U.S. patent application number 12/560339 was filed with the patent office on 2010-01-07 for method of preparing propionic acid-terminated polymers.
This patent application is currently assigned to Nektar Therapeutics. Invention is credited to Antoni Kozlowski, Samuel P. McManus.
Application Number | 20100004428 12/560339 |
Document ID | / |
Family ID | 34807154 |
Filed Date | 2010-01-07 |
United States Patent
Application |
20100004428 |
Kind Code |
A1 |
Kozlowski; Antoni ; et
al. |
January 7, 2010 |
Method of Preparing Propionic Acid-Terminated Polymers
Abstract
The invention provides methods for preparing polymers bearing a
terminal propionic acid. The method involves first reacting a water
soluble and non-peptidic polymer comprising at least one hydroxyl
group with a tertiary alkyl acrylate in the presence of a catalyst
to form a propionic acid ester of the polymer, wherein the polymer
has a weight average molecular weight of at least about 10,000 Da;
and then treating the propionic acid ester of the polymer with a
strong acid to form a propionic acid of the polymer.
Inventors: |
Kozlowski; Antoni;
(Huntsville, AL) ; McManus; Samuel P.;
(Huntsville, AL) |
Correspondence
Address: |
NEKTAR THERAPEUTICS
201 INDUSTRIAL ROAD
SAN CARLOS
CA
94070
US
|
Assignee: |
Nektar Therapeutics
San Carlos
CA
|
Family ID: |
34807154 |
Appl. No.: |
12/560339 |
Filed: |
September 15, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11040142 |
Jan 21, 2005 |
7608663 |
|
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12560339 |
|
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60538006 |
Jan 21, 2004 |
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Current U.S.
Class: |
530/351 ;
530/397; 530/399; 530/402; 548/259; 548/335.1; 548/542; 560/20 |
Current CPC
Class: |
C08F 8/14 20130101; C08G
65/3322 20130101 |
Class at
Publication: |
530/351 ;
548/542; 560/20; 548/259; 548/335.1; 530/402; 530/397; 530/399 |
International
Class: |
C07K 14/555 20060101
C07K014/555; C07D 207/46 20060101 C07D207/46; C07C 205/06 20060101
C07C205/06; C07D 249/18 20060101 C07D249/18; C07D 233/54 20060101
C07D233/54; C07K 14/715 20060101 C07K014/715; C07K 14/505 20060101
C07K014/505; C07K 14/61 20060101 C07K014/61 |
Claims
1. A composition comprised of a conjugate formed from the reaction
of an active ester of PEG and a biologically active molecule,
wherein the active ester of PEG is prepared by the method
comprising: i) reacting a PEG polymer with a tertiary alkyl
acrylate in the presence of a catalyst comprising a quaternary
ammonium salt to form a propionic acid ester of the PEG polymer,
wherein the PEG polymer comprises at least one hydroxyl group and
said PEG polymer is a monofunctional PEG, difunctional PEG, or
branched PEG molecule comprising 1 to about 25 hydroxyl groups and
having a weight average molecular weight of at least about 10,000
Da; and ii) treating the propionic acid ester of the PEG polymer
with a strong acid to form a PEG polymer functionalized with at
least one propionic acid group; iii) optionally,
chromatographically purifying the PEG polymer functionalized with
at least one propionic acid group; iv) derivatizing the PEG polymer
functionalized with at least one propionic acid group to form an
active ester of PEG; and v) optionally, chromatographically
purifying the active ester of PEG.
2. The composition of claim 1, wherein the active ester is selected
from the group consisting of N-succinimidyl ester, o-, m-, or
p-nitrophenyl ester, 1-benzotriazolyl ester, imidazolyl ester, and
N-sulfosuccinimidyl ester.
3. The composition of claim 1, wherein the active ester is selected
from the group consisting of: ##STR00024## wherein m is from 3 to
3000, and EC is a residue of a moiety selected from the group
consisting of fluorescein, biotin, acrylate, vinylsulfone,
maleimide, tert-butyl carbonyl, and 9-fluorenylmethoxycarbonyl.
4. The composition of claim 3, wherein the active ester comprises
the structure ##STR00025## wherein m is 3 to 3000.
5. The composition of claim 1, wherein the biologically active
molecule is selected from the group consisting of an interferon, a
TNFR, erythropoietin, human growth hormone, and variants and
mimetics of any of the foregoing.
6. The composition of claim 1, wherein the tertiary alkyl acrylate
is .alpha.- or .beta.-substituted.
7. The composition of claim 6, wherein the substituting group is
selected from the group consisting of halo, hydroxyl, thiol,
alkylthio, acyl, acyloxy, nitro, cyano, azido, trihalomethyl,
alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl,
alkoxy, substituted alkoxy, aryl, substituted aryl, heterocycle,
substituted heterocycle, heteroaryl, and substituted
heteroaryl.
8. The composition of claim 7, wherein the substituting group is
selected from the group consisting of methyl, ethyl, n-propyl,
isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, and
benzyl.
9. The composition of claim 1, wherein the tertiary alkyl group of
the tertiary alkyl acrylate is tert-butyl, tert-amyl,
.alpha.,.alpha.'-dimethylbenzyl, trityl, 1-adamantyl, or
2-methyl-2-adamantyl.
10. The composition of claim 1, wherein the tertiary alkyl acrylate
has the structure: ##STR00026## wherein: R.sub.1 and R.sub.2 are
each independently selected from the group consisting of hydrogen,
halo, hydroxyl, thiol, alkylthio, acyl, acyloxy, nitro, cyano,
azido, trihalomethyl, alkyl, substituted alkyl, cycloalkyl,
substituted cycloalkyl, alkoxy, substituted alkoxy, aryl,
substituted aryl, heterocycle, substituted heterocycle, heteroaryl,
and substituted heteroaryl; and R.sub.3-R.sub.5 are each
independently alkyl, substituted alkyl, aryl or substituted
aryl.
11. The composition of claim 10, wherein R.sub.3, R.sub.4, and
R.sub.5, are each methyl, ethyl, or phenyl.
12. The composition of claim 10, wherein R.sub.1 and R.sub.2 are
hydrogen.
13. The composition of claim 10, wherein R.sub.1 is hydrogen and
R.sub.2 is selected from the group consisting of methyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, and
benzyl,
14. The composition of claim 1, wherein the strong acid is
trifluoroacetic acid, trifluoromethancsulfonic acid, formic acid,
hydrochloric acid, or p-toluenesulfonic acid.
15. The composition of claim 1, wherein the catalyst comprising the
quaternary ammonium salt is selected from the group consisting of
tetramethyl ammonium halide, tetraethyl ammonium halide,
tetrapropyl ammonium halide, tetrabutyl ammonium halide,
tetramethyl ammonium hydroxide, tetraethyl ammonium hydroxide,
tetrapropyl ammonium hydroxide, and tetrabutyl ammonium
hydroxide.
16. The composition of claim 1, wherein the catalyst comprising the
quaternary ammonium salt has the structure: ##STR00027## wherein
each R is independently alkyl or substituted alkyl and X is a
counter ion.
17. The composition of claim 16, wherein each R is C1-C8 alkyl and
X is halo or hydroxide.
18. The composition of claim 1, wherein said reacting step and said
treating step are conducted in the presence of an organic
solvent.
19. The composition of claim 18, wherein the organic solvent is
selected from the group consisting of dichloromethane,
tetrahydrofuran, dimethylformamide, acetonitrile, toluene, xylene,
phenyl acetonitrile, nitrobenzene, tetrachloroethylene, anisole,
and chlorobenzene.
20. The composition of claim 1, wherein the weight average
molecular weight is from about 10,000 to about 100,000 Da.
21. The composition of claim 20, wherein the weight average
molecular weight is from about 20,000 to about 60,000 Da,
22. The composition of claim 1, wherein the step of
chromatographically purifying the PEG polymer functionalized with
at least one propionic acid group is performed.
23. The composition of claim 1, wherein the step of
chromatographically purifying the active ester of PEG is
performed.
24. The composition of claim 1, wherein the step of
chromatographically purifying the PEG polymer functionalized with
at least one propionic acid group is performed and the step of
chromatographically purifying the active ester of PEG is performed.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation application of U.S.
patent application Ser. No. 11/040,142; filed Jan. 21, 2005, which
application claims the benefit of priority to U.S. Provisional
Patent Application Ser. No. 60/538,006, filed Jan. 21, 2004, the
disclosures of which are incorporated herein by reference in their
entireties.
FIELD OF THE INVENTION
[0002] This invention relates to methods for preparing water
soluble and non-peptidic polymers carrying substituted or
unsubstituted propionic acid functional groups, particularly
propionic acid-terminated poly(ethylene glycol) polymers.
BACKGROUND OF THE INVENTION
[0003] Poly(ethylene glycol) (PEG) derivatives activated with
electrophilic groups are useful for coupling to amino groups of
biologically active molecules, such as proteins. In particular,
active esters of carboxylic acid derivatives of PEG have been used
to attach PEG to proteins bearing amino groups.
[0004] U.S. Pat. No. 5,672,662 discloses PEG derivatives having a
terminal propionic or butanoic acid moiety that can be used to
prepare active esters suitable for conjugation to proteins or other
molecules bearing amino groups. The synthetic method for propionic
acid-substituted PEG described in the patent involves Michael
addition of poly(ethylene glycol) to acrylonitrile followed by
hydrolysis of the nitrile to form the carboxyl group. Hydrolysis of
the nitrile requires severe reaction conditions, such as treatment
with concentrated sulfuric acid at 95.degree. C. or higher. The
ether linkages in PEG are sensitive to such conditions and
significant chain cleavage and reduction in yield can result from
this process, particularly when relatively high molecular weight
polymers are involved, such as polymers having a molecular weight
above about 10,000 Da.
[0005] U.S. Pat. No, 5,523,479 to Sanders et al. discloses a method
for forming ethercarboxylic acids by reacting an alcohol having a
molecular weight of 32 to 6,000 Da with a tertiary alkyl ester of
an .alpha.,.beta.-unsaturated carboxylic acid in the presence of a
catalyst, such as potassium hydroxide, followed by acid hydrolysis.
The Sanders et al, patent does not address the use of higher
molecular weight polymer reagents, such as PEG polymers having a
molecular weight of about 10,000 Da or higher.
[0006] There is a need in the art for alternative methods for
preparing propionic acid-terminated polymers, particularly high
molecular weight polymers, in high yield without utilizing harsh
reaction conditions that can cause chain cleavage within the
polymer backbone (e.g., at the terminal methoxy group in the
polymer backbone).
SUMMARY OF THE INVENTION
[0007] The present method avoids the harsh hydrolysis conditions
that characterize conventional methods for producing propionic
acid-terminated polymers. Instead, the method of the invention
first involves a Michael addition reaction between a tertiary alkyl
acrylate and a polymer functionalized with at least one hydroxyl
group, followed by removal of a tertiary alkyl group from the
terminal ester to form a carboxyl group using relatively mild
conditions, such as treatment with trifluoroacetic acid at about
50.degree. C.
[0008] In one aspect, the present invention provides a method for
preparing a water soluble and non-peptidic polymer functionalized
with at least one propionic acid group, the method comprising:
[0009] i) reacting a water soluble and non-peptidic polymer
comprising at least one hydroxyl group with a tertiary alkyl
acrylate or substituted tertiary alkyl acrylate in the presence of
a catalyst to form a substituted or unsubstituted propionic acid
ester of the polymer, wherein the polymer has a weight (or number)
average molecular weight of at least about 10,000 Da; and [0010]
ii) treating the substituted or unsubstituted propionic acid ester
of the polymer with a strong acid, such as (for example)
trifluoroacetic acid, trifluoromethanesulfonic acid, formic acid,
hydrochloric acid, or p-toluenesulfonic acid, to form a propionic
or substituted propionic acid of the polymer.
[0011] The tertiary alkyl acrylate can be .alpha.- or
.beta.-substituted and exemplary substituting groups include halo,
hydroxyl, thiol, alkylthio, acyl, acyloxy, nitro, cyano, azido,
trihalomethyl, alkyl, substituted alkyl, cycloalkyl, substituted
cycloalkyl, alkoxy, substituted alkoxy, aryl, substituted aryl,
heterocycle, substituted heterocycle, heteroaryl, and substituted
heteroaryl. In one preferred embodiment, the tertiary alkyl
acrylate is .alpha.- or .beta.-substituted, preferably
.alpha.-substituted, with methyl, ethyl, n-propyl, isopropyl,
n-butyl, isobutyl, sec-butyl, tert-butyl, or benzyl. Preferred
tertiary alkyl groups of the tertiary alkyl acrylate include
tert-butyl, tert-amyl, .alpha.,.alpha.'-dimethylbenzyl, trityl,
1-adamantyl, and 2-methyl-2-adamantyl.
[0012] In one or more embodiments, the tertiary alkyl acrylate has
the structure:
##STR00001##
wherein: [0013] R.sub.1 and R.sub.2 are each independently selected
from the group consisting of hydrogen, halo, hydroxyl, thiol,
alkylthio, acyl, acyloxy, nitro, cyano, azido, trihalomethyl,
alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl,
alkoxy, substituted alkoxy, aryl, substituted aryl, heterocycle,
substituted heterocycle, heteroaryl, and substituted heteroaryl;
and [0014] R.sub.3--R.sub.5 (that is, each of R.sub.3, R.sub.4 and
R.sub.5) are each independently alkyl, substituted alkyl, aryl or
substituted aryl.
[0015] Preferably, R.sub.3, R.sub.4, and R.sub.5, are each methyl,
ethyl, or phenyl, and R.sub.1 and R.sub.2 are hydrogen or R.sub.1
is hydrogen and R.sub.2 is methyl, ethyl, n-propyl, isopropyl,
n-butyl, isobutyl, sec-butyl, tert-butyl, or benzyl.
[0016] The catalyst used in the Michael addition reaction is
preferably a quaternary ammonium hydroxide, such as a tetraalkyl
ammonium halide or hydroxide (e.g., tetramethyl ammonium hydroxide,
tetraethyl ammonium hydroxide, tetrapropyl ammonium hydroxide, or
tetrabutyl ammonium hydroxide, as well as the corresponding
halides). In one or more embodiments, the quaternary ammonium
hydroxide has the structure:
##STR00002##
wherein each R is independently alkyl or substituted alkyl (e.g.,
C1-C8 alkyl). In addition, .sup.-OH counter ion can be substituted
for halo, wherein halo represents fluoro, chloro, bromo, and
iodo.
[0017] Both the reacting step i) and treating step ii) can be
conducted in the presence of an organic solvent, such as
dichloromethane (DCM), tetrahydrofuran (THF), dimethylformamide
(DMF), dimethylsulfoxide (DMSO), acetonitrile, toluene, benzene,
xylene, phenylacetonitrile, nitrobenzene, tetrachloroethylene,
anisole, chlorobenzene, and tert-butanol.
[0018] The polymer bearing at least one hydroxyl group is
preferably poly(ethylene glycol), but can be any other
water-soluble and non-peptidic polymer, such as other 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.
The polymer preferably has a weight (or number) average molecular
weight of about 10,000 to about 100,000 Da, more preferably about
10,000 to about 40,000 Da. The PEG polymer or other polymer can
have any of a variety of structures and geometric configurations,
including, for example, monofunctional PEG, difunctional PEG, and
branched PEG.
[0019] Following formation of the carboxylic acid group, the
propionic acid functionalized polymer can be derivatized to form an
acid derivative such as acyl halide, acyl pseudohalide, ester,
anhydride, amide, imide, or hydrazide. Furthermore, the acid or
certain functionalized polymers, e.g. active esters, can be used as
intermediates to react with appropriate reagents or other small
molecules or short polymeric species to form yet additional
reactive derivatives such as maleimides, thiols, reactive
disulfides, acetals, aldehydes and the like. In one embodiment, the
propionic acid-functionalized polymer is derivatized to form an
active ester. Exemplary active ester groups include
N-hydroxysuccinimidyl ester, o-, m-, or p-nitrophenyl ester,
1-hydroxybenzotriazolyl ester, imidazolyl ester, and
N-hydroxysulfosuccinimidyl ester.
[0020] In a preferred embodiment of the invention, the method for
preparing a poly(ethylene glycol) (PEG) polymer functionalized with
at least one propionic acid group comprises: [0021] i) reacting a
PEG polymer with a tertiary alkyl acrylate or substituted tertiary
alkyl acrylate in the presence of a quaternary ammonium hydroxide
to form a propionic acid or substituted propionic acid ester of
PEG, wherein the PEG polymer is a monofunctional PEG, difunctional
PEG, or branched PEG molecule comprising 1 to about 25 hydroxyl
groups and having a number average molecular weight of at least
about 10,000 Da; [0022] ii) treating the propionic acid or
substituted propionic acid ester of PEG with a strong acid, such as
(for example) trifluoroacetic acid, trifluoromethanesulfonic acid,
formic acid, hydrochloric acid, or p-toluenesulfonic acid, to form
a PEG polymer functionalized with at least one propionic acid or
substituted propionic acid group; [0023] iii) optionally,
chromatographically purifying the PEG polymer functionalized with
at least one propionic acid group; [0024] iii) optionally,
derivatizing the propionic acid or substituted propionic acid
functionalized PEG to form, for example, an active ester selected
from the group consisting of N-hydroxysuccinimidyl ester, o-, m-,
or p-nitrophenyl ester, 1- hydroxybenzotriazolyl ester, imidazolyl
ester, and N-hydroxysulfosuccinimidyl ester; and [0025] v)
optionally, chromatographically purifying the active ester of
PEG.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention now will be described more fully
hereinafter. 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.
[0027] I. Definitions
[0028] Before describing the present invention in detail, it is to
be understood that this invention is not limited to the particular
polymers, synthetic techniques, active agents, and the like as such
may vary. It is also to be understood that the terminology used
herein is for describing particular embodiments only, and is not
intended to be limiting.
[0029] It must be noted that, as used in this specification, the
singular fowls "a," "an," and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to a "polymer" includes a single polymer as well as two
or more of the same or different polymers, reference to a
"conjugate" refers to a single conjugate as well as two or more of
the same or different conjugates, reference to an "excipient"
includes a single excipient as well as two or more of the same or
different excipients, and the like.
[0030] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions described below.
[0031] "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 3 to 3000, and the terminal groups and architecture of
the overall PEG may vary. "PEG" means a polymer that contains a
majority, that is to say, greater than 50%, of subunits that are
--CH.sub.2CH.sub.2O--. One commonly employed PEG is end-capped PEG.
When PEG is defined as "--O(CH.sub.2CH.sub.2O).sub.m--" the end
capping group is generally a carbon-containing group typically
comprised of 1-20 carbons and is preferably alkyl (e.g., methyl,
ethyl or benzyl) although saturated and unsaturated forms thereof,
as well as aryl, hetcroaryl, cyclo, heterocyclo, and substituted
forms of any of the foregoing are also envisioned. When PEG is
defined as
"--CH.sub.2CH.sub.2O(CH.sub.2CH.sub.2O).sub.m--CH.sub.2CH.sub.2--,"
the end capping group is generally a carbon-containing group
typically comprised of 1-20 carbon atoms and an oxygen atom that is
covalently bonded to the group and is available for covalently
bonding to one terminus of the PEG. In this case, the group is
typically alkoxy (e.g., methoxy, ethoxy or benzyloxy) and with
respect to the carbon-containing group can optionally be saturated
and unsaturated, as well as aryl, heteroaryl, cyclo, heterocyclo,
and substituted forms of any of the foregoing. The other
("non-end-capped") terminus is a typically hydroxyl, amine or an
activated group that can be subjected to further chemical
modification when PEG is defined as
"--CH.sub.2CH.sub.2O(CH.sub.2CH.sub.2O).sub.m--CH.sub.2CH.sub.2-."
In addition, the end-capping group can also be a silane. Specific
PEG forms for use in the invention include PEGs having a variety of
molecular weights, structures or geometries (e.g., branched,
linear, forked PEGs, multifunctional, and the like), to be
described in greater detail below.
[0032] The end-capping group can also advantageously comprise a
detectable label. When the polymer has an end-capping group
comprising a detectable label, the amount or location of the
polymer and/or the moiety (e.g., active agent) to which the polymer
is attached can be determined by using a suitable detector. Such
labels include, without limitation, fluorescers, chemiluminescers,
moieties used in enzyme labeling, colorimetric (e.g., dyes), metal
ions, radioactive moieties, and the like.
[0033] "Molecular mass" in the context of a water-soluble,
non-peptidic polymer of the invention such as PEG, refers to the
weight average molecular weight of a polymer, typically determined
by size exclusion chromatography, light scattering techniques, or
intrinsic viscosity determination in an organic solvent like
1,2,4-trichlorobenzene. The polymers of the invention are typically
polydisperse, possessing low polydispersity values of less than
about 1.05.
[0034] "Activated carboxylic acid" means a functional derivative of
a carboxylic acid that is more reactive than the parent carboxylic
acid, in particular, with respect to nucleophilic acyl
substitution. Activated carboxylic acids include but are not
limited to acid halides (such as acid chlorides), anhydrides,
amides and esters.
[0035] The term "reactive" or "activated" when used in conjunction
with a particular functional group, refers to a reactive functional
group that reacts readily with an electrophile or a nucleophile on
another molecule. This is in contrast to those groups that require
strong catalysts or highly impractical reaction conditions in order
to react (i.e, a "nonreactive" or "inert" group).
[0036] The terms "protected" or "protecting group" or "protective
group" refer to the presence of a moiety (i.e., the protecting
group) 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).
[0037] As used herein, the term "functional group" or any synonym
thereof is meant to encompass protected forms thereof
[0038] The term "spacer" or "spacer moiety" is used herein to refer
to an atom or a collection of atoms optionally used to link
interconnecting moieties such as a terminus of a water-soluble
polymer portion and an electrophile. The spacer moieties of the
invention may be hydrolytically stable or may include a
physiologically hydrolyzable or enzymatically degradable
linkage.
[0039] "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
straight chain, although typically straight chain is preferred.
Exemplary alkyl groups include ethyl, propyl, butyl, pentyl,
2-methylbutyl, 2-methylpropyl (isobutyl), 3-methylpentyl, and the
like. As used herein, "alkyl" includes cycloalkyl or cycloalkylene
when three or more carbon atoms are referenced.
[0040] "Lower alkyl" refers to an alkyl group containing from 1 to
6 carbon atoms, and may be straight chain or branched, as
exemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl.
[0041] "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.
[0042] As used herein, "alkenyl" refers to a branched or unbranched
hydrocarbon group of 1 to 15 atoms in length, containing at least
one double bond, such as ethenyl, n-propenyl, isopropenyl,
n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, and the
like.
[0043] The term "alkynyl" as used herein refers to a branched or
unbranched hydrocarbon group of 2 to 15 atoms in length, containing
at least one triple bond, such as ethynyl, n-propynyl, isopentynyl,
n-butynyl, octynyl, decynyl, and so forth.
[0044] "Alkoxy" refers to an --O--R group, wherein R is alkyl or
substituted alkyl, preferably C1-C20 alkyl (e.g., methyl, ethyl,
propyl, benzyl, etc.), preferably C1-C8.
[0045] "Non-interfering substituents" are those groups that, when
present in a molecule, are typically non-reactive with other
functional groups contained within the molecule.
[0046] The term "substituted" as in, for example, "substituted
alkyl," refers to a moiety (e.g., an alkyl group) substituted with
one or more non-interfering substituents, such as, but not limited
to: C3-C8 cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like;
halo, e.g., fluoro, chloro, bromo, and iodo; cyano; alkoxy; phenyl;
substituted phenyl; and the like.
[0047] "Aryl" means one or more aromatic rings, each of 5 or 6 core
carbon atoms. Aryl includes multiple aryl rings that may be fused,
as in naphthyl or unfused, as in biphenyl. Aryl rings may also be
fused or unfused with one or more cyclic hydrocarbon, heteroaryl,
or heterocyclic rings. As used herein, "aryl" includes
heteroaryl.
[0048] "Substituted aryl" is aryl having one or more
non-interfering groups as a substituent. For substitutions on a
phenyl ring, the substituents may be in any orientation (i.e.,
ortho, meta, or para).
[0049] "Heterocycle" or "heterocyclic" means one or more rings of
5-12 atoms, preferably 5-7 atoms, with or without unsaturation or
aromatic character and having at least one ring atom which is not a
carbon. Preferred heteroatoms include sulfur, oxygen, and
nitrogen.
[0050] "Substituted heterocycle" is a heterocycle having one or
more side chains formed from non-interfering substituents.
[0051] "Heteroaryl" is an aryl group containing from one to four
heteroatoms, preferably N, O, or S, or a combination thereof.
Heteroaryl rings may also be fused with one or more cyclic
hydrocarbon, heterocyclic, aryl, or heteroaryl rings.
[0052] "Substituted heteroaryl" is heteroaryl having one or more
non-interfering groups as substituents.
[0053] "Electrophile" refers to an ion or atom or a neutral or
ionic collection of atoms having an electrophilic center, i.e., a
center that is electron seeking or capable of reacting with a
nucleophile.
[0054] "Nucleophile" refers to an ion or atom or a neutral or ionic
collection of atoms having a nucleophilic center, i.e., a center
that is seeking an electrophilic center or capable of reacting with
an electrophile.
[0055] 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, orthoesters, and
oligonucleotides.
[0056] An "enzymatically degradable linkage" means a linkage that
is subject to degradation by one or more enzymes.
[0057] A "hydrolytically stable" linkage or bond refers to a
chemical bond, typically a covalent bond, that is substantially
stable in water, that is to say, does not undergo hydrolysis under
physiological conditions to any appreciable extent over an extended
period of time. Examples of hydrolytically stable linkages include
but are not limited to the following: carbon-carbon bonds (e.g., in
aliphatic chains), ethers, amides, urethanes, and the like.
Generally, a hydrolytically stable linkage is one that exhibits a
rate of hydrolysis of less than about 1-2% per day under
physiological conditions. Hydrolysis rates of representative
chemical bonds can be found in most standard chemistry
textbooks.
[0058] "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 the patient.
[0059] "Pharmacologically effective amount," "physiologically
effective amount," and "therapeutically effective amount" are used
interchangeably herein to mean the amount of a PEG-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.
[0060] "Multifunctional" in the context of a polymer of the
invention means a polymer having 3 or more functional groups
contained therein, where the functional groups may be the same or
different. Multifunctional polymers of the invention 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 within the polymer backbone.
[0061] A "difunctional" polymer means a polymer having two
functional groups contained therein, either the same (i.e.,
homodifunctional) or different (i.e., heterodifunctional).
[0062] A basic or acidic reactant described herein includes
neutral, charged, and any corresponding salt forms thereof.
[0063] 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 conjugate, and includes both humans and
animals.
[0064] "Optional" or "optionally" means that the subsequently
described circumstance may or may not occur, so that the
description includes instances where the circumstance occurs and
instances where it does not.
[0065] Unless otherwise noted, molecular weight is expressed herein
as number average molecular weight (M.sub.n), which is defined
as
NiMi Ni , ##EQU00001##
wherein Ni is the number of polymer molecules (or the number of
moles of those molecules) having molecular weight Mi,
[0066] As used herein, "non-peptide" refers to a polymer backbone
substantially free of peptide linkages. However, the polymer
backbone 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.
[0067] II. Method of Preparing Propionic Acid Functionalized
Polymers
[0068] The method of the invention provides a synthetic route for
forming water soluble and non-peptidic polymers functionalized with
at least one propionic acid group. The method involves reaction of
a polymer comprising at least one hydroxyl group, such as
monofunctional, difunctional or multifunctional PEG molecules, with
a tertiary alkyl acrylate reagent in a Michael addition reaction,
which results in a polymer substituted with at least one tertiary
alkyl ester of propionic acid. The ester is then hydrolyzed under
relatively mild conditions as compared to the hydrolysis conditions
required for a nitrile group. The hydrolysis conditions used in the
method of the invention do not cause yield-reducing degradation and
chain cleavage of the polymer backbone, thereby making the method
particularly well-suited for higher molecular weight polymers, such
as polymers having a molecular weight of greater than about 10,000
Da.
[0069] In a preferred embodiment, a catalyst is used to promote the
Michael addition reaction. The choice of catalyst is particularly
important for higher molecular weight polymer starting materials
because, as indicated in Comparative Example 1, certain catalysts
in the art are unable to advance the Michael addition reaction to
any significant degree. A preferred catalyst comprises a quaternary
ammonium hydroxide. Exemplary quaternary ammonium hydroxides
include tetramethyl ammonium hydroxide, tetraethyl ammonium
hydroxide, tetrapropyl ammonium hydroxide, and tetrabutyl ammonium
hydroxide. Quaternary ammonium hydroxides can be used directly or
generated in situ from corresponding quaternary ammonium salts,
preferably halides. If a quaternary ammonium halide salt is used,
it is necessary to activate the ammonium salt by addition of an
alkali metal or alkaline earth metal hydroxide, such as KOH or
NaOH, as shown in Examples 1 and 3. The catalyst can be dissolved
in the same organic solvent as the reaction reagents or added in
the form of an aqueous solution.
[0070] In one embodiment, the quaternary ammonium hydroxide has the
structure:
##STR00003##
wherein each R is independently alkyl or substituted alkyl,
preferably substituted or unsubstituted C1-8 alkyl.
[0071] Hydrolysis of the tertiary alkyl ester group can be
accomplished by treatment with any strong acid, such as various
solutions of mineral acids (e.g., hydrohalic acids, sulfuric acid,
phosphorous acid, and the like) or organic acids. One preferred
acid is trifluoroacetic acid (TFA). Examples of other suitable
acids include formic acid, hydrochloric acid, p-toluenesulfonic
acid, and trifluoromethanesulfonic acid.
[0072] The reagents in both the Michael addition step and the
subsequent hydrolysis step are preferably dissolved in a suitable
organic solvent. Exemplary organic solvents include dichloromethane
(DCM), tetrahydrofuran (THF), dimethylformamide (DMF),
dimethylsulfoxide (DMSO), acetonitrile, toluene, benzene, xylene,
phenylacetonitrile, nitrobenzene, tetrachloroethylene, anisole,
chlorobenzene, tert-butanol, and the like.
[0073] The Michael addition reaction can be carried out at any
temperature that maintains the polymer starting material (i.e., the
polymeric alcohol) in a liquid state. Preferably, the temperature
is about 20 to about 120.degree. C., more preferably about 20 to
about 60.degree. C. Such temperatures are lower than those
typically suggested, and represents relatively gentler reaction
conditions, especially in the presence of basic catalyst, thereby
practically eliminating degradation and chain cleavage of the
polymer backbone. The Michael addition reaction time is typically
about 6 hours to about 24 hours. The polymer is typically reacted
with the tertiary alkyl acrylate using an excess of the acrylate
reagent (e.g., up to about 30 fold molar excess) in order to
promote substantially complete conversion of the polymeric alcohol.
The catalyst is typically present in an amount of about 0.05 weight
percent to about 20 weight percent based on the weight of the
starting polymer.
[0074] The acid-promoted hydrolysis step typically comprises
treating the propionic acid ester of the polymer with a strong
acid, preferably an organic acid, at a temperature of about 20 to
about 100.degree. C., preferably at the lower end of the range, for
about 0.5 hours to about 6 hours. The use of organic acids with
relatively lower temperatures represents milder reaction conditions
than required for the hydrolysis of, for example, nitriles as
evidenced in U.S. Pat. No. 5,672,662, with the result of
practically eliminating degradation and chain cleavage of the
polymer backbone. Following conversion of the ester to the desired
acid, any organic solvents or acids, such as trifluoroacetic acid,
can be removed by distillation. Thereafter, the desired product is
preferably dissolved in deionized water and treated with a strong
base to hydrolyze any esters of residual polymeric alcohol (e.g.,
PEG-OH) and acid (e.g., trifluoroacetic acid), followed by
treatment with a strong mineral acid (for pH adjustment) to convert
the resulting salt of the polymer-propionic acid (e.g.,
PEG-propionic acid) to the free acid form. The product is then
extracted using a chlorinated solvent such as dichloromethane and
concentrated. The desired product can then be purified using
methods known in the art for polymers of this type.
[0075] Using the method of the invention, propionic
acid-functionalized polymers can be produced in high yield with a
high degree of polymer substitution. Typically, the percentage of
polymer substitution of the propionic acid ester onto the polymeric
alcohol is at least about 70%, preferably at least about 80%, and
most preferably at least about 90% substitution. The product yield
is typically at least about 60%, more preferably at least about
70%, and most preferably at least about 80%.
[0076] A general reaction scheme of the present invention, denoted
as Reaction Scheme I, is shown below. As shown, an mPEG-OH molecule
is reacted with a tert-butyl acrylate substituted at the
.alpha.-carbon as described in greater detail below. A quaternary
ammonium halide activated with KOH is used as a catalyst for the
Michael addition step. Trifluoroacetic acid ("TFA") is used in an
acid-promoted hydrolysis step to remove the tert-butyl protecting
group. In Reaction Scheme I, "Me" represents methyl, "n" represents
the number of repeating ethylene oxide monomers, "t-Bu" represents
t-butyl, and R.sub.2 is as defined in Section II.B.
##STR00004##
[0077] In exemplary Reaction Scheme II below, a method according to
the invention is outlined that includes formation of an active NHS
ester following purification of the propionic acid functionalized
polymer.
##STR00005##
[0078] As explained more fully below, the polymeric alcohol
starting material may comprise any water soluble and non-peptidic
polymer having any of a wide variety of geometric configurations
(e.g., linear, branched, forked, and so forth). For the sake of
simplicity, the above reaction schemes illustrate use of a
monofunctional polymer having a single hydroxyl group.
[0079] However, 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).
[0080] If a methoxy-PEG-OH is used as the raw material, as shown in
Reaction Scheme II, this process produces the active ester with the
polymer backbone chain impact. That is, there is no observable
chain cleavage from the acid-catalyzed hydrolysis of the ester.
While not wishing to be bound by theory, it is believed the absence
of chain cleavage is the result of using relatively reduced
temperatures and a milder organic acid such as trifluoroacetic acid
followed by base treatment. This lack of chain cleavage and
especially the lack of terminal demethylation (thereby resulting in
a lack of demethylated product), results in a significant
difference and advantage over the propionic acid-terminated
polymers (and corresponding active esters) prepared in accordance
with the process described in the U.S. Pat. No. 5,672,662.
Additionally, the conjugates prepared from active reagents derived
from PEG-propionic acid manufactured using U.S. Pat. No. 5,672,662
would suffer by comparison to conjugates prepared using the present
method.
[0081] A. Water Soluble and Non-Peptidic Polymers
[0082] The polymer should be non-toxic and biocompatible, meaning
that the polymer is capable of coexistence with living tissues or
organisms without causing harm. When referring to the polymer, it
is to be understood that the polymer can be any of a number of
water soluble and non-peptidic polymers, such as those described
herein as suitable for use in the present invention. Preferably,
poly/ethylene glycol) (i.e., PEG) is the polymer. The term PEG
includes poly(ethylene glycol) in any of a number of geometries or
forms, including linear forms (e.g., methoxy-PEG-OH,
benzyloxy-PEG-OH, or HO-PEG-OH), branched or multi-arm forms (e.g.,
forked PEG or PEG attached to a polyol core), pendant PEG, or PEG
with degradable linkages therein, to be more fully described
below.
[0083] The polymer comprises at least one hydroxyl group capable of
reacting with a tertiary alkyl acrylate reagent in a Michael
addition reaction. In addition to the one or more hydroxyl groups,
the polymer may comprise other functional groups that would not
interfere with the Michael addition reaction, such as acetal of an
aldehyde having a carbon length of 1 to 25 carbons (e.g.,
acetaldehyde, propionaldehyde, and butyraldehyde), alkenyl,
acrylate, methacrylate, acrylamide, active sulfone, hydrazide,
isothiocyanate, maleimide, vinylsulfone, dithiopyridine,
vinylpyridinc, iodoacetamide, epoxide, glyoxal, biotin, dione,
mesylate, tosylate, and tresylate.
[0084] The number of hydroxyl groups carried by the polymer and the
position of the functional groups may vary. Typically, the polymer
will comprise 1 to about 25 hydroxyl groups, preferably 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10 hydroxyl groups. Linear polymers, such as PEG
polymers, will typically comprise one or two hydroxyl groups
positioned at the terminus of the polymer chain. If the PEG polymer
is monofunctional (i.e., mPEG), the polymer will include a single
hydroxyl group. If the PEG polymer is difunctional, the polymer may
contain two hydroxyl groups, one at each terminus of the polymer
chain, or may contain a single hydroxyl group and a different
functional group at the opposing terminus. As would be understood,
multi-arm or branched polymers may comprise a greater number of
hydroxyl groups.
[0085] Multi-armed or branched PEG molecules, such as those
described in U.S. Pat. No. 5,932,462, which is incorporated by
reference herein in its entirety, can also be used as the PEG
polymer. Generally speaking, a multi-armed or branched polymer
possesses two or more polymer "arms" extending from a central
branch point. For example, an exemplary branched PEG polymer has
the structure:
##STR00006##
[0086] wherein PEG.sub.1 and PEG.sub.2 are PEG polymers in any of
the forms or geometries described herein, and which can be the same
or different, and L' is a hydrolytically stable linkage. An
exemplary branched PEG of Formula III has the structure:
##STR00007##
wherein poly.sub.a and poly.sub.b are PEG backbones, such as
methoxy poly(ethylene glycol); R'' is a nonreactive moiety, such as
H, methyl or a PEG backbone; and P and Q are nonreactive linkages.
In a preferred embodiment, the branched PEG polymer is methoxy
poly(ethylene glycol) disubstituted lysine.
[0087] The branched PEG structure of Formula IV can be attached to
a third oligomer or polymer chain as shown below:
##STR00008##
wherein PEG.sub.3 is a third PEG oligomer or polymer chain, which
can be the same or different from PEG.sub.1 and PEG.sub.2.
[0088] 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. An exemplary
multi-arm polymer of this type has the structure:
R(-L''-PEG-OH).sub.q (Formula VI)
wherein:
[0089] R is the hydrocarbon chain of the polyol or polyamine core
molecule, typically comprising about 3 to about 150 carbon atoms,
preferably about 3 to about 50 carbon atoms, and most preferably
about 3 to about 10 carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, or
10), optionally substituted with one or more heteroatoms (e.g., O,
S, or N) in the hydrocarbon chain, and which may be linear or
cyclic;
[0090] L'' is a linkage formed from reaction of the polyol or
polyamine core molecule with the polymer arms (e.g., --O-- or
--NH--C(O)--), and can serve as a spacer moiety;
[0091] PEG is a poly(ethylene glycol) polymer segment; and
[0092] q is an integer from 3 to about 25, more preferably to about
10, most preferably 3 to about 8 (e.g., 3, 4, 5, 6, 7, or 8).
[0093] The central core molecule in the multi-arm embodiment
described above is derived from a molecule that provides a number
of polymer attachment sites equal to the desired number of water
soluble and non-peptidic polymer arms. Preferably, the central core
molecule of the multi-arm polymer structure is the residue of a
polyol or a polyamine bearing at least three hydroxyl or amino
groups available for polymer attachment. A "polyol" is a molecule
comprising a plurality of available hydroxyl groups. A "polyamine"
is a molecule comprising a plurality of available amino groups.
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). The polyol or polyamine may include
other protected or unprotected functional groups as well without
departing from the invention. Although the spacing between hydroxyl
or amino groups will vary, there is typically 1 to about 20 atoms,
such as carbon atoms, between each hydroxyl or amino group,
preferably 1 to about 5. The particular polyol or polyamine chosen
will depend on the desired number of hydroxyl or amino groups
needed for attachment to the polymer arms.
[0094] The polyol or polyamine core will typically have the
structure R--(OH).sub.p or R--(NH.sub.2).sub.p prior to reaction
with the polymer arms, wherein R is a hydrocarbon chain, typically
comprising about 3 to about 150 carbon atoms, preferably about 3 to
about 50 carbon atoms, and most preferably about 3 to about 10
carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, or 10), optionally
substituted with one or more heteroatoms (e.g., 0, S, or N) in the
hydrocarbon chain, and which may be linear or cyclic, and p is the
number of hydroxyl or amino groups and is typically 3 to about 25,
preferably 3 to about 10, more preferably 3 to about 8 (e.g., 3, 4,
5, 6, 7, or 8).
[0095] Polyols that are suitable for use as the polymer core are
nearly limitless. Aliphatic polyols having from 1 to about 10
carbon atoms and from 1 to about 10 hydroxyl groups may be used,
including ethylene glycol, alkane diols, alkyl glycols, alkylidene
alkyl diols, alkyl cycloalkane diols, 1,5-decalindiol,
4,8-bis(hydroxymethyl)tricyclodecane, cycloalkylidene diols,
dihydroxyalkanes, trihydroxyalkanes, and the like. Cycloaliphatic
polyols may also be employed, including straight chained or
closed-ring sugars and sugar alcohols, such as mannitol, sorbitol,
inositol, xylitol, quebrachitol, threitol, arabitol, erythritol,
adonitol, dulcitol, facose, ribose, arabinose, xylose, lyxose,
rhamnose, galactose, glucose, fructose, sorbose, mannose, pyranose,
altrose, talose, tagitose, pyranosides, sucrose, lactose, maltose,
and the like. More examples of aliphatic polyols include
derivatives of glyceraldehyde, glucose, ribose, mannose, galactose,
and related stereoisomers. Aromatic polyols may also be used, such
as 1,1,1-tris(4'-hydroxyphenyl) alkanes, such as
1,1,1-tris(4-hydroxyphenyl)ethane, (1,3-adamantanediyl)diphenol,
2,6-bis(hydroxyalkyl)cresols,
2,2'alkylene-bis(6-t-butyl-4-alkylphenols),
2,2'-alkylene-bis(t-butylphenols), catechol, alkylcatechols,
pyrogallol, fluoroglycinol, 1,2,4-benzenetriol, resorcinol,
alkylresorcinols, dialkylresorcinols, orcinol monohydrate,
olivetol, hydroquinone, alkylhydroquinones, 1,1-bi-2-naphthol,
phenyl hydroquinones, dihydroxynaphthalenes,
4,4'-(9-fluorenylidene)-diphenol, anthrarobin, dithranol, bis
(hydroxyphenyl) methane biphenols, dialkyistilbesterols,
bis(hydroxyphenyl)alkanes, bisphenol-A and derivatives thereof,
meso-hexesterol, nordihydroguaiaretic acid, calixarenes and
derivatives thereof, tannic acid, and the like. Other core polyols
that may be used include crown ether, cyclodextrins, dextrins and
other carbohydrates (e.g., monosaccharides, oligosaccharides, and
polysaccharides, starches and amylase).
[0096] Preferred polyols include glycerol, sugars such as sorbitol
or pentaerythritol, and glycerol oligomers, such as hexaglycerol. A
21-arm polymer can be synthesized using
hydroxypropyl-.beta.-cyclodextrin, which has 21 available hydroxyl
groups.
[0097] Exemplary polyamines include aliphatic polyamines such as
diethylene triamine, N,N',N''-trimethyldiethylene triamine,
pentamethyl diethylene triamine, triethylene tetramine,
tetraethylene pentamine, pentaethylene hexamine, dipropylene
triamine, tripropylene tetramine, bis-(3-aminopropyl)-amine,
bis-(3-aminopropyl)-methylamine, and
N,N-dimethyl-dipropylene-triamine. Naturally occurring polyamines
that can be used in the present invention include putrescine,
spermidine, and spermine. Numerous suitable pentamines, tetramines,
oligoamines, and pentamidine analogs suitable for use in the
present invention are described in Bacchi et al., Antimicrobial
Agents and Chemotherapy, January 2002, p. 55-61, Vol. 46, No. 1,
which is incorporated by reference herein.
[0098] 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. An example of a
forked PEG is represented by PEG-YCH(-L-Z).sub.2, where Y is a
linking group and Z is an activated terminal group for covalent
attachment to a biologically active agent. The Z group is linked to
CH by a linker, L, which is a chain of atoms of defined length.
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. The chain of atoms, L, linking the
Z functional groups (e.g., hydroxyl groups) to the branching carbon
atom serve as a tethering group and may comprise, for example, an
alkyl chain, ether linkage, ester linkage, amide linkage, or
combinations thereof.
[0099] The PEG polymer may comprise 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 alkylenc
group.
[0100] Although less preferred, in addition to the above-described
forms of PEG, the polymer can also be prepared with an
enzymatically degradable linkage or one or more hydrolytically
stable or degradable linkages in the polymer backbone, including
any of the above described polymers. For example, PEG can be
prepared with ester linkages in the polymer backbone that are
subject to hydrolysis. As shown below, this hydrolysis results in
cleavage of the polymer into fragments of lower molecular
weight:
-PEG-CO.sub.2-PEG-+H.sub.2O.fwdarw.-PEG-CO.sub.2H+HO-PEG-
[0101] Other hydrolytically degradable linkages, useful as a
degradable linkage within a polymer backbone, include carbonate
linkages; imine linkages resulting, for example, from reaction of
an amine and an aldehyde (see, e.g., Ouchi et al., Polymer
Preprints, 38(1):582-3 (1997), which is incorporated herein by
reference.); phosphate ester linkages formed, for example, by
reacting an alcohol with a phosphate group; hydrazone linkages
which are typically formed by reaction of a hydrazide and an
aldehyde; acetal linkages that are typically formed by reaction
between an aldehyde and an alcohol; ortho ester linkages that are,
for example, formed by reaction between acid derivatives and an
alcohol; and oligonucleotide linkages formed by, for example, a
phosphoramidite group, e.g., at the end of a polymer, and a 5'
hydroxyl group of an oligonucleotide.
[0102] It is understood by those skilled in the art that the term
poly(ethylene glycol) or PEG represents or includes all the above
forms of PEG. Any of a variety of other polymers comprising other
non-peptidic and water soluble polymer chains can also be used in
the present invention. The polymer can be linear, or can be in any
of the above-described forms (e.g., branched, forked, and the
like). Examples of suitable polymers include, but are not limited
to, other 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.
[0103] Different polymers can be incorporated into the same polymer
backbone. For example, one or more of the PEG molecules in the
branched structures shown in Formulas III-VI can be replaced with a
different polymer type. Any combination of water soluble and
non-peptidic polymers is encompassed within the present
invention.
[0104] 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 10,000 Da to about
100,000 Da are useful in the present invention, preferably about
10,000 Da to about 60,000 Da, and more preferably about 10,000 Da
to about 40,000 Da. Exemplary polymer embodiments have a molecular
weight of approximately 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da,
30,000 Da, 35,000 Da, and 40,000 Da. However, lower molecular
weight polymers can also be used without departing from the present
invention, such as polymers having a molecular weight as low as
about 100 Da (e.g., polymers having a molecular weight of about 250
Da, about 500 Da, about 750 Da, about 1,000 Da, about 1,500 Da,
about 2,500 Da, and about 5,000 Da).
[0105] Useful exemplary weight average molecular weights of the
polymers include about 100 Da, about 200 Da, about 300 Da, about
400 Da, about 500 Da, about 600 Da, about 700 Da, about 750 Da,
about 800 Da, about 900 Da, about 1,000 Da, about 2,000 Da, about
2,500 Da, about 3,000 Da, about 4,000 Da, about 5,000 Da, about
6,000 Da, about 7,000 Da, about 7,500 Da, about 8,000 Da, about
9,000 Da, about 10,000 Da, about 11,000 Da, about 12,000 Da, about
12,500 Da, about 15,000 Da, about 20,000 Da, about 25,000 Da, and
30,000 Da, about 40,000 Da, about 50,000 Da, about 60,000 Da, about
70,000 Da, about 75,000 Da, about 80,000 Da, about 85,000 Da, about
90,000 Da, about 100,000 Da, and about 120,000 Da.
[0106] With respect to singly branched versions of the polymer,
exemplary ranges of useful sizes for the total molecular weight of
the polymer (as based essentially on the combined weights of the
two water soluble polymer portions) include the following: from
about 200 Da to about 100,000 Da; from about 1,000 Da to about
80,000 Da; from about 2,000 Da to about 60,000 Da; from about 4,000
Daltons to about 50,000 Daltons; and from about 10,000 Da to about
40,000 Da. More particularly, total weight average molecular weight
of a singly branched version of the polymer of the invention
corresponds to one of the following: about 400; about 1,000; about
1,500; about 2,000; about 3000; about 4,000; about 10,000; about
15,000; about 20,000; about 30,000; about 40,000; about 50,000;
about 60,000; or about 80,000.
[0107] With respect to PEG, wherein a structure comprising a
repeating ethylene oxide monomer, such as
"--(CH.sub.2CH.sub.2O).sub.m--" or "--(OCH.sub.2CH.sub.2).sub.m--"
[as in, for example,
H.sub.3CO--(CH.sub.2CH.sub.2O).sub.m--CHR.sub.2--CHR.sub.2--C(O)--Y,
where R.sub.1 R.sub.2 and Y are as defined for Formula VIII]
exemplary values for m include: from about 3 to about 3,000; from
about 10 to about 3,000; from about 15 to about 3,000; from about
20 to about 3,000; from about 25 to about 3,000; from about 30 to
about 3,000; from about 40 to about 3,000; from about 50 to about
3,000; from about 55 to about 3,000; from about 75 to about 3,000;
from about 100 to about 3,000; and from about 225 to about
3,000.
[0108] B. Tertiary Alkyl Acrylate Reagent
[0109] The tertiary alkyl acrylate can be .alpha.- or
.beta.-substituted. Exemplary substituting groups include halo,
alkylthio, acyl, acyloxy, nitro, cyano, azido, trihalomethyl,
alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl,
alkoxy, substituted alkoxy, aryl, substituted aryl, heterocycle,
substituted heterocycle, heteroaryl, and substituted heteroaryl.
The tertiary alkyl acrylate is preferably unsubstituted at the
.beta. carbon. The acrylate reagent is advantageously substituted
at the a carbon with an alkyl or aryl group that provides steric
hindrance to the final carboxylic acid group. As taught in U.S.
Pat. No. 6,495,659, which is incorporated herein by reference in
its entirety, the steric effects of a side chain attached to the a
carbon can favorably affect the hydrolytic stability of drug
conjugates formed using the polymer acid. In one preferred
embodiment, the tertiary alkyl acrylate is .alpha.- or
.beta.-substituted, preferably .alpha.-substituted, with methyl,
ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,
tert-butyl, or benzyl.
[0110] In one embodiment, the tertiary alkyl acrylate has the
structure:
##STR00009##
wherein: [0111] R.sub.1 and R.sub.2 are each independently selected
from the group consisting of hydrogen, halo, alkylthio, acyl,
acyloxy, nitro, cyano, azido, trihalomethyl, alkyl, substituted
alkyl, cycloalkyl, substituted cycloalkyl, alkoxy, aryl,
substituted aryl, heterocycle, substituted heterocycle, heteroaryl,
and substituted heteroaryl; and [0112] R.sub.3-R.sub.5 are each
independently alkyl, substituted alkyl, aryl or substituted
aryl.
[0113] Preferably, R.sub.3, R.sub.4, and R.sub.5, are each methyl,
ethyl, or phenyl, and R.sub.1 and R.sub.2 are hydrogen or R.sub.1
is hydrogen and R.sub.2 is methyl, ethyl, n-propyl, isopropyl,
n-butyl, isobutyl, sec-butyl, tert-butyl, or benzyl. In one
preferred embodiment, the tertiary alkyl group is tert-butyl (i.e.,
each of R.sub.3-R.sub.5 is methyl). Other exemplary tertiary alkyl
groups include tert-amyl, .alpha.,.alpha.''-dimethylbenzyl, trityl,
1-adamantyl, and 2-methyl-2-adamantyl.
[0114] Preferred tertiary alkyl acrylates include tert-butyl
acrylate and tert-butyl methacrylate, which are commercially
available from Sigma-Aldrich Corporation, St. Louis, Mo. Other
exemplary tertiary alkyl acrylates include tert-butyl esters of
crotonic acid or isocrotonic acid. Additionally, other tertiary
alkyl or tertiary cycloalkyl acrylate or methacrylates are suitable
for use in the present invention.
[0115] C. The Polymer Bearing at Least One Propionic Acid Group
[0116] Following the method described herein, the water soluble and
non-peptidic polymer will bear at least one terminal propionic acid
group. An exemplary polymer will correspond to the following
structure:
POLY-CHR.sub.1--CHR.sub.2--COOH (Formula VII)
wherein POLY is the residue of a water soluble and non-peptidic
polymer (such as PEG), and R.sub.1 and R.sub.2 (as discussed above
in Section II.B) are each independently selected from the group
consisting of hydrogen, halo, alkylthio, acyl, acyloxy, nitro,
cyano, azido, trihalomethyl, alkyl, substituted alkyl, cycloalkyl,
substituted cycloalkyl, alkoxy, aryl, substituted aryl,
heterocycle, substituted heterocycle, heteroaryl, and substituted
heteroaryl. In terms of POLY, the corresponding water soluble and
non-peptidic polymers are discussed above in Section II.A, PEG
[e.g., "--(CH.sub.2CH.sub.2O).sub.m--" or
"--(OCH.sub.2CH.sub.2).sub.m--"] is a particularly preferred POLY
and is discussed above in Section II.A. In one or more embodiments,
branched versions of the polymer are preferred.
[0117] If desired, the propionic acid functionalized-polymer can be
further modified to form useful reactive derivatives of carboxylic
acids using methodology known in the art. Thus, the invention
includes polymers obtainable from and/or obtained from the
described propionic acid-functionalized polymers. For example, the
carboxylic acid can be further derivatized to form 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 addition, the
carboxylic acid can be reduced to form an aldehyde, either directly
from the carboxylic acid using a suitable reducing agent, or
indirectly through an amide, nitrile or ester using a suitable
reducing agent. Also, the acid or certain functionalized polymers,
e.g. active esters, may be used as intermediates to react with
appropriate reagents or other small molecules or short polymeric
species to form yet additional reactive derivatives such as
maleimides, thiols, reactive disulfides, acetals, aldehydes and the
like.
[0118] In a preferred embodiment, the propionic acid is esterified
to form an active ester, such as an N-hydroxysuccinimidyl ester,
o-, m-, or p-nitrophenyl ester, 1-hydroxybenzotriazolyl ester,
imidazolyl ester, or N-hydroxysulfosuccinimidyl ester. The
propionic acid or reactive derivative thereof attached to the
polymer preferably has the structure:
--CHR.sub.1--CHR.sub.2--C(O)--Y (Formula VIII)
wherein R.sub.1 and R.sub.2 (as discussed above in Section II.B)
are each independently selected from the group consisting of
hydrogen, halo, alkylthio, acyl, acyloxy, nitro, cyano, azido,
trihalomethyl, alkyl, substituted alkyl, cycloalkyl, substituted
cycloalkyl, alkoxy, aryl, substituted aryl, heterocycle,
substituted heterocycle, heteroaryl, and substituted heteroaryl,
and Y is selected from the group consisting of hydrogen, halo,
hydroxy, amino, substituted amino, --NCO, --NCS, N.sub.3, --CN, and
--O--R', wherein R' is N-succinimidyl, nitrophenyl, benzotriazolyl,
imidazolyl, N-sulfosuccinimidyl, N-phthalimidyl, N-glutarimidyl,
N-tetrahydrophthalimidyl, N-norbornene-2,3-dicarboximidyl, and
hydroxy-7-azabenzotriazolyl.
[0119] In one embodiment, Y is a substituted amino having the
structure --NHR.sub.6, wherein R.sub.6 is any organic group that
may contain additional reactive functional groups (e.g., aldehyde,
maleimide, mercapto, and the like) and where the additional
functional group or groups are separated from the carbonyl carbon
by an alkylene chain (e.g., C1-6 alkylene chain) and, optionally,
an additional linker, such as a short PEG chain and another
alkylene chain (e.g., alkylene-PEG-alkylene).
[0120] Exemplary polymers that can be prepared through the
described propionic acid-functionalized polymers include the
following:
##STR00010##
wherein m is defined as in Section II.A, above, and EC is a residue
of a moiety selected from the group consisting of fluorescein,
biotin, acrylate, vinylsulfone, maleimide, tert-butyl carbonyl
(t-Boc), and 9-fluorenylmethoxycarbonyl (Fmoc).
[0121] In some situations, it is preferred that the polymer bearing
a propionic acid is not methoxy PEG propionic acid having a weight
average molecular weight of about 20,000 Da [i.e.,
H.sub.3CO--(CH.sub.2CH.sub.2O).sub.m--CH.sub.2--CH.sub.2--COOH,
wherein m does not result in a weight average molecular weight of
about 20,000 Da], and not methoxy PEG propionic acid having a
weight average molecular weight of about 30,000 Da [i.e.,
H.sub.3CO--(CH.sub.2CH.sub.2O).sub.m--CH.sub.2--CH.sub.2--COOH,
wherein m does not result in a weight average molecular weight of
about 30,000 Da]. Furthermore, in some situations, it is preferred
that the polymer bearing a propionic acid is not methoxy PEG (or
other polymer) propionic acid having a weight average molecular
weight of between about 17,500 Da and 22,500 Da, and not methoxy
PEG propionic acid having a weight average molecular weight of
between about 27,500 Da and 32,500 Da. In still other situations,
it is preferred that the weight average molecular weight of the
methoxy PEG propionic acid is greater than 35,000 Da. Preferred
polymers bearing a propionic acid comprise branched (i.e., singly
branched or multi-branched) structures as previously discussed.
[0122] D. Biologically Active Molecules for Conjugation
[0123] The propionic acid-terminated 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 groups. 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, American Chemical Society, Washington, DC
(1997)).
[0124] With respect to polymers used in conjugation,
electrophilically-activated polymer derivatives, such as active
esters, are useful for conjugation to amino groups on proteins or
other biologically active molecules. Conjugation of a polymer
bearing an active carboxylic acid ester with an amino group on a
biologically active molecule results in formation of a stable amide
bond between the polymer and the biologically active molecule.
[0125] 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 antagnonists), 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,
antioxicants, 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.
[0126] Examples of active agents suitable for use in covalent
attachment to a polymer prepared by the method of the invention
include, but are not limited to, calcitonin, erythropoietin (EPO),
Factor VIII, Factor IX, ceredase, cerezyme, cyclosporin,
granulocyte colony stimulating factor (GCSF), thrombopoietin (TPO),
alpha-1 proteinase inhibitor, elcatonin, granulocyte macrophage
colony stimulating factor (GMCSF), growth hormone, human growth
hormone (HGH), growth hormone releasing hormone (GHRH), heparin,
low molecular weight heparin (LMWH), interferon alpha, interferon
beta, interferon gamma, interleukin-1 receptor, interleukin-2,
interleukin-1 receptor antagonist, interleukin-3, interleukin-4,
interleukin-6, luteinizing hormone releasing hormone (LHRH), factor
IX 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 hoimone (FSH), insulin-like
growth factor (IGF), insulintropin, macrophage colony stimulating
factor (M-CSF), nerve growth factor (NGF), tissue growth factors,
keratinocyte growth factor (KGF), glial growth factor (GGF), tumor
necrosis factor (TNF), endothelial growth factors, parathyroid
hormone (PTH), glucagon-like peptide thymosin alpha I, IIb/IIIa
inhibitor, alpha-1 antitrypsin, phosphodiesterase (PDE) compounds,
VLA-4 inhibitors, bisphosphonates, respiratory syncytial virus
antibody, cystic fibrosis transmembrane regulator (CFTR) gene,
deoxyreibonuclease (Dnase), bactericidal/permeability increasing
protein (BPI), anti-CMV antibody, 13-cis retinoic acid, 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, gramicidin, 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,
cefmetazole, 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, ergotamine tartrate and where
applicable, analogues, agonists, antagonists, inhibitors, and
pharmaceutically acceptable salt forms of the above. In reference
to peptides and proteins, the invention is intended to encompass
synthetic, native, glycosylated, unglycosylated, PEGylated forms,
and biologically active fragments and analogs thereof.
[0127] Thus, the invention includes a composition comprising a
conjugate of a propionic acid-terminated polymer (or a reactive
derivative thereof) and a biologically active molecule wherein the
propionic acid-terminated polymer is prepared in accordance with
the method described herein. Thus, for example, the conjugate in
the composition can be formed by reacting
##STR00011##
with an interferon, wherein m is defined as in Section II.A. In
addition, the conjugate in the composition can be formed by
reacting
##STR00012##
with a TNFR tumor necrosis factor receptor, wherein m is defined as
in Section II.A. Further, the conjugate in the composition can be
formed by reacting
##STR00013##
reacting with erythropoietin, wherein m is defined as in Section
II.A. Also, the conjugate in the composition can be formed by
reacting
##STR00014##
with human growth hormone, wherein m is defined as in Section II.A.
Variants and mimetics of the interferon, TNFR, erythropoietin, and
human growth hormone can be substituted as the biologically active
agent in these conjugates. An exemplary conjugate will comprise a
structure corresponding to Formula IX:
##STR00015##
wherein Active Agent represents a residue of an amine-containing
active agent and m is defined as in Section II.A.
[0128] Because the compositions include conjugates prepared from
propionic acid-terminated polymers (or a reactive derivative
thereof) formed by the presently described method--which results in
reduced degradation and chain cleavage of the polymer backbone--the
resulting conjugates and compositions likewise have reduced
degradation and chain cleavage of the polymer backbone, at least as
compared to conjugates prepared from propionic acid-terminated
polymers (or reactive derivatives thereof) prepared by alternate
methods (i.e., methods different from the presently described
method for synthesizing propionic acid-terminated polymers, or a
reactive derivatives thereof).
[0129] In particular, the presently described method unexpectedly
and advantageously provides propionic-acid terminated polymers (as
well as reactive derivatives and conjugates) that are more pure as
a result of reduced degradation and chain cleavage. The method
particularly improves the purity when the propionic acid-terminated
polymer comprises propionic-acid terminated poly(ethylene oxide)
bearing a methoxy end-cap.
[0130] For example, a propionic acid-terminated poly(ethylene
oxide) bearing a methoxy end-cap [e.g.,
CH.sub.3O--(CH.sub.2CH.sub.2O).sub.m--CH.sub.2CH.sub.2COOH)]
comprises ether groups, each ether group having an oxygen atom with
a certain sensitivity to cleavage via nucleophilic attack. While
not wishing to be bound by theory, it appears for at least three
reasons that the most sensitive ether group in the polymer prone to
cleave is the ether group associated with the methoxy end-cap.
[0131] First, for reasons of accessibility, the ether group
associated with the methoxy end-cap is the most exposed and
subsequently relatively available for a chain-cleaving nucleophilic
attack. Second, the oxygen in the ether group associated with the
methoxy end-cap is more basic than the oxygens in the ether groups
associated with the repeating ethylene oxide monomers. This is so
because the oxygens in the ether groups associated with the
repeating ethylene oxide monomers have the benefit of two
neighboring ether oxygens (and their electron-withdrawing effects)
while the oxygen associated with the methoxy end-cap has only a
single neighboring ether oxygen. Third, while the oxygen in the
ether group nearest the carboxylic acid group also only has a
single neighboring ether oxygen, this oxygen benefits from the
neighboring and stability-enhancing carboxylic acid group that
serves as a electron sink.
[0132] The above analysis can be illustrated in Schematic A, where
the arrows in the left structures show electron-donating and
electron-withdrawing effects of the attached groups and the
right-hand structures are compared for the relative stability of
the protonated forms. In Schematic A, m is defined as in Section
II.A.
##STR00016##
[0133] Again, while not wishing to be bound by theory, it is
believed that of the two right-hand structures in the above
schematic, a nucleophile is most likely to attack by backside
nucleophilic displacement at the carbon at the protonated methoxy
oxygen (top, right-hand structure in the above schematic). Reasons
for this preference include a lower degree of steric hindrance at
the methyl group (thereby favoring methyl group displacement) and a
higher degree of steric hindrance within the polymer backbone, as
shown in Schematic B. In Schematic B, m is defined as in Section
II.A.
##STR00017##
[0134] Thus, for example, if a hydrogen sulfate anion is the
nucleophile that will produce chain cleavage, the favored pathway
for cleavage is believed to be the loss of the methyl group of the
methoxy end-cap. See Schematic C, wherein m is defined as in
Section ILA. This process will lead to formation of a hydroxyl
end-cap in place of the methoxy end-cap (a "demethylated polymer").
This hydroxyl end-capped side product impurity was neither observed
nor reported in the "nitrile-based" method described in U.S. Pat.
No. 5,672,662 because the hydroxyl end-capped polymer has
essentially the same high performance liquid chromatography
("HPLC") retention properties as the corresponding methoxy
end-capped species as each has essentially the same molecular
weight. It will be recalled that the "nitrile-based" method
described for preparing propionic acid-terminated polymers in U.S.
Pat. No. 5,672,662 requires the use of relatively harsh conditions
such as one or more of (a) using strong concentrated mineral acids
like sulfuric acid, or hydrochloric acid, (b) high temperatures,
and (c) very long reaction times (exceeding 30 hours in the case of
hydrochloric acid-promoted hydrolysis).
##STR00018##
[0135] It is preferred, then, that a composition disclosed in the
invention is substantially free of side product impurities bearing
a hydroxyl end-cap (e.g., substantially free of
HO--(CH.sub.2CH.sub.2O).sub.m--CH.sub.2CH.sub.2COOH species). In
this regard, a composition that is substantially free of side
product impurities bearing a hydroxyl end-cap will contain less
than about 15% by weight, more preferably less than about 10% by
weight, more preferably less than about 5% by weight, still more
preferably less than about 3% by weight, yet still more preferably
less than about 2% by weight of side product impurities bearing a
hydroxyl end-cap, with less than about 1% by weight being most
preferred.
[0136] The side product impurity--bearing a hydroxyl end-cap as
show in Formula X--can introduce additional unwanted species in a
composition. In particular, a hydroxyl end-cap-containing impurity
(such as that shown in Foimula X) can (i) compete with a reactive
component of another molecule (e.g., the hydroxyl group of a
reagent used to form polymer derivatives), and/or (ii) ultimately
result in one or more hydroxyl end-capped conjugate species.
[0137] The side product impurity (such as that shown in Formula X)
can react with other molecules to form additional species that are
not desired to be present in the composition. For example, during
esterification to form a reactive ester, the side product impurity
bearing a hydroxyl end-cap will compete with the hydroxyl group of
the ester-forming N-hydroxysuccinimide reagent, thereby forming a
dimeric species wherein two polymers are linked. This unintended
reaction does occur when, for example, a side product impurity
corresponding to Formula X is present in the reaction mixture,
thereby resulting in the formulation of the dimeric species shown
in Formula XI:
##STR00019##
wherein m is defined as in Section II.A. In contrast to a simply
demethylated impurity, the dimeric species is readily observable by
gel permeation chromatography (GPC) or HPLC because it has a higher
molecular weight.
[0138] It is preferred, then, that a composition disclosed in the
invention is substantially free of species comprising a structure
corresponding to a dimeric species (e.g., hydroxyl end-capped and
methoxy end-capped as shown in Formula XI). In this regard, a
composition that is substantially free of dimeric species will
contain less than about 10% by weight, more preferably less than
about 5% by weight, more preferably less than about 4% by weight,
still more preferably less than about 3% by weight, yet still more
preferably less than about 2% by weight of dimeric species, with
less than about 1% by weight being most preferred.
[0139] As previously indicated, the hydroxyl end-cap-containing
impurity (such as the one shown in Formula X) can ultimately result
in one or more species of hydroxyl end-capped polymer-active agent
conjugates. Thus, for example, an impurity having a structure
comprising a structure corresponding to Formula X can--when subject
to an esterification reaction with N-hydroxysuccinimide--result in
a species comprising a structure corresponding to Formula XII:
##STR00020##
wherein m is defined as in Section II.A.
[0140] It should also be noted that a species comprising a
structure corresponding to Formula XII can result via an
esterification reaction if the original starting methoxy end-capped
poly(ethylene glycol) material used to prepare a propionic
acid-terminated polymer is contaminated with "diol" poly(ethylene
glycol), i.e., HO--(CH.sub.2CH.sub.2O).sub.m--H. In this regard, it
is preferred to use starting methoxy end-capped poly(ethylene
glycol) compositions comprising less than 2% by weight of "diol"
poly(ethylene glycol).
[0141] If a species comprising a structure corresponding to Formula
XII is subsequently combined with an amine-containing active agent,
then a hydroxyl end-capped conjugate having a species comprising a
structure corresponding to Formula XIII can be formed:
##STR00021##
wherein Active Agent represents a residue of an amine-containing
active agent and m is defined as in Section II.A. A species
comprising a structure corresponding to Formula XII could also lead
to a conjugate comprising a structure corresponding to Formula
XIV:
##STR00022##
wherein Active Agent represents a residue of an amine-containing
active agent and m is defined as in Section II.A. In addition, a
species comprising a structure corresponding to Formula XII could
result in a conjugate comprising a hydroxyl end-capped structure
corresponding to corresponding to Formula XV:
##STR00023##
wherein Active Agent represents a residue of an amine-containing
active agent and m is defined as in Section II.A.
[0142] Each of the impurities comprising a structure corresponding
to one of Formulae X, XII, and XIII include a hydroxyl end-cap (or
"demethylated" group). As pointed out above, the special concern
with a composition comprising a relatively large amount of one or
more of these or other hydroxyl end-capped species is the ability
of the impurity to react with an active agent when the composition
is used in a conjugation reaction. The result can be the formation
of second, third and fourth conjugate species, such as the intended
conjugate (e.g, a species comprising a structure corresponding to
Formula IX), and one or more unintended conjugates (e.g., one or
more species comprising a structure corresponding to Formulas XIII
or XIV. It has been found that mixtures of various conjugate
species reduces the consistency, performance, and reproducibility
of the resulting composition.
[0143] It is preferred that the composition is substantially free
of conjugate species corresponding to any one or combination of
Formulae XIII, XIV, and XV. In this regard, a composition that is
substantially free of conjugate species corresponding to any one or
combination of Formulae XIII, XIV and XV will contain less than
about 10% by weight, more preferably less than about 5% by weight,
more preferably less than about 4% by weight, still more preferably
less than about 3% by weight, yet still more preferably less than
about 2% by weight of conjugate species corresponding to any one or
combination of Formulae XIII, XIV, and XV, with less than about 1%
by weight being most preferred.
EXPERIMENTAL
[0144] The following examples are given to illustrate the
invention, but should not be considered in limitation of the
invention. For example, although monofunctional PEG reagents are
used in the examples to illustrate the invention, difunctional or
multifunctional PEG polymers could also be used in the present
invention, as well as other types of water soluble and non-peptide
polymers.
[0145] Unless otherwise noted, all PEG reagents referred to in the
appended examples are available from Nektar AL of Huntsville, Ala.
All NMR data was generated by a 300 or 400 MHz NMR spectrometer
manufactured by Bruker.
[0146] Example 1 illustrates formation of a 20,000 Da
mPEG-propionic acid polymer using tert-butyl acrylate as the
tertiary alkyl acrylate reagent and tetrabutylammonium hydroxide,
formed in situ from tetrabutylammonium bromide and potassium
hydroxide, as the Michael addition catalyst. TFA is used to cleave
the tert-butyl group. Example 2 is similar to Example 1, except a
20,000 Da henzyloxy-PEG-propionic acid polymer is formed using the
direct addition of tetrabutylammonium hydroxide as the catalyst.
Example 3 is similar to Example 1, except the mPEG has a molecular
weight of 30,000 Da.
[0147] Comparative Example 1 shows that the synthesis method
outlined in Example 1 of U.S. Pat. No. 5,523,479 to Sanders et al.
fails to produce the desired propionic acid, tert-butyl ester, when
using a 20,000 Da polymer starting material. It is believed that
the method disclosed in U.S. Pat. No. 5,523,479 is ineffective in
forming higher molecular weight propionic acid functionalized
polymers of the type utilized in the present invention. In
particular, it is believed that the catalysts suggested in the
Sanders patent are incapable of promoting the Michael addition
reaction to any significant degree when a higher molecular weight
polymer starting material is used.
[0148] Comparative Example 2 shows that the synthesis method
outlined in Example 1 of U.S. Pat. No. 5,672,662 to Harris et al.
fails to produce pure m-PEG(20,000 Da)-propionic acid, when using a
20,000 Da low diol contaminated methoxy-end-capped PEG starting
material, because the relatively harsh reaction conditions lead to
the demethylation and chain cleavage of methoxy-end-capped and
produce substantial amount of HO-PEG(20,000 Da)-propionic acid.
Example 1
Synthesis of mPEG(20,000 Da)-Propionic Acid
[0149] A. mPEG(20,000 Da)-Propionic Acid, tert-Butyl Ester
[0150] A solution of mPEG (20,000 Da) (35.0 g, 0.00175 moles) (NOF
Corporation) and tetrabutylainmonium bromide (0.6 g) in toluene
(125 ml) was azeotropically dried by distilling off 105 ml of
toluene. Potassium hydroxide (0.15 g) in form of fine powder was
added and the mixture was warmed up to 60.degree. C. under argon
atmosphere. Then tert-butyl acrylate (2.0 ml, 0.01365 moles, 7.8
fold excess) was added during 2 h and the mixture was stirred
overnight at 60.degree. C. under argon atmosphere. Next the solvent
was distilled off under reduced pressure and the residue was
dissolved in dichloromethane (400 ml). The resulting solution was
washed two times with deionized water (2.times.50 ml) and then
dried with anhydrous magnesium sulfate. Next the solvent was
distilled off under reduced pressure. Yield 28.5 g. NMR (d6-DMSO):
1.40 ppm (s, (CH.sub.3).sub.3C--, 9H), 2.41 ppm (t,
--CH.sub.2--COO--, 2H), 3.24 ppm (s, --OCH3, 3H), 3.51 ppm (s, PEG
backbone); substitution 69.1%.
[0151] B. mPEG(20,000 Da)-Propionic Acid
[0152] mPEG (20,000 Da)-Propionic Acid, tert-Butyl Ester (20g) from
Step A was dissolved in a dichloromethane/trifluoroacetic acid
mixture (1:1; 120 ml) and the solution was stirred 1 h at
60.degree. C. After cooling to room temperature, dichloromethane
(400 ml) was added to the reaction mixture and the resulting
solution was washed with deionized water (400 ml), and dried with
anhydrous magnesium sulfate. Next the solvent was distilled off
under reduced pressure. The crude product was dissolved in
deionized water (400 ml) and the pH of the solution was adjusted to
12 with 1.0 M NaOH. The solution was stirred 2 h at pH=12. Next
NaCl (40 g) was added and the pH was adjusted to 3 with 10-%
phosphoric acid. The product was extracted with dichloromethane,
the solution was dried with anhydrous magnesium sulfate, and the
solvent was distilled off under reduced pressure giving 16.5 g of
white solid product. Anion exchange chromatography showed that the
product contains: m-PEG(20,000 Da)-propionic acid 68.2% and
m-PEG-20K 31.8%. Next the product was chromatographically purified
using typical anion exchange chromatography media giving 100% pure
PEG(20,000 Da)-propionic acid (9.8 g). In this regard, "100%
PEG(20,000 Da)-propionic acid" means 100% pure PEG(20,000
Da)-monopropionic acid.
[0153] NMR (d.sub.6-DMSO): 2.43 ppm (t, --CH.sub.2--COO--, 2H),
3.24 ppm (s, --OCH.sub.3, 3H), 3.51 ppm (s, PEG backbone); No
PEG-OH groups were detected by NMR (no triplet at 4.58 ppm); This
experimental result means that the product was 100% pure
mPEG(20,000 Da)-monopropionic acid with no detectable HO-PEG(20,000
Da)-propionic acid present,
Example 2
Synthesis of Benzyloxy-PEG(20,000 Da)-Propionic Acid
[0154] A solution of benzyloxy-PEG (20,000 Da) (35.0 g, 0.00175
moles) (NOF Corporation) and tetrabutylammonium hydroxide (2.0 g of
40 wt % solution in water) in toluene (200 ml) was azeotropically
dried by distilling off 175 ml toluene. The obtained solution was
warmed up to 65.degree. C. under argon atmosphere. Then tert-butyl
acrylate (1.5 ml, 0.01024 moles, 5.85 fold excess) was added during
3.5 hours and the mixture was stirred overnight at 60-65.degree. C.
under argon atmosphere. Next the solvent was distilled off under
reduced pressure and the residue was dissolved in dichloromethane
(40 ml).
[0155] Trifluoroacetic acid (40 ml) was added and the solution was
heated to boiling for 2 h. Dichloromethane and trifluoroacetic acid
were distilled off under reduced pressure and the crude product was
dissolved in 400 ml deionized water. The pH was adjusted to 12 with
1.0M NaOH and the solution was stirred 2 h at pH=12. Next NaC1 (40
g) was added and the pH was adjusted to 3 with 10% phosphoric acid.
The product was extracted with dichloromethane, the extract was
dried with anhydrous magnesium sulfate, and the solvent was
distilled off under reduced pressure giving 28.5 g of white solid
product. Anion exchange chromatography showed that the product
contains: PEG(20,000 Da)-monopropionic acid 69.7% and PEG(20,000
Da) 30.3%. Next the product was chromatographically purified giving
100% pure PEG(20,000 Da)-monopropionic acid. NMR (d.sub.6-DMSO):
2.43 ppm (t, --CH.sub.2--COO--, 2H), 3.24 ppm (s, --OCH.sub.3, 3H),
3.51 ppm (s, PEG backbone), 4.49 ppm (s, --CH.sub.2--, benzyloxy,
2H), 7.33 ppm (m, C.sub.6H.sub.5--, 5H). No PEG-OH groups were
detected (no triplet at 4.58 ppm); This means that the product was
100% pure benzyloxy-PEG(20,000 Da)-propionic acid.
Example 3
Synthesis of mPEG(30,000 Da)-Propionic Acid
[0156] A. mPEG(30,000 Da)-Propionic Acid, tert-Butyl Ester
[0157] A solution of mPEG (30,000 Da) (50.0g, 0.00167 moles) (NOF
Corporation) and tetrabutylammonium bromide (0.8 g) in toluene (200
ml) was azeotropically dried by distilling off 100 ml toluene.
Potassium hydroxide (0.16 g) in form of fine powder was added and
the mixture was warmed up to 60.degree. C. under argon atmosphere.
Then tert-butyl acrylate (2.5 ml, 0.01707 moles, 10.2 fold excess)
was added during 4h and the mixture was stirred overnight at
60.degree. C. under argon atmosphere. Next the solvent was
distilled off under reduced pressure and the residue was dissolved
in dichloromethane (400 ml). The obtained solution was washed two
times with deionized water (2.times.100 ml) and then dried with
anhydrous magnesium sulfate. Next the solvent was distilled off
under reduced pressure giving 42.5 g of solid product. NMR
(d.sub.6-DMSO): 1.40 ppm (s, (CH.sub.3).sub.3C--, 9H) 2.41 ppm (t,
--CH.sub.2--COO--, 2H), 3.24 ppm (s, --OCH.sub.3, 3H), 3.51 ppm (s,
PEG backbone); substitution 73.8%.
[0158] B. mPEG(30,000 Da)-Propionic Acid
[0159] mPEG (30,000 Da)-Propionic Acid, tert-Butyl Ester (40 g)
from Step A was dissolved in a dichloromethane/trifluoroacetic acid
mixture (1:1; 150 ml) and the solution was stirred 1 h at
55.degree. C. After cooling to room temperature, dichloromethane
(600 ml) was added to the reaction mixture and the solution was
washed with deionized water (400 ml), and dried with anhydrous
magnesium sulfate. Next the solvent was distilled off under reduced
pressure. The crude product was dissolved in deionized water (800
ml) and the pH of the solution was adjusted to 12 with 1.0M NaOH.
The solution was stirred 2 h at pH=12. Next, NaCl (80 g) was added
and the pH was readjusted to 3 with 10% phosphoric acid. The
product was extracted with dichloromethane giving 33.5 g of white
solid product. Anion exchange chromatography showed that the
product contains: PEG(30,000 Da)-monopropionic acid 67.7% and
PEG(30,000 Da) 32.3%. Next, the product was chromatographically
purified using typical anion exchange chromatography media giving
100% pure PEG(30,000 Da)-monopropionic acid (25.3 g).
[0160] NMR (d.sub.6-DMSO): 2.43 ppm (t, --CH.sub.2--COO--, 2H),
3.24 ppm (s, --OCH.sub.3, 3H), 3.51 ppm (s, PEG backbone). No
PEG-OH groups were detected (no triplet at 4.58 ppm); This means
that the product was 100% pure mPEG(30,000 Da)-propionic acid.
Comparative Example 1
Attempted Synthesis of mPEG(20,000 Da)-Propionic Acid, tert-Butyl
Ester
[0161] Using the method outlined in Example 1 of U.S. Pat. No.
5,523,479, a solution of mPEG (20,000 Da) (35.0 g, 0.00175 moles)
(NOF Corporation) in toluene (125 ml) was azeotropically dried by
distilling off 105 ml of solvent. Potassium hydroxide (0.15 g) in
the form of fine powder was added and the mixture was warmed to
60.degree. C. under argon atmosphere. Then tert-butyl acrylatc (2.0
ml, 0.01365 moles, 7.8 fold excess) was added during 2 hours and
the mixture was stirred overnight at 60.degree. C. under argon
atmosphere. Next, the solvent was distilled off under reduced
pressure. Yield 36.5 g. NMR analysis showed that the polymer
starting material was unchanged: mPEG (20,000 Da). NMR
(d.sub.6-DMSO): 3.24 ppm (s, --OCH.sub.3, 3H), 3.51 ppm (s, PEG
backbone), 4.58 ppm (t, --OH, 1H).
Comparative Example 2
Synthesis of mPEG(20,000 Da)-Propionic Acid according to U.S. Pat.
No. 5,672,662
[0162] A. mPEG(20,000 Da)-Propionitrile
[0163] A mixture of mPEG (20,000 Da) (25.0 g, 0.00125 moles) (NOF
Corporation), distilled water (25.0 ml) and potassium hydroxide
(0.5 g) was cooled to 0-5.degree. C. in an ice bath. Acrylonitrile
(3.4 g) was added slowly, and the solution was stirred for three
hours at 0-5.degree. C. Ten percent NaCl solution (225 ml) was
added to the reaction mixture and the product was extracted with
dichloromethane (200, 100, and 50 ml). The organic layer was dried
over magnesium sulfate, and the solvent was distilled off under
reduced pressure. The crude product was dissolved in
dichloromethane (35 ml) and precipitated with isopropanol (225 ml)
at room temperature. The precipitate was removed by filtration and
dried under vacuum. Yield of M-PEG nitrile 23.5 g.
[0164] B. mPEG(20,000 Da)-Propionamide
[0165] A mixture of M-PEG nitrile from the above step (23.5 g) and
concentrated hydrochloric acid (117.5 g) was stirred at, room
temperature for 48 hours. The solution was diluted with one liter
of water and extracted with dichloromethane (200, 150, and 100 ml).
The combined organic extracts were washed twice with water, dried
over magnesium sulfate, filtered, and concentrated to dryness by
rotary evaporation. Yield of PEG amide 21.5 g.
[0166] C. mPEG(20,000 Da)-Propionic Acid
[0167] M-PEG amide from the above step (16.0 g) was dissolved in
1150 ml of distilled water, 100 g of potassium hydroxide was added,
and the solution was stirred for 22 hours at room temperature.
Sodium chloride (150 g) was added, and the solution was extracted
with dichloromethane (150 ml.times.3). The combined organic
extracts were washed with 5% phosphoric acid, water (twice), and
dried over magnesium sulfate. Next, the solvent was distilled off
under reduced pressure giving 14.0 g of white solid product. Anion
exchange chromatography showed that the product contains:
PEG(20,000 Da)-propionic acid 62.5%; and PEG-20K 37.5%. Next, the
product was chromatographically purified using typical anion
exchange chromatography media giving 100% pure PEG(20,000
Da)-monopropionic acid (6.5 g). NMR (d.sub.6-DMSO): 2.43 ppm (t,
--CH.sub.2--COO--, 2H), 3.24 ppm (s, --OCH.sub.3, 2.61 H), 3.51 ppm
(s, PEG backbone, 1725 H), 4.58 ppm (t, PEG-OH, 0.13 H). NMR
analysis (triplet at 4.58) showed that the product contained 13 mol
% of PEG-OH groups; this means that the product was a mixture of
the desired mPEG(20,000 Da)-Propionic Acid (87%) and
HO-PEG(20,000)-Propionic Acid (13%).
[0168] 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, it is to be understood that the
invention is not to be limited to the specific embodiments
disclosed and that modifications and other embodiments are intended
to be included. Although specific terms are employed herein, they
are used in a generic and descriptive sense only and not for
purposes of limitation.
[0169] All articles, books, patents and other publications
referenced herein are hereby incorporated by reference in their
entireties.
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