U.S. patent application number 11/744162 was filed with the patent office on 2008-04-10 for natriuretic peptide conjugate using carrier substance.
This patent application is currently assigned to HANMI PHARM. IND. CO., LTD.. Invention is credited to Sung Min Bae, Dae Jin Kim, Young Hoon Kim, Young Min Kim, Se Chang Kwon, Gwan Sun Lee, Chang Ki Lim, Dae Hae Song.
Application Number | 20080085862 11/744162 |
Document ID | / |
Family ID | 36589418 |
Filed Date | 2008-04-10 |
United States Patent
Application |
20080085862 |
Kind Code |
A1 |
Kim; Young Min ; et
al. |
April 10, 2008 |
NATRIURETIC PEPTIDE CONJUGATE USING CARRIER SUBSTANCE
Abstract
The present invention relates to an Natriuretic peptide
conjugate having improved in-vivo duration of efficacy and
stability, comprising an Natriuretic peptide, a non-peptidyl
polymer and a carrier substance, which are covalently linked to
each other, and a use of the same. The Natriuretic peptide
conjugate of the present invention has the in-vivo activity which
is maintained relatively high, and has remarkably increased blood
half-life, and thus it can be desirably employed in the development
of long-acting formulations of various peptide drugs.
Inventors: |
Kim; Young Min; (Yongin-si,
KR) ; Kim; Dae Jin; (Seocho-gu, KR) ; Bae;
Sung Min; (Seocho-gu, KR) ; Lim; Chang Ki;
(Suwon-si, KR) ; Kwon; Se Chang; (Gwangjin-gu,
KR) ; Lee; Gwan Sun; (Songpa-gu, KR) ; Song;
Dae Hae; (Gwanak-gu, KR) ; Kim; Young Hoon;
(Youngdeungpo-gu, KR) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 5400
SEATTLE
WA
98104
US
|
Assignee: |
HANMI PHARM. IND. CO., LTD.
Hwaseong-si
KR
|
Family ID: |
36589418 |
Appl. No.: |
11/744162 |
Filed: |
May 3, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10535232 |
Jun 19, 2006 |
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11744162 |
May 3, 2007 |
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PCT/KR04/02944 |
Nov 13, 2004 |
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10535232 |
Jun 19, 2006 |
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Current U.S.
Class: |
424/178.1 ;
514/1.7; 514/12.4; 514/15.7; 514/3.2; 514/7.4; 530/324 |
Current CPC
Class: |
A61P 3/10 20180101; A61P
7/06 20180101; A61P 15/00 20180101; C07K 14/505 20130101; C07K
16/46 20130101; C07K 2319/30 20130101; A61K 47/6835 20170801; A61P
35/00 20180101; C07K 14/56 20130101; A61P 43/00 20180101; A61P
37/00 20180101; C07K 2317/732 20130101; A61K 47/6813 20170801; A61P
5/10 20180101; A61P 7/00 20180101; C07K 19/00 20130101; C07K 14/535
20130101; A61P 5/06 20180101; A61K 47/68 20170801; C07K 2317/734
20130101; C07K 2317/52 20130101; A61P 9/00 20180101; C07K 14/61
20130101; A61P 3/04 20180101; A61K 47/6811 20170801 |
Class at
Publication: |
514/012 ;
530/324 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61P 9/00 20060101 A61P009/00; C07K 14/00 20060101
C07K014/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 13, 2003 |
KR |
10-2003-0080299 |
Claims
1. A Natriuretic peptide conjugate, comprising a Natriuretic
peptide and an immunoglobulin Fc region, which are linked by a
non-peptidyl polymer, wherein the non-peptidyl polymer is selected
from the group consisting of polyethylene glycol, polypropylene
glycol, copolymers of ethylene glycol and propylene glycol,
polyoxyethylated polyols, polyvinyl alcohol, polysaccharides,
dextran, polyvinyl ethyl ether, biodegradable polymers, lipid
polymers, chitins, hyaluronic acid, and combinations thereof.
2. The Natriuretic peptide conjugate according to claim 1, wherein
the Natriuretic peptide is selected from the group consisting of
Atrial Natriuretic Peptide(ANP), Brain Natriuretic peptide(BNP),
C-type Natriuretic peptide(CNP), Dendroaspis Natriuretic
peptide(DNP), and a derivative, a fragment and a variant
thereof.
3. The Natriuretic peptide conjugate according to claim 2, wherein
the Natriuretic peptide is native BNP
4. The Natriuretic peptide conjugate according to any one of claims
1 to 3, wherein the non-peptidyl polymer has both ends, each
binding to an amine group or a thiol group of the immunoglobulin Fc
region and native BNP.
5. The Natriuretic peptide conjugate according to claim 4, wherein
the non-peptidyl polymer has both ends, each binding to an
N-terminus of the immunoglobulin Fc region, and native BNP.
6. The Natriuretic peptide conjugate according to claim 1, wherein
the immunoglobulin Fc region is deglycosylated.
7. The Natriuretic peptide conjugate according to claim 1, wherein
the immunoglobulin Fc region is composed of one to four domains
selected from the group consisting of CH1, CH2, CH3 and CH4
domains.
8. The Natriuretic peptide conjugate according to claim 7, wherein
the immunoglobulin Fc region further includes a hinge region.
9. The Natriuretic peptide conjugate according to claim 1, wherein
the immunoglobulin Fc region is an Fc region derived from an
immunoglobulin selected from the group consisting of IgG, IgA, IgD,
IgE, or IgM.
10. The Natriuretic peptide conjugate according to claim 9, wherein
each domain of the immunoglobulin Fc region is a domain hybrid of a
different origin derived from an immunoglobulin selected from the
group consisting of IgG, IgA, IgD, IgE, and IgM.
11. The Natriuretic peptide conjugate according to claim 9, wherein
the immunoglobulin Fc region is a dimer or a multimer (a
combination of immunoglobulin Fc) composed of single-chain
immunoglobulin of the same origin.
12. The Natriuretic peptide conjugate according to claim 9, wherein
the immunoglobulin Fc region is an IgG4 Fc region.
13. The Natriuretic peptide conjugate according to claim 12,
wherein the immunoglobulin Fc region is a human deglycosylated IgG4
Fc region.
14. The Natriuretic peptide conjugate according to claim 1, wherein
the reactive group of the non-peptidyl polymer is selected from the
group consisting of an aldehyde group, a propione aldehyde group, a
butyl aldehyde group, a maleimide group, and a succinimide
derivative.
15. The Natriuretic peptide conjugate according to claim 14,
wherein the succinimide derivative is succinimidyl propionate,
succinimidyl carboxymethyl, hydroxy succinimidyl, or succinimidyl
carbonate.
16. The Natriuretic peptide conjugate according to claim 14,
wherein the non-peptidyl polymer has a reactive aldehyde group at
both ends.
17. The Natriuretic peptide conjugate according to claim 1, wherein
the non-peptidyl polymer is polyethylene glycol.
18. A method for preparing an Natriuretic peptide conjugate,
comprising the steps of: (1) Covalently linking a non-peptidyl
polymer having a reactive group selected from the group consisting
of aldehyde, maleimide, and succinimide derivatives at both ends
thereof, with an amine or thiol group of a Natriuretic peptide; (2)
Isolating a conjugate comprising the Natriuretic peptide, in which
the non-peptidyl polymer from the reaction mixture of (1) is linked
covalently to N-terminus; and (3) Covalently linking an
immunoglobulin Fc region to the other end of the non-peptidyl
polymer of the isolated conjugate to produce a peptide conjugate
comprising the immunoglobulin Fc region and the Natriuretic
peptide, which are linked to each end of the non-peptidyl
polymer.
19. The method for preparing the Natriuretic peptide conjugate
according to claim 18, wherein the non-peptidyl polymer is
polyethylene glycol.
20. The method for preparing the Natriuretic peptide conjugate
according to claim 18, wherein the Natriuretic peptide is native
BNP.
21. A pharmaceutical composition comprising the peptide conjugate
of any one of claims 1 to 17.
22. A method for treating acute or chronic congestive heart failure
(CHF), hypertension, asthma, inflammation related disease,
hyperlipidemia or impotence comprising administering the peptide
conjugate of any one of claims 1 to 17, or the pharmaceutical
composition of claim 21.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to the Natriuretic peptide
conjugate for a long-acting formulation. Specifically, the present
invention relates to a BNP conjugate having a remarkably improved
in-vivo duration of efficacy by selectively binding with a specific
amino acid residue, and modification of a specific amino acid
residue, in order to remarkably increase the blood half-life by
covalently linking the Natriuretic peptide with a non-peptidyl
polymer and a carrier substance, and a method for preparation
thereof.
[0003] 2. Description of the Related Art
[0004] Since peptides tend to be easily denatured due to their low
stability, degraded by in-vivo proteolytic enzymes, thus losing the
activity, and have a relatively small size, thereby easily passing
through the kidney. Accordingly, in order to maintain the blood
levels and the titers of a medicament in blood comprising a peptide
as a pharmaceutically effective component, it is necessary to
administer the peptide drug frequently to a patient to maintain
desired blood levels and titers. However, the peptide drugs are
usually administered in the form of injectable preparations, and
such frequent administration for maintaining the blood levels of
the physiologically active peptides cause severe pain for the
patients. To solve these problems, many efforts have been made. As
one of such efforts, there has been suggested an approach that
transmission through the biological membrane of the peptide drug is
increased, and then the peptide drug is transferred into the body
by oropharyngeal or nasopharyngeal inhalation. However, this
approach is still difficult in maintaining the in-vivo activity of
the peptide drug due to the remarkably lower in-vivo transfer
efficiency, as compared with injectable preparations.
[0005] Natriuretic peptide group consist of 4 kinds of structurally
similar polypeptides, which includes Atrial Natriuretic
Peptide(ANP), Brain Natriuretic peptide(BNP), C-type Natriuretic
peptide(CNP) and Dendroaspis Natriuretic peptide(DNP).
[0006] BNP (Natrecor, J&J) is the peptide of 3,464 Dalton
molecular weight, which consists of 32 amino acids and contains one
intra-disulfide bond. Binding to NPR-A to activate the production
of cGMP, which leads to reduction in the arterial blood pressure,
and as a result, BNP is used as congestive heart failure(CHF)
therapeutic agent. Because the blood half-life in Rat is about 1
min, which is very short, they use the troublesome administration
method by infusion for 48 hours period. (J. Pharmaceutical Sciences
95; 2499.about.2506(2006)).
[0007] Therefore many efforts have been made to improve the blood
stability of the peptide drug, and to maintain the drug in the
blood at a high level for a prolonged period of time, thereby
maximizing the pharmaceutical efficacy of the drug. The long-acting
preparation of such peptide drug therefore needs to increase the
stability of the peptide drug, and to maintain the titers at
sufficiently high levels without causing immune responses in
patients. These peptides have a problem, usually in that the size
of the peptide is small. Thus, they cannot be recovered in the
kidney, and are then extracorporeally discharged. Accordingly, a
method for chemically adding a polymeric substance having high
solubility, such as polyethylene glycol (PEG), onto the surface of
the peptide to inhibit the loss in the kidney, has been used. PEG
non-specifically binds to a specific site or various sites of a
target peptide to give an effect of increasing the molecular weight
of a peptide, and thus inhibiting the loss by the kidney, and
preventing hydrolysis, without causing any side-effects. For
example, WO2006/076471 describes that PEG binds to BNP, thereby
sustaining the physiological activity. WO05116655 present the
possibility of oral administration by making pegylated BNP.
However, this method increases the molecular weight of PEG, thereby
increasing the in-vivo residence time of the peptide drug, while as
the molecular weight is increased, the titer of the peptide drug is
remarkably reduced, and the reactivity with the peptide is also
reduced. Accordingly, it undesirably lowers the yield.
[0008] It was reported that albumin fusion BNP by using the
recombinant gene technology was produced (Pharmaceutical Research,
21(11):2105-2111(2004)). Though the blood half-life of mammalian
derived albumin fusion BNP in mouse increased about 12-19 hours, in
vitro activity of albumin fusion BNP was remarkably reduced to 1.6%
compared to that of native BNP. Therefore the fusion technology
could not overcome the low activity because of the albumin fusion.
On the other hand, using the peptide containing disulfide bond like
BNP, the possibility of misfolding is high, which cause the
application to be difficult.
[0009] In WO 04011498, BNP conjugate covalently bonded with
recombinant albumin and chemical linker was produced and its blood
half-life is about 14-16 hours, which dose not show remarkable
blood stability, proving the method of using albumin as long-acting
protein carrier is not appropriate.
[0010] Thus, the present inventors used a preparation method, in
which a immunoglobulin Fc region, a non-peptidyl polymer, and an
BNP are site-specifically linked as a method for maximizing the
effects of increasing the blood half-life of an BNP, and of
maintaining the in-vivo activity. They have found that the BNP
conjugate has a remarkably increased effect of in-vivo duration of
efficacy, thereby completing the present invention.
SUMMARY OF THE INVENTION
[0011] It is an object of the present invention to provide an
excellent long-acting preparation of Natriuretic peptide which
maintains the in-vivo activity of the Natriuretic peptide, while
extending the blood half-life.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows the results of reverse phase HPLC for
measurement of the purity of a BNP(N)-PEG-immunoglobulin Fc
conjugate;
[0013] FIG. 2 shows the results of reverse phase HPLC for
measurement of the purity of a BNP(Lys)-PEG-immunoglobulin Fc
conjugate; and
[0014] FIG. 3 shows the results of measurement of the purity of a
BNP(N)--PEG-immunoglobulin Fc conjugate by 12% SDS-PAGE
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0015] In one embodiment of the present invention for solving the
above-described problems, there is provided a long-acting BNP
conjugate, in which a BNP, a non-peptidyl polymer possessing a
reactive group at both ends thereof, and a immunoglobulin Fc region
are covalently linked to each other.
[0016] The BNP of the present invention, which holds blood vessel
extension function, decreases arterial blood pressure. These
peptides include a precursor, a derivative, a fragment, and a
variant, and preferably the peptide having over 95% amino acid
sequence homology.
[0017] The BNP of the present invention has 1 NPR-A binding motif
and one or more PEGylation site, and includes human BNP, rat BNP,
canine BNP and human ANP.
[0018] The sequence of the BNP(1-32) amino acids is as follows:
[0019] BNP(1-32)
[0020] Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-
[0021] Phe-Gly-Arg-Lys-Met-Asp-Arg-11e-Ser-Ser-
[0022] Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-
[0023] Arg-His
[0024] BNP derivative means a peptide which exhibits an amino acid
sequence homology of at least 80% with that of BNP, which may be in
the chemically modified form. The peptide may have some groups on
the amino acid residue chemically substituted (e.g.,
alpha-methylated, alpha-hydroxylatied), deleted (e.g., deaminated),
or modified (e.g., N-methylated).
[0025] The BNP fragment means one in the form in which one or more
amino acids are added or deleted at an amino terminus or a carboxyl
terminus of a native BNP, wherein the added amino acid is possibly
non-naturally occurring amino acid (e.g., D-type amino acid).
[0026] The BNP variant means a peptide, which has one or more amino
acid sequences different from those of a native BNP.
[0027] Each of the preparation methods for the BNP derivative, the
fragment, and the variant can be used individually or in
combination.
[0028] The native BNP used in the present invention and the
modified BNP can be synthesized using a solid phase synthesis
method, and most of the native peptides including a native BNP can
be produced by a recombination technology.
[0029] Further, the BNP used in the present invention can bind to
the non-peptidyl polymer on various sites.
[0030] The conjugate prepared according to the present invention
can have an activity which varies depending on the sites to be
linked to the BNP.
[0031] For example, it can be coupled with an amino terminus, and
other terminus other than the amino terminus, such as a carboxyl
terminus, respectively, which indicates difference in the in vitro
activity. The aldehyde reactive group selectively binds to an amino
terminus at a low pH, and can bind to a lysine residue to form a
covalent bond at a high pH, such as pH 9.0. A pegylation reaction
is allowed to proceed with varying pH, and then a positional isomer
can be separated from the reaction mixture using an ion exchange
column.
[0032] If the BNP peptide is to be coupled at a site other than the
amino terminus, a reactive thiol group can be introduced to the
site of amino acid residue to be modified in the native amino acid
sequence to form a covalent bond using a maleimide linker at the
non-peptidyl polymer.
[0033] Further, a reactive amine group can be introduced to the
site of amino acid residue to be modified in the native amino acid
sequence to form a covalent bond using an aldehyde linker at the
non-peptidyl polymer.
[0034] In one specific preferable embodiment, the present inventors
induced a pegylation reaction to link a PEG to N-terminus when
coupling the PEG with a native BNP at pH 6.0. After coupling with
carrier, it was found that in vitro activity is maintained at about
29% (Table 1). Further blood half-life of BNP is about 21 hour,
while native BNP was not titrated because of very short blood
half-life (table 1).
[0035] Therefore the blood half-life of the BNP-PEG-immunoglobulin
Fc conjugated in present invention was remarkably increased over 21
hours, minimizing the titer reduction by coupling the N-terminal
which does not affect the activity. As a result, a novel long-acing
BNP formulation having a remarkably improved effect of the in-vivo
efficacy sustainability could be prepared.
[0036] The BNP used in the present invention is linked with a
carrier substance and a non-peptidyl polymer.
[0037] The carrier substance which can be used in the present
invention can be selected from the group consisting of an
immunoglobulin Fc region, albumin, transferrin, and PEG, and
preferably it is an immunoglobulin Fc region.
[0038] The immunoglobulin Fc region is safe for use as a drug
carrier because it is a biodegradable polypeptide that is in vivo
metabolized. Also, the immunoglobulin Fc region has a relatively
low molecular weight, as compared to the whole immunoglobulin
molecules, and thus, it is advantageous in the preparation,
purification and yield of the conjugate. Since the immunoglobulin
Fc region does not contain a Fab fragment, whose amino acid
sequence differs according to the antibody subclasses and which
thus is highly non-homogenous, it can be expected that the
immunoglobulin Fc region may greatly increase the homogeneity of
substances and be less antigenic.
[0039] The term "immunoglobulin Fc region", as used herein, refers
to a protein that contains the heavy-chain constant region 2
(C.sub.H2) and the heavy-chain constant region 3 (C.sub.H3) of an
immunoglobulin, and not the variable regions of the heavy and light
chains, the heavy-chain constant region 1 (C.sub.H1) and the
light-chain constant region 1 (C.sub.L1) of the immunoglobulin. It
may further include a hinge region at the heavy-chain constant
region. Also, the immunoglobulin Fc region of the present invention
may contain a part or all of the Fc region including the
heavy-chain constant region 1 (C.sub.H1) and/or the light-chain
constant region 1 (C.sub.L1), except for the variable regions of
the heavy and light chains, as long as it has a physiological
function substantially similar to or better than the native
protein. Also, the IgG Fc region may be a fragment having a
deletion in a relatively long portion of the amino acid sequence of
C.sub.H2 and/or C.sub.H3. That is, the immunoglobulin Fc region of
the present invention may comprise 1) a C.sub.H1 domain, a C.sub.H2
domain, a C.sub.H3 domain and a C.sub.H4 domain, 2) a C.sub.H1
domain and a C.sub.H2 domain, 3) a C.sub.H1 domain and a C.sub.H3
domain, 4) a C.sub.H2 domain and a C.sub.H3 domain, 5) a
combination of one or more domains and an immunoglobulin hinge
region (or a portion of the hinge region), and 6) a dimer of each
domain of the heavy-chain constant regions and the light-chain
constant region.
[0040] The immunoglobulin Fc region of the present invention
includes a native amino acid sequence, and a sequence derivative
(mutant) thereof. An amino acid sequence derivative is a sequence
that is different from the native amino acid sequence due to a
deletion, an insertion, a non-conservative or conservative
substitution or combinations thereof of one or more amino acid
residues. For example, in an IgG Fc, amino acid residues known to
be important in binding, at positions 214 to 238, 297 to 299, 318
to 322, or 327 to 331, may be used as a suitable target for
modification. Also, other various derivatives are possible,
including one in which a region capable of forming a disulfide bond
is deleted, or certain amino acid residues are eliminated at the
N-terminal end of a native Fc form or a methionine residue is added
thereto. Further, to remove effector functions, a deletion may
occur in a complement-binding site, such as a C1q-binding site and
an ADCC site. Techniques of preparing such sequence derivatives of
the immunoglobulin Fc region are disclosed in International Pat.
Publication Nos. WO 97/34631 and WO 96/32478.
[0041] Amino acid exchanges in proteins and peptides, which do not
generally alter the activity of the proteins or peptides are known
in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press,
New York, 1979). The most commonly occurring exchanges are Ala/Ser,
Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, AlaNal,
Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, LeuNal,
Ala/Glu and Asp/Gly, in both directions.
[0042] In addition, the Fc region, if desired, may be modified by
phosphorylation, sulfation, acrylation, glycosylation, methylation,
farnesylation, acetylation, amidation, and the like.
[0043] The aforementioned Fc derivatives are derivatives that have
a biological activity identical to the Fc region of the present
invention or improved structural stability, for example, against
heat, pH, or the like.
[0044] In addition, these Fc regions may be obtained from native
forms isolated from humans and other animals including cows, goats,
swine, mice, rabbits, hamsters, rats and guinea pigs, or may be
recombinants or derivatives thereof, obtained from transformed
animal cells or microorganisms. Herein, they may be obtained from a
native immunoglobulin by isolating whole immunoglobulins from human
or animal organisms and treating them with a proteolytic enzyme.
Papain digests the native immunoglobulin into Fab and Fc regions,
and pepsin treatment results in the production of pF'c and F(ab')2
fragments. These fragments may be subjected, for example, to size
exclusion chromatography to isolate Fc or pF'c.
[0045] Preferably, a human-derived Fc region is a recombinant
immunoglobulin Fc region that is obtained from a microorganism.
[0046] In addition, the immunoglobulin Fc region of the present
invention may be in the form of having native sugar chains,
increased sugar chains compared to a native form or decreased sugar
chains compared to the native form, or may be in a deglycosylated
form. The increase, decrease or removal of the immunoglobulin Fc
sugar chains may be achieved by methods common in the art, such as
a chemical method, an enzymatic method and a genetic engineering
method using a microorganism. The removal of sugar chains from an
Fc region results in a sharp decrease in binding affinity to the
C1q part of the first complement component C1 and a decrease or
loss in antibody-dependent cell-mediated cytotoxicity (ADCC) or
complement-dependent cytotoxicity (CDC), thereby not inducing
unnecessary immune responses in-vivo. In this regard, an
immunoglobulin Fc region in a deglycosylated or aglycosylated form
may be more suitable to the object of the present invention as a
drug carrier.
[0047] As used herein, the term "deglycosylation" refers to
enzymatically remove sugar moieties from an Fc region, and the term
"aglycosylation" means that an Fc region is produced in an
unglycosylated form by a prokaryote, preferably E. coli.
[0048] On the other hand, the immunoglobulin Fc region may be
derived from humans or other animals including cows, goats, swine,
mice, rabbits, hamsters, rats and guinea pigs, and preferably
humans. In addition, the immunoglobulin Fc region may be an Fc
region that is derived from IgG, IgA, IgD, IgE and IgM, or that is
made by combinations thereof or hybrids thereof. Preferably, it is
derived from IgG or IgM, which is among the most abundant proteins
in human blood, and most preferably from IgG, which is known to
enhance the half-lives of ligand-binding proteins.
[0049] On the other hand, the term "combination", as used herein,
means that polypeptides encoding single-chain immunoglobulin Fc
regions of the same origin are linked to a single-chain polypeptide
of a different origin to form a dimer or multimer. That is, a dimer
or multimer may be formed from two or more fragments selected from
the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc
fragments.
[0050] The term "hybrid", as used herein, means that sequences
encoding two or more immunoglobulin Fc regions of different origin
are present in a single-chain immunoglobulin Fc region. In the
present invention, various types of hybrids are possible. That is,
domain hybrids may be composed of one to four domains selected from
the group consisting of CH1, CH2, CH3 and CH.sub.4 of IgG Fc, IgM
Fc, IgA Fc, IgE Fc and IgD Fc, and may include the hinge
region.
[0051] On the other hand, IgG is divided into IgG1, IgG2, IgG3 and
IgG4 subclasses, and the present invention includes combinations
and hybrids thereof. Preferred are IgG2 and IgG4 subclasses, and
most preferred is the Fc region of IgG4 rarely having effector
functions such as CDC (complement dependent cytotoxicity).
[0052] That is, as the drug carrier of the present invention, the
most preferable immunoglobulin Fc region is a human IgG4-derived
non-glycosylated Fc region. The human-derived Fc region is more
preferable than a non-human derived Fc region, which may act as an
antigen in the human body and cause undesirable immune responses
such as the production of a new antibody against the antigen.
[0053] The term "non-peptidyl polymer", as used herein, refers to a
biocompatible polymer including two or more repeating units linked
to each other by a covalent bond excluding a peptide bond.
[0054] The non-peptidyl polymer which can be used in the present
invention may be selected form the group consisting of polyethylene
glycol, polypropylene glycol, copolymers of ethylene glycol and
propylene glycol, polyoxyethylated polyols, polyvinyl alcohol,
polysaccharides, dextran, polyvinyl ethyl ether, biodegradable
polymers such as PLA (poly(lactic acid) and PLGA
(polylactic-glycolic acid), lipid polymers, chitins, hyaluronic
acid, and combinations thereof, and preferred is poly ethylene
glycol. Also, derivatives thereof well known in the art and being
easily prepared within the skill of the art are included in the
scope of the present invention.
[0055] The peptide linker which is used in the fusion protein
obtained by a conventional inframe fusion method has drawbacks that
it is easily in-vivo cleaved by a proteolytic enzyme, and thus a
sufficient effect of increasing the blood half-life of the active
drug by a carrier cannot be obtained as expected. However, in the
present invention, a polymer having resistance to the proteolytic
enzyme can be used to maintain the blood half-life of the peptide
to be similar to that of the carrier. Therefore, any non-peptidyl
polymer which can be used in the present invention can be used
without any limitation, as long as it is a polymer having the
aforementioned function, that is, a polymer having resistance to
the in-vivo proteolytic enzyme. The non-peptidyl polymer preferably
has a molecular weight in the range of 1 to 100 kDa, and preferably
of 1 to 20 kDa. Also, the non-peptidyl polymer of the present
invention, linked to the carrier substance, may be one polymer or a
combination of different types of polymers.
[0056] The non-peptidyl polymer used in the present invention has a
reactive group capable of binding to the carrier substance and the
protein drug.
[0057] The non-peptidyl polymer has a reactive group at both ends,
which is preferably selected from the group consisting of a
reactive aldehyde group, a propionaldehyde group, a butyraldehyde
group, a maleimide group and a succinimide derivative. The
succinimide derivative may be succinimidyl propionate, hydroxy
succinimidyl, succinimidyl carboxymethyl, or succinimidyl
carbonate. In particular, when the non-peptidyl polymer has a
reactive aldehyde group at both ends, it is effective in linking at
both ends with a physiologically active polypeptide and an
immunoglobulin Fc region with minimal non-specific reactions. A
final product generated by reductive alkylation by an aldehyde bond
is much more stable than when linked by an amide bond. The aldehyde
reactive group selectively binds to an amino terminus at a low pH,
and can bind to a lysine residue to form a covalent bond at a high
pH, such as pH 9.0.
[0058] The reactive groups at both ends of the non-peptidyl polymer
may be the same or different. For example, the non-peptidyl polymer
may possess a maleimide group at one end and, at the other end, an
aldehyde group, a propionaldehyde group or a butyraldehyde group.
When a polyethylene glycol having a reactive hydroxy group at both
ends thereof is used as the non-peptidyl polymer, the hydroxy group
may be activated to various reactive groups by known chemical
reactions, or a polyethylene glycol having a commercially available
modified reactive group may be used so as to prepare the
Natriuretic peptide conjugate of the present invention.
[0059] The Natriuretic peptide conjugate of the present invention
maintains the conventional in-vivo activities of the Natriuretic
peptide, such as extension of blood vessel and regulation of blood
pressure, and further remarkably increases the blood half-life of
the Natriuretic peptide, and hence the in-vivo efficacy sustaining
effect of the peptide, it is useful to treat the disease such as
acute or chronic congestive heart failure, hypertension, asthma,
inflammation related disease, hyperlipidemia, impotence and
etc.
[0060] In another embodiment, the present invention provides a
method for preparing a Natriuretic peptide conjugate, comprising
the steps of:
[0061] (1) Covalently linking a non-peptidyl polymer having an
aldehyde group at both ends thereof, with an amino terminus of the
Natriuretic peptide;
[0062] (2) Isolating a conjugate comprising the Natriuretic
peptide, in which the non-peptidyl polymer from the reaction
mixture of (1) is linked covalently to the amino terminus; and
[0063] (3) Covalently linking a carrier substance to the other end
of the non-peptidyl polymer of the isolated conjugate to produce a
peptide conjugate comprising the carrier substance and the
Natriuretic peptide, which are linked to each end of the
non-peptidyl polymer.
[0064] In a further embodiment, the present invention provides a
pharmaceutical composition for treating acute of chronic CHF,
comprising the Natriuretic peptide conjugate of the present
invention.
[0065] The pharmaceutical composition comprising the conjugate of
the present invention can further comprise a pharmaceutically
acceptable carrier. For oral administration, the pharmaceutically
acceptable carrier may include a binder, a lubricant, a
disintegrator, an excipient, a solubilizer, a dispersing agent, a
stabilizer, a suspending agent, a coloring agent, and a perfume.
For injectable preparations, the pharmaceutically acceptable
carrier may include a buffering agent, a preserving agent, an
analgesic, a solubilizer, an isotonic agent, and a stabilizer. For
preparations for topical administration, the pharmaceutically
acceptable carrier may include a base, an excipient, a lubricant,
and a preserving agent. The pharmaceutical composition of the
present invention may be formulated into a variety of dosage forms
in combination with the aforementioned pharmaceutically acceptable
carriers. For example, for oral administration, the pharmaceutical
composition may be formulated into tablets, troches, capsules,
elixirs, suspensions, syrups or wafers. For injectable
preparations, the pharmaceutical composition may be formulated into
a unit dosage form, such as a multidose container or an ampule as a
single-dose dosage form. The pharmaceutical composition may be also
formulated into solutions, suspensions, tablets, pills, capsules
and long-acting preparations.
[0066] On the other hand, examples of the carrier, the excipient,
and the diluent suitable for the pharmaceutical formulations
include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol,
erythritol, maltitol, starch, acacia rubber, alginate, gelatin,
calcium phosphate, calcium silicate, cellulose, methylcellulose,
microcrystalline cellulose, polyvinylpyrrolidone, water,
methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium
stearate and mineral oils. In addition, the pharmaceutical
formulations may further include fillers, anti-coagulating agents,
lubricants, humectants, perfumes, and antiseptics.
[0067] The administration frequency and dose of the pharmaceutical
composition of the present invention can be determined by several
related factors including the types of diseases to be treated,
administration routes, the patient's age, gender, weight and
severity of the illness, as well as by the types of the drug as an
active component. Since the pharmaceutical composition of the
present invention has excellent duration of in-vivo efficacy and
titer, it can remarkably reduce the administration frequency and
dose of pharmaceutical drugs of the present invention.
[0068] In a further embodiment, the present invention provides a
method for treating diabetes, obesity, acute coronary syndrome, or
polycystic ovary syndrome, comprising a step of administering the
Natriuretic peptide conjugate, or a pharmaceutical composition
containing the same.
[0069] The term "administration", as used herein, means
introduction of a predetermined amount of a substance into a
patient by a certain suitable method. The conjugate of the present
invention may be administered via any of the common routes, as long
as it is able to reach a desired tissue. A variety of modes of
administration are contemplated, including intraperitoneally,
intravenously, intramuscularly, subcutaneously, intradermally,
orally, topically, intranasally, intrapulmonarily and
intrarectally, but the present invention is not limited to these
exemplified modes of administration. However, since peptides are
digested upon oral administration, active ingredients of a
composition for oral administration should be coated or formulated
for protection against degradation in the stomach. Preferably, the
present composition may be administered in an injectable form. In
addition, the pharmaceutical composition of the present invention
may be administered using a certain apparatus capable of
transporting the active ingredients into a target cell.
[0070] A better understanding of the present invention may be
obtained through the following examples which are set forth to
illustrate, but are not to be construed as the limit of the present
invention.
Examples
Example 1
Pegylation of BNP, and Isolation of Positional Isomer
[0071] 3.4K ButyrALD(2) PEG (PEG having two butyraldehyde groups)
and the N-terminus of BNP(AP, USA) were subject to pegylation by
reacting the peptide and the PEG at 4.degree. C. for 60 min at a
molar ratio of 1:10, with a peptide concentration of 3 mg/ml. At
this time, the reaction was performed in a NaOAc buffer at pH 4.0
at a concentration of 100 mM, and 20 mM SCB (NaCNBH3) as a reducing
agent was added thereto to perform the reaction. 3.4K ButyrALD(2)
PEG and the lysine(Lys) residue of the BNP were subject to
pegylation by reacting the peptide and the PEG at 4.degree. C. for
90 min at a molar ratio of 1:30, with a peptide concentration of 3
mg/ml. At this time, the reaction was performed in a Na-Borate
buffer at pH 9.0 at a concentration of 100 mM, and 20 mM SCB as a
reducing agent was added thereto to perform the reaction. Isomers
were isolated from the each of the reaction solutions using SOURCE
S (XK 16 ml, Amersham Bioscience). It was found that a peak for
pegylation of the N-terminus was found earlier, and then two peaks
for pegylation of the lysine residues were found.
[0072] Column: SOURCE S (XK 16 ml, Amersham Bioscience)
[0073] Flow rate: 2.0 ml/min
[0074] Gradient: A 0.fwdarw.30% 60 min B (A: 20 mM Tris pH 8.0, B:
A +0.5 M NaCl)
Example 2
Preparation of BNP(N)-PEG-Immunoglobulin Fc Conjugate
[0075] Using the same method as described in EXAMPLE 1, 3.4K
ButyrALD(2) PEG and the N-terminus of the BNP were reacted, and
only the N-terminal isomers were purified, and then coupled with
immunoglobulin Fc. The reaction was performed at a ratio of
peptide:immunoglobulin Fc of 1:6, and a total concentration of
proteins of 50 mg/ml at 4.degree. C. for 18 hours. The reaction was
performed in a solution of 100 mM K--P (pH 6.0), and 20 mM SCB as a
reducing agent was added thereto. The coupling reaction solution
was purified using two purification columns. First, SOURCE S (XK 16
ml, Amersham Bioscience) was used to remove a large amount of
immunoglobulin Fc which had not participated in the coupling
reaction. Under the condition of 20 mM Na-P (pH 8.0), the
immunoglobulin Fc does not bind to the column and only
BNP-immunoglobulin Fc binds to it. Using 1 M NaCl with salt
gradient, high purity BNP-immunoglobulin Fc could be purified from
the peptide which does not participate the coupling reaction. As
the results of reverse phase HPLC, the purity was found to be
97.2%. [FIG. 1]
[0076] Column: SOURCE S (XK 16 ml, Amersham Bioscience)
[0077] Flow rate: 2.0 ml/min
[0078] Gradient: A 0.fwdarw.30% 60 min B (A: 20 mM Na--P (pH 8.0),
B: A+1 M NaCl)
Example 3
Preparation of BNP(LYS)-Immunoglobulin Fc Conjugate
[0079] Using the same method as described in EXAMPLE 1, 3.4K
ButyrALD(2) PEG and the lysine(Lys) of the BNP were reacted, and
only the Lys isomers were purified, and then coupled with
immunoglobulin Fc. The reaction was performed at a ratio of
peptide: immunoglobulin Fc of 1:6, and a total concentration of
proteins of 50 mg/ml at 4.degree. C. for 18 hours. The reaction was
performed in a solution of 100 mM K--P (pH 6.0), and 20 mM SCB as a
reducing agent was added thereto. After the coupling reaction, the
purification process using SOURCE S 16 ml was the same as in
EXAMPLE 2. As the results of reverse phase HPLC, the purity was
found to be 98.2%. [FIG. 1]
Example 4
Preparation of Conjugate using PropionALD Linker PEG
[0080] Using 3.4K PropionALD(2) PEG (PEG having two propionaldehyde
groups), 3.4K-BNP was prepared in the same method as described in
EXAMPLE 1. It was coupled immunoglobulin Fc in the same method as
described in EXAMPLE 2.
Example 5
Preparation of BNP(3-32)Fragment and BNP(3-32)-Immunoglobulin Fc
Conjugate
[0081] Under the treatment of Di-Peptidyl-Peptidase (DPP IV)
(Sigma, USA) to native BNP(1.about.32), BNP(3.about.32) is
produced, in which 2 amino acid at the amino terminus was
removed.
[0082] After stopping the enzyme reaction, BNP (3-32) was purified
from native BNP (1-32) by the method using the Source S 16 ml
column. As a result of reverse phase HPLC, the purity was found to
be over 95%. The molecular weight was measured by MALDI TOF to be
3,280 Dalton, 184 dalton less than that of native BNP (1-32).
[0083] Using the purified BNP (3-32), BNP (3-32)-immunoglobulin Fc
was prepared, using the same method as described in EXAMPLE 1, 2.
As a linker, 3.4K PropionALD(2) PEG was used, described in EXAMPLE
4. As the results of reverse phase HPLC, the purity of purified BNP
(3-32)-immunoglobulin Fc was found to be 97%.
Example 6
Measurement of In-Vitro Activity of Long-Acting BNP
[0084] To measure the efficacy of the long-acting BNP preparation,
a method for measuring the in-vitro cell activity was used.
Typically, in order to measure the in-vitro activity of BNP, human
aortic smooth muscle cell were separated, and whether cGMP's in the
cell was increased after treatment of BNP was determined.
[0085] Human aortic smooth muscle cell was treated with BNP and
test materials at varying concentrations. The occurrence of cAMP's,
which are secondary signaling molecules in the cells, by the test
materials, was measured, and hence EC50 values, and compared to
each other.
Example 7
Measurement of Pharmacodynamics of Long-Acting BNP
[0086] BNP and test materials were subcutaneously administrated to
3 SD Rat in each group with dosage of 100 .mu.g/kg. After
administration, blood sample was taken in 1, 6, 12, 24, 30, 48, 72,
96, 120 and 216 hours. To prevent the clotting, heparin containing
tube was used and cell was removed by the ependorf high speed
centrifugation for 5 min. the Concentration of peptide in serum was
titrated by ELISA using antibody. TABLE-US-00001 TABLE 1 Blood In
vitro half-life titer Test materials (hours) (%) BNP 0.5 100
BNP(N)-PGE-Fc 26 11.4 BNP(3-32)(N)-PGE-Fc 33 9.6
[0087] BNP(N)--PGE-Fc: Conjugate in which the N-terminus of the
BNP(1-32) and the Fc region were linked to PEG. [0088]
BNP(3-32)(N)-PGE-Fc: Conjugate in which the N-terminus of the
BNP(3-32) and the Fc region were linked to PEG.
EFFECTS OF THE INVENTION
[0089] The Natriuretic peptide conjugate of the present invention
has the in-vivo activity which is maintained relatively high, and
has remarkably increased blood half-life, and thus it can be
desirably employed in the development of long-acting formulations
of various peptide drugs.
[0090] All of the above U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications and non-patent publications referred to in this
specification and/or listed in the Application Data Sheet, are
incorporated herein by reference, in their entirety.
[0091] From the foregoing it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
* * * * *