Note: Descriptions are shown in the official language in which they were submitted.
W092~16555 PCT/US92/02~7
- 2101918
HYDRAZINE CONTAINING CONJUGATES OF
POLYPEPTIDES AND GLYCOPOLYPEPTIDES WITH POLYMERS
Technical Field
The present invention relates to biologically
active macromolecular conjugates, in particular, to
conjugates of biologically active polypeptides and
glycopolypeptides with water-soluble polymers.
Backaround Art
The conjugation of polypeptides with
water-soluble polymers such as polyethylene glycol (PEG)
is well known. The coupling of peptides and
polypeptides to PEG and similar water-soluble polymers
is disclosed by U.S. Patent No. 4,179,337 to Davis
et al.
Pavis et al. discloses that physiologically
active polypeptides modified with PEG exhibit
dramatically reduced immunogenicity and antigenicity.
Also, the PEG-protein conjugates, when injected into a
living organism, have been shown to remain in the
bloodstream considerably longer than the corresponding
native proteins. Accordingly, a number of
PEG-conjugated therapeutic proteins were developed
exhibiting reduced immunogenicity and antigenicity and
longer clearance times, while retaining a substantial
portion of the protein's physiological activity.
Significant PEG-conjugated therapeutic proteins include
tissue plasminogen activator, insulin, interleukin 2 and
hemoglobin. Furthermore, Dreborg et al., Crit. Rev.
Therap. Drua Carrier Svst., 6, 315-65 tl990) disclose
that covalent modification of potent allergen proteins
with PEG often can be effective in reducing their
allergenicity. Sehon, et al., Pharmacol. Toxicol.
Proteins, 65, 205-19 (1987) disclose that such
PEG-conjugated allergen proteins having reduced
allergenicity can then be utilized as tolerance
inducers.
In most instances, as exemplified by U.S.
Patent No. 4,179,337, covalent attachment of the polymer
W092/16555 PCT/US92/02~7
21 01 91 8 -2- _
is effected by reacting PEG-succinimide derivatives with
amino groups on the exterior of protein molecules.
However, the amino groups of many proteins are moieties
responsible for polypeptide activity that can be readily
inactivated as a result of such modification. The
conjugation of such proteins is not desirable, because
it results in the reduction of physiological activity.
Other proteins may have only a small number of available
amino groups, and consequently very few polymer
anchoring sites. As a result, many proteins of interest
cannot be conjugated with PEG in this manner.
The known alternatives to covalent attachment
of polymers to other functional groups on the exterior
of proteins have serious limitations. U.S. Patent No.
4,179,337 discloses, for example, that PEG-maleimide
derivatives can be used to covalently attach polymers to
protein sulfhydryl groups. However, this is of limited
versatility because very few proteins have free
sulfhydryl groups that are not required for biological
or enzymatic activity and would thus be available for
chemical modification.
U.S. Patent No. 4,179,337 discloses the
reaction of an amino-PEG derivative with
1-ethyl-3-(3-dimethylamino-propyl) carbodiimide(EDC)-
activated carboxylic acid groups of trypsin and otherproteins. The selectivity of this reaction is rather
poor because the reactivity of amino-PEG is similar to
that of the lysyl residues of proteins, with both the
amino-PEG and protein amino groups competing to react
with the activated carboxylic acid groups. This results
in intermolecular as well as intramolecular crosslinking
and a loss of protein activity.
In a similar reaction disclosed by Pollack et
al., JACS, 98, 289 (1976), p-aminobenzyl ethers of PEG
are coupled to carboxylic acid groups of
D-glucose-6-phosphate dehydrogenase by treatment with
EDC.
' W092/16~55 PCT/US92/~2~7
21 01918
--3--
A polymer derivative that protein amino groups
would not compete with for activated carboxylic acid
groups of proteins would be highly desirable. This
would eliminate intermolecular and intramolecular
crosslinking and improve the enzymatic activity of
polymer conjugates.
U.S. Patent No. 4,847,325 to Shadle et al.
suggests that glycosilated Colony Stimulating Factor-l
(CSF-1) could be covalently attached to PEG by reacting
PEG-amine, PEG-hydrazine or PEG-hydrazide with CSF-l
that had been oxidized with periodate to convert vicinaI
diols in the sugars to aldehydes. However, this
disclosure is silent regarding the details on
preparation of such conjugates and their reactivity.
The degree of polymer conjugation with amino
groups is ordinarily determined by assaying the
conjugate with trinitrobenzene sulfo,nic acid (TNBS) to
determine the number of free amino groups. For polymers
conjugated at protein amino groups, t,he difference
between the number of free amino groups, in the modified
protein and the number of free amino groups in the
native protein represents the degree of conjugation of
the protein.
The results from TNBS assays are meaningless
when determining the degree of conjugation of proteins
when the polymer is covalently attached to alternative
functional groups. In such instances, the number of
free amino groups will not vary between conjugated and
non-conjugated protein species.
The conjugated protein can also be digested in
small fragments with an enzyme and separated by column
chromatography followed by preparation of a peptide map
for comparison to a map of the unmodified protein, with
the fragments having altered elution times indicative of
the location of polymer attachments. However, this
procedure consumes large quantities of product and is
not suitable for use with polypeptides of limited
availability. Radioactive labeling represents another
WO 92tl6555 PCT/US92/02047
2i 01 91 8 -4-
alternative, but this alternative is not suitable for
materials being prepared for therapeutic end uses for
which the determination of degree of conjugation is most
critical.
Yamasaki et al., Aqric. Biol. Chem., 52~8~,
2125-7 (1988) disclose the preparation of
PEG-succinimide derivatives with norleucine and lysine
residues between the polymer and the succinimido moiety,
which residues permit the measurement of the amount of
PEG covalently attached to the amino groups of proteins
by amino acid analysis for the presence of norleucine or
lysine. Sartore et al., Proced. Intern. Sym. Control.
Rel. Bioact. Mater., 17, 208-9 (1990) also disclose the
use of a norleucine spacer in PEG-succinimide
derivatives covalently bonded to protein amino groups,
noting that the use of such an unnatural amino acid
helps in the characterization of the adduct because a
single amino acid analysis would give both protein
conc~ntration and number of polymer chains bound to the
~0 amino groups. In other words, in the purified
conjugate, each single norleucine residue acid
represents a polymer chain bound to an exterior amino
group.
There remains a need for methods to covalently
attach polymers to non-amino moieties of polypeptides
and glycopolypeptides without a loss of activity from
intermolecular crosslinking, as well as for methods of
assaying the degree of conjugation of the polymer to the
polypeptide at functional groups other than amino
groups.
Summary of the Invention
It has now been discovered that water-soluble
polymers can be conjugated with biologically active
polypeptides and glycopolypeptides utilizing acyl
hydrazine derivatives of the water-soluble polymers.
The acyl hydrazine derivatives of the water-soluble
polymers covalently link to either the oxidized
carbohydrate residues of the glycopolypeptides or the
W092/16555 2 i O 1 9 1 ~ /US92/02~7
--5--
reactive carbonyl or activated carboxylic acid groups of
peptide moieties of polypeptides or glycopolypeptides.
This invention extends the realm of water-soluble
polymer-peptide conjugation to those polypeptide and
glycopolypeptide materials that could not have been
modified heretofore by conventional methods.
Furthermore, under neutral or mildly acidic condition of
conjugation reactions, due to their low PKa (about 3)
acyl hydrazine containing polymers of this invention
possess higher reactivity than the amino groups of
polypeptides (PKa about lO.S), therefore minimizing and
in most cases eliminating the competing reactions of
these amino groups, thus preventing polypeptide
crosslinking and preserving the biological activity of
the conjugates.
In accordance with the present invention, a
biologically active macromolecular conjugate is provided
of a biologically active polypeptide or glycopolypeptide
and one or more water-soluble polymer molecules
covalently bonded thereto at a reactive carbonyl or
carboxylic acid group of a peptide moiety on the
polypeptide or glycopolypeptide by a linkage containing
a hydrazide or hydrazone functional group. The linkage
is formed by reacting an acyl hydrazine derivative of
the water-soluble polymer with a polypeptide or
glycopolypeptide having an activated carboxylic acid
group or a reactive carbonyl group generated thereon.
The present invention also provides a
biologically active macromolecular conjugate of a
biologically active glycopolypeptide and one or more
water-soluble polymer molecules covalently bonded
thereto at an oxidized carbohydrate moiety of the
glycopolypeptide by a linkage containing a hydrazide or
hydrazone functional group bound to the polymer via a
short peptide sequence. The oxidation of the
carbohydrate moiety produces reactive aldehydes. The
hydrazone linkage is formed by reacting an acyl
hydrazine derivative of the water-soluble polymer
W092/t6sss PCT/US92/02047
2101918 -6-
containing the peptide sequence with these aldehyde
groups. The hydrazone can be further stabilized by
reduction to a very ~table alkyl hydrazine derivative.
The peptide sequence influences the lability
of the linkage to proteolytic enzymes and also allows
convenient characterization of the polymer conjugates by
amino acid analysis of their hydrolysates. By using
state-of-the-art techniques of amino acid analysis, the
quantity of peptide sequences, and consequently the
degree of conjugation, can be determined for picomolar
concentrations of the conjugate.
Therefore, it is also in accordance with the
present invention that the peptide sequences also be
utilized with the polypeptide conjugates of the present
invention to bind the linkages containing a hydrazide or
hydrazone functional group to the water-soluble polymer.
Brief Description of the Drawinas
FIG. 1 i8 a GF-HPLC chromatogram comparison of
mPEG-beta-alanine-bovine serum albumin conjugate to
native bovine serum albumin.
FIG. 2 is a GF-HPLC chromatogram comparison of
mPEG-beta-alanine-ovalbumin conjugate to native
ovalbumin.
FIG. 3 is a GF-HPLC chromatogram comparison of
PEG-beta-alanine-IgG, conjugated via oxidized
carbohydrate moieties, to native IgG.
FIG. 4 is a GF-HPLC chromatogram comparison of
PEG-beta-alanine-rhG-CSF, conjugated via carboxylic acid
groups of rhG-CSF, to native rhG-CSF.
Best Mode of Carrvinq Out the Invention
The macromolecules of the present invention
are biologically active polypeptides or
glycopolypeptides having one or more water-soluble
polymer molecules covalently bonded thereto. The term
"biologically active" is used consistently with the
meaning commonly understood to those of ordinary skill
in the polypeptide and glycopolypeptide art, which
meaning is not limited to physiologically or
W092/1~555 PCT/US92/02~7
_7_i 21~1918
pharmacologically activities of the polypeptides or
glycopolypeptides in the therapeutic sense. For
example, many physiologically active polypeptides such
as enzymes, the water-soluble polymer conjugates of
which have therapeutic applications, are also able to
catalyze reactions in organic solvents. Likewise, while
therapeutic uses exist for water-soluble polymer
conjugates of proteins such as concanavalin A,
immunoglobulins, and the like, the polymer conjugates of
these proteins are also useful as laboratory diagnostic
tools.
Enzymes of interest, for both biological
applications in general and therapeutic applications in
particular include the oxidoreductases, transferases,
hydrolases, lyases, isomerases and ligases disclosed by
U.S. Patent No. 4,179,337, the disclosure of which is
hereby incorporated herein by reference thereto.
Without being limited to particular enzymes, examples of
specific enzymes of interest include asparaginase,
arginase, adenosine deaminase, superoxide dismutase,
catalase, chymotrypsin, lipase, uricase and bilirubin
oxidase. Carbohydrate-specific enzymes are also of
interest--for example, glucose oxidase, glucosidase,
galactosidase, glucocerebrosidase, glucuronidase, etc.
Examples of other proteins of general
biological or therapeutic interest include, but are not
limited to, Factor VIII and polypeptide hormones such as
insulin, ACTH, glucagon, somatostatin, somatotropins,
thymosin, parathyroid hormone, pigmentary hormones,
somatomedins, erythropoietin, luteinizing hormone,
hypothamic releasing factors, antidiuretic hormones and
prolactin.
Examples of glycopolypeptides of interest
include, but are not limited to, immunoglobulins,
chorionic gonadotrophin, follicle-stimulating hormone,
thyroid-stimulating hormone, ovalbumin, bovine serum
albumin (BSA), lectins, tissue plasminogen activator,
numerous enzymes and glycosilated interleukins,
W092/1655~ i PCT/US92/02~7
inter~e~Qa and colony stimulating factors.
Immunoglobulins of interest include IgG, IgE, IgM, IgA,
IgD and fragments thereof.
Many of the above glycopolypeptides such as
the interleukins, interferons and colony stimulating
factors also exist in non-glycosilated form, usually the
result of preparation by recombinant protein techniques.
The structure of such versions may not contain
carbohydrate moieties. However, the non-glycosilated
versions are still capable of conjugation at reactive
carbonyl or carboxylic acid groups of the peptide
moieties.
Examples of allergen proteins and
glycoproteins having reduced allerginicity when
conjugated with water-soluble polymers and consequently
suitable for use as tolerance inducers include those
allergens disclosed by Dreborg et al., Crit. Rev.
Thera~. Drua Carrier Syst., discussed above, the
teachings of which are hereby incorporated herein by
reference thereto. Among the allergens disclosed by
this article are Ragweed Antigen E, honey bee venom,
mite allergen, and the like.
The water-soluble polymers suitable for
attachment to the polypeptides and glycopolypeptide
include polyalkylene oxides, polyoxyethylenated polyols,
polyacrylamides, polyvinyl pyrrolidone, polyvinyl
alcohol, dextran, and other carbohydrate-based polymers.
To be suitable for use in the present invention, the
polymer must be soluble in water at room temperature.
Polyalkylene oxide homopolymers meeting this requirement
are polyethylene glycol (PEG) and copolymers thereof.
Block copolymers of PEG with polypropylene glycol or
polypropylene oxide are also suitable for use with the
present inYention, provided that the degree of block
copolymerization is not so great as to render the
polymer insoluble in water at room temperature.
Examples of polyoxyethylenated polyols include
W092/16~55 PcT/us92/02~7
'~101918
g
polyoxyethylenated glycerols, polyoxyethylenated
sorbitols, polyoxyethylenated glucoses, and the like.
The molecular weight of the polymer is not
critical, and will depend mainly upon the end use of a
particular polymer conjugate. Those of ordinary skill
in the art are capable of determining molecular weight
ranges suitable for their end use applications. In
general, the useful range of molecular weight is a
number average molecular weight between about 600 and
about 100,000 daltons, and preferably between
about 2,000 and about 20,000 daltons.
One or more polymer units can be attached
covalently to the polypeptide or glycopolypeptide by
reacting an acyl hydrazine derivative of the polymer
with a polypeptide or glycopolypeptide having a reactive
carbonyl group or an activated peptide carboxylic acid
group. For purposes of the present invention, the
reactive carbonyl group is defined as being either a
ketone or aldehyde group, excluding other
carboxyl-containing groups such as amides. Aldehyde
groups are preferred, because they are more reactive
than ketones.
The carbonyl group can be generated either on
a peptide or a saccharide unit. For example, Dixon,
J. Protéin Chem., 3, g9 (1984) has reviewed some of the
methods to generate reactive carbonyl groups on the
N-terminus of a polypeptide molecule. Carbonyl groups
can be generated on peptides, for example, by reacting a
polypeptide or glycopolypeptide with a suitable
heterobifunctional reagent such as a reactive ester of
formyl benzoic acid, disclosed by Xing et al.,
BiochemistrY, 25, 5774 (1986), the teachings of which
are hereby incorporated herein by reference thereto.
Carbonyl groups can be generated on saccharide units of
glycopolypeptides, for example, by oxidizing vicinal
diols of carbohydrate moieties of glycopolypeptides with
excess periodate or enzymatically e.g. by use of
galactose oxidase.
WO92/16S55 PCT/US92/02047
'21~1918 -lo- -
The polymer acyl hydrazine reacts with the
reactive carbonyl group on the polypeptide or
glycopolypeptide to form a hydrazone linkage between the
polymer and the polypeptide or glycopolypeptide. The
hydrazone can be reduced to a more stable alkyl
hydrazide by using for example NaBH4 or NaCNBH3.
The activated peptide carboxylic acid group
can be derived either from a C-terminus carboxylic acid
group or a carboxylic acid group of aspartic or glutamic
acid residues. The polymer acyl hydrazine reacts with
the activated peptide carboxylic acid group to form a
diacylhydrazine linkage between the polymer and the
polypeptide or glycopolypeptide.
Activated carboxylic acid groups are
carboxylic acid groups substituted with a suitable
leaving group capable of being displaced by the polymer
acyl hydrazine. Examples of suitable leaving groups are
disclosed by Bodanszky, Principles of Peptide Synthesis
(Springer-Verlag, New York, 1984), the disclosure of
Z0 which is hereby incorporated herein by reference
thereto. Such leaving groups, which are well-known in
the art of peptide chemistry, include, but are not
limited to, imidazolyl, triazolyl, N-hydroxysuccin-
imidyl, N-hydroxynorbornenedicarboximidyl and phenolic
leaving groups, and are substituted onto the peptide
carboxylic acid group by reacting the polypeptide or
glycopolypeptide in the presence of an activating
reagent with the corresponding imidazole, triazole ,
N-hydroxysuccinimide, N-hydroxynorbornene dicarboximide
and phenolic compounds.
Suitable activating reagents are also
well-known and disclosed by the above-cited Bodanszky,
Principles of Peptide Synthesis, the disclosure of which
is hereby incorporated herein by reference thereto.
3S Examples of such activating reagents include, but are
not limited to, water-soluble carbodiimides such as
ethyl dimethylamino-propyl carbodiimide (EDC)
and 3-[2-morpholinyl-(4)-ethyl] carbodiimide, p-toluene
W092/l65~5 PCT/US92/02~7
~101918
sulfonate, 5-substituted isoxazolium salts, such as
Woodward's Reagent K, and the like.
The acyl hydrazine polymer derivatives of the
present invention will have the general structure (I):
X-R-Z-C-NH-NH2 (I)
wherein R is one of the above-disclosed water-soluble
polymers, Z is 0, NH, S or a lower alkyl group
containing up to ten carbon atoms, and X is a terminal
group on the polymer. X can be a hydroxyl group, in
which case the polymer has two labile groups per polymer
moiety capable of reacting to form a derivative that can
be covalently linked with a polypeptide or
glycopolypeptide. X can therefore also be a group into
which the terminal hydroxyl group may be converted,
including the reactive derivatives of the prior art
disclosed in U.S. Patent Nos. 4,179,337 and 4,847,325,
the disclosures of which are hereby incorporated herein
by reference thereto, as well as the acyl hydrazine
derivatives of the present invention. Such heterobi-
functional polymers can be prepared by methods known to
those skilled in the art, including the methods
disclosed by the present specification with reference to
the preparation of acyl hydrazine derivatives, as well
as the methods disclosed by Zalipsky and Barany, Polvm.
Pre~r., 27(1~, 1 (1986) and Zalipsky and Barany, J.
Bioact. Compat. Polym., 5, 227 (1990), the disclosures
of which are hereby incorporated herein by reference
thereto.
Where X is a functional group useful for
covalently linking the polymer with a second polypeptide
or glycopolypeptide, X can be a solid support or a small
molecule such as a drug, or an acyl hydrazide derivative
of the formula (II):
~ -Z-C-NH-NH2 (II)
WO92/16555 PCT/US92/~2~7
., !
2 10 ~9 ~8 -12-
When Z is the same as disclosed above for acyl hydrazine
derivatives, the polymer will then be a symmetrical,
homobifunctional polymer derivative.
Such double polymer substitution can result in
either intra- or intermolecular crosslinking of the
polypeptide and glycopolypeptide moieties, which, in
some cases, can be useful. Such crosslinking can be
controlled by the amount of polymer used and the
concentration of reacting species, which methods are
well-known to those of ordinary skill in the art.
Crosslinking of the polypeptide or
glycopolypeptide moieties can also be prevented by using
a pre-blocked polymer having only one labile hydroxyl
group per polymer-moiety. With such polymers, X would
represent a blocking group such as an alkoxy group of
one to four carbon atoms. The preferred blocking group
is a methoxy group.
In any event, the selectivity of the acyl
hydrazines for the reactive carbonyl or activated
carboxylic acid groups over the peptlde amino group
prevents intermolecular crosslinking between peptide
amino groups and the reactive carbonyl groups and
activated carboxylic acid groups, limiting occurrences
of such crosslinking to instances when bifunctional
polymer derivatives are employed.
X can also represent an antibody or solid
support covalently coupled to the polymer by methods
known to those skilled in the art. Examples of solid
supports covalently coupled to water-soluble polymers
and methods of coupling water-soluble polymers to solid
supports are disclosed in Published European Patent
Application No. 295,073, the disclosure of which is
hereby incorporated herein by reference thereto.
The acyl hydrazine derivative is prepared by
reacting, for example, the terminal -OH group of
methoxylated PEG (mPEG-OH) with phosgene to form
mPEG-chloroformate as described in U.S. Patent Appln.
Ser. No. 340,928 by Zalipsky, filed April 19, 1989, the
W092/165S5 PCT/US92/02M7
-13- 21 01918
disclosure of which is hereby incorporated herein by
reference thereto. The reaction is carried out in
organic solvents in which the reactants are soluble,
such as methylene chloride, and will run to completion
overnight at room temperature. The solvents and excess
phosgene are removed and the residue of polymeric
chloroformate ,s then reacted with an excess of
hydrazine.
The preparation of acyl hydrazine polymer
derivatives is described with reference to mPEG for
purposes of illustration, not limitation. Similar
products would be obtained with any of the polymers
suitable for use with the present invention, and it will
be clear to those of ordinary skill in the art how this
preparation can be adapted to the other suitable
polymers.
A more preferred form of the present invention
uses polymer hydrazides of the general formula (III):
X-R-Z-~-AA-NH-NH2 (III)
wherein R represents the water-soluble polymers, Z
represents the groups described above with respect to
Formula I, X represents the polymer terminal groups
described above and AA represents an amino acid or a
peptide sequence. AA can be a peptide sequence of any
of the common amino acids, or at least one amino acid
residue. In the case of AA being one amino acid
residue, it is preferable that it is a residue that does
not appear naturally in proteins. Examples of such
unusual residues include, but are not limited to, alpha-
or gamma- amino butyric acid, norleucine, homoserine,
beta-alanine, epsilon-caproic acid, and the like.
When Z is oxygen, the linkage is a urethane
linkage, which is very stable at ambient temperature in
a variety of buffers, even at extreme pH's, but is
readily split under conditions normally used for protein
hydrolysis, thus allowing determination of amino acid
components of AA by amino acid analysis.
WO92/16555 PCT/US92/02~7
2~ o~9~8 _
-14-
The peptide sequence can serve two roles.
First, it can provide for convenient characterization of
the modified protein by quatitation of the sequence by
amino acid analysis. In this instance, the peptide
sequence preferably is as short as possible and
preferably contains unusual amino acid residues. For
characterization of the modified protein, the peptide
sequence most preferably contains but one amino acid.
In addition, AA can also contain a labeled
amino acid residue (chromophore, fluorophore, or
radioisotope containing), or an amino acid that could be
easily labeled (e.g. tyrosine can be iodinated). The
presence of such labels would facilitate the
experimental evaluation of the resulting polymer-
polypeptide conjugates.
Second, the peptide sequence can optimize thelability of the covalent linkage between the
water-soluble polymer and the polypeptide to proteolytic
enzymes. In ~his second instance, the peptide sequence
is preferably as long as possible and preferably
contains natural amino acid residues. By controlling
enzymatic lability in this manner, the polymer
conjugates can be used to deliver physiologically active
polypeptides or glycopolypeptides to specific sites,
such as cancer cells having elevated concentrations of
certain proteolytic enzymes to which the peptide
sequence is labile.
The length and sequence of the peptide in this
second instance can be fine-tuned depending on the
system of use and specificity of the target enzyme.
Usually, three to seven amino acid residues would be
required. Using modern techniques of peptide chemistry
such short peptide sequences can be readily assembled.
In symmetrical, homobifunctional polymer
derivatives, X can also contain a second peptide
sequence residue. For example, when X is an acyl
hydrazine derivative, X would have the general formula
(IV):
W092/16~55 PCT/US92/02~7
-15- 2101918
1l
-Z-C-AA-NH-NH2 (IV)
wherein Z and AA are as described above.
The acyl hydrazine polymer derivative
containing a peptide sequence can be synthesized by
first preparing the polymeric chloroformate as described
above. The polymeric chloroformate is then reacted with
the peptide or an amino acid derivative in a solvent in
which the polymeric chloroformate is soluble, such as
methylene chloride. The peptide or amino acid is
preferably in the form of the ester of the C-terminus
acid group, more preferably methyl or ethyl esters.
This reaction is also operative under mild
conditions and typically runs to completion at room
temperature and the resulting product can be readily
converted to a hydrazide by hydrazinolysis. The acyl
hydrazine polymer derivative containing a peptide
sequence is then recovered and purified by conventional
methods, such as repeated precipitation of the polymer-
product.
Alternatively, the acyl hydrazine polymerderivative containing a peptide seguence or an amino
acid can be prepared by reacting the peptide sequence
with a succinimidyl carbonate active ester of the
polymer, as disclosed by the above-mentioned Zalipsky,
U.S. Patent Appln. No. 340,928 or by directly reacting
isocyanate derivatives of an amino acid with the
terminal hydroxyl group of the polymer as disclosed by
Zalipsky et al., Int. J Pe~tide Protein Res., 30, 740
(1987), the disclosures of both of which are hereby
incorporated herein by reference thereto. Again, both
reactions are essentially conventional and operative
under mild conditions, running to completion at room
temperature in organic solvents in which the polymer is
solvent, such as methylene chloride. The reaction of
isocyanate derivatives of amino acid esters with
terminal hydroxyl groups of polymers is disclosed in the
above-cited Zalipsky and Barany, Polvm. Pre~r., as well
WO92tl6~5 PCT/US92/02~7
210~9~8 -16- ~
as in Zalipsky et al., Int._J Peptide~_Protein Res., the
teachings of both of which are hereby incorporated
herein by reference thereto. The succinimidyl carbonate
derivative of the polymer is formed by the known method
of reacting the above-disclosed polymeric chloroformate
with N-hydroxysuccinimide, as disclosed by the
above-cited Zalipsky, U.S. Patent Appln. No. 340,928,
the disclosure of which is hereby incorporated herein by
reference thereto.
Either of the above polymer-polypeptide
derivatives can be readily converted to a hydrazide by
the hydrazinolysis method disclosed above to yield an
acyl hydrazine. The preparation of peptide sequences is
essentially conventional and disclosed by the
above-cited Bodanszky, Principles of Peptide Synthesis,
the disclosure of which is hereby incorporated herein by
reference thereto.
The reaction of polymer acyl hydrazine
derivatives with carbonyl-containing polypeptides and
glycopolypeptides to form a hydrazone linkage is
illustrated by the reaction sequence of Scheme 1 in
which R represents the above-described water-soluble
polymers, Z is as described above with respect to
Formulae I-IV and either or both of R1 and R2 are
independently selected from oxidized carbohydrate
moieties of glycopolypeptides and peptide units of
polypeptides and glycopolypeptides on which reactive
carbonyl groups have been generated:
W092/1655~ PCT/US92/02~7
-17- 2101918
Scheme 1
~ }
R-Z-C--NH--NH2 + O=C
R2 \~
f 1
R-Z-C-NH-N=C~
R2
Hydrazone
Reduction
R lRl
R-Z-C-NH-NH-~CH
Hydrazide
The hydrazone can be reduced to the more
stable alkyl hydrazide by reacting the hydrazone with,
for example, NaBH4 or NaCNBH3.
The reaction of polymer acyl hydrazine
derivatives containing peptide sequences, with
carbonyl-containing polypeptides and glycopolypeptides
is shown in Scheme lA, in which R, Rl, R2 and Z are the
same as described above with respect to Scheme 1 and AA
represents the above-described peptide sequence:
Scheme lA
Il / 1
R-Z-C-AA-NH-NH2 + O=C
R2 --~
Rl ~ ll
~ R-Z-C-AA-NH-N=C
R2
/ Hydrazone
~ Reduction
R ,Rl
R-Z-C-AA-NH-NH-CH
R2
3S Hydrazide
The reaction of polymer acyl hydrazine
derivatives with activated peptide carboxylic acid
groups of polypeptides and glycopolypeptides to form
W092/l65~ 2 1 o ~ 9 t 8 PCT/US92/02047
-18-
diacylhydrazides is illustrated by the reaction sequence
of Scheme 2:
Scheme 2
O Q
1) ~ activation
R3-C-OH e.g., EDC R3-C-R4
P R ,, ~
2) R3 C-R4 + R-Z-C-NH-NH2> R-Z-C-NH-NH-C-R3
Diacylhydrazide
R again represents the above-described
water-soluble polymers, and Z is the same as described
above for Formulae I-IV. R3 represents a polypeptide
containing aspartic acid, glutamic acid or a C-terminus
carboxylic acid residues. R4 represents one of the
above-described leaving groups substituted on the
peptide carboxylic acid when the carboxylic acid group
is activated as described above.
The reaction oS polymer acyl hydrazine
derivatives containing peptide sequences, with activated
peptide carboxylic acid groups of polypeptides and
glycopolypeptides is shown in Scheme 2A, in which R, R3,
R4 and Z are the same as described above with respect to
Scheme 2, and AA represents the above-described peptide
sequence:
Scheme 2A
1) 1 activation ~ U
R3-C-OH e.g., EDC R3-C-R4
2) R3-~-R4 + R-Z-C-AA-NH-NH2 ) R-Z-~-AA-NH-NH-C-R3
Diacylhydrazide
Generally, the conjugation of a polypeptide or
glycopolypeptide with a water-soluble polymer first
involves either oxidizing carbohydrate moieties of the
glycopolypeptide or activating carboxylic acid groups of
peptide moieties of the polypeptides or
glycopolypeptides. The carbohydrate moieties can be
W092/16555 PCT/US9~/02~7
~101918
--19--
oxidized by reacting the glycopolypeptide in aqueous
solution with sodium periodate or enzymatically using
galactose oxidase or combination of neuraminidase and
galactose oxidase as disclosed by Solomon et al.,
J. Chromatoaraphv, 510, 321-9 (1990). The reaction runs
rapidly to completion at room temperature. The reaction
medium is preferably buffered, depending upon the
requirements of the polypeptide or glycopolypeptide.
The oxidized glycopolypeptide is then recovered and
separated from the excess periodate by column
chromatography.
Carboxylic acid groups of peptide moieties can
be activated by reacting the polypeptide or
glycopolypeptide with an activating reagent such as a
water-soluble carbodimide such as EDC. The reactants
are contacted in an aqueous reaction medium at a pH
between about 3.0 and 8.0, and preferably about 5.0,
which medium may be buffered to maintain the pH. This
reaction is taking place under mild conditions
(typically 4 to 37`C) that are tolerated well by most
proteins.
Polypeptides or glycopolypeptides having
peptide units on which reactive carbonyl groups have
been generated may be directly reacted with the acyl
hydrazine polymer derivatives in an aqueous reaction
medium. This reaction medium may also be buffered,
depending upon the pH requirements of the polypeptide or
glycopolypeptide and the optimum pH for the reaction,
which pH is generally between about 5.0 and about 7.0
and preferably about 6Ø
In all instances, the optimum reaction media
pH for the stability of particular polypeptides or
glycopolypeptides and for reaction efficiency, and the
buffer in which this can be achieved, is readily
determined within the above ranges by those of ordinary
skill in the art without undue experimentation. For
purposes of this application, the operativeness of the
within reactions under mild conditions is defined as
WO92/16555 PCT/US92/02047
21~19~8
-20-
meaning that the preferred temperature range is between
about 4 and about 37`C. Those of ordinary skill in the
art will understand that the reactions will run somewhat
faster to completion at higher temperatures, with the
proviso that the temperature of the reaction medium
cannot exceed the temperature at which the polypeptides
or glycopolypeptides begin to denature. Furthermore,
those of ordinary skill in the art will understand that
certain polypeptides and glycopolypeptides will require
reaction with the polymer acyl hydrazine derivatives at
reduced temperatures to minimize loss of activity and/or
prevent denaturing. The reduced temperature required by
particular polypeptides and glycopolypeptides is
preferably no lower than 4`C and in no event should this
temperature be lower than O`C. The reaction will still
take place, although longer reaction times may be
necessary.
Usually, the polypeptide or glycopolypeptide
i5 reacted in aqueous solution with a quantity of the
acyl hydrazine polymer derivative in excess of the
desired degree of conjugation. This reaction also
proceeds under mild conditions, typically at 4 to 37`C.
The reaction medium may be optionally buffered,
depending upon the requirements of the polypeptide or
the glycopolypeptide, and the optimum pH at which the
reaction takes place. Following the reaction, the
conjugated product is recovered and purified by
diafiltration, column chromatography or the like. When
the acyl hydrazine polymer derivative includes an amino
acid or a peptide seguence, the degree of polymer
conjugation of the polypeptide or glycopolypeptide can
then be determined by amino acid analysis.
In view of the foregoing, it can be readily
appreciated that the acyl hydrazine polymer derivatives
of the present invention possess the optimum balance of
reactivity and selectivity so that polymer conjugates
can be formed with non-amino functional groups of
polypeptides and glycopolypeptides with virtually no
WO92/165~ PCT/US92/02~7
21019i8
-21-
competition between the acyl hydrazines and the peptide
amino groups for the non-amino functional groups. Thus,
crosslinking is prevented and the activity of the
polypeptide or glycopolypeptide is preserved.
The following non-limiting examples set forth
hereinbelow illustrates certain aspects of the
invention. All parts and percentages are by weight
unless otherwise noted, and all temperatures are in
degrees Celsius.
EXPERIMENTAL
MATERIALS:
Methoxy-PEG (mPEG) is available from
Union Carbide. The solvents used, as well as
beta-alanine ethyl ester HCL, hydrazine, P2O5, EDC,
N-hydroxy-5-norbornene-2,3-dicarboximide ~HONb), NaCNBH3
and NaIO4 are available from Aldrich Chemicals of
Milwaukee, Wisconsin. Chymotrypsin was obtained from
Worthington Chemical. BSA, ovalbumin and human
immunoglobulin G (IgG) are available from Sigma Chemical
of St. Louis, Missouri. G-CSF was obtained from Amgen
of Thousand Oaks, California.
EXAMPLE 1
SYNTHESIS OF mPEG-HYDRAZIDE DERIVATIVE CONTAINING
BETA-ALANINE:
mPEG (MWn 5,000, 100 g, 20 mmol) was dissolved
in toluene (250 mL~ and azeotropically dried for two
hours under reflux. The solution was brought to 25`C,
diluted with methylene chloride (50 mL~ and then treated
with phosgene (30 mL of 20 percent toluene
solution, 56 mmol) overnight. The solvents and the
excess of phosgene were removed by rotary evaporation
under vacuum. The solid residue of polymeric
chl~roformate was dissolved in methylene chloride
(g0 mL) and treated with beta-alanine ethyl ester
hydrochloride (6.1 g, 40 mmol) predissolved in methylene
chloride (total volume 30 mL) followed by triethylamine
(8.4 mL, 60 mmol). Approximately 30 minutes later, the
so}ution was diluted with toluene (50 mL), filtered and
W092/1655~ PCT/US92/~2~7
210191~ -22-
evaporated to dryness. The crude product was dissolved
in warm (50`C) ethyl acetate (500 mL) and filtered
through celite. The filtrate was diluted with
isopropanol to a total volume of 1,000 mL and left
overnight at 25`C to facilitate precipitation of the
product. Another recrystallization of the product from
isopropanol was performed. The yield of dried
mPEG-beta-alanine ethyl ester was 98 g (9s~). The
following IR and NMR spectrum were then obtained:
IR (neat):3341 (N-H), 1723 (C=0, urethane) CM 1, lH-NMR
(CDC13):Delta 1.17 (t, CH3CH20), 2.44 ~t)CH2CH2 of
beta-alanine), 3.64 (PEG), 3.9 (t, NH (C=0) OCH2),
4.11(2,CH3CH20), 5.25 (broad, NH) ppm.
The mPEG-beta-alanine ethyl ester
(62 g, 12 mmol) was dissolved in pyridine (120 mL) and
treated with hydrazine (12 mL, 0.375 mole) under reflux
for six hours. The solution was rotary evaporated to
dryness and the residue crystallized twice from
isopropanol and dried in vacuo over P205. The yield
was 60 g (97%).
The absence of free hydrazine in the product
was ascertained by reverse-phase (C-18) thin-layer
chromatography in water/methanol (3:1) using TNBS
spraying solution for detection.
Colorimetric assay of hydrazide groups using
TNBS gave 0.2 mmol/g (103% of theoretical). The
beta-alanine content of the polymer was 0.205 mmol/g
(105% of theoretical) as determined ~y amino acid
analysis of a completely hydrolysed
(6N HCl, llO`C, 24 h) aliquot of the product. 13C-NMR
(CDCl3):delta 171.2 (C=O,hydrazide); 156.4
(C=O,urethane); 71.8 (CH30_H2); 70.0 (PEG); 68.5
(CH2CH20C-0); 63.7 (CH2_~20C=0); 58.9 (CH30); 37.1
(NHCH2CH2); 33.g (NHCH2CH2) ppm. IR (neat):3328
(NH); 1719 (C=O,urethane); 1671 (C=O,hydrazide) cm 1
W092/1655~ PCT/US92/02~7
-2i-` 2101918
EXAMPLE 2
COUPLING OF ~PEG-HYDRAZIDE DERIVATIVE CONTAINING
BETA-ALANINE TO EDC-ACTIVATED CARBOXYL GROUPS OF
CHYMOTRYPSIN:
Chymotrypsin (20 mg, 8.0 x 10-7 mole,
1.28 x 10 5 equiv. of carboxyl) and the
mPEG-beta-alanine-hydrazide derivative of Example 1
(800 mg, 0.16 mmol) were dissolved in 8 ml of 1 mM HCl,
the solution was brought to pH 5.0 and treated with EDC
(15 mg, 0.078 mmol). The reaction mixture was stirred
gently at 25'C overnight while pH 5.0 was maintained by
addition of 1.0 N HCl. Excess reagents were removed by
extensive diafiltration of the reaction solution at 4`C
against one mM HCl. In order to determine the extent of
the coupling reaction, an aliquot of the
PEG-chymotrypsin conjugate was completely hydrolyzed
(6 N HCl, 110`C, 24 hours) and amino acid analysis was
performed. The amount of beta-alanine corresponded
to 2.4 molecules of mPEG per molecule of chymotrypsin.
EXAMPLE 3
COUPLING OF mPEG-HYDRAZIDE DERIVATIV~ CONTAINING
BETA-ALANINE TO HONb ACTIVATED CARBO%YL GROUPS OF
C~YMOTRYPSIN:
The same conjugation protocol as Example 2 was
employed, in the presence of HONb (28.7 mg, 0.16 mmol).
The PEG-chymotrypsin obtained had an
average 2.7 molecules of mPEG per molecule of protein,
based on quantitation of beta-alanine by amino acid
analysis. This demonstrates that the conjugation
process is only slightly enhanced by the presence of
HONb.
EXAMPLE 4
COUPLING OF mPEG-HYDRAZIDE DERIVATIVE CONTAINING
BETA-ALANINE TO EDC-ACTIVATED CARBOXYL GROUPS OF BSA:
A solution of BSA (20 mg) and a
mPEG-beta-alanine hydrazide derivative of Example 1
(800 mg, 0.16 mmol) in 50 mM NaCl (10 mL) was treated
with EDC (15 mg, 0.078 mmol) overnight at pH 5.0, 25`C
WO92/16555 PCT/US92/02~7
~10191~ - -
-24-
as in Example 2. Excess reagents were removed by
extensive diafiltration of the reaction solution at 4`C
against phosphate buffer (50 mM, pH 7.7). The content
of beta-alanine in the conjugate corresponded to 8.1
residues of mPEG per molecule of BSA. A GF-HPLC
comparison of the PEG-conjugate to native BSA was
performed with a BIOSEP SEC 4000 column, the results of
which are depicted in FIG. 1. The elution conditions
were 10% ~vol/voll methanol/40 mM phosphate buffer.
FIG. 1 depicts good homogeneity of the PEG-conjugate 1,
with a substantially increased molecular weight as
compared to the native BSA 2.
EXAMPLE 5
COUPLING OF mPEG-HYDRAZIDE DERIVATIVE CONTAINING BETA-
ALANINE TO OXIDIZED CARBOHYDRATE MOIETIES OF OvALBUMIN:
Ovalbumin (20 mg, 4.4 x 10 7 mole) dissolved
in Phosphate Buffered Saline (PBS) buffer, pH 6.0
(1.8 mL) was treated with NaIO4 (0.2 mL of 200 mM
aqueous solution). The reaction was allowed to proceed
in the dark at 4`C. After one hour, the oxidized
glycoprotein was separated from the excess of periodate
by passing the reaction solution through a 12 mL
Sephadex G-25 column equilibrated with acetate buffer to
pH 5Ø Additional samples were prepared and the
procedure was repeated equilibrating the column with PBS
buffer at pH 6.0 and phosphate buffer at pH 7Ø This
resulted in three separate reaction mixtures having
different buffering systems. To each mixture was added
the mPEG-beta-alanine-hydrazide derivative of Example l
(150 mg, 2.9 x 10 5 mole). Each of the three reaction
mixtures was divided into two equal portions and NaCNBH3
(0.3 mL of 6.6 mg/mL solution, 3.15 x 10-5 mole) was
added to one portion of each. The reactions were
allowed to proceed overnight at 4`C. Each solution was
diafiltered using phosphate buffer pH 7.7 until all the
unreacted reagents were removed. The conjugates in the
solutions to which the NaCNBH3 was added formed
W092/l65~5 PCT/US92/02~7
-25- ~ 918
acylhydrazine linkages. Analyses of the mPEG-ovalbumin
coniuaates are summarized in Table I below.
Table I
- Buffer Tvpe of Linkaae # of mPEG in
_ Con~uaate*
5.0 acetate hydrazone 3.8
5.0 acetate acylhydrazine 3.6
..._ .._
6.0 PBS hydrazone 4.3
6.0 PBS acylhydrazine 2.4
7.0 phosphat~ hydrazone 3.1
7.0 phosphat~ acylhydrazine 3.0
* The average number of mPEG chains attached to an
ovalbumin molecule was calculated from the results of
amino acid analysis of the conjugates.
Depicted in FIG. 2 is the GF-HPLC analysis
using a TSK G 4000SW column and a 10% (vol/vol)
methanol/40 mM phosphate buffer pH 7.5 mobile phase,
which showed good homogeneity of the mPEG-ovalbumin
conjugate 3, and a substantially increased molecular
weight as compared to the native ovalbumin 4.
EXAMPLE 6
ATTACHMENT OF mPEG-HYDRAZI~E DERIVATIVE CONTAINING 8ETA-
ALANINE TO THE CARBOHYDRATE MOIETY OF IMMUNOGLOBULIN G:
Human immunoglobulin G (IgG)
(5 mg, 3.12 x 10-5 mmol) in PBS (0.8 mL, 50 mM, pH 6.0)
was treated with a freshly prepared solution of sodium
periodate (0.2 mL, 200 mM) in PBS. The resulting
solution was incubated at 4`C. After one hour, the
oxidized glycoprotein was separated from the excessive
periodate by passing the reaction solution through
a 12 mL Sephadex G-25 column. The oxidized IgG was
collected and treated with the mPEG-beta-alanine
hydrazide derivative of Example 1
(200 mg, 1.25 x 10 3 mmol) at 4`C overnight. Each
solution was diafiltered using phosphate buffer pH 7.7
WO92/16555 PCT/US92/02~7
2lol9l8
-26-
until all unreacted reagents were removed. A GF-HPLC
comparison of the conjugate to native IgG was performed
with a ZORBAX GF-450 column, the results of which are
depicted in FIG. 3. A 0.2 M phosphate buffer, pH 7.5
mobile phase was used. FIG. 3 depicts good homogeneity
of the PEG-conjugate 5, with a substantially increased
molecular weight as compared to the native IgG 6. The
amount of beta-alanine was determined by amino acid
analysis of a hydrolyzed (6 N HCl, 110`C, 24 h) aliquot
of the PEG-IgG conjugate to correspond to six residues
of mPEG per protein molecule.
EXAMPLE ?
ATTACHMENT OF mPEG-HYDRAZIDE DERIVATIVE CONTAINING
BETA-ALANINE TO THE CARBOHYDRATE MOIETY OF
IMMUNOGLOBULIN G WITHOUT REMOVAL OF EXCESS PERIODATE:
IgG (5-4 mg, 3.37 x 10 5 mmol) and PBS
(50 mM, 0.91 mL) was treated with a freshly prepared
solution of sodium periodate (0.09 mL of 110 mM) at 4`C
in the dark. After one hour, mPEG-beta-alanine
hydrazide (100 mg, 6.3 x 10 4 mmol) was added to the
reaction mixture, which was then incubated overnight
at 4`C. The solution was diafiltered against phosphate
buffer at pH 7.7 until all the unreacted reagents were
removed. Pure PEG-IgG was obtained, which was
determined by amino acid analysis of the beta-alanine
content of a hydrolyzed aliquot of the conjugate
(6 N HCl, 110`C, 24 h) to contain 8.6 residues of mPEG
per molecule of IgG.
In addition to requiring fewer manipulations,
it appears that this one-pot conjugation procedure is
more efficient than the one described in EXAMPLE 6.
EXAMPLE 8
ATTACHMENT OF mPEG-HYDRAZIDE DERIVATIVE TO CARBODIIMIDE-
ACTIVATED CARBOXYL GROUPS OF G-CSF:
The mPEG-beta-alanine-hydrazide of Example 1
(15.0 g, 2.9 mmol) was added to a solution of G-CSF
(86 mg, 4.78 x 10 6 mole) in 1 mM ~Cl (86 mL), followed
by EDC (128 mg, 0.667 mmol). The reaction mixture was
WO9~/165~5 ! PCT/US92/02047
-27-` ~101918
gently stirred at 25`C for 90 minutes while maintaining
the pH at about 4.7 to 5Ø Excess reagents were
removed by extensive diafiltration of the reaction
solution at 4`C against l mM HCl. A GF-HPLC comparison
of the PEG-conjugate to native G-CSF was performed using
a ZORBAX GF-450 column, the results of which are
depicted in FIG. 4. The mobile phase
was 0.2 M phosphate buffer pH 7.5. FIG. 4 depicts
PEG-conjugate 7, with a substantially increased
molecular weight as compared to native G-CSF 8.
The average number of mPEG residues in the
PEG-G-CSF was 5.8, as determined by measuring the amount
of beta-alanine in an hydrolyzed (6 N HCl, llO`C, 24 h)
aliquot of the conjugate. ~NBS assay confirmed that
both native and PEG-modified G-CSF-l had the same number
of amino groups, indicating that the EDC activated
carboxylic acid groups of the protein did not react with
amino groups of the protein. The preparation of
mPEG-G-CSP gave four separate bands on SDS-PAGE
(PhastGel-, ~omogenous 12.5, Pharmacia) in the range
from 29,000 to 67,000 daltons. Isoelectric Focusing
(PhastGel-, IEF 3-9, Pharmacia) of the mPEG-G-CSF-l
resulted in the separation of six bands with pI's
arranging between 6.8 and 9.0, noticeably higher than
the native protein (pI 5.2; 5.9). This clearly
indicates that the protein became more basic as a result
of the conjugation with the peptide carboxylic acid
groups without crosslinking of the activated carboxylic
acid groups with the peptide amino groups.
As will be readily appreciated, numerous
variations and combinations of the features set forth
above can be utilized without departing from the present
invention as set forth in the claims. Such variations
are not regarded as a departure from the spirit and
scope of the invention, and all such modifications are
intended to be included within the scope of the
following claims.
WO92/16555 . PCTIUS92/02047
2101918
. -28-
Industrial AlicabilitY
The present invention is applicable to the
production OI' polymers conjugated with various
biologically active and pharmaceutically active
compounds representing a novel form of drug delivery.
