KR20030017908A - multi-branched polymer used in conjugating protein or peptide, and resulting conjugator - Google Patents

multi-branched polymer used in conjugating protein or peptide, and resulting conjugator Download PDF

Info

Publication number
KR20030017908A
KR20030017908A KR1020010051528A KR20010051528A KR20030017908A KR 20030017908 A KR20030017908 A KR 20030017908A KR 1020010051528 A KR1020010051528 A KR 1020010051528A KR 20010051528 A KR20010051528 A KR 20010051528A KR 20030017908 A KR20030017908 A KR 20030017908A
Authority
KR
South Korea
Prior art keywords
protein
peg
peptide
molecular weight
polymer derivative
Prior art date
Application number
KR1020010051528A
Other languages
Korean (ko)
Other versions
KR100761652B1 (en
Inventor
조영우
김원근
임중인
박범수
이성희
김원배
이강춘
Original Assignee
동아제약 주식회사
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 동아제약 주식회사 filed Critical 동아제약 주식회사
Priority to KR1020010051528A priority Critical patent/KR100761652B1/en
Publication of KR20030017908A publication Critical patent/KR20030017908A/en
Application granted granted Critical
Publication of KR100761652B1 publication Critical patent/KR100761652B1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
    • 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
    • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1275—Lipoproteins; Chylomicrons; Artificial HDL, LDL, VLDL, protein-free species thereof; Precursors thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/34—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from hydroxy compounds or their metallic derivatives
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/34—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from hydroxy compounds or their metallic derivatives
    • C08G65/38—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from hydroxy compounds or their metallic derivatives derived from phenols
    • C08G65/40—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from hydroxy compounds or their metallic derivatives derived from phenols from phenols (I) and other compounds (II), e.g. OH-Ar-OH + X-Ar-X, where X is halogen atom, i.e. leaving group
    • C08G65/4012—Other compound (II) containing a ketone group, e.g. X-Ar-C(=O)-Ar-X for polyetherketones
    • C08G65/4031—(I) or (II) containing nitrogen
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/34—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from hydroxy compounds or their metallic derivatives
    • C08G65/48—Polymers modified by chemical after-treatment

Abstract

PURPOSE: A physiological highly active multibranched polymer derivative and a conjugate bonded with protein or peptide are provided to minimize the reduction of physiological activity of protein and to increase the solubility in vivo by reducing the number of the polymer derivative bonded to the physiological site of protein and to protect the molecular structure of protein against the enzyme, thereby improving the in vivo utilization rate of active protein or peptide. CONSTITUTION: The multibranched polymer derivative has a mean molecular weight of polyethylene glycol unit of 15,000-80,000 D and is represented by the formula 1, wherein R1, R2 and R3 are independent each another and are an alkyl group of C1-C6; n1, n2 and n3 are an integer of 100-500; and Z is an active radical. Preferably Z is COONHS or CONH-CH2CH2(OCH2CH2)n4OCOONHS, wherein n4 is an integer of 50-500. The protein-polymer or peptide-polymer joined compound is one obtained by joining the multibranched polymer derivative with protein or peptide. Preferably the bonding site of the joined compound is the amine group or N-terminal of lysine.

Description

단백질 또는 펩타이드에 결합되는 다가지의 고분자 유도체와 접합체{multi-branched polymer used in conjugating protein or peptide, and resulting conjugator}Multi-branched polymer used in conjugating protein or peptide, and resulting conjugator}

본 발명은 단백질 또는 펩타이드에 결합되는 다가지의 고분자 유도체 및 접합체에 관한 것으로서, 보다 상세하게는 고분자를 활성화하여 고반응성의 가지가 3개 달린 고분자 유도체를 합성하고, 그 유도체에 단백질이나 펩타이드에 연결되는 연결쇄를 접합시키므로써 얻어지는 단백질-고분자 또는 펩타이드-고분자 접합체에 관한 것이다.The present invention relates to various polymer derivatives and conjugates that bind to proteins or peptides, and more particularly, to activate polymers, to synthesize polymer derivatives having three highly reactive branches, and to connect the derivatives to proteins or peptides. It relates to a protein-polymer or a peptide-polymer conjugate obtained by conjugating the linking chain.

최근들어 산업적으로 급격히 발전되고 있는 화학적, 유전공학적 기법에 의해 많은 효소(enzyme), 단백질(protein), 호르몬(hormone) 및 펩타이드(peptide)들이 대량으로 생산되어 특이적 생체촉매 작용을 하는 생체촉매(biocatalysts) 또는 의약품으로 개발되고 사용되고 있으나, 이러한 단백질 또는 펩타이드를 의약품으로 사용하는데는 많은 제약이 있다. 이러한 제약들로는 첫째, 낮은안정성(stability)과 빠른 체내 소실(clearance)이 문제가 되는데, 많은 단백질들은 생체내로 투여되었을 때 순환계(circulation)에서 빠르게 소실(clearance)되며, 또한 효소에 의해 단시간에 파괴되거나 쉽게 가수분해된다. 둘째, 반복적인 투여에 의해 면역반응이 유도되어 항체가 생성되고 이 항체에 의해 과민반응(hypersensitivity)이 유발되어 생명을 위협하기도 한다. 또한 망상세포의 내피계(reticuloendothelial system, RES)에 의해 소실이 증가하게 되며, 이러한 문제는 자연적인 생산물뿐만 아니라 기초 구조가 같은 제조된 단백질에서도 나타난다.In recent years, a lot of enzymes, proteins, hormones and peptides are produced in large quantities by chemical and genetic engineering techniques that are rapidly developed in the industry. Although developed and used as biocatalysts or pharmaceuticals, there are many limitations in using such proteins or peptides as pharmaceuticals. These constraints include, firstly, low stability and fast clearance in the body. Many proteins are rapidly cleared in the circulation when administered in vivo, and are also destroyed by enzymes in a short time. Easily hydrolyzed. Secondly, repeated administrations induce an immune response to produce antibodies, which in turn cause hypersensitivity, which can be life threatening. In addition, the loss is increased by the reticuloendothelial system (RES) of the reticular cells, and this problem is not only a natural product but also a manufactured protein having the same basic structure.

지금까지 대부분의 단백질 또는 펩타이드 약물은 주로 주사제로 투여되고 있으며, 투여횟수는 1일 1회 또는 주 3회 등의 잦은 빈도로 투여되어 왔다. 약물의 빈번한 투여는 환자에게 고통을 줄 뿐만 아니라, 특히 장기간 치료가 필요한 환자에 있어서는 양질의 생활을 유지하기가 어려웠다. 따라서 상기와 같은 문제점이 개선된 보다 안정적이고 장기적으로 활성을 유지하는 약물의 개발이 요구되고 있다.Until now, most protein or peptide drugs are mainly administered by injection, and the frequency of administration has been frequently administered once a day or three times a week. Frequent administration of drugs not only suffers from the patients, but also makes it difficult to maintain a good quality of life, especially in patients in need of long-term treatment. Therefore, there is a need for the development of a drug that is more stable and maintains long-term activity with the above problems.

단백질 치료제의 대부분은 혈중에서의 짧은 반감기로 인하여 지속적인 임상 효능을 기대하기 어려운 면이 있다. 단백질 또는 약리 활성을 갖는 분자를 고분자와 결합시키는 것은 생체내(in vivo) 및 생체외(in vitro) 적용시 많은 이점을 제공할 수 있다. 생리활성 분자에 대한 고분자의 공유결합은 생체 내에서의 안정성을 증가시킬 뿐만 아니라 장관 시스템, 신장(kidney), 비장(spleen) 또는간(liver)에 의한 소실을 연장시킬 수 있으며, 생체적합성(biocompatibility)을 증가시켜서 면역 반응성을 감소시킬 수 있게 된다. 또한 분자의 표면특성 및 용해성을 변화시켜서 물 또는 유기용매에 대한 용해도를 증가시킬 수 있다.Most protein therapeutics are difficult to expect sustained clinical efficacy due to their short half-life in the blood. Coupling proteins or molecules with pharmacological activity with polymers can provide a number of advantages in in vivo and in vitro applications. Covalent binding of polymers to bioactive molecules not only increases their stability in vivo, but can also lead to prolonged loss by the intestinal system, kidney, spleen, or liver, resulting in biocompatibility. Increase immune response. It is also possible to increase the solubility in water or organic solvents by changing the surface properties and solubility of the molecules.

최근에는 폴리에틸렌 글리콜(polyethylene glycol, 이하 "PEG"라 칭한다.)-결합 생체분자가 임상적으로 유용한 특성을 갖는 것으로 나타났다. PEG-결합 생체분자가 증가된 물리적, 열적 안정성과 용해도를 가지고, 효소에 의해 쉽게 분해되는 것을 예방하고, 생체내 순환 반감기가 길어지며 제거율이 감소되는 여러 가지의 유익한 효능이 있는 것으로 보고되고 있다. 특히 생체내에서의 고분자의 제거는 그 분자량에 반비례 한다는 사실이 알려져 있다[Inada 등,J. Bioact and Compatible Polymers 5, 343(1990); Delgado 등,Critical Reviews in Therapeutic Drug Carrier Systems 9, 249(1992); Katre,Advanced Drug Delivery Systems 10, 91(1993)].Recently, polyethylene glycol (hereinafter referred to as "PEG")-binding biomolecules have been shown to have clinically useful properties. It has been reported that PEG-binding biomolecules have a variety of beneficial effects with increased physical and thermal stability and solubility, preventing easy degradation by enzymes, prolonged circulating half-lives and reduced elimination rates. In particular, it is known that removal of polymers in vivo is inversely proportional to their molecular weight [Inada et al., J. Bioact and Compatible Polymers 5 , 343 ( 1990 ); Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9 , 249 ( 1992 ); Katre, Advanced Drug Delivery Systems 10 , 91 ( 1993 ).

아부초브스키 등은 PEG와 결합된 여러 가지 단백질들이 혈장에서 체내 반감기가 증가되고 면역원성이 감소됨을 증명하였으며, 또한 데이비스 등은 유리카제(uricase)가 PEG와 결합하면 체내 반감기가 증가되고 요산의 대사시 부작용이 감소됨을 증명하였다[Abuchowski et al.,Cancer Biochem. Biophys.,7, 175-186,1984; Davis et al.,Lancet,2, 281-283,1981].Abuchosky et al. Demonstrated that PEG-associated proteins increase the body half-life and decrease immunogenicity in plasma. Davis et al. Also found that uricase combined with PEG increases the body half-life and metabolizes uric acid. Have been shown to reduce side effects during treatment [Abuchowski et al., Cancer Biochem. Biophys. , 7 , 175-186, 1984 ; Davis et al., Lancet , 2 , 281-283, 1981 ].

미국특허 제4179337호는 단백질과 폴리펩타이드(polypeptide)를 분자량이 500∼20,000의 PEG 또는 수용성 폴리머에 결합시켜 생물학적 활성을 유지하면서 항원성과 면역원성이 감소된 단백질-고분자 결합체에 대해 언급하였다.U.S. Pat.No. 4,417,337 mentions protein-polymer conjugates that reduce antigenicity and immunogenicity while maintaining biological activity by binding proteins and polypeptides to PEG or water-soluble polymers with molecular weights of 500 to 20,000.

Monfardini 등은 PEG-단백질 결합체에서 가지 달린 PEG 결합체는 선형 PEG 결합체보다 pH와 열적 안정성이 증가하고, 단백 분해 효소에 대한 안정성이 크게 증가한다는 것을 증명하였고, PEG-단백질의 경우는 면역원성 및 항원성 뿐만 아니라 독성도 감소됨을 보고하였다[Bioconjugate Chem.6, 62,1995].Monfardini et al. Demonstrated that branched PEG conjugates in PEG-protein conjugates have increased pH and thermal stability and greater stability to proteolytic enzymes than linear PEG conjugates, and immunogenicity and antigenicity in the case of PEG-proteins. As well as reduced toxicity [ Bioconjugate Chem . 6 , 62, 1995 ].

단백질 또는 펩타이드에 PEG를 결합시키는데는 여러 가지 방법이 있는데, 단백질 또는 펩타이드의 아미노(amino) 잔기, 올리고당의 히드록시기에 활성화된 PEG를 결합시키는 방법이 있다.There are various methods of binding PEG to a protein or peptide, and there are methods of binding PEG activated to an amino moiety of a protein or peptide and a hydroxyl group of an oligosaccharide.

활성화된 PEG와 결합하는 단백질 또는 펩타이드의 아미노 잔기로는 라이신(lysine)기와 N-말단을 들 수 있으며, PEG를 활성화시키는 방법은 다음과 같다. PEG의 한쪽 하이드록시기(hydroxy group)을 메칠 에테르기(methyl ether group)로 치환시키고, 나머지 한쪽 하이드록시기에 친전자성 물질(electrophile)을 함유한 작용기를 결합시킨다. 활성화된 고분자의 예로는 아마이드 결합(amide bond)을 가진 PEG-N-하이드록시석신이미드-활성화된 에스테르(PEG-N-Hydroxysuccinimide-activated esters), 알킬 결합(alkyl bond)을 가진 PEG-에폭사이드(PEG-epoxide)와 PEG-트레실레이트(PEG-tresylate), 우레탄 결합(urethane bond)을 가진 PEG-카보닐이미다졸(PEG-carbonyl imidazole), PEG-니트로페닐 카보네이트(PEG-nitrophenyl carbonates) 및 N-말단에 쉬프 베이스(Schiff base)를 가진 PEG-알데하이드(PEG-aldehyde) 등이 있다.An amino residue of a protein or peptide that binds to activated PEG includes a lysine group and an N-terminus, and a method of activating PEG is as follows. One hydroxyl group of the PEG is replaced with a methyl ether group, and the other hydroxyl group is bonded to a functional group containing an electrophile. Examples of activated polymers include PEG-N-Hydroxysuccinimide-activated esters with amide bonds and PEG-epoxides with alkyl bonds. (PEG-epoxide) and PEG-tresylate, PEG-carbonyl imidazole with urethane bonds, PEG-nitrophenyl carbonates And PEG-aldehyde having a Schiff base at the N-terminus.

일반적으로 단백질을 수식하는 모노메톡시 폴리에틸렌 글리콜(monomethoxy polyethylene glycol, 이하 "mPEG"라 칭한다.)은 선형 고분자로서 분자량은 1,000 내지 20,000 정도의 것이 사용되고 있다. 그런데, 단백질 또는 펩타이드의 생리활성 부위는 한정되어 있으므로, 활성을 유지하면서 다수의 선형 고분자를 상기 단백질 등에 접합시키는 데에는 한계가 있다. 특히 저분자량의 단백질의 경우 상대적으로 생리활성부위가 적어 고분자의 접합에 의해 급격히 활성이 저하되는 문제가 있다. 이에 단백질의 활성을 유지하면서 다수의 고분자 물질을 결합시키는 방법에 대한 연구가 진행되어 왔다. 기존의 선형 고분자를 접합시키는 방법은 이용하는데 한계가 있으므로 이를 극복하기 위하여 같은 분자량을 가지고 있으나 가지 달린 mPEG를 만드는 방법이 시도되었는데 이러한 방법은 와나 등의 보고에 따른 것으로서, 트리클로로트리아진(trichlorotriazine)을 이용하여 가지가 2개 달린 mPEG 유도체를 합성하고, 이를 사용하여 단백질에 접합하는 것이 기재되어 있다[Wana, H et al.,Ann. NY. Acad. Sci.613, 95-108,1990].In general, monomethoxy polyethylene glycol (hereinafter referred to as "mPEG") that modifies a protein is a linear polymer and molecular weight of about 1,000 to 20,000 is used. However, since the physiologically active site of the protein or peptide is limited, there is a limit in conjugating a large number of linear polymers to the protein or the like while maintaining the activity. In particular, a low molecular weight protein has a relatively low physiologically active site, and thus there is a problem in that activity is rapidly decreased due to the conjugation of a polymer. Accordingly, researches on a method of binding a plurality of polymer materials while maintaining protein activity have been conducted. In order to overcome the limitations of existing linear polymers, the method of making mPEG with the same molecular weight has been attempted to overcome this problem. This method is based on the report of Wana et al., Which is based on trichlorotriazine. It has been described to synthesize two branched mPEG derivatives and to use them to conjugate proteins [Wana, H et al., Ann. NY. Acad. Sci . 613 , 95-108, 1990 ].

최근에는 분자량 20,000의 mPEG 2개를 가지로 연결한 mPEG 고분자 유도체를 만드는 방법을 개발하여, 이를 사용하여 인터페론(Interferon)에 접합시켜 이 PEG-인터페론을 개발하였다는 특허가 있다[미합중국특허 제5932462호].Recently, there has been a patent for developing a method of making mPEG polymer derivatives having two mPEGs having a molecular weight of 20,000 and connecting them to an interferon to develop the PEG-interferon [US Patent No. 5932462]. ].

그러나 상기한 바와 같은 활성화된 가지 달린 고분자들도 아직까지 그 크기가 거대하여 수식된 단백질의 표면에 입체적 장애(steric hindrance)를 유발함으로써 단백질의 활성을 떨어뜨리는 문제가 있다.However, the activated branched polymers as described above still have a problem of degrading protein activity by causing steric hindrance on the surface of the modified protein due to its large size.

이에, 본 발명자는 상기한 문제점을 해결하고자 고분자 유도체에 연결쇄(linker)를 연결하여 단백질에 접합시킴으로써 생리활성부위를 방해하는 입체적 장애의 문제점을 최소화하여 생리활성의 감소를 극소화시키고, 보다 가지를 증가시켜 단백질에 대한 고분자의 크기를 증가시킴으로써 입체적 장애를 줄이는 동시에 생체내의 소실 속도를 감소시켜 작용 지속 시간을 증가시킬 수 있음을 알아내어 본 발명을 완성하였다.Thus, the present inventors minimize the problem of physiological activity by minimizing the problem of steric hindrance that interferes with the physiological activity site by connecting a linker to a polymer derivative and conjugating to a protein in order to solve the above problems. The present invention was completed by finding out that by increasing the size of the polymer for the protein to reduce steric hindrance and at the same time reduce the rate of disappearance in vivo to increase the duration of action.

본 발명의 목적은 가지가 3개 달린 생체적합성 고분자 유도체를 합성하고 이 유도체에 연결쇄를 연결한 가지가 4개 달린 고반응성의 고분자 유도체를 제공하는 것이다.An object of the present invention is to synthesize a biocompatible polymer derivative having three branches and to provide a highly reactive polymer derivative having four branches having a linking chain connected to the derivative.

또한, 본 발명의 목적은 상기 고분자 유도체를 이용하여 단백질 또는 펩타이드 고유의 활성을 유지한 단백질-고분자 또는 펩타이드-고분자 접합체를 제공하는 것이다.It is also an object of the present invention to provide a protein-polymer or peptide-polymer conjugate that maintains the inherent activity of a protein or peptide using the polymer derivative.

상기와 같은 목적을 달성하기 위하여, 본 발명의 목적은 가지가 3개 달린 생체적합성 고분자 유도체를 합성하고 이 유도체에 연결쇄를 연결한 가지가 4개 달린 고반응성의 고분자 유도체를 제공한다.In order to achieve the above object, an object of the present invention is to synthesize a biocompatible polymer derivative having three branches and provides a highly reactive polymer derivative having four branches having a linking chain connected to the derivative.

또한, 본 발명의 목적은 상기 고분자 유도체를 이용하여 단백질 또는 펩타이드 고유의 활성을 유지한 단백질-고분자 또는 펩타이드-고분자 접합체를 제공한다.It is also an object of the present invention to provide a protein-polymer or peptide-polymer conjugate that maintains the inherent activity of a protein or peptide using the polymer derivative.

이하 본 발명을 더욱 상세히 설명한다.Hereinafter, the present invention will be described in more detail.

본 발명은 하기화학식 1의 구조를 가진 다가지의 고분자 유도체를 포함한다.The present invention includes various polymer derivatives having the structure of Formula 1 below.

상기식에서,In the above formula,

R1, R2및 R3는 서로 독립적이며, C1∼C6인 알킬기이고,R 1 , R 2 and R 3 are independent of each other, are C 1 to C 6 alkyl group,

n1, n2 및 n3는 100∼500의 정수이며, 바람직하게는 200∼500의 정수이며,n1, n2 and n3 are integers of 100-500, Preferably they are integers of 200-500,

Z는 활성기를 나타낸다.Z represents an active group.

상기화학식 1은 고분자인 RCH2CH2(OCH2CH2)n가 3 개 이상 결합된 가지 달린 구조를 갖는 활성화된 고분자이다. Formula 1 is an activated polymer having a branched structure in which at least three RCH 2 CH 2 (OCH 2 CH 2 ) n polymers are bonded.

상기 고분자는 자연적이거나 합성에 의한 수용성 물질로서, 그 종류는 폴리에틸렌글리콜(polyethylene glycol, PEG), 폴리락트산(polylatic acid), 폴리프로필렌글리콜(polypropylene glycol), 폴리옥시에틸렌(polyoxyethylene, POE), 폴리비닐 알콜(polyvinyl alcohol), 폴리우레탄(polyurethane) 및 폴리알킬렌 옥사이드(polyalkylene oxide, PAO) 등의 수용성 고분자 및 덱스트란(dextran), 폴리아크릴 아마이드(polyacryl amide) 등의 비면역성 고분자 등이 사용될 수 있다.The polymer is a natural or synthetic water-soluble substance, the kind is polyethylene glycol (PEG), polylactic acid (polylatic acid), polypropylene glycol (polypropylene glycol), polyoxyethylene (polyoxyethylene, POE), polyvinyl Water-soluble polymers such as polyvinyl alcohol, polyurethane and polyalkylene oxide (PAO), and non-immune polymers such as dextran and polyacryl amide may be used. .

선형고분자의 분자량은 클수록 고분자 접합체의 약동력학은 개선되나 활성은 감소하게 된다. 가지 달린 고분자의 경우에는 이러한 활성의 감소는 완화시킬 수 있으나 현재 선형고분자의 합성이 분자량 20,000 정도로 한계가 되어있기 때문에 본 발명에서는 가지 달린 고분자 유도체의 합성에 사용되는 선형고분자의 분자량은 1,000∼20,000이며, 바림직하게는 3,000∼20,000이다.The higher the molecular weight of the linear polymer, the better the pharmacokinetics of the polymer conjugate but the lower the activity. In the case of branched polymers, such a decrease in activity can be alleviated, but since the synthesis of linear polymers is currently limited to a molecular weight of 20,000, the molecular weight of linear polymers used in the synthesis of branched polymer derivatives is 1,000 to 20,000. Preferably 3,000 to 20,000.

또한, 상기 화학식 1에서의 활성기를 나타내는 Z는 단백질 또는 펩타이드와 결합하는 연결쇄의 역할을 하여 고반응성의 고분자 유도체를 제공하는 것으로서, 바람직하게는 COONHS 또는 CONHCH2CH2(OCH2CH2)n4OCOONHS이며, 이 때 n4는 50∼500의 정수이며, 바람직하게는 50∼250의 정수이다. 활성화된 가지 달린 고분자 유도체의 연결쇄로 사용되는 고분자는 분자량이 바람직하게는 1,000∼10,000을 사용한다.In addition, Z representing the active group in Formula 1 serves as a linking chain to bind a protein or a peptide to provide a highly reactive polymer derivative, preferably COONHS or CONHCH 2 CH 2 (OCH 2 CH 2 ) n 4 OCOONHS, n4 is an integer of 50-500, Preferably it is an integer of 50-250. The polymer used as the linking chain of the activated branched polymer derivative has a molecular weight of preferably 1,000 to 10,000.

본 발명은 상기 화학식 1의 가지 달린 고분자 유도체의 제조방법을 포함한다.The present invention includes a method for producing a branched polymer derivative of Chemical Formula 1.

이하 본 발명에서 사용된 고분자로 모노메톡시 폴리에틸렌글리콜(monomethoxy polyethylene glycol, 이하 "mPEG"라 칭한다.)를 사용하였으며, N-하이드록시석신이미드 에스테르로 치환시켜 활성화시키는 반응식을하기 반응식 1과 반응식 2에 나타내었다.Monomethoxy polyethylene glycol (hereinafter referred to as "mPEG") was used as the polymer used in the present invention, and a reaction scheme for activating by substituting with N-hydroxysuccinimide ester was shown in Scheme 1 and Scheme 2 is shown.

상기 반응식 1은 3 개의 가지를 갖는 활성화된 고분자 유도체(이하 "Tri-PEG"라 칭한다.)의 합성과정을 단계적으로 나타낸 것으로, 상기 화학식 1의 가지 달린 고분자 유도체를 합성하기 위해서는 생체적합성 고분자가 활성화된 형태를 갖추도록 하여야 한다. 이를 위해서 고분자 유도체의 말단 작용기는 N-하이드록시석신이미드 에스테르(N-hydroxysuccinimide ester, NHS)로 치환시켜 활성화시켰다. 또한, 단위 PEG를 Lys-Lys-HCl을 사용하여 3개의 가지를 갖는 고분자 유도체를 제조한다.Scheme 1 shows a step-by-step synthesis process of the activated polymer derivative having three branches (hereinafter referred to as "Tri-PEG"), the biocompatible polymer is activated to synthesize the branched polymer derivative of Formula 1 It should be in the form of To this end, the terminal functional group of the polymer derivative was activated by replacing with N-hydroxysuccinimide ester (NHS). In addition, a unit PEG is used to prepare a polymer derivative having three branches using Lys-Lys-HCl.

상기 반응식 2는 활성화된 4개의 가지를 갖는 활성화된 고분자 유도체(이하"Tetra-PEG"라 칭한다.)를 합성하는 과정을 나타낸 것으로서, 활성화된 Tri-PEG의 Lys-Lys 말단에 긴 사슬의 활성화된 생체적합성 고분자를 단백질 또는 펩타이드에 대한 연결쇄로 사용하기 위해 제조한다.Reaction Scheme 2 shows a process of synthesizing an activated polymer derivative having four activated branches (hereinafter referred to as "Tetra-PEG"), wherein the long-chain activated at the Lys-Lys end of the activated Tri-PEG is activated. Biocompatible polymers are prepared for use as linking chains for proteins or peptides.

또한, 본 발명은 다가지 고분자 유도체를 단백질 또는 펩타이드와 결합시켜 제조되는 단백질-고분자 또는 펩타이드-고분자 접합체를 포함한다.The present invention also encompasses protein-polymers or peptide-polymer conjugates prepared by combining various polymer derivatives with proteins or peptides.

단백질 또는 펩타이드와 고분자의 결합에서 펩타이드 내 결합부위는 라이신의 아민기나 N-말단부위이며, 상기 단백질 또는 펩타이드는 인터페론-알파, 인터페론-베타, 인터페론-감마, 염기성 섬유아세포 성장 인자 또는 호종구 생성 자극 인자를 사용한다.In the binding of a protein or peptide to a polymer, the binding site in the peptide is an amine group or an N-terminal site of lysine, and the protein or peptide is an interferon-alpha, interferon-beta, interferon-gamma, basic fibroblast growth factor or neutrophil production stimulation. Use arguments.

상기 단백질 또는 펩타이드와 활성화된 고분자 유도체의 반응 몰비는 고분자 유도체:단백질 또는 펩타이드에 대해 1:1에서 1:50이며, 바람직하게는 1:5내지 1:20이다.The reaction molar ratio of the protein or peptide to the activated polymer derivative is from 1: 1 to 1:50, preferably from 1: 5 to 1:20, relative to the polymer derivative: protein or peptide.

본 발명의 고반응성 고분자 유도체에 단백질이나 펩타이드와의 접합체는 단백질이나 펩타이드 본래의 활성을 최대한 유지하면서 또한 체내로부터의 소실 및 분해를 더욱 감소시킨다. 일 예로 가지 달린 고분자 유도체-인터페론 접합체의 경우, T1/2가 인터페론보다 Tetra-PEG가 약 12배 정도 증가하였음을 알 수 있으며 mPEG 보다 약 3.5 배 증가함을 알 수 있다. 또한 생물학적 활성은 mPEG와 비교하여 약 4배 정도 감소하였다.Conjugates with proteins or peptides in the highly reactive polymer derivatives of the present invention maintain the inherent activity of the protein or peptide to the maximum and further reduce loss and degradation from the body. As an example, in the case of the branched polymer derivative-interferon conjugate, it can be seen that T 1/2 increased Tetra-PEG about 12 times than interferon and about 3.5 times increased than mPEG. In addition, biological activity was reduced by about four times compared to mPEG.

이하 본 발명의 실시예를 다음에 의하여 설명한다.An embodiment of the present invention will be described below.

그러나 본 발명이 실시예에 의해 한정되는 것은 아니다.However, the present invention is not limited by the examples.

<실시예 1> 분자량이 10,000인 활성화된 mPEG-OCHExample 1 Activated mPEG-OCH with a Molecular Weight of 10,000 22 COONHS의 합성Synthesis of COONHS

(단계 1) 분자량이 10,000인 mPEG-OCH2COOH의 합성(Step 1) Synthesis of mPEG-OCH 2 COOH having a molecular weight of 10,000

분자량이 10,000인 PEG를 이용하여 mPEG를 제조하고, 분자량 10,000인 mPEG-OH(20g, 2mM)를 질소기체하에서 THF에 녹인 후 실온에서 교반하면서 나트륨 및 나프탈렌 용액을 가하고 3시간 동안 실온에서 교반하였다. 브로모에틸렌아세테이트(bromoethylacetate)(1.0g, 6mM)를 실온에서 교반하면서 적하시키는 방법(dropwise)으로 넣어 주었다. 실온에서 15시간 이상 반응시킨 후 냉각조(ice bath)에서 아이스 에테르(ice ether)를 가해 응고시켰다. 생성된 고형분을 여과(solid filter)하여 회수한 후 이를 에테르로 세척하고, 진공 건조하였다. 그 후 상기 고형분을 물에 녹인 후 1N 소디움 하이드록사이드(NaOH)를 가해 pH를 11로 맞추고 55 ℃에서 24시간 동안 교반한 후 실온으로 식히고 HCl을 가해 pH를 3으로 조정한 다음 증발시켰다. 염화메틸렌(methylene chloride, 이하 "MC"라 칭한다.)에 녹이고 실온에 1시간 방치한 후 셀라이트 여과(celite filter)하여 증발시켰다. 그 다음 이소프로필 알코올(isopropyl alcohol, 이하 "IPA"라 칭한다.)을 넣어 녹인 후 냉각조하에서 결정화시켰다. 결정화되어 얻어진 연갈색 고형분을 여과하고 에테르로 세척한 후 진공 건조하였다.MPEG was prepared using PEG with a molecular weight of 10,000, and mPEG-OH (20 g, 2 mM) having a molecular weight of 10,000 was dissolved in THF under nitrogen gas, and sodium and naphthalene solutions were added with stirring at room temperature, followed by stirring at room temperature for 3 hours. Bromoethylacetate (bromoethylacetate) (1.0 g, 6 mM) was added dropwise while stirring at room temperature. After reacting at room temperature for 15 hours or more, ice ether was added and coagulated in an ice bath. The resulting solid was collected by filtration (solid filter), which was then washed with ether and dried in vacuo. Then, the solid was dissolved in water, 1N sodium hydroxide (NaOH) was added to adjust the pH to 11, stirred at 55 ° C. for 24 hours, cooled to room temperature, HCl was added to adjust pH to 3, and evaporated. It was dissolved in methylene chloride (hereinafter referred to as "MC"), left at room temperature for 1 hour, and evaporated by celite filtration. Then, isopropyl alcohol (hereinafter referred to as "IPA") was added thereto to dissolve and crystallized under a cooling bath. The light brown solid obtained by crystallization was filtered, washed with ether and dried in vacuo.

(단계 2) 분자량 10,000인 mPEG-OCH2COONHS의 합성(Step 2) Synthesis of mPEG-OCH2COONHS having a molecular weight of 10,000

단계 1에서 얻어진 분자량 10,000인 mPEG-OCH2COOH(6g, 0.6mM)를 MC에 녹인 후 교반하면서 NHS(0.2g, 1.8mM)와 N,N-디시클로헥실 카보디이미드(N,N-dicyclohexyl carbodiimide, DCC)(0.37g, 1.8mM)를 넣어 주었다. 30 ℃로 가열하면서 교반하여 18시간 동안 반응시킨 후 실온으로 식혀, 셀라이트 여과한 다음 활성탄소(charcoal)로 여과하여 증발시켰다. IPA를 넣어 녹인 후 냉각조하에서 결정화하였으며, 그 결과 얻은 백색의 고형분을 여과하고 에테르로 세척한 다음 진공 건조하였다.MPEG-OCH2COOH (6 g, 0.6 mM) having a molecular weight of 10,000 obtained in step 1 was dissolved in MC, followed by stirring with NHS (0.2 g, 1.8 mM) and N, N-dicyclohexyl carbodiimide (N, N-dicyclohexyl carbodiimide, DCC) (0.37g, 1.8mM) was added. The mixture was stirred with heating to 30 ° C., reacted for 18 hours, cooled to room temperature, filtered through Celite, and then filtered through activated carbon to evaporate. IPA was added to dissolve and crystallized in a cooling bath. The resulting white solid was filtered, washed with ether and dried in vacuo.

<실시예 2> 분자량 20,000인 활성화된 mPEG-OCHExample 2 Activated mPEG-OCH with Molecular Weight 20,000 22 COONHS의 합성Synthesis of COONHS

(단계 1) 분자량 20,000인 mPEG-OCH2COOH의 합성(Step 1) Synthesis of mPEG-OCH 2 COOH having a molecular weight of 20,000

분자량 20,000인 mPEG-OH(10g, 0.5mM)를 실시예 1의 단계 1과 동일하게 반응시켜 분자량 20,000인 mPEG-OCH2COOH에 대한 연갈색 고형분을 얻었다.MPEG-OH (10 g, 0.5 mM) having a molecular weight of 20,000 was reacted in the same manner as in Step 1 of Example 1 to obtain a light brown solid content for mPEG-OCH 2 COOH having a molecular weight of 20,000.

(단계 2) 분자량 20,000인 mPEG-OCH2COONHS의 합성(Step 2) Synthesis of mPEG-OCH 2 COONHS having a molecular weight of 20,000

분자량 20,000인 mPEG-OH(5g, 0.25mM)를 실시예 1의 단계 2와 동일하게 반응시켜 분자량 20,000인 mPEG-OCH2COONHS에 대한 백색 고형분을 얻었다.MPEG-OH (5 g, 0.25 mM) having a molecular weight of 20,000 was reacted in the same manner as in Step 2 of Example 1 to obtain a white solid for mPEG-OCH 2 COONHS having a molecular weight of 20,000.

<실시예 3> 분자량 30,000인 활성화된 가지 달린 고분자 유도체 Tri-PEG-NHS의 합성Example 3 Synthesis of Activated Branched Polymer Derivative Tri-PEG-NHS with Molecular Weight 30,000

(단계 1) 분자량 30,000인 Tri-PEG-COOH의 합성(Step 1) Synthesis of Tri-PEG-COOH having a Molecular Weight of 30,000

라이신-라이신-HCl(Lys-Lys-HCl)(0.32g, 0.0106mM)을 붕산염 완충용액(0.1M pH 8.5)에 녹인 후 교반하면서 분자량 10,000인 mPEG-OCH2COONHS(1.6g, 0.16mM)을 적하하여 넣어 주었다. 2일 동안 실온에서 반응시킨 후 물 30 mL을 넣고 옥살산을 가하여 pH 3으로 조정하였다. MC로 3회 추출한 뒤 MC 분획층에 Na2SO4를 가하여 건조시킨 후 여과하여 증발시켰다. 여과액을 3mL로 농축한 후 차가운 에틸에테르 20mL을 넣어 침전시킨다. 위 반응의 침전물 0.7g을 증류수(distilled water, DW)에 녹이고 DEAE Sepharose FF에 주입한다. DEAE Sepharose FF column은 500mL의 젤(gel)로 충진되어 있고, pH7의 0,5% 붕산(boric acid), 소디움 하이드록사이드(sodium hydroxide) 완충액 1500mL로 평형화하였다. 평형화가 끝난 후 물로 세척하여 사용하였다. mPEG-Lys-Lys, mPEG2-Lys-Lys 그리고 mPEG의 불순물은 컬럼을 물로 세척할 때 제거되는데 반해, mPEG3-COOH는 10mM 염화나트륨(NaCl)에서 용출되었다. 용출액에 옥살산을 가하여 pH 3으로 조정한 후 MC로 3회 추출한 뒤 MC 분획층에 Na2SO4를 가하여 건조시킨 후 여과하여 증발시켰다. IPA를 넣고 녹인 후 냉각조하에서 결정화하여 백색의 고형분을 얻어 여과하고 에테르로 세척한 다음 진공 건조하여 백색 고형분(Tri-PEG-COOH)을 얻었다. 상기 백색 고형분은 하기 화학식 2과 같은 구조식을 갖는다.(화학식 2에서 mPEG는 분자량이 10,000이다.)Lys-Lys-HCl (Lys-Lys-HCl) (0.32g, 0.0106mM) was dissolved in borate buffer solution (0.1M pH 8.5) and stirred with mPEG-OCH 2 COONHS (1.6g, 0.16mM). Dropped me in. After reacting at room temperature for 2 days, 30 mL of water was added thereto and adjusted to pH 3 by adding oxalic acid. After extraction three times with MC, Na 2 SO 4 was added to the MC fraction layer, dried and filtered and evaporated. The filtrate was concentrated to 3 mL and precipitated with 20 mL of cold ethyl ether. 0.7 g of the precipitate of the above reaction is dissolved in distilled water (DW) and injected into DEAE Sepharose FF. The DEAE Sepharose FF column was filled with 500 mL of gel and equilibrated with 1500 mL of sodium hydroxide buffer at pH 7, 0,5% boric acid. After equilibration was used to wash with water. Impurities in mPEG-Lys-Lys, mPEG2-Lys-Lys and mPEG were removed when the column was washed with water, whereas mPEG3-COOH was eluted in 10 mM sodium chloride (NaCl). Oxalic acid was added to the eluate, adjusted to pH 3, extracted three times with MC, dried over Na 2 SO 4 , and filtered and evaporated. IPA was added and dissolved, and then crystallized under a cooling bath to obtain a white solid, which was filtered, washed with ether and dried in vacuo to give a white solid (Tri-PEG-COOH). The white solid has a structure represented by the following Chemical Formula 2. (In Chemical Formula 2, mPEG has a molecular weight of 10,000.)

(단계 2) 분자량 30,000인 Tri-PEG-NHS의 합성(Step 2) Synthesis of Tri-PEG-NHS with Molecular Weight 30,000

상기 단계 1에서 얻은 분자량 30,000 Tri-PEG-COOH(1.2g, 0.043mM)를 NHS(0.02g, 0.174mM) 및 DCC(0.036g, 0.174mM)와 혼합하여 실시예 1의 단계 2와 동일한 방법으로 반응시켜 백색 고형분을 얻었다. 상기 백색 고형분(Tri-PEG-NHS)은 하기 화학식 3과 같은 구조식을 갖는다(화학식 3에서 mPEG는 분자량이 10,000이다.).The molecular weight of 30,000 Tri-PEG-COOH (1.2g, 0.043mM) obtained in step 1 was mixed with NHS (0.02g, 0.174mM) and DCC (0.036g, 0.174mM) in the same manner as in Step 2 of Example 1 It reacted and obtained white solid content. The white solid (Tri-PEG-NHS) has a structural formula (Formula 3 mPEG has a molecular weight of 10,000).

<실시예 4> 분자량 60,000인 활성화된 가지 달린 고분자 유도체 Tri-PEG-NHS의합성Example 4 Synthesis of Activated Branched Polymer Derivative Tri-PEG-NHS with Molecular Weight 60,000

(단계 1) 분자량 60,000인 Tri-PEG-COOH의 합성(Step 1) Synthesis of Tri-PEG-COOH having a Molecular Weight of 60,000

분자량 20,000인 mPEG-OCH2COONHS(1.6g, 0.08mM)를 실시예 3의 단계 1과 동일하게 반응시켜 백색 고형분을 얻었다. 결과 백색 고형분은 Tri-PEG-COOH로서 상기 화학식 2의 구조를 갖는 화합물이다(상기 화학식 2에서 mPEG의 분자량은 20,000이다.).MPEG-OCH2COONHS (1.6 g, 0.08 mM) having a molecular weight of 20,000 was reacted in the same manner as in Step 1 of Example 3, to obtain a white solid. The resulting white solid is Tri-PEG-COOH, a compound having the structure of Formula 2 (mPEG has a molecular weight of 20,000 in Formula 2).

(단계 2) 분자량 60,000인 Tri-PEG-NHS의 합성(Step 2) Synthesis of Tri-PEG-NHS having a molecular weight of 60,000

분자량 60,000인 Tri-PEG-COOH(1.2g, 0.02mM)을 실시예 3의 단계 2와 동일하게 반응시켜 백색 고형분을 얻었다. 결과 백색 고형분은 Tri-PEG-NHS로서 상기 화학식 3의 구조를 갖는 화합물이다(상기 화학식 2에서 mPEG의 분자량은 20,000이다.).Tri-PEG-COOH (1.2 g, 0.02 mM) having a molecular weight of 60,000 was reacted in the same manner as in Step 2 of Example 3, to obtain a white solid. The resulting white solid is Tri-PEG-NHS, a compound having the structure of Formula 3 (mPEG has a molecular weight of 20,000 in Formula 2).

<실시예 5> 긴 사슬의 연결쇄를 결합시킨 분자량 33,400인 활성화된 가지 달린 고분자 유도체 Tetra-PEG-NHS의 합성Example 5 Synthesis of Activated Branched Polymer Derivative Tetra-PEG-NHS with Molecular Weight 33,400 Linked with Long Chain Linked Chains

(단계 1) 분자량 33,400인 Tetra-PEG-COOH의 합성(Step 1) Synthesis of Tetra-PEG-COOH having a Molecular Weight of 33,400

실시예 3에서 얻은 분자량 30,000인 Tri-PEG-NHS(0.4g, 0.0133mM)를 MC에 녹이고 실온에서 교반하면서 분자량 3,400인 NH2PEG-COOH(0.048g, 0.014mM)를 첨가하였다. 40 ℃에서 2일 동안 반응시킨 후 셀라이트로 여과하여 증발시켰다. IPA를 넣고 녹인 후 냉각조하에서 결정화시켜 백색의 고형분을 얻었으며 이를 여과하여에테르로 세척한 후 진공건조하였다. 이렇게 얻어진 백색 고형분(Tetra-PEG-COOH)은 하기 화학식 4와 같은 구조를 가진다(화학식 4에서 mPEG는 분자량이 10,000이다.).Tri-PEG-NHS (0.4 g, 0.0133 mM) having a molecular weight of 30,000 obtained in Example 3 was dissolved in MC and NH 2 PEG-COOH (0.048 g, 0.014 mM) having a molecular weight of 3,400 was added while stirring at room temperature. After reacting at 40 ° C. for 2 days, the mixture was filtered through celite and evaporated. IPA was added to dissolve and crystallized in a cooling bath to obtain a white solid, which was filtered, washed with ether and dried in vacuo. The white solid thus obtained (Tetra-PEG-COOH) has a structure as shown in the following formula (4) (In formula 4 mPEG has a molecular weight of 10,000).

(단계 2) 분자량 33,400인 Tetra-PEG-NHS의 합성(Step 2) Synthesis of Tetra-PEG-NHS having a molecular weight of 33,400

단계 1에서 얻은 Tetra-PEG-COOH(0.4g, 0.012mM)를 NHS(0.0048g, 0.042mM) 및 DCC(0.0086g, 0.042mM)와 혼합하여 실시예 3의 단계 2와 동일한 방법으로 반응시켜 백색 고형분을 얻었다. 이 백색 고형분(Tetra-PEG-NHS)은 하기 화학식 5와 같은 구조를 가진다(화학식 5에서 mPEG는 분자량이 10,000이다.).Tetra-PEG-COOH (0.4 g, 0.012 mM) obtained in step 1 was mixed with NHS (0.0048 g, 0.042 mM) and DCC (0.0086 g, 0.042 mM) in the same manner as in step 2 of Example 3 to give a white Solid content was obtained. This white solid (Tetra-PEG-NHS) has a structure as shown in Formula 5 below (mPEG has a molecular weight of 10,000 in Formula 5).

<실시예 6> 긴 사슬의 연결쇄를 결합시킨 분자량 35,000인 활성화된 가지 달린고분자 유도체 Tetra-PEG-NHS의 합성Example 6 Synthesis of Activated Branched Polymer Derivative Tetra-PEG-NHS with Molecular Weight 35,000 Linked with Long Chain Linked Chains

실시예 3에서 얻은 분자량 30,000인 Tri-PEG-NHS(0.4g, 0.0133mM)과 분자량 5,000인 NH2PEG-COOH(0.07g, 0.014mM)를 실시예 5에서와 같이 반응하여 분자량 35,000의 tetra-PEG-NHS를 얻었다.Tri-PEG-NHS (0.4 g, 0.0133 mM) having a molecular weight of 30,000 obtained in Example 3 and NH 2 PEG-COOH (0.07 g, 0.014 mM) having a molecular weight of 5,000 were reacted as in Example 5 to produce tetra- having a molecular weight of 35,000. PEG-NHS was obtained.

<실시예 7> 긴 사슬의 연결쇄를 결합시킨 분자량 40,000인 활성화된 가지 달린 고분자 유도체 Tetra-PEG-NHS의 합성Example 7 Synthesis of Tetra-PEG-NHS Activated Branched Polymer Derivatives with a Molecular Weight of 40,000

실시예 3에서 얻은 분자량 30,000인 Tri-PEG-NHS(0.4g, 0.0133mM)과 분자량 10,000인 NH2PEG-COOH(0.14g, 0.014mM)를 실시예 5에서와 같이 반응하여 분자량 40,000의 tetra-PEG-NHS를 얻었다.Tri-PEG-NHS (0.4 g, 0.0133 mM) having a molecular weight of 30,000 obtained in Example 3 and NH 2 PEG-COOH (0.14 g, 0.014 mM) having a molecular weight of 10,000 were reacted as in Example 5 to give tetra- having a molecular weight of 40,000. PEG-NHS was obtained.

<실시예 8> 긴 사슬의 연결쇄를 결합시킨 분자량 63,400인 활성화된 가지 달린 고분자 유도체 Tetra-PEG-NHS의 합성Example 8 Synthesis of Tetra-PEG-NHS with Activated Branched Polymer Derivatives of Molecular Weight 63,400 Linked with Long Chain Linkages

(단계 1) 분자량 63,400인 Tetra-PEG-COOH의 합성(Step 1) Synthesis of Tetra-PEG-COOH having a Molecular Weight of 63,400

실시예 4에서 얻은 분자량 60,000인 Tri-PEG-NHS(0.6g, 0.01mM)를 실시예 5의 단계 1과 동일하게 반응시켜 상기 화학식 4의 구조을 갖는 백색 고형분, Tetra-PEG-COOH를 얻었다(상기 화학식 4에서 mPEG의 분자량은 20,000이다.).Tri-PEG-NHS having a molecular weight of 60,000 obtained in Example 4 (0.6 g, 0.01 mM) was reacted in the same manner as in Step 1 of Example 5 to obtain a white solid having a structure of Formula 4, Tetra-PEG-COOH (The MPEG has a molecular weight of 20,000.

(단계 2) T분자량 63,400인 Tetra-PEG-NHS의 합성(Step 2) Synthesis of Tetra-PEG-NHS with T molecular weight 63,400

분자량 63,400인 Tetra-PEG-COOH(0.45g, 0.0071mM)을 실시예 5의 단계 2와 동일하게 반응시켜 백색 고형분을 얻었다. 결과 백색 고형분은 Tetra-PEG-NHS로서 상기 화학식 5의 구조를 갖는 화합물이다(상기 화학식 5에서 mPEG의 분자량은 20,000이다.).Tetra-PEG-COOH (0.45 g, 0.0071 mM) having a molecular weight of 63,400 was reacted in the same manner as in Step 2 of Example 5, to obtain a white solid. The resulting white solid is Tetra-PEG-NHS, a compound having the structure of Formula 5 (mPEG has a molecular weight of 20,000 in Formula 5).

<실시예 9> 긴 사슬의 연결쇄를 결합시킨 분자량 65,000인 활성화된 가지 달린 고분자 유도체 Tetra-PEG-NHS의 합성Example 9 Synthesis of Activated Branched Polymer Derivative Tetra-PEG-NHS with Molecular Weight 65,000 Linked with Long Chain Linkage

실시예 4에서 얻은 분자량 60,000인 Tri-PEG-NHS(0.6g, 0.01mM)과 분자량 5,000인 NH2PEG-COOH(0.05g, 0.01mM)를 실시예 8에서와 같이 반응하여 분자량 65,000의 tetra-PEG-NHS를 얻었다.Tri-PEG-NHS (0.6 g, 0.01 mM) having a molecular weight of 60,000 obtained in Example 4 and NH 2 PEG-COOH (0.05 g, 0.01 mM) having a molecular weight of 5,000 were reacted as in Example 8 to give tetra- PEG-NHS was obtained.

<실시예 10> 긴 사슬의 연결쇄를 결합시킨 분자량 70,000인 활성화된 가지 달린 고분자 유도체 Tetra-PEG-NHS의 합성Example 10 Synthesis of Tetra-PEG-NHS Activated Branched Polymer Derivatives with a Molecular Weight of 70,000 by Linking Long Chain Linkages

실시예 4에서 얻은 분자량 60,000인 Tri-PEG-NHS(0.6g, 0.01mM)과 분자량 10,000인 NH2PEG-COOH(0.1g, 0.01mM)를 실시예 8에서와 같이 반응하여 분자량 70,000의 tetra-PEG-NHS를 얻었다.Tri-PEG-NHS (0.6 g, 0.01 mM) having a molecular weight of 60,000 obtained in Example 4 and NH 2 PEG-COOH (0.1 g, 0.01 mM) having a molecular weight of 10,000 were reacted as in Example 8 to obtain tetra- having a molecular weight of 70,000. PEG-NHS was obtained.

<실시예 11> 분자량 30,000인 Tri-PEG-인터페론 제조<Example 11> Tri-PEG-interferon having a molecular weight of 30,000

10mg의 인터페론(Interferon)을 pH8의 100mM 바이신(bicine) 완충액 으로 투석한 후, 실시예 3에서 제조된 분자량 30,000인 Tri-PEG-NHS 160mg을 넣어 실온에서 2시간 동안 교반하면서 반응시킨 후 0.1M 글리신(glycine)을 첨가하여 반응을 중단시켰다. pH4의 40mM 인산나트륨(NaH2PO4) 완충액으로 평형화 된 HiprepTM 26/10(Amersham Pharmacia Biotech)과 같은 De-salting column 에 반응액을 주입한 후 같은 완충액으로써 용출시켜 완충액을 교환하였다. 이때 반응시에 Tri-PEG-NHS에서 분리된 NHS가 제거된다. 용출액을 pH4의 40mM 인산나트륨(NaH2PO4) 완충액으로 평형화된 SP-세파로제(SP-sepharose) 음이온 교환수지(anion exchange chromatography, Amersham Pharmacia Biotech)에 주입한 후 PEG-인터페론을 분리하였다. 분리 과정에서 사용된 염화나트륨(NaCl)은 0∼300 mM의 농도구배를 주어 분획하였다. 분획된 용출액은 SDS-PAGE를 이용하여 크기와 형태를 확인한 후 인터페론에 Tri-PEG가 2개 이상 결합된 형태의 접합체와 반응하지 않은 인터페론을 배제하고 Tri-PEG가 하나만 접합된 인터페론만을 분리하였다. 반응 후 또는 분리된 Tri-PEG-인터페론은 SDS-PAGE, 크기배제 HPLC(size-exclusion HPLC) 또는 MALDI-TOF 질량 분석기(MALDI-TOF mass spectrometer)로 확인하였다.10 mg of Interferon was dialyzed with 100 mM bisine buffer at pH 8, and then 160 mg of Tri-PEG-NHS having a molecular weight of 30,000 prepared in Example 3 was added and reacted with stirring at room temperature for 2 hours. The reaction was stopped by adding glycine. The reaction solution was injected into a De-salting column such as HiprepTM 26/10 (Amersham Pharmacia Biotech) equilibrated with 40 mM sodium phosphate (NaH 2 PO 4 ) buffer at pH 4 and eluted with the same buffer to exchange buffers. At this time, NHS separated from Tri-PEG-NHS is removed. The eluate was injected into SP-sepharose anion exchange chromatography (Amersham Pharmacia Biotech) equilibrated with 40 mM sodium phosphate (NaH 2 PO 4 ) buffer at pH 4 and then PEG-interferon was isolated. Sodium chloride (NaCl) used in the separation process was fractionated by giving a concentration gradient of 0 ~ 300 mM. After confirming the size and shape of the fractionated eluate using SDS-PAGE, the interferon did not react with the conjugate of the form in which two or more tri-PEGs were bound to the interferon, and only the interferon in which only one tri-PEG was conjugated was separated. After reaction or isolated Tri-PEG-interferon was confirmed by SDS-PAGE, size-exclusion HPLC or MALDI-TOF mass spectrometer.

<실시예 12> 분자량 60,000인 Tri-PEG-인터페론 제조<Example 12> Tri-PEG-interferon having a molecular weight of 60,000

10mg의 인터페론(Interferon)을 pH8의 100mM 바이신(bicine) 완충액 으로 투석한 후, 실시예 4에서 제조된 Tri-PEG-NHS(60,000) 320mg을 넣어 실온에서 2시간 동안 교반하면서 반응시켰다. 그 다음은 실시예 11의 방법과 동일하게 하여 Tri-PEG(60,000)-인터페론을 얻었다.10 mg of Interferon was dialyzed with 100 mM bisine buffer at pH 8, and then, 320 mg of Tri-PEG-NHS (60,000) prepared in Example 4 was added and reacted with stirring at room temperature for 2 hours. Next, Tri-PEG (60,000) -interferon was obtained in the same manner as in Example 11.

<실시예 13> 분자량 33,400인 Tetra-PEG-인터페론 제조Example 13 Preparation of Tetra-PEG-Interferon with a Molecular Weight 33,400

10mg의 인터페론(Interferon)을 pH 8의 100mM 바이신(bicine) 완충액으로 투석한 후, 실시예 5에서 제조된 분자량 33,400인 Tetra-PEG-NHS 180mg을 넣어 실온에서 2시간 동안 교반하면서 반응시켰다. 그 다음은 실시예 11의 방법과 동일하게 하여 분자량 33,400인 Tetra-PEG-인터페론을 얻었다.10 mg of Interferon was dialyzed with 100 mM bisine buffer at pH 8, and then 180 mg of Tetra-PEG-NHS having a molecular weight of 33,400 prepared in Example 5 was added and reacted with stirring at room temperature for 2 hours. Then, in the same manner as in Example 11, Tetra-PEG-interferon having a molecular weight of 33,400 was obtained.

<실시예 14> 분자량 35,000인 Tetra-PEG-인터페론 제조Example 14 Tetra-PEG-interferon having a molecular weight of 35,000

10mg의 인터페론(Interferon)을 pH 8의 100mM 바이신(bicine) 완충액으로 투석한 후, 실시예 6에서 제조된 분자량 35,000인 Tetra-PEG-NHS 200mg을 넣어 실온에서 2시간 동안 교반하면서 반응시켰다. 그 다음은 실시예 11의 방법과 동일하게 하여 분자량 35,000인 Tetra-PEG-인터페론을 얻었다.10 mg of Interferon was dialyzed with 100 mM bisine buffer at pH 8, and then 200 mg of Tetra-PEG-NHS having a molecular weight of 35,000 prepared in Example 6 was added and reacted with stirring at room temperature for 2 hours. Then, in the same manner as in Example 11, Tetra-PEG-interferon having a molecular weight of 35,000 was obtained.

<실시예 15> 분자량 40,000인 Tetra-PEG-인터페론 제조Example 15 Preparation of Tetra-PEG-Interferon with a Molecular Weight of 40,000

10mg의 인터페론(Interferon)을 pH 8의 100mM 바이신(bicine) 완충액으로 투석한 후, 실시예 5에서 제조된 분자량 40,000인 Tetra-PEG-NHS 240mg을 넣어 실온에서 2시간 동안 교반하면서 반응시켰다. 그 다음은 실시예 11의 방법과 동일하게 하여 분자량 40,000인 Tetra-PEG-인터페론을 얻었다.10 mg of Interferon was dialyzed with 100 mM bisine buffer at pH 8, and then 240 mg of Tetra-PEG-NHS having a molecular weight of 40,000 prepared in Example 5 was added and reacted with stirring at room temperature for 2 hours. Then, in the same manner as in Example 11, Tetra-PEG-interferon having a molecular weight of 40,000 was obtained.

<실시예 16> 분자량 63,400인 Tetra-PEG-인터페론 제조Example 16 Preparation of Tetra-PEG-Interferon with Molecular Weight 63,400

10mg의 인터페론(Interferon)을 pH8의 100mM 바이신(bicine) 완충액 으로 투석한 후, 실시예 8에서 제조된 분자량 63,400인 Tetra-PEG-NHS 360mg을 넣어 실온에서 2시간 동안 교반하면서 반응시켰다. 그 다음은 실시예 11의 방법과 동일하게 하여 분자량 63,400인 Tetra-PEG-인터페론을 얻었다.10 mg of Interferon (Interferon) was dialyzed with 100 mM bisine (pHM) buffer of pH8, and then, 360 mg of Tetra-PEG-NHS having a molecular weight of 63,400 prepared in Example 8 was added thereto, and the mixture was stirred at room temperature for 2 hours. Then, in the same manner as in Example 11, Tetra-PEG-interferon having a molecular weight of 63,400 was obtained.

<실시예 17> 분자량 65,000인 Tetra-PEG-인터페론 제조Example 17 Preparation of Tetra-PEG-Interferon with a Molecular Weight of 65,000

10mg의 인터페론(Interferon)을 pH8의 100mM 바이신(bicine) 완충액으로 투석한 후, 실시예 9에서 제조된 분자량 65,000인 Tetra-PEG-NHS 400mg을 넣어 실온에서 2시간 동안 교반하면서 반응시켰다. 그 다음은 실시예 11의 방법과 동일하게 하여 분자량 65,000인 Tetra-PEG-인터페론을 얻었다.10 mg of Interferon was dialyzed with 100 mM bisine buffer at pH 8, and then 400 mg of Tetra-PEG-NHS having a molecular weight of 65,000 prepared in Example 9 was added and reacted with stirring at room temperature for 2 hours. Then, in the same manner as in Example 11, Tetra-PEG-interferon having a molecular weight of 65,000 was obtained.

<실시예 18> 분자량 70,000인 Tetra-PEG-인터페론 제조<Example 18> Preparation of Tetra-PEG-interferon having a molecular weight of 70,000

10mg의 인터페론(Interferon)을 pH8의 100mM 바이신(bicine) 완충액으로 투석한 후, 실시예 10에서 제조된 분자량 70,000인 Tetra-PEG-NHS 450mg을 넣어 실온에서 2시간 동안 교반하면서 반응시켰다. 그 다음은 실시예 11의 방법과 동일하게 하여 분자량 70,000인 Tetra-PEG-인터페론을 얻었다.10 mg of Interferon (Interferon) was dialyzed with 100 mM bisine (pHM) buffer at pH 8, and then 450 mg of Tetra-PEG-NHS having a molecular weight of 70,000 prepared in Example 10 was added and reacted with stirring at room temperature for 2 hours. Then, in the same manner as in Example 11, Tetra-PEG-interferon having a molecular weight of 70,000 was obtained.

<실험예 1> 활성화된 가지 달린 고분자 유도체의 활성측정Experimental Example 1 Activity Measurement of Activated Branched Polymer Derivative

상기에 합성된 가지 달린 고분자 유도체들의 단백질이나 펩타이드에 대한 반응성을 조사하기 위하여, 이들을 실시예 11∼18에서와 같이 인터페론에 접합시켰다. 실시예에서와 같이 정제과정을 실시하여 분리한 PEG가 하나만 접합된 인터페론(mono-PEG-인터페론)을 얻었다.In order to investigate the reactivity to the protein or peptide of the branched polymer derivatives synthesized above, they were conjugated to interferon as in Examples 11 to 18. As in Example, purification was performed to obtain an interferon (mono-PEG-interferon) in which only one isolated PEG was conjugated.

상기에서 얻어진 모노-PEG-인터페론을 수포성 구내염 바이러스 및 마딘-다비 소 신장 세포(Marbin-Darby Bovine Kidney,MDBK)를 이용한 인터페론 cytophatic effect inhibition(CPE) 분석을 실시하여 생물학적 활성을 측정하였다.The mono-PEG-interferon obtained above was subjected to interferon cytophatic effect inhibition (CPE) analysis using bullous stomatitis virus and Marbin-Darby Bovine Kidney (MDBK) to measure biological activity.

<실험예 2> 활성화된 가지 달린 고분자 유도체의 약물역학Experimental Example 2 Pharmacokinetics of Activated Branched Polymer Derivatives

인터페론과 PEG-인터페론 접합체를 한 시료당 각 3마리의 랫트(Sprague Dawley)에 300백만IU씩 피하주사하였고, 관찰시점은 약물 투여 후 0분, 5분, 15분, 30분, 1시간, 2시간, 4시간, 8시간, 24시간, 2일, 3일, 4일, 5일, 6일 그리고 7일째로 하여 약물역학을 실험하였다. 검정은 인터페론 cytophatic effect inhibition(CPE) 분석으로 측정된 생물학적 활성값을 사용하였다.Interferon and PEG-interferon conjugates were injected subcutaneously at 300 million IU into each of three rats (Sprague Dawley) per sample, and the time of observation was 0, 5, 15, 30, 1 hour, 2 after drug administration. Pharmacodynamics were tested at time, 4 hours, 8 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days and 7 days. The assay used biological activity measured by interferon cytophatic effect inhibition (CPE) assay.

하기표 1에 인터페론과 PEG-인터페론 접합체에 대한 T1/2값을 얻었다. Table 1 below shows the T 1/2 values for the interferon and PEG-interferon conjugates.

PEG-인터페론 접합체의 생물학적 활성과 T1/2 Biological Activity and T 1/2 of PEG-Interferon Conjugates 고분자 유도체의 종류Type of polymer derivative 생물학적 활성Biological activity T1/2T1 / 2 인터페론Interferon 100100 3.73.7 mPEG(MW 10,000)mPEG (MW 10,000) 3838 15.415.4 Di-PEG(MW 20,000)Di-PEG (MW 20,000) 1212 23.323.3 Tri-PEG(MW 30,000)Tri-PEG (MW 30,000) 6.26.2 32.532.5 Tetra-PEG(MW 33,400)Tetra-PEG (MW 33,400) 10.310.3 36.536.5 Tetra-PEG(MW 35,000)Tetra-PEG (MW 35,000) 1111 3737 Tetra-PEG(MW 40,000)Tetra-PEG (MW 40,000) 10.510.5 40.240.2 mPEG(MW 20,000)mPEG (MW 20,000) 3232 18.218.2 Di-PEG(MW 40,000)Di-PEG (MW 40,000) 77 33.833.8 Tri-PEG(MW 60,000)Tri-PEG (MW 60,000) 33 42.142.1 Tetra-PEG(MW 63,400)Tetra-PEG (MW 63,400) 6.56.5 43.343.3 Tetra-PEG(MW 65,000)Tetra-PEG (MW 65,000) 6.26.2 43.543.5 Tetra-PEG(MW 70,000)Tetra-PEG (MW 70,000) 7.17.1 44.944.9

상술한 바와 같이 본 발명은 단백질 또는 펩타이드를 수식하는데 여러 개의 긴 사슬구조를 가진 분자량이 증가된 고분자를 사용함으로써 체내에서 분해와 소실을 감소시키는 고반응성 유도체를 합성하고, 이 고반응성 유도체에 단백질이나 펩타이드와 접합체를 형성할 수 있는 하나의 긴 사슬을 더 연결하여 더욱 개선된 고활성 유도체를 합성하는데 이용될 수 있다. 이러한 개선된 고활성 유도체를 이용한 단백질이나 펩타이드 접합체는 단백질이나 펩타이드 본래의 활성을 최대한 유지하면서 또한 체내로부터의 소실 및 분해를 더욱 감소시킴으로써 작용시간이 길어지는 치료효과의 개선을 증가시켜 투여되는 약물의 횟수를 줄이고 치료 효과도 개선시킬 수 있을 뿐만 아니라 잦은 투여로 인한 환자의 불편을 줄일 수 있다.As described above, the present invention synthesizes a highly reactive derivative that reduces degradation and loss in the body by using a polymer having an increased molecular weight having a plurality of long chain structures in modifying a protein or a peptide. One long chain that can form a conjugate with the peptide can further be used to synthesize further improved high activity derivatives. Proteins or peptide conjugates using these improved high activity derivatives maintain the intact activity of proteins or peptides as much as possible and further reduce the loss and degradation from the body, thereby increasing the improvement of the therapeutic effect of prolonged action time. Not only can it reduce the number of times, improve the effectiveness of treatment, but also reduce the discomfort of patients due to frequent administration.

Claims (12)

폴리에틸렌 글리콜 단위의 평균 분자량이 15,000∼80,000돌턴(Dalton)인 하기 화학식 1로 표시되는 생리적으로 고활성인 고분자 유도체.A physiologically highly active polymer derivative represented by the following Chemical Formula 1, wherein the polyethylene glycol unit has an average molecular weight of 15,000 to 80,000 Daltons. 화학식 1Formula 1 상기식에서,In the above formula, R1, R2및 R3는 서로 독립적이며, C1∼C6인 알킬기이고,R 1 , R 2 and R 3 are independent of each other, are C 1 to C 6 alkyl group, n1, n2 및 n3는 100∼500의 정수이며,n1, n2 and n3 are integers of 100 to 500, Z는 활성기를 나타낸다.Z represents an active group. 제 1항에 있어서, 상기 Z는 COONHS 또는 CONH-CH2CH2(OCH2CH2)n4OCOONHS며, 이 때, n4가 50∼500사이의 정수값인 것을 특징으로 하는 생리적으로 고활성인 고분자 유도체.The method of claim 1, wherein Z is COONHS or CONH-CH 2 CH 2 (OCH 2 CH 2 ) n 4 OCOONHS, wherein n 4 is an integer between 50 and 500 physiologically high activity polymer derivative. 제 1항에 있어서, n1, n2 및 n3가 200∼500의 정수값인 것을 특징으로 하는 생리적으로 고활성인 고분자 유도체.The physiologically highly active polymer derivative according to claim 1, wherein n1, n2 and n3 are integer values of 200 to 500. 제 2항에 있어서, n4가 50∼250의 정수값인 것을 특징으로 하는 생리적으로 고활성인 고분자 유도체.The physiologically highly active polymer derivative according to claim 2, wherein n4 is an integer of 50 to 250. 제 1항에 있어서, 단위 폴리에틸렌 글리콜이 라이신-라이신(Lys-Lys-HCl)에 의해 3개가 연결된 것을 특징으로 하는 생리적으로 고활성인 고분자 유도체.The physiologically highly active polymer derivative according to claim 1, wherein three unit polyethylene glycols are linked by lysine-lysine (Lys-Lys-HCl). 제 1항의 활성화된 생체적합성 고분자 유도체에 단백질 또는 펩타이드가 결합된 단백질-고분자 또는 펩타이드-고분자 접합체.A protein-polymer or peptide-polymer conjugate in which a protein or peptide is bound to an activated biocompatible polymer derivative of claim 1. 제 6항에 있어서, 상기 단백질 또는 펩타이드:고분자 유도체의 반응 몰비가 약 1:1에서 1:50인 것을 특징으로 하는 접합체.7. The conjugate of claim 6, wherein the molar ratio of the protein or peptide: polymer derivative is from about 1: 1 to 1:50. 제 6항에 있어서, 상기 단백질 또는 펩타이드:고분자 유도체의 반응 몰비가 약 1:5에서 1:20인 것을 특징으로 하는 접합체.7. The conjugate of claim 6, wherein the molar ratio of the protein or peptide: polymer derivative is about 1: 5 to 1:20. 제 6항에 있어서, 단백질 또는 펩타이드와 고분자의 결합에서 펩타이드 내 결합부위는 라이신의 아민기나 N-말단인 것을 특징으로 하는 접합체7. The conjugate according to claim 6, wherein the binding site in the peptide at the binding of the protein or peptide to the polymer is an amine group or N-terminus of lysine. 제 6항에 있어서, 단백질 또는 펩타이드는 인터페론 알파, 베타,감마(Interferon -α, -β or -γ), 염기성 섬유아세포 성장 인자(basic fibroblast growth factor, bFGF) 또는 호중구 생성 자극 인자(granulocyte colony stimulating factor, G-CSF)중 하나를 특징으로 하는 접합체.The method of claim 6, wherein the protein or peptide is selected from the group consisting of interferon alpha, beta, gamma (Interferon -α, -β or -γ), basic fibroblast growth factor (bFGF) or neutrophil production stimulating factor (granulocyte colony stimulating factor). factor, G-CSF). 제 6항에 있어서, 접합체는 인터페론 알파와 고분자 유도체의 접합체인 것을 특징으로 하는 접합체.7. The conjugate of claim 6, wherein the conjugate is a conjugate of interferon alpha and a polymer derivative. 제 11항에 의한 접합체 및 치료적으로 불활성인 담체를 포함하는 인터페론 감수성 질환 치료용 또는 예방용 약학적 조성물.A pharmaceutical composition for treating or preventing interferon-sensitive diseases comprising the conjugate according to claim 11 and a therapeutically inert carrier.
KR1020010051528A 2001-08-25 2001-08-25 multi-branched polymer used in conjugating protein or peptide, and resulting conjugator KR100761652B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020010051528A KR100761652B1 (en) 2001-08-25 2001-08-25 multi-branched polymer used in conjugating protein or peptide, and resulting conjugator

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR1020010051528A KR100761652B1 (en) 2001-08-25 2001-08-25 multi-branched polymer used in conjugating protein or peptide, and resulting conjugator

Publications (2)

Publication Number Publication Date
KR20030017908A true KR20030017908A (en) 2003-03-04
KR100761652B1 KR100761652B1 (en) 2007-10-04

Family

ID=27720856

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020010051528A KR100761652B1 (en) 2001-08-25 2001-08-25 multi-branched polymer used in conjugating protein or peptide, and resulting conjugator

Country Status (1)

Country Link
KR (1) KR100761652B1 (en)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5919455A (en) * 1993-10-27 1999-07-06 Enzon, Inc. Non-antigenic branched polymer conjugates
US5932462A (en) * 1995-01-10 1999-08-03 Shearwater Polymers, Inc. Multiarmed, monofunctional, polymer for coupling to molecules and surfaces
KR100396983B1 (en) * 2000-07-29 2003-09-02 이강춘 Highly reactive branched polymer and proteins or peptides conjugated with the polymer

Also Published As

Publication number Publication date
KR100761652B1 (en) 2007-10-04

Similar Documents

Publication Publication Date Title
US9364553B2 (en) Synergistic biomolecule-polymer conjugates
KR100396983B1 (en) Highly reactive branched polymer and proteins or peptides conjugated with the polymer
EP1039922B1 (en) Improved interferon polymer conjugates
EP0247860B1 (en) Tumor necrosis factor formulation and its preparation
US7201897B2 (en) Interferon conjugates
CA2227891C (en) Conjugation-stabilized therapeutic agent compositions, delivery and diagnostic formulations
EP1039921B1 (en) Substantially pure histidine-linked protein polymer conjugates
US8372422B2 (en) Hydroxyapatite-targeting poly(ethylene glycol) and related polymers
HUT75533A (en) Improved interferon polymer conjugates
CA2720306C (en) Double-stranded polyethylene glycol modified growth hormone, preparation method and application thereof
US20030103934A1 (en) Drugs having long-term retention in target tissue
US8629104B2 (en) G-CSF and water-soluble polymer conjugate
KR100761652B1 (en) multi-branched polymer used in conjugating protein or peptide, and resulting conjugator
KR20030076417A (en) Preparation of G-CSF stoichiometrically conjugated with biocompatible polymers at cystein residue
KR100888371B1 (en) Antivirus agent comprising bridged co-polymer derivatives and interferon complexes
KR100480432B1 (en) Conjugates of granulocyte-colony stimulating factor and polyethylene glycol derivatives
JPWO2003000278A1 (en) Ointment

Legal Events

Date Code Title Description
A201 Request for examination
E902 Notification of reason for refusal
E701 Decision to grant or registration of patent right
GRNT Written decision to grant
FPAY Annual fee payment

Payment date: 20121231

Year of fee payment: 7

FPAY Annual fee payment

Payment date: 20131226

Year of fee payment: 8

FPAY Annual fee payment

Payment date: 20141211

Year of fee payment: 9

FPAY Annual fee payment

Payment date: 20151230

Year of fee payment: 10

FPAY Annual fee payment

Payment date: 20161222

Year of fee payment: 11

FPAY Annual fee payment

Payment date: 20171219

Year of fee payment: 12

FPAY Annual fee payment

Payment date: 20181217

Year of fee payment: 13

FPAY Annual fee payment

Payment date: 20191126

Year of fee payment: 14