U.S. patent application number 15/157343 was filed with the patent office on 2017-02-09 for polymer factor ix moiety conjugates.
The applicant listed for this patent is Nektar Therapeutics. Invention is credited to Mary J. Bossard, Gayle Stephenson Phillips.
Application Number | 20170035896 15/157343 |
Document ID | / |
Family ID | 35429498 |
Filed Date | 2017-02-09 |
United States Patent
Application |
20170035896 |
Kind Code |
A1 |
Bossard; Mary J. ; et
al. |
February 9, 2017 |
POLYMER FACTOR IX MOIETY CONJUGATES
Abstract
Conjugates of a Factor IX moiety and one or more water-soluble
polymers are provided. Typically, the water-soluble polymer is
poly(ethylene glycol) or a derivative thereof. Also provided (among
other things) are compositions comprising the conjugates, methods
of making the conjugates, and methods of administering to a patient
compositions comprising the conjugates.
Inventors: |
Bossard; Mary J.; (Madison,
AL) ; Stephenson Phillips; Gayle; (Harvest,
AL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Nektar Therapeutics |
San Francisco |
CA |
US |
|
|
Family ID: |
35429498 |
Appl. No.: |
15/157343 |
Filed: |
May 17, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14076116 |
Nov 8, 2013 |
9347054 |
|
|
15157343 |
|
|
|
|
12638811 |
Dec 15, 2009 |
8586711 |
|
|
14076116 |
|
|
|
|
12499770 |
Jul 8, 2009 |
|
|
|
12638811 |
|
|
|
|
11172459 |
Jun 30, 2005 |
7579444 |
|
|
12499770 |
|
|
|
|
60584505 |
Jun 30, 2004 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 7/04 20180101; C12N
9/644 20130101; A61K 38/4846 20130101; C12Y 304/21022 20130101;
A61K 47/60 20170801; C12N 9/96 20130101 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 9/00 20060101 A61K009/00; A61K 38/48 20060101
A61K038/48 |
Claims
1. A method for administration, the method comprising administering
to a subject in need thereof a composition comprising a plurality
of polyethylene glycol (PEG)-Factor IX moiety conjugates, the
conjugates comprising a branched polyethylene glycol covalently
attached via a spacer moiety comprised of one or more atoms to a
Factor IX moiety that is either Factor IX or Factor IXa, where at
least about 85% of the polyethylene glycol-Factor IX moiety
conjugates in the composition are monoPEGylated.
2. The method of claim 1, wherein the branched polyethylene glycol
comprises two polyethylene glycol chains attached to a polyol
core.
3. The method of claim 2, wherein the polyol core is a glycerol
core.
4. The method of claim 3, wherein the branched polyethylene glycol
has a weight average molecular weight of about 40,000 daltons.
5. The method of claim 4, wherein the branched polyethylene glycol
comprises two polyethylene glycol chains each having a weight
average molecular weight of about 20,000 Daltons.
6. The method of claim 1, wherein the spacer intervening between
the Factor IX moiety and the polyethylene glycol chains comprises a
carbamate group.
7. The method of claim 3, where the polyol core is a glycerol
core.
8. The method of claim 1, where at least about 95% of the
polyethylene glycol-Factor IX moiety conjugates in the composition
are monoPEGylated.
9. The method of claim 1, wherein at least about 99% of the
conjugates in the composition have from one to two of the branched
polyethylene glycols covalently attached to the Factor IX
moiety.
10. The method of claim 1, where the branched polyethylene glycol
is covalently attached to the Factor IX moiety via an amide
linkage.
11. The method of claim 1, wherein the Factor IX moiety is Factor
IX.
12. The method of claim 1, wherein the Factor IX moiety is Factor
IXa.
13. The method of claim 1, wherein the Factor IX moiety is
recombinantly derived.
14. The method of claim 1, wherein the Factor IX moiety is
blood-derived.
15. The method of claim 1, wherein the composition comprises a
pharmaceutically acceptable excipient.
16. The method of claim 1, wherein the composition is in a liquid
form suitable for injection.
17. The method of claim 16, wherein the administering is via
injection.
18. The method of claim 17, wherein the administering is via
intravenous injection.
19. The method of claim 1, wherein the subject has hemophilia B and
the composition is used for replacement therapy.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/076,116, filed Nov. 8, 2013, now U.S. Pat.
No. 9,347,054, which is a continuation of U.S. patent application
Ser. No. 12/638,811, filed Dec. 15, 2009, now U.S. Pat. No.
8,586,711, which is a continuation of U.S. patent application Ser.
No. 12/499,770, filed Jul. 8, 2009, now abandoned, which is a
continuation of U.S. patent application Ser. No. 11/172,459, filed
Jun. 30, 2005, now U.S. Pat. No. 7,579,444, which claims the
benefit of priority to U.S. Provisional Patent Application No.
60/584,505, filed Jun. 30, 2004, all of which are incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to conjugates
comprising a Factor IX moiety (i.e., a moiety having Factor IX
activity) and a polymer. In addition, the invention relates to
compositions comprising the conjugates, methods for synthesizing
the conjugates, methods for delivering the conjugates, and methods
for treating patients.
REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM
[0003] A Sequence Listing is being submitted electronically via EFS
in the form of a text file, created May 16, 2016 and named
"0915340434seqlist.txt" (4,458 bytes), the contents of which are
incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0004] Hemostasis is the process of arresting the outflow of blood
from an injured blood vessel. For mammals, as well as many other
organisms, the hemostatic process is critically important for
continued survival. Defects in the hemostatic process can result
in, for example, the inability to effectively form blood clots that
serve to stop the loss of blood following vascular injury. In
humans, individuals who suffer from an inability to form blood
clots are called hemophiliacs. Of particular concern for
hemophiliacs is the life-threatening risk that once started,
bleeding will never cease.
[0005] Generally, hemophiliacs lack the ability to produce
effective amounts of one or more substances ultimately required for
the transformation of soluble fibrinogen into insoluble fibrin. For
example, hemophiliacs who suffer from hemophilia B (also called
"congenital factor IX deficiency" and "Christmas disease") have an
inability to produce effective levels of Factor IX. Factor IX is a
key component of one of several "cascades" of reactions that result
in the formation of blood clots. Critical for the cascade of
reactions referred to as the "intrinsic pathway," Factor IX
ultimately influences the conversion of fibrinogen into the major
component of blood clots, fibrin.
[0006] Although the process by which blood clots are formed is
relatively complex, the role of Factor IX in the intrinsic pathway
can be described briefly. When blood comes into contact with
negatively charged surfaces and/or subendothelial connective
tissues (as a result of, for example, tissue damage associated with
a laceration), Factor XII (or Hageman factor) in the presence of
other substances is transformed into Factor XIIa. Factor XIIa
(along with other substances) transforms Factor XI into Factor XIa.
In turn, Factor XIa (along with other substances) transforms Factor
IX into Factor IXa. Factor VIII, Factor IXa, calcium ions and
phospholipid micelles form a lipoprotein complex with Factor X and
activate it to form Factor Xa. Thereafter, Factor Xa (along with
other substances) converts prothrombin into thrombin, with the
result that a relatively large amount of thrombin is produced over
time. Relatively large amounts of thrombin convert fibrinogen into
fibrin. Fibrin, in turn, forms the matrix or lattice responsible
for the formation of blood clots. Factor IX's role in the intrinsic
pathway of blood clotting is shown schematically in FIG. 13.
[0007] Affecting one out of 34,500 males, hemophilia B can result
from any one of a variety of mutations of the Factor IX gene, which
is located on the X-chromosome. Depending on the particular
mutation, hemophilia B can manifest itself as severe, moderate or
mild. Individuals suffering from the severest forms of hemophilia B
entirely lack the ability to express active forms of Factor IX.
Clinically, individuals affected with hemophilia B suffer from nose
bleeds, easy bruising, joint hemorrhage, and prolonged bleeding
from wounds. Current treatment of hemophilia B involves the
infusion of exogenous Factor IX concentrate collected from human
plasma or prepared via recombinant DNA techniques. Because these
treatments serve only to supplement the lack of effective levels of
Factor IX, individuals suffering from severe forms of hemophilia B
require regular injections (as often as three times a week) of
Factor IX concentrate throughout their lives. Patients suffering
from even more moderate forms of hemophilia B often require
injection of Factor IX concentrate before and/or following surgery
and dental work.
[0008] Several commercial forms of Factor IX concentrates are
available to provide replacement therapy for patients suffering
from hemophilia B. For example, blood-derived Factor IX complex
products (containing other factors) are sold under the BEBULIN
VH.RTM. (Baxter Healthcare, Vienna, Austria), KONYNE 80.RTM. (Bayer
Corporation, Eikhart Ind.), PROFILNINE SD.TM. (Alpha Therapeutic
Corporation, Los Angeles Calif.), and PROPLEX T.RTM. (Baxter
Healthcare, Glendale Calif.) brands. Somewhat more purified forms
of Factor IX products are sold under the ALPHANINE SD.RTM. (Alpha
Therapeutic Corporation, Los Angeles Calif.) and MONONINE.RTM.
(Aventis Behring, Kankakee Ill.) brands. With respect to
recombinantly prepared Factor IX concentrates, one product is
currently available under the BENEFIX.RTM. (Wyeth/Genetics
Institute, Cambridge Mass.) brand.
[0009] Generally, the recombinant source of Factor IX concentrate
is favored over blood-derived sources since the latter involves the
risk of transmitting viruses and/or other diseases. In addition,
purity is often higher with the recombinant source, thereby
avoiding potential problems arising from administering unwanted
blood factors and other proteins generally present in blood-derived
sources.
[0010] Notwithstanding the benefits of administering a
recombinant-based formulation, the processing of recombinant-based
products often requires the presence of certain proteins such as
albumin, which can be present in the final formulation administered
to the patient. As a result, patients who receive such formulations
develop allergic reactions to these foreign proteins. In any event,
both blood-derived and recombinant-based products suffer from the
disadvantage of repeated administration.
[0011] PEGylation, or the attachment of a poly(ethylene glycol)
derivative to a protein, has been described as a means to reduce
immunogenicity as well as a means to prolong a protein's in vivo
half-life. With respect to Factor IX, however, previous approaches
for forming protein-polymer conjugates suffered from several
deficiencies.
[0012] For example, U.S. Pat. No. 5,969,040 describes a process
comprising the step of oxidizing vicinal diols of carbohydrate
moieties in the activation region of Factor IX to form aldehydes.
Following the oxidizing step, the described process includes the
step of covalently attaching one or more non-antigenic polymers
[such as a hydrazine-bearing poly(ethylene glycol) derivative] to
the oxidized carbohydrate moieties. A problem with this approach,
however, is the increased complexity attributed to the additional
steps required to obtain an oxidized form of Factor IX. In
addition, any oxidizers that may remain following the oxidation
step may degrade the polymer associated with the conjugate.
Finally, this approach is limited to conjugation using specific
polymers (i.e., hydrazide-containing polymers) and specific regions
on Factor IX (i.e., vicinal diols of carbohydrate moieties in the
activation region of Factor IX).
[0013] The presence of oxidizers (present either as a result of the
process described in U.S. Pat. No. 5,969,040, or from other causes)
introduces additional challenges with respect to providing an
acceptable pharmaceutical product of a polymer conjugated to Factor
IX. Specifically, methionine and other hydroxyl-containing amino
acids may be subject to unwanted oxidation in the presence of
oxidizers, thereby introducing aldehyde groups. Any residual
aldehydes not reacted with the polymeric reagent will be reactive
and can potentially damage the protein. In order to address this
problem, unreacted aldehydes need to be capped with glycine or
other small molecule to stabilize the protein. In doing so,
however, an analytical problem arises in that for regulatory
purposes, a product should be readily defined; the introduction of
additional components can frustrate otherwise straightforward
product definition. In particular, the use of capping agents would
present a particularly difficult challenge.
[0014] U.S. Pat. No. 6,037,452 describes attachment of a
poly(alkylene oxide) to Factor IX, wherein attachment to Factor IX
is effected through a poly(alkylene oxide) bearing one of the
following reactive groups: triazine, acetyl, hydrazine, diazonium,
amino, and succinimidyl ester. Again, however, the reference lacks
disclosure of effecting attachment through polymers bearing
reactive groups other than triazine, acetyl, hydrazine, diazonium,
amino, or succinimidyl ester.
[0015] Thus, there remains a need in the art to provide additional
conjugates between water-soluble polymers and moieties having
Factor IX activity. In particular, there is a need to provide more
simple processes for conjugating a polymer to a moiety having
Factor IX activity. The present invention is therefore directed to
such conjugates as well as compositions comprising the conjugates
and related methods as described herein, which are believed to be
new and completely unsuggested by the art.
[0016] Accordingly, in one or more embodiments of the invention, a
conjugate is provided, the conjugate comprising a Factor IX moiety
covalently attached, either directly or through a spacer moiety
comprised of one or more atoms, to a water-soluble polymer, wherein
the molecular weight of the water-soluble polymer is greater than
5,000 Daltons and less than about 150,000 Daltons.
[0017] In one or more embodiments of the invention, a conjugate is
provided, the conjugate comprising a Factor IX moiety covalently
attached at an amino acid residue, either directly or through a
spacer moiety comprised of one or more atoms, to a water-soluble
polymer, wherein the amino acid residue is not attached, either
directly or through the spacer moiety, via a --CH.sub.2--C(O)--O--,
--N(H)--C(O)CH.sub.2--O--, --C(O)--N(H)--,
--N(H)--C(O)--CH.sub.2--O--, --C(O)--CH.sub.2--O--,
--C(O)--CH.sub.2--CH.sub.2--C(O)--O--, diazo, or triazine
linkage.
[0018] In one or more embodiments of the invention, a conjugate is
provided, the conjugate comprising a Factor IX moiety covalently
attached, either directly or through a spacer moiety comprising of
one or more atoms to a non-linear water-soluble polymer.
[0019] In one or more embodiments of the invention, a composition
is provided, the composition comprising a plurality of conjugates,
wherein at least about 80% of all conjugates in the composition are
each comprised of a Factor IX moiety covalently attached to one,
two, three or four water-soluble polymers, and further wherein for
each water-soluble polymer in the conjugate, the Factor IX moiety
is attached either directly or through a spacer moiety comprised of
one or more atoms. The compositions encompass all types of
formulations and in particular those that are suited for injection
such as powders that can be reconstituted, as well as liquids
(e.g., suspensions and solutions).
[0020] In one or more embodiments of the invention, a method for
preparing a conjugate is provided, the method comprising adding a
polymeric reagent composition to a Factor IX composition under
conditions sufficient to result in a conjugate comprising a Factor
IX moiety covalently attached, either directly or through a spacer
moiety comprised of one or more atoms, to a water-soluble
polymer.
[0021] In one or more embodiments of the invention, a method for
delivering a conjugate is provided, the method comprising
administering to the patient a composition comprising a conjugate
as described herein. The step of administering the conjugate can be
effected by injection (e.g., intramuscular injection, intravenous
injection, subcutaneous injection, and so forth) or other
approach.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1 and FIG. 2 are copies of gels resulting from sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
analysis of samples described in Examples 1 through 4.
[0023] FIG. 3 is a copy of a gel resulting from SDS-PAGE analysis
of samples described in Examples 5, 6, 10 and 11.
[0024] FIG. 4 is a copy of a gel resulting from SDS-PAGE analysis
of samples described in Examples 2, 9 and 12 through 16.
[0025] FIG. 5 is a plot corresponding to the resulting conjugation
solution of Example 1.
[0026] FIG. 6 is a plot corresponding to the resulting conjugation
solution of Example 2.
[0027] FIG. 7 is a plot corresponding to the resulting conjugation
solution of Example 3.
[0028] FIG. 8 is a plot corresponding to the resulting conjugation
solution of Example 4
[0029] FIG. 9 is a plot corresponding to the resulting conjugation
solution of Example 12.
[0030] FIG. 10 is a plot corresponding to the resulting conjugation
solution of Example 13.
[0031] FIG. 11 is a plot corresponding to the resulting conjugation
solution of Example 14.
[0032] FIG. 12 is a plot corresponding to the resulting conjugation
solution of Example 15.
[0033] FIG. 13 demonstrates the role of Factor IX in the intrinsic
pathway of blood clotting.
DETAILED DESCRIPTION OF THE INVENTION
[0034] Before describing one or more embodiments of the present
invention in detail, it is to be understood that this invention is
not limited to the particular polymers, synthetic techniques,
Factor IX moieties, and the like, as such may vary.
[0035] It must be noted that, as used in this specification and the
claims, the singular forms "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 "an
optional excipient" refers to a single optional excipient as well
as two or more of the same or different optional excipients, and
the like.
[0036] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions described below.
[0037] "PEG," "polyethylene glycol" and "poly(ethylene glycol)" as
used herein, are interchangeable. Typically, PEGs for use in
accordance with the invention comprise the following structure
"--(OCH.sub.2CH.sub.2)--" where (n) is 2 to 4000. As used herein,
PEG also includes
"--CH.sub.2CH.sub.2--O(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--"
and "--(OCH.sub.2CH.sub.2).sub.nO--," depending upon whether or not
the terminal oxygens have been displaced. Throughout the
specification and claims, it should be remembered that the term
"PEG" includes structures having various terminal or "end capping"
groups and so forth. The term "PEG" also means a polymer that
contains a majority, that is to say, greater than 50%, of
--OCH.sub.2CH.sub.2-- repeating subunits. With respect to specific
forms, the PEG can take any number of a variety of molecular
weights, as well as structures or geometries such as "branched,"
"linear," "forked," "multifunctional," and the like, to be
described in greater detail below.
[0038] The terms "end-capped" and "terminally capped" are
interchangeably used herein to refer to a terminal or endpoint of a
polymer having an end-capping moiety. Typically, although not
necessarily, the end-capping moiety comprises a hydroxy or
C.sub.1-20 alkoxy group, more preferably a C.sub.1-10 alkoxy group,
and still more preferably a C.sub.1-5 alkoxy group. Thus, examples
of end-capping moieties include alkoxy (e.g., methoxy, ethoxy and
benzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and
the like. It must be remembered that the end-capping moiety may
include one or more atoms of the terminal monomer in the polymer
[e.g., the end-capping moiety "methoxy" in
CH.sub.3O(CH.sub.2CH.sub.2O).sub.n--] or not [e.g., "CH.sub.3" in
CH.sub.3(OCH.sub.2CH.sub.2).sub.n--] In addition, saturated,
unsaturated, substituted and unsubstituted forms of each of the
foregoing are envisioned. Moreover, the end-capping group can also
be a silane. 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 coupled can be determined by using a suitable detector. Such
labels include, without limitation, fluorescers, chemiluminescers,
moieties used in enzyme labeling, colorimetric moieties (e.g.,
dyes), metal ions, radioactive moieties, and the like. Suitable
detectors include photometers, films, spectrometers, and the like.
The end-capping group can also advantageously comprise a
phospholipid. When the polymer has an end-capping group comprising
a phospholipid, unique properties are imparted to the polymer and
any resulting conjugate. Exemplary phospholipids include, without
limitation, those selected from the class of phospholipids called
phosphatidylcholines. Specific phospholipids include, without
limitation, those selected from the group consisting of
dilauroylphosphatidylcholine, dioleylphosphatidylcholine,
dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine,
behenoylphosphatidylcholine, arachidoylphosphatidylcholine, and
lecithin.
[0039] "Non-naturally occurring" with respect to a polymer as
described herein, means a polymer that in its entirety is not found
in nature. A non-naturally occurring polymer may, however, contain
one or more monomers or segments of monomers that are naturally
occurring, so long as the overall polymer structure is not found in
nature.
[0040] The term "water soluble" as in a "water-soluble polymer" is
any polymer that is soluble in water at room temperature.
Typically, a water-soluble polymer will transmit at least about
75%, more preferably at least about 95%, of light transmitted by
the same solution after filtering. On a weight basis, a
water-soluble polymer will preferably be at least about 35% (by
weight) soluble in water, more preferably at least about 50% (by
weight) soluble in water, still more preferably about 70% (by
weight) soluble in water, and still more preferably about 85% (by
weight) soluble in water. It is most preferred, however, that the
water-soluble polymer is about 95% (by weight) soluble in water or
completely soluble in water.
[0041] Molecular weight in the context of a water-soluble polymer,
such as PEG, can be expressed as either a number-average molecular
weight or a weight-average molecular weight. Unless otherwise
indicated, all references to molecular weight herein refer to the
weight-average molecular weight. Both molecular weight
determinations, number-average and weight-average, can be measured
using gel permeation chromatography or other liquid chromatography
techniques. Other methods for measuring molecular weight values can
also be used, such as the use of end-group analysis or the
measurement of colligative properties (e.g., freezing-point
depression, boiling-point elevation, or osmotic pressure) to
determine number-average molecular weight or the use of light
scattering techniques, ultracentrifugation or viscometry to
determine weight-average molecular weight. The polymers of the
invention are typically polydisperse (i.e., number-average
molecular weight and weight-average molecular weight of the
polymers are not equal), possessing low polydispersity values of
preferably less than about 1.2, more preferably less than about
1.15, still more preferably less than about 1.10, yet still more
preferably less than about 1.05, and most preferably less than
about 1.03. As used herein, references will at times be made to a
single water-soluble polymer having either a weight-average
molecular weight or number-average molecular weight; such
references will be understood to mean that the single-water soluble
polymer was obtained from a composition of water-soluble polymers
having the stated molecular weight.
[0042] The terms "active" or "activated" when used in conjunction
with a particular functional group, refer 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 "non-reactive" or "inert" group).
[0043] As used herein, the term "functional group" or any synonym
thereof is meant to encompass protected forms thereof as well as
unprotected forms.
[0044] The terms "spacer moiety," "linkage" and "linker" are 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 and a Factor IX moiety or an electrophile or
nucleophile of a Factor IX moiety. The spacer moiety may be
hydrolytically stable or may include a physiologically hydrolyzable
or enzymatically degradable linkage.
[0045] "Alkyl" refers to a hydrocarbon chain, typically ranging
from about 1 to 15 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 methyl, ethyl, propyl, butyl,
pentyl, 1-methylbutyl, 1-ethylpropyl, 3-methylpentyl, and the like.
As used herein, "alkyl" includes cycloalkyl as well as
cycloalkylene-containing alkyl.
[0046] "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, and t-butyl.
[0047] "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 carbon atoms. "Cycloalkylene" refers to a
cycloalkyl group that is inserted into an alkyl chain by bonding of
the chain at any two carbons in the cyclic ring system.
[0048] "Alkoxy" refers to an --O--R group, wherein R is alkyl or
substituted alkyl, preferably C.sub.1-6 alkyl (e.g., methoxy,
ethoxy, propyloxy, and so forth).
[0049] The term "substituted" as in, for example, "substituted
alkyl," refers to a moiety (e.g., an alkyl group) substituted with
one or more noninterfering substituents, such as, but not limited
to: alkyl, C.sub.3-8 cycloalkyl, e.g., cyclopropyl, cyclobutyl, and
the like; halo, e.g., fluoro, chloro, bromo, and iodo; cyano;
alkoxy, lower phenyl; substituted phenyl; and the like.
"Substituted aryl" is aryl having one or more noninterfering groups
as a substituent. For substitutions on a phenyl ring, the
substituents may be in any orientation (i.e., ortho, meta, or
para).
[0050] "Noninterfering substituents" are those groups that, when
present in a molecule, are typically non-reactive with other
functional groups contained within the molecule.
[0051] "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.
[0052] "Heteroaryl" is an aryl group containing from one to four
heteroatoms, preferably sulfur, oxygen, or nitrogen, or a
combination thereof. Heteroaryl rings may also be fused with one or
more cyclic hydrocarbon, heterocyclic, aryl, or heteroaryl
rings.
[0053] "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 that is not a
carbon. Preferred heteroatoms include sulfur, oxygen, and
nitrogen.
[0054] "Substituted heteroaryl" is heteroaryl having one or more
noninterfering groups as substituents.
[0055] "Substituted heterocycle" is a heterocycle having one or
more side chains formed from noninterfering substituents.
[0056] An "organic radical" as used herein shall include alkyl,
substituted alkyl, aryl and substituted aryl.
[0057] "Electrophile" and "electrophilic group" refer to an ion or
atom or collection of atoms, that may be ionic, having an
electrophilic center, i.e., a center that is electron seeking,
capable of reacting with a nucleophile.
[0058] "Nucleophile" and "nucelophilic group" refers to an ion or
atom or collection of atoms that may be ionic having a nucleophilic
center, i.e., a center that is seeking an electrophilic center or
with an electrophile.
[0059] A "physiologically cleavable" or "hydrolyzable" bond is a
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, peptides and oligonucleotides.
[0060] An "enzymatically degradable linkage" means a linkage that
is subject to degradation by one or more enzymes.
[0061] 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.
[0062] "Pharmaceutically acceptable excipient" refers to an
excipient that may optionally be included in the compositions of
the invention and that causes no significant adverse toxicological
effects to the patient.
[0063] "Therapeutically effective amount" is used herein to mean
the amount of a polymer-Factor IX moiety conjugate that is needed
to provide a desired level of the conjugate (or corresponding
unconjugated Factor IX moiety) in the bloodstream or in the target
tissue. The precise amount will depend upon numerous factors, e.g.,
the particular Factor IX moiety, the components and physical
characteristics of the therapeutic composition, intended patient
population, mode of delivery, individual patient considerations,
and the like, and can readily be determined by one skilled in the
art, based upon the information provided herein.
[0064] "Multi-functional" means a polymer having three or more
functional groups contained therein, where the functional groups
may be the same or different. Multi-functional polymeric reagents
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.
[0065] The term "Factor IX moiety," as used herein, refers to a
moiety having Factor IX activity. The Factor IX moiety will also
have at least one electrophilic group or nucleophilic group suited
for reaction with a polymeric reagent. Typically, although not
necessarily, the Factor IX moiety is a protein. In addition, the
term "Factor IX moiety" encompasses both the Factor IX moiety prior
to conjugation as well as the Factor IX moiety residue following
conjugation. As will be explained in further detail below, one of
ordinary skill in the art can determine whether any given moiety
has Factor IX activity. A protein comprising an amino acid sequence
corresponding to SEQ ID NO: 1 is a Factor IX moiety, as well as any
protein or polypeptide substantially homologous thereto, whose
biological properties result in the activity of Factor IX. As used
herein, the term "Factor IX moiety" includes proteins modified
deliberately, as for example, by site directed mutagenesis or
accidentally through mutations. The term "Factor IX moiety" also
includes derivatives having from 1 to 6 additional glycosylation
sites, derivatives having at least one additional amino acid at the
carboxy terminal end of the protein wherein the additional amino
acid(s) includes at least one glycosylation site, and derivatives
having an amino acid sequence which includes at least one
glycosylation site.
[0066] The term "substantially homologous" means that a particular
subject sequence, for example, a mutant sequence, varies from a
reference sequence by one or more substitutions, deletions, or
additions, the net effect of which does not result in an adverse
functional dissimilarity between the reference and subject
sequences. For purposes of the present invention, sequences having
greater than 95 percent homology, equivalent biological properties
(although potentiality different degrees of activity), and
equivalent expression characteristics are considered substantially
homologous. For purposes of determining homology, truncation of the
mature sequence should be disregarded. Sequences having lesser
degrees of homology, comparable bioactivity, and equivalent
expression characteristics are considered substantial equivalents.
Exemplary Factor IX moieties for use herein include those proteins
having a sequence that is substantially homologous to SEQ ID NO:
1.
[0067] The term "fragment" means any protein or polypeptide having
the amino acid sequence of a portion of a Factor IX moiety that
retains some degree of Factor IX activity. Fragments include
proteins or polypeptides produced by proteolytic degradation of the
Factor IX protein or produced by chemical synthesis by methods
routine in the art. Determining whether a particular fragment has
the biological activity of Factor IX can carried out by
conventional, well known tests utilized for such purposes on one or
more species of mammals. An appropriate test which can be utilized
to demonstrate such biological activity is described herein.
[0068] A "deletion variant" of a Factor IX moiety is peptide or
protein in which one amino acid residue of the Factor IX moiety has
been deleted and the amino acid residues preceding and following
the deleted amino acid residue are connected via an amide bond
(except in instances where the deleted amino acid residue was
located on a terminus of the peptide or protein). Deletion variants
include instances where only a single amino acid residue has been
deleted, as well as instances where two amino acids are deleted,
three amino acids are deleted, four amino acids are deleted, and so
forth. Each deletion variant must, however, retain some degree of
Factor IX activity.
[0069] A "substitution variant" of a Factor IX moiety is peptide or
protein in which one amino acid residue of the Factor IX moiety has
been deleted and a different amino acid residue has taken its
place. Substitution variants include instances where only a single
amino acid residue has been substituted, as well as instances where
two amino acids are substituted, three amino acids are substituted,
four amino acids are substituted, and so forth. Each substitution
variant must, however, have some degree of Factor IX activity.
[0070] An "addition variant" of a Factor IX moiety is peptide or
protein in which one amino acid residue of the Factor IX moiety has
been added into an amino acid sequence and adjacent amino acid
residues are attached to the added amino acid residue by way of
amide bonds (except in instances where the added amino acid residue
is located on a terminus of the peptide or protein, wherein only a
single amide bond attaches the added amino acid residue). Addition
variants include instances where only a single amino acid residue
has been added, as well as instances where two amino acids are
added, three amino acids are added, four amino acids are added, and
so forth. Each addition variant must, however, have some degree of
Factor IX activity.
[0071] The term "patient," refers to a living organism suffering
from or prone to a condition that can be prevented or treated by
administration of an active agent (e.g., conjugate), and includes
both humans and animals.
[0072] "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.
[0073] "Substantially" (unless specifically defined for a
particular context elsewhere or the context clearly dictates
otherwise) means nearly totally or completely, for instance,
satisfying one or more of the following: greater than 50%, 51% or
greater, 75% or greater, 80% or greater, 90% or greater, and 95% or
greater of the condition.
[0074] Unless the context clearly dictates otherwise, when the term
"about" precedes a numerical value, the numerical value is
understood to mean .+-.10% of the stated numerical value.
[0075] Amino acid residues in peptides are abbreviated as follows:
Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile
or I; Methionine is Met or M; Valine is Val or V; Serine is Ser or
S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A;
Tyrosine is Tyr or Y; Histidine is His or H; Glutarnine is Gln or
Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp
or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan
is Trp or W; Arginine is Arg or R; and Glycine is Gly or G.
[0076] Turning to one or more embodiments of the invention, a
conjugate is provided, the conjugate comprising a Factor IX moiety
covalently attached, either directly or through a spacer moiety
comprised of one or more atoms, to a water-soluble polymer. The
conjugates of the invention will have one or more of the following
features.
The Factor IX Moiety
[0077] As previously stated, the term "Factor IX moiety" shall
include the Factor IX moiety prior to conjugation as well as to the
Factor IX moiety following attachment to a water-soluble polymer.
It is understood, however, that when the Factor IX moiety is
attached to a nonpeptidic water-soluble polymer, the Factor IX
moiety is slightly altered due to the presence of one or more
covalent bonds associated with linkage to the polymer (or spacer
moiety that is attached to the polymer). Often, this slightly
altered form of the Factor IX moiety attached to another molecule
is referred to a "residue" of the Factor IX moiety.
[0078] The Factor IX moiety can be derived from either
non-recombinant methods or from recombinant methods and the
invention is not limited in this regard. In addition, the Factor IX
moiety can be derived from human sources or from animal
sources.
[0079] The Factor IX moiety can be derived non-recombinantly. For
example, the Factor IX moiety can be obtained from blood-derived
sources. In particular, Factor IX can be fractionated from human
plasma using precipitation and centrifugation techniques known to
those of ordinary skill in the art. See, for example, Wickerhauser
(1976) Transfusion 16(4):345-350 and Slichter et al. (1976)
Transfusion 16(6):616-626. Factor IX can also be isolated from
human granulocytes. See Szmitkoski et al. (1977) Haematologia
(Budap.) 11(1-2):177-187.
[0080] The Factor IX moiety can be derived from recombinant
methods. For example, the cDNA coding for native Factor IX, which
is a Factor IX moiety, has been isolated, characterized, and cloned
into expression vectors. See, e.g., Choo et al. (1982) "Molecular
Cloning of the Gene for Human Anti-hemophilic Factor IX," Nature,
Vol. 299: 178-180, and Kurachi et al. (1982) "Isolation and
Characterization of a cDNA Coding for Human Factor IX," Proc. Natl.
Acad. Sci. U.S.A., Vol. 79: 6461-65.
[0081] Once expressed, native Factor IX is a single chain
glycoprotein of about 55,000 Daltons. It can structurally be
considered as having four domains: the Gla or gamma
carboxyglutamate-rich domain; the EGF-like regions; the activation
peptide; and the active site. The expressed amino acid sequence is
provided as SEQ ID NO: 1. Unless specifically noted, all
assignments of a numeric location of an amino acid residue as
provided herein are based on SEQ ID NO: 1.
[0082] Exemplary recombinant methods used to prepare a Factor IX
moiety (whether native Factor IX or a different protein having
Factor IX activity) can be briefly described. Such methods involve
constructing the nucleic acid encoding the desired polypeptide or
fragment, cloning the nucleic acid into an expression vector,
transforming a host cell (e.g., plant, bacteria such as E. coli,
yeast such as Saccharomyces cerevisiae, or mammalian cell such as
Chinese hamster ovary cell or baby hamster kidney cell), and
expressing the nucleic acid to produce the desired polypeptide or
fragment. The expression can occur via exogenous expression (when
the host cell naturally contains the desired genetic coding) or via
endogenous expression. Methods for producing and expressing
recombinant polypeptides in vitro and in prokaryotic and eukaryotic
host cells are known to those of ordinary skill in the art. See,
for example, U.S. Pat. No. 4,868,122.
[0083] To facilitate identification and purification of the
recombinant polypeptide, nucleic acid sequences that encode for an
epitope tag or other affinity binding sequence can be inserted or
added in-frame with the coding sequence, thereby producing a fusion
protein comprised of the desired polypeptide and a polypeptide
suited for binding. Fusion proteins can be identified and purified
by first running a mixture containing the fusion protein through an
affinity column bearing binding moieties (e.g., antibodies)
directed against the epitope tag or other binding sequence in the
fusion proteins, thereby binding the fusion protein within the
column. Thereafter, the fusion protein can be recovered by washing
the column with the appropriate solution (e.g., acid) to release
the bound fusion protein. The recombinant polypeptide can also be
identified and purified by lysing the host cells, separating the
polypeptide, e.g., by size exclusion chromatography, and collecting
the polypeptide. These and other methods for identifying and
purifying recombinant polypeptides are known to those of ordinary
skill in the art. In one or more embodiments of the present
invention, however, it is preferred that the Factor IX moiety is
not in the form of a fusion protein.
[0084] Depending on the system used to express proteins having
Factor IX activity, the Factor IX moiety can be unglycosylated or
glycosylated and either may be used. That is, the Factor IX moiety
can be unglycosylated or the Factor IX moiety can be glycosylated.
In one or more embodiments of the invention, it is preferred that
the Factor IX moiety is glycosylated.
[0085] The moiety having Factor IX activity can advantageously be
modified to include one or more amino acid residues such as, for
example, lysine, cysteine and/or arginine, in order to provide
facile attachment of a polymer to an atom within an amino acid. In
addition, the Factor IX moiety can be modified to include a
non-naturally occurring amino acid residue. Techniques for adding
amino acid residues and non-naturally occurring amino acid residues
are well known to those of ordinary skill in the art. Reference is
made to J. March, Advanced Organic Chemistry: Reactions Mechanisms
and Structure, 4th Ed. (New York: Wiley-Interscience, 1992).
[0086] In addition, the Factor IX moiety can advantageously be
modified to include attachment of a functional group (other than
through addition of a functional group-containing amino acid
residue). For example, the Factor IX moiety can be modified to
include a thiol group. In addition, the Factor IX moiety can be
modified to include an N-terminal alpha carbon. In addition, the
Factor IX moiety can be modified to include one or more
carbohydrate moieties. Factor IX moieties modified to contain an
aminoxy, aldehyde or other functional group can also be used.
[0087] Nonlimiting examples of Factor IX moieties include the
following: Factor IX; Factor IXa; truncated versions of Factor IX;
hybrid proteins, and peptide mimetics having Factor IX activity.
Biologically active fragments, deletion variants, substitution
variants or addition variants of any of the foregoing that maintain
at least some degree of Factor IX activity can also serve as a
Factor IX moiety.
[0088] For any given moiety, it is possible to determine whether
that moiety has Factor IX activity. For example, several animal
lines have been intentionally bred with the genetic mutation for
hemophilia such that an animal produced from such a line has very
low and insufficient levels of Factor IX. Such lines are available
from a variety of sources such as, without limitation, the Division
of Laboratories and Research, New York Department of Public Health,
Albany, N.Y. and the Department of Pathology, University of North
Carolina, Chapel Hill, N.C. Both of these sources, for example,
provide canines suffering from canine hemophilia B. In order to
test the Factor IX activity of any given moiety in question, the
moiety is injected into the diseased animal, a small cut made and
bleeding time compared to a healthy control. Another method useful
for determining Factor IX activity is to determine cofactor and
procoagulant activity. See, for example, Mertens et al. (1993)
Brit. J. Haematol. 85:133-42. Other methods known to those of
ordinary skill in the art can also be used to determine whether a
given moiety has Factor IX activity. Such methods are useful for
determining the Factor IX activity of both a proposed Factor IX
moiety as well as the corresponding polymer-Factor IX moiety
conjugate.
The Water-Soluble Polymer
[0089] As previously discussed, each conjugate comprises a Factor
IX moiety attached to a water-soluble polymer. With respect to the
water-soluble polymer, the water-soluble polymer is nonpeptidic,
nontoxic, non-naturally occurring and biocompatible. With respect
to biocompatibility, a substance is considered biocompatible if the
beneficial effects associated with use of the substance alone or
with another substance (e.g., an active agent such a Factor IX
moiety) in connection with living tissues (e.g., administration to
a patient) outweighs any deleterious effects as evaluated by a
clinician, e.g., a physician. With respect to non-immunogenicity, a
substance is considered nonimmunogenic if the intended use of the
substance in vivo does not produce an undesired immune response
(e.g., the formation of antibodies) or, if an immune response is
produced, that such a response is not deemed clinically significant
or important as evaluated by a clinician. It is particularly
preferred that the water-soluble polymer is biocompatible and
nonimmunogenic.
[0090] Further the polymer is typically characterized as having
from 2 to about 300 termini. Examples of such polymers include, but
are not limited to, poly(alkylene glycols) such as polyethylene
glycol (PEG), poly(propylene glycol) ("PPG"), copolymers of
ethylene glycol and propylene glycol and the like,
poly(oxyethylated polyol), poly(olefinic alcohol),
poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),
poly(hydroxyalkylmethacrylate), poly(saccharides),
poly(.alpha.-hydroxy acid), poly(vinyl alcohol), polyphosphazene,
polyoxazoline, poly(N-acryloylmorpholine), and combinations of any
of the foregoing.
[0091] The polymer is not limited in a particular structure and can
be linear (e.g., alkoxy PEG or bifunctional PEG), or non-linear
such as branched, forked, multi-armed (e.g., PEGs attached to a
polyol core), and dendritic. Moreover, the internal structure of
the polymer can be organized in any number of different patterns
and can be selected from the group consisting of homopolymer,
alternating copolymer, random copolymer, block copolymer,
alternating tripolymer, random tripolymer, and block
tripolymer.
[0092] Typically, activated PEG and other activated water-soluble
polymers (i.e., polymeric reagents) are activated with a suitable
activating group appropriate for coupling to a desired site on the
Factor IX moiety. Thus, a polymeric reagent will possess a reactive
group for reaction with the Factor IX moiety. Representative
polymeric reagents and methods for conjugating these polymers to an
active moiety are known in the art and further described in
Zalipsky, S., et al., "Use of Functionalized Poly(Ethylene Glycols)
for Modification of Polypeptides" in Polyethylene Glycol Chemistry:
Biotechnical and Biomedical Applications, J. M. Harris, Plenus
Press, New York (1992), and in Zalipsky (1995) Advanced Drug
Reviews16:157-182.
[0093] Typically, the weight-average molecular weight of the
water-soluble polymer in the conjugate is from about 100 Daltons to
about 150,000 Daltons. Exemplary ranges, however, include
weight-average molecular weights in the range of greater than 5,000
Daltons to about 100,000 Daltons, in the range of from about 6,000
Daltons to about 90,000 Daltons, in the range of from about 10,000
Daltons to about 85,000 Daltons, in the range of greater than
10,000 Daltons to about 85,000 Daltons, in the range of from about
20,000 Daltons to about 85,000 Daltons, in the range of from about
53,000 Daltons to about 85,000 Daltons, in the range of from about
25,000 Daltons to about 120,000 Daltons, in the range of from about
29,000 Daltons to about 120,000 Daltons, in the range of from about
35,000 Daltons to about 120,000 Daltons, and in the range of from
about 40,000 Daltons to about 120,000 Daltons. For any given
water-soluble polymer, PEGs having a molecular weight in one or
more of these ranges are preferred.
[0094] Exemplary weight-average molecular weights for the
water-soluble polymer include about 100 Daltons, about 200 Daltons,
about 300 Daltons, about 400 Daltons, about 500 Daltons, about 600
Daltons, about 700 Daltons, about 750 Daltons, about 800 Daltons,
about 900 Daltons, about 1,000 Daltons, about 1,500 Daltons, about
2,000 Daltons, about 2,200 Daltons, about 2,500 Daltons, about
3,000 Daltons, about 4,000 Daltons, about 4,400 Daltons, about
4,500 Daltons, about 5,000 Daltons, about 5,500 Daltons, about
6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons, about
8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about
11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about
14,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about
22,500 Daltons, about 25,000 Daltons, about 30,000 Daltons, about
35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about
50,000 Daltons, about 55,000 Daltons, about 60,000 Daltons, about
65,000 Daltons, about 70,000 Daltons, and about 75,000 Daltons.
Branched versions of the water-soluble polymer (e.g., a branched
40,000 Dalton water-soluble polymer comprised of two 20,000 Dalton
polymers) having a total molecular weight of any of the foregoing
can also be used. In one or more embodiments, the conjugate will
not have any PEG moieties attached, either directly or indirectly,
with a PEG having a weight-average molecular weight of less than
about 6,000 Daltons.
[0095] When used as the polymer, PEGs will typically comprise a
number of (OCH.sub.2CH.sub.2) monomers [or (CH.sub.2CH.sub.2O)
monomers, depending on how the PEG is defined]. As used throughout
the description, the number of repeating units is identified by the
subscript "n" in "(OCH.sub.2CH.sub.2).sub.n." Thus, the value of
(n) typically falls within one or more of the following ranges:
from 2 to about 3400, from about 100 to about 2300, from about 100
to about 2270, from about 136 to about 2050, from about 225 to
about 1930, from about 450 to about 1930, from about 1200 to about
1930, from about 568 to about 2727, from about 660 to about 2730,
from about 795 to about 2730, from about 795 to about 2730, from
about 909 to about 2730, and from about 1,200 to about 1,900. For
any given polymer in which the molecular weight is known, it is
possible to determine the number of repeating units (i.e., "n") by
dividing the total weight-average molecular weight of the polymer
by the molecular weight of the repeating monomer.
[0096] With regard to the molecular weight of the water-soluble
polymer, in or more embodiments of the invention, a conjugate is
provided, the conjugate comprising a Factor IX moiety covalently
attached, either directly or through a spacer moiety comprised of
one or more atoms, to a water-soluble polymer, wherein the
molecular weight of the water-soluble polymer is greater than 5,000
Daltons and less than about 150,000 Daltons.
[0097] One particularly preferred polymer for use in the invention
is an end-capped polymer, that is, a polymer having at least one
terminus capped with a relatively inert group, such as a lower
C.sub.1-6 alkoxy group, although a hydroxyl group can also be used.
When the polymer is PEG, for example, it is preferred to use a
methoxy-PEG (commonly referred to as mPEG), which is a linear form
of PEG wherein one terminus of the polymer has a methoxy
(--OCH.sub.3) group, while the other terminus is a hydroxyl or
other functional group that can be optionally chemically
modified.
[0098] In one form useful in the present invention, free or unbound
PEG is a linear polymer terminated at each end with hydroxyl
groups:
HO--CH.sub.2CH.sub.2O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--OH,
wherein (n) typically ranges from zero to about 4,000.
[0099] The above polymer, alpha-, omega-dihydroxylpoly(ethylene
glycol), can be represented in brief form as HO-PEG-OH where it is
understood that the -PEG- symbol can represent the following
structural unit:
--CH.sub.2CH.sub.2O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--,
wherein (n) is as defined as above.
[0100] Another type of PEG useful in the present invention is
methoxy-PEG-OH, or mPEG in brief, in which one terminus is the
relatively inert methoxy group, while the other terminus is a
hydroxyl group. The structure of mPEG is given below.
CH.sub.3O--CH.sub.2CH.sub.2O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.-
2--OH
wherein (n) is as described above.
[0101] Multi-armed or branched PEG molecules, such as those
described in U.S. Pat. No. 5,932,462, can also be used as the PEG
polymer. For example, PEG can have the structure:
##STR00001##
wherein:
[0102] poly.sub.a and poly.sub.b are PEG backbones (either the same
or different), such as methoxy poly(ethylene glycol);
[0103] R'' is a non-reactive moiety, such as H, methyl or a PEG
backbone; and
[0104] P and Q are non-reactive linkages. In a one or more
embodiments, the branched PEG polymer is methoxy poly(ethylene
glycol) disubstituted lysine. Depending on the specific Factor IX
moiety used, the reactive ester functional group of the
disubstituted lysine may be further modified to form a functional
group suitable for reaction with the target group within the Factor
IX moiety.
[0105] In addition, the PEG can comprise a forked PEG. An example
of a forked PEG is represented by the following structure:
##STR00002##
wherein X is a spacer moiety of one or more atoms and each Z is an
activated terminal group linked to CH by a chain of atoms of
defined length. International Application No. PCT/US99/05333,
discloses various forked PEG structures capable of use in one or
more embodiments of the present invention. The chain of atoms
linking the Z functional groups to the branching carbon atom serve
as a tethering group and may comprise, for example, alkyl chains,
ether chains, ester chains, amide chains and combinations
thereof.
[0106] The PEG polymer may comprise a pendant PEG molecule having
reactive groups, such as carboxyl, covalently attached along the
length of the PEG rather than at the end of the PEG chain. The
pendant reactive groups can be attached to the PEG directly or
through a spacer moiety, such as an alkylene group.
[0107] In addition to the above-described forms of PEG, the polymer
can also be prepared with one or more weak or degradable linkages
(such as a hydrolytically degradable linkage) in the polymer,
including any of the above described polymers. For example, PEG can
be prepared with ester linkages in the polymer 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-
[0108] 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. (1997) Polymer
Preprints 38(1):582-3); 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; orthoester linkages that are,
for example, formed by reaction between a formate and an alcohol;
amide linkages formed by an amine group, e.g., at an end of a
polymer such as PEG, and a carboxyl group of another PEG chain;
urethane linkages formed from reaction of, e.g., a PEG with a
terminal isocyanate group and a PEG alcohol; peptide linkages
formed by an amine group, e.g., at an end of a polymer such as PEG,
and a carboxyl group of a peptide; 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.
[0109] Such optional features of the polymer conjugate, i.e., the
introduction of one or more degradable linkages into the polymer
chain, may provide for additional control over the final desired
pharmacological properties of the conjugate upon administration.
For example, a large and relatively inert conjugate (e.g., having
one or more high molecular weight PEG chains attached to a Factor
IX moiety, for example, one or more PEG chains having a molecular
weight greater than about 10,000, wherein the conjugate possesses
essentially no bioactivity) may be administered, which is
hydrolyzed to generate a bioactive conjugate possessing a portion
of the original PEG chain. In this way, the properties of the
conjugate can be more effectively tailored to balance the
bioactivity of the conjugate over time.
[0110] Those of ordinary skill in the art will recognize that the
foregoing discussion concerning substantially water-soluble polymer
segments is by no means exhaustive and is merely illustrative, and
that all polymeric materials having the qualities described above
are contemplated. As used herein, the term "polymeric reagent"
generally refers to an entire molecule, which can comprise a
water-soluble polymer segment and a functional group.
Conjugates
[0111] As described above, a conjugate of the invention comprises a
water-soluble polymer covalently attached (either directly or
through a spacer moiety) to a Factor IX moiety. Typically, for any
given conjugate, there will be one to four water-soluble polymers
covalently attached to a Factor IX moiety (wherein for each
water-soluble polymer, the water soluble polymer can be attached
either directly to the Factor IX moiety or through a spacer
moiety). In some instances, however, the conjugate may have 1, 2,
3, 4, 5, 6, 7, 8 or more water-soluble polymers individually
attached to a Factor IX moiety. In addition, the conjugate may
include not more than 8 water-soluble polymers individually
attached to a Factor IX moiety, not more than 7 water-soluble
polymers individually attached to a Factor IX moiety, not more than
6 water-soluble polymers individually attached to a Factor IX
moiety, not more than 5 water-soluble polymers individually
attached to a Factor IX moiety, not more than 4 water-soluble
polymers individually attached to a Factor IX moiety, not more than
3 water-soluble polymers individually attached to a Factor IX
moiety, and not more than 2 water-soluble polymers individually
attached to a Factor IX moiety.
[0112] The particular linkage between the Factor IX moiety and the
polymer (or the spacer moiety that is attached to the polymer)
depends on a number of factors. Such factors include, for example,
the particular linkage chemistry employed, the particular Factor IX
moiety, the available functional groups within the Factor IX moiety
(either for attachment to a polymer or conversion to a suitable
attachment site), the possible presence of additional reactive
functional groups within the Factor IX moiety, and the like.
[0113] In one or more embodiments of the invention, the linkage
between the Factor IX moiety and the polymer (or the spacer moiety
that is attached to the polymer) is a hydrolytically stable
linkage, such as an amide, urethane (also known as carbamate),
amine, thioether (also known as sulfide), or urea (also known as
carbamide). In one or more embodiments, the linkage does not result
from reaction of the polymeric reagent bearing triazine, acetyl,
hydrazine, diazonium, amino, or succinimidyl ester functional group
with the Factor IX moiety. In some cases, it is preferred that the
linkage is not a carbamate linkage and not a carbamide linkage, and
furthermore, that no linkage is formed based on the reaction of a
polymer derivative bearing an isocyanate or isothiocyanate species
to a Factor IX moiety. Again, a preferred hydrolytically stable
linkage is an amide. An amide can be readily prepared by reaction
of a carboxyl group contained within the Factor IX moiety (e.g.,
the terminal carboxyl of a peptidic moiety having Factor IX
activity) with an amino-terminated polymer.
[0114] In one or more embodiments of the invention, the linkage
between the Factor IX moiety and the polymer (or the spacer moiety
that is attached to the polymer) is a degradable linkage. In this
way, the linkage of the water-soluble polymer (and any spacer
moiety) is "cleavable." That is, the water-soluble polymer (and any
spacer moiety) cleaves (either through hydrolysis, enzymatic
processes, or otherwise), thereby resulting in the native or
unconjugated Factor IX moiety. Preferably, cleavable linkages
result in the polymer (and any spacer moiety) detaching from the
Factor IX moiety in vivo without leaving any fragment of the
water-soluble polymer (and any spacer moiety). Exemplary degradable
linkages include carbonate, carboxylate ester, phosphate ester,
thiolester, anhydrides, acetals, ketals, acyloxyalkyl ether,
imines, and orthoesters. Such linkages can be readily prepared by
appropriate modification of either the Factor IX moiety (e.g., the
carboxyl group C terminus of the protein or a side chain hydroxyl
group of an amino acid such as serine or threonine contained within
the protein) and/or the polymeric reagent using coupling methods
commonly employed in the art. Most preferred, however, are
hydrolyzable linkages that are readily formed by reaction of a
suitably activated polymer with a non-modified functional group
contained within the moiety having Factor IX activity.
[0115] With regard to linkages, in one more embodiments of the
invention, a conjugate is provided, comprising a Factor IX moiety
covalently attached at an amino acid residue, either directly or
through a spacer moiety comprised of one or more atoms, to a
water-soluble polymer, wherein the amino acid residue is not
attached, either directly or through the spacer moiety, via a
CH.sub.2--C(O)--O--, --N(H)--C(O)CH.sub.2--O--, --C(O)--N(H)--,
--N(H)--C(O)--CH.sub.2--O--, --C(O)--CH.sub.2--O--,
--C(O)--CH.sub.2--CH.sub.2--C(O)--O--, diazo, or triazine
linkage.
[0116] The conjugates (as opposed to an unconjugated Factor IX
moiety) may or may not possess a measurable degree of Factor IX
activity. That is to say, a conjugate in accordance with the
invention will possesses anywhere from about 0% to about 100% or
more of the bioactivity of the unmodified parent Factor IX moiety.
Preferably, compounds possessing little or no Factor IX activity
typically contain a hydrolyzable linkage connecting the polymer to
the moiety, so that regardless of the lack of activity in the
conjugate, the active parent molecule (or a derivative thereof
having Factor IX activity) is released upon aqueous-induced
cleavage of the linkage. Such activity may be determined using a
suitable in-vivo or in-vitro model, depending upon the known
activity of the particular moiety having Factor IX activity
employed.
[0117] Optimally, cleavage of each water-soluble polymer portion is
facilitated through the use of physiologically cleavable and/or
enzymatically degradable linkages such as urethane, amide,
carbonate or ester-containing linkages. In this way, clearance of
the conjugate [via cleavage of individual water-soluble polymer(s)]
can be modulated by selecting the polymer molecular size and the
type functional group that would provide the desired clearance
properties. One of ordinary skill in the art can determine the
proper molecular size of the polymer as well as the cleavable
functional group. For example, one of ordinary skill in the art,
using routine experimentation, can determine a proper molecular
size and cleavable functional group by first preparing a variety of
polymer-Factor IX conjugates with different polymer weights and
cleavable functional groups, and then obtaining the clearance
profile for each conjugate by administering the conjugate to a
patient and taking periodic blood and/or urine sampling. Once a
series of clearance profiles have been obtained for each tested
conjugate, a conjugate having the desired clearance can be
identified.
[0118] For conjugates possessing a hydrolytically stable linkage
that couples the Factor IX moiety to the polymer, the conjugate
will typically possess a measurable degree of Factor IX activity.
For instance, such conjugates are typically characterized as having
a bioactivity satisfying one or more of the following percentages
relative to that of the unconjugated Factor IX moiety: at least
about 2%, at least about 5%, at least about 10%, at least about
15%, at least about 25%, at least about 30%, at least about 40%, at
least about 50%, at least about 60%, at least about 80%, at least
about 85%, at least about 90%, at least about 95%, at least about
97%, at least about 100%, and more than 105% (when measured in a
suitable model, such as those presented here and/or well known in
the art). Preferably, conjugates having a hydrolytically stable
linkage (e.g., an amide linkage) will possess at least some degree
of the bioactivity of the unmodified parent Factor IX moiety.
[0119] Exemplary conjugates will now be described. The Factor IX
moiety is expected to share (at least in part) an amino acid
sequence similar or related to native Factor IX. Thus, while
reference will be made to specific locations or atoms within the
native Factor IX protein, such a reference is for convenience only
and one having ordinary skill in the art will be able to readily
determine the corresponding location or atom in other moieties
having Factor IX activity. In particular, the description provided
herein for native Factor IX is often applicable to Factor IXa, as
well as fragments, deletion variants, substitution variants or
addition variants of any of the foregoing.
[0120] Amino groups on Factor IX moieties can provide a point of
attachment between the Factor IX moiety and the water-soluble
polymer. Native Factor IX comprises 27 lysine residues, each having
an .epsilon.-amino group that may be available for conjugation, as
well as one amino terminus. Thus, exemplary attachment points of
such Factor IX moieties include attachment at an amino acid
(through the amine-containing side chain of lysine) at any one or
more of positions 39, 45, 51, 68, 89, 109, 127, 137, 146, 168, 189,
234, 247, 260, 274, 293, 311, 339, 347, 362, 387, 438, 440, 446,
455, 457, and 459. Further, the N-terminal amine of any protein
having Factor IX activity can also serve as a point of
attachment.
[0121] There are a number of examples of suitable water-soluble
polymeric reagents useful for forming covalent linkages with
available amines of a Factor IX moiety. Specific examples, along
with the corresponding conjugates, are provided in Table 1, below.
In the table, the variable (n) represents the number of repeating
monomeric units and "--NH--F9" represents the Factor IX moiety
following conjugation to the water-soluble polymer. While each
polymeric portion [e.g., (OCH.sub.2CH.sub.2).sub.n or
(CH.sub.2CH.sub.2O).sub.n] presented in Table 1 terminates in a
"CH.sub.3" group, other groups (such as H and benzyl) can be
substituted therefor.
TABLE-US-00001 TABLE 1 Amine-Specific Polymeric Reagents and the
Factor IX Moiety Conjugate Formed Therefrom Polymeric Reagent
##STR00003## mPEG-Oxycarbonylimidazole Derivative ##STR00004## mPEG
Nitrophenyl Derivative ##STR00005## mPEG-Trichlorophenyl Carbonates
##STR00006## mPEG-Succinimidyl Derivative ##STR00007##
Homobifunctional PEG-Succinimidyl Derivative ##STR00008##
Heterobifunctional PEG-Succinimidyl Derivative ##STR00009##
mPEG-Succinimidyl Derivative ##STR00010## mPEG-Succinimdyl
Derivative ##STR00011## mPEG Succinimidyl Derivative ##STR00012##
mPEG-Succinimidyl Derivative ##STR00013## mPEG-Benzotriazole
Carbonate Derivative ##STR00014## mPEG-Succinimidyl Derivative
##STR00015## mPEG-Succinimidyl Derivative ##STR00016## mPEG
Succinimidyl Derivative ##STR00017## Branched
mPEG2-N-Hydroxysuccinimide Derivative ##STR00018## Branched
mPEG2-Aldehyde Derivative ##STR00019## mPEG-Succinimidyl Derivative
##STR00020## mPEG-Succinimidyl Derivative ##STR00021##
Homobifunctional PEG-Succinimidyl Derivative ##STR00022##
mPEG-Succinimidyl Derivative ##STR00023## Homobifunctional
PEG-Succinimidyl Propionate Derivative ##STR00024##
mPEG-Succinimidyl Derivative ##STR00025## Branched
mPEG2-N-Hydroxysuccinimide Derivative ##STR00026## Branched
mPEG2-N-Hydroxysuccinimide Derivative ##STR00027## mPEG-Thioester
Derivative ##STR00028## Homobifunctional PEG Propionaldehyde
Derivative ##STR00029## mPEG Propionaldehyde Derivative
##STR00030## Homobifunctional PEG Butyraldehyde Derivative
##STR00031## mPEG Butryaldehyde Derivative ##STR00032## mPEG
Butryaldehyde Derivative ##STR00033## Homobifunctional PEG
Butryaldehyde Derivative ##STR00034## Branched mPEG2 Butyraldehyde
Derivative ##STR00035## Branched mPEG2 Butyraldehyde Derivative
##STR00036## mPEG Acetal Derivative ##STR00037## mPEG Piperidone
Derivative ##STR00038## mPEG Methylketone Derivative ##STR00039##
mPEG tresylate ##STR00040## mPEG Maleimide Derivative (under
certain reaction conditions such as pH >8) ##STR00041## mPEG
Maleimide Derivative (under certain reaction conditions such as pH
>8) ##STR00042## mPEG Maleimide Derivative (under certain
reaction conditions such as pH >8) ##STR00043## mPEG Forked
Maleimide Derivative (under certain reaction conditions such as pH
>8) ##STR00044## branched mPEG2 Maleimide Derivative (under
certain reaction conditions such as pH >8) Corresponding
Conjugate ##STR00045## Carbamate Linkage ##STR00046## Carbamate
Linkage ##STR00047## Carbamate Linkage ##STR00048## Amide Linkage
##STR00049## Amide Linkages ##STR00050## Amide Linkage ##STR00051##
Amide Linkage ##STR00052## Amide Linkage ##STR00053## Amide Linkage
##STR00054## Amide Linkage ##STR00055## Carbamate Linkage
##STR00056## Carbamate Linkage ##STR00057## Amide Linkage
##STR00058## Amide Linkage ##STR00059## Amide Linkage ##STR00060##
Secondary Amine Linkage ##STR00061## Amide Linkage
##STR00062## Amide Linkage ##STR00063## Amide Linkages ##STR00064##
Amide Linkage ##STR00065## Amide Linkages ##STR00066## Amide
Linkage ##STR00067## Amide Linkage ##STR00068## Amide Linkage
##STR00069## Amide Linkage (typically to Factor IX moiety having an
N-terminal cysteine or histidine) ##STR00070## Secondary Amine
Linkages
H.sub.3C--(OCH.sub.2CH.sub.2).sub.n--O--CH.sub.2CH.sub.2--CH.sub.2--NH--F9
Secondary Amine Linkage ##STR00071## Secondary Amine Linkages
H.sub.3C--(OCH.sub.2CH.sub.2).sub.n--O--CH.sub.2CH.sub.2CH.sub.2--CH.sub.2-
--NH--F9 Secondary Amine Linkage ##STR00072## Secondary Amine
Linkage ##STR00073## Secondary Amine Linkages ##STR00074##
Secondary Amine Linkage ##STR00075## Secondary Amine Linkage
H.sub.3C--(OCH.sub.2CH.sub.2).sub.n--O--CH.sub.2CH.sub.2--NH--F9
Secondary Amine Linkage ##STR00076## Secondary Amine Linkage (to a
secondary carbon) ##STR00077## secondary amine linkage (to a
secondary carbon)
H.sub.3CO--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--NH--F9
Secondary Amine Linkage ##STR00078## Secondary Amine Linkage
##STR00079## Secondary Amine Linkage ##STR00080## Secondary Amine
Linkage ##STR00081## Secondary Amine Linkages ##STR00082##
Secondary Amine Linkage
[0122] Conjugation of a polymeric reagent to an amine group of a
Factor IX moiety can be accomplished by a variety of techniques. In
one approach, a Factor IX moiety can be conjugated to a polymeric
reagent functionalized with a succinimidyl derivative (or other
activated ester group, wherein approaches similar to those
described for a succinimidyl derivative can be used for other
activated ester group-containing polymeric reagents). In this
approach, the polymeric reagent bearing a succinimidyl group can be
attached to the Factor IX moiety in aqueous media at a pH of 7.0 to
9.0, although different reaction conditions (e.g., a lower pH such
as 6 to 7, or different temperatures and/or less than 15.degree.
C.) can result in the attachment of a polymer to a different
location on the Factor IX moiety. In addition, an amide linkage can
be formed by reacting an amine-terminated non-peptidic,
water-soluble polymer with a Factor IX moiety bearing an aldehyde
or an activated carboxylic acid group.
[0123] An exemplary conjugate comprises the following structure
##STR00083##
wherein:
[0124] (n) is an integer having a value of from 2 to 3400;
[0125] X is a spacer moiety, preferably one of methylene
("--CH.sub.2--"), ethylene ("--CH.sub.2CH.sub.2--") and propylene
("--CH.sub.2CH.sub.2CH.sub.2--");
[0126] R.sup.1 is an organic radical, preferably H or methyl
("--CH.sub.3"); and
[0127] F9 is a Factor IX moiety.
[0128] Typical of another approach useful for conjugating the
Factor IX moiety to a polymeric reagent is the use of a reductive
amination reaction to conjugate a primary amine of a Factor IX
moiety with a polymer functionalized with a ketone, aldehyde or a
hydrated form thereof (e.g., ketone hydrate and aldehyde hydrate).
In this approach, the primary amine from the Factor IX moiety
reacts with the carbonyl group of the aldehyde or ketone (or the
corresponding hydroxy-containing group of a hydrated aldehyde or
ketone), thereby forming a Schiff base. The Schiff base, in turn,
can then be reductively converted to a stable conjugate through use
of a reducing agent such as sodium borohydride. Selective reactions
(e.g., at the N-terminus are possible) are possible, particularly
with a polymer functionalized with a ketone or an alpha-methyl
branched aldehyde and/or under specific reaction conditions (e.g.,
reduced pH).
[0129] Carboxyl groups represent another functional group that can
serve as a point of attachment on the Factor IX moiety.
Structurally, the conjugate will comprise the following:
##STR00084##
where F9 and the adjacent carbonyl group corresponds to the
carboxyl-containing Factor IX moiety, X is a spacer moiety,
preferably a heteroatom selected from O, N(H), and S, and POLY is a
water-soluble polymer such as PEG, optionally terminating in an
end-capping moiety.
[0130] The C(O)--X linkage results from the reaction between a
polymeric derivative bearing a terminal functional group and a
carboxyl-containing Factor IX moiety. As discussed above, the
specific linkage will depend on the type of functional group
utilized. If the polymer is end-functionalized or "activated" with
a hydroxyl group, the resulting linkage will be a carboxylic acid
ester and X will be O. If the polymer backbone is functionalized
with a thiol group, the resulting linkage will be a thioester and X
will be S. When certain multi-arm, branched or forked polymers are
employed, the C(O)X moiety, and in particular the X moiety, may be
relatively more complex and may include a longer linkage
structure.
[0131] Polymeric reagents containing a hydrazide moiety are also
useful for conjugation at a carbonyl. To the extent that the Factor
IX moiety does not contain a carbonyl moiety, a carbonyl moiety can
be introduced by reducing any carboxylic acids (e.g., the
C-terminal carboxylic acid) and/or by providing glycosylated or
glycated (wherein the added sugars have a carbonyl moiety) versions
of the Factor IX moiety. Specific examples of polymeric reagents
comprising a hydrazide moiety, along with the corresponding
conjugates, are provided in Table 2, below. In addition, any
polymeric reagent comprising an activated ester (e.g., a
succinimidyl group) can be converted to contain a hydrazide moiety
by reacting the polymeric reagent comprising the activated ester
with hydrazine (NH.sub.2--NH.sub.2) or tert-butyl carbazate
[NH.sub.2NHCO.sub.2C(CH.sub.3).sub.3]. In the table, the variable
(n) represents the number of repeating monomeric units and
".dbd.C--F9" represents the Factor IX moiety following conjugation
to the polymeric reagent. Optionally, the hydrazone linkage can be
reduced using a suitable reducing agent. While each polymeric
portion [e.g., (OCH.sub.2CH.sub.2).sub.n or
(CH.sub.2CH.sub.2O).sub.n] presented in Table 1 terminates in a
"CH.sub.3" group, other groups (such as H and benzyl) can be
substituted therefor.
TABLE-US-00002 TABLE 2 Carboxyl-Specific Polymeric Reagents and the
Factor IX Moiety Conjugate Formed Therefrom Polymeric Reagent
##STR00085## mPEG-Hydrazine Derivative ##STR00086## mPEG-Hydrazine
Derivative ##STR00087## mPEG-Hydrazine Derivative ##STR00088##
mPEG-Hydrazine Derivative ##STR00089## mPEG-Hydrazine Derivative
##STR00090## mPEG-Hydrazine Derivative ##STR00091## mPEG-Hydrazine
Derivative ##STR00092## mPEG-Hydrazine Derivative Corresponding
Conjugate ##STR00093## Hydrazone Linkage ##STR00094## Hydrazone
Linkage ##STR00095## Hydrazone Linkage ##STR00096## Hydrazone
Linkage ##STR00097## Hydrazone Linkage ##STR00098## Hydrazone
Linkage ##STR00099## Hydrazone Linkage ##STR00100## Hydrazone
Linkage
[0132] Thiol groups contained within the Factor IX moiety can serve
as effective sites of attachment for the water-soluble polymer. In
particular, cysteine residues provide thiol groups when the Factor
IX moiety is a protein. The thiol groups in such cysteine residues
can be reacted with an activated PEG that is specific for reaction
with thiol groups, e.g., an N-maleimidyl polymer or other
derivative, as described in U.S. Pat. No. 5,739,208 and in
International Patent Publication No. WO 01/62827.
[0133] While not wishing to be bound by theory, it is believed that
all of the cysteine residues within Factor IX participate in
disulfide bonding. As a consequence, conjugation to a cysteine
residue participating in disulfide bonding may disrupt the tertiary
structure of Factor IX and potentially significantly decrease its
overall activity. Thus, to the extent that any particular Factor IX
moiety lacks a thiol group or disruption of disulfide bonds is to
be avoided, it is possible to add a cysteine residue to the Factor
IX moiety using conventional synthetic techniques. See, for
example, the procedure described in International Patent
Publication WO 90/12874 for adding cysteine residues, wherein such
a procedure can be adapted for a Factor IX moiety. In addition,
conventional genetic engineering processes can also be used to
introduce a cysteine residue into the Factor IX moiety.
[0134] Specific examples, along with the corresponding conjugates,
are provided in Table 3, below. In the table, the variable (n)
represents the number of repeating monomeric units and "--S--F9"
represents the Factor IX moiety following conjugation to the
water-soluble polymer. While each polymeric portion [e.g.,
(OCH.sub.2CH.sub.2).sub.n or (CH.sub.2CH.sub.2O).sub.n] presented
in Table 3 terminates in a "CH.sub.3" group, other groups (such as
H and benzyl) can be substituted therefor.
TABLE-US-00003 TABLE 3 Thiol-Specific Polymeric Reagents and the
Factor IX Moiety Conjugate Formed Therefrom Polymeric Reagent
##STR00101## mPEG Maleimide Derivative ##STR00102## mPEG Maleimide
Derivative ##STR00103## mPEG Maleimide Derivative ##STR00104##
Homobifunctional mPEG Maleimide Derivative ##STR00105## mPEG
Maleimide Derivative ##STR00106## mPEG Maleimide Derivative
##STR00107## mPEG Forked Maleimide Derivative ##STR00108## branched
mPEG2 Maleimide Derivative ##STR00109## branched mPEG2 Maleimide
Derivative ##STR00110## Branched mPEG2 Forked Maleimide Derivative
##STR00111## Branched mPEG2 Forked Maleimide Derivative
##STR00112## mPEG Vinyl Sulfone Derivative ##STR00113## mPEG Thiol
Derivative ##STR00114## Homobifunctional PEG Thiol Derivative
##STR00115## mPEG Disulfide Derivative ##STR00116##
Homobifunctional PEG Disulfide Derivative Corresponding Conjugate
##STR00117## Thioether Linkage ##STR00118## Thioether Linkage
##STR00119## Thioether Linkage ##STR00120## Thioether Linkages
##STR00121## Thioether Linkage ##STR00122## Thioether Linkage
##STR00123## Thioether Linkage ##STR00124## Thioether Linkage
##STR00125## Thioether Linkage ##STR00126## Thioether Linkages
##STR00127## Thioether Linkages ##STR00128## Thioether Linkage
##STR00129## Disulfide Linkage ##STR00130## Disulfide Linkages
H.sub.3CO--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--S--
-S--F9 Disulfide Linkage
F9--S--S--CH.sub.2CH.sub.2--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2CH.-
sub.2CH.sub.2--S--S--F9 Disulfide Linkages
[0135] With respect to conjugates formed from water-soluble
polymers bearing one or more maleimide functional groups
(regardless of whether the maleimide reacts with an amine or thiol
group on the Factor IX moiety), the corresponding maleamic acid
form(s) of the water-soluble polymer can also react with the Factor
IX moiety. Under certain conditions (e.g., a pH of about 7-9 and in
the presence of water), the maleimide ring will "open" to form the
corresponding maleamic acid. The maleamic acid, in turn, can react
with an amine or thiol group of a Factor IX moiety. Exemplary
maleamic acid-based reactions are schematically shown below. POLY
represents the water-soluble polymer, and F9 represents the Factor
IX moiety.
##STR00131##
[0136] A representative conjugate in accordance with the invention
can have the following structure:
POLY-L.sub.0,1-C(O)Z--Y--S--S--F9
wherein POLY is a water-soluble polymer, L is an optional linker, Z
is a heteroatom selected from the group consisting of O, NH, and S,
and Y is selected from the group consisting of C.sub.2-10 alkyl,
C.sub.2-10 substituted alkyl, aryl, and substituted aryl, and F9 is
a Factor IX moiety. Polymeric reagents that can be reacted with a
Factor IX moiety and result in this type of conjugate are described
in U.S. Patent Application Publication No. 2005/0014903.
[0137] With respect to polymeric reagents, those described here and
elsewhere can be purchased from commercial sources (e.g., Nektar
Therapeutics, Huntsville Ala.). In addition, methods for preparing
the polymeric reagents are described in the literature.
[0138] The attachment between the Factor IX moiety and
water-soluble polymer can be direct, wherein no intervening atoms
are located between the Factor IX moiety can the polymer, or
indirect, wherein one or more atoms are located between the Factor
IX moiety and polymer. With respect to the indirect attachment, a
"spacer moiety" serves as a link between the Factor IX moiety and
the water-soluble polymer. The one or more atoms making up the
spacer moiety can include one or more of carbon atoms, nitrogen
atoms, sulfur atoms, oxygen atoms, and combinations thereof. The
spacer moiety can comprise an amide, secondary amine, carbamate,
thioether, and/or disulfide group. Nonlimiting examples of specific
spacer moieties include those selected from the group consisting of
--O--, --S--, --C(O)--, --C(O)--NH--, --NH--C(O)--NH--,
--O--C(O)--NH--, --C(S)--, --CH.sub.2--, --CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--, --O--CH.sub.2--,
--CH.sub.2--O--, --O--CH.sub.2--CH.sub.2--,
--CH.sub.2--O--CH.sub.2--, --CH.sub.2--CH.sub.2--O--,
--O--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--O--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--O--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--O--,
--O--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--O--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--O--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--O--,
--C(O)--NH--CH.sub.2--, --C(O)--NH--CH.sub.2--CH.sub.2--,
--CH.sub.2--C(O)--NH--CH.sub.2--, --CH.sub.2--CH.sub.2--C(O)--NH--,
--C(O)--NH--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--,
--C(O)--NH--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--,
--C(O)--O--CH.sub.2--, --CH.sub.2--C(O)--O--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--O--CH.sub.2--,
--C(O)--O--CH.sub.2--CH.sub.2--, --NH--C(O)--CH.sub.2--,
--CH.sub.2--NH--C(O)--CH.sub.2--,
--CH.sub.2--CH.sub.2--NH--C(O)--CH.sub.2--,
--NH--C(O)--CH.sub.2--CH.sub.2--,
--CH.sub.2--NH--C(O)--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--NH--C(O)--CH.sub.2--CH.sub.2--,
--C(O)--NH--CH.sub.2--, --C(O)--NH--CH.sub.2--CH.sub.2--,
--O--C(O)--NH--CH.sub.2--, --O--C(O)--NH--CH.sub.2--CH.sub.2--,
--NH--CH.sub.2--, --NH--CH.sub.2--CH.sub.2--,
--CH.sub.2--NH--CH.sub.2--, --CH.sub.2--CH.sub.2--NH--CH.sub.2--,
--C(O)--CH.sub.2--, --C(O)--CH.sub.2--CH.sub.2--,
--CH.sub.2--C(O)--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--C(O)--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--NH--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--NH--C(O)--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--NH--C(O)--C-
H.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--C(O)--NH--CH.sub.2--CH.sub.2--N-
H--C(O)--CH.sub.2--CH.sub.2--,
--O--C(O)--NH--[CH.sub.2].sub.h--(OCH2CH2).sub.j--, bivalent
cycloalkyl group, --O--, --S--, an amino acid, --N(R.sup.6)--, and
combinations of two or more of any of the foregoing, wherein
R.sup.6 is H or an organic radical selected from the group
consisting of alkyl, substituted alkyl, alkenyl, substituted
alkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl,
(h) is zero to six, and (j) is zero to 20. Other specific spacer
moieties have the following structures:
--C(O)--NH--(CH.sub.2).sub.1-6--NH--C(O)--,
--NH--C(O)--NH--(CH.sub.2).sub.1-6--NH--C(O)--, and
--O--C(O)--NH--(CH.sub.2).sub.1-6--NH--C(O)--, wherein the
subscript values following each methylene indicate the number of
methylenes contained in the structure, e.g., (CH.sub.2).sub.1-6
means that the structure can contain 1, 2, 3, 4, 5 or 6 methylenes.
Additionally, any of the above spacer moieties may further include
an ethylene oxide oligomer chain comprising 1 to 20 ethylene oxide
monomer units [i.e., --(CH.sub.2CH.sub.2O).sub.1-20]. That is, the
ethylene oxide oligomer chain can occur before or after the spacer
moiety, and optionally in between any two atoms of a spacer moiety
comprised of two or more atoms. Also, the oligomer chain would not
be considered part of the spacer moiety if the oligomer is adjacent
to a polymer segment and merely represent an extension of the
polymer segment. The spacer moiety does not include sugars or
carbohydrates and it is preferred that the conjugate lacks
substantially any water-soluble polymers attached directly, or
through a spacer moiety, to a sugar or carbohydrate that, in turn,
is attached to a Factor IX moiety.
[0139] In some instances, the conjugate may only have a single
water-soluble polymer associated with a single Factor IX moiety. As
a consequence, it may be desirous to have the water-soluble polymer
be a non-linear water-soluble polymer (and prepare the conjugate
using a non-linear polymeric reagent). A preferred non-linear
water-soluble polymer is a branched water-soluble polymer, although
multi-branched water-soluble polymers are included. By
incorporating a branched water-soluble polymer, it is possible, for
example, to double the effective molecular weight for each
attachment site as compared to a single polymer.
[0140] Exemplary conjugates of the invention wherein the
water-soluble polymer is in a branched form, include branched forms
comprising a branched polymer and a branched approach comprising
the structure:
##STR00132##
wherein each (n) is independently an integer having a value of from
2 to 3400.
[0141] Exemplary conjugates of the invention comprise the following
structure:
##STR00133##
wherein:
[0142] each (n) is independently an integer having a value of from
2 to 3400;
[0143] X is spacer moiety;
[0144] (b) is an integer having a value 2 through 6;
[0145] (c) is an integer having a value 2 through 6;
[0146] R.sup.2, in each occurrence, is independently H or lower
alkyl; and
[0147] F9 is a Factor IX moiety.
[0148] An exemplary conjugate of the invention comprises the
following structure:
##STR00134##
wherein:
[0149] each (n) is independently an integer having a value of from
2 to 3400; and
[0150] F9 is a Factor IX moiety.
[0151] Another exemplary conjugate of the invention comprises the
following structure:
##STR00135##
wherein:
[0152] each (n) is independently an integer having a value of from
2 to 3400;
[0153] (a) is either zero or one;
[0154] X, when present, is a spacer moiety comprised of one or more
atoms;
[0155] (b') is zero or an integer having a value of one through
ten;
[0156] (c) is an integer having a value of one through ten;
[0157] R.sup.2, in each occurrence, is independently H or an
organic radical;
[0158] R.sup.3, in each occurrence, is independently H or an
organic radical; and
[0159] F9 is a Factor IX moiety.
[0160] An exemplary conjugates of the invention comprises the
following structure:
##STR00136##
wherein:
[0161] each (n) is independently an integer having a value of from
2 to 3400; and
[0162] F9 is a Factor IX moiety.
Compositions
[0163] The conjugates are typically part of a composition.
Generally, the composition comprises a plurality of conjugates,
preferably although not necessarily, each having one, two, three or
four water-soluble polymers separately covalently attached (either
directly or through a spacer moiety) to one Factor IX moiety. The
compositions, however, can also comprise other conjugates having
four, five, six, seven, eight or more polymers attached to any
given moiety having Factor IX activity. In addition, the invention
includes instances wherein the composition comprises a plurality of
conjugates, each conjugate comprising one water-soluble polymer
covalently attached to one Factor IX moiety, as well as
compositions comprising two, three, four, five, six, seven, eight,
or more water-soluble polymers covalently attached to one Factor IX
moiety.
[0164] In one or more embodiments of the invention, a composition
is provided, the composition comprising a plurality of conjugates,
wherein at least about 80% of all conjugates in the composition are
each comprised of a Factor IX moiety covalently attached to one,
two, three or four water-soluble polymers, and further wherein for
each water-soluble polymer in the conjugate, the Factor IX moiety
is attached to the water-soluble polymer either directly or through
a spacer moiety comprised of one or more atoms.
[0165] With respect to the conjugates in the composition, the
composition will typically satisfy one or more of the following
characteristics: at least about 85% of the conjugates in the
composition will have from one to five polymers attached to the
Factor IX moiety; at least about 85% of the conjugates in the
composition will have from one to four polymers attached to the
Factor IX moiety; at least about 85% of the conjugates in the
composition will have from one to three polymers attached to the
Factor IX moiety; at least about 85% of the conjugates in the
composition will have from one to two polymers attached to the
Factor IX moiety; at least about 85% of the conjugates in the
composition will have one polymer attached to the Factor IX moiety
(i.e., be monoPEGylated); at least about 95% of the conjugates in
the composition will have from one to five polymers attached to the
Factor IX moiety; at least about 95% of the conjugates in the
composition will have from one to four polymers attached to the
Factor IX moiety; at least about 95% of the conjugates in the
composition will have from one to three polymers attached to the
Factor IX moiety; at least about 95% of the conjugates in the
composition will have from one to two polymers attached to the
Factor IX moiety; at least about 95% of the conjugates in the
composition will have one polymer attached to the Factor IX moiety
(i.e., be monoPEGylated); at least about 99% of the conjugates in
the composition will have from one to five polymers attached to the
Factor IX moiety; at least about 99% of the conjugates in the
composition will have from one to four polymers attached to the
Factor IX moiety; at least about 99% of the conjugates in the
composition will have from one to three polymers attached to the
Factor IX moiety; at least about 99% of the conjugates in the
composition will have from one to two polymers attached to the
Factor IX moiety; and at least about 99% of the conjugates in the
composition will have one polymer attached to the Factor IX moiety
(i.e., be monoPEGylated).
[0166] In one or more embodiments, it is preferred that the
conjugate-containing composition is free or substantially free of
albumin. It is also preferred that the composition is free or
substantially free of proteins that do not have Factor IX activity.
Thus, it is preferred that the composition is 85%, more preferably
95%, and most preferably 99% free of albumin. Additionally, it is
preferred that the composition is 85%, more preferably 95%, and
most preferably 99% free of any protein that does not have Factor
IX activity. To the extent that albumin is present in the
composition, exemplary compositions of the invention are
substantially free of conjugates comprising a poly(ethylene glycol)
polymer linking a residue of a Factor IX moiety to albumin.
[0167] Control of the desired number of polymers for any given
moiety can be achieved by selecting the proper polymeric reagent,
the ratio of polymeric reagent to the Factor IX moiety,
temperature, pH conditions, and other aspects of the conjugation
reaction. In addition, reduction or elimination of the undesired
conjugates (e.g., those conjugates having four or more attached
polymers) can be achieved through purification means.
[0168] For example, the polymer-Factor IX moiety conjugates can be
purified to obtain/isolate different conjugated species.
Specifically, the product mixture can be purified to obtain an
average of anywhere from one, two, three, four, five or more PEGs
per Factor IX moiety, typically one, two or three PEGs per Factor
IX moiety. The strategy for purification of the final conjugate
reaction mixture will depend upon a number of factors, including,
for example, the molecular weight of the polymeric reagent
employed, the particular Factor IX moiety, the desired dosing
regimen, and the residual activity and in vivo properties of the
individual conjugate(s).
[0169] If desired, conjugates having different molecular weights
can be isolated using gel filtration chromatography and/or ion
exchange chromatography. That is to say, gel filtration
chromatography is used to fractionate differently numbered
polymer-to-Factor IX moiety ratios (e.g., 1-mer, 2-mer, 3-mer, and
so forth, wherein "1-mer" indicates 1 polymer attached to a Factor
IX moiety, "2-mer" indicates two polymers attached to Factor IX
moiety, and so on) on the basis of their differing molecular
weights (where the difference corresponds essentially to the
average molecular weight of the water-soluble polymer portion). For
example, in an exemplary reaction where a 55,000 Dalton protein is
randomly conjugated to a polymeric reagent having a molecular
weight of about 20,000 Daltons, the resulting reaction mixture may
contain unmodified protein (having a molecular weight of about
55,000 Daltons), monoPEGylated protein (or "1-mer") (having a
molecular weight of about 75,000 Daltons), diPEGylated protein (or
2-mer" (having a molecular weight of about 95,000 Daltons), and so
forth.
[0170] While this approach can be used to separate PEG and other
polymer-Factor IX moiety conjugates having different molecular
weights, this approach is generally ineffective for separating
positional isomers having different polymer attachment sites within
the Factor IX moiety. For example, gel filtration chromatography
can be used to separate from each other mixtures of 1-mers, 2-mers,
3-mers, and so forth, although each of the recovered PEG-mer
compositions may contain PEGs attached to different reactive amino
groups (e.g., lysine residues) within Factor IX moiety.
[0171] Gel filtration columns suitable for carrying out this type
of separation include Superdex.TM. and Sephadex.TM. columns
available from Amersham Biosciences (Piscataway, N.J.). Selection
of a particular column will depend upon the desired fractionation
range desired. Elution is generally carried out using a suitable
buffer, such as phosphate, acetate, or the like. The collected
fractions may be analyzed by a number of different methods, for
example, (i) absorbance at 280 nm for protein content, (ii)
dye-based protein analysis using bovine serum albumin as a
standard, (iii) iodine testing for PEG content (Sims et al. (1980)
Anal. Biochem, 107:60-63), (iv) sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS PAGE), followed by staining
with barium iodide, and higher performance liquid
chromatography.
[0172] Separation of positional isomers can be carried out by
reverse phase chromatography using reverse phase-high performance
liquid chromatography (RP-HPLC) methods using for example a C18
column or C3 column (Amersham Biosciences or Vydac) or by ion
exchange chromatography using an ion exchange column, e.g., a
Sepharose.TM. ion exchange column available from Amersham
Biosciences. Either approach can be used to separate polymer-active
agent isomers having the same molecular weight (positional
isomers).
[0173] The compositions are preferably substantially free of
proteins that do not have Factor IX activity. In addition, the
compositions preferably are substantially free of all other
noncovalently attached water-soluble polymers. In some
circumstances, however, the composition can contain a mixture of
polymer-Factor IX moiety conjugates and unconjugated Factor IX.
[0174] Optionally, the composition of the invention further
comprises a phamaceutically acceptable excipient. If desired, the
pharmaceutically acceptable excipient can be added to a conjugate
to form a composition.
[0175] Exemplary excipients include, without limitation, those
selected from the group consisting of carbohydrates, inorganic
salts, antimicrobial agents, antioxidants, surfactants, buffers,
acids, bases, and combinations thereof.
[0176] A carbohydrate such as a sugar, a derivatized sugar such as
an alditol, aldonic acid, an esterified sugar, and/or a sugar
polymer may be present as an excipient. Specific carbohydrate
excipients include, for example: monosaccharides, such as fructose,
maltose, galactose, glucose, D-mannose, sorbose, and the like;
disaccharides, such as lactose, sucrose, trehalose, cellobiose, and
the like; polysaccharides, such as raffinose, melezitose,
maltodextrins, dextrans, starches, and the like; and alditols, such
as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol
(glucitol), pyranosyl sorbitol, myoinositol, and the like.
[0177] The excipient can also include an inorganic salt or buffer
such as citric acid, sodium chloride, potassium chloride, sodium
sulfate, potassium nitrate, sodium phosphate monobasic, sodium
phosphate dibasic, and combinations thereof.
[0178] The composition can also include an antimicrobial agent for
preventing or deterring microbial growth. Nonlimiting examples of
antimicrobial agents suitable for the present invention include
benzalkonium chloride, benzethonium chloride, benzyl alcohol,
cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl
alcohol, phenylmercuric nitrate, thimersol, and combinations
thereof.
[0179] An antioxidant can be present in the composition as well.
Antioxidants are used to prevent oxidation, thereby preventing the
deterioration of the conjugate or other components of the
preparation. Suitable antioxidants for use in the present invention
include, for example, ascorbyl palmitate, butylated hydroxyanisole,
butylated hydroxytoluene, hypophosphorous acid, monothioglycerol,
propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate,
sodium metabisulfite, and combinations thereof.
[0180] A surfactant can be present as an excipient. Exemplary
surfactants include: polysorbates, such as "Tween 20" and "Tween
80," and pluronics such as F68 and F88 (both of which are available
from BASF, Mount Olive, N.J.); sorbitan esters; lipids, such as
phospholipids such as lecithin and other phosphatidylcholines,
phosphatidylethanolamines (although preferably not in liposomal
form), fatty acids and fatty esters; steroids, such as cholesterol;
and chelating agents, such as EDTA, zinc and other such suitable
cations.
[0181] Acids or bases can be present as an excipient in the
composition. Nonlimiting examples of acids that can be used include
those acids selected from the group consisting of hydrochloric
acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic
acid, formic acid, trichloroacetic acid, nitric acid, perchloric
acid, phosphoric acid, sulfuric acid, fumaric acid, and
combinations thereof. Examples of suitable bases include, without
limitation, bases selected from the group consisting of sodium
hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide,
ammonium acetate, potassium acetate, sodium phosphate, potassium
phosphate, sodium citrate, sodium formate, sodium sulfate,
potassium sulfate, potassium fumerate, and combinations
thereof.
[0182] The amount of the conjugate (i.e., the conjugate formed
between the active agent and the polymeric reagent) in the
composition will vary depending on a number of factors, but will
optimally be a therapeutically effective amount when the
composition is stored in a unit dose container (e.g., a vial). In
addition, the pharmaceutical preparation can be housed in a
syringe. A therapeutically effective amount can be determined
experimentally by repeated administration of increasing amounts of
the conjugate in order to determine which amount produces a
clinically desired endpoint.
[0183] The amount of any individual excipient in the composition
will vary depending on the activity of the excipient and particular
needs of the composition. Typically, the optimal amount of any
individual excipient is determined through routine experimentation,
i.e., by preparing compositions containing varying amounts of the
excipient (ranging from low to high), examining the stability and
other parameters, and then determining the range at which optimal
performance is attained with no significant adverse effects.
[0184] Generally, however, the excipient will be present in the
composition in an amount of about 1% to about 99% by weight,
preferably from about 5% to about 98% by weight, more preferably
from about 15 to about 95% by weight of the excipient, with
concentrations less than 30% by weight most preferred.
[0185] These foregoing pharmaceutical excipients along with other
excipients are described in "Remington: The Science & Practice
of Pharmacy", 19.sup.th ed., Williams & Williams, (1995), the
"Physician's Desk Reference", 52.sup.nd ed., Medical Economics,
Montvale, N.J. (1998), and Kibbe, A. H., Handbook of Pharmaceutical
Excipients, 3.sup.rd Edition, American Pharmaceutical Association,
Washington, D.C., 2000.
[0186] The compositions encompass all types of formulations and in
particular those that are suited for injection, e.g., powders or
lyophilates that can be reconstituted as well as liquids. Examples
of suitable diluents for reconstituting solid compositions prior to
injection include bacteriostatic water for injection, dextrose 5%
in water, phosphate-buffered saline, Ringer's solution, saline,
sterile water, deionized water, and combinations thereof. With
respect to liquid pharmaceutical compositions, solutions and
suspensions are envisioned.
[0187] The compositions of the present invention are typically,
although not necessarily, administered via injection and are
therefore generally liquid solutions or suspensions immediately
prior to administration. The pharmaceutical preparation can also
take other forms such as syrups, creams, ointments, tablets,
powders, and the like. Other modes of administration are also
included, such as pulmonary, rectal, transdermal, transmucosal,
oral, intrathecal, subcutaneous, intra-arterial, and so forth.
[0188] The invention also provides a method for delivering a
conjugate as provided herein to a patient suffering from a
condition that is responsive to treatment with conjugate. The
method comprises delivering, generally via injection, a
therapeutically effective amount of the conjugate (preferably
provided as part of a pharmaceutical composition). The conjugates
(typically as part of a pharmaceutical composition) can be
delivered by, for example, intravenous injection, intramuscular
injection, subcutaneous injection, and so forth. Suitable
formulation types for parenteral administration include
ready-for-injection solutions, dry powders for combination with a
solvent prior to use, suspensions ready for injection, dry
insoluble compositions for combination with a vehicle prior to use,
and emulsions and liquid concentrates for dilution prior to
administration, among others.
[0189] The method of delivering may be used to treat a patient
having a condition that can be remedied or prevented by
administration of the conjugate. Those of ordinary skill in the art
appreciate which conditions a specific conjugate can effectively
treat. For example, the conjugates can be used to treat individuals
suffering from hemophilia B, either as a replacement therapy or on
a prophylaxis basis. Administration of the conjugate for
prophylaxis includes situations where a patient suffering from
hemophilia B is about to undergo surgery and the conjugate is
administered between one to four hours prior to surgery. In
addition, the conjugates are suited for use as a prophylactic
against uncontrolled bleeding, optionally in patients not suffering
from hemophilia. Thus, for example, the conjugate can be
administered to a patient at risk for uncontrolled bleeding prior
to surgery.
[0190] The actual dose to be administered will vary depend upon the
age, weight, and general condition of the subject as well as the
severity of the condition being treated, the judgment of the health
care professional, and conjugate being administered.
Therapeutically effective amounts are known to those skilled in the
art and/or are described in the pertinent reference texts and
literature. Generally, on a weight basis, a therapeutically
effective amount will range from about 0.001 mg to 100 mg,
preferably in doses from 0.01 mg/day to 75 mg/day, and more
preferably in doses from 0.10 mg/day to 50 mg/day. On an activity
basis, corresponding doses based on international units of activity
can be calculated by one of ordinary skill in the art.
[0191] The unit dosage of any given conjugate (again, preferably
provided as part of a pharmaceutical composition) can be
administered in a variety of dosing schedules depending on the
judgment of the clinician, needs of the patient, and so forth. The
specific dosing schedule will be known by those of ordinary skill
in the art or can be determined experimentally using routine
methods. Exemplary dosing schedules include, without limitation,
administration five times a day, four times a day, three times a
day, twice daily, once daily, three times weekly, twice weekly,
once weekly, twice monthly, once monthly, and any combination
thereof. Once the clinical endpoint has been achieved, dosing of
the composition is halted.
[0192] It is to be understood that while the invention has been
described in conjunction with the preferred specific embodiments
thereof, that the foregoing description as well as the examples
that follow are intended to illustrate and not limit the scope of
the invention. Other aspects, advantages and modifications within
the scope of the invention will be apparent to those skilled in the
art to which the invention pertains.
[0193] All articles, books, patents and other publications
referenced herein are hereby incorporated by reference in their
entireties.
EXPERIMENTAL
[0194] The practice of the invention will employ, unless otherwise
indicated, conventional techniques of organic synthesis and the
like, which are within the skill of the art. Such techniques are
fully explained in the literature. Reagents and materials are
commercially available unless specifically stated to the contrary.
See, for example, J. March, Advanced Organic Chemistry: Reactions
Mechanisms and Structure, 4th Ed. (New York: Wiley-Interscience,
1992), supra.
[0195] In the following examples, efforts have been made to ensure
accuracy with respect to numbers used (e.g., amounts, temperatures,
etc.) but some experimental error and deviation should be accounted
for. Unless indicated otherwise, temperature is in degrees C. and
pressure is at or near atmospheric pressure at sea level.
[0196] Although other abbreviations known by one having ordinary
skill in the art will be referenced, other reagents and materials
will be used, and other methods known by one having ordinary skill
in the art will be used, the following list and methods description
is provided for the sake of convenience.
[0197] NaCNBH.sub.3 sodium cyanoborohydride, 95% (Aldrich)
[0198] HCl hydrochloric acid, glacial (Fisher)
[0199] K or kDa kilodaltons
[0200] Acetonitrile (Fisher Optima)
[0201] TFA Trifluoroacetic acid, HPLC grade (JT Baker)
[0202] PBS Phosphate buffered saline (Sigma)
[0203] SEC Size exclusion chromatography
[0204] HPLC high performance liquid chromatography
[0205] SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel
electrophoresis
[0206] HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]
biotechnology performance certified, 99.5+% (Sigma)
[0207] Ethyl alcohol, USP, Absolute-200 Proof (AAPER)
[0208] NuPAGE.RTM. MES [2-(N-morpholino)ethane sulfonic acid] SDS
running buffer (Invitrogen Corporation, Carlsbad Calif.)
[0209] NuPAGE.RTM. 4.times. LDS (lithium docecyl sulfate) sample
buffer (Invitrogen Corporation, Carlsbad Calif.)
[0210] SigmaMarker, low range (M.W. 6,500-66,000) (Sigma)
[0211] SigmaMarker, high range (M.W. 36,000-205,000) (Sigma)
[0212] NuPAGE.RTM. Novex Bis-Tris
[Bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane-HCl] gel
(Invitrogen Corporation, Carlsbad Calif.)
SEC-HPLC Analysis
[0213] Size exclusion chromatography (SEC) was performed on an
Agilent 1100 HPLC system (Agilent). For those samples analyzed
using SEC-HPLC, each sample was analyzed using a SHODEX protein
KW-804 column (Showa Denko KK, Tokyo Japan), at pH 7.2. The flow
rate for the column was set at 0.5 mL/minute. Eluted protein and
PEG-protein conjugates were detected using an UV-based approach
having a wavelength set at 280 nm.
SDS-PAGE Analysis
[0214] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) was performed using an XCELL SURELOCK Mini-Cell
electrophoresis system (Invitrogen Corporation, Carlsbad Calif.).
For those samples analyzed using SDS-PAGE, each sample was mixed
with 4.times. LDS Sample Buffer (Invitrogen Corporation, Carlsbad
Calif.). The prepared samples were then loaded onto a NuPAGE Novex
4-12% Bis-Tris gel and run for approximately thirty minutes at 200
V using NuPAGE.RTM. MES running buffer (Invitrogen Corporation,
Carlsbad Calif.).
RP-HPLC Analysis
[0215] Reverse phase-high performance liquid chromatography was
performed using a C3 reverse column (Hamilton, Zorbax). A 30-80%
gradient of acetonitrile was used along with an elevated
temperature over thirty minutes at 0.5 mL/minute.
[0216] Recombinant Factor IX corresponding to the amino acid
sequence of SEQ. ID. NO. 1. was used in Examples 1-16. Factor IX
was obtained in a buffer containing both L-histidine and glycine.
Because the amine groups associated with L-histidine and glycine in
the buffer would compete for amine groups associated with Factor
IX, it was necessary to exchange the amine-containing buffer for an
amine-free buffer to improve the Factor IX conjugation yield when
amine-directed polymeric reagents were used to effect
conjugation.
[0217] Briefly, the amine-containing buffer was exchanged for an
amine-free buffer by one of two approaches, depending on the volume
of buffer to be exchanged. For relatively small volumes of buffer,
a 500 .mu.L Zeba Desalt centrifuge column (Pierce Biotechnology,
Rockford Ill.) was used according to the protocol provided by the
manufacturer. For relatively large volumes of buffer, a 2 mL
CENTRICON.RTM. centrifugal filter device (Millipore Corporation,
Billerica Mass.) with a 10,000 or 30,000 Dalton molecular weight
cutoff was used according to the protocol provided by the
manufacturer. All samples used in the Examples without ethanol were
changed to a 1.times. PBS buffer having a pH of 7.5, while all
samples used in the Examples with ethanol were exchanged to a
1.times. PBS buffer having a pH of 7.5 with ethanol added to form a
10% ethanol-containing Factor IX reaction mixture.
[0218] The amine-free buffer containing recombinant Factor IX
corresponding to the amino acid sequence of SEQ. ID. NO. 1. (the
"Factor IX stock solution") was used in Examples 1-16. The Factor
IX stock solution contained about 0.2 mg/mL to 0.55 mg/mL of Factor
IX.
EXAMPLE 1
PEGylation of Factor IX with mPEG-SMB, 30 kDa
1:1 Polymer to Factor IX Ratio; Without Ethanol
##STR00137##
[0220] mPEG-SMB, 30 kDa, stored at -20.degree. C. under argon, was
warmed to ambient temperature. The warmed mPEG-SMB (4.1 mg) was
dissolved in 1 mL of 2 mM HCl to form an mPEG-SMB solution. The
mPEG-SMB solution was added to an aliquot of the Factor IX stock
solution containing 0.07 mg of Factor IX until a one:one molar
ratio of mPEG-SMB relative to Factor IX was reached. After the
addition of the mPEG-SMB, the pH of the reaction was tested to
ensure a pH of 7.2. to 7.5, and mixed well. To allow for coupling
of the mPEG-SMB to Factor IX via an amide linkage, the reaction
solution was stirred for three hours at room temperature, after
which SDS PAGE was run on the sample, which confirmed the presence
of monoconjugated material ("1-mer"). See the lane labeled as "1:1
30K SMB" in the gel provided as FIG. 1. Thereafter, coupling was
allowed to continue by stirring the reaction solution for fifteen
hours at 4.degree. C., thereby resulting in a conjugate
solution.
[0221] RP-HPLC (C.sub.3) and a second SDS PAGE were used for the
characterization of the resulting conjugate solution. Based on the
second SDS PAGE result, conjugation was shown. See the lane labeled
as "1:1 30K SMB" in the gel provided as FIG. 2. RP-HPLC (C.sub.3)
was used to separate the components of the resulting conjugate
solution and the resulting chromatogram indicated a yield of 0.54%
(representing 100% monoPEGyled or "1-mer" species). See the
chromatagram provided as FIG. 5.
[0222] It is expected that longer reactions times, increase
temperatures and/or multiple additions of the polymeric reagent
could increase yields. Using this same approach, other conjugates
can be prepared using mPEG-SMB having other weight-average
molecular weights.
EXAMPLE 2
PEGylation of Factor IX with mPEG-SMB, 30 kDa
10:1 Polymer to Factor IX Ratio; Without Ethanol
##STR00138##
[0224] mPEG-SMB, 30 kDa, stored at -20.degree. C. under argon, was
warmed to ambient temperature. The warmed mPEG-SMB (4.1 mg) was
dissolved in 1 mL of 2 mM to form an mPEG-SMB solution. The
mPEG-SMB solution was added to an aliquot of the Factor IX stock
solution containing 0.07 mg of Factor IX until a ten molar excess
of mPEG-SMB relative to Factor IX was reached. After the addition
of the mPEG-SMB, the pH of the reaction mixture was tested to
ensure a pH of 7.2 to 7.5, and mixed well. To allow for coupling of
the mPEG-SMB to Factor IX via an amide linkage, the reaction
solution was stirred for three hours at room temperature, after
which SDS PAGE was run on the sample, which confirmed the presence
of monoconjugated material ("1-mer"). See the lane labeled as "10:1
30K SMB" in the gel provided as FIG. 1. Thereafter, coupling was
allowed to continue by stirring the reaction solution for fifteen
hours at 4.degree. C., thereby resulting in a conjugate
solution.
[0225] RP-HPLC (C.sub.3) and a second SDS PAGE were used for the
characterization of the resulting conjugate solution. Based on the
second SDS PAGE results, conjugation was shown. See the lane
labeled as "10:1 30K SMB" in the gel provided as FIG. 2. RP-HPLC
(C.sub.3) was used to separate the components of the resulting
conjugate solution and the resulting chromatogram indicated a yield
of 6.4% (representing 100% monoPEGyled or "1-mer" species). See the
chromatagram provided as FIG. 6.
[0226] It is expected that longer reactions times, increased
temperatures and/or multiple additions of the polymeric reagent
could increase yields. Thus, when this experiment was repeated for
an extended time at room temperature prior to continuing the
reaction overnight at 4.degree. C., an increase conjugate yield
resulted as evidenced by a darker band as seen in an SDS PAGE gel.
See the lane labeled as "10:1 30K SMB" in FIG. 4. Using the same
approaches described here, other conjugates can be prepared using
mPEG-SMB having other weight-average molecular weights.
EXAMPLE 3
PEGylation of Factor IX with Branched mPEG2-N-Hydroxysuccinimide,
40 kDa
1:1 Polymer to Factor IX Ratio; Without Ethanol
##STR00139##
[0228] Branched mPEG2-N-hydroxysuccinimide, 40 kDa, stored at
-20.degree. C. under argon, was warmed to ambient temperature. The
warmed branched mPEG2-N-hydroxysuccinimide (2.0 mg) was dissolved
in 1 mL of 2 mM HCl to form a branched mPEG2-N-hydroxysuccinimide
solution. The branched mPEG2-N-hydroxysuccinimide solution was
added to an aliquot of the Factor IX stock solution containing 0.07
mg of Factor IX until a one:one molar ratio of branched
mPEG2-N-hydroxysuccinimide relative to Factor IX was reached. After
the addition of branched mPEG2-N-hydroxysuccinimide, the pH of the
reaction mixture was tested to ensure a pH of 7.2 to 7.5, and mixed
well. To allow for coupling of the branched
mPEG2-N-hydroxysuccinimide to Factor IX via an amide linkage, the
reaction solution was stirred for three hours at room temperature,
after which SDS PAGE was run on the sample, which showed no
detectable conjugation. See the lane labeled as "1:1 40K NHS" in
the gel provided as FIG. 1. Thereafter, addition time for
conjugation was provided by stirring the reaction solution for
fifteen hours at 4.degree. C., thereby resulting in a conjugate
solution.
[0229] RP-HPLC (C.sub.3) and a second SIDS PAGE were used for the
characterization of the resulting conjugate solution. Based on the
second SDS PAGE results, conjugation was shown. See the lane
labeled as "1:1 40K NHS" in the gel provided as FIG. 2. RP-HPLC
(C.sub.3) was used to separate the components of the resulting
conjugate solution and the resulting chromatogram indicated a yield
of 0.1% (representing 100% monoPEGyled or "1-mer" species). See the
chromatagram provided as FIG. 7.
[0230] It is expected that longer reactions times, increased
temperatures and/or multiple additions of the polymeric reagent
could increase yields. Using this same approach, other conjugates
can be prepared using branched mPEG2-N-hydroxysuccinimide having
other weight-average molecular weights.
EXAMPLE 4
PEGylation of Factor IX with Branched mPEG2-N-Hydroxysuccinimide,
40 kDa
10:1 Polymer to Factor IX Ratio; Without Ethanol
##STR00140##
[0232] Branched mPEG2-N-hydroxysuccinimide, 40 kDa, stored at
-20.degree. C. under argon, was warmed to ambient temperature. The
warmed branched mPEG2-N-hydroxysuccinimide (2.0 mg) was dissolved
in 1 mL of 2 mM HCl to form a branched mPEG2-N-hydroxysuccinimide
solution. The branched mPEG2-N-hydroxysuccinimide solution was
added to an aliquot of the Factor IX stock solution containing 0.07
mg of Factor IX until a ten molar excess of branched
mPEG2-N-hydroxysuccinimide relative to Factor IX was reached. After
the addition of branched mPEG2-N-hydroxysuccinimide, the pH of the
reaction mixture was tested to ensure a pH of 7.2 to 7.5, and mixed
well. To allow for coupling of the branched
mPEG2-N-hydroxysuccinimide to Factor IX via an amide linkage, the
reaction solution was stirred for three hours at room temperature,
after which SDS PAGE was run on the sample, which showed no
detectable conjugation. See the lane labeled as 10:1 40 k NHS'' in
the gel provided as FIG. 1. Thereafter, coupling was allowed to
continue by stirring the reaction solution for fifteen hours at
4.degree. C., thereby resulting in a conjugate solution.
[0233] RP-HPLC (C.sub.3) and a second SDS PAGE were used for the
characterization of the resulting conjugate solution. Based on the
second SDS PAGE results, conjugation was still not detectable. See
the lane labeled as "10:1 40K NHS" in the gel provided as FIG. 2.
RP-HPLC (C.sub.3) was used to separate the components of the
resulting conjugate solution and the resulting chromatogram
indicated no detectable conjugate yield. See the chromatagram
provided as FIG. 8.
[0234] It is expected that longer reactions times, increased
temperatures and/or multiple additions of the polymeric reagent
could increase yields. Using this same approach, other conjugates
can be prepared using branched mPEG2-N-hydroxysuccinimide having
other weight-average molecular weights.
EXAMPLE 5
PEGylation of Factor IX with mPEG-SMB, 30 kDa
10:1 Polymer to Factor IX Ratio; with Ethanol
##STR00141##
[0236] As ethanol is believed to increase the structural
flexibility of certain proteins, ethanol was introduced into the
buffer and reaction system. mPEG-SMB, 30 kDa, stored at -20.degree.
C. under argon, was warmed to ambient temperature. The warmed
mPEG-SMB (10.0 mg) was dissolved in 0.5 mL of 2 mM HCl with ethanol
added to form a 10% ethanol-containing mPEG-SMB solution. The 10%
ethanol-containing mPEG-SMB solution was added to the 10%
ethanol-containing Factor IX reaction mixture until a ten molar
excess of mPEG-SMB relative to Factor IX was reached. After the
addition of the mPEG-SMB, the pH of the reaction mixture was tested
to ensure a pH of 7.2 to 7.5, and mixed well. To allow for coupling
of the mPEG-SMB to Factor IX via an amide linkage, the reaction
solution was stirred for three hours at room temperature. Coupling
was allowed to continue by stirring the reaction solution overnight
at 4.degree. C., thereby resulting in a conjugate solution.
[0237] SDS PAGE was used for the characterization of the resulting
conjugate solution. Based on the SDS PAGE results, conjugation was
not detected. See the lane labeled as "10:1 30 K SMB+EtOH" in the
gel provided as FIG. 3. It is now believed that the introduction of
ethanol does not increase the structural flexibility of Factor IX
to allow for increased conjugation of mPEG-SMB, 30 kDa.
[0238] It is expected that longer reactions times, increased
temperatures and/or multiple additions of the polymeric reagent
could increase yields. Using this same approach, other conjugates
can be prepared using mPEG-SMB having other weight-average
molecular weights.
EXAMPLE 6
PEGylation of Factor IX with mPEG-SMB, 30 kDa
20:1 Polymer to Factor IX Ratio; with Ethanol
##STR00142##
[0240] As ethanol is believed to increase the structural
flexibility of certain proteins, ethanol was introduced into the
buffer and reaction system. mPEG-SMB, 30 kDa, stored at -20.degree.
C. under argon, was warmed to ambient temperature. The warmed
mPEG-SMB (10.0 mg) was dissolved in 0.5 mL of 2 mM HCl with ethanol
added to form a 10% ethanol-containing mPEG-SMB solution. The 10%
ethanol-containing mPEG-SMB solution was added to the 10%
ethanol-containing Factor IX reaction mixture until a twenty molar
excess of mPEG-SMB relative to Factor IX was reached. After the
addition of the mPEG-SMB, the pH of the reaction mixture was tested
to ensure a pH of 7.2 to 7.5, and mixed well. To allow for coupling
of the mPEG -SMB to Factor IX via an amide linkage, the reaction
solution was stirred for three hours at room temperature. Coupling
was allowed to continue by stirring the reaction solution overnight
at 4.degree. C., thereby resulting in a conjugate solution.
[0241] SDS PAGE was used for the characterization of the resulting
conjugate solution. Based on the SDS PAGE results, conjugation was
not detected. See the lane labeled as "20:1 30 K SMB+EtOH" in the
gel provided as FIG. 3. It is now believed that the introduction of
ethanol does not increase the structural flexibility of Factor IX
to allow for increased conjugation of mPEG-SMB, 30 kDa.
[0242] It is expected that longer reactions times, increased
temperatures and/or multiple additions of the polymeric reagent
could increase yields. Using this same approach, other conjugates
can be prepared using mPEG-SMB having other weight-average
molecular weights.
[0243] EXAMPLE 7
PEGylation of Factor IX with Branched mPEG2-N-Hydroxysuccinimide,
40 kDa
10:1 Polymer to Factor IX Ratio; with Ethanol
##STR00143##
[0245] As ethanol is believed to increase the structural
flexibility of certain proteins, ethanol was introduced into the
buffer and reaction system. Branched mPEG2-N-hydroxysuccinimide, 40
kDa, stored at -20.degree. C. under argon, was warmed to ambient
temperature. The warmed branched mPEG2-N-hydroxysuccinimide (2.0
mg) was dissolved in 1.0 mL of 2 mM HCl with ethanol added to form
a 10% ethanol-containing branched mPEG2-N-hydroxysuccinimide
solution. The 10% ethanol-containing branched
mPEG2-N-hydroxysuccinimide solution was added to the 10%
ethanol-containing Factor IX reaction mixture until a ten molar
excess of branched mPEG2-N-hydroxysuccinimide relative to Factor IX
was reached. After the addition of branched
mPEG2-N-hydroxysuccinimide, the pH of the reaction mixture was
tested to ensure a pH of 7.2 to 7.5, and mixed well. To allow for
coupling of the branched mPEG2-N-hydroxysuccinimide to Factor IX
via an amide linkage, the reaction solution was stirred for three
hours at room temperature. Coupling was allowed to continue by
stirring the reaction solution overnight at 4.degree. C., thereby
resulting in a conjugate solution.
[0246] SDS PAGE was used for the characterization of the resulting
conjugate solution. Based on the SDS PAGE results, conjugation was
not detected (results not shown). It is now believed that the
introduction of ethanol does not increase the structural
flexibility of Factor IX to allow for increased conjugation of
branched mPEG2-N-hydroxysuccinimide, 40 kDa.
[0247] It is expected that longer reactions times, increased
temperatures and/or multiple additions of the polymeric reagent
could increase yields. Using this same approach, other conjugates
can be prepared using branched mPEG2-N-hydroxysuccinimide having
other weight-average molecular weights.
EXAMPLE 8
PEGylation of Factor IX with Branched mPEG2-N-Hydroxysuccinimide,
40 kDa
20:1 Polymer to Factor IX Ratio; with Ethanol
##STR00144##
[0249] As ethanol is believed to increase the structural
flexibility of certain proteins, ethanol was introduced into the
buffer and reaction system. Branched mPEG2-N-hydroxysuccinimide, 40
kDa, stored at -20.degree. C. under argon, was warmed to ambient
temperature. The warmed branched mPEG2-N-hydroxysuccinimide (2.0
mg) was dissolved in 1.0 mL of 2 mM HCl with ethanol added to form
a 10% ethanol-containing branched mPEG2-N-hydroxysuccinimide
solution. The 10% ethanol-containing branched
mPEG2-N-hydroxysuccinimide solution was added to the 10%
ethanol-containing Factor IX reaction mixture until a twenty molar
excess of branched mPEG2-N-hydroxysuccinimide relative to Factor IX
was reached. After the addition of branched
mPEG2-N-hydroxysuccinimide, the pH of the reaction mixture was
tested to ensure a pH of 7.2 to 7.5, and mixed well. To allow for
coupling of the branched mPEG2-N-hydroxysuccinimide to Factor IX
via an amide linkage, the reaction solution was stirred for three
hours at room temperature. Coupling was allowed to continue by
stirring the reaction solution overnight at 4.degree. C., thereby
resulting in a conjugate solution.
[0250] SDS PAGE was used for the characterization of the resulting
conjugate solution. Based on the SDS PAGE results, conjugation was
not detected (results not shown). It is now believed that the
introduction of ethanol does not increase the structural
flexibility of Factor IX to allow for increased conjugation of
branched mPEG2-N-hydroxysuccinimide, 40 kDa.
[0251] It is expected that longer reactions times, increased
temperatures and/or multiple additions of the polymeric reagent
could increase yields. Using this same approach, other conjugates
can be prepared using branched mPEG2-N-hydroxysuccinimide having
other weight-average molecular weights.
EXAMPLE 9
PEGylation of Factor IX with mPEG-SMB, 30 kDa
20:1 Polymer to Factor IX Ratio; without Ethanol
##STR00145##
[0253] mPEG-SMB, 30 kDa, stored at -20.degree. C. under argon, was
warmed to ambient temperature. The warmed mPEG-SMB (8.6 mg) was
dissolved in 1.0 mL of 2 mM HCl to form an mPEG-SMB solution. The
mPEG-SMB solution was added to an aliquot of the Factor IX stock
solution containing 0.07 mg of Factor IX until a twenty molar
excess of mPEG-SMB relative to Factor IX was reached. After the
addition of the mPEG-SMB, the pH of the reaction mixture was tested
to ensure a pH of 7.2 to 7.5. To allow for coupling of the mPEG-SMB
to Factor IX via an amide linkage, the reaction solution was
stirred for two hours at room temperature. Coupling was allowed to
continue by stirring the reaction solution overnight at 4.degree.
C., thereby resulting in a conjugate solution. Thereafter, coupling
was allowed to continue by stirring overnight at 4.degree. C.,
thereby resulting in a conjugate solution.
[0254] SDS PAGE was used for the characterization. Based on the
second SDS PAGE results, conjugation was shown. See the lane
labeled as "20:1 30 K SMB" in the gel provided as FIG. 4.
[0255] It is expected that longer reactions times, increased
temperatures and/or multiple additions of the polymeric reagent
could increase yields. Using this same approach, other conjugates
can be prepared using branched mPEG-SMB having other weight-average
molecular weights.
EXAMPLE 10
PEGylation of Factor IX with mPEG-SPA, 20 kDa
20:1 Polymer to Factor IX Ratio; with Ethanol
##STR00146##
[0257] As ethanol is believed to increase the structural
flexibility of certain proteins, ethanol was introduced into the
buffer and reaction system. mPEG-SPA, 20 kDa, stored at -20.degree.
C. under argon, was warmed to ambient temperature. The warmed
mPEG-SPA (10.0 mg) was dissolved in 0.5 mL of 2 mM HCl with ethanol
added to form a 10% ethanol-containing mPEG-SPA solution. The 10%
ethanol-containing mPEG-SPA solution was added to the 10%
ethanol-containing Factor IX reaction mixture until a twenty molar
excess of mPEG-SPA relative to Factor IX was reached. After the
addition of the mPEG-SPA, the pH of the reaction mixture was tested
to ensure a pH of 7.2 to 7.5, and mixed well. To allow for coupling
of the mPEG-SPA to Factor IX via an amide linkage, the reaction
solution was stirred for two hours at room temperature. Coupling
was allowed to continue by stirring the reaction solution.
[0258] Based on SDS PAGE results, conjugation was not detected. See
the lane labeled as "20:1 20 K SPA+EtOH" in the gel provided as
FIG. 3. It is now believed that the introduction of ethanol does
not increase the structural flexibility of Factor IX to allow for
increased conjugation of mPEG-SPA, 20 kDa.
[0259] It is expected that longer reactions times, increased
temperatures and/or multiple additions of the polymeric reagent
could increase yields. Using this same approach, other conjugates
can be prepared using mPEG-SPA having other weight-average
molecular weights.
EXAMPLE 11
PEGylation of Factor IX with mPEG-SPA, 20 kDa
40:1 Polymer to Factor IX Ratio; with Ethanol
##STR00147##
[0261] As ethanol is believed to increase the structural
flexibility of certain proteins, ethanol was introduced into the
buffer and reaction system. mPEG-SPA, 20 kDa, stored at -20.degree.
C. under argon, was warmed to ambient temperature. The warmed
mPEG-SPA (10.0 mg) was dissolved in 0.5 mL of 2 mM HCl with ethanol
added to form a 10% ethanol-containing mPEG-SPA solution. The 10%
ethanol-containing mPEG-SPA solution was added to the 10%
ethanol-containing Factor IX reaction mixture until a forty molar
excess of mPEG-SPA relative to Factor IX was reached. After the
addition of the mPEG-SPA, the pH of the reaction mixture was tested
to ensure a pH of 7.2 to 7.5, and mixed well. To allow for coupling
of the mPEG-SPA to Factor IX via an amide linkage, the reaction
solution was stirred for two hours at room temperature. Coupling
was allowed to continue by stirring the reaction solution.
[0262] Based on SDS PAGE results, conjugation was not detected. See
the lane labeled as "40:1 20 K SPA+EtOH" in the gel provided as
FIG. 3. It is now believed that the introduction of ethanol does
not increase the structural flexibility of Factor IX to allow for
increased conjugation of mPEG-SPA, 20 kDa.
[0263] It is expected that longer reactions times, increased
temperatures and/or multiple additions of the polymeric reagent
could increase yields. Using this same approach, other conjugates
can be prepared using mPEG-SPA having other weight-average
molecular weights.
EXAMPLE 12
PEGylation of Factor IX with Branched mPEG-Butyraldehyde, 20
kDa
10:1 Polymer to Factor IX Ratio; without Ethanol
##STR00148##
[0265] Branched mPEG2-Butyraldehyde, 20 kDa, stored at -20.degree.
C. under argon, was warmed to ambient temperature. The warmed
branched mPEG2-butyraldehyde (10.9 mg) was dissolved in 1 mL of 2
mM HCl to form a branched mPEG2-butyraldehyde solution. The
branched mPEG2-butryaldehyde solution was added to an aliquot of
the Factor IX stock solution containing 0.07 mg of Factor IX until
a ten molar excess of branched mPEG2-butryaldehyde to Factor IX was
reached. After thirty minutes of mixing, a reducing agent,
NaCNBH.sub.3 (dissolved in 1.times. PBS), was added at excess
relative to the branched mPEG2-butyraldehyde (with the pH tested
and adjusted as necessary to ensure reduction to the secondary
amine). The solution was then stirred overnight at 4.degree. C. to
ensure coupling via an amine linkage.
[0266] RP-HPLC (C.sub.3) and a SDS PAGE were used for the
characterization of the resulting conjugate solution. Based on the
SDS PAGE results, conjugation was not detected. See the lane
labeled as "10:1 20 K BYA" in the gel provided as FIG. 4. RP-HPLC
(C.sub.3) was used to separate the components of the resulting
conjugate solution and the resulting chromatogram did not confirm
the presence of conjugated material. See the chromatagram provided
as FIG. 9.
[0267] It is expected that longer reactions times, increased
temperatures and/or multiple additions of the polymeric reagent
could increase yields. Using this same approach, other conjugates
can be prepared using branched mPEG2-butyraldehyde having other
weight-average molecular weights.
EXAMPLE 13
PEGylation of Factor IX with Branched mPEG-Butyraldehyde, 20
kDa
20:1 Polymer to Factor IX Ratio; without Ethanol
##STR00149##
[0269] Branched mPEG2-Butyraldehyde, 20 kDa, stored at -20.degree.
C. under argon, was warmed to ambient temperature. The warmed
branched mPEG2-butyraldehyde (10.9 mg) was dissolved in 1 mL of 2
mM HCl to form a branched mPEG2-butyraldehyde solution. The
branched mPEG2-butryaldehyde solution was added to an aliquot of
the Factor IX stock solution containing 0.07 mg of Factor IX until
a twenty molar excess of branched mPEG2-butryaldehyde to Factor IX
was reached. After thirty minutes of mixing, a reducing agent,
NaCNBH.sub.3 (dissolved in 1.times. PBS), was added at excess
relative to the branched mPEG2-butyraldehyde (with the pH tested
and adjusted as necessary to ensure reduction to the secondary
amine). The solution was then stirred overnight at 4.degree. C. to
ensure coupling via an amine linkage.
[0270] RP-HPLC (C.sub.3) and a SDS PAGE were used for the
characterization of the resulting conjugate solution. Based on the
SDS PAGE results, conjugation was not detected. See the lane
labeled as "20:1 20 K BYA" in the gel provided as FIG. 4. RP-HPLC
(C.sub.3) was used to separate the components of the resulting
conjugate solution and the resulting chromatogram did not confirm
the presence of conjugated material. See the chromatogram provided
as FIG. 10.
[0271] It is expected that longer reactions times, increased
temperatures and/or multiple additions of the polymeric reagent
could increase yields. Using this same approach, other conjugates
can be prepared using branched mPEG2-butyraldehyde having other
weight-average molecular weights.
EXAMPLE 14
PEGylation of Factor IX with mPEG-SPA, 20 kDa
53:1 Polymer to Factor IX Ratio; without Ethanol
##STR00150##
[0273] mPEG-SPA, 20 kDa, stored at -20.degree. C. under argon, was
warmed to ambient temperature. The warmed mPEG-SPA (5.4 mg) was
dissolved in 1 mL of 2 mM HCl to form an mPEG-SPA solution. The
mPEG-SPA solution was added to an aliquot of the Factor IX stock
solution containing 0.07 mg of Factor IX until a fifty-three molar
excess of mPEG-SPA relative to Factor IX was reached. After the
addition of the mPEG-SPA, the pH of the reaction mixture was tested
to ensure a pH of 7.2 to 7.5. To allow for coupling of the mPEG-SPA
to Factor IX via an amide linkage, the reaction solution was
stirred for two hours at room temperature. Coupling was allowed to
continue by stirring the reaction solution overnight at 4.degree.
C., thereby resulting in a conjugate solution.
[0274] RP-HPLC (C.sub.3) and a second SDS PAGE were used for the
characterization of the resulting conjugate solution. Based on the
SDS PAGE results, conjugation was verified. See the lane labeled
"53:1 20 K SPA" in the gel provided as FIG. 4. RP-HPLC (C.sub.3)
was used to separate the components of the resulting conjugate
solution and the resulting chromatogram indicated approximately 60%
conjugation yield (comprising 51.9% monoPEGylated or "1-mer"
species and 8% diPEGylated or "2-mer" species). See the
chromatogram provided as FIG. 11. It is believed, however, that the
actual yield may be somewhat lower due to the relatively large
excess of polymeric reagent.
[0275] Using this same approach, other conjugates can be prepared
using mPEG-SPA having other weight-average molecular weights.
EXAMPLE 15
PEGylation of Factor IX with mPEG-SPA, 20 kDa
110:1 Polymer to Factor IX Ratio; without Ethanol
##STR00151##
[0277] mPEG-SPA, 20 kDa, stored at -20.degree. C. under argon, was
warmed to ambient temperature. The warmed mPEG-SPA (5.4 mg) was
dissolved in 1 mL of 2 mM HCl to form an mPEG-SPA solution. The
mPEG-SPA solution was added to an aliquot of the Factor IX stock
solution containing 0.07 mg of Factor IX until a one hundred-ten
molar excess of mPEG-SPA relative to Factor IX was reached. After
the addition of the mPEG-SPA, the pH of the reaction mixture was
tested to ensure a pH of 7.2 to 7.5. To allow for coupling of the
mPEG-SPA to Factor IX via an amide linkage, the reaction solution
was stirred for two hours at room temperature. Coupling was allowed
to continue by stirring the reaction solution overnight at
4.degree. C., thereby resulting in a conjugate solution.
[0278] RP-HPLC (C.sub.3) and a second SDS PAGE were used for the
characterization of the resulting conjugate solution. Based on the
SDS PAGE results, conjugation was verified. See the lane labeled
"110:1 20 K SPA" in the gel provided as FIG. 4. RP-HPLC (C.sub.3)
was used to separate the components of the resulting conjugate
solution and the resulting chromatogram indicated approximately 44%
conjugation yield (representing approximately 100% monoPEGylated or
"1-mer" species). See the chromatogram provided as FIG. 12. It is
believed, however, that the actual yield may be somewhat lower due
to the relatively large excess of polymeric reagent.
[0279] Using this same approach, other conjugates can be prepared
using mPEG-SPA having other weight-average molecular weights.
EXAMPLE 16
PEGylation of Factor IX with Branched mPEG-Butyraldehyde, 20
kDa
20:1 Polymer to Factor IX Ratio; with Ethanol
##STR00152##
[0281] As ethanol is believed to increase the structural
flexibility of certain proteins, ethanol was introduced into the
buffer and reaction system. Branched mPEG2-Butyraldehyde, 20kDa,
stored at -20.degree. C. under argon, was warmed to ambient
temperature. The warmed branched mPEG2-butyraldehyde (10.9 mg) was
dissolved in 1 mL of 2 mM HCl with ethanol added to form a 10%
ethanol-containing branched mPEG2-butyraldehyde solution. The 10%
ethanol-containing branched mPEG2-butryaldehyde solution was added
to an aliquot of the Factor IX stock solution containing 0.07 mg of
Factor IX until a twenty molar excess of branched
mPEG2-butryaldehyde to Factor IX was reached. After thirty minutes
of mixing, a reducing agent, NaCNBH.sub.3 (dissolved in 1.times.
PBS), was added at excess relative to the branched
mPEG2-butyraldehyde (with the pH tested and adjusted as necessary
to ensure reduction to the secondary amine). The solution was then
stirred overnight at 4.degree. C. to ensure coupling via an amine
linkage.
[0282] RP-HPLC (C.sub.3) and a SDS PAGE were used for the
characterization of the resulting conjugate solution. Based on the
SDS PAGE results, conjugation was not detected. See the lane
labeled as "20:1 20 K BYA+EtOH" in the gel provided as FIG. 4.
RP-HPLC (C.sub.3) confirmed the absence of detectable conjugated
material (results not shown). It is now believed that the
introduction of ethanol does not increase the structural
flexibility of Factor IX to allow for increased conjugation of
branched mPEG2-butyraldehyde.
[0283] It is expected that longer reactions times, increased
temperatures and/or multiple additions of the polymeric reagent
could increase yields. Using this same approach, other conjugates
can be prepared using branched mPEG2-butyraldehyde having other
weight-average molecular weights.
EXAMPLE 17
PEGylation of Factor IXa with mPEG-SBA
[0284] mPEG-Succinimidyl butanoate having a molecular weight of
10,000 Daltons is obtained from Nektar Therapeutics, (Huntsville,
Ala.). The basic structure of the polymer reagent is provided
below:
##STR00153##
[0285] If lyophilized, Factor IXa is dissolved in amine-free buffer
such as phosphate to result in a final pH to 7.2-9. To this
solution is then added a 1.5 to 10-fold molar excess of mPEG-SBA.
The resulting mixture is stirred at room temperature for several
hours.
[0286] The reaction mixture is analyzed by SDS-PAGE to determine
the degree of PEGylation of the protein.
EXAMPLE 18
PEGylation of Factor IX with mPEG-PIP, 5K
##STR00154##
[0288] The above polymeric reagent, shown as both the ketone and
corresponding ketal, is prepared as described in U.S. Patent
Application Publication No. 2005/0031576.
[0289] To prepare the above polymeric reagent, to a solution of
methoxy-polyethylene glycol-succinimidyl propionate having a
weight-average molecular weight of 5,000 Daltons (1.0 g, 0.002
moles) in methylene chloride (20 ml), triethyl amine (0.084 ml,
0.006 moles) and 4-piperidone monohydrate hydrochloride (0.077 g,
0.005 moles) are added. The reaction mixture is stirred at room
temperature under a nitrogen atmosphere overnight and then purified
prior to conjugation. Alternatively, the polymer reagent may be
purchased from Nektar Therapeutics.
[0290] To effect conjugation, to a solution of Factor IX in aqueous
buffer is added a 20-fold molar excess of mPEG-PIP, 5K. The
resulting solution is placed on a Roto Mix.TM. orbital shaker
(Thermolyne Corp., Dubuque, Iowa) set at slow speed to facilitate
reaction at room temperature. After 15 minutes, aqueous
NaCNBH.sub.3 is added in an amount equal to a 50 fold-molar excess
relative to Factor IX. Aliquots are withdrawn at timed intervals
from the reaction mixture and are analyzed by SDS-PAGE (using gels
available from Bio-Rad Laboratories, Hercules, Calif.).
[0291] SDS-PAGE analysis indicates the presence of PEG derivatives
of Factor IX having 1, 2, and 3 PEG moieties attached.
EXAMPLE 19
Conjugation of Cysteine-Inserted Factor IX with mPEG-MAL, 20K
[0292] Factor IX is inserted with one or more cysteine residues
according to the process described in WO 90/12874.
[0293] Prior to the conjugation, a buffer exchange for Factor IX is
performed to replace histidine with HEPES.
[0294] mPEG-MAL, 20K, stored at -20.degree. C. under argon, is
warmed to ambient temperature. The warmed mPEG-MAL reagent (4.4 mg)
is dissolved in 0.044 ml of HEPES buffer [50 mM HEPES (or other
suitable buffer) pH 7.0] to make a 10% mPEG-MAL solution. The
mPEG-MAL solution is quickly added to 4 ml of Factor IX solution
[0.4324 mg/ml in 50 mM HEPES (or other suitable formulation) pH
7.0] and is mixed well. After 30 minutes of reaction at room
temperature, the reaction vial is transferred to the cold room
(4.degree. C.), and another 0.044 ml of mPEG-MAL solution is added
to the reaction mixture, followed by the addition of three more
aliquots of 0.044 ml of mPEG-MAL solution over the course of two
hours. The pH is determined (pH 7.0.+-.0.2). The molar ratio of
mPEG-MAL to protein is 50:1. The final mPEG-MAL concentration is
5.213 mg/ml, and the final Factor IX concentration is 0.410 mg/ml.
The reaction is allowed to proceed overnight at 4.degree. C. on
Rotomix (slow speed, Thermolyne).
[0295] The conjugate mixture is purified using gel filtration
chromatography. A size exclusion chromatography method is developed
for analyzing the reaction mixtures, and the final products.
SDS-PAGE analysis is also used for the characterization of the
samples.
EXAMPLE 20
In-Vitro Activity of Exemplary Factor IX-PEG Conjugates
[0296] The biological activity of each of the Factor IX-PEG
conjugates described in the Examples 1, 2, 3, 9, 14 and 15 are
determined. All of the Factor IX-PEG conjugates tested are
determined to have some degree of Factor IX activity.
Sequence CWU 1
1
11461PRTHomo sapiens 1Met Gln Arg Val Asn Met Ile Met Ala Glu Ser
Pro Gly Leu Ile Thr 1 5 10 15 Ile Cys Leu Leu Gly Tyr Leu Leu Ser
Ala Glu Cys Thr Val Phe Leu 20 25 30 Asp His Glu Asn Ala Asn Lys
Ile Leu Asn Arg Pro Lys Arg Tyr Asn 35 40 45 Ser Gly Lys Leu Glu
Glu Phe Val Gln Gly Asn Leu Glu Arg Glu Cys 50 55 60 Met Glu Glu
Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe Glu Asn 65 70 75 80Thr
Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly Asp Gln 85 90
95 Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp Asp Ile
100 105 110 Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys
Asn Cys 115 120 125 Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg
Cys Glu Gln Phe 130 135 140 Cys Lys Asn Ser Ala Asp Asn Lys Val Val
Cys Ser Cys Thr Glu Gly 145 150 155 160Tyr Arg Leu Ala Glu Asn Gln
Lys Ser Cys Glu Pro Ala Val Pro Phe 165 170 175 Pro Cys Gly Arg Val
Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala 180 185 190 Glu Ala Val
Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala Glu 195 200 205 Thr
Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn Asp Phe 210 215
220 Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp
225 230 235 240Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly
Gly Ser Ile 245 250 255 Val Asn Glu Lys Trp Ile Val Thr Ala Ala His
Cys Val Glu Thr Gly 260 265 270 Val Lys Ile Thr Val Val Ala Gly Glu
His Asn Ile Glu Glu Thr Glu 275 280 285 His Thr Glu Gln Lys Arg Asn
Val Ile Arg Ile Ile Pro His His Asn 290 295 300 Tyr Asn Ala Ala Ile
Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu 305 310 315 320Leu Asp
Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys Ile 325 330 335
Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser Gly Tyr 340
345 350 Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala Leu
Val 355 360 365 Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr
Cys Leu Arg 370 375 380 Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe
Cys Ala Gly Phe His 385 390 395 400Glu Gly Gly Arg Asp Ser Cys Gln
Gly Asp Ser Gly Gly Pro His Val 405 410 415 Thr Glu Val Glu Gly Thr
Ser Phe Leu Thr Gly Ile Ile Ser Trp Gly 420 425 430 Glu Glu Cys Ala
Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Ser 435 440 445 Arg Tyr
Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr 450 455 460
* * * * *