic, water soluble polymers can
be used in the present invention. The polymer should be non-toxic
and biocompatible, meaning that it is capable of coexistence with
living tissues or organisms without causing harm. Examples of
suitable polymers include, but are not limited to, poly(alkylene
glycols), copolymers of ethylene glycol and propylene glycol,
poly(olefinic alcohol), poly(vinylpyrrolidone),
poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),
poly(saccharides), poly(.alpha.-hydroxyacetic acid), poly(acrylic
acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines,
poly(N-acryloylmorpholine), such as described in U.S. Pat. No.
5,629,384, which is incorporated by reference herein in its
entirety, and copolymers, terpolymers, and mixtures thereof.
[0113] The molecular weight of the polymer will vary depending on
the desired application, the configuration of the polymer
structure, the degree of branching, and the like. Generally,
polymers having a molecular weight of about 100 Da to about 100,000
Da are useful in the present invention, preferably about 200 Da to
about 60,000 Da, and more preferably about 300 Da to about 40,000
Da. Exemplary polymer embodiments have a molecular weight of
approximately 200 Da, 350 Da, 550 Da, 750 Da, 1,000 Da, 2,000 Da,
3,000 Da, 4,000 Da, 5,000 Da, 7,500 Da, 10,000 Da, 15,000 Da,
20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 50,000 Da,
55,000 Da, and 60,000 Da.
[0114] The polymer preferably comprises at least one hydroxyl
group, capable of reacting with a tertiary ester reagent carrying a
leaving group, as described herein, in a nucleophilic substitution
reaction. However, other functional groups capable of reacting with
a functional group of the tertiary ester reagent could also be
used. These include other nucleophilic groups, such as amine,
hydrazide (--C(.dbd.O)NH.sub.2), and thiol; and electrophilic
groups, such as carboxylic ester, including imide ester,
orthoester, carbonate, isocyanate, isothiocyanate, aldehyde,
ketone, thione, alkenyl, acrylate, methacrylate, acrylamide,
sulfone, maleimide, disulfide, iodo, epoxy, sulfonate,
thiosulfonate, silane, alkoxysilane, halosilane, and
phosphoramidate. More specific examples of these groups include
succinimidyl ester or carbonate, imidazolyl ester or carbonate,
benzotriazole ester or carbonate, p-nitrophenyl carbonate, vinyl
sulfone, chloroethylsulfone, vinylpyridine, pyridyl disulfide,
iodoacetamide, glyoxal, dione, mesylate, tosylate, and tresylate.
Also included are other activated carboxylic acid derivatives, as
well as hydrates or protected derivatives of any of the above
moieties (e.g. aldehyde hydrate, hemiacetal, acetal, ketone
hydrate, hemiketal, ketal, thioketal, thioacetal). Preferred
electrophilic groups include succinimidyl carbonate, succinimidyl
ester, maleimide, benzotriazole carbonate, glycidyl ether,
imidazoyl ester, p-nitrophenyl carbonate, acrylate, tresylate,
aldehyde, and orthopyridyl disulfide.
[0115] The functional groups are selected such that a nucleophilic
group on the polymer reacts with an electrophilic group on the
tertiary ester reagent, or vice versa. The reaction between the two
functional groups is preferably a displacement reaction of a
leaving group by a nucleophile, but could also be, for example, a
condensation or addition reaction.
[0116] The polymer preferably comprises at least one nucleophilic
group, such as a hydroxyl group. For ease of reference, hydroxyl
groups are discussed below, although other functional groups could
be used. A polymer may also include different functional groups
within the same molecule. Preferably these have similar
functionality, e.g. both nucleophilic, such as a hydroxyl group and
an amino group.
[0117] Preferably, the polymer is a poly(ethylene glycol) (i.e.,
PEG)polymer. As noted above, the term PEG includes poly(ethylene
glycol) in any of a number of geometries or forms, including linear
forms, branched or multi-arm forms (e.g., forked PEG or PEG
attached to a polyol core), pendant PEG, or PEG with degradable
linkages therein.
[0118] The number and position of hydroxyl groups (and/or other
functional groups) carried by the polymer may vary. Typically, the
polymer comprises 1 to about 25 hydroxyl groups, preferably 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10 hydroxyl groups. Linear polymers, such
as linear PEG polymers, typically comprise one or two hydroxyl
groups, each positioned at a terminus of the polymer chain. If the
PEG polymer is monofunctional (i.e., mPEG), the polymer includes a
single hydroxyl group. If the PEG polymer is difunctional, the
polymer contains two hydroxyl groups, one at each terminus of the
polymer chain, or it contains a single hydroxyl group and a
different functional group at the opposing terminus. Multi-arm or
branched polymers may comprise a greater number of hydroxyl
groups.
[0119] Multi-armed or branched PEG molecules are described, for
example, in U.S. Pat. No. 5,932,462, which is incorporated by
reference herein in its entirety. Generally speaking, a multi-armed
or branched polymer possesses two or more polymer "arms" extending
from a central branch point, which preferably comprise a
hydrolytically stable linking structure. An exemplary branched PEG
polymer is methoxy poly(ethylene glycol) disubstituted lysine.
[0120] In another multi-arm embodiment, the polymer comprises a
central core molecule derived from a polyol or polyamine, the
central core molecule providing a plurality of attachments sites
suitable for covalently attaching polymer arms to the core molecule
in order to form a multi-arm polymer structure. Depending on the
desired number of polymer arms, the polyol or polyamine will
typically comprise 3 to about 25 hydroxyl or amino groups,
preferably 3 to about 10, most preferably 3 to about 8 (e.g., 3, 4,
5, 6, 7, or 8).
[0121] Multi-armed polymers are further described, for example, in
co-owned U.S. Patent Appn. Nos. 2002/0156047 and 2002/0156047,
which are incorporated herein by reference.
[0122] The PEG polymer may alternatively comprise a forked PEG.
Generally speaking, a polymer having a forked structure is
characterized as having a polymer chain attached to two or more
functional groups via covalent linkages extending from a
hydrolytically stable branch point in the polymer. U.S. Pat. No.
6,362,254, the contents of which are incorporated by reference
herein, discloses various forked PEG structures capable of use in
the present invention.
[0123] The PEG polymer may also be a pendant PEG molecule, having
reactive groups (e.g., hydroxyl groups) covalently attached along
the length of the PEG backbone rather than at the end of the PEG
chain. The pendant reactive groups can be attached to the PEG
backbone directly or through a linking moiety, such as an alkylene
group.
[0124] Different polymers can be incorporated into the same polymer
backbone. For example, one or more of the PEG molecules in the
branched structures described above can be replaced with a
different polymer type.
[0125] The polymer can also be prepared with one or more
hydrolytically stable or degradable linkages in the polymer
backbone. For example, PEG can be prepared with ester linkages in
the polymer backbone that are subject to hydrolysis. Other
hydrolytically degradable linkages that may be incorporated include
carbonate, imine, phosphate ester, hydrazone, acetal, orthoester,
and phosphoramidate linkages.
[0126] The term poly(ethylene glycol) or PEG includes any or all
the above described variations. Generally preferred PEG structures
include linear monofunctional, branched monofunctional, and linear,
branched or forked difunctional or trifunctional PEGs.
[0127] Because end-capped polyethylene glycol starting materials,
such as mPEG (methoxy-PEG) or bPEG (benzyloxy-PEG), can contain
detectable amounts of PEG diol impurity, leading to side products
that are often difficult to analyze or separate, the PEG starting
material is, in one preferred embodiment, a diol-free benzyloxy-PEG
as described in co-owned U.S. Pat. No. 6,448,369.
[0128] IV. Further Derivatization and Conjugation of Carboxylic
Acid Functionalized Polymers
[0129] A. Overview
[0130] If desired, a carboxylic acid functionalized polymer
prepared by the method of the invention can be further modified to
form useful reactive derivatives of carboxylic acids using
methodology known in the art. Preparation of such derivatives is
facilitated by the high purity of the carboxylic acid
functionalized polymers of the invention, as compared to prior art
products containing, for example, residual starting material
polymer and/or residual reagents such as TFA. This is a significant
benefit, particularly for a pharmaceutical product, since the
presence and amounts of such contaminants can be highly variable,
thus leading to irreproducibility of the product.
[0131] Accordingly, the method of the invention, wherein a
carboxylic acid functionalized polymer is prepared, may further
comprise the steps of (i) modifying the carboxylic acid to form a
reactive derivative and (ii) conjugating the reactive derivative to
a pharmacologically relevant molecule having a corresponding
reactive functional group. The steps (i) and (ii) may be performed
in situ, where the carboxylic acid is converted to an activated
derivative using one of many activating reagents known in the art,
then immediately reacted with the molecule to be conjugated.
[0132] The carboxylic acid can be derivatized to form, for example,
acyl halides, acyl pseudohalides, such as acyl cyanide, acyl
isocyanate, and acyl azide, neutral salts, such as alkali metal or
alkaline-earth metal salts (e.g. calcium, sodium, or barium salts),
esters, anhydrides, amides, imides, hydrazides, and the like. In a
preferred embodiment, the acid is esterified to form an active
ester, such as an N-succinimidyl ester, o-, m-, or p-nitrophenyl
ester, 1-benzotriazolyl ester, imidazolyl ester, or
N-sulfosuccinimidyl ester.
[0133] In one embodiment, the further derivatized polymer is a PEG
polymer having the structure:
mPEG-O--(CR.sup.1R.sup.2).sub.n--C(.dbd.O)-Z (V)
[0134] wherein R.sub.1, R.sub.2 and n are as described above. The
moiety Z is preferably selected from the group consisting of halo,
amino, substituted amino, --NCO, --NCS, N.sub.3, --CN, and --OR',
wherein R' is selected from N-succinimidyl, nitrophenyl,
benzotriazolyl, imidazolyl, N-sulfosuccinimidyl, N-phthalimidyl,
N-glutarimidyl, N-tetrahydrophthalimidyl,
N-norbornene-2,3-dicarboximidyl, and
hydroxy-7-azabenzotriazolyl.
[0135] The carboxyl-containing polymer produced by the method of
the invention, or a reactive derivative thereof, can be used to
form conjugates with biologically active molecules, particularly
biologically active molecules carrying nucleophilic functional
groups, such as amino, hydroxyl, or mercapto (thiol) groups.
[0136] Frequently, the molecule to be conjugated is a protein.
Proteins are conjugated via reactive amino acids, such as lysine,
histidine, arginine, aspartic acid, glutamic acid, serine,
threonine, tyrosine, cysteine, the N-terminal amino group, and the
C-terminal carboxylic acid. Carbohydrate moieties on glycosylated
proteins may also be employed as conjugation sites. For reaction
with an activated carboxylic acid, the most suitable groups are the
N-terminal amino group, amine-containing side chains on lysine,
histidine, and arginine, hydroxyl-containing side chains on serine,
threonine, and tyrosine, and thiol side chains on cysteine.
[0137] Although the preferred methods of conjugation of the
carboxyl-containing polymers of the invention employ activated
carboxylic acid derivatives, which react with nucleophilic groups
on the molecule to be conjugated, it is also possible to derivatize
the terminal carboxyl group to contain any variety of functional
groups. For example, in one embodiment, the moiety Z in structure
(V) above has the structure --NHR.sub.6, wherein R.sub.6 is an
organic group that contains a reactive functional group (e.g.,
aldehyde, maleimide, mercapto, hydroxyl, amino, etc.), the
functional group(s) being separated from the nitrogen atom by an
alkylene chain (e.g., C1-6) and, optionally, an additional linker,
such as a short PEG chain and another alkylene chain (e.g.,
alkylene-PEG-alkylene).
[0138] B. Exemplary Methods of Conjugation
[0139] Such polymer conjugates can be formed using known techniques
for covalent attachment of an activated polymer, such as an
activated PEG, to a biologically active agent. See, for example,
Poly(ethylene glycol): Chemistry and Biological Applications, J. M.
Harris and S. Zalipsky, editors, American Chemical Society,
Washington, D.C. (1997) or Bioconjugate Techniques, G. T.
Hermanson, Academic Press (1996). In general, conjugation reactions
are typically carried out in a buffer, such as a phosphate or
acetate buffer, at or near room temperature, although conditions
will depend on the particular reaction being carried out. An excess
of the polymeric reagent is typically combined with the active
agent. In some cases, however, it is preferred to have
stoichiometric amounts of the reactive groups on the polymeric
reagent and on the active agent.
[0140] Progress of a conjugation reaction can be monitored by
SDS-PAGE, MALDI-TOF mass spectrometry, or any other suitable
analytical method. Once a plateau is reached with respect to the
amount of conjugate formed or the amount of unconjugated polymer
remaining, the reaction is assumed to be complete. The product
mixture is purified, if necessary, to separate excess reagents,
unconjugated reactants (e.g., active agent) undesired
multi-conjugated species, and/or unreacted polymer, using known
methods.
[0141] For example, conjugates having different molecular weights
can be separated using gel filtration chromatography. Fractions may
be analyzed by a number of different methods, e.g. (i) OD at 280 nm
for protein content, (ii) BSA protein analysis, (iii) iodine
testing for PEG content (Sims et al., Anal. Biochem. 107:60-63,
1980), or (iv) SDS-PAGE, followed by staining with barium
iodide.
[0142] Separation of positional isomers (that is, conjugates of the
same or substantially the same molecular weight having a polymer
attached at different positions on a molecule) can be carried out
by reverse phase HPLC or ion exchange chromatography.
[0143] The conjugated product may be lyophilized for storage, with
or without residual buffer. In some instances, it is preferable to
exchange a buffer used for conjugation, such as sodium acetate, for
a volatile buffer, such as ammonium carbonate or ammonium acetate,
that can be readily removed during lyophilization. Alternatively, a
buffer exchange step may be used using a formulation buffer, so
that the lyophilized conjugate is in a form suitable for
reconstitution into a formulation buffer and ultimately for
administration to a mammal.
[0144] C. Exemplary Agents for Conjugation
[0145] A biologically active agent for use in coupling to polymer
formed by the method of the invention may be any one or more of the
following. Suitable agents may be selected from, for example,
hypnotics and sedatives, psychic energizers, tranquilizers,
respiratory drugs, anticonvulsants, muscle relaxants, antiparkinson
agents (dopamine antagonists), analgesics, anti-inflammatories,
antianxiety drugs (anxiolytics), appetite suppressants,
antimigraine agents, muscle contractants, anti-infectives
(antibiotics, antivirals, antifungals, vaccines) antiarthritics,
antimalarials, antiemetics, anepileptics, bronchodilators,
cytokines, growth factors, anti-cancer agents, antithrombotic
agents, antihypertensives, cardiovascular drugs, antiarrhythmics,
antioxidants, anti-asthma agents, hormonal agents including
contraceptives, sympathomimetics, diuretics, lipid regulating
agents, antiandrogenic agents, antiparasitics, anticoagulants,
neoplastics, antineoplastics, hypoglycemics, nutritional agents and
supplements, growth supplements, antienteritis agents, vaccines,
antibodies, diagnostic agents, and contrasting agents.
[0146] More particularly, the active agent may fall into one of a
number of structural classes, including but not limited to small
molecules, peptides, polypeptides, proteins, antibodies,
polysaccharides, steroids, nucleotides, oligonucleotides,
polynucleotides, fats, electrolytes, and the like. Preferably, an
active agent for coupling to a carboxyl-containing polymer of the
invention possesses a native amino, hydroxyl, or thiol group, or is
modified to contain at least one such group.
[0147] Specific examples of active agents include but are not
limited to aspariginase, amdoxovir (DAPD), antide, becaplermin,
calcitonins, cyanovirin, denileukin diftitox, erythropoietin (EPO),
EPO agonists (e.g., peptides from about 10-40 amino acids in length
and comprising a particular core sequence as described in WO
96/40749), dornase alpha, erythropoiesis stimulating protein
(NESP), coagulation factors such as Factor V, Factor VII, Factor
VIIa, Factor VIII, Factor IX, Factor X, Factor XII, Factor XIII,
von Willebrand factor; ceredase, cerezyme, alpha-glucosidase,
collagen, cyclosporin, alpha defensins, beta defensins, exedin-4,
granulocyte colony stimulating factor (GCSF), thrombopoietin (TPO),
alpha-1 proteinase inhibitor, elcatonin, granulocyte macrophage
colony stimulating factor (GMCSF), fibrinogen, filgrastim, growth
hormones, human growth hormone (hGH), growth hormone releasing
hormone (GHRH), GRO-beta, GRO-beta antibody, bone morphogenic
proteins such as bone morphogenic protein-2, bone morphogenic
protein-6, OP-1; acidic fibroblast growth factor, basic fibroblast
growth factor, CD-40 ligand, heparin, human serum albumin, low
molecular weight heparin (LMWH), interferons such as interferon
alpha, interferon beta, interferon gamma, interferon omega,
interferon tau, consensus interferon; interleukins and interleukin
receptors such as interleukin-1 receptor, interleukin-2,
interluekin-2 fusion proteins, interleukin-1 receptor antagonist,
interleukin-3, interleukin-4, interleukin-4 receptor,
interleukin-6, interleukin-8, interleukin-12, interleukin-13
receptor, interleukin-17 receptor; lactoferrin and lactoferrin
fragments, luteinizing hormone releasing hormone (LHRH), insulin,
pro-insulin, insulin analogues (e.g., mono-acylated insulin as
described in U.S. Pat. No. 5,922,675), amylin, C-peptide,
somatostatin, somatostatin analogs including octreotide,
vasopressin, follicle stimulating hormone (FSH), influenza vaccine,
insulin-like growth factor (IGF), insulintropin, macrophage colony
stimulating factor (M-CSF), plasminogen activators such as
alteplase, urokinase, reteplase, streptokinase, pamiteplase,
lanoteplase, and teneteplase; nerve growth factor (NGF),
osteoprotegerin, platelet-derived growth factor, tissue growth
factors, transforming growth factor-1, vascular endothelial growth
factor, leukemia inhibiting factor, keratinocyte growth factor
(KGF), glial growth factor (GGF), T Cell receptors, CD
molecules/antigens, tumor necrosis factor (TNF), monocyte
chemoattractant protein-1, endothelial growth factors, parathyroid
hormone (PTH), glucagon-like peptide, somatotropin, thymosin alpha
1, thymosin alpha 1 IIb/IIIa inhibitor, thymosin beta 10, thymosin
beta 9, thymosin beta 4, alpha-1 antitrypsin, phosphodiesterase
(PDE) compounds, VLA-4 (very late antigen-4), VLA-4 inhibitors,
bisphosponates, respiratory syncytial virus antibody, cystic
fibrosis transmembrane regulator (CFTR) gene, deoxyreibonuclease
(Dnase), bactericidal/permeability increasing protein (BPI), and
anti-CMV antibody. Exemplary monoclonal antibodies include
etanercept (a dimeric fusion protein consisting of the extracehular
ligand-binding portion of the human 75 kD TNF receptor linked to
the Fc portion of IgG1), abciximab, afeliomomab, basiliximab,
daclizumab, infliximab, ibritumomab tiuexetan, mitumomab,
muromonab-CD3, iodine 131 tositumomab conjugate, olizumab,
rituximab, and trastuzumab (herceptin).
[0148] Additional agents suitable for covalent attachment to a
polymer include amifostine, amiodarone, aminocaproic acid,
aminohippurate sodium, aminoglutethimide, aminolevulinic acid,
aminosalicylic acid, amsacrine, anagrelide, anastrozole,
asparaginase, anthracyclines, bexarotene, bicalutamide, bleomycin,
buserelin, busulfan, cabergoline, capecitabine, carboplatin,
carmustine, chlorambucin, cilastatin sodium, cisplatin, cladribine,
clodronate, cyclophosphamide, cyproterone, cytarabine,
camptothecins, 13-cis retinoic acid, all trans retinoic acid,
dacarbazine, dactinomycin, daunorubicin, deferoxamine,
dexamethasone, diclofenac, diethylstilbestrol, docetaxel,
doxorubicin, epirubicin, estramustine, etoposide, exemestane,
fexofenadine, fludarabine, fludrocortisone, fluorouracil,
fluoxymesterone, flutamide, gemcitabine, epinephrine, L-Dopa,
hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan,
itraconazole, goserelin, letrozole, leucovorin, levamisole,
lisinopril, lovothyroxine sodium, lomustine, mechlorethamine,
medroxyprogesterone, megestrol, melphalan, mercaptopurine,
metaraminol bitartrate, methotrexate, metoclopramide, mexiletine,
mitomycin, mitotane, mitoxantrone, naloxone, nicotine, nilutamide,
octreotide, oxaliplatin, pamidronate, pentostatin, pilcamycin,
porfimer, prednisone, procarbazine, prochlorperazine, ondansetron,
raltitrexed, sirolimus, streptozocin, tacrolimus, tamoxifen,
temozolomide, teniposide, testosterone, tetrahydrocannabinol,
thalidomide, thioguanine, thiotepa, topotecan, tretinoin,
valrubicin, vinblastine, vincristine, vindesine, vinorelbine,
dolasetron, granisetron; formoterol, fluticasone, leuprolide,
midazolam, alprazolam, amphotericin B, podophylotoxins, nucleoside
antivirals, aroyl hydrazones, sumatriptan; macrolides such as
erythromycin, oleandomycin, troleandomycin, roxithromycin,
clarithromycin, davercin, azithromycin, flurithromycin,
dirithromycin, josamycin, spiromycin, midecamycin, leucomycin,
miocamycin, rokitamycin, andazithromycin, and swinolide A;
fluoroquinolones such as ciprofloxacin, ofloxacin, levofloxacin,
trovafloxacin, alatrofloxacin, moxifloxicin, norfloxacin, enoxacin,
grepafloxacin, gatifloxacin, lomefloxacin, sparfloxacin,
temafloxacin, pefloxacin, amifloxacin, fleroxacin, tosufloxacin,
prulifloxacin, irloxacin, pazufloxacin, clinafloxacin, and
sitafloxacin; aminoglycosides such as gentamicin, netilmicin,
paramecin, tobramycin, amikacin, kanamycin, neomycin, and
streptomycin, vancomycin, teicoplanin, rampolanin, mideplanin,
colistin, daptomycin, granicidin, colistimethate; polymixins such
as polymixin B, capreomycin, bacitracin, penems; penicillins
including penicllinase-sensitive agents like penicillin G,
penicillin V; penicllinase-resistant agents like methicillin,
oxacillin, cloxacillin, dicloxacillin, floxacillin, nafcillin; gram
negative microorganism active agents like ampicillin, amoxicillin,
and hetacillin, cillin, and galampicillin; antipseudomonal
penicillins like carbenicillin, ticarcillin, azlocillin,
mezlocillin, and piperacillin; cephalosporins like cefpodoxime,
cefprozil, ceftbuten, ceftizoxime, ceftriaxone, cephalothin,
cephapirin, cephalexin, cephradrine, cefoxitin, cefamandole,
cefazolin, cephaloridine, cefaclor, cefadroxil, cephaloglycin,
cefuroxime, ceforanide, cefotaxime, cefatrizine, cephacetrile,
cefepime, cefixime, cefonicid, cefoperazone, cefotetan,
cefinetazole, ceftazidime, loracarbef, and moxalactam, monobactams
like aztreonam; and carbapenems such as imipenem, meropenem,
pentamidine isethiouate, albuterol sulfate, lidocaine,
metaproterenol sulfate, beclomethasone diprepionate, triamcinolone
acetamide, budesonide acetonide, fluticasone, ipratropium bromide,
flunisolide, cromolyn sodium, and ergotamine tartrate; taxanes such
as paclitaxel; SN-38, and tyrphostines.
[0149] The above exemplary biologically active agents are meant to
encompass, where applicable, analogues, agonists, antagonists,
inhibitors, isomers, and pharmaceutically acceptable salt forms
thereof. In reference to peptides and proteins, the invention is
intended to encompass synthetic, recombinant, native, glycosylated,
and non-glycosylated forms, as well as biologically active
fragments thereof. The above biologically active proteins are
additionally meant to encompass variants having one or more amino
acids substituted (e.g., cysteine), deleted, or the like, as long
as the resulting variant protein possesses at least a certain
degree of activity of the parent (native) protein.
[0150] V. Pharmaceutical Compositions and Administration
Methods
[0151] The present invention also includes pharmaceutical
preparations comprising a conjugate as provided herein in
combination with a pharmaceutical excipient. Exemplary excipients
include, without limitation, those selected from the group
consisting of carbohydrates, antimicrobial agents, antioxidants,
surfactants, buffers, and combinations thereof.
[0152] A carbohydrate such as a sugar, a derivatized sugar such as
an alditol, aldonic acid, an esterified sugar, and/or a sugar
polymer may be present as an excipient. Specific carbohydrate
excipients include, for example: monosaccharides, such as fructose,
maltose, galactose, glucose, D-mannose, sorbose, and the like;
disaccharides, such as lactose, sucrose, trehalose, cellobiose, and
the like; polysaccharides, such as raffinose, melezitose,
maltodextrins, dextrans, starches, and the like; and alditols, such
as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol
(glucitol), pyranosyl sorbitol, myoinositol, and the like.
[0153] The excipient can also include an inorganic salt or buffer
such as citric acid, sodium chloride, potassium chloride, sodium
sulfate, potassium nitrate, sodium phosphate monobasic, sodium
phosphate dibasic, and combinations thereof.
[0154] The preparation may also include an antimicrobial agent for
preventing or deterring microbial growth. Nonlimiting examples of
antimicrobial agents suitable for the present invention include
benzalkonium chloride, benzethonium chloride, benzyl alcohol,
cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl
alcohol, phenylmercuric nitrate, thimersol, and combinations
thereof.
[0155] An antioxidant can be present in the preparation as well.
Antioxidants are used to prevent oxidation, thereby preventing the
deterioration of the conjugate or other components of the
preparation. Suitable antioxidants for use in the present invention
include, for example, ascorbyl palmitate, butylated hydroxyanisole,
butylated hydroxytoluene, hypophosphorous acid, monothioglycerol,
propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate,
sodium metabisulfite, and combinations thereof.
[0156] A surfactant may be present as an excipient. Exemplary
surfactants include: polysorbates, such as "Tween 20" and "Tween
80," and pluronics such as F68 and F88 (both of which are available
from BASF, Mount Olive, N.J.); sorbitan esters; lipids, such as
phospholipids such as lecithin and other phosphatidylcholines,
phosphatidylethanolamines (although preferably not in liposomal
form), fatty acids and fatty esters; steroids, such as cholesterol;
and chelating agents, such as EDTA, zinc and other such suitable
cations.
[0157] Acids or bases may be present as an excipient in the
preparation. Nonlimiting examples of acids that can be used include
those acids selected from the group consisting of hydrochloric
acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic
acid, formic acid, trichloroacetic acid, nitric acid, perchloric
acid, phosphoric acid, sulfuric acid, fumaric acid, and
combinations thereof. Examples of suitable bases include, without
limitation, bases selected from the group consisting of sodium
hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide,
ammonium acetate, potassium acetate, sodium phosphate, potassium
phosphate, sodium citrate, sodium formate, sodium sulfate,
potassium sulfate, potassium fumarate, and combinations
thereof.
[0158] The pharmaceutical preparations encompass all types of
formulations and in particular those that are suited for injection,
e.g., powders that can be reconstituted as well as suspensions and
solutions. The amount of the conjugate (i.e., the conjugate formed
between the active agent and the polymer described herein) in the
composition will vary depending on a number of factors, but will
optimally be a therapeutically effective dose when the composition
is stored in a unit dose container (e.g., a vial). In addition, the
pharmaceutical preparation can be housed in a syringe. A
therapeutically effective dose can be determined experimentally by
repeated administration of increasing amounts of the conjugate in
order to determine which amount produces a clinically desired
endpoint.
[0159] The amount of any individual excipient in the composition
will vary depending on the activity of the excipient and particular
needs of the composition. Typically, the optimal amount of any
individual excipient is determined through routine experimentation,
i.e., by preparing compositions containing varying amounts of the
excipient (ranging from low to high), examining the stability and
other parameters, and then determining the range at which optimal
performance is attained with no significant adverse effects.
Generally, however, the excipient will be present in the
composition in an amount of about 1% to about 99% by weight,
preferably from about 5%-98% by weight, more preferably from about
15-95% by weight of the excipient, with concentrations less than
30% by weight most preferred.
[0160] The foregoing pharmaceutical excipients and others are
described in Remington: The Science & Practice of Pharmacy, 19'
ed., Williams & Williams, (1995), the Physician's Desk
Reference, 52.sup.nd ed., Medical Economics, Montvale, N.J. (1998),
and Kibbe, A. H., Handbook of Pharmaceutical Excipients, 3.sup.rd
Edition, American Pharmaceutical Association, Washington, D.C.,
2000.
[0161] The pharmaceutical preparations of the present invention are
typically, although not necessarily, administered via injection and
are therefore generally liquid solutions or suspensions immediately
prior to administration. The pharmaceutical preparation can also
take other forms such as syrups, creams, ointments, tablets,
powders, and the like. Other modes of administration are also
included, such as pulmonary, rectal, transdermal, transmucosal,
oral, intrathecal, subcutaneous, intra-arterial, and so forth.
Suitable formulation types for parenteral administration include
ready-for-injection solutions, dry powders for combination with a
solvent prior to use, suspensions ready for injection, dry
insoluble compositions for combination with a vehicle prior to use,
and emulsions and liquid concentrates for dilution prior to
administration, among others.
[0162] The invention also provides a method for administering a
conjugate as provided herein to a patient suffering from a
condition that is responsive to treatment with conjugate, as
determined by those skilled in the art. The method comprises
administering, generally via injection, a therapeutically effective
amount of the conjugate, preferably provided as part of a
pharmaceutical preparation.
[0163] The actual dose to be administered will vary depend upon the
age, weight, and general condition of the subject, as well as the
severity of the condition being treated, the judgment of the health
care professional, and the conjugate being administered.
Therapeutically effective amounts of particular drugs are known to
those skilled in the art and/or are described in the pertinent
reference texts and literature. Generally, a therapeutically
effective amount of conjugate will range from about 0.001 mg to 100
mg, preferably in doses from 0.01 mg/day to 75 mg/day, and more
preferably in doses from 0.10 mg/day to 50 mg/day. The unit dosage
of any given conjugate (again, preferably provided as part of a
pharmaceutical preparation) can be administered in a variety of
dosing schedules depending on the judgment of the clinician, needs
of the patient, and so forth.
EXAMPLES
[0164] The following examples are provided to illustrate the
invention but should not be considered in limitation of the
invention. For example, although PEG is used in the Examples, the
use of other water soluble, non-peptidic polymers is encompassed by
the invention, as discussed above.
[0165] All PEG reagents referred to in these Examples are available
from Nektar AL, Huntsville, Ala. All NMR data was generated by a
300 or 400 MHz NMR spectrometer manufactured by Bruker.
[0166] Example 1 illustrates reaction of mPEG-OH with tert-butyl
bromoacetate in the presence of a base to form a tert-butyl ester
terminated polymer. Thereafter, the polymer is subjected to
base-promoted hydrolysis using NaOH as the base, followed by
acidification using phosphoric acid, to form the final carboxylic
acid terminated polymer.
[0167] Examples 2 and 3 exemplify similar reaction of a
difunctional PEG starting material (PEG diol; HO-PEG-OH). Example 4
illustrates reaction of a multifunctional, 4-armed PEG starting
material, based on a pentaerythritol core and having four reactive
hydroxyls, one at the terminus of each PEG "arm".
Example 1
mPEG(30,000)-carboxylic acid
[0168] A solution of mPEG-30,000 (50 g, 0.00167 moles) (NOF
Corporation) in toluene (600 ml) was azeotropically dried by
distilling off 300 ml toluene. t-Butanol (70 ml), potassium
tert-butoxide (95%, 1.75 g, 0.0148 moles, 8.9 fold excess) and
tert-butyl bromoacetate (3.3 g, 0.0169 moles, 10.1 fold excess)
were added, and the mixture was stirred overnight at 45.degree. C.
under argon atmosphere. The solvent was distilled off under reduced
pressure, and the residue was dissolved in distilled water (1000
ml).
[0169] The pH of the aqueous solution was adjusted to 12 with 1 M
sodium hydroxide, and the solution was stirred for 18 h, keeping
the pH at 12 by periodic addition of 1M sodium hydroxide.
[0170] The pH was adjusted to 3 with 5% phosphoric acid, and the
product was extracted with dichloromethane. The extract was dried
with anhydrous magnesium sulfate and added to ethyl ether. The
precipitated product was filtered off and dried under reduced
pressure, giving a yield of 46.6 g.
[0171] NMR (d.sub.6-DMSO): 3.24 ppm (s, --OCH.sub.3), 3.51 ppm (s,
PEG backbone), 4.01 ppm (s, --CH.sub.2--COO--).
[0172] Anion exchange chromatographic analysis:
mPEG(30,000)-carboxylic acid 100%. This analysis showed that
essentially no starting material or other polymeric impurity was
present in the ether-precipitated product.
Example 2
PEG(10,000)-dicarboxylic acid
[0173] PEG-10,000 (35.25 g, 0.00705 eq) (NOF Corporation)
(terminated at both ends with hydroxyl) was dissolved in toluene
(600 ml) and azeotropically dried by distilling off toluene. The
residue was redissolved into anhydrous toluene (500 ml).
tert-Butanol (40 ml), potassium tert-butoxide (4 g, 0.0356 moles,
5.1 fold excess) and anhydrous toluene (40 ml) were combined and
added to the above reaction mixture, followed by stirring for about
3.5 hours. t-Butyl bromoacetate (7 ml, 0.0474 moles, 6.7 fold
excess) was added, and the mixture was stirred overnight at
40.degree. C. under argon atmosphere. The solvent was distilled off
under reduced pressure, and the residue was dissolved in distilled
water (1000 ml).
[0174] The pH of the aqueous solution was adjusted to 12.1 with 1M
sodium hydroxide, and the solution was stirred overnight, keeping
the pH at 12.1 by periodic addition of 1M sodium hydroxide.
[0175] The pH was adjusted to 1.0 with 1M hydrochloric acid, and
the product was extracted with dichloromethane. The extract was
dried with anhydrous sodium sulfate, concentrated, and added to
ethyl ether. The precipitated product was filtered off and dried
under reduced pressure, to yield 33 g.
[0176] NMR (d.sub.6-DMSO): 3.51 ppm (s, PEG backbone), 4.01 ppm (s,
--CH.sub.2--COO--).
[0177] Anion exchange chromatographic analysis:
PEG(10,000)-dicarboxylic acid 100%.
Example 3
PEG(5,000)-dicarboxylic acid
[0178] A solution of PEG-5,000 (35 g, 0.01400 equivalents) (NOF
Corporation) in acetonitrile (800 ml) was azeotropically dried by
distilling off acetonitrile, and the residue was redissolved into
anhydrous toluene (300 ml). t-Butanol (50 ml), potassium
tert-butoxide (4.7 g, 0.0419 moles, 2.99 fold excess), and
anhydrous toluene (50 ml) were combined and added to the above
reaction mixture, followed by about 3.5 hours of stirring. t-Butyl
bromoacetate (7.2 ml, 0.0488 moles, 3.48 fold excess) was added,
and the mixture was stirred 20 hrs at room temperature under an
argon atmosphere. The solvent was distilled off under reduced
pressure, and the residue was dissolved in distilled water (1000
ml).
[0179] The pH of the aqueous solution was adjusted to 12.0 with 1M
sodium hydroxide, and the solution was stirred overnight, keeping
the pH at 12.0 by periodic addition of 1M sodium hydroxide.
[0180] The pH was adjusted to 2.0 with 1M hydrochloric acid and the
product was extracted with dichloromethane. The extract was dried
with anhydrous sodium sulfate, concentrated and added to ethyl
ether. The precipitated product was filtered off and dried under
reduced pressure, to yield 32 g.
[0181] NMR (d.sub.6-DMSO): 3.51 ppm (s, PEG backbone), 4.01 ppm (s,
--CH.sub.2--COO--).
[0182] Anion exchange chromatographic analysis:
PEG(5,000)-dicarboxylic acid 100%.
Example 4
4-Arm-PEG(10,000)-tetracarboxylic acid
[0183] A solution of Multi-arm PEG (4-Arm), MW 10 kDa (Nektar,
Huntsville Ala.) (160 g, 0.064 equivalents) in toluene (2,300 ml)
was azeotropically dried by distilling off 1,000 ml of toluene at
80.degree. C. under reduced pressure. In another vessel,
tert-butanol (17.3 ml) and potassium tert-butoxide (7.18 g, 0.128
moles, 2.00 fold excess) were mixed and then added to the dried
toluene solution from above. The resulting solution was stirred for
about 3.5 hours at 45.degree. C. t-Butyl bromoacetate (20.8 ml,
0.141 moles, 2.20 fold excess) was added, and the mixture was
stirred 12 hrs at 45.degree. C. under an argon atmosphere. The
solvent was distilled off under reduced pressure, and the residue
was dissolved in distilled water (1,600 ml).
[0184] The pH of the aqueous solution was adjusted to 12.0 with 1M
sodium hydroxide, and the solution was stirred for 17 hr while
keeping the pH at 12.0 by periodic addition of 1M sodium
hydroxide.
[0185] The pH was then adjusted to 1.5 with 1M phosphoric acid, and
the product was extracted with dichloromethane. The extract was
dried with anhydrous sodium sulfate, concentrated and added to
ethyl ether. The precipitated product was filtered off and dried
under reduced pressure, to yield 15.5 g.
[0186] NMR (d.sub.6-DMSO): 3.51 ppm (s, PEG backbone), 4.01 ppm (s,
--CH.sub.2--COO--), substitution 100%.
[0187] After 8 months of storage at -20.degree. C., GPC analysis
was identical to the original product. Therefore, no detectable
degradation occurred during storage.
[0188] Many modifications and other embodiments of the invention
will come to mind to one skilled in the art to which this invention
pertains having the benefit of the teachings presented in the
foregoing description. Therefore, the invention is not to be
limited to the specific embodiments disclosed, and modifications
and other embodiments are intended to be included, within the scope
of the appended claims.
* * * * *