Note: Descriptions are shown in the official language in which they were submitted.
43
Field of the Invention
This invention relates to water-soluble
polymers, such as monomethoxypoly(ethylene glycol),
that are modified to form a hydrazone linkage with an
aldehyde group on a protein, and the invention also
relates to protein molecules modified by these water-
soluble polymers.
This invention further relates to such water-
soluble polymers that are modified to form an oxime
linkage, and protein molecules modified thereby.
I Backqround Art
I Protein and other similar organic molecules may
! 15 be chemically modified by covalent conjugation to
I water-soluble organic polymers such as polyethylene
! glycol (PEG) The production of such protein
conjugates is of interest because of the desirable
properties conferred on polypeptides by the
attachment of the water-soluble polymers. These
desirable properties include increased solubility in
aqueous solutions, increased stability during
storage, reduced immunogenicity, increased resistance
.
.. ~. ~ , .
, ~ . .
,~"..: ~
21iO~3
--2
to enzymatic degradation, compatibility with a wider
variety of drug administration systems, and increased
in vivo half-life. These properties that are brought
about by the derivatization of polypeptides with PEG
or other waker-soluble polymers are especially of
interest when the polypeptide is to be used as a
therapeu*ic agent injected into the body or when the
polypeptide is to be used in assays, usually
immunoassays, for the detection and/or quantification
of a compound of interest.
The attachment of reporter groups, ligands, etc.
to proteins through a glycoprotein's carbohydrate
moiety has been described [Weber, P. and Hof, L.
(1975) Biochem. Biophys. Res. Commun. 65, 1298-1302;
O'Shannessy, D.J., Dobersen, M.J., and Quarles, R.H.
(1984) Immunol. Lett. 8, 273-277; O'Shannessy, D.J.
and Quarles, R.H. (1985) J. Appl. Biochem. 7,
347-355; Chua, M.-M., Fan, S.-T., and Karush, F.
(1984) Biochim. Biophys. Acta 800, 291-300,
O'Shannessy, D.J. and Wilchek, M. (1990) Anal.
Biochem. 191, 1-8; Koppel, G.A. (1990) Bioconjugate
Chem. 1, 13-23]. A number of groups have been
covalently attached in this manner including biotin,
fluorescent probes, anticancer compounds, and solid
supports.
U.S. Patent No. 4,847,325 describes the
possibility of synthesizing a PEG-amine,
PEG-hydrazide or PEG-hydrazine and attaching it to a
glycoprotein. However, no experimental evidence was
given that these PEG-derivatives had been
synthesized, that these PEG-derivatives could modify
an oxidized glycoprotein, and what the resulting
biological properties of these putative PEG-proteins
might be.
. ~
.,,I,i,,
,^,~
, ,- .
~,, .
.,
43
The publication by Kogan, T.P., Synthetic
Communications, 22(16), 2417-2424 (1992), describes
the synthesis of a monomethoxypoly(ethylene glycol)-
hydrazide.
The enzyme peroxidase has been modified with PEG
through its carbohydrate groups [Urrutigoity, M. and
Souppe, J. (1989) Biocatalysis 2, 145-149~. In this
modification PEG-diamine was reacted with oxidized
peroxidase, and the resulting imine was reduced with
borohydride to form a stable bond between PEG and the
carbohydrate group on the protein. Three molecules
of PEG-20,000 were attached to the enzyme. A
possible problem in using PEG-diamine in this manner
is intermoleeular cross-linking taking place between
the protein molecules with PEG diamine functioning as
the cross-linker. Another drawback of using PEG-
diamine is the consumption of two available aldehyde
groups for each PEG-diamine attached to the protein,
thus lowering the potential number of sites for PEG
incorporation.
Besides the hydrazone forming mPEG derivatives,
also synthesized is a series of oxime forming mPEG
derivatives. Oximes are formed by the reaction of
hydroxylamine or oxylamine derivatives with aldehyde
or ketone groups. Polystyrene substituted
benzophenone oximes have been used as supports for
solid-phase peptide synthesis [DeGrado, W.F., and
; Kaiser, E.T. (1980) J. Org. Chem. 45, 1295-1300]. In
this example the growing peptide chain is coupled to
the oxime group via an ester linkage. The
substituted oxime bond is quite stable, and even
unsubstituted aldoximes show good stability towards
the Beckmann rearrangement requiring 60 hr at 100 in
¦ the presence of silica gel to yield the reaction
~ '
~,
'~ . '
,.,,, , ~
, -
, ,,~,,j;,
_4_
[March, J. (1985) Advanced Organic Chemistry, New
York-John Wiley & Sons, pp 987-989]. The oxime
linkage has been used to couple morpholinodoxorubicin
to an antibody [Mueller, B.M., Wrasidlo, W.A., and
Reisfeld, R.A. (1990) Bioconjugate Chem. 1, 325-330].
In this example the ketone group of
morpholinodoxorubicin was reacted with aminooxyacetic
acid. The newly coupled free acid group was
activated, and morpholinodoxorubicin was linked to
the free amino groups of lysine on a monoclonal
antibody.
A number of proteins have been modified by PEG.
For a review see Inada, Y., Yoshimoto, T. Matsushima,
A., and Saito, Y. (1986) Trends Biotechnol. 4: 68-73.
A number of patents have issued and applications
published in this field as listed below: U.S. Pat.
No. 4,179,337; U.S. Pat. No. 4,609,546; U.S. Pat.
No. 4,261,973; U.S. Pat. No. 4,055,635; U.S. Pat. No.
3,960,830; U.S. Pat. No. 4,415,665; U.S. Pat. No.
4,412,989; U.S. Pat. No. 4,002,531; U.S. Pat. No.
4,414,147; U.S. Pat. No. 3,788,948; U.S. Pat. No.
4,732,863; U.S. Pat. No. 4,745,180; EP No. 152,847;
EP No. 98,110 published January 11, 1984. The above
patents and patent publications also describe the use
of other water-soluble polymer protein modifying
reagents including but not restricted to
polypropylene glycol (PPG), polyoxyethylated polyol
(POP), heparin, heparin fragments, dextran,
polysaccharides, polyamino acids including proline,
polyvinyl alcohol (PVA) and other water-soluble
- organic polymers.
; A recent patent publication (WO90/12874)
describes the preparation of an mPEG-EPO in which the
EPO contains a cysteine residue introduced by genetic
,
,
...
;,', ': ~
2~ o~ 43
engineering. A cysteine specific mPEG-reagent is
then covalently attached to the genetically
engineered free sulfhydryl group. Only one mPEG
molecule could be incorporated into EPO and no
evidence of this incorporation was presented. Also
no biological or biophysical properties of the
resulting mPEG-EPO were described.
Erythropoietin is a glycoprotein which regulates
red blood cell production. Erythropoietin exerts its
biological effect by binding to receptors on
erythroid precursors (Krantz, S.B., Blood 77: 419-434
(1991)). The binding of erythropoietin to its
receptor causes erythroid precursors to proliferate
and differentiate into mature red blood cells. Other
growth factors such as interleukin 3 or
granulocyte-macrophage colony-stimulating factor also
are involved in erythropoiesis along with cofactors
such as iron, folic acid, and vitamin B12. Currently
erythropoietin is approved for use in anemia of
chronic renal failure in both dialysis and
predialysis patients and for the anemia of ~IV
infection and in combination with zidovudine therapy.
Current uses for erythropoietin under study include
anemia of cancer, presurgical autologous blood
donation, and perisurgical adjuvant therapy.
Erythropoietin consists of 155 amino acids which
includes two disulfide bridges. Erythropoietin has
four carbohydrate chains emanating from the protein
backbone. Three of the carbohydrate groups are
N-linked and are attached to asparagines 24, 38, and
83. Also there is one O-linked carbohydrate group
secured to serine 126. The carbohydrate chains are
branched and consist of fucose, galactose,
N-acetylgalactosamine, N-acetylglucosamine, mannose,
., .
:. .
.
y~
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.. ~,, .
~1~0~3
--6--
and sialic acid. The carbohydrate composition of
erythropoietin is heterogeneous as determined by
Sasaki, H., Bothner, B., Dell, A., and Fukuda, M.
(1987) J. Biol. Chem. 262, 12059-12076. Carbohydrate
groups account for about 40~ of the protein's weight.
The carbohydrate groups on erythropoietin are
believed to increase the solubility of erythropoietin
and prolong its serum half-life.
Several limitations exist with respect to which
~0 polypeptides may be covalently conjugated to water-
soluble polymers and the extent to which the
polypeptides can be modified. Different water-
soluble polymer reagents vary with respect to the
functional groups that provide for coupling to amino
acid residues in polypeptides of interest. Specific
functional groups provide for the coupling of water-
soluble polymers to specific amino acid residues.
Modification of lysine residues using different
mPEG-reagents having different properties such as
succinimidyl carbonate-PEG, succinimidyl succinate-
PEG, imidate-PEG, cyanuric chloride-PEG,
carbonyldiimidazole-PEG, and PEG-phenylcarbonate
derivatives (4-nitrophenol and 2,4,5-trichlorophenol)
have been described. Each reagent has its own
specific property. The subject application involves
new carbohydrate PEG modifying agents with different
specificities to oxidized carbohydrate groups
analogous to the different lysine modifying PEG-
derivatives.
Glycoproteins, i.e., polypeptides covalently
joined to a carbohydrate molecule or molecules,
provide additional opportunities for providing
different methods of water-soluble polymer
derivatization of a polypeptide because of the
."
~- , ,,., ~.
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,,,
~, .
7 ~o~43
presence of the carbohydrate moieties on the
polypeptide. Water-soluble polymer reagents may be
coupled directly to the carbohydrate moieties of
glycoproteins as opposed to the amino acid
polypeptide backbone, i.e., various functional groups
present on the polypeptide, o~ the glycoprotein. It
may be advantageous to couple water-soluble reagents
to the carbohydrate moiety of a glycoprotein rather
than t~ the polypeptide backbone amino acids because
of differences in charge displacement, steric
hinderance, amino acid residues at active sites, and
other problems that may disrupt the structure and
function of the polypeptide component of the water-
soluble polymer modified glycoprotein.
By providing for water-soluble polymer reagents
that may be coupled to the carbohydrate moiety of
glycoproteins it may be possible to covalently
conjugate water-soluble polymers to proteins without
substantially adversely affecting the biological
activity of proteins that would be adversely affected
through coupling at other amino acid residues.
SUM~ARY OF THE INVENTION
~he present invention provides methods and
compositions for modifying polypeptides with
derivatives of water-soluble organic polymers, i.e.,
water-soluble polymer reagents, that form a hydrazone
linkage with an aldehyde group or group with similar
chemical reactivity, e.g., ketones, lactols,
activated carboxylic acids or activated carboxylic
acid derivatives on a polypeptide. Novel hydrazide,
semicarbazide, aryl hydrazide, thiosemicarbazide,
hydrazide carboxylate, carbonic acid dihydrazide,
carbazide, and thiocarbazide derivatives of
~;,~; .~, :
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~., -' ' '
2~ 3 ::--8--
polyethylene glycol (PEG) and other wat~r-soluble
polymers are provided. One or more of the water-
soluble polymer reagents may be coupled to individual
polypeptides or similar organic molecules to form
hydrazones that link the polypeptide to water-soluble
polymers.
Another aspect of the subject invention is to
provide for proteins, particularly glycoproteins,
modified by the covalent attachment of hydrazone
linkage water-soluble polymer derivatives.
Also disclosed are methods and compositions for
modifying polypeptides with derivatives of water-
soluble organic polymers that form an oxime linkage
with the above-mentioned aldehyde or similarly
reactive groups. Novel oxylamine derivatives as
listed hereinbelow, of polyethylene glycol (PEG) and
other water-soluble polymers are provided and wherein
one or ~ore of the water-soluble polymer reagents may
be coupled to individual polypeptides or similar
organic molecules to form oximes that link the
polypeptide to water-soluble polymers.
Another aspect of the subject invention is to
provide for proteins, particularly glycoproteins,
modified by the covalent attachment of oxylamine
water-soluble polymer derivatives.
The water-soluble polymer reagents of the
subject invention include hydrazone linkage and oxime
linkage forming derivatives of polyethylene glycol
homopolymers, polypropylene glycol homopolymers,
copolymers of ethylene glycol with propylene glycol,
wherein said homopolymers and copolymers are
unsubstituted or substituted at one end with an alkyl
group, polyoxyethylated polyols, polyvinyl alcohol,
polysaccharides, polyvinyl ethyl ethers, and
'
2~ 3
Poly[(2-hydroxyethyl)-DL-aspartamide] and other
water-soluble organic polymers. Polyethylene glycol
water-soluble polymers include polyethylene glycol
where one of the terminal hydroxyl group is modified
with an R group, i.e., R0-PEG, where R may be alkyl,
aryl, alkyaryl, aroyl, alkanoyl, benzoyl,
arylalkylethers, cycloalkyl, cycloalkylaryl, and the
like. The water-soluble polymers listed are only
exemplary of water-soluble polymers represented by P.
lo Various derivatives of the specifically recited
water-soluble polymers are also contemplated,
provided that the derivatives are water-soluble.
More preferably, the water-soluble polymer P is
selected from the group consisting of polyethylene
glycol and derivatives thereof, the monomethyl ether
of polyethylene glycol (mPEG) being particularly
preferred (so as to avoid cross-linking between
proteins).
Polypeptides of interest for water-soluble
polymer derivatization by the subject water-soluble
polymer include hormones, lymphokines, cytokines,
growth factors, enzymes, vaccine antigens, and
antibodies. Water-solùble polymer derivatization of
erythropoietin (EP0), especially recombinant
erythropoietin, and precursors, intermediates and
mimetics thereof, are of particular interest.
Another aspect of the invention is to provide
erythropoietin that has been partially oxidized and
subsequently combined with (i) a semicarbazide
derivative of the monomethoxypoly (ethylene glycol)
(mPEG), so as to produce mPEG derivatized
erythropoietin molecules containing 17-25 mPEG
molecules/molecule of erythropoietin (joined through
hydrazone linkages), (ii) a carboxylate hydrazide
,
.. . . .
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.,~'
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--10--
derivative of mPEG so as to produce derivatized EPO
containing about 22-32 mPEGs/EPO (joined through
hydrazone linkages), and (iii) oxylamine derivatives
of mPEG so as to produce derivatized EPO containing
about 3-36 mPEGs/EPO (joined through oxime linkages),
all as measured by gel filtration retention time.
Another aspect of the invention is to provide
methods of activating polypeptides for covalent
conjugation with the subject water-soluble polymer
reagents.
DESCRIPTION OF THE DRAWINGS
Figure la shows an HPLC chromatogram of EPO.
Figure lb shows an HPLC chromatogram of EPO modified
with a hydrazine derivative of mPEG5000. Figure lc
shows an HPLC chromatogram of EPO modified with a
succinimide ester of mPEG5000.
Figure 2 is a graph of the hematocrit level of
mice treated with mPEG5000-EPO containing different
amounts of attached mPEG (28, 18 and 12
mPEGs/molecule of EPO). The EPO derivatized with 18
or 28 mPEG5000/molecule are derivatized using the
subject semicarbazide compound. EPO derivatized with
12 mPEG5000/molecule is derivatized using the subject
hydrazide compound.
Figure 3 is a graph showing the ability of EPO,
hydrazide mPEG5000 EPO (12 PEG/EPO), hydrazide
mPEG12000-EPO (6 PEG/EPO), thiosemicarbazide
mPEG5000-EPO (25 PEG/EPO), semicarbazide mPEG12000-
EPO (14 PEG/EPO), and semicarbazide mPEG12000-EPO (29
PEG/EPO) to bind a monoclonal antibody specific for
EPO in an ELISA assay.
Figure 4 is a graph showing a comparison of the
biological activity of EPO when modified with either
,
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2 ~ ~ ~
mPEG-Hydrazide (HY) or mPEG-Semicarbazide (SC). Two
different molecular weights (8500 and 5000) of mPEG
were used in the comparison. Mouse albumin is used
as the control.
Figure 5 is a plot showing the hematocrit level
of mice treated with mPEG8500-EPO containing
different amounts of attached mPEG (34, 20 and 12
mPEGs). EPO derivatized with 34 or 20
mPEG85000/molecule are derivatized using the subject
semicarbazide compound. EPO derivatized with 12
mPEG85000/molecule is derivatized using the subject
hydrazide compound. Mouse albumin is used as the
control.
Figure 6 is a graph showing the results of ELISA
assays for mPEG modified EPO using a Clinigen0 EP0
EIA test kit. In the legend, SC5-24 refers to
semicarbazide mPEG5000 modified EP0 with 24 molecules
of mPEG/molecule of EPO, SC5-18 refers to the
semicarbazide mPEG5000 modified EP0 with 18 molecules
of mPEG semicarbazide /molecule of EP0.
Figure 7 is a graph showing the circulating
half-life of EPO in plasma. In the legend SC5-18-iv
refers to the semicarbazide mPEG5000 modified EPO
with 18 molecules of mPEG /molecule of EPO and
injected intravenously, EPo-iv refers to injected
intravenously.
Figure 8 is a graph showing changes in
hematocrit level in response to injection with EPO.
In the legend, the term SC5-1~ refers to the
semicarbazide mPEG5000 modified EPO with 18 molecules
of mPEG /molecule of EPO, the term SC5-22 refers to
' the semicarbazide mPEG5000 modified EPO with 22
molecules of mPEG5000 /molecule of EPO, the term HY5-
!
, .
;,,.
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,,~:r.jr
."} ~
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2~543
-12-
8 refers to the hydrazide mPEG5000 modified EP0 with
8 molecules of mPEG5000 /molecule of EP0.
Figure 9 is a graph showing changes in
hematocrit level in response to injection with EPO.
In the legend, the term SC5-24 refers to the
semicarbazide mPEG5000 modified EP0 with 24 molecules
of mPEG Imolecule of EPO, the term TS5-25 refers to
the thiosemicarbazide mPEG5000 modified EP0 with 25
molecules of mPEG5000 /molecule of EPO, the term ~H5-
22 refers to the dihydrazide mPEG5000 modified EPO
with 22 molecules of mPEG5000 /molecule of EPO, M.A.
refers to the mouse albumin control.
Figure 10 is a graph showing changes in
hematocrit level in response to injection with EPO.
In the legend, the term TS5-17 refers to the
thiosemicarbazide mPEG5000 modified EP0 with 17
molecules of mPEG5000 /molecule of EPO, the term
SC8.5-12 refers to the semihydrazide mPEG8500
modified EP0 modified with 12 molecules of mPEG8500
/molecule of EP0, SC2-15 refers to the semicarbaæide
mPEG2000 modified EPO with 15 molecules of mPEG2000
/molecule of EPO, M.A. refers to the mouse albumin
control.
Figure 11 is a graph showing changes in
hematocrit level in response to injection with EP0.
The legend is as follows: the term SC5-18 refers to
the mPEG5000 semicarbazide modified EPO with 18
molecules of mPEG5000 /molecule of EPO, the term
SC12-14 refers to the semicarbazide mPEG12,000
` 30 modified EPO with 14 molecules of mPEG12,000
/molecule of EP0, the term SC5-28 refers to the
semihydrazide mPEG5000 modified EPO with 28 molecules
of mPEG5000 /molecule of EP0, the term HY12 6 refers
to the hydrazide mPEG12,000 modified EP0 with 6
" ,,
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.-. ,s,, :
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-13-
molecules of mPEG12,000 /molecule of EPO, M.A. refers
to the mouse albumin control.
Figure 12 is a graph showing changes in
hematocrit level in response to injection with EPO.
In the legend, the term SC8.5-34 refers to the
semicarbazide mPEG8500 modified EPO with 34 molecules
of mPEG8500 /molecule of EPO, the term SC12-29 refers
to the semicarbazide mPEG12,000 modified EPO with 29
molecules of mPEG12,000 /molecule of EPO, M.A. refers
to the mouse albumin control.
Figure 13 is a graph of hematocrit levels in
mice injected subcutaneously or intravenously EPO and
EPO derivatives. The legend is as follows: EPO-SC
refers to EPO injected subcutaneously, EPo-iv refers
to EPO injected intravenously, SC5-28-SC refers to
semicarbazide mPEG5000 modified EPO with 28 molecules
of mPEG5000 /molecule of EPO injected subcutaneously, .
SC5-28-iv refers to semicarbazide mPEG5000 modified
EPO with 28 molecules of mPEG5000 /molecule of EPO
in~ected intravenously, HY12-6-SC refers to hydrazide
mPEG12,000 modified EPO with 6 molecules of
mPEG12,000 /molecule of EPO injected subcutaneously,
HY12-6-iv refers to hydrazide mPEG12,000 modified EPO
with 6 molecules of mPEG12,000 /molecule of EPO
injected intravenously, M.A.-sc refers to mouse
albumin control injected subcutaneously, M.A.-iv
refers to mouse albumin control injected
intravenously.
Figure 14 is a graph showing hematocrit levels
in mice injected with multiple versus single doses of
.1 micrograms of EPO. The legend is as follows:
EPOx3sc refers to EPO injected subcutaneously three
times a week, EPox3iv refers to EPO injected
intravenously three times a week, SC5-22xlsc refers
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-14- 2i~ 43
to semicarbazide mPEG5000 modified EPG with 22
molecules of mPEG5000 /molecule of EPO injected
subcutaneously once a week, SG5-22xliv refers to
semicarbazide mPEG5000 modified EPO with 22 molecules
of mPEG5000 /molecule of EPO injected intravenously
once a week, M.A.x3 refers to control mouse albumin
injected intravenously three times a week.
Figure 15 is a graph showing hematocrit levels
in mice with tumor necrosis factor ~(TNF~)-induced
anemia and injected with EPO and derivatives of EP0.
The legend is as follows: TNF(5) refers to TNF
injected over five days, T+EP0(5) refers to TNF and
EPO injected simultaneously over a period of five
days, T+EPO(2) refers to TNF injected over a period
of five days and EPO injected on days 1 and 4,
T+SC5-18(5) refers to TNF injected over a period of
five days simultaneously with semicarbazide mPEG5000
modified EP0 with 18 molecules of mPEG5000 /molecule
of EPO, T+SC5-18(2) refers to TNF injected over a
period of five days simultaneously with semicarbazide
mPEG5000 modified EPO with 18 molecules of mPEG5000
/molecule of EPO injected on days 1 and 4, M-A refers
to the mouse albumin control.
Figure 16 is a graph showing hematocrit changes
in response to injection with EPO. In the legend,
18PEG-A refers to EPO modified with mPEG-0-CH2CH2-NH-
CO-ONH2, (formula XXI of the invention), with 18 mPEG
molecules per molecule of EP0; 31 PEG-C refers to EPO
modified with mPEG-0-CH2CH2-NH-CO-CH2-ONH2 (formula
XXIV of the invention), with 31 mPRG/EPO; 25PEG-C
refers to EP0 modified with mPEG-0-CH2-CH2-NH-C0-CH2-
ONH2 (formula XXIV of the invention), with 25
molecules of mPEG/molecule EPO.
, . . .
:,
:
,,
.`' :
".~-,
2110~3
-15-
Figure 17 is a graph showing hematocrit changes
in response to injection with EPO. In the legend, 22
PEG-B refers to EPO modified with the oxime-
derivatized mPEG-0-CH2CH2-ONH2 (formula XXIII of the
invention), with 22 mPEG molecules/molecule of EPO;
17 PEG-B refers to EPO modified with the same oxime
linker having 17 mPEG molecules/molecule of EPO, and
12 PEG-B refers to EPO modified with the same oxime
linker having 12 mPEG molecules/molecule of EPO.
Figure 18 is a graph showing the ability of EPO,
m-PEG-O-CH2CH2-NH-CO-ONH2 ("A") mPEG5000 EPO (1~
PEG/EPO), mPEG-0-CH2CH2-ONH2 ("B") mPEG5000 EPO (22
PEG/EPO), mPEG-0-CH2CH2-ONH2("B") mPEG5000 EPO (17
PEGIEPO),mPEG-O-CH2CH2-ONH2 ("B") mPEG5000 EPO (12
PEG/EPO), mPEG-O-CH2CH2-NH-CO-CH2-ONH2 ("C") mPEG5000
EPO (31 PEG/EPO), and mPEG-O-CH2CH2-NH-CO-CH2-ONH2
("C") mPEG5000 EPO (25 PEG/EPO) to bind a monoclonal
antibody specific for EPO in an ELISA assay.
Figure 19 is a graph showing EPO dependent cell
proliferation using mPEG-EPOs. In the legend 18 PEG-
A refers to EPO modified with mPEG-0-CH2CH2-NH-CO-0NH2
(formula XXI) at 18 PEG/EPO, 22 PEG-B refers to EPO
modified with mPEG-0-CH2CH2-ONH2 (formula XXIII) at 22
PEG/EPO, 17 PEG-B refers to formula XXIII at 17
PEG/EPO, 12 PEG-B refers to formula XXIII at 12
PEG/EPO, 31 PEG-C refers to EPO modified with mPEG-O-
CH2CH2-NH-CO-CH2-ONH2 (formula XXIV) at 31 PEG/EPO, and
25 PEG-C refers to formula XXIV at 25 PEG/EPO.
Figure 20 is a graph showing hematocrit chang~s
in response to injection with EPO. In the legend, 31
mPEGs refers to EPO modified with the oxime-
derivatized mPEG-O-CO-NHNH~ (formula II of the
invention), having 31 mPEG molecules/molecule EPO.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
~,
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,,:
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-16-
Definitions
The term "water-soluble polymer reagent" as used
herein, refers to a water-soluble polymer modified so
as to contain a functional group that provides for
the covalent conjugation of the water-soluble poly~er
to a polypeptide.
The term "polypeptide" as used herein, refers to
polypeptides of various sizes, including larger
polypeptides (frequently referred to as proteins),
small peptides, and glycoproteins.
The term "oxidation activatable group" as used
herein, refers to functional groups such as alcohols,
polyols, lactols, amines, phenols, carboxylic acids,
or carboxylic acid derivatives that react with the
hydrazide portion or the oxylamine portion of the
subject compounds after the functional group has been
exposed to oxidative conditions. Oxidation
activatable groups present on a polypeptide that is a
glycoprotein may be present on the carbohydrate
portion of the glycoprotein or on the amino acid
residue portion of the glycoprotein. Exemplary, but
not exclusive, of oxidation activatable groups are
hydroxyl groups present on the carbohydrate portion
of glycoproteins. The hydroxyl groups may be
oxidized to hydrazide reactive aldehydes or oxylamine
reactive aldehydes, depending on the derivative
employed.
The term "partial oxidation" as used herein,
refers to the processes of oxidation that proceed to
an extent that does not completely abolish the
biological activity of the polypeptide being
oxidized.
The term "activated for conjugation" as used
herein with respect to polypeptides, refers to the
:~,
:i,',:,,
17 2~0543
partial oxidation of a polypeptide, where the extent
of oxidation is sufficient to convert at least one
oxidation activatable group to a functional group
capable of chemically reacting with the hydrazide
portion or oxylamine portion (or similar functional
group portion) of one of the subject water-soluble
polymer reagents.
The term "biological activity" as used herein,
refers to biologically relevant properties of a
compound including: enzymatic activity, the ability
to bind to receptors (including antibodies), the
ability to bind ligands, the ability to induce an
immune response, therapeutic activity and the like.
The term "antibodies," as used herein, includes
i5 both polyclonal and monoclonal antibodies with
natural immunoglobulin sequences, synthetic antibody
derivatives, and the like; antibodies may be modified
so as to be joined to any of a variety of labels,
p fluorescent, radioactive, enzymatic, biotin/avidin or
the like. Synthetic antibody derivatives include
natural immunoglobulin sequences that have been
mutated and selected for altered binding specificity,
s various immunoglobulin gene derived polypeptides,
typically single chain, produced by genetically
modified bacteria, antibodies modified so as to
; contain modified constant regions and the like; a
~ review of such synthetic antibody derivatives based
- o~ the principles of antibody formation is provided
in Winter and Milstein, Nature. 349: 293-299 (1991).
i
The Invention
The subject invention provides novel polypeptide
modifying reagents that are hydrazine or oxylamine
derivatives of water-soluble polymers such as PEG,
i
,,
, .
;
.. ,. :,. . . .
: .,-
"~
~.7,."
P,
~iio~3
i.e, polyethylene glycol, for use in modifying
polypeptides so as to be bound to water-soluble
polymers. The water-soluble polymer reagents of the
subject invention may be used to covalently attach a
variety of water-soluble polymers to polypeptides of
interest. The subject hydrazine and oxylamine
derivatives of water-soluble polymers, i.e., water-
soluble polymer reagents, may be covalently attached
to proteins through reactions with aldehyde groups or
other suitable functional groups present on the
protein of interest. Aldehyde groups may be
introduced by partially oxidizing the hydroxyl groups
(or other oxidation activatable groups) on the
polypeptide. Examples of oxidation activable groups
include the hydroxyl groups present on the
carbohydrate moieties of a glycoprotein. Suitable
methods of oxidation, i.e., partial oxidation,
include treating the polypeptide of interest with an
oxidizing agent such as periodate or other oxidation
agents known to those of skill in the art, or adding
an enzyme capable of catalyzing oxidation reactions
on portions of the protein of interest, e.g.,
galactose oxidase. Another aspect of the subject
invention is to provide polypeptides modified by the
reagent molecules, i.e., the subject water-soluble
polymer hydra~ine or oxylamine derivati~es, so as to
be covalently bonded to one or more water-soluble
polypeptides.
Preferred formulae of the compounds useful for
coupling water-soluble polymers to polypeptides are
as follows:
HYDRAZINE DERIV~TIVES
(I) p-0-CH2-CO-
.j,, .
, .
.y "
,~"' ~,
2110 j~3
--19--
a hydrazine derivative;
(II) P-O-CO-NHNH2,
a hydrazine carboxylate derivative;
(III) P-NH-CO-NHNH2
a semicarbazide derivative;
(IV) P-NH-CS-NHNH2,
a thiosemicarbazide derivative;
(V) P-NHCO-NHNHCO-NHNHz
a carbonic acid dihydrazide derivative;
(VI) P-NHNHCONHNH2,
a carbazide derivative;
(VII) . P-NHNHCSNHNH2,
a thiocarbazide derivative;
(VIII) p-NH-co-c6H4-NHNH2l
an aryl hydrazide derivative;
(IX) p-o-co-cH2cH2-co-NHNH
' a hydrazide derivative;
OXYLAMINE DERIVATIVES
(XIX) p-o-cH2cH2-co-oNH2;
.
(XX) p-O-CH2CH2-0-cO-oNH2;
(XXI) p-O-CH2CH2-NH-cO-oNH2;
.
:. .
.
, ~,f~"
, .~,. . . .
21~ 3
-20-
(XXII) p-O-CH2CH2-NH-cs-
(XXIII) p-o-cH2cH2-oNH
(XXIV) P-o-cH2cH2-NH
(XXV) P-o-cH2c~2-o-co-cH2-oNH2i
(XXVI) P-O-CH2CH2-CH(OH)-C~2-ONH2; and
(XXVII) P-o-cH2cH2-co-cH2-oNH2-
P represents a water-soluble organic polymer in
the above formulae. Water-soluble organic polymers
of interest have hydroxyl groups appended to the
10 polymer backbone and may be selected from known
water-soluble polymers including but not limited to:
(a) dextran and dextran derivatives, including
dextran sulfate, P-amino cross linked dextrin, and
carboxymethyl dextrin (b) cellulose and cellulose
derivatives, including methylcellulose and
carboxymethyl cellulose (c) starch and dextrines, and
derivatives and hydroylactes of starch (d)
polyalklyene glycol and derivatives thereof,
including polyethylene glycol, methoxypolyethylene
glycol, polyethylene glycol homopolymers,
polypropylene glycol homopolymers, copolymers of
ethylene glycol with propylene glycol, wherein said
~ homopolymers and copolymers are unsubstituted or
substituted at one end with an alkyl group (e)
~`' 25 heparin and fragments of heparin, (f) polyvinyl
alcohol and polyvinyl ethyl ethers, (g)
polyvinylpyrrolidone, (h) ~ Poly[(2-hydroxyethyl)-
DL-aspartamide, and (i) polyoxyethylated polyols.
.
,~ ,
,, .
., ~, . . .
.. ~ ' , : " ~ -
;.~ ~ .
2~ 3
-21-
Preferably, the water-soluble polymer P is selected
from dextran and dextran derivatives, dextrine and
dextrine derivatives, and more preferably
polyethylene glycol and derivatives thereof.
Polyethylene glycol water-soluble polymers include
polyethylene glycol where one of the terminal
hydroxyl group is modified with an R group, i.e., R0-
PEG, where R may be alkyl, aryl, alkyaryl, aroyl,
alkanoyl, benzoyl, arylalkylethers, cycloalkyl,
cycloalkylaryl, and the like. The water-soluble
polymers listed are only exemplary of water-soluble
polymers represented by P. Various derivatives of
the specifically recited water-soluble polymers are
also contemplated, provided that the derivatives are
water-soluble. More preferably, the water-soluble
polymer P is selected from the group consisting of
polyethylene glycol and derivatives thereof, the
monomethyl ether of polyethylene glycol (mPEG) being
particularly preferred (so as to avoid cross-linking
between proteins). When polypeptides modified by the
water-soluble polymer reagents of the subject
invention are to be used as pharmaceuticals, polymer
P should be non-toxic.
The compounds of formulae I-IX may be
represented generally by the formula:
Q
P-Y-l=X
wherein X is O or S; Q is selected from the group
consisting of -NHNH2, and -C6H4-NHNH2; and Y is
selected from the group consisting of -0-, -OCH2-, -
NH-, -NHNH-, -O-C0-CH2CH2- and -NHCO-N-NHNH-; and P is
a water- soluble organic polymer (as in compounds
I-IX).
ij, . . . . . . . . ...
;~ ' ' ' ~', ,: . '
~ , . ..
,'~ . ,
"-, .
'~liO~3
-22-
The compounds of formulae XIX-XXVII may be
represented generally by the formula:
P-Y-X-Q
wherein X is C=o, C=S, CH2 or CHOH; Q is selected from
the group consisting of -ONH2-, and -CH2-ONH2-, and Y
is selected from the group consisting of -O-CH2CH2-, -
O-CH2CH2-O-, -O-CH2CH2-N-, O-CH2CH2-S, and -O-CH2CH2CH-;
and P is a water soluble organic polymer (as in
compounds XIX-XXVII).
In addition to the molecules of formulae I, II,
III, IV, V, VI, VII, VIII, and IX the subject
invention also includes polypeptides modified by
reaction with the molecules of formulae I, II, III,
IV, V, VI, VII, VIII, and IX. Polypeptides modified
by the water-soluble polymer reagents of formulae I,
II, III, IV, VI, VII, VIII, and IX may be represented
by formulae X, XI, XII, XIII, XIV, XV, XVI, XVII, and
XVIII, respectively:
HYDRAZIDE-MODIFIED POLYPEPTIDES
(X) [P-O-CH2-CO-NHN=CH-]o~Z,
~l
( XI ) ~ p--O--CO--NHN=CH--] n~ Z
~t
(XII) [P--NH-CO-NHN=CH-]n--Z,
(XIII ) [ P--NH-CS-NHN=CH- ] o~Z,
;(
1 . '
~ :~
2110~3
(XIV) [ P--NHCO--N--NIINHCO--NlIN=CI{--] n~z
( XV ) [ P -NHN CON=CH ] D- Z ~
( XVI ) [ P-NHNCSN=CH- ] ~- Z,
(XVII) [P-NH-CO-C6H4~NHN=CH-]n-Z, and
(XVIII) [P-o-co-cH2cH2-co-NHN=cH-]n-zl
wherein P is a water-soluble polymer as previously
described, Z represents a polypeptide, as described
above, and n represents a number in the range 1 to x,
where x is the maximum number of oxidation
activatable groups present in polypeptide Z. The C
in the hydrazone linkage formed between the water-
soluble polymer reagent and Z was originally present
on Z, not the water-soluble polymer reagent.
In addition to the molecules of formulae XIX,
XX, XXI, XXII, XXIII, XXIV, XXV, XXVI and XXVII the
subject invention also includes polypeptides modified
by reaction with the molecules of formulae XIX, XX,
XXI, XXII, XXIII, XXIV, XXV, XXVI and XXVII.
Polypeptides modified by the water-soluble polymer
reagents of formulae XIX, XX, XXI, XXII, XXIII, XXIV,
XXV, XXVI and XXVII may be represented by formulae
XXVIII, XXIX, XXX, XXXI, XXXII, XXXIII, XXXIV, XXXV
and XXXVI, respectively:
OXYLAMINE-MODIFIED POLYPEPTIDES
2 5 ~ XXVI I I ) [ P-O-CH2CH2-CO-ON=CH- ] n~ Z;
(XXIX) [p--O--CH2CH2--O--CO--ON=CH--] n~Z;
:~,
: ~ :
'v~
2 ~ 3
-24-
(XXX) [P-O-CH2CH2-NH-CO-ON=CH-]~-Z;
(XXXI) [P-O-CH2CH2-NH-CS-ON=CH-]~-Z;
(XXXII) [P-O-CH2CH2-ON=CH-]n-Z;
(XXXIII) [P~O~CH2CH2~NH~CO~CH2~0N=cH~]n-z;
(XXXIV) [p-o-cH2cH2-o-co-cH2-oN=cH-]n-z;
(XXXV) [P-O-CH2CH2-CH(OH)-CH2-ON=CH-]n-Z; and
(XXXVI) [P-o-cH2cH2-co-cH2-oN=cH-]n~Z~
wherein P is a water-soluble polymer as previously
described, Z represents a polypeptide, as described
above, and n represents a number in the range 1 to X,
where X is the maximum number of oxidation and
activatable groups present in polypeptide Z. The
carbon atom in the oxime linkage formed between the
water-soluble polymer reagent and Z was originally
present on Z, not the water-soluble polymer reagent.
Although polypeptides may be modified by the
coupling of up to x water-soluble polymers per
polypeptide molecule, it may be desirable to modify a
given polypeptide by less than x water-soluble
polymer molecules. It may be undesirable to
derivatize a polypeptide with the maximum number of
water-soluble polymers, i.e., x water-soluble
polymers/polypeptide molecule, because for some
polypeptides, increasing the number of water-soluble
polymers per molecule of polypeptide may diminish
biological activities as compared the unmodified
polypeptide. For example see figures 2, 4, 5, 9, 10,
. ?~ , .
~",
2~ 43
-25-
and 11, for some results obtained with water-soluble
polymer modified EPo.
Different methods of measuring the number of
water-soluble polymer molecules attached to a
glycoprotein molecule, as in hydrazone linked
compounds of formulae X, XI, XII, XIII, XIV, XV, XVI,
XVII, and XVIII and as in oxime linked compounds of
formulae XXVIII, XXIX, XXX, XXXI, XXXII, XXXIII,
XXXIV, XXXV and XXXVI may give different results.
For the purpose of this application, when a
polypeptide is said to be derivatized by a given
number of water-soluble polymer molecules/molecule of
protein, the number of water-soluble polymers given
is the empirically determined figure measured by gel
filtxation chromatography retention time.
The synthesis of compo~lnds of formulae X, XI,
XII, XIII, XIV, XV, XVI, XVII, and XVIII may result
in the creation of a mixture of reaction products
differing from one another with respect to the exact
number of water-soluble polymers attached to the
polypeptide through hydrazone linkages and the sites
on the polypeptide where these hydrazone linkages are
present. Similarly, the synthesis of compounds of
formulae XXVIII, XXIX, XXX, XXXI, XXXII, XXXIII,
XXXIV, XXXV and XXXVI may result in the creation of a
mixture of reaction products differing from one
another with respect to the exact number of water-
solu~le polymers attached to the polypeptide through
oxime linkage and the sites on the polypeptide where
these oxime linkages are present. As the polymer P -
I comprises multiple identical units of varying
¦ amounts, it will be appreciated that the molecular
weight of P may vary considerably. Further~ore, when
P is s~id to have a given molecular weight, that
S.~:7',"" " ' : '
~'"~, " ~ " '
.~ , - ,
~`~
2~ ~o~3
-26-
molecular weight may only be approximate, reflecting
the average molecular weight of a population of
molecules P differing with respect to one another in
regards to the number of subunits present in the
molecule. In general, P will have a molecular weight
of about 200 to 200,000, preferably in the range of
700 to 30,000, more preferably in the range of 2,000-
12,000. Suitable molecular weights for P, when the
molecules of formulae I, II, III, IV, V, VI, VII,
VIII, and IX and the molecules of formulae XXVIII,
XXIX, XXX, XXXI, XXXII, XXXIII, XXXIV, XXXV and XXXVI
are to be coupled to a polypeptide will vary in
accordance with the specific polypeptide to be
modified and the specific water-soluble polymer
selected. Individual polypeptide molecules may be
derivatized by one or more different water-soluble
polymers by means of reaction with different
embodiments of the compounds of formulae I, II, III,
IV, V, VI, VII, VIII, and IX (the hydrazones), or the
compounds of formulae XIX, XX, XXI, XXII, XXIII,
XXIV, XXV, XXVI and XXVII (the oximes), or any
combination of the hydrazones and the oximes.
An advantage of the subject invention is that
polypeptides may be modified by the attachment of
water-soluble polymers without substantially reducing
the biological activity of the polypeptide, or
reducing the biological activity to a lesser extent
than the biological activity would be reduced by the
attachment of a similar number of the same water-
soluble polymers/polypeptide molecule by means of
previously known chemical coupling methods and
compounds. Aspects of the biological activity of EPO
include the stimulation of red blood cell formation.
A detailed description of the biological activity of
~':
.~. ,
,',~ , ,
-27- 21~0 ~4~
EPo can be found in Krantz, s.s., slood 77: 419-434
(1991),
Another advantage of the subject invention is
that polypeptides modified by the compounds of
formulae I, II, III, IV, V, VI, VII, VIII, and IX or
the compounds of formulae XIX, XX, XXI, XXII, XXIII,
XXIV, XXV, XXVI and XXVII may then retain a greater
degree of their biological activity than when the
same polypeptide is modified to the same degree by
lo joining water-soluble polymers to polypeptides
employing the frequently used (prior to the subject
invention) active esters of mPEG for lysine
modification. Thus, the subject invention provides
for modified polypeptides that possess the advantages
associated with the covalent conjugation of water-
soluble polymers while minimizing the loss of
biological activity associated with the modification.
Consequently, polypeptides that may be more highly
derivatized by water-soluble polymers, and thus or
otherwise possess the advantages associated with the
~ higher degree of derivatization, may be produced that
;jj have the same level or a higher level of biological
activity as polypeptides derivatized by water-soluble
polymers to a lesser extent using conventional
methodology.
'i Another advantage of using a hydrazone forming
derivatives of PEG (and other water-soluble polymers)
instead of PEG-amine for coupling PEG to a protein is
that coupling of a hydrazide or oxylamine (or similar
compounds) to an aldehyde yields a hydrazone or oxime
respectively, while coupling through an amine gives
~;~ an imine, which is less stable than a hydrazone or an
oxime and needs to be reduced to give a stable
derivative. Thus an extra step is required when
,.. . .
.,
. "
',
., ,~.
. ,,
, ~ ...
,, ,.
21~0~3
-28-
using an amine instead of a hydrazone forming
compound.
Another advantage of the subject invention is
that higher levels of water-soluble polymers may be
attached to glycoproteins than with other water~
soluble polymer derivatives. The semicarbazide
(formula III), thiosemicarbazide (formula IV), and
carbonic acid dihydrazide (formula V) derivatives are
of particular interest because of their higher
reactivity than comparable hydrazide derivatives of
the subject invention. Reactions involving the mPEG
derivatization of EP0 with semicarbazides,
thiosemicarbazides and carboxylate hydrazide
described herein have resulted the addition of up to
about 31-34 mPEG molecules for each molecule of EP0,
whereas similar reactions using corresponding
hydrazide derivatives of EPO have resulted in the
addition of about 6-12 molecules of mPEG to each
molecule of EPO. Reactions between carbonic acid
dihydrazide and hydrazide carboxylate derivatives and
EP0 have resulted in the addition of up to 22 mPEG
molecules to each molecule of EPO. In order for
hydrazide derivatives of mPEG to incorporate about 20
mPEG molecules to each molecules of EPO, very strong
oxidation conditions were required, e.g., 50 mM
periodate, 60 minutes incubation at room temperature.
The subject mPEG semicarbazide and thiosemicarbazide
derivatives could be used to provide EP0 modified
with PEG to a similar extent, but under more mild
oxidation conditions, e.g., 10 mM periodate, for 5-15
minutes at 0C. Strong oxidizing conditions may have
an adverse effect on the structural and biological
properties of many polypeptides, thus PEG
semicarbazide, carbonic acid dihydrazide, hydrazide
,.," . . . ,- , . ~
~' ,,~
...,~," .
;~
.,,: ,
, ............ .
...
fi .:
, ~,
~;
21~0~3
-29-
carboxylate, and thiosemicarbazide derivatives may be
particularly useful compounds for modifying
polypeptides with PEG (or other water-soluble
polymers).
A novel series of oxylamine derivatives of mPEG
have been synthesized and have been reacted to the
oxidized carbohydrate groups of EPO. Some of the
mPEG-oxylamines showed high reactivity to the
oxidized carbohydrates. Also a lower number of mPEGs
could be incorporated onto EPO and still give the
high ln vivo activity as seen with the semicarbazide
and carboxylate hydrazide (hydrazone forming) mPEG-
derivatives. This lower number for mPEG
incorporation is advantageous in that shorter and
milder oxidation conditions can be used in the
modification. Also lesser amounts of mPEG-derivative
can be used in the modification reaction.
Similarly, particularly high levels of water-
soluble polymers are attached to glycoproteins when
the compounds of formula XXI, formula XXIV, and
formula XXIII are employed as compared to comparable
formula XIX oxylamine derivative. Reactions
involving derivatization of EPO with formula XXI and
formula XXIV described herein have resulted in the
addition of up to 18-l9 mPEGS/EPO, and 31 mPEG
molecules for every molecule of EPO, respectively,
whereas similar reactions using corresponding formula
XXII and XIX oxylamine derivatives of EPO have
resulted in the addition of about 3-4 molecules of
mPEG to each molecule of EP0. Perhaps more
importantly, the bioactivity of resulting oxylamine-
derivatized PEG-EP0 is surprisingly high even at more
moderate levels of attachment of water-soluble
polymer to EPO (see hereinbelow and Figure 17). In
~}~,
,
'
."
~ ~ .
21 10~3
-30-
this regard, bioactivity of a 12 mPEG isolated
fraction of formula XXIII is of particular interest
(See Figures 16 & 17).
The ability to generate long acting mPEG-EPO
with high activity via coupling mPEG to the oxidized
carbohydrate groups depends on the mPEG-derivative
chosen. Some mPEG carbohydrate modifying derivatives
are not reactive enough to attach an optimum amount
of mPEG onto EPO. Some mPEG-derivatives require a
high amount of incorporation onto EPO due to the
stability of the resulting bond. An optimal amount
of mPEG incorporation for the semicarbazide
derivative is about 17-25, more preferably about 22;
for the carboxylate hydrazide derivative, about 22-
32, more preferably about 31. Reactivity of the
mPEG-oxylamines is about 3-36 mPEGs/EPO.
The water-soluble polymer reagents of the
I subject invention may be used to modify a variety of
¦ polypeptides or similar molecules that contain
aldehydes or functional groups with similar chemical
reactivity, e.g., ketones, lactols, activated
carboxylic acids or activated carboxylic acid
derivatives, capable of chemically reacting with the
hydrazide portion (or similar functional portion) of
the subject water-soluble polymer reagent derived
from the oxidation of hydroxyl groups, the oxidation
of other oxidation activatable groups present on the
polypeptide of interest (including carbohydrate
moieties when the polypeptide is a glycoprotein, and
¦ 30 amino acid residues in the primary sequence, e.g.,
¦ the N-terminus of serine, threonine, hydroxylysines),
J or hydrazine or oxylamine reactive group present on
polypeptides prior to or after any oxidative
/
?
' :1
r"~
~, . . .
~'., ~',, " :
,
~, :
':'~' '
2110~ ~3
treatment. Polypeptides of interest include
antibodies, monoclonal and polyclonal, cytokines,
growth factors, hormones, enzymes, protein or peptide
ligands and the like. Polypeptides of interest for
modification by hydrazone linkage or oxime linkage
forming water-soluble polymer reagent molecules of
the subject invention may be isolated from their
natural sources, genetically engineered cells, e.g.,
CHO cells transformed with expression vectors for the
production of EPO, or produced by various ln vitro
synthesis methods. A particularly preferred
polypeptide for the purposes of the instant invention
is EPO, and precursors, intermediates and mimetics
thereof, whether human or recombinant.
While the water-soluble polymer reagents of the
subject invention may be used to modify most
polypeptides, it is of particular interest to modify
(1) polypeptides for use as drugs, and (2)
polypeptides for use in assays. Polypeptide for use
in assays include specific binding proteins,
polypeptides recognized by specific-binding proteins,
and enzymes. By specific-binding proteins it is
intended antibodies, hormone receptors, lectins, and
the like.
Various polypeptides may be modified by the
subject water-soluble polymer reagents and the
subject methods for their use so as to be coupled to
different water-soluble polymers and to differing
degrees or modification. Varying parameters such as
3~ (1) the number of water-soluble polymers coupled to
an individual polypeptide molecule, which will depend
upon the reactivity of the derivatized mPEGs to the
EPO, and the bioactivity of the resulting mPEG-EPO;
e.g., reactivity from about 3-36 molecules of
,:
;.
,-
,,~, .
. .
2~10~3
-32-
mPEG/EPO (2) the molecular weight of the water-
soluble polymer, e.g., 2,000-12,000 daltons (3) the
structure of the water-soluble polymer, e.g.,
monomethoxypoly(ethylene ylycol) (4) the reaction
conditions under which the reaction between the
water-soluble polymer reagent and the polypeptide of
interest, e.g., temperature and duration, and (5) the
oxidation conditions under which the polypeptide for
modification is activated for covalent conjugation,
e.g., periodate at a concentration in the range of
10-40 ~mol/mg of protein, may influence the
biological properties of the resultant water-soluble
polymer modified polypeptide.
In a preferred embodiment of the invention,
activation of polypeptides for covalent conjugation
is performed by mixing the protein for modification
with periodate (0.1-1,000 ~mole/mg protein) for a
period of time in the range of one minute to three
days, more preferably 0.5-50 ~mole periodate/mg
protein, for a time period in the range of 5 minutes
to 180 minutes. In a preferred embodiment of the
invention, activation for conjugation is performed by
mixing the protein for modification with periodate at
a temperature in the range of -10--50-C, more
preferably in the range of 0 -30 C.
In a preferred embodiment of the subject
invention when the protein for modification is EP0,
EP0 is derivatized with the compounds of formulae II-
VIII, more preferably the compounds of formulae II-V,
the compound of formula III, the semicarbazide, and
formula II, the carboxylate hydrazide, being
particularly preferred, where the water-soluble
polymer P is methoxypolyethylene glycol (mPEG) and
each molecule of EP0 is derivatized by 3-36, more
.^,,
:,:
~, r~
j
'~10~43
preferably 17-25 molecules of methoxypolyethylene
glycol (in the case of the semicarbazide), and more
preferably 22-32 molecules of methoxypolyethylene
glycol (in the case of the carboxylate hydrazide) and
the mPEG used has an average molecular weight of
about 5000 daltons. Preferred reaction conditions
for the production of the mPEG5000 semicarbazide
modified EPO are at 0-30C, for 5 to 60 minutes, and
0.5-50 ~moles periodate/mg of EPO (with periodate as
oxidizing agent). EPO for modification by the
subject water-soluble polymer reagents and methods is
preferably obtained from genetically engineered
cells, more preferably from CHO cells genetically
modified to produce EPO. By employing the preferred
water soluble polymer reagent and conditions for
modifying EP0, unexpectedly prolonged biological
half-life of EP0 is obtained and increased hematocrit
levels çan be seen, for example, see Figures 2, 4 and
5.
In another preferred embodiment, EP0 is
derivatized with the compounds of formulae XX-XXVII,
more preferably the compounds of formulae XXI, XXIV,
and XXIII, wherein the water-soluble polymer P is
mPEG and each molecule of EPO is derivatized by about
18-19 mPEG~, 31 mPEGS, and 17 mPEGs, respectively,
for formulae XXI, XXIV and XXIII. These results were
obtained when the mPEG used has an average molecular
weight of about 5000 daltons. The compound of
formula XXIII had a 12mPEG fraction, which had ln
yivo bioactivity comparable to 22mPEG semicarbazide
and 31 mPEG carboxylate hydrazide. Reactivity
(molecules of mPEG/EPO) for formulae XXI, XXIV and
XXIII was in excess of that for PEG hydrazide under
the same reaction conditions. Preferred reaction
~ - .
, . ,
: ::
'~L~0~43
-34-
conditions for the production of the mPEG 5000 oxime
may require more mild oxidation conditions such as a
shorter oxidizing time or lower concentrations of
oxidant than with the corresponding hydrazide to
produce higher in vivo bioactivity.
The subject invention also provides methods of
activating polypeptides for conjugation, i.e.,
covalent conjugation, with the subject water-soluble
polymer reagents. These methods of activating
polypeptides for conjugation comprise the step of
partially oxidizing the polypeptides of interest.
Partial oxidation may be achieved by adding an
oxidizing agent such as periodate and other oxidation
agents known to those of skill in the art, or by
adding an enzyme capable of catalyzing oxidation
reactions on portions of the polypeptide of interest,
e.g., galactose oxidase. The preferred method of
partially oxidizing a polypeptide for activation for
conjugation is by the addition of periodate in a
concentration in the range of 0.1-1,000 ~mole/mg
protein, for a period of time in the range of one
minute to three days, more preferably 0.5-50 ~mole
periodate/mg protein, for a time period in the range
of 5 minutes to 180 minutes. The temperature at which
the activation is performed is preferably in the
range of -10--50-C, more preferably in the range of
-30 C.
Salts of any of the macromolecules described
herein, e.g., polypeptides, water-soluble polymers
and derivatives thereof, will naturally occur when
such molecules are present in (or isolated from)
agueous solutions of various pHs. All salts of
polypeptides and other macromolecules having the
indicated biological activity are considered to be
~' ~ ' ' ' , : ,
,~,, .
~;
-
2 ~ 10~43
~ithin the scope of the present invention. Examples
include alkali, alkaline earth, and other metal salts
of carboxylic acid residues, acid addition salts
(e.g., HCl) of amino residues, and zwitterions formed
by reactions between carboxylic acid and amino
residues within the same molecule.
The mode of administration of the preparations
of the invention may determine the sites and/or cells
in the organism to which the compound(s) will be
delivered. The compounds of the invention can be
administered alone but will generally be administered
in admixture with a pharmaceutical carrier or diluent
selected with regard to the intended route of
administration and standard pharmaceutical practice.
! 15 The preparations may be injected parenterally, for
; example, intra-arterially or intravenously. The
preparations may also be delivered via oral,
subcutaneous, or intramuscular routes. For
parenteral administration, they can be used, for
example, in the form of a sterile, aqueous solution
which may contain other solutes, for example, enough
salts or glucose to make the solution isotonic.
~ For the oral mode of administration, the EPO
;~ compositions of the invention can be used in the form
of tablets, capsules, lozenges, powders, syrups,
~ elixirs, aqueous solutions and suspensions and the
`7, like. In the case of tablets, carriers which can be
used include lactose, sodium citrate, and salts of
i phosphoric acid. Various disintegrants such as
starch, and lubricating agents such as magnesium
stearate are commonly used in tablets. For
administration in capsule form, useful diluents are
lactose and high molecular weight polyethylene
glycols. When aqueous solutions are required for
,~,, , ~
:
. ~ .. , . -
' ~i - , . :: .: .
''~: ' ' - :
,~ ~ , . .
.. .
2110~3
-36-
oral use, certain sweetening and/or flavoring agents
can be added.
For administration to humans in the treatment of
disease states responding to EPO therapy, the
prescribing physician will ultimately determine the
appropriate dosage for a given human subject, and
this can be expected to vary according to the weight,
age and response of the individual as well as the
nature and severity of the patient's disease. The
dosage of the drug in pegylated form may generally be
about that employed for native drug, however, it may
in some cases be preferable or necessary to
administer dosages outside these limits.
It is also of interest to supply the
water-soluble polymer reagents of formulae I, II,
III, IV V, VI, VII, VIII, and IX, and XIX, XX, XXI,
XXII, XXIII, XXIV, XXV, XXVI and XXVII separately or
in various combinations, in the form of a kit, so as
to provide for the convenient and reproducible
derivatiz~tion of polypeptides of interest. Kits of
interest may contain solutions comprising the water-
soluble polymer reagent of formulae I, II, III, IV,
V, VI, VII, VIII, or IX, or those of formulae XIX,
XX, XXI, XXII, XXIII, XXIV, XXV, XXVI, XXVII,
buffers, oxidizing agents, reaction indicator
compounds, protein concentration measurement
reagents, e.g., for Bradford assays, and the like.
Compounds included in kits are preferably provided in
pre-measured portions and pre-mixed solutions so as
to provide for reproducibility and minimize error.
Kits also preferably contain instructions.
Instructions are directed to various steps in
performing the subject methods.
.,
.... ..
~,...
: ,,
":-, .
2110~43
-37-
SYNTHESIS OF WATER-SOLUBLE POLYMER DERIVATIVES
The following syntheses of the subject compounds
are exemplary and are not included for the purpose of
limiting the invention. The person of average skill
in the art of organic chemistry can devise variations
on tne exemplified syntheses.
SYNTHESIS OF HYDRAZONE FORMING m-PEG
Synthesis of mPEG-HYdrazide
There are several ways to synthesize
mPEG-hydrazide. Two methods are presented.
mPEG5000-acid (20.8 g, 4 mmol) was dissolved in
30 ml dichloromethane and t-butyl carbazate (2.64 g,
8 mmol) in 15 ml dichloromethane was added followed
by 1.68 g (8 mmol) dicyclohexylcarbodiimide which was
dissolved in 10 ml dimethylformamide. After running
the reaction over night at room temperature the
reaction mixture was filtered. The filtrate was
concentrated, and the resulting residue was taken up
in dichloromethane. Ether was added to precipitate
the mPEG-t-butyl-carbazide which was filtered and
dried. The product was placed in a dicholormethane/
trifluoroacetic acid (1:1) mixture. After 40 minutes
the solution was concentrated, redissolved in
dicholormethane and ether was added. The product
was recovered by filtration. Yield 17.7 g. IR:
(C-O) 1730, 1700. Analysis. Calcd. for N, 0.55.
Found: N, 0.44.
An alternative method shown below converts
mPEG-alcohol to an ester. The mPEG-ester then i5
hydrolyzed with hydrazine to give mPEG-hydrazide.
The synthesis of mPEG-ester is similar to the
procedure of Royer, G.P., and Anantharmaiah, G.M.
(1979) J. Amer. Chem. Soc. 101, 3394-3395.
' ~
.... . .
2110~3
-38-
NaH
mPEG-OH -------------------->mPEG-O-CH2-CO2C6H5
Br-CH2-c02c6Hs
H2NNH2
mPEG-O-C~2-CO2C6H~ ---------->mPEG-O-CH2-CO-NHNH2
In a typical synthesis mPEG-OH was dried for
about five hr at 85 in a high vacuum oven. After
cooling 5 g mPEG-OH (MW = 5000, 1 mmol) was dissolved
in 5 ml dry tetrahydrofuran. To 26.4 mg (1.1 mmol)
sodium hydride was added 1 ml dry tetrahydrofuran.
The mPEG5000 solution was added to the NaH dropwise.
The mixture was stirred for one hr at room
temperature in an argon atmosphere. During this time
the solution became orange in color. Bromoacetyl
acid benzyl ester (2.29 g, 10 mmol) was dissolved in
1 ml dry tetrahydrofuran, and this solution was added
~ dropwise to the mPEG5000 mixture. The reaction was
s stirred overnight at room temperature under an argon
i atmosphere after which time it was filtered. Cold
ether was added to the filtrate to precipitate the
mPEG5000-benzyl ester, and the solid was collected
and dried. Yield 4.5 g. IR (C=O) 1752. The
, compound was further purified by gel filtration on a
LH-20 column eluting with methanol/methylene chloride
(5:1).
~ The mPEG5000-benzyl ester was converted to the
,, hydrazide by treatment with hydrazine. In a typical
, experiment 1.0 g mPEG5000-benzyl ester (0.194 mmol)
was dissolved in 3 ml methanol/methylene chloride
(5:1) in an argon atmosphere. Hydrazine (0.091 ml,
' 2.91 mmol) was added and the solution was stirred at
rj room temperature for around 70 hr. The mixture was
~' placed on a LH-20 column eluting with
i methanol/methylene chloride (5:1). The
,~,
~! ,
,~
~' ~
::,
: ,..,~
: -~'~ . - .
" j,.
2~0~3
-39-
mPEG5000-hydrazide was separated from the hydrazide
and was precipitated with ether. The solid was
collected by filtration. Yield 0.78 g. IR (H2N-C=O):
1669. Analysis. Calcd. for N, 0.55. Found: N,
0.34.
Also synthesized were mPEG2000-hydrazide,
mPEG6000-hydrazide, mPEG8500-hydrazide, and
mPEG12000-hydrazide using the procedures described
above.
Synthesis of mPEG-Hydrazine Carboxylate
H2NNH2
mPEG-O-CO-Im ---------------> mPEG-O-CO-NHNH2
The above mPEG5000-hydrazine carboxylate was
synthesized as follows. Methoxypolyoxyethylene
imidazolyl carbonyl (from Sigma Chemical, 2.5 g, 0.49
mmol) was treated with hydrazine ( 0.077 ml, 2.45
mmol) in 10 ml methylene chloride. After 4 hr at
room temperature the reaction mixture was filtered,
` and the filtrate was treated with cold ether. The
resulting precipitate was collected. Yield 2.22 g.
IR (C=0): 1718. Analysis. Calcd. for N, 0.55.
Found: N, 0.595.
Synthesis of mPEG-Semicarbazide
COCl2
mPEG-NH2 ----~ ----------> mPEG-NH-CO-Cl
H2NNH2
mPEG-NH-CO~Cl -~ --------> mPEG-NH-C0-NHNH2
...
. .
S~,r
2il~43
-40-
The above mPEG5000-semicarbazide was synthesized
as follows. mPEG5000-amine was ~ynthesized as
described by Rajasekharan Pillai, V.N., and Mutter,
M. (1980) J. Org. Chem. 45, 53G4-5370. The
mPEG5000-amine (2 g, 0.4 mmol) was dissolved in 9 ml
dichloromethane and 0.28 ml triethylamine was added.
To the mixture in an argon atmosphere was added
phosgene (in toluene, 0.42 ml, 0.8 mmol). The
reaction went overnight and then was bubbled with
argon to remove any excess phosgene. The solution
was concentrated, and the residue was dissolved in
dicchloromethane and 0.063 ml hydrazine (2 mmol) was
added followed by 2 ml methanol. The reaction went
for 4 hr after which time cold ether was added, and
the precipitate was removed by filtration and dried.
Yield 1.51 g. IR (C=O): 1683. Analysis. Calcd.
¦ for N, 0.83. Found: N, 0.56.
I Also synthesized were the semicarbazides of
i mPEG2000, mPEG6000, mPEG8500, and mPEG12000 using
the procedures described above.
¦ Synthesis of mPEG-Thiosemicarbazide
To 1.5 g mPEG5000-amine (0.3 mmol) in 5 ml
dichloromethane was added 0.1 ml triethylamine (0.75
mmol) and 0.071 g (0.3 mmol)
di-2-pyridylthionocarbonate. The reaction went
overnight, where upon 0.047 ml (0.3 mmol) hydrazine
was added. After 4 hr the mixture was filtered, and
i the filtrate was treated with cold ether. The
J product was collected by filtration. Yield 1.34 g.IR (N-H stretch): 3332. Analysis. Calcd. fo~ N,
0.83. Found: N, 0.255.
!
,~ I
,5''
,r"~
~, ............. .
5 43
-41-
Synthesis of mPEG-Carbonic Acid Dihvdrazide
In a reaetion flask purged with argon was added
t-butyl carbazate (0.04 g, 0.3 mmol) in 2 ml
dichloromethane, 0.084 ml triethylamine (0.6 mmol),
and 0.03 g triphosgene (0.1 mmol). After 5 minutes
1.5 g mPEG5000-amine (0.3 mmol) in 4 ml
dieh~oromethane was added. The reaction went
overnight after which time cold ether was added to
precipitate the product. The product was isolated by
filtration. Yield 1.44 g. The protected dihydrazide
(0.61 g) was treated with 2 ml trifluoroacetic acid
at room temperature for 10 minutes. The
trifluoroacetic acid was removed, and the resulting
oil was dissolved in methylene chloride and
eoncentrated. This step was repeated. The oil was
dissolved in methylene chloride and the product was
preeipitated with eold ether. The produet was
isolated by filtration. Yield 0.41 g. IR (C=0):1695.
Analysis ealcd. for N, 1.37. Found: 0.41.
Synthesis of mPEG-Arvlhvdrazide
Boc-NHNH-C6H4-COOH
mPEG-NH2 -------------------> mPEG-NH-CO-C6H4-NHNH2
The above mPEG5000-arylhydrazine was synthesized
as follows. Boc-NHNH-C6H4-COOH was prepared by
reaeting 4-hydrazinobenzoie acid with di-tert-butyl
pyrocarbonate in dioxane in the presence of base at
0C. The protected aryl acid hydrazine (0.378 g, 1.5
mmol) was reacted with mPEG-amine (1.5g, 0.3 mmol) in
a diehloromethane/dimethylformamide solution (4ml,
1:1~. Also added was dicyclohexylcarbodiimide
(0.31g, 1.5 mmol), 1-hydroxybenzotriazole (0.2g, 1.5
., ~
."~ .
~,
si~
~/
2110~43
-42-
mmol), and triethylamine (0.21ml, 1.5mmol). The
reaction went overnight, after which time the
contents were filtered. The filtrate was treated
with ether, and the precipitate was collected. The
precipitate was treated with trifluoroacetic acid,
and after 30 min, the trifluoroacetic acid was
removed. Ether was added to the oily residue to
precipitate the mPEG-arylhydrazine product. The
product was isolated by filtration. Yield l.l9g. IR
(N-H): 3267; (C=O): 1655; (C=C): 1606. Analysis.
Calcd. for N, 0.82. Found: N, 0.50.
SYNTHESIS OF OXIME-FORMING m-PEG
synthesis of CH3O-(CH2cH2O) n CH2cH2-co-oNH2
1. BocNHOH/TEA
mPEG-CO-NHS -----~ -----> mPEG-O-CH2CH2-CO-ONH2
? 2. TFA
mPEG~5000-succinimide ester (NHS) (2.0g, 0.4
~ mmol) was dissolved in 10ml dichloromethane. t-Butyl
i N-hydroxycarbamate (0.53g, 4 mmol) was added followed
by 0.7ml triethylamine ~5 mmol). After running the
reaction overnight cold ether was added, and the
resulting precepitate was collected by filtration,
' washed, and dried. The product (1.5g) was placed in
a dicholormethane/trifluoroacetic acid (1:1) mixture.
After 60 minutes the solution was concentrated. The
?, compound was further purified by gel filtration on a
~ LH-20 column eluting with methanol/methylene chloride
-3 (5:1). Yield 1.2g IR: (C=O) 1741. Analysis Calcd.
for N,0.28. Found: N,0.13.
Synthesis of CH~O-(CH2CH2O)n-CH2CH2-O-CO-ONH2;
~ , ~
?~
"~,
.'~.,,
''
' '
, '
A~ 1 1 0 ~ 4 3
-43-
1. BocNHOH/TEA
mPEG-O-CO-Im ---------------> mPEG-O-CH2CH2-O-CO-ONH2
2. TFA
mPEG-5000-oxycarbonylimidazole (2.0g, 0.4 mmol)
was dissolved in 10ml dichloromethane. t-Butyl N-
hydroxycarbamate (0.53g, 4 mmol) was added followed
by 0.7ml triethylamine (5 mmol). After running the
reaction overnight cold ether was added, and the
resulting precipitate was collected by filtration,
washed, and dried. The product (1.5g) was placed in
a dichloromethane/trifluoroacetic acid (1:2) mixture.
After 60 minutes the solution was concentrated. The
compound was further purified by gel filtration on a
LH-20 column eluting with methanol/methylene chloride
~5 (f:1). Yield 1.2g. IR:(C=0) 1692. Analysis Calcd.
for N, 0.28. Found: N, 0.15.
Svnthesis of_CH30-(CH2CH2O)~-CH2CH~-NH-CO-ONH2
1. Im-CO-Im
mPEG-NH2 ---------------> mPEG-O-CH2CH2-NH-CO-ONH2
2. BocNHOH/TEA
3. TFA
mPEG-5000-amine (2.0g, 0.4 mmol) was dissolved
, in 10ml chloroform along with carbonyldimidazole
(o.23gm, 1.45 mmol). This reaction followed the
procedure for activation of mPEG-alcohol with
s carbonyldimidazole as described by Ranucci, E., and
Feruti, P. (1991) Macromolecules 24, 3747-3752. The
reaction was stirred for two hours at room
temperature after which time 7ml water was addedt and
the organic layer was extracted. The water
extraction was repeated five times. The organic
layer was dried over sodium sulfate, and the salt was
. ,.
,
. . ' ~
i'.
~,
~ . .
2110 a ~3
-44-
filtered. Added to the filtrate was t-butyl N-
hydroxycarbamate (0.53g, 4 mmol) along with 0.7ml
triethylamine (5 mmol~. The reaction was stirred
overnight after which time cold ether was added, and
the resulting precipitate was collected by
filtration, washed, and dried. The product (1.5g~
was placed in a dicholormethane/trifluoroacetic acid
(1:2) mixture. After 60 minutes the solution was
concentrated. The compound further purified by gel
filtration on a LH-20 column eluting with
methanol/methylene chloride (5:1). Yield 1.3g. IR:
(C=0):1726. Analysis. Calcd. for N, 0.55. Found: N,
0.43.
Synthesis of CH3O-(CH2CH2O),~-CH2CH2-NH-CS-ONH2;
1- (CsH4NO)2cs
mPEG-NH2 ---------------> mPEG-O-CH2CH2-NH-CS-ONH2
2. BocNHOH/TEA
3. TFA
mPEG-5000-amine (1.5g, 0.3 mmol) was dissolved
l 20 in 30ml dichloromethane. Triethylamine (0.lml, 0.75
;~, mmol) was added followed by di-2-
pyridylthionocarbonate (0.082g, 0.35 mmol). The
reaction was stirred for two hours at room
temperature after which time t-butyl N-
hydroxycarbamate (0.53g, 4 mmol) was added along with
0.5ml triethylamine (3.75 mmol). The reaction was
stirred overnight after which time cold ether was
added, and the resulting precipitate was collected by
) filtration, washed, aNd dried. The product (1.6g~
'; 30 was placed in dicholormethane/trifluoroacetic acid
1) mixture. After 30 minutes the solution was
concentrated. The compound was further purified by
''~ . ,.
:'
;.;.,
r'
' '.,~ '
i,.
"',',
'' ~ ,'
;
'~" '
2110~3
-45-
gel filtration on a LH-20 column eluting with
methanol/methylene chloride (5:1). Yield 0.72g. IR:
(C=S) : 1684. Analysis. Calcd. for N, 0.55. Found:
N, 0.30.
Svnthesis of CH3O-(CH~ 2CH2-ONH2
1. BocNHOH/TEA
mPEG-O-Trs ---------------> mPEG-O-CH2CH2-ONH2
2. TFA
mPEG-5000-tresylate (2.0g, 0.4 mmol) was
dissolved in 10ml dichloromethane to which t-butyl N-
hydroxycarbamate (0.53g, 4 mmol) and 0.7ml
triethylamine (5mmol) were added. The mixture was
heated to 45 (reflux), and the reaction ran
overnight. Cold ether was added to the reaction
mixture, and the resulting precipitate was collected
by filtration, washed, and dried. The product (1.5g)
was placed in a dicholormethane/trifluoroacetic acid
(3:7) mixture. After 60 minutes the solution was
concentrated. The compound was further purified by
gel filtration on a LH-20 column eluting with
methanol/methylene chloride (5:1). Yield 1.6g.
Analysis. Calcd. for N, 0.28. Found: N, 0.19.
An alternative synthesis is the following. t-
Butyl N-hydroxycarbamate (0.53g, 4 mmol) was placed
in lml tetrahydrofuran followed by NaH (78 mg, 3.25
mmol). After a few minutes this solution was added
to mPEG-5000-tresylate (1.0g, 0.2 mmol) which was in
5ml tetrahydrofuran. The mixture was heated to 40,
and the reaction went overnight. Cold ether was
added to the reaction mixture, and the resulting
precipitate was collected by filtration, washed and
dried. the product (1.5g) was placed in a
,
."
s~'
.`,f,,; .
~' ,
5 43
-46-
dicholormethane/trifluoroacetic acid (3:7) mixture.
After 60 minutes the solution was concentrated. The
compound was further purified by gel filtration on ~
LH-20 column eluting with methanol/methylene chloride
(5:1). Yield 0.75g. Analysis. Calcd. for N, 0.28.
Found: N, 0.10.
Synthesis of CH O-(CH2cH2O~ 2cH2-NH-cO-cH~ 2
1. BocNHOCH2COOH
PyBOP/DIEA
0 mPEG-NH2 ---------------> mPEG-O-CH2CH2-NH-CO-CH2ONH2
2. TFA
I mPEG-5000-amine (2.0g, 0.4 mmol) and soc-
¦ aminoxyacetic acid (0.2g, 1.05 mmol) were dissolved
; in 20ml dichloromethane. Benzotriazol-1-yl-
oxytripyrrolidinophosphonium hexafluorophosphate
(l.lg, 2mmol) was added followed by
diisopropylethylamine (0.7ml, 3.9 mmol). The
reaction was stirred for about 72 hours at room
temperature after which time cold ether was added,
and the resulting precipitate was collected by
filtration, washed, and dried. Half of the collected
precipitate was paced in a
dicholormethane/trifluoroacetic acid (3:7) mixture.
After 60 minutes the solution was concentrated. The
compound was further purified by gel filtration on a
LH-20 column eluting with methanol/methylene chloride
d~ (5:1). The resulting yellow precipitate was taken up
in water and was treated with decolorizing carbon.
After about 24 hr. the decolorizing carbon was
filtered, and the clear filtrate was concentrated.
The residue was dissolved in dichloromethane, dried
with sodium sulfate, filtered, and the filtrate was
: . ,,
.,
,~
:~
: .- :
, .~,;~ . , ~
~ r~
. ,""
~1~0~3
-47-
treated with cold ether. A white product was
obtained. Yield 0.5g. IR: (C=0): 1676. Analysis.
Calcd. for N, 0.55. Found: N, 0.50.
Svnthesis of CH3O-(CH2cH2O~ 2cH2-O-co-cH2-oNH2
1. BocNHOCH~COOH
PyBOP/DIEA/DMAP
mPEG-OH ---------------> mPEG-O-CH2CH2-O-CO-CH2ONH2
2. TFA
mPEG-5000-alcohol (2.0g, 0.4 mmol) and Boc-
aminooxyacetic acid (0.2g, 1.05 mmol) were dissolved
in 20 ml dichloromethane. Benzotriazol-1-yl-
oxytripyrrolidinophosphonium hexaflurophosphate
(l.lg, 2mmol) was added followed by
diisopropylethylamine (0.7ml, 3.09 mmol). The
reaction was stirred for about 2 hours at room
temperature after which time dimethylaminopyridine
(0.244g, 2 mmol) was added. After about four days
cold ether was added, and the resulting precipitate
was collected by filtration, washed, and dried. The
precipitate was taken up in water and was treated
with decolorizing carbon. After about 24 hr. the
decolorizing carbon was filtered, and the clear
filtrate was concentrated. The residue was dissolved
in dichloromethane, dried with sodium sulfate,
filtered, and the filtrate was treated with cold
ether. Analysis. Calcd. for N, 0.28. Found: N,
0.14.
Half of the collected precipitate was placed in a
dicholormethane/trifluoroacetic acid (2:1) mixture.
After 30 minutes the solution was concentrated. The
compound was further purified by gel filtration on a
, ~
.,
..
2i~0~43
-48-
LH-20 column eluting with methanol/methylene chloride
(5:1). Yield 0.3g. IR: (C=0): 1734.
Synthesis of CH30-(CH2CH~ H2CH2-CH!OH)-CH2-ONH2
O 1. BocNHOCH2COOH
mPEG~CH-CH2-------------> mPEG-O-CH2CH2-CH(OH)-CH2-ONH2
2. TFA
mPEG-5000-epoxide (1.0g, 0.2 mmol) was dissolved
in 10ml 0.lM NaOH. t-Butyl N-hydroxycarbamate (0.53g,
4 mmol) was added. After running the reaction
overnight the reaction mixture was extracted with
dichloromethane. Sodium sulfate was added and was
filtered. Cold ether was added the dichloromethane
solution, and the resulting precipitate was collected
by ~iltration, washed, and dried. The compound was
further purified by gel filtration on a LH-20 column
eluting with methanol/methylene chloride (5:1). The
protected mPEG-derivative (0.25g) was placed in a
dicholormethane/trifluoroacetic acid (1:1) mixture.
After 30 minutes the solution was concentrated and
taken up in dichloromethane. The compound was
isolated by precipitation from ether. Yield 0.2g.
IR: (~-H): 3447. Analysis. Calcd. for N, 0.28.
Found: N, 0.21.
Synthesis of CH30-(CH2CH20)~2CH2-CO-CH2-ONH2
1. DMSOIAc2O
mPEG-O-CH2CH2-CH(OH)-CH2-ONH-Boc 2. TFA
mPEG-O-CH2CH2-cO~cH2~0NH2
~o~3
-49-
mPEG-O-CH2CH2-CH(OH)-CH2-ON~-Boc (0.3g, 0.6 mmol)
was placed in 3 ml dry dimethyl sulfoxide followed by
the addition of 2 ml dry acetic anhydride. The
reaction went for about 24 hr. at room temperature
after which time cold ether was added. The resulting
precipitate was collected by filtration, washed and
dried. The product was placed in a
dicholormethane/trifluoroacetic acid (1:1) mixture.
After 30 minutes the solution was concentrated. The
compound was isolated by precipitation from cold
ether. Yield 0.18g. IR: (C=0): 1698. Analysis.
Calcd. for N, 0.28. Found: N, 0.20.
~nthesis of mPEG-Ornithine Semicarbazide
mPEG-5000-amine (5.0 g, 1. mmol) was dissolved
in 5 ml dry methylene chloride and 10 ml dry
dimethylformamide was added. Fmoc-Orn(Boc)-OPfp (3.1
g, 5 mmole) was added. After 1 hr. at room
temperature the solution was concentrated. Water was
added to the residue, and the solution was filtered,
centrifugred, and filtered to remove the dispersed
solid in the aqueous solution. The filtered aqueous
solution was concentrated, and the residue was ta~en
up in methylene chloride and was dried with sodium
~ sulfate. The solution was filtered. The filtrate
¦ 25 was treated with cold ether. The resulting
I precipitate was colleced by filtration, washed, and
dried. Yield 3.3 g. IR: (C=0):1713, 16~0. The Fmoc
group was removed by treating the compound with 25%
piperidine (in methylene chloride) for 30 min. Cold
ether was added to the solution to precipitate the
mPEG-derivative. The precipitate was collected,
washed, and dried. The free alpha amino group was
acetylated by dissolving 1.4 g of the mPEG-derivative
,,. , - .
-.,., ~
2 ~ 3
-50-
with 3 ml methylene chloride and adding 1 ml acetic
anhydride. After about 1.7 hr. at room temperature
the solution was concentrated. The resulting solid
was treated with trifluoroacetic acid in methylene
chloride (3:5) for 1 hr. at room temperature. The
solvent was removed and resulting oil was taken up in
methylene chloride and cold ether was added. The
precipitate formed was collected, washed, and dried.
Yield 1.1 g. IR: (C=O): 1685. The mPEG-derivative
was dissolved in 3 ml dry methylene chloride and
triethylamine (0.54 ml, 3.84 mmole) was added
followed by 1 ml phosgene in toluene (1.92 mmole) and
an additional 2 ml dry methylene chloride. The
reaction went overnight at room temperature after
which time the solvent was removed. The residue was
dissolved in 3 ml dry methylene ch loride and 0.2 ml
' hydrazone (5.76 mmole) was added. Dry methanol was
added until the solution became clear (3.4 ml).
After 4 hr. at room temperature the solution was
clarified by centrifugation and was concentrated.
The compound was purified by gel filtxation on a LH-
3 20 column eluting with methanol/methylene chloride
(5:1). Yield 0.7 g. IR: (C=O): 1675.
,
The invention having been described, the
following examples are offered by way of
illustration, not by way of limitation, of the
subject invention.
; EXAMPLES
' Modification of EPO rHydrazide Method)
In a typical experiment, EPO (0.5-1.0 mg)
(obtained from Ortho Biotech) was placed in 100 mM
; sodium acetate, pH 5.6, total volume 0.786 ml.
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-51-
Enough 10 mg/ml solution of sodium periodate was
added to give a final concentration of sodium
periodate at 10 mMol. The oxidation went for 30 min
at 0C in the dark after which time 0.33 ml 80 mMol
Na2SO3 was added. After 5 min the solution was
concentrated and washed three times with 100 mMol
sodium acetate, pH 4.2 in a microconcentrator. After
the final concentration the oxidized EPO solution was
brought up to 1.0 ml with 100 mM sodium acetate.
mPEG5000-hydrazide (50 mg) was added to the oxidized
EPO. The mixture was stirred over night at room
temperature. The mPEG5000-EP0 was purified by gel
filtration using a Sephacryl S-200-HR column (1 mm x
45 mm) eluting with a phosphate buffer containing
0.05% sodium azide. The amount of mPEG modifying EPO
was determined by HPLC gel filtration using a either
a Zorbax0 GF-250 or GF-450 column using a 0.1 M
phosphate buffer, pH 7. From 6-12 molecules mPEGs
were found to be attached to each molecule of EPO.
Modification of EPO rSemicarbazide Method)
The same procedure as above for the hydrazide
method was performed, except the reaction time for
'i oxidation was decreased to 5 minutes and a decreased
amount of mPEG-semicarbazide (10 mg) was used
compared to the amount of mPEG-hydrazide (50 mg).
Even with the decreased oxidation time and less mPEG
` added, more (about 18) mPEG molecules were attached
to EPO. If longer oxidation times (15 min) and more
mPEG-semicarbazide is added, around 30 mPEG molecules
càn be attached to EP0 depending on the molecular
weight of mPEG used. Thus mPEG-semicarbazide appears
~ to be more reactive than mPEG-hydrazide and attaches
:,
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2~0a~3
-52-
many more mPEG molecules to EPO than mPEG-hydrazide
is able to under similar reaction conditions.
A comparison of the effect of modifying EP0 with
mPEG on either the carbohydrate groups or on the
amino acid side chains is shown in Fig. 1.
Analytical HPLC gel filtration conditions are the
same as described above. The chromatogram of
unmodified EP0 is presented in Fig. la. A single
peak with a retention time of 10.5 min is found.
When EPO is modified with mPEG5000 on its
carbohydrate groups (Fig. lb), a single large peak
with a retention time of 9.4 minutes is seen for the
unpurified reaction product. When EPO is reaeted
I with a succinimide ester of mPEG5000 whieh reacts
¦ 15 with the side ehain of lysine, a heterogeneous
i mixture of reaetion produets is obtained (peaks from
7.5 - 10.2 minutes). A similar heterogeneous pattern
for mPEG modification using succinimide coupling to
CSF-l, interleukin-2, and ~-interferon has been found
; 20 (U.S. Pat No. 4,847,325 and 4,9117,888). There are
also more low molecular weight impurities present
, with succinimide coupling (Fig. le). Active ester
eoupling using sueeinimide derivatives of mPEG has
been the preferred method for attaehing mPEG to
proteins [Nueci, M.L., Shorr, R., and Abuehowski, A.
(1991~ Adv. Drug Delivery Rev., 6, 133-151]. EPO was
also derivatized with mPEG8500 using the above-
deseribed semiearbazide method, see Figure 4 for
results of biological experiments. The above-
described semiearbazide method was also used to
obtain EP0 modified with mPEG12000 (see Figure 3) and
EPO modified with mPEG2000 (see Figure 10).
EPO also was modified with thiosemicarbazide,
, hydrazide earboxylate, and carbonie aeid dihydrazide
,
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,
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2il~3
-53-
derivatives of mPEG. These derivatives of mPEG
performed like the semicarbazide derivatives of mPEG
in that high levels of coupling mPEG to EPO could be
obtained using these derivatives when compared to the
hydrazide derivatives of mPEG. Other conditions for
the oxidation of EPO can be used such as increased
temperature, increased concentration of sodium
periodate, and increased or decreased reaction times
as long as these oxidation conditions do not impair
the biological activity of EPO.
Lar~e-Scale Modification of EPO (Semicarbazide or
Carboxylate Hydrazide Method)
EPO (12 . Omg) (obtained from Ortho Biotech) was
placed in 100 mM sodium acetate, pH 5.5, total volume
1.8 ml. Sodium periodate (0.215 ml) at a
concentration of 40 mg/ml was added. The oxidation
went for 20 minutes at 0C in the dark after which
time 0.02 ml of ethylene glycol was added, and the
admixture stirred for 10 minutes at 0C. The
oxidized-EPO was purified by gel filtration using a
Sephadex~ G-25 column (2 . 5 cm X 9 cm) and eluted with
100mM sodium acetate buffer, pH 4.3. Eluted oxidized
EPO (10-llml) was pooled. mPEG5000 semicarbazide
(100 mg) was added to the purified oxidized EPO. The
mixture was stirred overnight at room temperature.
The mPEG-5000 EPO was purified by gel filtration
using a Sephacryl~ S-200-HR column eluting eith
buffer consisting of 0.2M NaCl, 0.02 M sodium
citrate, 0.025% sodium azide, pH 7Ø
, 30 The above modification was repeated using 200 mg
i of mPEG5000 semicarbazide. Reactivity was about 22
mPEG molecules per molecule of EPO.
The above modification was repeated employing
half the amount of the reactants as specified
i
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2~,10~3
-54-
hereinabove, using 200 mg of carboxylate hydrazide.
Reactivity was about 30 mPEG molecules per molecule
of EPO.
Modification of EPO (Oxime Method)
A. mPEG-CH~_2-NH-CO-CH2-ONH2
The same procedure as above for the large-scale
semicarbazide method was performed. However, instead
of addition of mPEG5000 semicarbazide, mPEG5000-CH2-
CH2-NH-C0-CH2-ONH2 (50 mg) was admixed with 2.15 ml of
oxidized EPO at room temperature overnight. The
mPEG5000-EPO was purified by gel filtration using a
Sephacryl~ S-200-HR column eluting with buffer
consisting of 0.2M NaCl, 0.02 M sodium citrate,
0.025% sodium azide, pH 7. About 31 molecules of
mPEG5000 were found to be attached to each molecule
of EPO as determined by HPLC gel filtration using a
Phenomenex Biosep-Sec-S4000 column (30 cm X .017 cm).
A minor fraction consisting of 25 molecules of mPEG-
; EPO was also isolated, and was used for biological testing. See Figures 16, 18 and 19.
i B. mPEG-O-CH2_2-NH-CO-ONH2
The above modification of EPO was repeated using
mPEG-O-CH2CH2-NH-CO-ONH2. About 18-19 molecules of
mPEG5000 were found to be attached to each molecule
~ 25 of PEG as determined using the methods as in
-~ modification A above. Biological data is shown in Figures 16, 18 and 19.
c . mPEG-O-CH2CH2-ONH2
The above modification of EPO was repeated using
, 30 mPEG-O-CH2CH2-ONH2. About 17 molecules of mPEG5000-~ were found to be attached to each molecule of PEG as
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-55-
determined using the methods as in modification A
above. Two minor fractions of 22mPEG, 12mPEG were
also isolated(Figure 17, 18, 19).
D. mPEG-O-CH2cH2-cO-oNH2
The above modification of EPO is repeated using
the PEG-oxime derivative mPEG-O-CH2CH2-CO-ONH2. About
3 molecules of mPEG5000 were found to be attached to
each molecule of EPO, as determined using the methods
as for modification A above.
E. mPEG-O-CH2CH2-CH(OH)-CH2-ONH2
, The above modification of EPO is repeated using
i the PEG-oxime derivative mPEG-O-CH2CH2-CH(OH)-CH2-ONH2.
j About 31 molecules of mPEG5000 were found to be
attached to each molecule of EPO, as determined using
the methods as for modification A above.
F._ mPEG-O-CH2CH2-NH-CS-ONH2
The above modification of EPO is repeated using
the PEG-oxime derivative mPEG-O-CH2CH2-NH-CS-ONH2.
About 4 molecules of mPEG5000 were found to be
~' 20 attached to each molecule of EPO, as determined using
the methods as for modification A above.
~i
Bioloqical ActivitY of mPEG-EPO
The mPEG-EPO derivatives were assayed for
biological activity in vivo by measuring the increase
~! 25 in erythrocytes generated after injection of the
',~3 modified protein (Egrie, J.C., Strickland, T.W.,
Lane, J., Aoki, K., Cohen, A.M., Smalling, R., Trail,
G., Lin, F.K., Browne, J.K., and Hines, D.K. (1986)
Immunobiol. 172: 213-224~. Briefly, mice (female
CD-l, eight weeks old) were injected either
.
i:
2~ ~3 ~3
-56-
intraperitoneally or subcutaneously with 0.4 ~g
protein once a day for two consecutive days. Blood
was withdrawn on predetermined days for hematocrit
readings.
The in vivo biological activities (hematocrit
levels) of the mPEG-EPOs linked via hydrazide
derivatives of mPEG is presented in Figures 2, 4 and
5 and Tables I and II. To summariæe the findings,
Figures 2 and 5 show that the optimal number of mPEG
coupling is not obvious and has to be determined by
synthesis and biological testing in order to give the
best mPEG-EPO. Figure 4 compares mPEG coupling using
hydrazide and semicarbazide linkers. Higher and
longer hematocrit levels for mPEG-EPO could be
obtained using the semicarbazide linker. The
observed results are due, in part, to the higher
level of incorporation of mPEG which could be
obtained using the semicarbazide linker. Table I
shows the biological activity of the mPEG-EPOs as a
function of the number of mPEGs incorporated and the
¦ molecular weight of mPEG used.
Table I summarizes the biological activity of
different hydrazide mPEG-EPOs comparing molecular
weight of mPEG used and the amount of mPEG coupled.
~ 25 Table II shows a compar'~son of the different
3 mPEG hydrazide linkers used. Table II summarizes the
biological activities of EPO modified with different
mPEG5000-hydrazide derivatives where optimal amounts
of mPEG were incorporated. Not all the carbohydrate
i 30 mPEG-derivatives give the same biological activity
¦ when coupled to EPO due to the inability to
sufficiently couple an optimal amount of mPEG or
other factors. Biological activities are the best
values obtained for each linker.
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-57- 2110 ~ 43
TABL13 I
Tablo l: ~lolo~lc~l Actlvlly ol Dlll~rr~n~ mPE~-EPOa
I~DII1CUIhl Durt~llDn ol ~cllvlly
~Q~- ~x. ll~mnlocllJ (~)" llol~llYo lo EPO (I)~y~
16 Z000 00 14
~ # OnO0 63 C
12 ~ 6000 61 7
1 û 6000 111 10
22 ~000 ~ 1 20
24 6000 ~;â 13
28 6000 Ei4 14
~l 17 ~ooo 6a 1~
12 ~ 86uo 64 7
M600 68 14
7 7
1 2000 6~ 7
14 120W 63 11
~0 ~2000 61 7
J
x ^ /~c Delormlncd by 81ro Exclu~hn Cllrom~agr~phy
r - ~hloDlGt I ~ ay Dn-cllbed ~n Expr~rlJnonl~l 8ecllon
D-yc trom EP0 ~I-xlmum H~malocllt Le~ol l48 on D~y 4) R~ul~od lo Ro~ch EPO'- Ma~ nurn
~1 ~moloctlt Lovel ~llo~ ~tlilrlnD Ib O~rn U~xlmum Hcn~lucrll Lovol
,$ ~ Hydl021d- LlnkorU~ llt)lhot- U~od Sumlcnrba2kbLlnhot.
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Tablr~ 8iolo~1cal Actlvliy ot mPE~i5000 EPOs Comparln~ ;)il~erent Carbohydr~te Modlfying mPEG-Link6t~
Dur~tion o1 Actlvity
Llnkot l.IBX. Hr rral~ ' RelAllvo lo EFO (Dr~ys)"
CH~ (OCH2CH2)r~ 0 CH2-CO-NHNH2 (Hydrazld0) 54 j3
CH~-(OCH2CH2)n-NH-CO-NHNH2 ~50micrArbe21dr~) 61 20
CH3-(0CH~CH2)~ NH-CS-NHNH2 (Thlosomlcrlrb~21de) 5~ 14
CH3-~OCH2CI12)~-NH-CO-NHNH-CO-NHNH2 (CerbonloAc;d Dlhydrezid~) 57 12
CH~-(OCH2CH2)n-0-CO-NHNH2 ~Hydro~lde C~ri~olyl~lle, 22 mpr-r~3c) 60
CH3-(0CH2CH~)o-NH CO-CoH~-NtiNH2 ~Arylhy~razldo~ 52 7
^ i3ioloolcxl As-cy Oogcrlb0d Irl Exporlmr~n~rll Seclion
' D~y~ 1rom Ei'O M0~dmum Horr~rAlocrlt Lewl (4~% on Ooy 4) Roqulred lo Ro~ch EPO'~ Mcximum Horn~ltoorli Lovol Alter
A~srilnlnj;; Ir~ o~n ~axlmurr. HcmrA~oorit Level
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The hematocrit levels for EPO and mPEG5000-~POs
in mice are presented in Fig. 2. The 12PEG-EPO was
made by coupling mPEG5000-hydrazide and reflects the
maximum incorporation which could be achieved by this
mPEG derivative under the experimental conditions
given above. The 18PEG and 28PEG EPOs were made by
coupling with mPEG5000-semicarbazide. The
semicarbazide derivatives of mPEG result in much
better biological activity than the hydrazide
derivatives of mPEG due to the larger amounts of mPEG
which can be incorporated using this mPEG derivative.
All three mPEG5000-EPOs show increased maximum and
prolonged activity when compared to native EPO. Thus
modification of a protein's carbohydrate groups with
PEG can yield a much more potent therapeutic protein.
For additional data on the effects of various
mPEG hydrazide-modified EPOs employed in the
experiments on hematocrit levels, see Figures 8-15.
The mPEG modified EPO employed in the experiments
depicted in Figures 8-15 were prepared using the
appropriate water-soluble polymer reagent essentially
as described for the other mPEG modified EPO
molecules used the experiments depicted in Figures 2-
5.
2~ The in vivo biological activities (hematocrit
levels) of the mPEG-EPOs linked via oxime-forming
derivatives of mPEG is presented in Figures 16 and
17. To summarize, Figure 16 compares mPEG coupling
using mPEG-O-CH2CH2-NH-CO-ONH2 ("A") and mPEG-O-
CH2CH2-NH-CO-CH2-ONH2 ("C") linkers. Higher
hematocrit levels of mPEG-EPO could be obtained using
the "~" linker (corresponding to Formula XXX herein)
having 18 mPEG molecules per molecule of EP0 as
compared to the "C" linker (corresponding to Formula
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XXXIII herein) having 31 mPEG molecules per molecule
of EPO. Also notable was the higher hematocrit
activity of the "C" linker (Formula XXXIII herein~
having 25 mPEG molecules per molecule of EPO as
compared to the same linker having 31 molecules of
mPEG per molecule of EPO.
Figure 17 compares mPEG coupling using mPEG-O-
C~2CH2-ONH2 (formula XXIII) ("B") linker at 22, 17,
and 12 mPEG molecules per molecule of EPO. Highest
hematocrit levels are obtained at the lowest degree
of pegylation, and hematocrit was decreased inversely
proportional to degree of pegylation. In all mPEG
$ linkers using mPEG-O-CH2CH2-ONH2 oxime derivative
however, hematocrits were higher and of increased
duration as compared to native EPO.
Especially noteworthy is the biological activity
of the 12mPEG-EPO of formula XXIII. Hydrazide
derivitized 12 mPEG-EPO produces neither the degree
nor duration of hematocrit elevation as that of the
oxylamine derivitized EPO.
Antibody bindinq to mPEG-EPO
The antigenicity of the mPEG-EPOs was determined
by using the ClinigenT~ erythropoietin (EPO) EIA test
kit. Briefly, the assay consists of a micro titre
plate coated with a monoclonal antibody to EPO. EPO
or mPEG-EPO is allowed to interact with the c~ated
plate. After washing the plate a labeled polyclonal
antibody to EPO is incubated on the plate. After
substrate development the plate is read.
The results of the ELISA assay for hydrazide
derivatized EPO, are presented in Figure 3. The
mPEG-EPOs are presented as the approximate number of
mPEGs attached for a given molecular weight of mPEG.
,
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2~0~3
-61-
For example, 12PEG-5k means about 12 mPEG molecules
of a molecular weight of about 5000 were coupled to
each molecule of EPO. The data indicates that as the
number of mPEGs coupled to EPO are increased! the
antigenicity of the protein is decreased. Similarly
as the molecular weight of mPEG is increased, the
antigenicity, i.e. the binding of the antibody, of
the modified EP0 also is decreased. Reacting
~ oxidized EPO with a hydrazide derivative of mPEG did
not reach the high coupling levels seen with
semicarbazide, thiosemicarbazide, and carbonic acid
dihydrazide PEG derivatives and thus could not give
the large decreases in immunogenicity as seen with
these other mPEG derivatives. Decreasing the
antigenicity of a protein correlates to a decrease in
the immunogenicity of a protein as well. Thus
mPEG-EP0 coupled to the carbohydrate groups of EPO
may reduce any potential immunogenicity related to
the protein with those derivatives of mPEG able to be
coupled at high levels being the most effective.
For additional ELISA data with mPEG hydrazide
derivatives see Figure 6.
The results of the ELISA assay for oxime
derivatized EP0 are presented in Figure 18. The data
indicates that as the number of mPEGs coupled to EPO
are increased, the antigenicity of the protein is
decreased. Reacting oxidized EP0 with a linker that
resulted in comparatively low coupling levels (18
PEG-A, 17 PEG-B, 12 PEG-B) did not give the large
decreases in immunogencity as seen with the
comparatively high coupling level formulations (22
PEG-B, 25 PEG-C, and 31 PEG-C. Note that these
differences in a linker's ability to decrease
immunogenicity appear to be determined largely based
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on the coupling level (e.g. compare 12 PEG-B and
22PEG-B). Thus, mPEG coupled to carbohydrate groups
of EP0 through oxime linkages may reduce potential
immunogencity related to the protein, with those
derivatives of mPEG able to be coupled at high levels
being the most effective.
Modification of Horseradish Peroxidase: ComParison
of HYdrazide Versus Semicarbazide Cou~linq
Horseradish peroxidase (HRP) is a glycoprotein
enzyme (oxido-reductase). HRP was modified with
either mPEG5000-hydrazide or mPEG5000-semicarbazide
in order to see whether another glycoprotein besides
EPO could show the difference in modification between
the two different carbohydrate modification reagents.
In a typical experiment, horseradish peroxidase (2
mg) was placed in 100 mM sodium acetate, pH 5.6,
total volume 0.8 ml. Enough 10 mg/ml solution of
sodium periodate was added to give a final
concentration of sodium periodate at 10 mMol. The
oxidation went for 15 min at ooc in the dark, after
which time 0.33 ml 80 mMol Na2SO3 was added. After 5
minutes, the solution was concentrated and washed
', three times with 100 mMol sodium acetate, pH 4.2, in
,, a micro concentrator. The oxidized horseradish
, 25 peroxidase solution was then split in half, with one
'l half receiving mg PEG5000-hydrazide and the other
half receiving 30 mg mPEG5000-semicarbazide. The two
~', oxidized horseradish peroxidase solutions were then
'' stirred overnight at room temperature. The extent of
, 30 PEG modification was determined by HPLC gel
~ filtration using a Zorbax~ GF-250 column using,a 0.1
`, M phosphate buffer, pH 7. The mPEG5000-hydrazide
, modified horseradish peroxidase had approximately 7
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211~3
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PEG molecules/HRP; the mPEG5000-semicarbazide
modified horseradish peroxidase had approximately 19
PE& molecules/HRP. Thus modification of horseradish
peroxidase with PEG being attached to its
carbohydrate groups is more effective using a
semicarbazide derivative of PEG than a hydrazide
under the same experimental conditions.
}lalf-Life Determinations
Half-Life experiments were done in male Sprague-
Dawley rats weighing about 0.3 kg. Three rats were
used for each compound. The experimental details are
as follows. Each rat was injected IV (intravenously)
with 1 ~g EP0 or mPEG-EPO. For the hydrazide
derivatized mPEG-EPO, the mPEG-EPO used was mPEG500
semicarbazide-18, prepared essentially as described
in the section above on EPO modification with
semicarbazides. Blood was withdrawn from each rat at
2, 5, 15, 45, 90 minutes and 3, 6, 24, 48, 54 hour
time points. The blood was collected in heparinized
tubes, and the plasma was isolated. The isolated
plasma was tested for EP0 biological activity in an
EPO dependent cell proliferation assay. The in vitro
l assay employed used an FDC-P1/ER cell line. This
murine cell line incorporates the EPO receptor and is
dependent on EPO for grow~h. The assay was
j performed as follows. The cells were grown (106/ml)
in the absence of EP0 for 24 hr after which time
either EPO or mPEG-EPO at different concentrations is
' added to the cells. The cells were incubated for 42
J 30 hr, and then tritiated thymidine was added to the
cells. After 6 hr the cells were harvested and
counted. Cell growth was determined by the increased
up-take of thymidine. Results are given in figure 7.
,,
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-64-
The EPO dependent cell proliferation assay was
performed as described above using oxime-derivatized
mPEG-EP0. Results are given in Figure 19.
In Vivo Assav: Anemic Mouse Model
In this assay mice are rendered anemic by
injections for five consecutive days with TNF-alpha.
To overcome the anemia the mice were injected SC
(subcutaneously) with either EP0 or mPEG-EPO (at 0.03
~g/dose) over the same five days or on just two of
the five days that the mice receive TNF-alpha.
Results are given in figure 15.
Eauivalents
All publications and patents mentioned in the
above specification are herein incorporated by
reference~ The foregoing written specification is
considered to be sufficient to enable one skilled in
the art to practice the invention. Indeed, various
modifications of the above-described modes for
carrying out the invention which are obvious to those
skilled in the field of molecular biology or related
fields are intended to be within the scope of the
following claims.
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