JPH02231077A - Superoxide dismutase modified with heparin - Google Patents
Superoxide dismutase modified with heparinInfo
- Publication number
- JPH02231077A JPH02231077A JP1050004A JP5000489A JPH02231077A JP H02231077 A JPH02231077 A JP H02231077A JP 1050004 A JP1050004 A JP 1050004A JP 5000489 A JP5000489 A JP 5000489A JP H02231077 A JPH02231077 A JP H02231077A
- Authority
- JP
- Japan
- Prior art keywords
- sod
- heparin
- modified
- enzyme
- life
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
- 102000019197 Superoxide Dismutase Human genes 0.000 title claims abstract description 44
- 108010012715 Superoxide dismutase Proteins 0.000 title claims abstract description 44
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical group OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 title abstract description 24
- 229920000669 heparin Polymers 0.000 title abstract description 22
- 229960002897 heparin Drugs 0.000 title abstract description 22
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 claims abstract description 7
- 102000004190 Enzymes Human genes 0.000 abstract description 9
- 108090000790 Enzymes Proteins 0.000 abstract description 9
- 210000004369 blood Anatomy 0.000 abstract description 9
- 239000008280 blood Substances 0.000 abstract description 9
- 125000003277 amino group Chemical group 0.000 abstract description 5
- MGNCLNQXLYJVJD-UHFFFAOYSA-N cyanuric chloride Chemical compound ClC1=NC(Cl)=NC(Cl)=N1 MGNCLNQXLYJVJD-UHFFFAOYSA-N 0.000 abstract description 5
- 125000002887 hydroxy group Chemical group [H]O* 0.000 abstract description 4
- 230000000144 pharmacologic effect Effects 0.000 abstract description 4
- 230000000451 tissue damage Effects 0.000 abstract description 4
- 231100000827 tissue damage Toxicity 0.000 abstract description 4
- 230000002255 enzymatic effect Effects 0.000 abstract description 2
- JYEUMXHLPRZUAT-UHFFFAOYSA-N 1,2,3-triazine Chemical group C1=CN=NN=C1 JYEUMXHLPRZUAT-UHFFFAOYSA-N 0.000 abstract 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 abstract 1
- 230000001747 exhibiting effect Effects 0.000 abstract 1
- 238000010253 intravenous injection Methods 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 16
- 230000014759 maintenance of location Effects 0.000 description 7
- 238000000034 method Methods 0.000 description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 208000002177 Cataract Diseases 0.000 description 1
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 229920001202 Inulin Polymers 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 206010038933 Retinopathy of prematurity Diseases 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- 241000219315 Spinacia Species 0.000 description 1
- 235000009337 Spinacia oleracea Nutrition 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 229940009456 adriamycin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000006854 communication Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000007323 disproportionation reaction Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(OC[C@]3(OC[C@]4(OC[C@]5(OC[C@]6(OC[C@]7(OC[C@]8(OC[C@]9(OC[C@]%10(OC[C@]%11(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 description 1
- 229940029339 inulin Drugs 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000004731 jugular vein Anatomy 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- -1 osteoarthritis Chemical compound 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 229920001515 polyalkylene glycol Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- OVARTBFNCCXQKS-UHFFFAOYSA-N propan-2-one;hydrate Chemical compound O.CC(C)=O OVARTBFNCCXQKS-UHFFFAOYSA-N 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229940014800 succinic anhydride Drugs 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、生体内の酸素分子から発生したスーパーオキ
シド（０■）による組織障害の治療に有用なヘパリンで
修飾されたスーパーオキシドジスムターゼ（以下、ＳＯ
Ｄと略す）に関するものである．
〔従来技術の説明〕
スーパーオキシドジスムターゼ（ＳＯＤ）は、下式に示
す不均化反応によってスーパーオキシド（０；）を消失
させる作用を持つ酵素である．ＳＯＤ
２０富＋２ Ｈ Ｏｘ ＋Ｈｚ Ｏｘし
た０丁による組織障害、例えば、変形性関節炎、慢性関
節リウマチ、放射線照射による障害、紫外線による障害
、未熟児酸素網膜症、白内障、アドリアマイシンなどの
制癌剤の副作用、虚血部分への血流再開に伴う障害など
に対する有効な治療薬として注目されている．
このようにＳＯＤが医薬として有望であるにもかかわら
ず、ＳＯＤの血流内半減期が非常に短い（約５分）ため
に、その薬理活性が充分に発揮されない場合が多い．
ＳＯＤの血流内半減期が非常に短い原因としては、その
分子量（３２．０００）が腎糸球体の濾遇限界値（分子
量で約５０，０００）よりも小さいために血中から速や
かに消失し、尿中に排泄されることが考えられている．
従って、ＳＯＤの薬理活性を充分に発揮させるために、
ポリエチレングリコール（Ｐｙａｔｏｋ，Ｐ．Ｓ．ｅｔ
ａｌ，；Ｒｅｓｅａｒｃｈ Ｃｏｍｍｕｎｉｃａ
ｔｉｏｎｓ ｉｎ Ｃｈｅｍｉｃａｌ Ｐａｔｈ
ｏｌｏｇｙ ａｎｄ Ｐｈａｒｍ従って、ＳＯＤは
、生体内で酸素分子から発生ａｃｏｌｏｇｙ．２９．１
１３ （１９８０））、ラフトアルブミン（Ｗｏｎｇ，
Ｋ，ｅｔ ａｌ，；Ａｇｅｎｔ ａｎｄ Ａｃｔ
ｉｏｎｓ，上立，２３１ （１９８０））、フイコー
ル（ＭｃＣｏｒｄ，Ｊ，Ｍ，ｅｔ ａｌ．；Ｐｒｏｃ
ｅｅｄｉｎｇｓ ｏｆ Ｎａｔｉｏｎａｌ Ａｃ
ａｄｅｍｙｏｆ Ｓｃｉｅｎｃｅｓ，Ｕ．Ｓ．Ａ．，
１１．１１５９ （１９８０）］、ポリアルキレングリ
コール（特開昭６１−２４９３８８）やイヌリン（特開
昭５８−３２８２６）などを用１，ＮてＳＯＤを巨大分
子化させ、ＳＯＤの血中半減期を増加させる試みがなさ
れている．
ところで、巨大分子化したＳＯＤを通常の静脈投与剤と
して使用する場合には、その血中半減期が長くなるのみ
ならず、その酵素活性保持率が高く、かつ医薬としての
安全性が高いことが望ましい．
しかしながら、これらの巨大分子化ＳＯＤには、酵素活
性保持率、血中半減期の長さ、抗原性および医薬として
の安全性に対して十分には満足できないという問題があ
る．
〔発明が解決しようとする問題点〕
本発明の目的は、生体内の酸素分子から発生したＯ；に
よる組織障害の治療に有用なＳＯＤと医薬として安全性
が確認されているヘパリンとを結合させることによって
得られた修飾ＳＯＤ（以下、ｒ修飾ＳＯＤＪと略す）を
提供するものである．〔問題点を解決するための手段〕
本発明者らは、前記の問題点を解決するために鋭意研究
した結果、本発明の『修飾ＳＯＤＪは、ＳＯＤ修飾に伴
うＳＯＤ活性の低下は殆ど認められず、また、ｒ修飾Ｓ
ＯＤＪの血中半減期も顕著に長くなることを見出し、本
発明を完成するに至った．
即ち、本発明は、ヘパリンで修飾されたＳＯＤに関する
ものである．
以下、本発明について詳しく説明する．本発明の『修飾
ＳＯＤＪは、ＳＯＤと多糖類であるヘパリンとを化学的
に結合させて得られたものであり、約９０％の酵素活性
保持率と約１３時間の血中半減期を示すものである．
本発明のｒ修飾ＳＯＤＪの作製に用いるＳＯＤとしては
、ウシ、ヒトなどの動物、ホウレン草などの植物、及び
セラチアなどの微生物に由来するものを挙げることがで
きるが、ヒトに対する抗原性を考慮した医薬のｒ修飾Ｓ
ＯＤＪとしては、ヒ｝ＳＯＤを用いることが好まルい．
そのようなヒトＳＯＤとしては、ヒト赤血球、胎盤など
のＳＯＤを用いることもできるが、近年、遺伝子工学技
術を応用して生産されたヒ｝ＳＯＤ（例えば、特開昭６
１−１１１６９０など）を用いると、大量に安定した試
料を得られるのでさらに好ましい．
本発明のｒ修飾ＳＯＤＪの製造に用いるヘパリンとして
は、分子量が５，０００〜２０．０００のものが好まし
い．
本発明のｒ修飾ＳＯＤＪの製造におけるＳＯＤとヘバリ
ンとの結合割合は、１分子のＳＯＤ当たり１〜２０分子
がよ《、好ましくは２〜１２分子がよい．
本発明のｒ修飾ＳＯＤＪは、ＳＯＤの官能基（例えば、
アミノ基またはカルボキシル基）とヘパリンの官能基（
例えば、カルボキシル基、アミノ基または水酸基）とを
利用して、好まし《はＰＨ ６． ０〜１０、さらに好
まし《はｐ Ｈ ７． ０〜８．５で０．１〜１０％の
濃度としたＳＯＤと活性化ヘパリンを結合させたもので
あり、例えば、■ヘパリンの水酸基に塩化シアヌルを反
応させた後、これをそのトリアジン環を介してＳＯＤの
アミノ基に結合させることによってＳＯＤとヘバリンと
を結合させる方法
■ヘパリンのカルボキシル基をＮ−ヒドロキシコハク酸
とジシクロへキシルカルボジイミドとを用いてエステル
を導入し、これをＳＯＤのアミノ基に結合させることに
よってＳＯＤとヘバリンとを結合させる方法
■ヘパリンの水酸基に無水コハク酸を用いてカルボキシ
ル基を導入し、さらにそのカルボキシル基をＮ−ヒドロ
キシコハク酸とジシクロへキシルカルボジイミドとを用
いてエステルを導入し、これをＳＯＤのアミノ基に結合
させることによってＳＯＤとヘバリンとを結合させる方
法
などの方法で作製することができる。Detailed Description of the Invention [Industrial Field of Application] The present invention provides heparin-modified superoxide dismutase (hereinafter referred to as "superoxide dismutase") useful for the treatment of tissue damage caused by superoxide (0) generated from oxygen molecules in the body. , S.O.
(abbreviated as D). [Description of the Prior Art] Superoxide dismutase (SOD) is an enzyme that has the effect of eliminating superoxide (0;) by a dismutation reaction shown in the following formula. SOD 20 rich + 2 H Ox +Hz Tissue damage caused by oxygen, such as osteoarthritis, chronic rheumatoid arthritis, damage caused by radiation, damage caused by ultraviolet rays, oxygen retinopathy of prematurity, cataracts, side effects of anticancer drugs such as adriamycin, and depression. It is attracting attention as an effective treatment for disorders associated with the resumption of blood flow to blood areas. Although SOD is promising as a medicine, its pharmacological activity is often not fully demonstrated because its half-life in the bloodstream is very short (approximately 5 minutes). The reason why SOD has a very short half-life in the bloodstream is that its molecular weight (32,000) is smaller than the filtering limit of the renal glomerulus (approximately 50,000 molecular weight), so it disappears from the blood quickly. It is thought that it is excreted in the urine. Therefore, in order to fully demonstrate the pharmacological activity of SOD,
Polyethylene glycol (Pyatok, P.S.et
al,; Research Communica
tions in Chemical Path
Therefore, SOD is generated from oxygen molecules in the living body. 29.1
13 (1980)), raftalbumin (Wong,
K, et al; Agent and Act
ions, Kamidate, 231 (1980)), McCord, J, M, et al.; Proc.
eedings of National Ac
ademyofSciences,U. S. A. ，
11.1159 (1980)], polyalkylene glycol (JP-A No. 61-249388), inulin (JP-A No. 58-32826), etc. were used to make SOD into a macromolecule, and the half-life of SOD in the blood was reduced. Attempts are being made to increase it. By the way, when using macromolecular SOD as a regular intravenous drug, it not only has a long half-life in the blood, but also has a high enzyme activity retention rate and is highly safe as a medicine. desirable. However, these macromolecular SODs have problems in that they are not fully satisfactory in terms of enzyme activity retention, blood half-life, antigenicity, and pharmaceutical safety. [Problems to be Solved by the Invention] The purpose of the present invention is to combine SOD, which is useful in the treatment of tissue damage caused by O generated from oxygen molecules in the living body, with heparin, which has been confirmed to be safe as a medicine. The modified SOD obtained by this method (hereinafter abbreviated as r-modified SODJ) is provided. [Means for Solving the Problems] As a result of intensive research to solve the above-mentioned problems, the present inventors have found that the "modified SODJ" of the present invention shows almost no decrease in SOD activity due to SOD modification. Also, r modification S
The inventors discovered that the half-life of ODJ in the blood was also significantly longer, leading to the completion of the present invention. That is, the present invention relates to SOD modified with heparin. The present invention will be explained in detail below. The "modified SODJ" of the present invention is obtained by chemically bonding SOD with heparin, a polysaccharide, and exhibits an enzyme activity retention rate of about 90% and a blood half-life of about 13 hours. It is. Examples of the SOD used in the production of the r-modified SODJ of the present invention include those derived from animals such as cows and humans, plants such as spinach, and microorganisms such as Serratia. r modification S
It is preferable to use SOD as ODJ. As such human SOD, human SOD such as human red blood cells and placenta can be used, but in recent years, human SOD produced by applying genetic engineering technology (for example,
1-111690, etc.) is more preferable because a stable sample can be obtained in large quantities. The heparin used for producing the r-modified SODJ of the present invention preferably has a molecular weight of 5,000 to 20,000. The binding ratio of SOD and heparin in the production of r-modified SODJ of the present invention is preferably 1 to 20 molecules, preferably 2 to 12 molecules per molecule of SOD. The r-modified SODJ of the present invention has functional groups of SOD (for example,
amino group or carboxyl group) and the functional group of heparin (
For example, by using a carboxyl group, an amino group, or a hydroxyl group, preferably << is PH 6. 0 to 10, more preferably pH 7. It combines SOD with a concentration of 0 to 8.5 and activated heparin at a concentration of 0.1 to 10%. For example, 1. After reacting the hydroxyl group of heparin with cyanuric chloride, A method for bonding SOD and heparin by bonding to the amino group of SOD through Method for bonding SOD and heparin by bonding ■ Introducing a carboxyl group to the hydroxyl group of heparin using succinic anhydride, and then converting the carboxyl group into an ester using N-hydroxysuccinic acid and dicyclohexylcarbodiimide. It can be produced by a method such as a method in which SOD and hevarin are bonded by introducing and bonding this to the amino group of SOD.
このようにして、ヘパリンとＳＯＤとを結合することに
よって得られる『修飾ＳＯＤＪは、８７％の酵素活性保
持率を有するものである．〔実施例〕
以下、本発明を実施例によって具体的に説明する．なお
、これらの実施例は、本発明を例示するためのものであ
って、本発明の範囲を限定するものではない．
本発明の実施例に示した『修飾ＳＯＤＪの活性保持率は
、大柳の示した方法（Ｏｙａｎａｇｕｉ，Ｙ．；Ａｎａ
ｌｙｔｉｃａｌ Ｂｉｏｃｈｅｍｉｓｔｒｙ．１４２
，２９０−２９６ （１９８４）〕に準じてヘパリンの
修飾前後におけるＳＯＤの比活性を求め、その変化から
求めた．また、ヘパリンがＳＯＤ１分子当たり何分子結
合するかは、分析用の高速ゲル濾過力ラムである３ ０
０ ０ ＰＷおよび５０００ＰＷ（いづれも、東ソー
社製）を用いてｒ修飾ＳＯＤＪの分子量を測定し、その
分子量とＳＯＤの分子量とを比較することによって決定
した．
実施例１
１００ｍｇのヘパリン（ミドリ十字社製のヘパリン注射
剤を透析して得た。分子量は５，０００〜２０、０００
．）を２ｍｆの水に溶解し、これに室温で塩化シアヌル
液〔１ｍｌの水−アセトン溶液（１対２の混合体積比）
に塩化シアヌル１０ｍｇ溶解．〕を０． Ｉ Ｎの水酸
化ナトリウムを用いてｐ Ｈ ９． ５±１．０に調整
しながら１０分間で滴下した後、０． ０ ５ Ｎの塩
酸を加えてｐＨを３以下に調整し、さらに、その１０倍
容量のアセトンを加えて生じた沈澱を濾過分取し、それ
をアセトンで洗浄することによって７ ０ｍｇの乾燥活
性化ヘパリンを得た．
このようにして得られた５ ０ｍｇの活性化ヘパリンを
ヒトＣｕ，Ｚｎ−ＳＯＤ溶液〔特開昭６１−１１１６９
０号に示されたヒトＣｕ，Ｚｎ−ＳＯＤ生産菌Ｅ，ｃｏ
ｌｔ Ｗ３１１０ （ｐＵＢＥ２）で生産し、精製し
て得られた５ｍｇを２ｍｌの０．１Ｍホウ酸緩衝液（ｐ
｝１９．２）に溶解。〕に徐々に加えて４゜Ｃで穏やか
に１２時間攪拌した。In this way, the ``modified SODJ'' obtained by combining heparin and SOD has an enzymatic activity retention rate of 87%. [Example] Hereinafter, the present invention will be specifically explained with reference to Examples. It should be noted that these Examples are for illustrating the present invention, and are not intended to limit the scope of the present invention. The activity retention rate of the modified SODJ shown in the Examples of the present invention was determined by the method shown by Oyanagui (Oyanagui, Y.;
Lytical Biochemistry. 142
, 290-296 (1984)], the specific activity of SOD was determined before and after modification of heparin, and the specific activity was determined from the change. In addition, the number of molecules of heparin bound per SOD molecule can be determined using a high-speed gel filtration ram for analysis.
The molecular weight of r-modified SODJ was measured using 00 PW and 5000 PW (both manufactured by Tosoh Corporation), and the molecular weight was determined by comparing the molecular weight with that of SOD. Example 1 100 mg of heparin (obtained by dialysis of heparin injection manufactured by Midori Juji Co., Ltd.) Molecular weight: 5,000 to 20,000
．． ) in 2 mf of water, and add cyanuric chloride solution [1 ml of water-acetone solution (mixed volume ratio of 1:2) to this at room temperature.
Dissolve 10 mg of cyanuric chloride in. ]0. pH 9 using IN sodium hydroxide. After dropping for 10 minutes while adjusting to 5±1.0, 0. The pH was adjusted to 3 or less by adding 0 5 N hydrochloric acid, and then 10 times the volume of acetone was added, the resulting precipitate was collected by filtration, and washed with acetone to obtain 70 mg of dry activation. I got heparin. 50 mg of activated heparin thus obtained was added to a human Cu, Zn-SOD solution
Human Cu, Zn-SOD producing bacteria E, co shown in No. 0
lt W3110 (pUBE2), 5 mg obtained by purification was added to 2 ml of 0.1M borate buffer (pUBE2).
}19.2). ] and gently stirred at 4°C for 12 hours.
その後、水に対して透析し、さらに凍結乾燥することに
よて１ ５ｍｇのｒ修飾ＳＯＤＪを得た。Thereafter, 15 mg of r-modified SODJ was obtained by dialysis against water and further freeze-drying.
ヘパリンはＳＯＤＩ分子当たり約３〜１０分子結合して
おり、その酵素活性保持率は８７％であった．
実施例２
ウイスタ一系ラット（♂、体重は２５０±３０ｇ）の２
匹に、生理食塩水に溶解した実施例１のｒ修飾ＳＯＤＪ
溶液を、１匹当たりＳＯＤの蛋白質量として０． ６
ｍ ｇづつ総頚静脈へ注入した。Approximately 3 to 10 molecules of heparin were bound per SODI molecule, and the retention rate of the enzyme activity was 87%. Example 2 Wista strain rat (male, weight 250±30g) 2
r-modified SODJ of Example 1 dissolved in physiological saline.
The solution was expressed as a protein amount of SOD per animal of 0. 6
mg each was injected into the common jugular vein.
注入から、２分、５分、１５分、３０分、６０分、９０
分、１２０分、１５０分、１８０分経過時に０． ４
ｍ ｌづつ採血し、その血漿中のＳＯＤ量を前記の大柳
のＳＯＤ活性測定法で測定した．ｒ修飾ＳＯＤＪの血流
内半減期を求めるために、これらの血漿中のＳＯＤの相
対活性を時間に対してプロットした結果、その半減期は
、約１３時間であった．
比較例１
未修飾のＳＯＤの血流内半減期を求めるために、実施例
２の場合と同様にして、ウィスタ一系ラット（♂、体重
は２５０±３０ｇ）を用いて血漿中のＳＯＤの相対活性
及び相対濃度を時間に対してプロットした結果、その半
減期は、相対活性及び相対濃度のいずれの場合において
も約５分であった．
〔発明の効果〕
本発明の酵素活性保持率が高く、かつ血流内半減期が改
善されたｒ修飾ＳＯＤＪは、静脈投与剤としてのＳＯＤ
の薬理効果を高めるものである．特許出願人 宇部興
産株式会社2 minutes, 5 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes after injection
0 minutes, 120 minutes, 150 minutes, 180 minutes elapsed. 4
Blood was collected in ml portions, and the amount of SOD in the plasma was measured using the Oyanagi SOD activity assay method described above. To determine the half-life of r-modified SODJ in the bloodstream, the relative activity of these SODs in plasma was plotted against time, and the half-life was approximately 13 hours. Comparative Example 1 In order to determine the half-life of unmodified SOD in the bloodstream, the relative amount of SOD in plasma was determined using Wista strain rats (male, weight 250±30 g) in the same manner as in Example 2. As a result of plotting the activity and relative concentration against time, the half-life was approximately 5 minutes for both relative activity and relative concentration. [Effects of the Invention] The r-modified SODJ of the present invention, which has a high enzyme activity retention rate and an improved half-life in the bloodstream, can be used as an intravenous agent for SOD.
It enhances the pharmacological effects of. Patent applicant: Ube Industries, Ltd.
Claims (1)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1050004A JPH02231077A (en) | 1989-03-03 | 1989-03-03 | Superoxide dismutase modified with heparin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1050004A JPH02231077A (en) | 1989-03-03 | 1989-03-03 | Superoxide dismutase modified with heparin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH02231077A true JPH02231077A (en) | 1990-09-13 |
Family
ID=12846856
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1050004A Pending JPH02231077A (en) | 1989-03-03 | 1989-03-03 | Superoxide dismutase modified with heparin |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02231077A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5834273A (en) * | 1991-03-28 | 1998-11-10 | Wako Pure Chemical Industries, Ltd. | Heat-stable and water soluble modified enzymes |
| US8106007B2 (en) | 2005-02-01 | 2012-01-31 | N.V. Organon | Conjugates of a polypeptide and a pentasaccharide |
-
1989
- 1989-03-03 JP JP1050004A patent/JPH02231077A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5834273A (en) * | 1991-03-28 | 1998-11-10 | Wako Pure Chemical Industries, Ltd. | Heat-stable and water soluble modified enzymes |
| US8106007B2 (en) | 2005-02-01 | 2012-01-31 | N.V. Organon | Conjugates of a polypeptide and a pentasaccharide |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2015242970B2 (en) | Glycopolysialylation of non-blood coagulation proteins | |
| US5177059A (en) | Polymyxin B conjugates | |
| JP2022058911A (en) | Glycopolysialylation of non-blood coagulation protein | |
| ES2284670T3 (en) | PROCEDURE FOR MANUFACTURE OF SYNTHETIC OXYGEN VEHICLES FROM COVALENTLY RETICULATED HEMOGLOBINS WITH FUNCTIONAL PROPERTIES IMPROVED BY RETICULATION IN THE PRESENCE OF EFFECTS THAT DO NOT CHEMICALLY REACT THE OXYGEN BODY AFFINITY. | |
| JPH10504347A (en) | Water-soluble polyene complex | |
| JP2515389B2 (en) | Superoxide dismutase conjugate | |
| KR0184858B1 (en) | Polymyxin conjugates | |
| JPH02231078A (en) | Superoxide dismutase modified with hyaluronic acid | |
| JPH03147784A (en) | Production of modified superoxide dismutase | |
| ES2395894T3 (en) | Mammalian hemoglobins compatible with blood plasma, cross-linked and conjugated with poly (alkylene oxides) as artificial medical oxygen carriers, their preparation and use | |
| JPH03167102A (en) | Antimicrobial agent | |
| CA2314716C (en) | Modified polysaccharides exhibiting altered biological recognition | |
| JPH02231077A (en) | Superoxide dismutase modified with heparin | |
| JP3209338B2 (en) | Polyethylene glycol-modified arginine deiminase and method for producing the same | |
| JPH02231075A (en) | Superoxide dismutase modified with dextran sulfate | |
| JPH02231076A (en) | Superoxide dismutase modified with dextran | |
| JP2545729B2 (en) | Polymer conjugate of methotrexate derivative and pyran copolymer and method for producing the same | |
| AU711200B2 (en) | Polymyxin conjugates | |
| JPH0195774A (en) | Modified superoxide dismutase | |
| US20030236224A1 (en) | Modified polysaccharides exhibiting altered biological recognition | |
| US7307150B2 (en) | Dextran-hemoglobin conjugates as blood substitutes | |
| CA2380908A1 (en) | Plasma substitute composition | |
| CA2561394A1 (en) | Production and use of the methotrexate-albumin conjugate as an immunosuppressive agent in gvhd | |
| RU2556378C2 (en) | Conjugate of glycoprotein, possessing erythropoietin activity, with poly-1,4-ethylenepiperazine n-oxide derivatives (versions), pharmaceutical composition and method of conjugate obtaining | |
| JPS63313581A (en) | Modified superoxide dismutase |