WO2008136611A1 - Natriuretic peptide conjugate using carrier substance - Google Patents

Natriuretic peptide conjugate using carrier substance Download PDF

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Publication number
WO2008136611A1
WO2008136611A1 PCT/KR2008/002502 KR2008002502W WO2008136611A1 WO 2008136611 A1 WO2008136611 A1 WO 2008136611A1 KR 2008002502 W KR2008002502 W KR 2008002502W WO 2008136611 A1 WO2008136611 A1 WO 2008136611A1
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WIPO (PCT)
Prior art keywords
natriuretic peptide
immunoglobulin
region
peptide conjugate
group
Prior art date
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PCT/KR2008/002502
Other languages
French (fr)
Inventor
Dae Hae Song
Chang Ki Lim
Young Hoon Kim
Se Chang Kwon
Gwan Sun Lee
Dae Jin Kim
Sung Min Bae
Original Assignee
Hanmi Pharmaceutical Co., Ltd.
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Priority claimed from US11/744,162 external-priority patent/US20080085862A1/en
Application filed by Hanmi Pharmaceutical Co., Ltd. filed Critical Hanmi Pharmaceutical Co., Ltd.
Publication of WO2008136611A1 publication Critical patent/WO2008136611A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment

Definitions

  • 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.
  • 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.
  • 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.
  • 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).
  • ANP Atrial Natriuretic Peptide
  • BNP Brain Natriuretic peptide
  • CNP C- type Natriuretic peptide
  • DNP Dendroaspis Natriuretic peptide
  • 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 ⁇ 2506(2006)).
  • 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.
  • WO2006/076471 describes that PEG binds to BNP, thereby sustaining the physiological activity.
  • WO05116655 present the possibility of oral administration by making pegylated BNP.
  • 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.
  • albumin fusion BNP by using the recombinant gene technology was produced (Pharmaceutical Research, 21(11):2105-2111(2004)).
  • 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.
  • using the peptide containing disulfide bond like BNP the possibility of misfolding is high, which cause the application to be difficult.
  • 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.
  • Fig. 1 shows the results of reverse phase HPLC for measurement of the purity of a
  • Fig. 2 shows the results of reverse phase HPLC for measurement of the purity of a
  • Fig. 3 shows the results of measurement of the purity of a
  • 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.
  • 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.
  • the BNP of the present invention has 1 NPR-A binding motif and one or more
  • PEGylation site includes human BNP, rat BNP, canine BNP and human ANP.
  • 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).
  • 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).
  • the BNP variant means a peptide, which has one or more amino acid sequences different from those of a native BNP.
  • 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.
  • the BNP used in the present invention can bind to the non-peptidyl polymer on various sites.
  • the conjugate prepared according to the present invention can have an activity which varies depending on the sites to be linked to the BNP.
  • 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.
  • 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.
  • 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.
  • 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).
  • the BNP used in the present invention is linked with a carrier substance and a non- peptidyl polymer.
  • 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.
  • 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.
  • immunoglobulin Fc region refers to a protein that contains the heavy-chain constant region 2 (C H 2) and the heavy-chain constant region 3 (C H 3) of an immunoglobulin, and not the variable regions of the heavy and light chains, the heavy-chain constant region 1 (C H 1) and the light-chain constant region 1 (C L 1) of the immunoglobulin. It may further include a hinge region at the heavy-chain constant region.
  • 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 H 1) and/or the light-chain constant region 1 (C L 1), 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.
  • the IgG Fc region may be a fragment having a deletion in a relatively long portion of the amino acid sequence of C H 2 and/or C H 3.
  • the immunoglobulin Fc region of the present invention may comprise 1) a C H 1 domain, a C H 2 domain, a C H 3 domain and a C H 4 domain, 2) a C H 1 domain and a C H 2 domain, 3) a C H 1 domain and a C H 3 domain, 4) a C H 2 domain and a C H 3 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.
  • the immunoglobulin Fc region of the present invention includes a native amino acid sequence, and a sequence derivative (mutant) thereof.
  • 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.
  • 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.
  • 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.
  • a deletion may occur in a complement-binding site, such as a Clq-binding site and an ADCC site.
  • a complement-binding site such as a Clq-binding site and an ADCC site.
  • the Fc region may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.
  • 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.
  • 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.
  • 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.
  • a human-derived Fc region is a recombinant immunoglobulin Fc region that is obtained from a microorganism.
  • 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.
  • 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.
  • deglycosylation refers to enzymatically remove sugar moieties from an Fc region
  • amino acid sequence preferably E. coli
  • 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.
  • 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.
  • 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.
  • 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.
  • hybrid means that sequences encoding two or more immunoglobulin Fc regions of different origin are present in a single-chain immunoglobulin Fc region.
  • domain hybrids may be composed of one to four domains selected from the group consisting of C H 1, C H 2, C H 3 and C H 4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc, and may include the hinge region.
  • IgG is divided into IgGl, 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).
  • 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.
  • non-peptidyl polymer refers to a biocompatible polymer including two or more repeating units linked to each other by a covalent bond excluding a peptide bond.
  • 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.
  • 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.
  • 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.
  • 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.
  • the non-peptidyl polymer used in the present invention has a reactive group capable of binding to the carrier substance and the protein drug.
  • 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, suc- cinimidyl carboxymethyl, or succinimidyl carbonate.
  • 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.
  • the reactive groups at both ends of the non-peptidyl polymer may be the same or different.
  • 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 butyr- aldehyde group.
  • 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.
  • 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.
  • the present invention provides a method for preparing a
  • Natriuretic peptide conjugate comprising the steps of:
  • the present invention provides a pharmaceutical composition for treating acute of chronic CHF, comprising the Natriuretic peptide conjugate of the present invention.
  • the pharmaceutical composition comprising the conjugate of the present invention can further comprise a pharmaceutically acceptable carrier.
  • 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.
  • the pharmaceutically acceptable carrier may include a buffering agent, a preserving agent, an analgesic, a solubilizer, an isotonic agent, and a stabilizer.
  • 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.
  • the pharmaceutical composition may be formulated into tablets, troches, capsules, elixirs, suspensions, syrups or wafers.
  • 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.
  • 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.
  • the pharmaceutical formulations may further include fillers, anti-coagulating agents, lubricants, humectants, perfumes, and antiseptics.
  • 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.
  • 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.
  • 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, in- tradermally, orally, topically, intranasally, intrapulmonarily and intrarectally, but the present invention is not limited to these exemplified modes of administration.
  • active ingredients of a composition for oral administration should be coated or formulated for protection against degradation in the stomach.
  • the present composition may be administered in an injectable form.
  • the pharmaceutical composition of the present invention may be administered using a certain apparatus capable of transporting the active ingredients into a target cell.
  • EXAMPLE 1 Pegylation of BNP. and isolation of positional isomer.
  • 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.
  • BNP(1 ⁇ 32), BNP(3 ⁇ 32) is produced, in which 2 amino acid at the amino terminus was removed.
  • BNP (3-32) was purified from native BNP
  • BNP (3-32)-immunoglobulin Fc was prepared, using the same method as described in EXAMPLE 1, 2.
  • a linker 3.4K PropionALD(2) PEG was used, described in EXAMPLE 4.
  • 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 [108] 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.
  • EXAMPLE 7 Measurement of pharmacodynamics of long-acting BNP [112] BNP and test materials were subcutaneously administrated to 3 SD Rat in each group with dosage of lOO ⁇ 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.
  • - BNP(N)-PGE-Fc Conjugate in which the N-terminus of the BNP(I -32) and the Fc region were linked to PEG.
  • - 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.
  • 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.

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.

Description

Description
NATRIURETIC PEPTIDE CONJUGATE USING CARRIER
SUBSTANCE
Technical Field
[1] 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.
[2]
Background Art
[3] 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.
[4] 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).
[5] 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;2499~2506(2006)).
[6] 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 extracor- poreally 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.
[7] 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.
[8] 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. Disclosure of Invention Technical Problem
[9] 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. Technical Solution
[10] 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
[11] Fig. 1 shows the results of reverse phase HPLC for measurement of the purity of a
BNP(N)-PEG-immunoglobulin Fc conjugate;
[12] Fig. 2 shows the results of reverse phase HPLC for measurement of the purity of a
BNP(Lys)-PEG-immunoglobulin Fc conjugate; and
[13] Fig. 3 shows the results of measurement of the purity of a
BNP(N)-PEG-immunoglobulin Fc conjugate by 12% SDS-PAGE. Best Mode for Carrying Out the Invention
[14] 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.
[15] 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.
[16] 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.
[17]
[18] The sequence of the BNP(l-32) amino acids is as follows:
[19]
[20] BNP(I -32)
[21] Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-
[22] Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-
[23] Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-
[24] Arg-His
[25]
[26] 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).
[27] 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).
[28] The BNP variant means a peptide, which has one or more amino acid sequences different from those of a native BNP.
[29] Each of the preparation methods for the BNP derivative, the fragment, and the variant can be used individually or in combination.
[30] 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.
[31] Further, the BNP used in the present invention can bind to the non-peptidyl polymer on various sites.
[32]
[33] The conjugate prepared according to the present invention can have an activity which varies depending on the sites to be linked to the BNP.
[34] 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.
[35] 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.
[36] 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.
[37]
[38] 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).
[39]
[40] 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.
[41]
[42] The BNP used in the present invention is linked with a carrier substance and a non- peptidyl polymer.
[43] 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.
[44] 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.
[45] The term "immunoglobulin Fc region", as used herein, refers to a protein that contains the heavy-chain constant region 2 (CH2) and the heavy-chain constant region 3 (CH3) of an immunoglobulin, and not the variable regions of the heavy and light chains, the heavy-chain constant region 1 (CH1) and the light-chain constant region 1 (CL1) 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 (CH1) and/or the light-chain constant region 1 (CL1), 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 CH2 and/or CH3. That is, the immunoglobulin Fc region of the present invention may comprise 1) a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 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. [46] 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 Clq-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.
[47] 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, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly, in both directions.
[48] In addition, the Fc region, if desired, may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.
[49] 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.
[50] 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.
[51] Preferably, a human-derived Fc region is a recombinant immunoglobulin Fc region that is obtained from a microorganism.
[52] 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 CIq part of the first complement component Cl 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.
[53] 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.
[54] 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.
[55] 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.
[56] 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 CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc, and may include the hinge region.
[57] On the other hand, IgG is divided into IgGl, 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).
[58] 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.
[59] 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.
[60] 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.
[61] 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.
[62] The non-peptidyl polymer used in the present invention has a reactive group capable of binding to the carrier substance and the protein drug.
[63] 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, suc- cinimidyl 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.
[64] 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 butyr- aldehyde 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.
[65] 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.
[66]
[67] In another embodiment, the present invention provides a method for preparing a
Natriuretic peptide conjugate, comprising the steps of:
[68] (1) Covalently linking a non-peptidyl polymer having an aldehyde group at both ends thereof, with an amino terminus of the Natriuretic peptide;
[69] (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
[70] (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.
[71]
[72] 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.
[73] 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.
[74] 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.
[75] 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.
[76] 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.
[77] 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, in- tradermally, 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.
[78] 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.
[79]
[80]
Mode for the Invention
[81] EXAMPLE 1. Pegylation of BNP. and isolation of positional isomer.
[82] 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 40C 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 40C 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.
[83]
[84] Column: SOURCE S (XK 16 ml, Amersham Bioscience)
[85] Flow rate: 2.0 ml/min
[86] : A 0 30% 60 min B (A: 20 mM Tris pH 8.0, B: A + 0.5 M NaCl)
[87]
[88] EXAMPLE 2. Preparation of BNP CNVPEG-immunoglobulin Fc conjugate
[89] 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 40C 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 IM 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]
[90]
[91] Column: SOURCE S (XK 16 ml, Amersham Bioscience)
[92] Flow rate: 2.0 ml/min
[93] Gradient: A 0 → 30% 60 min B (A: 20 mM Na-P (pH 8.0), B: A + 1 M NaCl)
[94]
[95]
[96] EXAMPLE 3. Preparation of BNPfLysVimmunoglobulin Fc conjugate
[97] 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 40C 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]
[98]
[99] EXAMPLE 4. Preparation of conjugate using propionALD linker PEG
[100] 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.
[101]
[102] EXAMPLE 5. Preparation of BNP(3-32s)fragment and BNPG-32Vimmunogloburin Fc
[103] Under the treatment of Di-Peptidyl-Peptidase (DPP IV) (Sigma, USA) to native
BNP(1~32), BNP(3~32) is produced, in which 2 amino acid at the amino terminus was removed.
[104] After stopping the enzyme reaction, BNP (3-32) was purified from native BNP
(1-32) by the method using the Source S 16ml 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).
[105] 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%.
[106] [107] EXAMPLE 6. Measurement of in- vitro activity of long-acting BNP [108] 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.
[109] 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.
[HO] [111] EXAMPLE 7. Measurement of pharmacodynamics of long-acting BNP [112] BNP and test materials were subcutaneously administrated to 3 SD Rat in each group with dosage of lOOμg/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.
[113] Table 1 [Table 1] [Table ]
Figure imgf000014_0001
[114] - BNP(N)-PGE-Fc: Conjugate in which the N-terminus of the BNP(I -32) and the Fc region were linked to PEG. [115] - 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. [116]
Industrial Applicability [117] 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.

Claims

Claims
[I] 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 claims 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.
[I I] 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.
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