U.S. patent application number 10/399254 was filed with the patent office on 2004-04-29 for peg-modified erythropoietin.
Invention is credited to Kawata, Hiromitsu, Machida, Minoru, Miyamoto, Hajime, Nakamura, Teruo, Sekimori, Yasuo.
Application Number | 20040082765 10/399254 |
Document ID | / |
Family ID | 18794529 |
Filed Date | 2004-04-29 |
United States Patent
Application |
20040082765 |
Kind Code |
A1 |
Nakamura, Teruo ; et
al. |
April 29, 2004 |
Peg-modified erythropoietin
Abstract
The present invention provides a polyethylene glycol-conjugated
erythropoietin (PEG-conjugated EPO) prepared by PEG conjugation on
the lysine residue at position 52 of native erythropoietin (native
EPO). In order to achieve more sustained efficacy without losing
physiological activities of native EPO, a glycoprotein rich in
sugar chains, there has been a need to develop a PEG-conjugated EPO
with significantly sustained efficacy by introducing a controlled
number of PEG molecules at controlled positions. This
PEG-conjugated EPO addresses such a need and provides more
sustained efficacy.
Inventors: |
Nakamura, Teruo; (Shizuoka,
JP) ; Sekimori, Yasuo; (Shizuoka, JP) ;
Machida, Minoru; (Shizuoka, JP) ; Kawata,
Hiromitsu; (Shizuoka, JP) ; Miyamoto, Hajime;
(Shizuoka, JP) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.
624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Family ID: |
18794529 |
Appl. No.: |
10/399254 |
Filed: |
April 16, 2003 |
PCT Filed: |
September 28, 2001 |
PCT NO: |
PCT/JP01/08539 |
Current U.S.
Class: |
530/399 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 38/1816 20130101; C07K 14/505 20130101; A61K 47/60 20170801;
A61P 7/06 20180101 |
Class at
Publication: |
530/399 |
International
Class: |
A61K 038/24; C07K
014/23 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 16, 2000 |
JP |
2000315421 |
Claims
1. A mono-PEG-conjugated EPO, which comprises native human
erythropoietin (native EPO) chemically conjugated with polyethylene
glycol (PEG).
2. The mono-PEG-conjugated EPO according to claim 1, wherein PEG
conjugation occurs on the lysine residue at position 52 of the
native EPO.
3. The mono-PEG-conjugated EPO according to claim 1 or 2, wherein
the polyethylene glycol (PEG) used for conjugation of the native
human erythropoietin (native EPO) has a molecular weight of 5 to 40
kDa and the apparent molecular weight of said mono-PEG-conjugated
EPO in an aqueous medium is 100 to 900 kDa per molecule, as
measured by gel filtration column chromatography.
4. A mono-PEG-conjugated EPO composition, which comprises the
mono-PEG-conjugated EPO according to any one of claims 1 to 3.
5. A PEG-conjugated EPO composition, which further comprises a
PEG-conjugated EPO having PEG molecules attached to at least two
amino acid residues in the mono-PEG-conjugated EPO according to any
one of claims 1 to 3 and/or native EPO, wherein the apparent
molecular weight of said PEG-conjugated EPO in an aqueous medium is
100 to 900 kDa per molecule, as measured by gel filtration column
chromatography.
6. A sustained-action erythropoietin formulation, which comprises
the PEG-conjugated EPO according to any one of claims 1 to 3 as an
active ingredient.
7. A sustained-action erythropoietin formulation, which comprises
the PEG-conjugated EPO composition according to claim 4 or 5 as an
active ingredient.
8. A method for preparing the PEG-conjugated erythropoietin
composition according to claim 4, which comprises reacting native
EPO with a succinimidyl ester derivative of PEG.
Description
TECHNICAL FIELD
[0001] The present invention relates to native human erythropoietin
chemically conjugated with polyethylene glycol (PEG). More
specifically, the present invention relates to mono-PEG-conjugated
erythropoietin having a PEG molecule primarily attached to the
lysine residue at position 52, which is obtained by reacting
recombinant human erythropoietin (rhEPO) produced in animal host
cells with an amino reactive derivative of PEG; a composition
comprising the conjugate; and a sustained-action erythropoietin
formulation comprising the conjugate or composition as an active
ingredient.
BACKGROUND ART
[0002] Erythropoietin (EPO) is a polypeptide rich in sugar chains,
which is predominantly produced in the kidneys and acts on
precursor cells of hematopoietic tissue to stimulate their
differentiation and proliferation into erythrocytes. EPO is
currently commercially available as human EPO recombinantly
produced in animal host cells, and its main use is as a therapeutic
agent for various types of anaemia, including renal anemia caused
by the underproduction of EPO resulting from nephropathy.
[0003] As used her in, the term "native EPO" is intended to
encompass human urine-derived EPO, such as those extracted,
isolated and purified by various techniques, and recombinant human
EPO (rhEPO) having the same sugar chains as human-derived EPO, such
as those recombinantly produced in animal host cells (e.g., CHO
cells, COS cells), as well as their variants modified to include
substitution or deletion of one or more amino acids constituting
such EPOs or modified to include addition of one or more amino
acids.
[0004] Currently used EPO is administered, e.g., by intravenous,
subcutaneous or intramuscular route. The percentage of
reticulocytes (erythrocyte precursors) in total erythrocytes can be
used as an indicator for EPO activity. The activity of native EPO
observed as the percentage of reticulocytes will reach a peak 3 to
5 days after administration by any route mentioned above, followed
by a rapid decline. Thus, native EPO should be injected twice or
three times per week to ensure sufficient therapeutic effects in
anaemia patients. This not only causes pain in the patients, but
also provides additional load on overworked doctors and other
medical staff members. Further, a decreased number of injections
required within a fixed period of time will save medical costs.
[0005] On the other hand, there are reports on proteins or
glycoproteins conjugated with water-soluble polymers (e.g., PEG)
having a hydrazide or oxylamine moiety capable of covalent bonding
through chemical reaction with an oxidizable functional group such
as polyol, lactol, amine, carboxylic acid or a carboxylic acid
derivative on the proteins or glycoproteins (s e, e.g., JP 7-196925
A). According to this report, a water-soluble polymer, such as PEG,
can be attached to various free radicals on amino acids or sugar
residues constituting a protein or glycoprotein to yield a coupling
product with 6 to 34 PEG molecules (molecular weight: 2000 to
12000) per protein molecule. In such a PEG-conjugated protein or
glycoprotein with many PEG molecules, it is difficult to control
the positions and number of PEG molecules attached to the protein
or glycoprotein and it is hard to obtain an uniform PEG-conjugated
protein or glycoprotein. Thus, there is a problem when the
PEG-conjugated protein or glycoprotein thus prepared is formulated
into pharmaceuticals.
[0006] There is also a report disclosing a sulfonate
ester-activated polymer (e.g., sulfonate ester-activated PEG) and
proposing a method in which this sulfonate ester-activated polymer
is reacted with a target material (e.g., a protein or glycoprotein)
to prepare a polymer-conjugated target material (see JP 9-504515
A). Examples of a reactive group on a target material which reacts
with this sulfonate ester-activated polymer include a primary or
secondary amino group, a thiol group and an aromatic hydroxyl
group. In such a sulfonate ester-activated polymer capable of
reacting with various reactive groups, it is therefore regarded as
difficult to control the number and positions of conjugatable
polymer molecules which are introduced. Also, such a polymer
clearly has the possibility of sulfonate amide formation, which
allows much higher heterogeneity for products.
[0007] Further, there is a report on a branched p lymer (e.g.,
branched PEG) which is attached to a target material (e.g., a
protein or glycoprotein) to give a branched polymer conjugate of
the target material (see JP 9-504299 A). Although this branched
polymer-conjugated target material also successfully sustains its
efficacy, it has been desired to develop polymer-conjugated EPOs
with more sustained efficacy.
[0008] Also, it has been believed that conjugation with higher
molecular weight PEG would result in more sustained efficacy.
[0009] Conjugation with higher molecular weight PEG will cause a
larger decrease in the in vitro activity of EPO. However, it has
been believed that EPO conjugated with higher molecular weight PEG
would show significantly improved plasma retention and hence
sustained in vivo activity, resulting in greater and more sustained
activity with increase in the molecular weight of PEG (Polyethylene
glycol-conjugated pharmaceutical proteins; PSTT Vol. 1, No. 8,
1998, 352-356). For example, in the case of a PEG conjugate of
G-CSF mutaine, it is known that its in vivo activity increases in
proportion to the calculated molecular weight of PEG in the range
of about 10 kDa up to 70 kDa (PCT/US00/01264, WO 00/44785).
[0010] Conventional PEG conjugates of EPO were designed to have
many PEG molecules with a relatively low molecular weight of around
5 kDa in order to sustain their efficacy. However, in EPO rich in
sugar chains, conjugation was limited exclusively to unglycosylated
amino acid residues involved in receptor binding, making it
impossible to avoid a decrease in in vivo activity and difficult to
balance sustained efficacy and decreased in vivo activity. Also,
even in a case where amino groups or sugar chains are conjugated
with PEG of greater than 10 kDa as stated above, there has been a
difficulty in practically formulating PEG-conjugated EPO into drugs
because the problem of controlling the number of PEG molecules
still remains.
DISCLOSURE OF THE INVENTION
[0011] For these reasons, in order to achieve more sustained
efficacy without losing physiological activities of EPO, a
glycoprotein rich in sugar chains, there is a need to develop a
PEG-conjugated EPO with significantly sustained efficacy by
introducing a controlled number of PEG molecules at controlled
positions.
[0012] As stated above, the general recognition of the relationship
between the molecular weight of PEG used for conjugation and in
vivo activity of EPO was that EPO conjugated with higher molecular
weight PEG showed more improved plasma retention and hence greater
and more sustained efficacy. However, the inventors of the present
invention have clarified that such general recognition does not
apply to PEG conjugation for native (recombinant) EPO with sugar
chains because conjugation with extremely high molecular weight PEG
also reduces the in vivo activity of EPO. As a result, they have
found that higher molecular weight PEG does not always produce a
better result, but rather there is an optimal range for the
molecular weight of PEG to have a balance between sustained
efficacy and in vivo activity, as well as finding that a particular
number of PEG molecules provides the most sustained efficacy. These
findings led to the completion of the present invention.
[0013] The inventors of the present invention have now prepared a
PEG-conjugated EPO by reacting recombinant human EPO with a
polyethylene glycol (PEG) derivative having an active ester at one
end, such as a methoxy-PEG-succinimidyl lower fatty acid ester.
This PEG-conjugated EPO comprises a composition of conjugates
having 1 to 3 linear PEG molecules per rhEPO molecule. A
mono-methoxy PEG-EPO (mono-mPEG-EPO) conjugate has one linear PEG
molecule per rhEPO molecule, which is primarily attached to the
.epsilon.-amino group of the lysine residue at position 52 of
rhEPO. As further described below, the inventors of the present
invention first confirmed that the PEG-conjugated EPO of the
present invention had significantly sustained efficacy, as compared
with unconjugated native EPO.
[0014] Further, the inventors of the present invention determined
the apparent molecular weight of PEG-conjugated EPO by gel
filtration column chromatography in an aqueous medium using
globular proteins as molecular weight markers, as detailed in
Example 2, for the purpose of predicting the in vivo apparent size
behavior of PEG-conjugated EPO molecules. As a result, it was
surprisingly found that PEG conjugates of native EPO rich in sugar
chains had an apparent molecular weight exceeding 100 kDa, and more
specifically that a PEG conjugate of native EPO prepared using 40
kDa branched PEG had an apparent molecular weight of approximately
800 kDa or more. In view of such a surprisingly high value of the
apparent molecular weight focused here, along with the fact that
receptor binding in native EPO was limited to exposed protein
structural moieties because of abundant sugar chains on EPO, the
inventors of the present invention concluded that PEG conjugation
to native EPO should be optimized for efficacy by controlling the
molecular weight and the number of PEG molecules used for
conjugation.
[0015] Thus, the inventors of the present invention further
prepared a mono-methoxy PEG-EPO (mono-mPEG-EPO) conjugate having
one linear PEG molecule of 5 kDa, 10 kDa, 15 kDa, 20 kDa or 30 kDa
per native EPO molecule, a di-methoxy PEG-EPO (di-mPEG-EPO)
conjugate having two linear PEG molecules of 5 kDa, 10 kDa, 15 kDa,
20 kDa or 30 kDa per native EPO molecule, and a mono-branched
methoxy PEG-EPO (mono-mPEG2-EPO) conjugate having one
double-branched PEG molecule of 10 kDa, 20 kDa or 40 kDa per native
EPO molecule. As further described in the Examples below, a
comparison among these three types of PEG-conjugated EPO indicated
that the plasma retention was longer in the di-methoxy PEG-EPO
(di-mPEG-EPO) and mono-branched methoxy PEG-EPO (mono-mPEG2-EPO)
conjugates than in the mono-methoxy PEG-EPO conjugate, whereas the
in vivo erythropoietic effect was greater in the mono-methoxy
PEG-EPO conjugate. This supported the above-mentioned inventors'
view that there was an optimal range for PEG conjugation to native
EPO.
BRIEF DESCRIPTION OF DRAWINGS
[0016] FIG. 1 shows an SDS-PAGE pattern of the PEG-conjugated EPOs
(mono-mPEG-EPO and di-mPEG-EPO) prepared in the present invention,
unconjugated EPO and others.
[0017] FIG. 2 shows a chromato pattern of the mono-mPEG-EPO
conjugate according to the present invention, which is digested
with endoprotease Lys-C and mapped by liquid chromatography.
[0018] FIG. 3 graphically shows cell growth activity on
EPO-dependent cells in the presence of PEG-conjugated EPOs,
including the mono-mPEG-EPO conjugate according to the present
invention.
[0019] FIG. 4 graphically shows the time course of peripheral
reticulocyte counts after administration of PEG-conjugated EPOs,
including the mono-mPEG-EPO conjugate according to the present
invention.
[0020] FIG. 5 graphically shows the time course of hemoglobin
levels after administration of PEG-conjugated EPOs, including the
mono-mPEG-EPO conjugate according to the present invention.
[0021] FIG. 6 graphically shows the time course of peripheral
reticulocyte counts after administration of PEG-conjugated EPOs,
including the mono-mPEG-EPO and mono-branched mPEG-EPO conjugates
according to the present invention.
[0022] FIG. 7 graphically shows the time course of hemoglobin
levels after administration of PEG-conjugated EPOs, including the
mono-mPEG-EPO and mono-branched mPEG-EPO conjugates according to
the present invention.
[0023] FIG. 8 graphically shows the dose-dependency of the
mono-mPEG-EPO (PEG(1)-EPO) conjugate according to the present
invention, as determined by its hemoglobin level.
[0024] FIG. 9 graphically shows a comparison between 5-day
continuous injection of EPO and single injection of the
mono-mPEG-EPO (PEG(1)-EPO) conjugate according to the present
invention.
[0025] FIG. 10 graphically shows cell growth activity on
EPO-dependent cells in the presence of PEG-conjugated EPOs,
including the mono-mPEG10K-EPO, mono-mPEG15K-EPO and
mono-mPEG20K-EPO conjugates according to the present invention.
[0026] FIG. 11 graphically shows cell growth activity on
EPO-dependent cells in the presence of PEG-conjugated EPOs,
including the mono-mPEG5K-EPO, mono-mPEG20K-EPO and
mono-mPEG30K-EPO conjugates according to the present invention.
[0027] FIG. 12 graphically shows the time course of peripheral
reticulocyte counts after administration of PEG-conjugated EPOs,
including the mono-mPEG5K-EPO, mono-mPEG10K-EPO, mono-mPEG15K-EPO,
mono-mPEG20K-EPO and mono-mPEG30K-EPO conjugates according to the
present invention.
[0028] FIG. 13 graphically shows the time course of hemoglobin
levels after administration of PEG-conjugated EPOs, including the
mono-mPEG5K-EPO, mono-mPEG10K-EPO, mono-mPEG15K-EPO,
mono-mPEG20K-EPO and mono-mPEG30K-EPO conjugates according to the
present invention.
[0029] FIG. 14 graphically shows the AUC of peripheral reticulocyte
counts after administration of PEG-conjugated EPOs, including the
mono-mPEG5K-EPO, mono-mPEG10K-EPO, mono-mPEG15K-EPO,
mono-mPEG20K-EPO and mono-mPEG30K-EPO conjugates according to the
present invention.
PREFERRED MODE FOR CARRYING OUT THE INVENTION
[0030] In view of the foregoing, for example, a highly purified
mono-methoxy PEG-EPO (mono-mPEG-EPO) conjugate, which has a PEG
molecule primarily attached to the amino group of the lysine
residue at position 52 of native EPO, is desirable for use in
formulating the PEG-conjugated EPO of the present invention
intopharmaceuticals. It may also be possible to formulate the
mono-methoxy PEG-EPO (mono-mPEG-EPO) conjugate as a main component,
together with its activity-related byproducts generated during
conjugation (e.g., a di-methoxy PEG-EPO (di-mPEG-EPO)
conjugate).
[0031] EPO available for use in PEG conjugation of the present
invention is intended to encompass commercially available
recombinant human EPOs produced in animal host cells, as well as
their variants modified to include substitution or deletion of one
or more amino acids constituting these EPOs or modified to include
addition of one or more amino acids.
[0032] In the present invention, any PEG derivative having one
methoxylated end can be used for PEG conjugation. In addition, the
other unmethoxylated end of such a PEG derivative may be converted
into a succinimidyl lower fatty acid ester, preferably succinimidyl
propionate or succinimidyl butyrate (SPA-PEG or SBA-PEG,
respectively), for reaction with an amino group of EPO. These PEG
derivatives can provide stable PEG conjugates because they have no
ester moiety in their backbone, except for their end activated for
reaction.
[0033] In order that native EPO binds to the EPO receptors on
erythrocyte precursor cells and transmits signals driving
differentiation of the cells into erythrocytes, the protein
structural regions essential for this purpose should be exposed.
However, in native EPO, the regions essential for receptor binding
and signal transduction are localized in small exposed protein
moieties because native EPO has a much higher sugar content than
other recombinant pharmaceutical proteins. Thus, when conjugation,
particularly with a polymer compound, occurs in such limited
exposed protein moieties, the conjugation would reduce the EPO
activity and ultimately fall to achieve the initial goal that
sustained-action drugs are developed.
[0034] In the present-invention, the inventors completed the
invention by determining the desired molecular weight and site for
PEG conjugation in order to provide native EPO under particular
conditions, as mentioned above, with greater and more sustained in
vivo efficacy than those of native EPO although the EPO activity
was slightly affected at the receptor binding level.
[0035] Although native EPO contains several lysine residues as
amino acid residues capable of PEG conjugation, particularly
preferred is a PEG-conjugated EPO where PEG conjugation occurs on
the amino group of the lysine residue at position 52. In one
aspect, the present invention encompasses a composition of
PEG-conjugated EPOs where PEG conjugation occurs primarily on the
amino group of the lysine residue at position 52.
[0036] In this cases the PEG-conjugated EPO composition comprises a
mono-PEG-conjugated EPO where PEG conjugation occurs on the amino
group of the lysine residue at position 52. It may further comprise
a mono-PEG-conjugated EPO having one PEG molecule on an amino acid
residue at other position and/or a PEG-conjugated EPO where PEG
conjugation occurs on two or more amino acid residues in native
EPO. The PEG-conjugated EPO contained in this composition
preferably has one to three PEG molecules per EPO molecule, more
preferably one PEG molecule per EPO molecule.
[0037] This PEG-conjugated EPO composition preferably comprises a
mono-PEG-conjugated EPO where PEG conjugation occurs on the amino
group of the lysine residue at position 52 as well as a
mono-PEG-conjugated EPO having one PEG molecule on an amino acid
residue at other position and/or a PEG-conjugated EPO where PEG
conjugation occurs on two or more amino acid residues in native
EPO.
[0038] The molecular weight of PEG used for conjugation can be
changed as appropriate for the degree of sustained efficacy
required by the resulting PEG-conjugated EPO, the degree of
decreased EPO activity, etc. The molecular weight of one PEG
molecule is 5 to 40 kDa, preferably 10 to 30 kDa, more preferably
20 to 30 kDa, and linear PEG is preferred to branched PEG if they
have the same molecular weight. The apparent molecular weight of
PEG-conjugated EPO In an aqueous medium is 100 to 900 kDa,
preferably 150 to 650 kDa, more preferably 400 to 650 kDa, as
measured by gel filtration column chromatography under the
conditions described in Example 2.
[0039] PEG molecules used for conjugation preferably have the
linear form, but it is possible to use branched or star-shaped PEG
molecules as long as the apparent molecular weight of the resulting
PEG-conjugated EPO is in the range mentioned above. In the case of
branched or star-shaped PEG, PEG conjugation also occurs on the
same amino acid residues as mentioned above.
[0040] Techniques for purification of PEG-conjugated EPO include
PEG/dextran two-phase partition, gel filtration chromatography,
ion-exchange chromatography, hydrophobic interaction
chromatography, reverse phase chromatography and affinity
chromatography.
[0041] With respect to the physiological activities and sustained
efficacy of the PEG-conjugated EPOs prepared in the present
invention, a comparison test was made for the following individual
items between vehicle and PEG-conjugated EPOs, including
unconjugated native EPO, di-methoxy PEG-EPO and mono-branched
PEG-EPO, confirming that the PEG-conjugated EPOs of the present
invention had greater and more sustained in vivo efficacy than
unconjugated native EPO. Further, the greatest and most sustained
in vivo efficacy was confirmed in a mono-methoxy PEG-EPO conjugate
with an apparent molecular weight of 400 to 650 kDa (see Examples 2
and 9) where PEG conjugation occurred primarily on the amino group
of the lysine residue at position 52 using linear PEG of 20 to 30
kDa.
[0042] More specifically, unconjugated native EPO and the
PEG-conjugated EPOs of the present invention were tested for their
plasma retention in rats after a single tail vein injection,
indicating that longer plasma retention was provided by a
PEG-conjugated EPO having a larger number of higher molecular
weight PEG molecules and having a higher apparent molecular weight,
as normally expected.
[0043] On the other hand, the PEG-conjugated EPOs were assayed for
their in vitro cell growth activity on EPO-dependent cells
(BaF/EpoR cells), indicating that the PEG-conjugated EPOs of the
present invention still retained in vitro cell growth activity
although they showed some decrease in the activity as compared with
unconjugated native EPO. This suggests that PEG conjugation affects
the binding affinity to the receptor, but does not completely block
the region directly binding to the receptor. Among the
PEG-conjugated EPOs of the present invention, mono-methoxy PEG-EPO
showed much higher activity than the other PEG-conjugated EPOs (see
Example 4).
[0044] Further, an in vivo test was performed on rats to monitor
changes in reticulocyte counts and hemoglobin levels after a single
tail vein injection of unconjugated native EPO or each
PEG-conjugated EPO of the present invention, confirming that the
PEG-conjugated EPOs of the present invention produced good results
in both reticulocyte counts and hemoglobin levels. Unlike common
PEG-conjugated proteins, the PEG-conjugated EPO of the present
invention has been confirmed to produce extremely good results,
particularly when PEG conjugation occurs primarily on the amino
group of the lysine residue at position 52 using 10-30 kDa PEG to
give a mono-methoxy PEG-EPO conjugate with an apparent molecular
weight of 150 to 650 kDa, as shown in the data on reticulocyte
counts and hemoglobin levels (see Examples 5 and 11).
[0045] The use of the PEG-conjugated EPOs (preferably, mono-methoxy
PEG-EPO) according to the present invention allows an extended
interval for erythropoietin administration. For example, the
interval can be extended to once per week from 2-3 times per week
or extended to every 10 days to every 2 weeks from every week.
Therefore, it is possible not only to ease the physical and time
burden on patients by reducing the numbers of hospital visits and
painful injections, but also to save medical costs by reducing the
load on medical staff members.
[0046] The dose will vary depending on and should be adjusted as
appropriate for the cause and severity of anaemia, the age and
erythropoietin sensitivity of individual patients, etc. For
example, for once-a-week intravenous injection, the dose will be 1
to 100 .mu.g, preferably 5 to 50 .mu.g per adult.
[0047] The PEG-conjugated EPO of the present invention can be
administered to patients by various routes such as intravenous
injection, intravenous drip infusion, subcutaneous injection,
transmucosal (e.g., transpulmonary, transnasal) application and
percutaneous application.
[0048] Depending on the employed route of administration, the
PEG-conjugated EPO of the present invention may be formulated into
the desired dosage form such as solutions, lyophilized
preparations, prefilled syringe-, painless needle- or
needleless-systems for subcutaneous administration, or sustained
release preparations for intracutaneous implantation (e.g.,
microcapsules, polymeric micelles, polymer-based gelatinous
preparations, liposomes), resulting in EPO formulations more stable
than native erythropoietin. Sustained release preparations of
PEG-conjugated EPO are more advantageous than those of native
erythropoietin, in that PEG conjugation stabilizes erythropoietin
activity in the sustained release preparations and that
PEG-conjugated erythropoietin shows much longer plasma retention
than native erythropoietin.
EXAMPLES
[0049] The present invention will be further described in the
following Examples, which are not intended to limit the scope of
the invention.
Example 1
Preparation of PEG-conjugated EPOs (1)
[0050] (Preparation of mono-mPEG-EPO and di-mPEG-EPO)
[0051] To a solution of rhEPO (in 100 mM phosphate buffer, pH 8.0),
methoxy PEG-succinimidyl propionate (PEG Mw: about 20 kDa,
Shearwater Polymers, Inc.)(hereinafter referred to as mPEG-SPA) was
added and stirred at room temperature for 30 minutes. {fraction
(1/10)} volumes of a 100 mM glycine solution was added and stirred
at room temperature for an additional 30 minutes to deactivate the
active ester. Four reactions were carried out in the same manner as
shown above. Table 1 shows the concentration and volume of the
rhEPO solution and the molar ratio of added mPEG-SPA to rhEPO in
each reaction. Each reaction solution was concentrated through a
Centricon-50 (Millipore) simultaneously with replacement of the
solvent by 20 mM phosphate buffer-150 mM NaCl (pH 7.4).
1TABLE 1 Reactions of PEG-conjugated EPO Reaction 1 Reaction 2
Reaction 3 Reaction 4 EPO concentration 2.94 1.75 2.81 2.45 (mg/mL)
EPO volume (mL) 0.50 2.31 2.50 2.04 Molar ratio of 3.97 5.00 5.08
5.03 mPEG-SPA (per mole of EPO)
[0052] Each concentrated solution was purified by gel filtration on
Superdex 200 HR 10/30 (Pharmacia Biotech) to collect both
mono-mPEG-EPO and di-mPEG-EPO fractions, provided that the
concentrated solution from Reaction 1 in Table 1 was used as such
for purification, while the others were purified in 2 to 4 divided
portions. In the case of Reactions 2 to 4, an intermediate fraction
including mono-mPEG-EPO and di-mPEG-EPO, which was eluted as a
mixture of both conjugates, was collected separately and purified
again on Superdex 200 HR 10/30 to separate mono-mPEG-EPO and
di-mPEG-EPO fractions. The fractions obtained through a series of
purification steps were respectively combined for each conjugate to
collect mono-mPEG-EPO (3.8 mg) and di-PEG-EPO (1.6 mg). Each of the
collected mono-mPEG-EPO and di-mPEG-EPO was filtered through a 0.22
.mu.m Milex filter (Millipore) to give sterilized di-mPEG-EPO (1.6
mg) and mono-mPEG-EPO (3.8 mg). These samples (about 0.75 mg/mL, 1
.mu.L each loaded into a sample well of an electrophoretic gel)
were SDS-PAGE electrophoresed on PastGel Gradient 4-15 (Pharmacia
Biotech) and stained with PhastGel Blue R (Pharmacia Biotech)(see
FIG. 1, in which EPO is intended to mean rhEPO).
[0053] In this example, rhEPO and PEG-conjugated EPOs were
quantified based on their absorbance at a wavelength of 279 nm,
given that a 1 mg/mL rhEPO solution had an absorbance of 0.93 at a
wavelength of 279 nm (found in Biochemical Data Book). In other
examples, quantification was performed, given that a 1 mg/mL rhEPO
solution had an absorbance of 1.31 at 281 nm.
Example 2
Molecular Weight Determination of PEG-conjugated EPOs (1)
[0054] (Molecular Weight Determination of Linear PEG-conjugated
EPOs)
[0055] To a solution of rhEPO (960 .mu.L, 0.49 mg/mL in 100 mM
phosphate buffer, pH 8.0), mPEG-SPA (5.32 mg, PEG Mw: about 20 kDa,
mPEG-SPA/rhEPO=10.2 (mol/mol)) was added and stirred gently at room
temperature for 1 hour. After addition of a 1M Gly solution (100
.mu.L), the reaction solution was diluted in PBS (500 .mu.L) and
concentrated to 390 .mu.L through a Centricon-50. The concentrated
solution was applied to two Superose 6 HR 10/30 (1.0.times.30 cm,
bed volume: 24 mL, Pharmacia Biotech) columns in series and eluted
with PBS to collect both di- and mono-mPEG-EPO fractions.
[0056] Each of the resulting di- and mono-mPEG-EPO fractions was
concentrated through a Centricon-50 simultaneously with replacement
of the solvent by Milli-Q water. The molecular weight of each
conjugate was then calibrated from BSA standards in ToF-MS analysis
using sinapinic acid/50% MeCN (4.05 mg/405 .mu.L) as a matrix and
at a sample/matrix ratio of 5 .mu.L/5 .mu.L.
[0057] For use in molecular weight determination by gel filtration,
each sample solution provided for ToF-MS analysis (30-35 .mu.L) was
diluted in PBS (200 .mu.L), applied to two Superose 6 HR 10/30
(1.0.times.30 cm, bed volume: 24 ml, Pharmacia Biotech) columns in
series and then eluted with PBS to measure the respective elution
times of unconjugated rhEPO and di- and mono-mPEG-EPOs. A molecular
weight calibration kit for gel filtration (Amersham Pharmacia
Biotech) was used to prepare the following mixed standard
solutions: (i) Thyroglobulin (Mw 669000), Aldolase (Mw 158000) and
Chymotripsinogen A (Mw 25000) and (ii) Ferritin (Mw 440000) and
Ovalbumin (Mw 43000), which were then subjected to gel filtration
under the same conditions. A calibration curve was prepared from
the elution times of individual standards and used to determine the
respective molecular weights of rhEPO and di- and
mono-mPEG-EPOs.
[0058] (Preparation and Molecular Weight Determination of Branched
PEG-conjugated EPOs)
[0059] To a solution of rhEPO (500 .mu.L, 2.27 mg/mL in 100 mM
phosphate buffer, pH 8.0), branched mPEG2-NHS (5.32 mg, PEG Mw:
about 40 kDa (20 kDa.times.2), branched mPEG2-NHS/rhEPO=2.99
(mol/mol)) was added and stirred gently at room temperature for 30
minutes. After addition of a 1M Gly solution (50 .mu.L, in 20 mM
Tris HCl, pH 8.0), stirring was continued at room temperature for
an additional 30 minutes. The solvent was replaced by 20 mM Tris
HCl (pH 8.0) through a Centricon-50 and adjusted to a final volume
of 750 .mu.L. The resulting solution was applied to ion-exchange
chromatography on Resourse Q (1 mL, Pharmacia Biotech) and eluted
with a gradient of 0-50% Eluent B (pH 8.0, 20 mM Tris HCl, 0.5 M
NaCl) in Eluent A (pH 8.0, 20 mM Tris HCl) to collect fractions
containing branched PEG-conjugated EPOS, which were then
concentrated to 375 .mu.L through a Centricon-50. After addition of
PBS (25 .mu.L), the solution was applied to two Superose 6 HR 10/30
(1.0.times.30 cm, bed volume: 24 mL, Pharmacia Biotech) columns in
series and then eluted with PBS to collect a di-mPEG2-EPO
(di-branched mPEG-EPO) and tri-mPEG2-EPO (tri-branched mPEG-EPO)
fraction as well as a mono-mPEG2-EPO (mono-branched mPEG-EPO)
fraction. Each of the resulting two fractions was concentrated
through a Centricon-50 simultaneously with replacement of the
solvent by Milli-Q water. The molecular weight of each conjugate
was then calibrated from BSA standards in ToF-MS analysis using
sinapinic acid/50% MeCN (4.05 mg/405 .mu.L) as a matrix and at a
sample/matrix ratio of 5 .mu.L/5 .mu.L.
[0060] For use in molecular weight determination by gel filtration
chromatography, each sample solution provided for ToF-MS analysis
was diluted in PBS (200 .mu.L for di-/tri-, 150 .mu.L for mono-),
applied to two Superose 6 HR 10/30 (1.0.times.30 cm, bed volume: 24
mL, Pharmacia Biotech) columns in series and then eluted with PBS
to measure the respective elution times of unconjugated rhEPO and
di- and mono-mPEG-EPOs. A molecular weight calibration kit for gel
filtration (Amersham Pharmacia Biotech) was used to prepare the
following mixed standard solutions: (i) Thyroglobulin (Mw 669000),
Aldolase (Mw 158000) and Chymotripsinogen A (Mw 25000) and (ii)
Ferritin (Mw 440000) and Ovalbumin (Mw 43000), which were then
subjected to gel filtration chromatography under the same
conditions. A calibration curve was prepared from the elution times
of individual standards and used to determine the molecular weight
of mono-mPEG2-EPO (mono-branched mPEG-EPO).
[0061] Table 2 shows the molecular weights of rhEPO, di- and
mono-mPEG-EPO, and mono-mPEG2-EPO (mono-branched mPEG-EPO) (in the
table, EPO, Linear and Branched are intended to mean rhEPO, linear
and branched, respectively).
2TABLE 2 Molecular weights of PEG-conjugated EPOs Calculated
Measured Mw (Da) Mw (Da) ToF-MS Gel filtration EPO 29000.sup.1)
28400 70000 Linear mono-mPEG-EPO 50000 49800 402000 PEG di-mPEG-EPO
71000 71300 914000 Branched mono-mPEG.sub.2-EPO 71000 71200 823000
PEG di-mPEG.sub.2-EPO 113000 113000 (2080000).sup.2)
.sup.1)putative molecular weight of EPOCH (chemical analysis) found
in Journal of clinical therapeutics & medicine, Vol. 6, Suppl.
2 (May) 1990, p. 24 .sup.2)out of calibration range (shown for
reference)
Example 3
Identification of Sites for PEG Conjugation
[0062] The PEG-conjugated EPO (mono-mPEG-EPO) from Example 1 was
analyzed to identify its site for PEG conjugation.
[0063] Mono-mPEG-EPO (denoted as PEG(1)-EPO in FIG. 2) and
unconjugated rhEPO (denoted as intact EPO in FIG. 2) were digested
with endoprotease Lys-C, followed by peptide mapping on a RP/C18
column.
[0064] (Experimental Method)
[0065] Mono-mPEG-EPO (786 .mu.g/mL, PEG Mw: about 20 kDa) and rhEPO
(1 mg/mL) were used as samples.
[0066] a) Reduced Carboxymethylation (RCM) and Lys-C Digestion
[0067] A denaturing solution was 300 mM phosphate buffer (pH 8.0)/6
M guanidine hydrochloride/6 mM EDTA. Five volumes of the denaturing
solution was added to each sample (50 .mu.g as EPO) to denature and
reduce EPO overnight at room temperature in the presence of DTT
(1.5 .mu.mol, 50-fold more than Cys). After addition of
monoiodoacetic acid (3.15 .mu.mol, 2.1-fold more than DTT), the
reaction mixture was carboxymethylated in the dark at room
temperature for 45 minutes and then dialyzed against 50 mM Tris.HCl
(pH 8.5). Digestion was carried out overnight at 37.degree. C. in
the presence of Lys-C (1 .mu.g, substrate:enzyme=50:1), followed by
addition of {fraction (1/10)} volumes of 10% TFA for peptide
mapping.
[0068] b) Peptide Mapping
[0069] A Vydac C18 (218TP52, 2.1 mm.times.250 mm) was used as a
column and a SMART system (Pharmacia) was used for LC. The flow
rate was set to 100 .mu.L/minute and detection was performed at 220
nm and 280 nm. The solvent and gradient for elution were set as
follows:
[0070] Solvent A: 0.1% TFA
[0071] Solvent B: 90% MeCN/0.1% TFA
[0072] Gradient: 5% B/15 min, 5-100% B/85 min, 100% B/10 min. FIG.
2 shows the chromato pattern obtained.
[0073] A comparison between mono-mPEG-EPO (denoted as PEG(1)-EPO in
FIG. 2) and unconjugated rhEPO (denoted as intact EPO in FIG. 2)
indicates a substantial loss of L4 and a significant decrease in L3
(L3 & L6 peak in FIG. 2). This suggests that PEG conjugation is
more likely to occur on Lys52 at the boundary between L3 and L4. In
addition, a new peak appears between L5 and L4, which may be
relevant to a PEG-conjugated peptide. Further, in view of the fact
that L4 does not completely disappear and L1 slightly decreases,
there is a potential molecular species where PEG conjugation occurs
at the N-terminal (Alal) indicated by L1. The chromato pattern
suggests that 70% to 80% of PEG conjugation occurs on Lys52.
Example 4
Assay for Cell Growth Activity of PEG-conjugated EPOs on
EPO-dependent cells (1)
[0074] BaF/EpoR cells showing EPO-dependent growth (Blood, 90,
1867-, 1997, PNAS, 93, 9471-, 1996) were washed by repeating 4
cycles of resuspension in 2% FCS-containing RPMI and
centrifugation. The cells were then suspended in 10% FCS-containing
RPMI at a density of 1.times.10.sup.6 cells/10 mL and dispensed
into each well of 96-well plates in a volume of 50 .mu.L per well.
Meanwhile, test samples were prepared using 10% FCS-containing RPMI
as a diluent to give the respective dilution series of EPO as well
as mono-mPEG-EPO, di-mPEG-EPO and mono-branched mPEG-EPO prepared
in Examples 1 and 2. Each sample solution (50 .mu.L/well, n=3) was
added to and mixed with the BaF/EpoR cells in the 96-well plates,
followed by incubation at 37.degree. C. under a humidified
atmosphere of 5% CO.sub.2 for 24 hours. Each well was supplemented
with 10 .mu.L Cell Count Reagent SF (Nacalai Tesque, Inc.) and
measured for its absorbance at wavelengths of 450 nm and 620 nm,
which was defined as the value at time 0. The plates were allowed
to stand at room temperature for an additional 6 hours, followed by
measurement of absorbance at wavelengths of 450 nm and 620 nm to
plot the data in graph form (see FIG. 3, in which mono, di,
branched and Epo are intended to mean mono-mPEG-EPO, di-mPEG-EPO,
mono-branched mPEG-EPO and rhEPO, respectively). Among the data
obtained, 3 points showing a linear dose-response relationship were
analyzed by linear regression to determine ED50 for each sample and
its 95% confidence interval using a SAS program. The results are
summarized in Table 3 (in which branched-mPEG-EPO denotes
mono-branched mPEG-EPO).
3TABLE 3 Cell growth activity on EPO-dependent cells 95% Confidence
ED50 (ng/mL) interval Specific activity rhEPO 0.12 0.10-0.14 1.0
mono-mPEG-EPO 0.94 0.73-1.2 0.13 di-mPEG-EPO 15 13-18 0.0079
branched mPEG-EPO 3.0 2.4-3.8 0.040
Example 5
Assay for Sustained Efficacy of mono-mPEG-EPO, di-mPEG-EPO and
rhEPO
[0075] Mono-mPEG-EPO (786 .mu.g/mL, having one mPEG molecule per
rhEPO molecule) and di-mPEG-EPO (640 .mu.g/mL, having two mPEG
molecules per rhEPO molecule) prepared in Example 1 were used as
test materials. Each of them was PEGylated by attaching
approximately 20 kDa mPEG, which had been ester-activated at one
end, to an amino group in rhEPO, followed by purification via gel
filtration chromatography.
[0076] Each PEG-conjugated EPO or rhEPO was diluted to 12.5
.mu.g/mL in physiological saline containing 0.05% rat serum albumin
and 0.05% Tween 20. Administration of the medium (vehicle) alone
was defined as a negative control. These administration solutions
were prepared on the first day of administration. In this Example.
Slc:SD male rats (Japan SLC, Inc) were provided for the experiment
when 7 weeks old. On the day of test material administration, the
rats were divided into 4 groups of 4 rats such that all groups had
substantially the same erythrocyte count.
[0077] These 4 groups of 4 rats were set as follows: vehicle group,
rhEPO group, mono-mPEG-EPO group and di-mPEG-EPO group. Each rat
received a single tail vein injection of 2 mL/kg (sample
concentration: 25 .mu.g/kg).
[0078] On the first day of administration and 2, 4, 7, 10, 14, 17,
21, 25, 29, 32, 35 and 39 days after administration, each rat was
fixed on a retainer under unanesthetized conditions and blood was
sampled from a wound made by sticking an injection needle into the
tail vein. Each blood sample was assayed for its reticulocyte count
and hemoglobin level. The hemoglobin level was determined using a
micro cell counter (Sysmex F-800, Toa Medical Electronics Co.,
Ltd.).
[0079] At individual time points, the hemoglobin level and
reticulocyte count were compared between the vehicle group and the
rhEPO, mono-mPEG-EPO or di-mPEG-EPO group by Dunnett's multiple
comparison test at 5% significance level to verify the significance
of differences in the mean values.
[0080] The results are shown in FIG. 4 (time course of peripheral
reticulocyte counts) and FIG. 5 (time course of hemoglobin levels).
In addition, the sustained period of erythropoiesis stimulation is
summarized in Table 4.
4TABLE 4 Effective period of erythropoiesis stimulation induced by
PEG-conjugated EPOs Effective period (days) as determined by Test
group hemoglobin level, > vehicle.sup.a rhEPO 1 (Day 4)
mono-mPEG-EPO 22 (Day 4-25) di-mPEG-EPO 18 (Day 4-21)
.sup.asignificant difference from vehicle control (P < 0.05)
[0081] On Day 2 after administration, all the test groups showed
significantly increased reticulocyte counts as compared with the
vehicle group. In the rhEPO group, the reticulocyte count reached a
peak on Day 4 after administration and decreased below the vehicle
group on Day 7 after administration. In contrast, the mono-mPEG-EPO
and di-mPEG-EPO groups showed a significantly higher value than the
vehicle group by Day 10 and Day 7 after administration,
respectively.
[0082] The hemoglobin level of the rhEPO group reached a peak on
Day 4 after administration and decreased to substantially the same
level as the vehicle group on Day 7 after administration. In
contrast, the mono-mPEG-EPO and di-mPEG-EPO groups showed a peak on
Day 14 and Day 10 after administration, respectively. These groups
also showed a significantly higher level than the vehicle group by
Day 25 and Day 21 after administration, respectively. The period,
during which the test group showed a significantly higher level
than the vehicle group, was only one day (Day 4) in the rhEPO
group, whereas it was 22 days (Day 4-25) and 18 days (Day 4-21) in
the mono-mPEG-EPO and di-mPEG-EPO groups, respectively (Table
4).
Example 6
Assay for Sustained Efficacy of mono-mPEG-EPO, mono-branched
mPEG-EPO and rhEPO
[0083] The same comparison experiment as shown in Example 5 was
repeated, except for the following point. The erythropoietic effect
was compared between the following conjugates when rats received a
single tail vein injection of 5 or 1 .mu.g/kg: mono-branched
PEG-EPO (br-mPEG-EPO, having one branched PEG molecule) prepared
through the reaction between an ester-activated (methoxy PEG
20000).sub.2 derivative of approximately 40 kDa
((mPEG).sub.2-succinimidyl propionate) and an amino group in rhEPO;
and linear mono-PEG-EPO (mono-mPEG-EPO, having one linear PEG
molecule) prepared through the reaction between an ester-activated
mPEG derivative of approximately 20 kDa (mPEG-succinimidyl
propionate) and an amino group in rhEPO. On the day of test
material administration, rats were divided into groups of five such
that all groups had substantially the same hemoglobin level.
[0084] On the first day of administration and 2, 4, 7, 10, 14, 21,
28 and 35 days after administration, each rat was fixed on a
retainer under unanesthetized conditions and blood was sampled from
a wound made by sticking an injection needle into the tail
vein.
[0085] The results are shown in FIG. 6 (time course of peripheral
reticulocyte counts) and FIG. 7 (time course of hemoglobin levels).
In addition, the sustained period of erythropoiesis stimulation is
summarized in Table 5. In FIGS. 6 and 7 and Table 5, br-mPEG-EPO
denotes mono-branched mPEG-EPO.
5TABLE 5 Doses and sustained erythropoietic effect of
PEG-conjugated EPOs Effective period (days) as determined Test
group by hemoglobin level, > vehicle.sup.a mono-mPEG-EPO; 5
.mu.g/kg 13 (Day 2-14) mono-mPEG-EPO; 1 .mu.g/kg 8 (Day 7-14)
br-mPEG-EPO; 5 .mu.g/kg 11 (Day 4-14) br-mPEG-EPO; 1 .mu.g/kg
--.sup.b .sup.asignificant difference from vehicle control (P <
0.05) .sup.bno significant difference from vehicle control
[0086] On Day 2 after administration, all the test groups showed
significantly increased reticulocyte counts as compared with the
vehicle group, and a peak was observed on Day 4 after
administration in each group. When peak reticulocyte count was
compared among the test groups, the mono-mPEG-EPO (5 .mu.g/kg)
group was the highest of all, the mono-branched mPEG-EPO (5
.mu.g/kg) group and the mono-mPEG-EPO (1 .mu.g/kg) group were of
the same level, and the mono-branched mPEG-EPO (1 .mu.g/kg) group
was the lowest. The period, during which the test group showed
significantly higher reticulocyte counts than the vehicle group,
was 6 days (Day 2-7) in the 5 .mu.g/kg groups for both
mono-mPEG-EPO and mono-branched mPEG-EPO, whereas it was 3 days
(Day 2-4) in the 1 .mu.g/kg groups (FIG. 6).
[0087] The hemoglobin level of the mono-mPEG-EPO (5 .mu.g/kg) group
reached a peak on Day 7 after administration, and the period,
during which the group showed a significant increase over the
vehicle group, was 13 days (Day 2-14). The mono-branched MPEG-EPO
(5 .mu.g/kg) group and the mono-mPEG-EPO (1 .mu.g/kg) group both
showed a peak on Day 4 after administration, and the period, during
which the groups showed a significant increase over the vehicle
group, was 11 days (Day 4-14) and 8 days (Day 7-14), respectively.
In contrast, the mono-branched mPEG-EPO (1 .mu.g/kg) group showed
no significant difference from the vehicle group over the period of
the experiment. (FIG. 7, Table 5).
Example 7
Dose-Dependent Efficacy of rhEPO and PEG-conjugated EPO and
Sustained Efficacy of PEG-conjugated EPO
[0088] Mono-mPEG-EPO prepared in Example 1 is verified for its
efficacy at doses of 25 .mu.g/kg or less, along with a comparison
between single tail vein injection of mono-mPEG-EPO and continuous
tail vein injection of unconjugated EPO.
[0089] (Dose-Dependent Efficacy of PEG-conjugated EPO)
[0090] Mono-mPEG-EPO was examined for the time- and dose-dependency
of its erythropoietic effect when 8-week-old male rats (n=5)
received a single tail vein injection of the conjugate at doses of
25 .mu.g/kg, 5 .mu.g/kg, 1 .mu.g/kg and 0.2 .mu.g/kg.
[0091] FIG. 8 shows-changes in hemoglobin levels of the rats which
received a single tail vein injection of mono-mPEG-EPO at doses of
25 .mu.g/kg, 5 .mu.g/kg, 1 .mu.g/kg and 0.2 .mu.g/kg (in the
figure, mono-mPEG-EPO is denoted as PEG(1)-EPO). When observed by
Day 28 after administration, mono-mPEG-EPO was found to provide
substantially the same peak hemoglobin level and sustained efficacy
in the 25 .mu.g/kg and 5 .mu.g/kg groups, whereas the 1 .mu.g/kg
and 0.2 .mu.g/kg groups were found to show dose-dependent decreases
in peak hemoglobin level and sustained efficacy.
[0092] (Comparison of Efficacy Between Single Tail Vein Injection
of mono-mPEG-EPO and 5-Day Continuous Tail Vein Injection of
rhEPO)
[0093] A comparison of efficacy was made between single tail vein
injection of mono-mPEG-EPO and 5-day continuous tail vein injection
of rhEPO. FIG. 9 shows the results obtained (in the figure,
mono-mPEG-EPO is denoted as PEG(1)-EPO). A comparison between
single injection of mono-mPEG-EPO (5 .mu.g/kg) and 5-day continuous
injection of EPO (1 .mu.g/kg per day, 5 .mu.g/kg in total)
indicated that the mono-mPEG-EPO group (5 .mu.g/kg, single
injection) showed a higher peak hemoglobin level and more sustained
efficacy. In contrast, a comparison between single injection of
mono-mPEG-EPO (1 .mu.g/kg) and 5-day continuous injection of EPO
(0.2 .mu.g/kg per day, 1 .mu.g/kg in total) indicated that both the
groups showed substantially the same changes in hemoglobin levels.
This strongly suggested that mono-mPEG-EPO could be administered
with less frequency than rhEPO.
Example 8
Preparation of PEG-conjugated EPOs (2)
[0094] (Preparation of mPEG5K-EPO)
[0095] EPO (1.3 mL, 2.27 mg/mL in 100 mM phosphate buffer, pH 8.0)
was added to mPEG5K-SPA (3.38 mg, PEG/EPO=4.09 (mol/mol)) and
stirred gently at room temperature for 30 minutes. After addition
of a 1 M Gly solution in water (130 .mu.L), stirring was continued
at room temperature for an additional 30 minutes to deactivate
active ester. The reaction solution was diluted in PBS (550 .mu.L)
and concentrated to 325 .mu.L through a Centricon-50. After
addition of PBS (150 .mu.L), the solution was purified by gel
filtration on two Superdex 200 HR 10/30 columns in series to
collect three potential fractions predominantly rich in
mono-mPEG5K-EPO, di-mPEG5K-EPO and a mixture thereof. Each fraction
was concentrated through a Centricon-50 and subjected again to gel
filtration under the same conditions to separate mono-mPEG5K-EPO
and di-mPEG5K-EPO fractions, which were then respectively combined
for each conjugate to give mono-mPEG5K-EPO (210 .mu.g) and
di-mPEG5K-EPO (218 .mu.g). SDS-PAGE was performed to confirm the
agreement of molecular weight between reaction products and
collected samples.
[0096] (Preparation of mPEG10K-EPO, mPEG15K-EPO, mPEG30K-EPO,
br-mPEG10K-EPO and br-mPEG20K-EPO)
[0097] Procedures for preparation will be shown below, with the
details of reaction conditions ((i) to (iv)) being summarized in
Table 6.
[0098] The indicated volume (ii) of an EPO solution (in 100 mM
phosphate buffer, pH 8.0) at the indicated concentration (i) was
added to the indicated volume (iii) of MPEG-SPA (linear) or
br-mPEG-NHS (branched) at the indicated PEG/EPO ratio (iv), and
they were stirred gently at room temperature for 30 minutes. After
addition of 1/10 volumes (based on the EPO solution provided for
the reaction) of a 1 M Gly solution in water, stirring was
continued at room temperature for an additional 30 minutes to
deactivate active ester. The solvent was replaced by 20 mM Tris (pH
8.0) through a Centricon-50. The resulting solution was applied to
ion-exchange chromatography on RESOURCE Q (1 mL) and eluted with a
gradient of 0-100% of the same Eluent B as used in Example 2 to
collect the respective fractions containing PEG-conjugated EPO and
EPO, followed by replacement of the solvent by PBS through a
Centricon-50. Each solution was subjected to gel filtration under
the same conditions as described for preparation of mPEG5K-EPO,
except that two Superose 6 HR 10/30 (1.0 cm.phi.-30 cm, Pharmacia
Biotech) columns in series were used instead in the preparation of
mPEG15K-EPO, mPEG30K-EPO and br-mPEG20K-EPO. Fractions which were
expected to contain EPO conjugated with one or two mPEG or br-mPEG
molecules were collected and concentrated. Fractions containing
mixed mPEG-EPOs with different PEG conjugation patterns were
concentrated again and then subjected to gel filtration to collect
mono-mPEG-EPO and di-mPEG-EPO.
[0099] SDS-PAGE was performed to confirm the agreement of molecular
weight between reaction products and collected samples. The yields
of the resulting samples were as shown in Table 7.
6TABLE 6 Reaction conditions for PEG-conjugated EPOs mPEG- EPO EPO
SPA or PEG/EPO Molecular weight concentration volume mPEG- ratio
mPEG-SPA or (i) (ii) NHS (iv) br-mPEG-NHS (mg/mL) (mL) (iii) (mg)
(mol/mol) Linear 10 kDa 1.75 1.0 2.91 3.12 Linear 15 kDa 1.75 1.0
4.43 2.97 Linear 30 kDa 1.82 1.1 10.95 3.16 Branched (br)10 kDa
2.43 0.7 5.07 4.93 Branched (br)20 kDa 2.43 0.7 9.67 4.93
[0100]
7TABLE 7 Yields of PEG-conjugated EPOs Yield (.mu.g EPO) Type of
PEG mono-PEGylated di-PEGylated mPEG10 K- 257 146 mPEG15 K- 277 135
mPEG30 K- 300 167 br-mPEG10 K- 247 138 br-mPEG20 K- 265 150
Example 9
Molecular Weight Determination of PEG-conjugated EPOs (2)
[0101] Various PEG-conjugated EPO solutions prepared in the same
manner as shown in Example 8 (Preparation of PEG-conjugated EPOs)
were concentrated through a Centricon-50 simultaneously with
solvent replacement by Milli-Q water. MALDI-ToF-MS analysis was
performed under the same conditions as shown in Example 2. Also,
each sample residue provided for ToF-MS analysis was diluted in PBS
and subjected to gel filtration under the same conditions as shown
in Example 2. Under the same conditions as described for mPEG 10K-
and mPEG15K- in Example 8, gel filtration column chromatography
(GPC) was performed on each sample to measure its elution time.
[0102] In the case of mPEG30K-EPO, its concentrated solution was
divided into two aliquots, one of which was provided for ToF-MS
analysis and the other for GPC. In the case of br-mPEG10K-EPOs and
br-mPEG20K-EPOs, the fraction at the elution time in GPC was
collected and provided for MALDI-ToF-MS analysis after solvent
replacement by Milli-Q water.
[0103] Table 8 Show the Results Obtained.
8TABLE 8 Molecular weights of PEG-conjugated EPOs Molecular weight
(Da) Cal- Sample culated ToF-MS GPC PEG EPO 29000.sup.1) 28400
70000 Linear mono-mPEG5000-EPO 34100 33800 114000 PEG
di-mPEG5000-EPO 39200 38900 161000 mono-mPEG20000-EPO 50000 49800
402000 di-mPEG20000-EPO 71000 71300 914000 mono-mPEG30000-EPO 60500
59900 606000 di-mPEG30000-EPO 92000 93000 (1620000).sup.2) Branched
mono-br- 39600 39600 161000 PEG mPEG10000-EPO di-br-mPEG10000-EPO
50200 50300 267000 mono-br- 50000 49700 316000 mPEG20000-EPO
di-br-mPEG20000-EPO 71000 71500 649000 mono-br- 71000 71200 823000
mPEG40000-EPO di-br-mPEG40000-EPO 113000 113000 (2080000).sup.2)
.sup.1)putative molecular weight of EPOCH (chemical analysis) found
in Journal of clinical therapeutics & medicine, Vol. 6, Suppl.
2 (May) 1990, p. 24 .sup.2)out of calibration range (shown for
reference)
Example 10
Assay for Cell Growth Activity of PEG-conjugated EPOs on
EPO-Dependent Cells (2)
[0104] BaF/EpoR cells showing EPO-dependent growth (Blod, 90,
1867-, 1997, PNAS, 93, 9471-, 1996) were washed by repeating 2
cycles of resuspension in 1% FCS-containing RPMI and
centrifugation. The cells were then suspended in 10% FCS-containing
RPMI at a density of 1.times.10.sup.5 cells/20 mL and dispensed
into each well of 96-well plates in a volume of 50 .mu.L per well.
Meanwhile, test samples were prepared using 10% FCS-containing RFMI
as a diluent to give the respective dilution series of EPO and
mono-mPEG-EPOs. Each sample solution (50 .mu.L/well, n=3) was added
to and mixed with the BaF/EpoR cells in the 96-well plates,
followed by incubation at 37.degree. C. under a humidified
atmosphere of 5% CO.sub.2 for 24 hours. Each well was supplemented
with 10 .mu.L Cell Count Reagent SF (Nacalai Tesque, Inc.) and
measured for its absorbance at wavelengths of 450 nm and 620 nm,
which was defined as the value at time 0. The plates were allowed
to stand at room temperature for an additional 5 hours, followed by
measurement of absorbance at wavelengths of 450 nm and 620 nm to
plot the data in graph form (see FIGS. 10 and 11, in which mono and
Epo are intended to mean mono-mPEG-EPO and rhEPO, respectively).
Among the data obtained, 2 points sandwiching the 50% activity
point in the dose-response relationship were analyzed by linear
regression to determine ED50 for each sample. The results are
summarized in Tables 9 and 10.
9 TABLE 9 ED50(ng/mL) vs EPO(%) EPO 0.134 100.0 mono-mPEG10000
0.255 52.5 mono-mPEG15000 0.240 55.6 mono-mPEG20000 0.557 24.0
[0105]
10 TABLE 10 ED50 (ng/mL) mono-mPEG5000 0.158 mono-mPEG30000 0.348
mono-mPEG20000 0.374 EPO 0.072
Example 11
Assay for Sustained Efficacy of mono-mPEG-EPOs with Different
Molecular Weights
[0106] Each PEG-conjugated EPO or rhEPO was diluted to 2.5 .mu.g/mL
in physiological saline containing 0.05% rat serum albumin and
0.05% Tween 20. Administration of the medium (vehicle) alone was
defined as a negative control. These administration solutions were
prepared on the first day of administration. In this Example,
Slc:SD male rats (Japan SLC. Inc) were provided for the experiment
when 7-8 weeks old. On the day of test material administration, the
rats were divided into 7 groups of 5 rats such that all groups had
substantially the same erythrocyte count.
[0107] These 7 groups of 5 rats were set as follows: vehicle group,
rhEPO group, mono-mPEG5K-EPO group, mono-mPEG10K-EPO group,
mono-mPEG15K-EPO group, mono-mPEG20K-EPO group and mono-mPEG30K-EPO
group. Each rat received a single tail vein injection of 2 mL/kg
(sample concentration: 5 .mu.g/kg).
[0108] On the first day of administration and 2, 4, 5, 7, 10, 15,
21, 29 and 35 days after administration, each rat was fixed on a
retainer under unanesthetized conditions and blood was sampled from
a wound made by sticking an injection needle into the tail vein.
Each blood sample was assayed for its reticulocyte count and
hemoglobin level. The hemoglobin level was determined using a micro
cell counter (Sysmex F-800, To a Medical Electronics Co.,
Ltd.).
[0109] Further, as an efficacy indicator reflecting both an
increase in reticulocyte counts and a period during which the
increase was observed, the AUC of reticulocyte counts (Reti-AUC) by
Day 15 after administration was calculated using the trapezoidal
rule for each group.
[0110] The results are shown in FIG. 12 (time course of peripheral
reticulocyte counts), FIG. 13 (time course of hemoglobin levels)
and FIG. 14 (AUC of reticulocyte counts).
INDUSTRIAL APPLICABILITY
[0111] In order to achieve more sustained efficacy without losing
physiological activities of native EPO, a glycoprotein rich in
sugar chains, there was developed a PEG-conjugated EPO with
significantly sustained efficacy, prepared by introducing a
controlled number of PEG molecules at controlled positions. The
ability of this PEG-conjugated EPO to provide more sustained
efficacy without affecting erythropoietic effect inherent to native
EPO allows, for example, a significant decrease in the number of
administrations to patients, a decrease in pain associated with
administration to patients, a decrease in the physical and time
burden on diseased patients by reducing the number of hospital
visits, as well as decreases in the load and hours on overworked
medical staff members, including doctors, nurses and pharmacists,
who work under extremely harsh conditions. Therefore, the
PEG-conjugated EPO achieves medical cost savings in all
aspects.
* * * * *