KR20030045414A - Conjugates of interferon-beta and polyethylene glycol derivatives - Google Patents
Conjugates of interferon-beta and polyethylene glycol derivatives Download PDFInfo
- Publication number
- KR20030045414A KR20030045414A KR1020010076130A KR20010076130A KR20030045414A KR 20030045414 A KR20030045414 A KR 20030045414A KR 1020010076130 A KR1020010076130 A KR 1020010076130A KR 20010076130 A KR20010076130 A KR 20010076130A KR 20030045414 A KR20030045414 A KR 20030045414A
- Authority
- KR
- South Korea
- Prior art keywords
- interferon
- beta
- propionaldehyde
- peg
- derivative
- Prior art date
Links
- 229920001223 polyethylene glycol Polymers 0.000 title claims abstract description 54
- 239000002202 Polyethylene glycol Substances 0.000 title claims abstract description 52
- 108090000467 Interferon-beta Proteins 0.000 title claims abstract description 43
- 102000003996 Interferon-beta Human genes 0.000 title claims abstract description 41
- 229960001388 interferon-beta Drugs 0.000 title claims abstract description 41
- 150000002334 glycols Chemical class 0.000 title 1
- 108010005716 Interferon beta-1a Proteins 0.000 claims abstract description 9
- 229960004461 interferon beta-1a Drugs 0.000 claims abstract description 9
- -1 and hence Proteins 0.000 claims abstract description 8
- 108010005714 Interferon beta-1b Proteins 0.000 claims abstract description 4
- 229960003161 interferon beta-1b Drugs 0.000 claims abstract description 4
- 150000001875 compounds Chemical class 0.000 claims description 18
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 12
- NBBJYMSMWIIQGU-UHFFFAOYSA-N Propionic aldehyde Chemical class CCC=O NBBJYMSMWIIQGU-UHFFFAOYSA-N 0.000 claims description 8
- RDZMTGDJXULLHG-UHFFFAOYSA-N C(CC)=O.NC(=O)OCC Chemical class C(CC)=O.NC(=O)OCC RDZMTGDJXULLHG-UHFFFAOYSA-N 0.000 claims description 5
- 102100026720 Interferon beta Human genes 0.000 claims description 2
- 238000000034 method Methods 0.000 claims description 2
- 201000006417 multiple sclerosis Diseases 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 7
- 239000004698 Polyethylene Substances 0.000 abstract description 3
- 230000005847 immunogenicity Effects 0.000 abstract description 3
- 230000000144 pharmacologic effect Effects 0.000 abstract description 3
- 229920000573 polyethylene Polymers 0.000 abstract description 3
- 230000004071 biological effect Effects 0.000 abstract description 2
- 230000003612 virological effect Effects 0.000 abstract description 2
- 206010028980 Neoplasm Diseases 0.000 abstract 1
- 201000011510 cancer Diseases 0.000 abstract 1
- 230000006866 deterioration Effects 0.000 abstract 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 45
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 45
- 230000002829 reductive effect Effects 0.000 description 41
- 239000011541 reaction mixture Substances 0.000 description 27
- 238000006243 chemical reaction Methods 0.000 description 26
- 239000000203 mixture Substances 0.000 description 25
- XEKOWRVHYACXOJ-UHFFFAOYSA-N ethyl acetate Substances CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 20
- 102000004169 proteins and genes Human genes 0.000 description 20
- 108090000623 proteins and genes Proteins 0.000 description 20
- 235000018102 proteins Nutrition 0.000 description 19
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 15
- 102000014150 Interferons Human genes 0.000 description 12
- 108010050904 Interferons Proteins 0.000 description 12
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 12
- 239000000843 powder Substances 0.000 description 12
- 239000003960 organic solvent Substances 0.000 description 10
- 238000001556 precipitation Methods 0.000 description 10
- 229940079322 interferon Drugs 0.000 description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 8
- 229940093499 ethyl acetate Drugs 0.000 description 8
- 229920000642 polymer Polymers 0.000 description 7
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 6
- 235000019341 magnesium sulphate Nutrition 0.000 description 6
- 102000005962 receptors Human genes 0.000 description 6
- 108020003175 receptors Proteins 0.000 description 6
- 229920001734 PEG propionaldehyde Polymers 0.000 description 5
- 239000012044 organic layer Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- BKIMMITUMNQMOS-UHFFFAOYSA-N nonane Chemical compound CCCCCCCCC BKIMMITUMNQMOS-UHFFFAOYSA-N 0.000 description 4
- 229920002401 polyacrylamide Polymers 0.000 description 4
- 230000009257 reactivity Effects 0.000 description 4
- PXXMSHBZYAOHBD-UHFFFAOYSA-N 3,3-diethoxypropan-1-amine Chemical compound CCOC(CCN)OCC PXXMSHBZYAOHBD-UHFFFAOYSA-N 0.000 description 3
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 3
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 3
- XBDQKXXYIPTUBI-UHFFFAOYSA-N Propionic acid Substances CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 239000012267 brine Substances 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 3
- 230000001766 physiological effect Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000004952 protein activity Effects 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 235000017557 sodium bicarbonate Nutrition 0.000 description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 3
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 3
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- ADFXKUOMJKEIND-UHFFFAOYSA-N 1,3-dicyclohexylurea Chemical compound C1CCCCC1NC(=O)NC1CCCCC1 ADFXKUOMJKEIND-UHFFFAOYSA-N 0.000 description 2
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 2
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Substances CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 125000003172 aldehyde group Chemical group 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- SWXVUIWOUIDPGS-UHFFFAOYSA-N diacetone alcohol Natural products CC(=O)CC(C)(C)O SWXVUIWOUIDPGS-UHFFFAOYSA-N 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000001747 exhibiting effect Effects 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 229940047124 interferons Drugs 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 229920001427 mPEG Polymers 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- LPNYRYFBWFDTMA-UHFFFAOYSA-N potassium tert-butoxide Chemical compound [K+].CC(C)(C)[O-] LPNYRYFBWFDTMA-UHFFFAOYSA-N 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000005062 synaptic transmission Effects 0.000 description 2
- QEQBMZQFDDDTPN-UHFFFAOYSA-N (2-methylpropan-2-yl)oxy benzenecarboperoxoate Chemical compound CC(C)(C)OOOC(=O)C1=CC=CC=C1 QEQBMZQFDDDTPN-UHFFFAOYSA-N 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Chemical compound OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000746367 Homo sapiens Granulocyte colony-stimulating factor Proteins 0.000 description 1
- 101001054334 Homo sapiens Interferon beta Proteins 0.000 description 1
- 102100040018 Interferon alpha-2 Human genes 0.000 description 1
- 108010047761 Interferon-alpha Proteins 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
- 108010079944 Interferon-alpha2b Proteins 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102000006386 Myelin Proteins Human genes 0.000 description 1
- 108010083674 Myelin Proteins Proteins 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- YGYAWVDWMABLBF-UHFFFAOYSA-N Phosgene Chemical compound ClC(Cl)=O YGYAWVDWMABLBF-UHFFFAOYSA-N 0.000 description 1
- 241000219061 Rheum Species 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 239000002262 Schiff base Substances 0.000 description 1
- 150000004753 Schiff bases Chemical class 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 102000019197 Superoxide Dismutase Human genes 0.000 description 1
- 108010012715 Superoxide dismutase Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- ATMLPEJAVWINOF-UHFFFAOYSA-N acrylic acid acrylic acid Chemical compound OC(=O)C=C.OC(=O)C=C ATMLPEJAVWINOF-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 238000005882 aldol condensation reaction Methods 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- ZTQSAGDEMFDKMZ-UHFFFAOYSA-N butyric aldehyde Natural products CCCC=O ZTQSAGDEMFDKMZ-UHFFFAOYSA-N 0.000 description 1
- 239000012830 cancer therapeutic Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 208000015114 central nervous system disease Diseases 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- PQJJJMRNHATNKG-UHFFFAOYSA-N ethyl bromoacetate Chemical compound CCOC(=O)CBr PQJJJMRNHATNKG-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 108091005608 glycosylated proteins Proteins 0.000 description 1
- 102000035122 glycosylated proteins Human genes 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 229960003130 interferon gamma Drugs 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 1
- 210000005012 myelin Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000035407 negative regulation of cell proliferation Effects 0.000 description 1
- 230000007830 nerve conduction Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000006320 pegylation Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- ZVCDLGYNFYZZOK-UHFFFAOYSA-M sodium cyanate Chemical compound [Na]OC#N ZVCDLGYNFYZZOK-UHFFFAOYSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- UCPYLLCMEDAXFR-UHFFFAOYSA-N triphosgene Chemical compound ClC(Cl)(Cl)OC(=O)OC(Cl)(Cl)Cl UCPYLLCMEDAXFR-UHFFFAOYSA-N 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000005727 virus proliferation Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/565—IFN-beta
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
본 발명은 인터페론-베타(Interferon-β)의 아미노 말단(amino-terminus, N-terminus)에 신규한 형태의 메톡시폴리에틸렌글리콜-프로피온알데히드 유도체를 결합시킨 폴리에틸렌글리콜-인터페론 베타 배합체(conjugate)에 관한 것이다.The present invention relates to a polyethylene glycol-interferon beta conjugate in which a novel type of methoxy polyethylene glycol-propionaldehyde derivative is bound to the amino terminus (amino-terminus, N-terminus) of Interferon-beta. It is about.
인터페론(interferon, IFN)은 핵이 존재하는 대부분의 세포로부터 유래하는 당단백질로, 바이러스의 복제를 억제함으로써 항 바이러스 성질을 가지며, 세포 증식을 억제하고 면역 반응을 조절한다. 인터페론은 세포 표면 세포막의 특정 수용체와 결합하여 일련의 세포내 반응을 개시하는데, 이러한 세포내 반응에는 특정 효소들의 활성화 유도, 세포 증식의 억제 등이 포함되고, 면역조절 반응으로서 대식세포(macrophage)의 식세포(phagocytosis) 활성의 증가, 표적 세포에 대한 임파구들의 세포 독성 증가, 그리고 바이러스에 감염된 세포들의 바이러스 증식 억제 등이 포함된다.Interferon (IFN) is a glycoprotein derived from most cells in which the nucleus is present. It has antiviral properties by inhibiting the replication of the virus, inhibits cell proliferation and modulates the immune response. Interferon binds to specific receptors on cell surface cell membranes and initiates a series of intracellular responses. These intracellular responses include the activation of specific enzymes, inhibition of cell proliferation, and the like. Increased phagocytosis activity, increased cytotoxicity of lymphocytes to target cells, and inhibition of virus proliferation of virus infected cells.
인간의 인터페론은 현재 5 종류가 알려져 있다. 인터페론-알파는 말초 혈액 백혈구나 림프 모세포에 의해 분비하고, 인터페론-베타는 섬유모세포(섬유아세포,fibroblast)가, 그리고 인터페론-감마는 B 세포가 분비하고 있으며, 이외에도 오메가 및 타우 인터페론이 존재한다.Five kinds of human interferons are known at present. Interferon-alpha is secreted by peripheral blood leukocytes or lymphoblasts, interferon-beta is secreted by fibroblasts (fibroblasts), and interferon-gamma is secreted by B cells, in addition to omega and tau interferons.
인터페론-베타는 바이러스 감염이나 다른 생물제제들에 반응해서 생체내 대부분의 세포들에서 생산되는 사이토카인(cytokine)으로, 특히 다발성 경화증 (multiple sclerosis, MS)에 상당한 치료 효과를 보이므로 그 유용성이 높이 평가되고 있다. 다발성 경화증은 뇌와 척수에서 신경세포섬유들을 절연하고 신경전도를 촉진하는 수초(myelin)들의 감소를 수반하는 중추신경계 질환이다. 즉, 신경세포섬유들이 신경전달을 관장하는데, 다발성 경화증 환자들은 신경전달이 감소하거나 완전히 차단되어 기능이 감소하거나 상실되는 것이다.Interferon-beta is a cytokine produced by most cells in vivo in response to viral infections and other biologics, especially because it has a significant therapeutic effect on multiple sclerosis (MS). It is evaluated. Multiple sclerosis is a central nervous system disease that involves the reduction of myelin, which insulates nerve cell fibers and promotes nerve conduction in the brain and spinal cord. In other words, nerve cell fibers govern neurotransmission, in patients with multiple sclerosis, neurotransmission is reduced or completely blocked, leading to decreased or lost function.
현재 미국에는 다발성 경화증 치료를 위한 2 가지 종류의 인터페론-베타가 존재하는데, 첫 번째로 인터페론-베타-1a(Interferon-β-1a, IFN-β-1a)는 인간 인터페론-베타 유전자를 포함하는 포유류 세포주로부터 생산되는 166 개 아미노산 잔기로 구성된 당화(glycosylated) 단백질이다. 두 번째 인터페론-베타-1b (Interferon-β-1b, IFN-β-1b)는 인터페론-베타의 당이 결여된(non-glycosylated) 재조합 단백질로, 대장균(E. coli)으로부터 생산되는 165 개 아미노산 잔기로 구성되는데, 아미노산 1 번 메티오닌(methionine) 잔기가 결실되어 있고, 17 번 시스테인(cysteine) 잔기가 세린(Serine)으로 치환되어 있다.Currently, there are two types of interferon-beta in the United States for the treatment of multiple sclerosis. First, interferon-beta-1a (Interferon-β-1a, IFN-β-1a) is a mammal containing the human interferon-beta gene. It is a glycosylated protein consisting of 166 amino acid residues produced from cell lines. The second interferon-beta-1b (IFN-β-1b) is a non-glycosylated recombinant protein of interferon-beta, a 165 amino acid produced from E. coli . It consists of residues, amino acid No. 1 methionine residues are deleted, and cysteine residues 17 are substituted with Serine.
폴리에틸렌 글리콜(polyethylene glycol, 이하 PEG)은 HO-(-CH2CH2O-)n-H의 구조를 갖는 고분자 화합물로, 친수성이 강하기 때문에 의약 단백질에 결합시켜 그용해도를 증가시킬 수 있다. 또한 적절하게 결합시키면 효소활성, 수용체 결합과 같은 주요 생물학적 기능들을 유지하면서 결합된 단백질의 분자량을 증가시키는 것에 의해, 신장여과를 감소시키고 외부항원을 인식하는 세포와 항체로부터 단백질을 보호하며 분해효소에 의한 단백질의 분해도 감소시킬 수 있다. 이와 같이 단백질에 결합 가능한 PEG의 분자량 범위는 대략 1,000∼100,000으로, PEG 분자량이 1,000 이상일 경우에는 독성이 상당히 낮은 편으로 알려져 있다. PEG의 분자량 범위가 1,000∼6,000인 것은 전신에 분포하고 신장을 통해 대사되며, 특히 분자량 40,000의 분지 PEG는 혈액과 간을 포함한 기관들에 분포되고 대사는 간에서 이루어지는 것으로 알려져 있다.Polyethylene glycol (PEG) is a high-molecular compound having a structure of HO-(-CH 2 CH 2 O-) n -H. Because of its high hydrophilicity, polyethylene glycol can increase its solubility by binding to a pharmaceutical protein. Proper binding also increases the molecular weight of the bound protein while maintaining key biological functions such as enzymatic activity and receptor binding, thereby reducing kidney filtration, protecting the protein from cells and antibodies that recognize foreign antigens, Can also reduce the degradation of proteins. As such, the molecular weight range of PEG that can be bound to proteins is approximately 1,000 to 100,000, and when the molecular weight of PEG is 1,000 or more, it is known that the toxicity is quite low. Its molecular weight ranges from 1,000 to 6,000 and is distributed throughout the body and metabolized through the kidneys. In particular, branched PEGs with a molecular weight of 40,000 are known to be distributed in organs including blood and liver, and metabolism in the liver.
비경구(parenteral) 경로를 통해 투여되는 의학적, 약리학적으로 유용한 단백질들은 항원성을 가지며, 대체로 수용성이 낮고 체내 잔존기간이 짧다는 단점이 있어 이를 극복하고자 하는 연구가 수행되고 있다. 데이비스(Frank F. Davis) 등의 미국특허 제4,179,337호에서는, PEG와 결합된 단백질 및 효소 등을 치료제로 사용할 경우, PEG가 갖는 장점인 항원성의 감소, 수용성의 증가, 체내 잔류 기간 증가 등의 효과를 얻을 수 있음을 개시하고 있다. 이 특허 이후, 생리활성 단백질을 PEG와 결합시켜 그 단점을 극복하고자 하는 시도가 이루어지고 있는데, 예를 들어, 베로니즈 등(Veroneseet al.,Applied Biochem. and Biotech. 11:141-152, 1985)은 리보뉴클레아제(ribonuclease)와 수퍼옥사이드 디스뮤타제(superoxide dismutase)를 PEG와 결합시키고 있다. 또한, 카터 등(Katreet al.)은 미국특허 제4,766,106호와 제4,917,888호에서 단백질들에 PEG를 포함한 폴리머(polymer, 중합체)를 결합시켜 단백질의 수용성을 증가시킨 내용을 개시하고 있으며, 나이테키 등(Niteckiet al.)은 미국특허 제4,902,502호에서 PEG나 다른 중합체들을 재조합 단백질에 결합시켜 항원성을 줄이고 체내 잔존 기간을 증가시키고 있다.Medically and pharmacologically useful proteins administered through the parenteral route have antigenicity, and are generally poor in water solubility and have a short remaining period in the body. In US Patent No. 4,179,337 to Frank F. Davis et al., The use of PEG-binding proteins and enzymes as therapeutic agents has the advantages of reducing antigenicity, increasing water solubility, and increasing the length of stay in the body, which are advantages of PEG. It is disclosed that can be obtained. Since this patent, attempts have been made to overcome the drawbacks by combining bioactive proteins with PEG, for example, Veronese et al. , Applied Biochem. And Biotech . 11: 141-152, 1985. ) Combines ribonuclease and superoxide dismutase with PEG. In addition, Carter et al. , U.S. Patent Nos. 4,766,106 and 4,917,888 discloses the binding of a polymer containing PEG to a protein to increase the water solubility of the protein. Nitecki et al. , In US Pat. No. 4,902,502, bind PEG or other polymers to recombinant proteins to reduce antigenicity and increase the duration of life in the body.
PEG와 단백질의 결합에는 이와 같은 장점 외에 결점도 존재한다. 즉, PEG는 대개 결합할 단백질의 하나 또는 그 이상의 자유 라이신(lysine, Lys) 잔기에 공유결합을 통해 결합하게 되는데, 이때 단백질의 표면 부위중 단백질의 활성도와 직접적인 관계가 있는 부위가 PEG와 결합할 경우, 그 부위는 더 이상 생물학적 기능을 수행할 수 없게 되어 단백질의 활성도가 감소하게 된다. 또한, PEG와 라이신 잔기의 결합은 대개 무작위적으로 일어나게 되므로 결합 위치에 따라 많은 종류의 PEG-단백질 배합체(conjugate)들이 혼합물로 존재하게 되고, 따라서 원하는 배합체를 순수 분리하는 과정이 복잡하고 어려워지게 된다. 예를 들어, 인터페론-알파 2b의 경우에는 단백질 표면에 8 개의 자유 라이신 잔기가 존재하는데, 이 잔기들 중에서 인터페론의 생물학적 활성에 영향을 미치지 않는 아미노산 부위에 위치 선택적인 방법으로 PEG를 결합시켜야만 의학적으로 유용한 배합체를 얻을 수 있게 된다.In addition to these advantages, there are drawbacks to PEG and protein binding. That is, PEG usually binds covalently to one or more free lysine (lys) residues of the protein to be bound, where a site directly related to protein activity on the surface of the protein is bound to PEG. In that case, the site will no longer be able to perform biological functions, resulting in reduced protein activity. In addition, the binding of PEG and lysine residues usually occurs randomly, so that many kinds of PEG-protein conjugates exist as a mixture depending on the binding position, and thus, the process of pure separation of the desired combination is complicated and difficult. You lose. For example, in the case of interferon-alpha 2b, there are eight free lysine residues on the surface of the protein, which must be PEG-linked in a position-selective manner to amino acid sites that do not affect the biological activity of interferon. Useful formulations can be obtained.
여러 저분자량 단백질 의약품과 마찬가지로 인터페론 치료요법에서도 비교적 짧은 혈중 반감기를 극복하려는 시도가 있었는데, 이중 성공적인 것의 하나가 단백질 의약품에 대한 PEG 폴리머의 결합(pegylation)에 의해 혈관잔류(vascular retention) 증가 등 변화된 약동학적 성질을 갖도록 한 것이다(Francis,et al., J. Drug Target, 3: 321-340, 1996). 이와 같은 페길레이션(pegylation)으로 얻어지는 효과들은 대상 단백질에 따라 큰 차이가 나는데, PEG의 결합 부위, 배합체 형성에 사용되는 화학반응, 그리고 PEG 폴리머의 분자량과 형태 등에 따라 더 현저한 차이가 발생할 수 있다(Delgadoet al., Pharm. Sci, 3: 59-66, 1997).As with many low-molecular-weight protein drugs, there have been attempts to overcome relatively short blood half-lives in interferon therapy. One of the most successful has been altered pharmacokinetics, such as increased vascular retention due to PEG polymerization of protein drugs. Of the nature of the drug (Francis, et al., J. Drug Target, 3 : 321-340, 1996). The effects of pegylation vary greatly depending on the protein of interest, which may be more pronounced depending on the binding site of PEG, the chemical reaction used to form the compound, and the molecular weight and form of the PEG polymer. (Delgado et al., Pharm. Sci, 3 : 59-66, 1997).
바이오젠(Biogen)사는 인터페론-베타-1a에서 수용체 결합부위와 물리적인 거리가 멀고 이에 따라 생리활성의 소실 없이 PEG 폴리머를 결합시킬 수 있는 부위로서 아미노 말단(N-terminus)을 선택하였다. 즉, 분자량 20,000의 PEG-알데히드 (aldehyde)를 인터페론-베타-1a와 실온, pH 6.0의 완충용액 중에서 20 시간 동안 반응시켜, 인터페론-베타-1a의 아미노 말단에 위치 특이적으로 PEG 폴리머를 결합시켰다. 이 배합체는 72 시간 동안 비교적 장시간 체내에 잔존하였으며, PEG가 인터페론-베타-1a의 항바이러스 활성에 악영향을 미치지는 않은 것으로 보고하고 있다(Pepinskyet al., J Pharm. and Exp. Therapeutics, 297: 1059-1066, 2001).Biogen chose the amino terminus (N-terminus) as the site where the interferon-beta-1a is physically separated from the receptor binding site and thus the PEG polymer can be bound without loss of physiological activity. That is, PEG-aldehyde (aldehyde) having a molecular weight of 20,000 was reacted with interferon-beta-1a in a buffer solution at room temperature and pH 6.0 for 20 hours to bind the PEG polymer to the amino terminal of the interferon-beta-1a in a specific position. . This combination remained in the body for a relatively long time for 72 hours, and it was reported that PEG did not adversely affect the antiviral activity of interferon-beta-1a (Pepinsky et al., J Pharm. And Exp. Therapeutics, 297). : 1059-1066, 2001).
아미노 말단의 알파-아미노(α-amino)기에 선택적으로 결합할 수 있는 PEG 폴리머로는 메톡시PEG-알데히드, 메톡시PEG-아세트알데히드(acetaldehyde), 메톡시 PEG-프로피온알데히드(propionaldehyde) 등이 사용되어 왔는데, 이는 알데히드 그룹이 아미노 말단에 선택적으로 반응하기 때문이다. 또한, 메톡시PEG-아세트알데히드는 알돌 축합(aldol condensation)에 의한 이합체 형성(dimerization)에 민감하기 때문에, 아세트알데히드 보다 프로피온알데히드 형태가 합성과 사용이 수월하다고 알려져 있다. 결합 반응은 쉬프(Schiff) 염기를 통해 알파-아미노기와 알데히드 그룹 간에 안정한 2차 아민 결합이 형성되어 PEG와 단백질의 배합체를 형성하게 되는 것이다. 메톡시PEG-프로피온알데히드를 사용한 예로서는, 재조합 인간 G-CSF(granulocyte-colony stimulating factor)의 아미노 말단(Kinstleret al.,Pharm Res., 13(7): 996-1002, 1996)과, 재조합 인간 TNF(tumor necrosis factor) 수용체 타입 1의 아미노 말단(Edwardset al., Ann. Rheum. Dis., 58(S1): I73-I81, 1999)에 PEG 폴리머를 결합시킨 것을 들 수 있다.As the PEG polymer capable of selectively binding to the amino terminal alpha-amino group, methoxy PEG-aldehyde, methoxy PEG-acetaldehyde, methoxy PEG-propionaldehyde, etc. are used. This is because the aldehyde group selectively reacts at the amino terminus. In addition, since methoxyPEG-acetaldehyde is sensitive to dimerization by aldol condensation, propionaldehyde forms are known to be easier to synthesize and use than acetaldehyde. The coupling reaction involves the formation of a stable secondary amine bond between the alpha-amino group and the aldehyde group via the Schiff base to form a combination of PEG and protein. Examples of using methoxyPEG-propionaldehyde include the amino terminus of recombinant human granulocyte-colony stimulating factor (K-CSF) (Kinstler et al., Pharm Res., 13 (7) : 996-1002, 1996), and recombinant humans. A PEG polymer is bound to the amino terminus of Edward necrosis factor (TNF) receptor type 1 (Edwards et al., Ann. Rheum. Dis., 58 (S1) : I73-I81, 1999).
이와 같은 종래의 PEG-단백질 간의 결합을 고려해볼 때, 다발성 경화증에 상당한 치료 효과를 보이는 인터페론-베타에 있어서 수용체 결합에 관여하지 않는 부위의 특정 아미노산 특정 잔기에 선택적인 반응성을 나타내는 신규한 형태의 PEG 유도체를 제공하고, 이러한 신규의 PEG 유도체와 인터페론-베타와의 배합체를 제공할 수 있다면 매우 유용하게 사용될 수 있을 것이다.Considering such conventional PEG-protein binding, a novel form of PEG that exhibits selective reactivity to specific amino acid specific residues at sites not involved in receptor binding in interferon-beta, which has a significant therapeutic effect on multiple sclerosis It would be very useful if one could provide a derivative and a combination of such novel PEG derivative with interferon-beta.
본 발명에서는 야생형(wild-type) 또는 재조합 인터페론-베타의 아미노 말단 (N-terminus)에 신규한 형태의 메톡시PEG-프로피온알데히드 유도체를 선택적으로 결합시킴으로써, 인터페론-베타의 항원유발성(immunogenicity)을 감소시키고 생리활성 저하를 최대한 감소시키면서 체내 잔존 시간을 증가시켜, 야생형이나 재조합 인터페론-베타 단백질의 치료효과에 필요한 투여량(dose)이나 투여횟수를 줄일 수 있도록 향상된 약동학 프로필(pharmacokinetic profile)과 약리적 성질을 갖도록 변형된 인터페론-베타와 PEG의 배합체를 제공하는 것을 그 목적으로 한다.In the present invention, by selectively binding a novel type of methoxyPEG-propionaldehyde derivative to the amino-terminus (N-terminus) of the wild-type or recombinant interferon-beta, the immunogenicity of interferon-beta Pharmacokinetic profile and pharmacology to reduce the dose or frequency required for the therapeutic effect of wild type or recombinant interferon-beta protein by increasing the body's remaining time while reducing physiological activity and reducing physiological activity as much as possible. It is an object to provide a combination of interferon-beta and PEG modified to have properties.
상기 목적을 달성하기 위하여 본 발명에서는, 인터페론-베타(Interferon-β; IFN-β)의 아미노 말단(amino-terminus, N-terminus)의 알파-아미노기(α-amino group)에 메톡시폴리에틸렌 글리콜-프로피온알데히드(methoxypolyethylene glycol-propionaldehyde) 유도체가 결합된 배합체(conjugate)를 제공한다.In order to achieve the above object, in the present invention, methoxypolyethylene glycol- at the alpha-amino group of the amino terminal (amino-terminus, N-terminus) of Interferon-beta (IFN-β) Provided is a conjugate to which a methoxypolyethylene glycol-propionaldehyde derivative is bound.
여기에서 인터페론-베타는 야생형 또는 재조합 인터페론-베타일 수 있으며, 또한 인터페론-베타-1a 또는 인터페론-베타-1b인 것이 바람직하다.The interferon-beta here may be wild type or recombinant interferon-beta, and also preferably interferon-beta-1a or interferon-beta-1b.
또한, 상기 메톡시폴리에틸렌글리콜(PEG)-프로피온알데히드 유도체는 인터페론-베타의 아미노 말단의 알파-아미노기(α-amino group)에 선택적인 반응성을 나타내는 신규한 형태의 메톡시PEG-프로피온알데히드 유도체로서, 직선(linear)형의 메톡시PEG-아미드(amide)-프로피온알데히드 유도체 및 메톡시PEG-우레탄(urethane) -프로피온알데히드 유도체, 그리고 펜던트(pendant)형의 PEG-아미드-프로피온알데히드 유도체 및 PEG-우레탄-프로피온알데히드 유도체의 적어도 하나일 수 있다.In addition, the methoxy polyethylene glycol (PEG) -propionaldehyde derivative is a novel form of methoxyPEG-propionaldehyde derivative exhibiting selective reactivity to the alpha-amino group of the amino terminal of the interferon-beta, Linear methoxyPEG-amide-propionaldehyde derivatives and methoxyPEG-urethane-propionaldehyde derivatives, and pendant PEG-amide-propionaldehyde derivatives and PEG-urethanes At least one of propionaldehyde derivatives.
여기에서, 메톡시폴리에틸렌글리콜-프로피온알데히드 유도체는 분자량 범위가 1,000∼1,000,000인 것이 바람직하다. 구체적으로, 직선형의 메톡시PEG-프로피온알데히드 유도체는 분자량 범위가 1,000∼100,000인 것이 바람직하고, 1,000∼40,000인 것이 더욱 바람직하다. 또한, 펜던트형의 PEG-프로피온알데히드 유도체는 PEG 뼈대의 분자량 범위가 5,000∼1,000,000인 것이 바람직하고, 5,000∼100,000인 것이 더욱 바람직하다. 그리고, 펜던트 그룹인 아미드-프로피온알데히드나 우레탄-프로피온알데히드의 개수는 1∼20인 것이 바람직하다.Here, it is preferable that the methoxy polyethylene glycol propionaldehyde derivative has a molecular weight range of 1,000 to 1,000,000. Specifically, the linear methoxyPEG-propionaldehyde derivative preferably has a molecular weight range of 1,000 to 100,000, more preferably 1,000 to 40,000. Moreover, it is preferable that the molecular weight range of PEG skeleton of a pendant PEG-propionaldehyde derivative is 5,000-1,000,000, and it is more preferable that it is 5,000-100,000. The number of amide-propionaldehyde or urethane-propionaldehyde as pendant groups is preferably 1 to 20.
본 발명에서는, 인터페론-베타의 아미노 말단의 알파-아미노기에 선택적인 반응성을 나타내는 신규한 형태의 메톡시PEG-프로피온알데히드 유도체를 결합시킴으로써, 이에 의해 형성되는 배합체의 종류를 한정시키는 동시에 단백질 활성도 감소를 최대한 억제하고 있다. 즉, 단백질의 2, 3 차 구조상에서 라이신(lysine) 잔기의 곁사슬(side chain)에 존재하는 입실론-아미노기에 반응성을 갖는 PEG 유도체들은 단백질 표면에 노출된 다수의 입실론-아미노기와 반응함으로써 단백질의 활성부위를 저해하여 활성도를 감소시키지만, 본 발명에 따른 메톡시PEG-프로피온알데히드 유도체는 아미노 말단의 알파-아미노기에 선택적인 반응성을 나타내기 때문에 목적하는 단백질에 하나의 PEG 유도체만을 결합시킬 수 있다. 또한, 이 아미노 말단이 수용체와의 결합에 관여하지 않는 부위라면, PEG 유도체와의 배합체 형성에 의한 야생형의 활성도 감소를 최대한 억제할 수 있다.In the present invention, by binding a novel form of methoxyPEG-propionaldehyde derivative exhibiting selective reactivity to the alpha-amino group of the amino terminus of interferon-beta, it limits the type of the combination formed thereby and reduces protein activity. Is suppressed as much as possible. That is, PEG derivatives that are reactive with epsilon-amino groups in the side chain of lysine residues on the secondary and tertiary structures of the protein react with a number of epsilon-amino groups exposed on the surface of the protein. Although the site is inhibited to decrease the activity, the methoxyPEG-propionaldehyde derivative according to the present invention exhibits selective reactivity to the alpha-amino group of the amino terminus so that only one PEG derivative can be bound to the protein of interest. In addition, as long as this amino terminal is a site which does not participate in binding with a receptor, the reduction of the activity of a wild type by formation of a compound with a PEG derivative can be suppressed as much as possible.
본 발명에 따르면, 인터페론-베타와의 배합체 형성에 사용되는 메톡시PEG-프로피온알데히드 유도체의 양(amount)은 적어도 인터페론-베타와 동일한 당량 (equimolar)이어야 하며, 아미노 말단의 알파-아미노 그룹과의 완전한 반응을 유도하기 위하여 메톡시PEG-프로피온알데히드 유도체를 과량(인터페론 단백질 1 몰당 PEG 유도체의 몰비가 1∼10 배의 범위)으로 가해주는 것이 바람직하다. 또한, 인터페론-베타와 메톡시PEG-프로피온알데히드의 배합반응은 pH 6.0∼7.0 범위에서 실온 조건 하에 약 5∼20 시간이 소요된다.According to the invention, the amount of the methoxyPEG-propionaldehyde derivative used to form the combination with interferon-beta should be at least the same equivalent as the interferon-beta, and the alpha-amino group at the amino terminus. In order to induce a complete reaction of the methoxyPEG-propionaldehyde derivative is preferably added in excess (mole ratio of PEG derivatives per mole of interferon protein in the range of 1 to 10 times). In addition, the compounding reaction of interferon-beta and methoxyPEG-propionaldehyde takes about 5 to 20 hours at room temperature under the pH range of 6.0 to 7.0.
이와 같이, 인터페론-베타의 아미노 말단의 알파-아미노기에 메톡시PEG-프로피온알데히드 유도체가 결합되어 얻어지는 인터페론-베타와 PEG 유도체의 배합체는, 인터페론-베타의 항원유발성(immunogenicity)이 감소되고 생리활성 저하가 억제되면서 체내 잔존 시간이 증가되어 향상된 약동학 프로필과 약리적 성질을 갖게 된다.As such, the combination of interferon-beta and PEG derivatives obtained by binding a methoxyPEG-propionaldehyde derivative to the alpha-amino group of the amino terminal of the interferon-beta, reduces the immunogenicity of interferon-beta and physiologically As the degradation of activity is suppressed, the remaining time in the body increases, resulting in improved pharmacokinetic profiles and pharmacological properties.
이하, 실시예를 통해 본 발명을 더욱 상세히 설명한다. 단, 이들 실시예는본 발명의 일부 실험방법과 조성을 나타낸 예시일 뿐, 본 발명의 범위가 이들만으로 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are only examples showing some experimental methods and compositions of the present invention, but the scope of the present invention is not limited thereto.
다음 실시예에서 사용된 PEG 유도체들은 선바이오(주)에 의해 합성된 제품을 이용한 것이다.The PEG derivatives used in the following examples are those using products synthesized by Sun Bio Co., Ltd.
[실시예 1] 메톡시PEG-아미드(amide)-프로피온알데히드의 제조Example 1 Preparation of MethoxyPEG-amide-Propionaldehyde
메톡시PEG(mPEG-OH)(MW. 20,000)과 포타슘 t-부톡사이드(potassium t-butoxide)를 t-부틸알콜(t-buthyl alcohol)에 넣고 60 ℃ 조건 하에서 교반하였다. 이 혼합물에 에틸브로모아세테이트(ethyl bromoacetate)를 천천히 첨가하고 80∼85 ℃ 조건 하에 15 시간 동안 교반하였다. 반응 혼합물을 여과한 후 여액을 감압증류하여 유기용매를 제거하고 증류수를 가하여 녹였다. 디에틸에테르(diethyl ether)로 1 회 세척하고 디클로로메탄(dichloromethane)으로 2 회 추출하였다. 추출된 유기층을 황산마그네슘(magnesium sulfate)으로 건조한 후 감압증류하여 유기용매를 제거하였다. 농축된 반응 혼합물에 디에틸에테르를 첨가하여 침전을 유도하고, 침전된 화합물은 감압 여과후 진공 감압 하에 건조하여 백색 분말 형태의 mPEG-에틸아세테이트 화합물을 얻었다. 이상의 반응 과정을 다음 반응식 1에 나타내었다.Methoxy PEG (mPEG-OH) (MW. 20,000) and potassium t-butoxide (potassium t-butoxide) was added to t- butyl alcohol (t-buthyl alcohol) and stirred under the conditions of 60 ℃. Ethyl bromoacetate was slowly added to the mixture and stirred for 15 hours under the conditions of 80 to 85 ° C. After the reaction mixture was filtered, the filtrate was distilled under reduced pressure to remove the organic solvent and dissolved by adding distilled water. Washed once with diethyl ether and extracted twice with dichloromethane. The extracted organic layer was dried over magnesium sulfate (magnesium sulfate) and distilled under reduced pressure to remove the organic solvent. Diethyl ether was added to the concentrated reaction mixture to induce precipitation, and the precipitated compound was filtered under reduced pressure and dried under vacuum reduced pressure to obtain a mPEG-ethylacetate compound in the form of a white powder. The above reaction process is shown in the following scheme 1.
여기에서, n=22∼2273으로, 22∼909가 바람직하다. 이는 반응식 1∼5까지 적용된다.Here, n = 22-2273 and 22-909 are preferable. This applies to schemes 1-5.
상기 mPEG-에틸아세테이트를 1 N 수산화나트륨 수용액에 녹여 상온에서 15 시간 동안 교반하였다. 1 N 염산수용액으로 반응 수용액의 pH를 2로 산성화시키고 디클로로메탄으로 2 회 추출하였다. 추출된 유기층을 황산마그네슘으로 건조하고, 유기용매를 감압증류하여 제거하였다. 농축된 반응 혼합물에 디에틸에테르를 첨가하여 침전을 유도하고, 침전된 화합물은 감압 여과후 진공 감압 하에 건조하여 백색 분말 형태의 mPEG-아세트산(mPEG-acetic acid) 화합물을 얻었다. 이상의 반응 과정을 다음 반응식 2에 나타내었다.The mPEG-ethyl acetate was dissolved in 1 N aqueous sodium hydroxide solution and stirred at room temperature for 15 hours. The pH of the reaction solution was acidified to 2 with 1 N aqueous hydrochloric acid and extracted twice with dichloromethane. The extracted organic layer was dried over magnesium sulfate, and the organic solvent was removed by distillation under reduced pressure. Diethyl ether was added to the concentrated reaction mixture to induce precipitation, and the precipitated compound was filtered under reduced pressure and dried under vacuum reduced pressure to obtain an mPEG-acetic acid compound in the form of a white powder. The above reaction process is shown in the following scheme 2.
상기 mPEG-아세트산을 디클로로메탄에 녹여 0∼5 ℃ 조건 하에 교반하였다. 이 혼합물에 N-하이드록시석시니미드(N-hydroxysuccinimide)를 첨가한 다음, 디사이클로헥실카르보디이미드(dicyclohexylcarbodiimide)를 디클로로메탄에 녹여 0∼5 ℃ 조건 하에 천천히 첨가하였다. 반응 혼합물을 상온에서 약 15 시간 동안 교반하였다. 반응 혼합물을 감압 여과하여 부산물인 디사이클로헥실우레아 (dicyclohexylurea)를 제거하고 감압 증류하여 유기용매를 제거하였다. 농축된 반응 혼합물은 에틸 아세테이트로 재결정하였다. 재결정 화합물은 감압 여과후 디에틸에테르로 2 회 세척하고, 진공 감압 하에 12 시간 동안 건조하여 백색 분말 형태의 mPEG-석시니미딜 아세테이트(mPEG-succinimidyl acetate) 화합물을 얻었다. 이상의 반응 과정을 다음 반응식 3에 나타내었다.The mPEG-acetic acid was dissolved in dichloromethane and stirred under 0-5 ° C. conditions. N-hydroxysuccinimide was added to this mixture, and then dicyclohexylcarbodiimide was dissolved in dichloromethane and slowly added under 0 to 5 ° C. The reaction mixture was stirred at room temperature for about 15 hours. The reaction mixture was filtered under reduced pressure to remove byproduct dicyclohexylurea and distilled under reduced pressure to remove the organic solvent. The concentrated reaction mixture was recrystallized from ethyl acetate. The recrystallized compound was washed twice with diethyl ether after filtration under reduced pressure, and dried under vacuum reduced pressure for 12 hours to obtain mPEG-succinimidyl acetate compound in the form of a white powder. The above reaction process is shown in the following scheme 3.
상기 mPEG-석시니미딜 아세테이트를 디클로로메탄에 녹여 상온에서 교반하였다. 이 혼합물에 1-아미노-3,3-디에톡시프로판(1-amino-3,3-diethoxypropane)을 첨가하였다. 반응 혼합물을 상온에서 2 시간 동안 교반하였다. 반응 혼합물에 디에틸에테르를 첨가하여 침전을 유도하고, 침전된 혼합물은 감압 여과한 후 에틸아세테이트로 재결정하였다. 재결정 화합물은 감압 여과하고 디에틸에테르로 2 회 세척후 진공 감압 하에 12 시간 동안 건조하여 백색 분말 형태의 mPEG-프로피온알데히드디에틸아세탈(mPEG-propionaldehydediethylacetal) 화합물을 얻었다. 이상의 반응 과정을 다음 반응식 4에 나타내었다.The mPEG-succinimidyl acetate was dissolved in dichloromethane and stirred at room temperature. To this mixture was added 1-amino-3,3-diethoxypropane. The reaction mixture was stirred at room temperature for 2 hours. Precipitation was induced by adding diethyl ether to the reaction mixture, and the precipitated mixture was filtered under reduced pressure and recrystallized with ethyl acetate. The recrystallized compound was filtered under reduced pressure, washed twice with diethyl ether, and dried under vacuum reduced pressure for 12 hours to obtain mPEG-propionaldehyde diethylacetal (mPEG-propionaldehydediethylacetal) compound in the form of a white powder. The above reaction process is shown in the following scheme 4.
상기 mPEG-프로피온알데히드디에틸아세탈을 인산(phosphoric acid, pH 1) 수용액에 녹여 40∼50 ℃ 조건 하에 2 시간 동안 교반하였다. 반응 혼합물을 상온으로 식힌 다음, 5 % 중탄산나트륨(sodium bicarbonate) 수용액으로 pH를 6으로 조정하고 브라인(brine)을 넣어준 후 디클로로메탄으로 2 회 추출하였다. 추출된 유기층은 황산마그네슘으로 건조하고 유기용매를 감압 증류하여 제거하였다. 농축된 반응 혼합물에 디에틸에테르를 첨가하여 침전을 유도하고, 침전된 화합물을 감압 여과후 진공 감압하에 건조하여 백색 분말 형태의 메톡시PEG-아미드-프로피온알데히드 화합물을 얻었다. 이상의 반응 과정을 다음 반응식 5에 나타내었다.The mPEG-propionaldehyde diethyl acetal was dissolved in an aqueous solution of phosphoric acid (phosphoric acid, pH 1) and stirred for 2 hours under 40 to 50 ° C. The reaction mixture was cooled to room temperature, adjusted to pH 6 with 5% sodium bicarbonate aqueous solution, and brine was added thereto, and then extracted twice with dichloromethane. The extracted organic layer was dried over magnesium sulfate, and the organic solvent was distilled off under reduced pressure. Diethyl ether was added to the concentrated reaction mixture to induce precipitation, and the precipitated compound was filtered under reduced pressure and dried under vacuum reduced pressure to obtain a methoxyPEG-amide-propionaldehyde compound in the form of a white powder. The above reaction process is shown in the following scheme 5.
[실시예 2] 메톡시PEG-우레탄(urethane)-프로피온알데히드의 제조Example 2 Preparation of MethoxyPEG-Urethane-Propionaldehyde
메톡시PEG(mPEG-OH)(MW. 20,000)를 디클로로메탄에 넣고 상온에서 교반하였다. 이 혼합물에, 디클로로메탄에 녹인 트라이포스겐(triphosgene)를 천천히 첨가하고 상온에서 15 시간 동안 교반하였다. 반응 혼합물을 감압 증류하여 유기용매와 남아있는 포스겐을 제거하였다. 감압 증류한 혼합물을 디클로로메탄에 녹여서 교반하였다. 이 반응 혼합물에 N-하이드록시석시니미드를 첨가한 다음, 트리에틸아민을 넣고 상온 조건에서 3 시간 동안 교반하였다. 반응 혼합물을 여과한 후 여액을 감압 증류하여 유기용매를 제거하고, 따뜻한(50 ℃) 에틸아세테이트에 녹였다. 반응혼합물을 여과한 여액을 저온에서 침전을 유도하고, 침전된 화합물은 감압 여과후 진공 감압하에 건조하여 백색 분말 형태의 mPEG-석시니미딜카보네이트(mPEG-succinimidylcarbonate) 화합물을 얻었다. 이상의 반응 과정을 다음 반응식 6에 나타내었다.MethoxyPEG (mPEG-OH) (MW. 20,000) was added to dichloromethane and stirred at room temperature. To this mixture, triphosgene dissolved in dichloromethane was slowly added and stirred at room temperature for 15 hours. The reaction mixture was distilled under reduced pressure to remove the organic solvent and remaining phosgene. The mixture distilled under reduced pressure was dissolved in dichloromethane and stirred. N-hydroxysuccinimide was added to the reaction mixture, triethylamine was added thereto, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and the filtrate was distilled under reduced pressure to remove the organic solvent, which was dissolved in warm (50 ° C.) ethyl acetate. The filtrate of the reaction mixture was filtered to induce precipitation at low temperature, and the precipitated compound was filtered under reduced pressure and dried under vacuum pressure to obtain mPEG-succinimidylcarbonate compound as a white powder. The above reaction process is shown in the following Scheme 6.
다음 반응식에서, n=22∼2273으로, 22∼909가 바람직하다. 이는 반응식 6∼8까지 적용된다.In the following reaction scheme, n = 22 to 2273, and 22 to 909 are preferable. This applies to Schemes 6-8.
상기 mPEG-석시니미딜카보네이트를 디클로로메탄에 녹여 상온에서 교반하였다. 이 혼합물에 1-아미노-3,3-디에톡시프로판(1-amino-3,3-diethoxypropane)을 첨가하였다. 반응 혼합물을 상온에서 2 시간 동안 교반하였다. 반응 혼합물에 디에틸에테르를 첨가하여 침전을 유도하고, 침전된 혼합물은 감압 여과한 후 에틸아세테이트로 재결정하였다. 재결정 화합물은 감압 여과하고 디에틸에테르로 2 회 세척후 진공 감압 하에 12 시간 동안 건조하여 백색 분말 형태의 mPEG-프로피온알데히드디에틸아세탈 화합물을 얻었다. 이상의 반응 과정을 다음 반응식 7에 나타내었다.The mPEG-succinimidyl carbonate was dissolved in dichloromethane and stirred at room temperature. To this mixture was added 1-amino-3,3-diethoxypropane. The reaction mixture was stirred at room temperature for 2 hours. Precipitation was induced by adding diethyl ether to the reaction mixture, and the precipitated mixture was filtered under reduced pressure and recrystallized with ethyl acetate. The recrystallized compound was filtered under reduced pressure, washed twice with diethyl ether, and dried under vacuum reduced pressure for 12 hours to obtain mPEG-propionaldehyde diethylacetal compound in the form of a white powder. The above reaction process is shown in the following scheme 7.
상기 mPEG-프로피온알데히드디에틸아세탈을 인산(pH 1) 수용액에 녹여 40∼50 ℃ 조건 하에 2 시간 동안 교반하였다. 반응 혼합물을 상온으로 식힌 다음, 5 % 중탄산나트륨 수용액으로 pH를 6으로 조정하고 브라인(brine)을 넣어준 후 디클로로메탄으로 2 회 추출하였다. 추출된 유기층은 황산마그네슘으로 건조하고 유기용매를 감압 증류하여 제거하였다. 농축된 반응 혼합물에 디에틸에테르를 첨가하여 침전을 유도하고, 침전된 화합물을 감압 여과후 진공 감압 하에 건조하여 백색분말 형태의 메톡시PEG-우레탄-프로피온알데히드 화합물을 얻었다. 이상의 반응 과정을 다음 반응식 8에 나타내었다.The mPEG-propionaldehyde diethyl acetal was dissolved in an aqueous solution of phosphoric acid (pH 1) and stirred for 2 hours under 40 to 50 ° C. After the reaction mixture was cooled to room temperature, the pH was adjusted to 6 with 5% aqueous sodium bicarbonate solution and brine was added thereto, and then extracted twice with dichloromethane. The extracted organic layer was dried over magnesium sulfate, and the organic solvent was distilled off under reduced pressure. Diethyl ether was added to the concentrated reaction mixture to induce precipitation, and the precipitated compound was filtered under reduced pressure and dried under vacuum pressure to obtain methoxyPEG-urethane-propionaldehyde compound in the form of white powder. The above reaction process is shown in the following scheme 8.
[실시예 3] 펜던트 PEG-아미드-프로피온알데히드의 제조Example 3 Preparation of Pendant PEG-amide-Propionaldehyde
메톡시PEG(MW 20,000) 또는 PEG(MW 20,000)이 들어있는 반응 용기에 노난(nonane)을 넣고 140∼145 ℃ 온도 조건으로 상승시키면서 교반하였다. 이 반응 혼합물에 아크릴산(acrylic acid)과 반응 개시제 t-부틸 퍼옥시벤조에이트(t-butyl peroxybenzoate)를 각각 1.5 시간 동안 천천히 첨가하였다. 반응물 첨가후 약 1 시간 동안 140∼145 ℃ 조건 하에서 교반하였다. 반응 혼합물을 감압 증류하여 노난을 제거하고 메탄올을 가하여 균일한 액상이 되도록 가열한 후 교반하였다. 이 혼합물을 여과지로 여과한 후 메탄올과 증류수의 혼합용액(9:1)을 가하고 팔 필트론 울트라필트레이션 장치(Pall Filtron Ultrafiltration system)로 정제하였다. 정제된 혼합물을 감압 증류하여 용매를 제거한 후 아세톤과 이소프로필알콜의 혼합용액(1:1)을 가하고 가열하여 균일한 용액을 얻었다. 이 혼합 용액을 0 ℃ 조건에서 12 시간 동안 방치하여 침전을 유도하였다. 침전된 화합물을 아세톤과 이소프로필알콜의 혼합용액(1:1)으로 3 회 및 디에틸에테르로 1 회 세척하면서 감압 여과하고, 진공 감압하에 건조하여 백색 분말 형태의 펜던트-PEG-프로피온산(pendant-PEG-propionic acid) 화합물을 얻었다. 이상의 반응 과정을 다음 반응식 9에 나타내었다.Nonane was added to a reaction vessel containing methoxy PEG (MW 20,000) or PEG (MW 20,000), and the mixture was stirred while rising to a temperature of 140 to 145 ° C. Acrylic acid (acrylic acid) and the reaction initiator t-butyl peroxybenzoate were slowly added to the reaction mixture for 1.5 hours, respectively. After the addition of the reaction, the mixture was stirred under the conditions of 140 to 145 ° C for about 1 hour. The reaction mixture was distilled under reduced pressure to remove nonane, and methanol was added thereto, followed by heating to a uniform liquid phase, followed by stirring. The mixture was filtered through a filter paper, and a mixed solution of methanol and distilled water (9: 1) was added thereto, and the mixture was purified by a Pall Filtron Ultrafiltration system. The purified mixture was distilled under reduced pressure to remove the solvent, and then a mixed solution of acetone and isopropyl alcohol (1: 1) was added thereto and heated to obtain a uniform solution. The mixed solution was left at 0 ° C. for 12 hours to induce precipitation. The precipitated compound was filtered under reduced pressure, washed three times with a mixed solution of acetone and isopropyl alcohol (1: 1) and once with diethyl ether, and dried under vacuum reduced pressure to obtain a pendant-PEG-propionic acid in the form of a white powder. PEG-propionic acid) compound was obtained. The above reaction process is shown in the following scheme 9.
다음 반응식에서 n=113∼22,728로, 113∼2273이 바람직하고. m=1∼20이다. 이는 반응식 9∼12까지 적용된다.In the following reaction scheme, n = 113 to 22,728 and 113 to 2273 are preferable. m = 1-20. This applies to schemes 9-12.
상기 펜던트-PEG-프로피온산을 디클로로메탄에 녹여 0∼5 ℃ 조건 하에 교반하였다. 이 혼합물에 N-하이드록시석시니미드를 첨가한 다음, 디사이클로헥실카르보디이미드(dicyclohexylcarbodiimide, DCC)와 4-(디메틸아미노)피리딘[4-(dimethylamino)pyridine, DMAP]을 디클로로메탄에 녹여 0∼5 ℃ 조건 하에 천천히 첨가하였다. 반응 혼합물을 상온에서 약 15 시간 동안 교반하였다. 반응 혼합물을 감압 여과하여 부산물인 디사이클로헥실우레아(dicyclohexylurea)를 제거하고 감압 증류하여 유기용매를 제거하였다. 농축된 반응 혼합물은 에틸아세테이트로 재결정하였다. 재결정 화합물은 감압 여과하고 디에틸에테르로 2 회 세척후 진공 감압 하에 12 시간 동안 건조하여 백색 분말 형태의 펜던트-PEG-석시니미딜 프로피오네이트(pendant PEG-succinimidyl propionate) 화합물을 얻었다. 이상의 반응 과정을 다음 반응식 10에 나타내었다.The pendant-PEG-propionic acid was dissolved in dichloromethane and stirred under 0-5 ° C. conditions. N-hydroxysuccinimide was added to the mixture, followed by dicyclohexylcarbodiimide (DCC) and 4- (dimethylamino) pyridine (DMAP) in dichloromethane. It was added slowly under ˜5 ° C. conditions. The reaction mixture was stirred at room temperature for about 15 hours. The reaction mixture was filtered under reduced pressure to remove byproduct dicyclohexylurea and distilled under reduced pressure to remove the organic solvent. The concentrated reaction mixture was recrystallized from ethyl acetate. The recrystallized compound was filtered under reduced pressure, washed twice with diethyl ether, and dried under vacuum reduced pressure for 12 hours to obtain a pendant PEG-succinimidyl propionate compound in the form of a white powder. The above reaction process is shown in the following Scheme 10.
상기 펜던트-PEG-석시니미딜 프로피오네이트를 디클로로메탄에 녹여 상온에서 교반하였다. 이 혼합물에 1-아미노-3,3-디에톡시프로판(1-amino-3,3-diethoxypropane)을 첨가하였다. 반응 혼합물을 상온에서 2 시간 동안 교반하였다. 반응 혼합물에 디에틸에테르를 첨가하여 침전을 유도하고, 침전된 혼합물은 감압 여과한 후 에틸아세테이트로 재결정하였다. 재결정 화합물은 감압 여과하고 디에틸에테르로 2 회 세척후 진공 감압 하에 12 시간 동안 건조하여 백색 분말 형태의 펜던트-PEG-프로피온알데히드디에틸아세탈 화합물을 얻었다. 이상의 반응 과정을 다음 반응식 11에 나타내었다.The pendant-PEG-succinimidyl propionate was dissolved in dichloromethane and stirred at room temperature. To this mixture was added 1-amino-3,3-diethoxypropane. The reaction mixture was stirred at room temperature for 2 hours. Precipitation was induced by adding diethyl ether to the reaction mixture, and the precipitated mixture was filtered under reduced pressure and recrystallized with ethyl acetate. The recrystallized compound was filtered under reduced pressure, washed twice with diethyl ether, and dried under vacuum reduced pressure for 12 hours to obtain a pendant-PEG-propionaldehyde diethylacetal compound in the form of a white powder. The above reaction process is shown in Scheme 11 below.
상기 펜던트-PEG-프로피온알데히드디에틸아세탈을 인산(pH 1) 수용액에 녹여 40∼50 ℃ 조건 하에 2 시간 동안 교반하였다. 반응 혼합물을 상온으로 식힌 다음, 5 % 중탄산나트륨 수용액으로 pH를 6으로 조정하고 브라인(brine)을 넣어준 후 디클로로메탄으로 2 회 추출하였다. 추출된 유기층은 황산마그네슘으로 건조하고 유기용매를 감압 증류하여 제거하였다. 농축된 반응 혼합물에 디에틸에테르를 첨가하여 침전을 유도하고, 침전된 화합물을 감압 여과후 진공 감압하에 건조하여 백색 분말 형태의 펜던트-PEG-아미드-프로피온알데히드(pendant PEG-amide propionaldehyde) 화합물을 얻었다. 이상의 반응 과정을 다음 반응식 12에 나타내었다.The pendant-PEG-propionaldehyde diethylacetal was dissolved in an aqueous solution of phosphoric acid (pH 1) and stirred for 2 hours under 40 to 50 ° C. After the reaction mixture was cooled to room temperature, the pH was adjusted to 6 with 5% aqueous sodium bicarbonate solution and brine was added thereto, and then extracted twice with dichloromethane. The extracted organic layer was dried over magnesium sulfate, and the organic solvent was distilled off under reduced pressure. Diethyl ether was added to the concentrated reaction mixture to induce precipitation, and the precipitated compound was filtered under reduced pressure and dried under vacuum pressure to obtain a pendant PEG-amide propionaldehyde compound in the form of a white powder. . The above reaction process is shown in Scheme 12 below.
[실시예 4]Example 4
인터페론-베타와 직선형 메톡시PEG-아미드-프로피온알데히드의 배합(conjugation)Conjugation of Interferon-Beta with Linear MethoxyPEG-amide-Propionaldehyde
위 실시예 1에서 제조한 분자량 1,000∼40,000의 직선형 메톡시PEG-아미드-프로피온알데히드와 인터페론-베타 단백질을 배합하는데, 인터페론:PEG의 몰비율 (molar ratio)이 1:1∼1:5가 되도록 준비하고 소디움 시아노보로하이드라이드를 포함하는 NaH2PO4, pH 6.0 완충용액에서 반응시켜 PEG-인터페론-베타 배합체를 형성하였다. 반응은 실온에서 5∼20 시간 동안 수행하며, 배합체의 완성은 SDS-폴리아크릴아미드 전기영동(SDS-polyacrylamide electrophoresis)으로 확인하였다.A linear methoxy PEG-amide-propionaldehyde and an interferon-beta protein having a molecular weight of 1,000 to 40,000 prepared in Example 1 were combined, so that the molar ratio of interferon: PEG was 1: 1 to 1: 5. Prepared and reacted in NaH 2 PO 4 , pH 6.0 buffer with sodium cyanoborohydride to form a PEG-interferon-beta combination. The reaction was carried out at room temperature for 5-20 hours, and the completion of the mixture was confirmed by SDS-polyacrylamide electrophoresis.
[실시예 5]Example 5
인터페론-베타와 직선형 메톡시PEG-우레탄-프로피온알데히드의 배합Combination of Interferon-beta and Linear MethoxyPEG-Urethane-Propionaldehyde
위 실시예 2에서 제조한 분자량 1,000∼40,000의 직선형 메톡시PEG-우레탄-프로피온알데히드와 인터페론-베타 단백질을 배합하는데, 인터페론:PEG의 몰비율이 1:1∼1:5가 되도록 준비하고 소디움 시아노보로하이드라이드를 포함하는 NaH2PO4, pH 6.0 완충용액에서 반응시켜 PEG-인터페론-베타 배합체를 형성하였다. 반응은 실온에서 5∼20 시간 동안 수행하며, 배합체의 완성은 SDS-폴리아크릴아미드 전기영동으로 확인하였다.A linear methoxy PEG-urethane-propionaldehyde and an interferon-beta protein having a molecular weight of 1,000 to 40,000 prepared in Example 2 were prepared, and the interferon: PEG was prepared to have a molar ratio of 1: 1 to 1: 5 and sodium cyanate. The reaction was performed in NaH 2 PO 4 , pH 6.0 buffer containing noborohydride to form a PEG-interferon-beta combination. The reaction was carried out at room temperature for 5-20 hours and the completion of the formulation was confirmed by SDS-polyacrylamide electrophoresis.
[실시예 6]Example 6
인터페론-베타와 펜던트형 PEG-아미드-프로피온알데히드의 배합Combination of Interferon-beta and Pendant-type PEG-amide-Propionaldehyde
위 실시예 3에서 제조한 분자량 5,000∼1,000,000의 PEG 뼈대에 1∼20 개의 아미드-프로피온알데히드 그룹을 갖는 펜던트 PEG-아미드-프로피온알데히드와 인터페론-베타 단백질을 배합하는데, 인터페론:PEG의 몰비율이 1:1∼1:5가 되도록 준비하고 소디움 시아노보로하이드라이드를 포함하는 NaH2PO4, pH 6.0 완충용액에서 반응시켜 PEG-인터페론-베타 배합체를 형성하였다. 반응은 실온에서 5∼20 시간 동안 수행하며, 배합체의 완성은 SDS-폴리아크릴아미드 전기영동으로 확인하였다.A PEG PEG-amide-propionaldehyde having 1 to 20 amide-propionaldehyde groups and an interferon-beta protein are mixed with a PEG skeleton having a molecular weight of 5,000 to 1,000,000 prepared in Example 3, wherein the molar ratio of interferon: PEG is 1 It was prepared to be 1: 1: 5 and reacted in NaH 2 PO 4 , pH 6.0 buffer containing sodium cyanoborohydride to form a PEG-interferon-beta blend. The reaction was carried out at room temperature for 5-20 hours and the completion of the formulation was confirmed by SDS-polyacrylamide electrophoresis.
[실시예 7]Example 7
인터페론-베타와 펜던트형 PEG-우레탄-프로피온알데히드의 배합Combination of interferon-beta and pendant PEG-urethane-propionaldehyde
분자량 5,000∼1,000,000의 PEG 뼈대에 1∼20 개의 우레탄-프로피온알데히드 그룹을 갖는 펜던트 PEG-우레탄-프로피온알데히드와 인터페론-베타 단백질을 배합하는데, 인터페론:PEG의 몰비율이 1:1∼1:5가 되도록 준비하고 소디움 시아노보로하이드라이드를 포함하는 NaH2PO4, pH 6.0 완충용액에서 반응시켜 PEG-인터페론-베타 배합체를 형성하였다. 반응은 실온에서 5∼20 시간 동안 수행하며, 배합체의 완성은 SDS-폴리아크릴아미드 전기영동으로 확인하였다.A pendant PEG-urethane-propionaldehyde having 1 to 20 urethane-propionaldehyde groups and an interferon-beta protein are mixed in a PEG skeleton having a molecular weight of 5,000 to 1,000,000. The molar ratio of interferon: PEG is 1: 1 to 1: 5. Was prepared and reacted in NaH 2 PO 4 , pH 6.0 buffer with sodium cyanoborohydride to form a PEG-interferon-beta combination. The reaction was carried out at room temperature for 5-20 hours and the completion of the formulation was confirmed by SDS-polyacrylamide electrophoresis.
본 발명에 따라 제조된 인터페론-베타의 아미노 말단의 알파-아미노기에 메톡시PEG-프로피온알데히드 유도체가 결합된 배합체는, 인터페론-베타의 항원유발성을 감소시키고 생리활성 저하를 최대한 감소시키면서 체내 잔존 시간을 증가시켜 향상된 약동학 프로필(pharmacokinetic profile)과 약리적 성질을 갖게 되므로, 다발성 경화증의 치료제 및 바이러스성 암 치료제 등으로 유용하게 사용될 수 있다.The combination in which the methoxyPEG-propionaldehyde derivative is bonded to the alpha-amino group of the amino terminus of the interferon-beta prepared according to the present invention, remains in the body while reducing the antigen-induced activity of interferon-beta and maximally reducing the degradation of bioactivity Since the pharmacokinetic profile and pharmacological properties are improved by increasing the time, it may be usefully used as a therapeutic agent for multiple sclerosis and a viral cancer therapeutic agent.
Claims (5)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2001-0076130A KR100480430B1 (en) | 2001-12-04 | 2001-12-04 | Conjugates of interferon-beta and polyethylene glycol derivatives |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2001-0076130A KR100480430B1 (en) | 2001-12-04 | 2001-12-04 | Conjugates of interferon-beta and polyethylene glycol derivatives |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20030045414A true KR20030045414A (en) | 2003-06-11 |
| KR100480430B1 KR100480430B1 (en) | 2005-04-06 |
Family
ID=29572847
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR10-2001-0076130A KR100480430B1 (en) | 2001-12-04 | 2001-12-04 | Conjugates of interferon-beta and polyethylene glycol derivatives |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR100480430B1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100488351B1 (en) * | 2001-12-11 | 2005-05-11 | 선바이오(주) | Novel polyethylene glycol-propionaldehyde derivatives |
| WO2006116948A1 (en) * | 2005-04-30 | 2006-11-09 | Chengdu Institute Of Biological Products | Interleukin-6 polyethylene glycol conjugate and its preparing method and use |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5252714A (en) * | 1990-11-28 | 1993-10-12 | The University Of Alabama In Huntsville | Preparation and use of polyethylene glycol propionaldehyde |
| US5951974A (en) * | 1993-11-10 | 1999-09-14 | Enzon, Inc. | Interferon polymer conjugates |
| US5738846A (en) * | 1994-11-10 | 1998-04-14 | Enzon, Inc. | Interferon polymer conjugates and process for preparing the same |
| KR100511749B1 (en) * | 2001-11-06 | 2005-09-02 | 선바이오(주) | Modified interferon-beta, and chemically modified conjugates thereof |
-
2001
- 2001-12-04 KR KR10-2001-0076130A patent/KR100480430B1/en active IP Right Grant
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100488351B1 (en) * | 2001-12-11 | 2005-05-11 | 선바이오(주) | Novel polyethylene glycol-propionaldehyde derivatives |
| WO2006116948A1 (en) * | 2005-04-30 | 2006-11-09 | Chengdu Institute Of Biological Products | Interleukin-6 polyethylene glycol conjugate and its preparing method and use |
Also Published As
| Publication number | Publication date |
|---|---|
| KR100480430B1 (en) | 2005-04-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9364553B2 (en) | Synergistic biomolecule-polymer conjugates | |
| US7201897B2 (en) | Interferon conjugates | |
| EP1039921B1 (en) | Substantially pure histidine-linked protein polymer conjugates | |
| EP1039922B1 (en) | Improved interferon polymer conjugates | |
| EP0730470B1 (en) | Improved interferon polymer conjugates | |
| PL196533B1 (en) | Method for the stepwise attachment of polyethylene glycol (peg) in series to polypeptide | |
| US20030103934A1 (en) | Drugs having long-term retention in target tissue | |
| US20090117077A1 (en) | Polyethylene glycol-interferon alpha conjugate | |
| KR100480430B1 (en) | Conjugates of interferon-beta and polyethylene glycol derivatives | |
| KR100480429B1 (en) | Conjugates of interferon-alpha and polyethylene glycol derivatives | |
| KR100480432B1 (en) | Conjugates of granulocyte-colony stimulating factor and polyethylene glycol derivatives | |
| KR100480423B1 (en) | Conjugates of erythropoietin and polyethylene glycol derivatives | |
| KR100888371B1 (en) | Antivirus agent comprising bridged co-polymer derivatives and interferon complexes | |
| KR100761652B1 (en) | multi-branched polymer used in conjugating protein or peptide, and resulting conjugator | |
| JPWO2003000278A1 (en) | Ointment | |
| KR20070110162A (en) | Polyethylene glycol-interferon alpha conjugate | |
| MXPA00010223A (en) | Polyol-ifn-beta conjugates |
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: 20130311 Year of fee payment: 9 |
|
| FPAY | Annual fee payment |
Payment date: 20140324 Year of fee payment: 10 |
|
| FPAY | Annual fee payment |
Payment date: 20160323 Year of fee payment: 12 |
|
| FPAY | Annual fee payment |
Payment date: 20170323 Year of fee payment: 13 |
|
| FPAY | Annual fee payment |
Payment date: 20180323 Year of fee payment: 14 |
|
| FPAY | Annual fee payment |
Payment date: 20190318 Year of fee payment: 15 |