U.S. patent application number 11/716866 was filed with the patent office on 2007-09-20 for chemically modified g-csf.
This patent application is currently assigned to KIRIN-AMGEN, INC.. Invention is credited to Rika Ishikawa, Makoto Kakitani, Yuji Okada.
Application Number | 20070219357 11/716866 |
Document ID | / |
Family ID | 26511383 |
Filed Date | 2007-09-20 |
United States Patent
Application |
20070219357 |
Kind Code |
A1 |
Ishikawa; Rika ; et
al. |
September 20, 2007 |
Chemically modified G-CSF
Abstract
The present invention provides a chemically-modified protein
prepared by binding polyethylene glycol to a polypeptide
characterized by being the product of expression by a host cell of
an exogenous DNA sequence and substantially having the following
amino acid sequence: TABLE-US-00001 (Met).sub.n Thr Pro Leu Gly Pro
Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg
Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr
Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile
Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly
Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln
Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln
Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu
Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala
Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln
Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro (n
= 0 (SEQ ID NO: 1) or 1 (SEQ ID NO: 2)) The chemically-modified
protein according to the present invention has a
neutrophils-increasing activity much more lasted than that of the
intact human G-CSF, enabling fewer numbers of administration with a
lower dose.
Inventors: |
Ishikawa; Rika; (Tokyo,
JP) ; Okada; Yuji; (Gunma-Ken, JP) ; Kakitani;
Makoto; (Gunma-Ken, JP) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
233 S. WACKER DRIVE, SUITE 6300
SEARS TOWER
CHICAGO
IL
60606
US
|
Assignee: |
KIRIN-AMGEN, INC.
Thousand Oaks
CA
|
Family ID: |
26511383 |
Appl. No.: |
11/716866 |
Filed: |
March 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11342519 |
Jan 30, 2006 |
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11716866 |
Mar 12, 2007 |
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10436784 |
May 12, 2003 |
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11342519 |
Jan 30, 2006 |
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09921114 |
Aug 2, 2001 |
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10436784 |
May 12, 2003 |
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09518896 |
Mar 6, 2000 |
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09921114 |
Aug 2, 2001 |
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08957719 |
Oct 27, 1997 |
6166183 |
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09518896 |
Mar 6, 2000 |
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07983620 |
Nov 30, 1992 |
5824778 |
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08957719 |
Oct 27, 1997 |
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07566451 |
Oct 1, 1990 |
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07983620 |
Nov 30, 1992 |
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Current U.S.
Class: |
530/410 |
Current CPC
Class: |
A61K 38/00 20130101;
A61K 47/60 20170801; Y10S 930/145 20130101; C07K 14/535 20130101;
C07K 17/08 20130101 |
Class at
Publication: |
530/410 |
International
Class: |
C07K 14/475 20060101
C07K014/475 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 22, 1988 |
JP |
324747/88 |
Jul 31, 1989 |
JP |
199176/89 |
Claims
1. A chemically-modified protein prepared by binding polyethylene
glycol to a polypeptide characterized by being the product of
expression by a host cell of an exogenous DNA sequence and
substantially having the following amino acid sequence:
TABLE-US-00009 (Met).sub.n Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro
Gln Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp
Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro
Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu
Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu
His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile
Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp
Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala
Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg
Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val
Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro (n = 0 (SEQ ID NO:1) or
1 (SEQ ID NO:2))
2. The chemically-modified protein according to claim 1 wherein
polyethylene glycol is bound through an amino group of the amino
acid(s) of the polypeptide.
3. The chemically-modified protein according to claim 1 wherein
polyethylene glycol is bound through a carboxyl group of the amino
acid(s) of the polypeptide.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 11/342,519, filed Jan. 30, 2006, which in turn
is a Continuation of U.S. patent application Ser. No. 10/436,784,
filed May 12, 2003, which is a Divisional application of U.S.
patent application Ser. No. 09/921,114, filed Aug. 2, 2001, which
is a Continuation of U.S. patent application Ser. No. 09/518,896
filed Mar. 6, 2000, which is a Continuation of U.S. patent
application Ser. No. 08/957,719 filed Oct. 27, 1997, which is a
Continuation of U.S. patent application Ser. No. 07/983,620 filed
Nov. 30, 1992, which is a Continuation of U.S. patent application
Ser. No. 07/566,451 filed Oct. 1, 1990, which is the U.S. National
Stage of PCT/JP89/01292.
TECHNICAL FIELD
[0002] The present invention relates to a chemical modification of
granulocyte colony-stimulating factor (G-CSF), by which the
chemical and/or physiological properties of G-CSF can be
changed.
BACKGROUND ART
[0003] Human G-CSF is one of the haematopoietic growth factors. It
has been shown to be present in the conditioned medium of a human
bladder carcinoma cell line denominated 5637 (ATCC HT8-9) (Welte et
al., Proc. Natl. Acad. Sci. (USA), 82, pp. 1526-1530, (1985)). The
determination of a DNA sequence encoding human G-CSF (Japanese
Patent Application Laying Open KOHYO No. 500636/88) has enabled the
production of human G-CSF by means of recombinant genetic
techniques.
[0004] Human G-CSF may be useful in the treatment of general
haematopoietic disorders including those arising from chemotherapy
or from radiation therapy. It may be also useful in bone marrow
transplantation. Wound healing burn treatment and the treatment of
bacterial inflammation may also benefit from the application of
human G-CSF (Welte et al., supra.).
[0005] It is generally observed that physiologically-active
proteins administered into a body can show their pharmacological
activity only for a short period of time due to their high
clearance rate in the body. Furthermore, high hydrophobicity of the
proteins reduces their stability.
[0006] For the purpose of decreasing the clearance rate, improving
stability or abolishing antigenicity of the proteins, some methods
have been proposed wherein the proteins are chemically modified by
using polyethylene glycol. Japanese Patent Application Laying Open
KOHYO No. 289522/87, for EXAMPLE, discloses the reduction in
immunogenicity of TNF which has been modified by polyethylene
glycol. Japanese Patent Application Laying Open KOHYO No. 503171/87
discloses with respect to IL-2 and IFN-.beta. the reduction in
immunogenicity and aggregating tendencies in an aqueous solution,
and the prolongation of half-life in blood. In addition, there are
disclosed the prolongation of half-life in blood and the
disappearance of antigenicity or immunogenicity owing to the
modification by polyethylene glycol with respect to a plasminogen
activator (Japanese Patent Application Laying Open KOHYO No.
60938/88), IL-2, IFN-.gamma. and SOD (Japanese Patent Application
Laying Open KOHYO No. 10800/88), and IAP (Japanese Patent
Application Laying Open KOHYO No. 126900/88).
[0007] However, these prior art publications have not disclosed an
improvement in biological activity and pharmacokinetics, which may
be expected as a result of the modification of human G-CSF by
polyethylene glycol.
[0008] Accordingly, it has been desired to prolong the half-life of
human G-CSF in the body so as to enhance its effects, as may be
expected. Furthermore, a G-CSF product which can accelerate
recovery from neutropenia has been desired.
DISCLOSURE OF INVENTION
[0009] After vigorous investigations in order to solve the above
problems, the present inventors have now found that their solution
can be realized by binding polyethylene glycol to human G-CSF, and
have completed the present invention.
[0010] Any purified and isolated human G-CSF which is produced by
host cells such as E. coli and animal cells transformed by using
recombinant genetic techniques may be used in the present
invention.
[0011] Among them, the human G-CSF which is produced by the
transformed E. coli is particularly preferable. Such human G-CSF
may be obtained in large quantities with high purity and
homogeneity and substantially has the following amino acid
sequence: TABLE-US-00002 (Met)n Thr Pro Leu Gly Pro Ala Ser Ser Leu
Pro Gln Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly
Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His
Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro
Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln
Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly
lle Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro
Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln
Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu
Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro [n = 0(SEQ ID NO:1)
or n = 1 (SEQ ID NO:2)]
[0012] The above human G-CSF may, for example, be prepared
according to a method disclosed in Japanese Patent Application
Laying Open KOHYO No.500636/88. The term "substantially has the
following amino acid sequence" means that the above amino acid
sequence may include one or more amino-acid changes (deletion,
addition, insertion or replacement) as long as such changes will
not cause any disadvantageous non-similarity in function to a
naturally-occurring human G-CSF.
[0013] It is more preferable to use the human G-CSF substantially
having the above amino acid sequence, in which at least one lysine,
aspartic acid or glutamic acid residue is included.
[0014] According to the present invention, polyethylene glycol is
covalently bound through amino acid residues of the polypeptide of
human G-CSF. The amino acid residue may be any reactive one having,
for example, free amino or carboxyl groups, to which a terminal
reactive group of an activated polyethylene glycol may be bound.
The amino acid residues having the free amino groups may include
lysine residues and N-terminal amino acid residue, and those having
the free carboxyl group may include aspartic acid, glutamic acid
residues and C-terminal amino acid residue.
[0015] A molecular weight of the polyethylene glycol used in the
present invention is not restricted to any particular range, being,
however, normally of from 500-20,000 and preferably of from
4,000-10,000.
[0016] Polyethylene glycol is bound onto human G-CSF via a terminal
reactive group (or "a spacer"). Polyethylene glycol having the
spacer is hereinafter referred to as "an activated polyethylene
glycol". The spacer, for example, is a terminal reactive group
which mediates a bond between the free amino or carboxyl groups and
polyethylene glycol. The activated polyethylene glycol which may be
bound to the free amino group includes N-hydroxysuccinylimide
polyethylene glycol having the following formula: ##STR1## which
may be prepared by activating succinic acid ester of polyethylene
glycol with N-hydroxysuccinylimide. Another activated polyethylene
glycol which may be bound to free amino group is
2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine having the
following formula: ##STR2## which had been prepared by reacting
polyethylene glycol monomethyl ether with cyanuric chloride. The
activated polyethylene glycol which is bound to the free carboxyl
group includes polyoxyethylenediamine having the following formula:
H.sub.2NCH.sub.2CH.sub.2CH.sub.2O(C.sub.2H.sub.4O).sub.nCH.sub.2CH.sub.2C-
H.sub.2NH.sub.2
[0017] The chemical modification through a covalent bond may be
performed under any suitable condition generally adopted in a
reaction of a biologically active substance with the activated
polyethylene glycol. In case where the reactive amino acid residues
in human G-CSF have free amino groups, the above modification is
preferably carried out in a buffer solution such as phosphate and
borate (pH 7.5-10.0) for 1-5 hrs at 4-37.degree. C. The activated
polyethylene glycol may be used in 1-200 times, preferably 5-50
times the molar amount of the number of free amino groups of human
G-CSF. On the other hand, where the reactive amino acid residues in
human G-CSF have the free carboxyl groups, the above modification
is preferably carried out in pH 3.5-5.5, for example, the
modification with polyoxyethylenediamine is carried out at the
presence of carbodiimide (pH 4.0-5.0) for 1-24 hrs at 4-37.degree.
C. The activated polyethylene glycol may be used in 1-200 times the
molar amount of the number of free carboxyl groups of human
G-CSF.
[0018] The extent of the modification of the amino acid residues
may be optionally controlled depending on an amount of the
activated polyethylene glycol used in the modification.
[0019] A polyethylene glycol-modified human G-CSF, namely
chemically modified protein according to the present invention, may
be purified from a reaction mixture by conventional methods which
are used for purification of proteins, such as dialysis,
salting-out, ultrafiltration, ion-exchange chromatography, gel
chromatography and electrophoresis. Ion-exchange chromatography is
particularly effective in removing unreacted polyethylene glycol
and human G-CSF.
[0020] The present polyethylene glycol-modified human G-CSF has a
more enduring pharmacological effect, which may be possibly
attributed to its prolonged half-life in body.
[0021] Furthermore, it is observed that the present polyethylene
glycol-modified human G-CSF may accelerate recovery from
neutropenia.
[0022] The present polyethylene glycol-modified human G-CSF has
essentially the same biological activity as an intact human G-CSF
and may accordingly be used in the same application as that. The
polyethylene glycol-modified human G-CSF has an activity for
increasing the number of neutrophils, and it is therefore useful in
the treatment of general haematopoietic disorders including those
arising from chemotherapy or from radiation therapy. It may be also
useful in the treatment of infection and under receiving the
therapy of bone marrow transplantation.
[0023] The present polyethylene glycol-modified human G-CSF may be
formulated into pharmaceuticals containing also a pharmaceutically
acceptable diluent, an agent for preparing an isotonic solution, a
pH-conditioner and the like in order to administer them into a
patient.
[0024] The above pharmaceuticals may be administered
subcutaneously, intramuscularly, intravenously or orally, depending
on a purpose of treatment. A dose may be also based on the kind and
condition of the disorder of a patient to be treated, being
normally between 0.1 .mu.g and 5 mg by injection and between 0.1 mg
and 5 g in an oral administration for an adult
BRIEF DESCRIPTION OF DRAWINGS
[0025] FIG. 1 shows scanning patterns of PEG (4,500) G-CSF obtained
by SDS-PAGE. The molar ratio of the activated PEG to the free amino
groups of the human G-CSF is 0 for (a), 1 for (b), 5 for (c), 10
for (d) and 50 for (e), respectively. The peak of the intact human
G-CSF is marked with *.
[0026] FIG. 2 shows the time course of the change in number of
neutrophils in mice after administration with human G-CSF or
PEG-modified G-CSF. Each point represents an average value obtained
from six mice with a standard deviation.
[0027] FIG. 3 shows an accelerating effect of PEG-modified human
G-CSF on the recovery from neutropenia induced by cyclophosphamide.
Each point represents an average value obtained from six mice with
a standard deviation.
[0028] FIG. 4 shows an accelerating effect of PEG-modified G-CSF on
the recovery from neutropenia induced by 5-FU. Each point
represents an average value obtained from six mice with a standard
deviation.
[0029] FIG. 5 shows the results obtained in the study of half-life
in serum of PEG (10,000) G-CSF (.largecircle.) and human G-CSF
(.circle-solid.). Each point represents an average value from three
rats with a standard deviation.
BEST MODE FOR CARRYING OUT THE INVENTION
[0030] The present invention will be further illustrated by
referring to the following EXAMPLEs which, however, are not be
construed as limiting the scope of the present invention.
EXAMPLE 1
Preparation of PEG (4,500) G-CSF
[0031] Recombinant human G-CSF (Japanese Patent Application Laying
Open KOHYO No. 500636/88) having the following amino acid sequence
(SEQ ID NO: 2) was used for the chemical modification according to
the present invention: TABLE-US-00003 (SEQ ID NO: 2) Met Thr Pro
Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu Glu
Gln Val Arg Lys lle Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys
Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser
Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln
Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly
Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp
Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met
Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro
Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser
His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala
Gln Pro
[0032] The activated polyethylene glycol (PEG) used was
Methoxypolyethyleneglycol-Succinimydyl Succinate (Nippon Oil and
Fats, Co., Ltd.) which had been prepared by activating a succinic
acid ester of polyethylene glycol with an average molecular weight
of about 4,500 with N-hydroxysuccinylimide.
[0033] The human G-CSF was incubated in 0.25M sodium borate buffer
(pH 8.0) for 1 hr at 4.degree. C. with the activated PEG in 1-50
times the molar amount of the number of the free amino groups in
the human G-CSF. The resulting product was applied to Sephadex G25
which had been equilibrated with 10 mM NH.sub.4 HCO.sub.3 for
buffer-exchange, and then to DEAE ion-exchange chromatography so as
to separate the PEG-modified human G-CSF from the agent and, if
necessary, an unreacted human G-CSF. The resultant PEG-modified
human G-CSF is hereinafter referred to as "PEG (4,500) G-CSF".
EXAMPLE 2
Characterization of PEG (4,500) G-CSF
[0034] PEG (4,500) G-CSF prepared in EXAMPLE 1 was characterized by
the number of unmodified amino groups and a molecular weight
estimated by SDS-PAGE.
[0035] The number of the unmodified amino groups was determined by
reacting them with 0.1% TNBS in 4% NaHCO.sub.3 followed by
measurement of absorbance at 335 nm (Habeeb et al., Anal. Biochem.,
14, pp. 328-336, (1966)).
[0036] The molecular weight of PEG (4,500) G-CSF was determined by
SDS-PAGE (16% gel, CBB staining) according to a method of Laemli,
Nature, 227, p. 680, 1970. Each lane on the gel was scanned by
using a chromato-scanner (SHIMADZU CORPORATION: CS-930) after
staining.
[0037] When a molar ratio of the activated PEG to the number of
free amino groups of human G-CSF increased, the extent of the
modification also increased. The product prepared in said molar
ratio of 1 has in addition to a band corresponding to an intact
human G-CSF (19K) another band with an apparent molecular weight of
about 26K (FIG. 1). With respect to the product prepared in the
molar ratio of 5 or more, a band with a higher molecular weight was
observed besides the above two bands. By scanning the resulting
gel, a content of each band was determined. From the result in
TABLE 1, it is estimated that the band of 26K consists of human
G-CSF wherein one human G-CSF molecule is bound with one activated
PEG molecule and that a band of 34K consists of human G-SCF wherein
one human G-CSF molecule is bound with two activated PEG molecules.
TABLE-US-00004 TABLE 1 Characterization of PEG (4,500) G-CSF
Unmodified NH.sub.2 Distribution Modified NH.sub.2 (an average
PEG/NH.sub.2 19K 26K 34K (%) number) 1 86 12 5 4.8 2 68 31 1 15 4.3
3 56 42 2 15 4.3 4 36 48 16 20 4.0 5 31 49 20 27 3.7 6 25 50 25 27
3.7 7 20 50 28 27 3.7
[0038] It was found that based on patterns obtained by SDS-PAGE of
the fractions from the ion-exchange chromatography (shown in FIG.
1) that the human G-CSF with a higher modification extent was
eluted faster from a column and that the fraction finally eluted
therefrom contained the intact human G-CSF.
[0039] The scanning patterns by SDS-PAGE of PEG (4,500) G-CSFs
including those obtained with a higher molar ratio of PEG/NH.sub.2
are shown in FIG. 1.
EXAMPLE 3
Preparation of PEG (10,000) G-CSF
[0040] The same human G-CSF as used in EXAMPLE 1 was modified by an
activated polyethylene glycol (an activated PEG 2; Seikagaku Kogyo
K.K.) with a molecular weight of about 10,000 having the following
formula: ##STR3## which had been prepared by reacting polyethylene
glycol monomethyl ether with cyanuric chloride.
[0041] The human G-CSF was incubated with the activated PEG 2 at 5
times the molar amount of free amino groups of the human G-CSF in
0.25M sodium borate buffer solution (pH 10.0) for 1 hr at room
temperature. The resulting product was applied to Sephadex G25
which had been equilibrated with 10 mM NH.sub.4 HCO.sub.3 for
buffer-exchange, and then to DEAE ion-exchange chromatography to
separate the PEG-modified human G-CSF from an unreacted human G-CSF
and reagent. The estimation of a molecular weight of the product by
SDS-PAGE as in EXAMPLE 2 has revealed that its average molecular
weight is about 45K with distributed among 30K (10%), 40K (70%) and
66K (20%). The resultant PEG-modified human G-CSF is hereinafter
referred to as "PEG (10,000) G-CSF".
[0042] Moreover, human G-CSF was incubated with the activated PEG 2
at 10 times the molar amount of free amino groups of the human
G-CSF in 0.25M sodium borate buffer solution (pH 10.0) for 2 hrs at
room temperature. The resulting product was subjected to separation
in the same manner as stated above.
[0043] It is estimated in the same manner as in EXAMPLE 2 that the
product of 30K consists of human G-CSF wherein one human G-CSF
molecule is coupled with one activated PEG molecule.
[0044] Furthermore, the human G-CSF was incubated with the
activated PEG 2 at 50 times the molar amount of free amino groups
of the human G-CSF.
[0045] The estimation of a molecular weight of the resulting
products by SDS-PAGE as in EXAMPLE 2 has revealed that its average
molecular weight is about 51K with distributed among 40K (58 %) and
66K (42%).
EXAMPLE 4
Preparation of PEG (4,000) G-CSF
[0046] PEG-modified human G-CSF was prepared by covalently binding
an activated polyethylene glycol, or polyoxyethylenediamine with an
average molecular weight of 4,000 (Nippon Oil and Fats Co., Ltd.)
to the above human G-CSF through the free carboxyl group
thereof.
[0047] The human G-CSF and the activated polyethylene glycol at 60
times the molar amount of the free carboxyl groups of the human
G-CSF were incubated in the presence of 0.05M
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a room temperature
for overnight. The reaction was terminated by adding 1M sodium
acetate (pH 4.75) and further incubated at 25.degree. C. in the
presence of 0.5M hydroxyamine for 5 hrs in order to regenerate
tyrosine residues. The resulting product was subjected to gel
chromatography on TSK G3000SW which had been equilibrated with 10
mM sodium acetate (pH 5.5) to separate the PEG-modified human G-CSF
from an unreacted human G-CSF and reagent. The estimation of a
molecular weight of the product by SDS-PAGE as in EXAMPLE 2 has
revealed that its molecular weight is distributed among 27K (70%),
35K (20%) and 42K (10%). The resultant PEG-modified human G-CSF is
hereinafter referred to as "PEG (4,000) G-CSF".
EXAMPLE 5
In Vivo Biological Assay of PEG (4,500) G-CSF
[0048] Male ICR mice (Experiment I: 4 weeks old, Experiment II: 8
weeks old) were used for in vivo assays for pharmacological
activity of PEG (4,500) G-CSF obtained in EXAMPLE 1. Samples of the
intact human G-CSF and PEG (4,500) G-CSF were intravenously
injected into mice at a dose of 10 .mu.g or 100 .mu.g protein/kg.
At 24 hrs (10 .mu.g protein/kg) or 32 hrs (100 .mu.g protein/kg)
after the injection, blood was collected from orbital vein and
leukocytes were counted by an automated blood cell counter E-2000
(Toa Medical Electronics, Japan). At the same time, a blood smear
was subjected to Wright-Giemsa stain and the leukocyte fraction was
determined by an automated blood cell analyzer MICROX (OMRON
TATEISI ELECTRONICS CO.) to count the number of neutrophils. The
results are summarized in TABLE 2 below.
[0049] In TABLE 2, PEG (4,500) G-CSF (1) is a product obtained in
the reaction wherein the molar ratio of the activated PEG/the free
amino group was five (FIG. 1,C), PEG (4,500) G-CSF (2) is a 26K
fraction obtained from DEAE ion-exchange chromatography, and PEG
(4,500) G-CSF (3) is a high molecular fraction (26K: 14%, 34K: 55%,
>34K: 28%) obtained from said DEAE ion-exchange
chromatography.
[0050] From the above results, it is observed that the increase in
the number of neutrophils in the mice injected with PEG (4,500)
G-CSFs (1), (2) and (3) was much larger than the increase in the
number of neutrophils in the mice injected with the intact G-CSF.
Especially, PEG (4,500) G-CSFs (1) and (3) with a higher extent of
the modification showed a remarkable increase in the number of
neutrophils.
[0051] When human G-CSF is injected into mice at a dose of 10 .mu.g
protein/kg, the number of neutrophils increases, and generally at
6-12 hrs after the injection, it reaches maximum. After that, the
number of neutrophils decreases slowly to a basal level about 30
hrs after injection. The number of neutrophils decreases to almost
a basal level after 24 hrs. Based on the foregoing time periods, it
was determined that in the case of 100 .mu.g protein/kg injections,
the time for collection of blood was 32 hrs after injection.
Accordingly, the above result that the numbers of neutrophils in
the mice injected with PEG (4,500) G-CSFs (1), (2) and (3) are
higher than those in the mice injected with the intact hG-CSF may
indicate that the activity of human G-CSF in mice has been extended
by the present modification.
[0052] An unreacted mixture of human G-CSF and PEG only showed the
same result as the intact human G-CSF (Data are not shown).
TABLE-US-00005 TABLE 2 Pharmacological activity (in vivo) of
PEG-modified human G-CSF Neutrophils Ratio Group N
(.times.10.sup.2/.mu.l) (to vehicle) a. 10 .mu.g/kg <Exp. I>
Vehicle 5 5.6 .+-. 1.0 1.0 Control G-CSF 6 9.6 .+-. 1.4 1.7
PEG(4500) G-CSF(1) 6 20.8 .+-. 2.6 3.7 PEG(4500) G-CSF(2) 6 17.5
.+-. 3.0 3.1 <Exp. II> Vehicle 6 12.3 .+-. 1.7 1.0 Control
G-CSF 6 27.1 .+-. 4.6 2.2 PEG(4500) G-CSF(3) 6 54.0 .+-. 7.2 4.4 b.
100 .mu.g/kg <Exp. I> Vehicle 6 6.6 .+-. 0.7 1.0 Control
G-CSF 6 18.5 .+-. 2.3 2.8 PEG(4500) G-CSF(1) 6 42.9 .+-. 4.3 6.5
PEG(4500) G-CSF(2) 6 22.6 .+-. 1.9 3.4
EXAMPLE 6
In Vivo Biological Assay of PEG (4,000) G-CSF
[0053] Male ICR mice (7 weeks old) were used for in vivo assays for
pharmacological activity of PEG (4,000) G-CSF obtained in EXAMPLE
4. Samples of the intact human G-CSF and PEG (4,000) G-CSF were
intravenously injected into mice at a dose of 10 .mu.g protein/kg.
At 24 hrs after the injection, blood was collected from the orbital
vein and the number of neutrophils was counted as in EXAMPLE 5. The
results are shown in TABLE 3.
[0054] It has been determined that PEG (4,000) G-CSF in which the
activated PEG is bound through the free carboxyl group also
increased the number of neutrophils more than intact human G-CSF.
TABLE-US-00006 TABLE 3 Pharmacological activity (in vivo) of PEG
(4,000) G-CSF Number of Number of Ratio Group Animals Neutrophils
(.times.10.sup.2/.mu.l) (to vehicle) Vehicle 6 10.9 + 1.0 1.0
G-CSF(control) 6 16.4 + 1.4 1.5 PEG(4,000) G-CSF 6 23.3 + 2.5
2.1
EXAMPLE 7
Effects of PEG-Modified Human G-CSFs on Increasing Mice
Neutrophils
[0055] Male ICR mice (7 weeks old) were used for in vivo assays for
pharmacological activity of PEG (4,500) G-CSF and PEG (10,000)
G-CSF obtained in EXAMPLEs 1 and 3, respectively. The PEG (4,500)
G-CSF used is a high molecular fraction from DEAE ion-exchange
chromatography of a product obtained in the reaction wherein the
molar ratio of the activated PEG/the free amino group was fifty (an
average molecular weight of 60K; 38K: 20%, 58K: 54%, 80K: 27%).
Samples of the human G-CSF, PEG (4,500) G-CSF and PEG (10,000)
G-CSF were intravenously injected into mice at a dose of 10 .mu.g
protein/kg. At 6, 24, 32, 48 and 72 hrs after the injection, blood
was collected from orbital vein and the number of neutrophils was
counted as in EXAMPLE 5, except for using an automated blood cell
counter CC180-A (Toa Medical Electronics, Japan).
[0056] As shown in FIG. 2, in the case of the intact human G-CSF,
the number of neutrophils decreases to a basal level 24 hrs after
the injection. On the other hand, a significant increase of
neutrophils was observed over 32 hrs and 48 hrs after the injection
for PEG (4,500) G-CSF and PEG (10,000) G-CSF, respectively.
[0057] Moreover, male ICR mice (8 weeks old) were intravenously
administered with the PEG (10,000) G-CSFs obtained in EXAMPLE 3;
(a) an average molecular weight of 30K, (b) an average molecular
weight of 51K; 40K: 58%, 66K: 42% at a dose of 10 .mu.g protein/kg.
At 24 hours after the injection the number of neutrophils was
counted as in EXAMPLE 5. The results are shown in TABLE 4.
TABLE-US-00007 TABLE 4 Pharmacological activity (in vivo) of PEG
(10,000) G-CSF Number Number of Neutrophils Ratio Group of Animals
(.times.10.sup.2/.mu.l) (to vehicle) Vehicle 5 7.4 + 0.6 1.0 G-CSF
5 16.4 + 3.1 2.2 PEG(10,000) G-CSF(a) 5 68.9 + 10.5 9.3 PEG(10,000)
G-CSF(b) 5 95.8 + 6.4 12.9
[0058] Both PEG (10,000) G-CSF (a) and (b) increased the number of
neutrophils more than intact human G-CSF. Especially, PEG (10,000)
G-CSF, with a higher extent of the modification, showed a more
remarkable increase in the number of neutrophils, as was the case
with PEG (4,500) G-CSF.
EXAMPLE 8
Effects of PEG-Modified Human G-CSF on Cyclophosphamide-Induced
Neutropenic Mice
[0059] Male ICR mice (7 weeks old) were intraperitoneally injected
with 200 mg/kg cyclophosphamide (CY) to induce neutropenia. Once a
day for 4 successive days starting from one day after the CY
injection, PEG (4,500) G-CSF and PEG (10,000) G-CSF as used in
EXAMPLE 7 were intravenously injected into the neutropenic mice at
a dose of 10 .mu.g protein/kg. At 6, 24 and 48 hrs after the last
injection, blood was collected from the orbital vein and
neutrophils were counted as in EXAMPLE 5.
[0060] As shown in FIG. 3, PEG-modified G-CSFs accelerated the
recovery from neutropenia induced by the injection of
cyclophosphamide as early or earlier than the intact G-CSF.
Especially, PEG (10,000) G-CSF effected a significant increase in
the number of neutrophils.
EXAMPLE 9
Effects of PEG-Modified Human G-CSF on 5-FU-Induced Neutropenic
Mice
[0061] Female BDF.sub.1 mice (7 weeks old, JAPAN SLC Co.,) were
intravenously injected with 200 mg/kg 5-FU to induce neutropenia.
At a dose of 10 .mu.g protein/kg either once a day for 11
successive days (PEG-1), or every other day (at day 1, 3, 5, 7, 9
and 11; PEG-2) and every third day (at day 1, 4, 7 and 10; PEG-3)
starting from one day after the 5-FU injection, the same PEG
(10,000) G-CSF as used in EXAMPLE 7 the intact human G-CSF were
subcutaneously injected into the neutropenic mice. At day 7, 8, 9,
10, 11, 12, 14 and 17, blood was collected from orbital vein and
neutrophils were counted as in EXAMPLE 5.
[0062] As shown in FIG. 4, it took about 14 days for recovery of
neutrophil counts of mice injected with only 5-FU to a basal level.
On the other hand, it took about 11 days and 9 days for recovery of
neutrophil counts of mice injected also with the intact human
G-CSF, and PEG-1, 2 and 3, respectively. Thus, PEG-modified G-CSFs
accelerated recovery from neutropenia induced by the injection of
5-FU earlier than intact G-CSF. Moreover, even with fewer
injections of the PEG-modified G-CSFs than the intact human G-CSF,
the same phenomena as the above could be observed.
EXAMPLE 10
Acute Toxicity of PEG-Modified Human G-CSF
[0063] Male and female Slc:IR mice (5 weeks old) in groups
consisting 6 mice each were intravenously administered with the
same PEG (4,500) G-CSF or PEG (10,000) G-CSF as used in EXAMPLE 7
at a dose of 10 .mu.g protein/kg, or with vehicle at a dose of 12
ml/kg. General conditions and survival of the treated mice were
observed as often as possible for 6 hrs immediately after
administration and once a day for the following 14 days. The body
weight was checked at the day of injection, 5, 8, 12 and 15th days.
Surviving mice were bled to death under ether anesthesia and
subjected to pathologic autopsy.
[0064] As shown in TABLE 5, no mouse died for the observed period.
LD 50 for both PEG (4,500) G-CSF and PEG (10,000) G-CSF was
estimated over 3,000 .mu.g protein/kg in both male and female mice.
No remarkable change in general condition, body weight or opinion
of the autopsy was observed for PEG (4,500) G-CSF or PEG (10,000)
G-CSF. These results may suggest that the acute toxicity of
PEG-modified human G-CSF is very weak, as the intact human G-CSF
is. TABLE-US-00008 TABLE 5 Mortality of male and female mice Dose
Number of deaths on day LD.sub.50 Sex Compound (.mu.g/kg) 1 2 3 4 5
6 7 8 9 10 11 12 13 14 15 Mortality.sup..dagger-dbl. (.mu.g/kg)
Male Vehicle -- 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0/6 -- PEG4500-G-CSF
3,000 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0/6 >3,000 PEG10000-G-CSF
3,000 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0/6 >3,000 Female Vehicle --
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0/6 -- PEG4500-G-CSF 3,000 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0/6 >3,000 PEG10000-G-CSF 3,000 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0/6 >3,000
EXAMPLE 11
Determination of Half-Life of PEG-Modified hG-CSF
[0065] Male Sprague-Dawley rats (7 weeks old) were used for study
of pharmacokinetics of the intact human G-CSF and PEG (10,000)
G-CSF prepared in EXAMPLE 3. Samples were intravenously injected
into rats at a dose of 100 .mu.g protein/kg. At 10 min, 2, 4, 8, 24
and 48 hrs after the injection, about 6-7 ml of blood from each of
three rats was collected from the abdominal aorta into a
polypropylene tube of about 15 ml volume and centrifuged
(18,000.times.g) at 4.degree. C. for 5 min to prepare a serum
fraction. The amount of the active human G-CSFs contained in the
serum fraction was determined by a bioassay for proliferation
induction of mouse bone marrow cells on the basis of incorporation
of .sup.3H-thymidine (Ralph et al., Blood 66, pp. 633-639, (1988)).
The time course of serum concentration is shown in FIG. 5. The
results indicate that the half lives of the intact human G-CSF and
PEG (10,000) G-CSF are 1.79 hrs and 7.05 hrs, respectively, and
AUCs are also 2,000 ng protein hrs/ml and 16,195 ng protein hrs/ml,
respectively. Accordingly, it is demonstrated that the clearance
rate of PEG (10,000) G-CSF in the body is less than that of the
intact human G-CSF has.
INDUSTRIAL APPLICABILITY
[0066] It is expected that the present PEG-modified human G-CSF may
make a great contribution to the treatment with human G-CSF because
it has a much longer lasting neutrophil-increasing activity than
that of the intact human G-CSF, enabling fewer numbers of
administration with a lower dose.
Sequence CWU 1
1
2 1 174 PRT Homo sapiens 1 Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro
Gln Ser Phe Leu Leu Lys 1 5 10 15 Cys Leu Glu Gln Val Arg Lys Ile
Gln Gly Asp Gly Ala Ala Leu Gln 20 25 30 Glu Lys Leu Cys Ala Thr
Tyr Lys Leu Cys His Pro Glu Glu Leu Val 35 40 45 Leu Leu Gly His
Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys 50 55 60 Pro Ser
Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser 65 70 75 80
Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser 85
90 95 Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
Asp 100 105 110 Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly
Met Ala Pro 115 120 125 Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala
Phe Ala Ser Ala Phe 130 135 140 Gln Arg Arg Ala Gly Gly Val Leu Val
Ala Ser His Leu Gln Ser Phe 145 150 155 160 Leu Glu Val Ser Tyr Arg
Val Leu Arg His Leu Ala Gln Pro 165 170 2 175 PRT Homo sapiens 2
Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu 1 5
10 15 Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala
Leu 20 25 30 Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro
Glu Glu Leu 35 40 45 Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp
Ala Pro Leu Ser Ser 50 55 60 Cys Pro Ser Gln Ala Leu Gln Leu Ala
Gly Cys Leu Ser Gln Leu His 65 70 75 80 Ser Gly Leu Phe Leu Tyr Gln
Gly Leu Leu Gln Ala Leu Glu Gly Ile 85 90 95 Ser Pro Glu Leu Gly
Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala 100 105 110 Asp Phe Ala
Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala 115 120 125 Pro
Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala 130 135
140 Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser
145 150 155 160 Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala
Gln Pro 165 170 175
* * * * *