JPH0785717B2 - Method for producing highly active superoxide dismutase polymer derivative - Google Patents
Method for producing highly active superoxide dismutase polymer derivativeInfo
- Publication number
- JPH0785717B2 JPH0785717B2 JP3155575A JP15557591A JPH0785717B2 JP H0785717 B2 JPH0785717 B2 JP H0785717B2 JP 3155575 A JP3155575 A JP 3155575A JP 15557591 A JP15557591 A JP 15557591A JP H0785717 B2 JPH0785717 B2 JP H0785717B2
- Authority
- JP
- Japan
- Prior art keywords
- sod
- formula
- integer
- polymer
- superoxide dismutase
- 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.)
- Expired - Lifetime
Links
- 102000019197 Superoxide Dismutase Human genes 0.000 title claims description 71
- 108010012715 Superoxide dismutase Proteins 0.000 title claims description 71
- 229920000642 polymer Polymers 0.000 title claims description 29
- 238000004519 manufacturing process Methods 0.000 title claims description 6
- 230000000694 effects Effects 0.000 claims description 22
- 239000000243 solution Substances 0.000 claims description 15
- 229920001223 polyethylene glycol Polymers 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 11
- 125000003277 amino group Chemical group 0.000 claims description 10
- 238000009739 binding Methods 0.000 claims description 10
- 239000002202 Polyethylene glycol Substances 0.000 claims description 6
- 229920001577 copolymer Polymers 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 5
- 150000001875 compounds Chemical class 0.000 claims description 4
- MFGALGYVFGDXIX-UHFFFAOYSA-N 2,3-Dimethylmaleic anhydride Chemical compound CC1=C(C)C(=O)OC1=O MFGALGYVFGDXIX-UHFFFAOYSA-N 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 230000002378 acidificating effect Effects 0.000 claims description 2
- VYVFOKZBASRWJJ-UHFFFAOYSA-N 3-(2-ethenylbut-3-enoxymethyl)penta-1,4-diene furan-2,5-dione Chemical compound O=C1OC(=O)C=C1.C=CC(C=C)COCC(C=C)C=C VYVFOKZBASRWJJ-UHFFFAOYSA-N 0.000 claims 1
- 101000731004 Rattus norvegicus Membrane-associated progesterone receptor component 1 Proteins 0.000 claims 1
- 241001627203 Vema Species 0.000 claims 1
- 238000000746 purification Methods 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 description 14
- 108090000790 Enzymes Proteins 0.000 description 14
- 229940088598 enzyme Drugs 0.000 description 14
- 238000001727 in vivo Methods 0.000 description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000000126 substance Substances 0.000 description 7
- QYKIQEUNHZKYBP-UHFFFAOYSA-N Vinyl ether Chemical compound C=COC=C QYKIQEUNHZKYBP-UHFFFAOYSA-N 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 229960000834 vinyl ether Drugs 0.000 description 6
- 241000700159 Rattus Species 0.000 description 5
- 230000003110 anti-inflammatory effect Effects 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 4
- 102000035195 Peptidases Human genes 0.000 description 4
- 108091005804 Peptidases Proteins 0.000 description 4
- CGBYBGVMDAPUIH-UHFFFAOYSA-N acide dimethylmaleique Natural products OC(=O)C(C)=C(C)C(O)=O CGBYBGVMDAPUIH-UHFFFAOYSA-N 0.000 description 4
- CGBYBGVMDAPUIH-ARJAWSKDSA-N dimethylmaleic acid Chemical compound OC(=O)C(/C)=C(/C)C(O)=O CGBYBGVMDAPUIH-ARJAWSKDSA-N 0.000 description 4
- DQJJMWZRDSGUJP-UHFFFAOYSA-N ethenoxyethene;furan-2,5-dione Chemical compound C=COC=C.O=C1OC(=O)C=C1 DQJJMWZRDSGUJP-UHFFFAOYSA-N 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 239000003223 protective agent Substances 0.000 description 4
- 238000000108 ultra-filtration Methods 0.000 description 4
- 229920000147 Styrene maleic anhydride Polymers 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 208000027866 inflammatory disease Diseases 0.000 description 3
- FPYJFEHAWHCUMM-UHFFFAOYSA-N maleic anhydride Chemical compound O=C1OC(=O)C=C1 FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 description 3
- 125000006239 protecting group Chemical group 0.000 description 3
- 230000000717 retained effect Effects 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- PYSRRFNXTXNWCD-UHFFFAOYSA-N 3-(2-phenylethenyl)furan-2,5-dione Chemical compound O=C1OC(=O)C(C=CC=2C=CC=CC=2)=C1 PYSRRFNXTXNWCD-UHFFFAOYSA-N 0.000 description 1
- 208000009137 Behcet syndrome Diseases 0.000 description 1
- 206010009900 Colitis ulcerative Diseases 0.000 description 1
- 208000011231 Crohn disease Diseases 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
- 241001465754 Metazoa Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 206010063837 Reperfusion injury Diseases 0.000 description 1
- 201000006704 Ulcerative Colitis Diseases 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 229940124599 anti-inflammatory drug Drugs 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000004534 cecum Anatomy 0.000 description 1
- 206010008118 cerebral infarction Diseases 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229910001867 inorganic solvent Inorganic materials 0.000 description 1
- 239000003049 inorganic solvent Substances 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 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 1
- 230000003211 malignant effect Effects 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 206010034674 peritonitis Diseases 0.000 description 1
- -1 polyethylene Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000010410 reperfusion Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
【０００１】[0001]
【産業上の利用分野】本発明は、高活性で低抗癌性ので
表されるスーパーオキシドジスムターゼ（ＳＯＤ）高分
子誘導体の製造方法に関するものであり、抗炎症薬とし
ての応用が期待される。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a superoxide dismutase (SOD) polymer derivative having high activity and low anticancer property, and is expected to be applied as an anti-inflammatory drug.
【０００２】[0002]
【従来の技術】スーパーオキシドジスムターゼ（ＳＯ
Ｄ）は生体内の炎症障害の原因となる活性酵素（スーパ
ーオキシド）を分解する分子量約3.2万の酵素である。
そのため炎症に起因する多くの難治性疾患に対して有効
性が期待されている。例えば、腎臓や心臓の移植・心筋
梗塞や脳梗塞の治療後血液の循環を開始した時に起こる
再還流障害、ベーチェット病、悪性リウマチ、クローン
病、潰瘍性大腸炎などの炎症性疾患及び放射線障害防御
などにＳＯＤは有効と考えられている。しかし、ＳＯＤ
を静脈内注射で生体内に投与した時の半減期は約5分と
短く、期待されたような抗炎症作用は生体内でほとんど
発揮されなかった。ＳＯＤの生体内での半減期を延長す
るために皮下あるいは筋肉内注射などの投与経路の変更
あるいは、脂質小包体のリポソーム中への包理などが試
みられているが、有用性を確立できていない。ＳＯＤの
生体内半減期を延長する有効な方法として合成高分子を
結合する方法があり、既にポリエチレングリコール（Ｐ
ＥＧ）やスチレン−無水マレイン酸共重合体（ＳＭＡ）
との結合体が知られている。（ＰＥＧに関してはChem.P
athol.Pharmacol,29,113(1980)）、ＳＭＡに関しては
（Bioindust,4,302(1987)）しかしこの両結合体につい
て現在まで生体内での抗炎症作用発現の報告はない。最
近ジビニルエーテルと無水マレイン酸共重合体（ＤＩＶ
ＥＭＡ）とＳＯＤの結合体に関して、インフルエンザウ
ィルス感染症に起因するマウスの炎症に有効との初めて
の報告があった。（Science,244,974(1989)）2. Description of the Related Art Superoxide dismutase (SO
D) is an enzyme having a molecular weight of about 32,000 that decomposes an active enzyme (superoxide) that causes inflammatory disorders in the living body.
Therefore, it is expected to be effective against many intractable diseases caused by inflammation. For example, reperfusion disorder, Behcet's disease, malignant rheumatism, Crohn's disease, ulcerative colitis, and other inflammatory diseases and radiation damage protection that occur when blood circulation is started after kidney or heart transplantation / treatment of myocardial infarction or cerebral infarction For example, SOD is considered to be effective. However, SOD
The half-life when administered by intravenous injection in vivo was as short as about 5 minutes, and the expected anti-inflammatory effect was hardly exhibited in vivo. In order to prolong the half-life of SOD in vivo, attempts have been made to change the administration route such as subcutaneous or intramuscular injection, or to embed lipid vesicles in liposomes, but the usefulness has been established. Absent. As an effective method for extending the in vivo half-life of SOD, there is a method of binding a synthetic polymer, and polyethylene glycol (P
EG) and styrene-maleic anhydride copolymer (SMA)
A combination with is known. (Regarding PEG, Chem.P
Athol.Pharmacol, 29 , 113 (1980)), and SMA (Bioindust, 4 , 302 (1987)), however, there has been no report on the expression of anti-inflammatory action in vivo for both conjugates. Recently, divinyl ether and maleic anhydride copolymer (DIV
For the first time, it was reported that the conjugate of EMA) and SOD was effective in inflammation of mice due to influenza virus infection. (Science, 244 , 974 (1989))
【０００３】[0003]
【発明が解決しようとする課題】このようにスーパーオ
キシドジスムターゼ（ＳＯＤ）を高分子と結合すること
により生体内半減期を延長し、生体内で抗炎症作用を発
現する例のあることも見いだされた。しかし、一般的に
ＳＯＤのような酵素を高分子と化学的に結合すると酵素
活性は50%以下へと著しく低下することが知られてい
る。ＳＯＤを薬剤として検討する実用的見地からは、Ｓ
ＯＤの酵素活性をできる限り保持して高分子との結合体
を作成する一般的合成法の開発が不可欠である。It has also been found that there is an example in which the half-life in vivo is extended by binding superoxide dismutase (SOD) to a polymer as described above, and an anti-inflammatory action is expressed in vivo. It was However, it is generally known that when an enzyme such as SOD is chemically bound to a polymer, the enzyme activity is significantly reduced to 50% or less. From the practical viewpoint of considering SOD as a drug, S
It is indispensable to develop a general synthetic method for preparing a conjugate with a polymer while retaining the OD enzyme activity as much as possible.
【０００４】[0004]
【課題を解決するための手段】スーパーオキシドジスム
ターゼ（ＳＯＤ）の三次元的構造についてはＸ線による
解析が進み、活性中心周辺について考察することが可能
である。それによればアミノ酸配列の134番目にあるリ
ジンの正電荷を帯びたアミノ基が負の電荷を帯びた活性
酵素（スーパーオキシドアニオンラジカル）を静電的相
互作用で引きつけ、活性中心の溝に導くことが重要とさ
れている。したがってこのアミノ基が高分子との結合に
用いられれば、酵素活性は著しく低下するはずである。
もしこのアミノ基を保護して高分子との結合反応を行
い、その後に保護基をはずすことができれば、活性を行
ったＳＯＤ高分子誘導体の合成が可能なはずである。た
だ現在の技術で134番目のリジン残基のアミノ基のみを
保護することはほとんど不可能である。しかし一般的に
ＳＯＤのアミノ基をまず低分子で保護し、高分子と反応
させ、その後低分子の保護基をはずすことは可能である
と考えられている。この考えに基づき、ＳＯＤをpH8の
弱塩基性緩衝液中で保護剤のジメチル無水マレイン酸と
反応させ、次に水と相容性の高い有機溶媒に溶解した高
分子（例えばジビニルエーテルと無水マレイン酸共重合
体(ＤＩＶＥＭＡ)あるいはポリエチレングリコール(Ｐ
ＥＧ2)等の高分子）と均一系で反応を行った。高分子と
の反応後溶液のpHを6として保護基のジメチルマレイン
酸を速やかに離脱させ、限外ろ過膜で除去・精製した。
このようにpHによって容易に除去可能な保護剤を用いる
手法の導入により、90%以上ＳＯＤとしての酵素活性を
保持したＳＯＤ高分子誘導体を合成できることを見いだ
し。この知見に基づいた本発明を完成するに至った。す
なわち、本発明はスーパーオキシドジスムターゼ（ＳＯ
Ｄ）を弱塩基性無機溶媒緩衝液中に溶解し、式（１）[Means for Solving the Problems] The three-dimensional structure of superoxide dismutase (SOD) has been analyzed by X-rays, and it is possible to consider the periphery of the active center. According to the report, the positively charged amino group of lysine at the 134th position in the amino acid sequence attracts the negatively charged active enzyme (superoxide anion radical) by electrostatic interaction, leading to the groove of the active center. Is considered important. Therefore, if this amino group is used for binding to a polymer, the enzymatic activity should be significantly reduced.
If this amino group can be protected to undergo a binding reaction with a polymer and then the protecting group can be removed, it should be possible to synthesize an activated SOD polymer derivative. However, it is almost impossible to protect only the amino group of the lysine residue at position 134 with the current technology. However, it is generally believed that it is possible to first protect the amino group of SOD with a low molecular weight compound, react it with a polymer, and then remove the low molecular weight protecting group. Based on this idea, SOD was reacted with a protective agent, dimethyl maleic anhydride, in a weakly basic buffer solution of pH 8, and then dissolved in an organic solvent highly compatible with water (for example, divinyl ether and maleic anhydride). Acid copolymer (DIVEMA) or polyethylene glycol (P
The reaction was carried out in a homogeneous system with a polymer such as EG 2 ). After the reaction with the polymer, the pH of the solution was adjusted to 6 and the protecting group dimethylmaleic acid was quickly removed, and the residue was removed and purified with an ultrafiltration membrane.
As described above, it was found that the introduction of the method using a protective agent that can be easily removed by pH makes it possible to synthesize an SOD polymer derivative having 90% or more SOD enzyme activity. The present invention has been completed based on this finding. That is, the present invention relates to superoxide dismutase (SO
D) was dissolved in a weakly basic inorganic solvent buffer solution to give a compound of formula (1)
【化７】 で表されるジメチル無水マレイン酸の有機溶媒を滴下し
て反応させた後、一般式（２）[Chemical 7] The organic solvent of dimethyl maleic anhydride represented by
【化８】 （式中のmは20から500までの整数である）で示されるジ
ビニルエーテルと無水マレイン酸の共重合体（ＤＩＶＥ
ＭＡ）あるいは、一般式（３）[Chemical 8] A copolymer of divinyl ether represented by the formula (m is an integer from 20 to 500) and maleic anhydride (DIVE
MA) or general formula (3)
【化９】 （式中のnは10から700までの整数である）で示されるポ
リエチレングリコール（ＰＥＧ2）等の高分子の有機溶
媒を加えて結合反応をおこなわせた後、反応溶液を弱酸
性としてジメチルマレイン酸を遊離せしめ、続いて精製
を行うことを特徴とする、一般式（４）[Chemical 9] (In the formula, n is an integer from 10 to 700) A polymer organic solvent such as polyethylene glycol (PEG 2 ) is added to carry out the binding reaction, and then the reaction solution is made weakly acidic to dimethylmalein. A compound of the general formula (4), characterized in that the acid is liberated and then purified.
【化１０】 （ただし、式中のiは1から22までの整数であり、Polyme
rとは一般式（５）[Chemical 10] (However, i in the formula is an integer from 1 to 22, and Polyme
r is the general formula (5)
【化１１】 （式中のmは20から500までの整数であり、Xの位置でＳ
ＯＤのアミノ基とアミド結合している）あるいは一般式
（３）[Chemical 11] (M in the formula is an integer from 20 to 500, and S at the position of X
Amide bond with amino group of OD) or general formula (3)
【化１２】 （式中のnは10から700までの整数であり、Yの位置でＳ
ＯＤのアミノ基と共有結合している）等の高分子であ
る）で表される高活性スーパーオキシドジスムターゼ
（ＳＯＤ）高分子誘導体の製造方法を提供するものであ
る。ＳＯＤと高分子の結合比率はＳＯＤがアミノ基を22
個有することから1分子のＳＯＤに対し結合できる高分
子は1から22分子（一般式（４）中のi）に限定される。
ＳＯＤ高分子誘導体中のＳＯＤ含有量は誘導体を加水分
解しアミノ酸分析を行うことにより定量的に決定した。
本発明に用いた一般式（２）のジビニルエーテル無水マ
レイン酸共重合体は、原料のジビニルエーテルと無水マ
レイン酸を公知の方法（MacromolecularSynthesis,8,89
(1982)）に従って共重合させる事によって得られる。Ｄ
ＩＶＥＭＡの分子量が5千以下ではＳＯＤとの結合で高
分子としての効果が得にくいこと、また10万以上では溶
血作用などの副作用が起こるから、一般式（２）中の重
合度mは20から500までの整数が適当である。また一般式
（３）に用いたポリエチレングリコール（ＰＥＧ2）は
典型的なポリエチレングリコール化試薬として知られる
（Trends in Biotech ,4,68,(1986)）ポリエチレングリ
コールの分子量は500以下では効果を得にくいこと、３
万以上では有効性に変化のないことから、一般式（３）
中のnは10から700までの整数が適当である。ＳＯＤのよ
うなタンパク質が生体内半減期が短い原因の一つは、体
内のタンパク質分解酵素により速やかに加水分解される
ことにある。本発明により作成されたＳＯＤ高分子誘導
体のタンパク質分解酵素に対する安定性をヒト血漿中で
検討した。未修飾のＳＯＤが３時間で酵素活性が２分の
１以下に低下するのに対し、ＳＯＤ高分子誘導体は６日
間経過後も活性はほとんど低下しなかった。この事は高
分子誘導体とすることによりＳＯＤは血漿中のタンパク
質分解酵素に対し抵抗性となったことを示すものであ
る。また、生体内の活性については、本発明により作成
されたＳＯＤ高分子誘導体は再還流障害及び炎症疾患の
動物実験モデルにおいて有効性が示された。以上のよう
に、本発明により作成されたＳＯＤ高分子誘導体はアミ
ノ基保護剤の作用によりＳＯＤの酵素活性が90%以上保
持され、血漿中タンパク質分解酵素に対して抵抗性で、
生体内での抗炎症作用も発現するという優れた効果を示
した。[Chemical 12] (N in the formula is an integer from 10 to 700, and S at the Y position
The present invention provides a method for producing a high activity superoxide dismutase (SOD) polymer derivative represented by () which is a polymer (covalently bonded to an amino group of OD). The binding ratio of SOD and polymer is such that SOD has 22 amino groups.
Since they have one, the number of polymers capable of binding to one molecule of SOD is limited to 1 to 22 molecules (i in the general formula (4)).
The SOD content in the SOD polymer derivative was quantitatively determined by hydrolyzing the derivative and performing amino acid analysis.
The divinyl ether / maleic anhydride copolymer of the general formula (2) used in the present invention is prepared by a known method (Macromolecular Synthesis, 8 , 89) from the raw material divinyl ether and maleic anhydride.
(1982)). D
If the molecular weight of IVEMA is 5,000 or less, it is difficult to obtain the effect as a polymer by binding with SOD, and if it is 100,000 or more, side effects such as hemolytic action occur. Therefore, the degree of polymerization m in the general formula (2) is 20 or more. An integer up to 500 is suitable. The polyethylene glycol (PEG 2 ) used in the general formula (3) is known as a typical polyethylene glycolation reagent (Trends in Biotech, 4 , 68, (1986)). Difficulty, 3
In the case of 10,000 or more, there is no change in effectiveness, so the general formula (3)
It is appropriate that n is an integer from 10 to 700. One of the reasons why proteins such as SOD have a short half-life in vivo is that they are rapidly hydrolyzed by proteolytic enzymes in the body. The stability of the SOD polymer derivative prepared by the present invention against proteolytic enzyme was examined in human plasma. The unmodified SOD showed a decrease in enzyme activity to less than half in 3 hours, whereas the SOD polymer derivative showed almost no decrease in activity even after 6 days. This means that SOD became resistant to proteolytic enzymes in plasma by using a polymer derivative. Regarding the in vivo activity, the SOD polymer derivative prepared by the present invention was shown to be effective in animal experimental models of reperfusion injury and inflammatory disease. As described above, the SOD polymer derivative prepared according to the present invention retains 90% or more of the SOD enzyme activity by the action of the amino group-protecting agent, is resistant to the proteolytic enzyme in plasma,
The excellent effect that the anti-inflammatory effect is also expressed in vivo was shown.
【０００５】[0005]
【実施例】次に実施例によって本発明をさらに詳細に説
明する。なおスーパーオキシドジスムターゼを「ＳＯ
Ｄ」、一般式（２）のジビニルエーテル−無水マレイン
酸共重合体を「ＤＩＶＥＭＡ」、一般式（３）のポリエ
チレングリコールを「ＰＥＧ2」と省略して記す。 実施例１ ＳＯＤ200mgを25mlの0.2molホウ酸緩衝液に溶解し0。Cに
冷却する。ジメチル無水マレイン酸1.8gを6mlのＮ−メ
チルピロリドン（ＮＭＰ）に溶解し、0。CでＳＯＤ溶液
に滴下する。反応液のpHを１mol水酸化ナトリウム水溶
液で8.0に調整し、滴下後１時間反応させる。ＤＩＶＥ
ＭＡ（平均分子量3.0万）1.0gを5mlのＮＭＰに溶解し、
0。C攪拌下にＳＯＤ反応溶液に加える。溶液のpHを１mol
水酸化ナトリウム水溶液で8.0とし、pHが一定になるま
で反応を続ける。溶液を蒸留水で10倍に希釈し、0。Cで
１mol塩酸でpHを6.0に調整し、室温で１時間攪拌する。
未反応物・ジメチルマレイン酸・塩類をミリホ゜ア社PTHK限
外ろ過膜（分画分子量10万）により除去し、ＧＰＣによ
りフリーのＳＯＤを含まない913mgのＳＯＤ−ＤＩＶＥ
ＭＡ結合体を淡青色綿状の凍結乾燥固体として得た。Ｓ
ＯＤ−ＤＩＶＥＭＡ結合中のＳＯＤ含有量は結合体を加
水分解してＳＯＤのアミノ酸を定量する事によって決定
された（8.7重量%）。この結果は１分子のＳＯＤに対し
約11個のＤＩＶＥＭＡが結合していることを示す。ＳＯ
Ｄ−ＤＩＶＥＭＡ結合体の酵素活性は、ニトロブルー・
テトラゾニウム（ＮＢＴ）法によって測定し（Anal.Bio
chem.,44,276(1971)）、ＳＯＤ当たり92.4%の活性を保
持していることが示された。 実施例２ 実施例１と同様にしてＳＯＤ200mgとＤＩＶＥＭＡ（平
均分子量3.0万）500mgを反応させ、ＳＯＤ−ＤＩＶＥＭ
Ａ結合体560mgを得た。この結合体中のＳＯＤ含有率は1
4.3重量%であり、1分子のＳＯＤに対し約6個のＤＩＶＥ
ＭＡが結合していた。結合体のＳＯＤ当たりの酵素活性
は91.5%保持されていた。 実施例３ 実施例１と同様にして、ＳＯＤ200mgとＤＩＶＥＭＡ
（平均分子量5千）200mgを反応させ、限外ろ過膜として
アミコン社PM30（分画分子量3万）で精製し、ＧＰＣにより
分取後凍結乾燥して淡青色粉末状固体208mgを得た。結
合体中のＳＯＤ含有率は30.2重量%であり、１分子のＳ
ＯＤに対し約14個のＤＩＶＥＭＡが結合していた。この
結合体のＳＯＤ当たりの酵素活性は92.1%保持されてい
た。 実施例４ ＳＯＤ50mgを10mlの0.2molホウ酸緩衝液に溶解し0。Cに
冷却する。450mgのジメチルマレイン酸を2mlのＮＭＰに
溶解し、0。CでＳＯＤ溶液に滴下する。反応液のpHを１m
ol水酸化ナトリウム水溶液で8.0に調整し、滴下後１時
間反応させる。粉末状のＰＥＧ2（平均分子量1万）500m
gをＳＯＤ溶液に加え、0。Cで１時間室温で反応させる。
反応後溶液を蒸留水で10倍に希釈し、0。Cで１mol塩酸に
よりpHを6.0とし、室温で１時間攪拌する。反応精製物
をアミコンPM30限外ろ過膜（分画分子量3万）で精製し、Ｇ
ＰＣでフリーのＳＯＤを除去した後凍結乾燥し、淡青色
綿状固体340mgを得た。ＳＯＤ−ＰＥＧ2結合体中のＳＯ
Ｄ含有量は実施例１と同様にしてアミノ酸分析により決
定した。結合体は19.4重量%のＳＯＤを含み、１分子の
ＳＯＤにＰＥＧ2が約13個結合していることが示され
た。この結合体のＳＯＤ当たりの酵素活性は90.7%保持
されていた。 参考例１ ヒト血漿500ml中にＳＯＤ3.4mgあるいは実施例１で得た
ＳＯＤ−ＤＩＶＥＭＡ結合体42.3mgを溶解し、37。Cで保
温した。一定時間経過後100μlを取り、ニトロブルー・
テトラゾニウム法によりＳＯＤとしての酵素活性を測定
した。ＳＯＤは図１に示すように３時間で酵素活性が２
分の１以下に低下するのに対し、ＳＯＤ−ＤＩＶＥＭＡ
結合体は６日間経過後も活性はほとんど低下しなかっ
た。 参考例２ ラットの腹部を開腹し盲腸を絹糸で結さつし、18Ｇの注
射針で１回刺して穴をあけた後閉腹する。穴から糞便が
腹腔内にもれるため、ラットは腹膜炎を起こし72時間で
86%のラットが死亡する。しかし、表１に示すようにＳ
ＯＤで治療を行うと35%のラットが72時間生存し、さら
にＳＯＤ−ＤＩＶＥＭＡ結合体では86%生存した。The present invention will be described in more detail with reference to Examples. In addition, superoxide dismutase is "SO
It referred maleic anhydride copolymer "DIVEMA", polyethylene glycol of the general formula (3) abbreviated as "PEG 2" - D ", the formula divinyl ether (2). Example 1 200 mg of SOD was dissolved in 25 ml of 0.2 mol borate buffer . Cool to C. Dimethyl maleic acid 1.8g were dissolved in N- methylpyrrolidone 6ml (NMP), 0. C is added dropwise to the SOD solution. The pH of the reaction solution is adjusted to 8.0 with a 1 mol aqueous sodium hydroxide solution, and the reaction is continued for 1 hour after dropping. DIVE
Dissolve 1.0 g of MA (average molecular weight of 30,000) in 5 ml of NMP,
0 . C Add to SOD reaction solution under stirring. PH of solution is 1 mol
Adjust to 8.0 with aqueous sodium hydroxide solution and continue the reaction until the pH becomes constant. Dilute the solution 10-fold with distilled water to 0 . Adjust the pH to 6.0 with 1 mol hydrochloric acid with C and stir at room temperature for 1 hour.
Unreacted substances, dimethylmaleic acid, salts were removed by Millipore PTHK ultrafiltration membrane (molecular weight cutoff of 100,000), and 913 mg of SOD-DIVE containing no free SOD by GPC.
The MA conjugate was obtained as a pale blue flocculent lyophilized solid. S
The SOD content in the OD-DIVEMA bond was determined by hydrolyzing the conjugate and quantifying the amino acids in SOD (8.7% by weight). This result shows that about 11 DIVEMAs are bound to one molecule of SOD. SO
The enzyme activity of the D-DIVEMA conjugate was
Measured by tetrazonium (NBT) method (Anal.Bio
Chem., 44 , 276 (1971)), and was shown to retain 92.4% activity per SOD. Example 2 In the same manner as in Example 1, SOD 200 mg and DIVEMA (average molecular weight 30,000) 500 mg were reacted to give SOD-DIVEM.
560 mg of A-conjugate was obtained. The SOD content in this conjugate is 1
It is 4.3% by weight, and about 6 units of SIVE per molecule of SOD
MA was bound. The enzyme activity per SOD of the conjugate was retained at 91.5%. Example 3 In the same manner as in Example 1, 200 mg of SOD and DIVEMA
200 mg of (average molecular weight of 5,000) was reacted, purified with PM30 (molecular weight cut off of 30,000) manufactured by Amicon as an ultrafiltration membrane, fractionated by GPC and freeze-dried to obtain 208 mg of a pale blue powdery solid. The SOD content in the conjugate is 30.2% by weight, and one molecule of S
About 14 DIVAMAs were bound to the OD. The enzyme activity per SOD of this conjugate was retained at 92.1%. Example 4 50 mg of SOD was dissolved in 10 ml of 0.2 mol borate buffer . Cool to C. Dissolve 450 mg of dimethyl maleic acid in 2 ml of NMP . C is added dropwise to the SOD solution. PH of reaction solution is 1m
The pH is adjusted to 8.0 with an aqueous solution of sodium hydroxide, and the mixture is reacted for 1 hour after dropping. Powdered PEG 2 (average molecular weight 10,000) 500m
g to the SOD solution, 0 . React at C for 1 hour at room temperature.
After the reaction, dilute the solution 10-fold with distilled water to 0 . The pH is adjusted to 6.0 with 1 mol hydrochloric acid at C, and the mixture is stirred at room temperature for 1 hour. The reaction purified product was purified with Amicon PM30 ultrafiltration membrane (molecular weight cutoff of 30,000) to obtain G
After removing the free SOD with PC, it was freeze-dried to obtain 340 mg of a pale blue flocculent solid. SO in SOD-PEG 2 conjugate
The D content was determined by amino acid analysis in the same manner as in Example 1. The conjugate contained 19.4% by weight of SOD, indicating that about 13 PEG 2 were attached to one molecule of SOD. The enzyme activity per SOD of this conjugate was retained at 90.7%. Reference Example 1 3.4 mg of SOD or 42.3 mg of the SOD-DIVEMA conjugate obtained in Example 1 was dissolved in 500 ml of human plasma . It was kept warm at C. After a certain period of time, take 100 μl and
The enzyme activity as SOD was measured by the tetrazonium method. As shown in Fig. 1, SOD showed an enzyme activity of 2 after 3 hours.
SOD-DIVEMA is reduced to less than 1 /
The activity of the conjugate hardly decreased after 6 days. Reference Example 2 The abdomen of a rat is laparotomized, the cecum is tied with a silk thread, and the rat is punctured once by puncturing once with an 18G injection needle and then closed. Rats developed peritonitis in 72 hours because feces leaked from the hole into the abdominal cavity.
86% of rats die. However, as shown in Table 1, S
When treated with OD, 35% of the rats survived for 72 hours and 86% with the SOD-DIVEMA conjugate.
【表１】 [Table 1]
【００６】[0096]
【発明の効果】本発明のＳＯＤ高分子誘導体の製造方法
は、脱離可能なアミノ基保護剤を用いることにより、高
分子との結合後ＳＯＤの酵素活性を90%以上保持できる
という製造方法であり、生体内での抗炎症作用もＳＯＤ
に比較して著しく高活性なＳＯＤ高分子誘導体を可能と
するものである。INDUSTRIAL APPLICABILITY The method for producing a SOD polymer derivative of the present invention is a production method in which the enzymatic activity of SOD after binding to a polymer can be maintained at 90% or more by using a removable amino group-protecting agent. Yes, the anti-inflammatory effect in vivo is SOD
It enables a highly active SOD polymer derivative as compared with the above.
【００７】[0097]
【図１】実施例１のＳＯＤ−ＤＩＶＥＭＡ結合体のヒト
血漿中の酵素活性の安定持続性を示すグラフ。FIG. 1 is a graph showing the stable durability of enzyme activity in human plasma of the SOD-DIVEMA conjugate of Example 1.
Claims (1)
を弱塩基性無機緩衝液中に溶解し、式（１） 【化１】 で示されるジメチル無水マレイン酸の有機溶媒溶液を摘
下して反応させた後、１般式（２） 【化２】 （式中のmは20から500までの整数である）で示されるジ
ビニルエチルエーテル無水マレイン酸の共重合体（ＤＩ
ＶＥＭＡ）あるいは一般式（３） 【化３】 （式中のnは10から700までの整数である）で示されるポ
リエチレングリコール（ＰＥＧ2）等の高分子の有機溶
媒を加えて結合反応を行わせた後、反応溶液を弱酸性と
してジメチルマレイン酸を遊離せしめ、続いて精製を行
うことを特徴とする、一般式（４） 【化４】 （ただし式中のiは1から22までの整数であり、Polymer
とは一般式（５） 【化５】 （式中のmは20から500までの整数であり、Xの位置でＳ
ＯＤのアミノ基とアミド結合している）あるいは、一般
式（６） 【化６】 （式中のnは10から700までの整数であり、Yの位置でＳ
ＯＤのアミノ基と結合している）等の高分子である）で
表される高活性スーパーオキシドジスムターゼ（ＳＯ
Ｄ）高分子誘導体の製造方法。1. Superoxide dismutase (SOD)
Is dissolved in a weakly basic inorganic buffer solution to obtain the compound of formula (1) An organic solvent solution of dimethyl maleic anhydride represented by (M in the formula is an integer from 20 to 500) a copolymer of divinylethyl ether maleic anhydride (DI
VEMA) or general formula (3) (N in the formula is an integer from 10 to 700) A high molecular weight organic solvent such as polyethylene glycol (PEG 2 ) is added to carry out the binding reaction, and then the reaction solution is made weakly acidic to dimethylmalein. The compound of the general formula (4) is characterized in that the acid is liberated and the purification is carried out subsequently. (However, i in the formula is an integer from 1 to 22,
Is the general formula (5) (M in the formula is an integer from 20 to 500, and S at the position of X
(Bonded to the amino group of OD through an amide bond) or represented by the general formula (6): (N in the formula is an integer from 10 to 700, and S at the Y position
A high activity superoxide dismutase (SO) which is a polymer (bonded to the amino group of OD) or the like)
D) A method for producing a polymer derivative.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3155575A JPH0785717B2 (en) | 1991-05-30 | 1991-05-30 | Method for producing highly active superoxide dismutase polymer derivative |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3155575A JPH0785717B2 (en) | 1991-05-30 | 1991-05-30 | Method for producing highly active superoxide dismutase polymer derivative |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0698766A JPH0698766A (en) | 1994-04-12 |
| JPH0785717B2 true JPH0785717B2 (en) | 1995-09-20 |
Family
ID=15609050
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3155575A Expired - Lifetime JPH0785717B2 (en) | 1991-05-30 | 1991-05-30 | Method for producing highly active superoxide dismutase polymer derivative |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0785717B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE69533987T2 (en) * | 1994-05-20 | 2006-03-16 | Hisamitsu Pharmaceutical Co., Inc., Tosu | PROTEIN OR POLYPEPTIDE, PROCESS FOR ITS PRODUCTION AND CORRESPONDING PRODUCTS |
| CN119837842B (en) * | 2024-03-25 | 2025-11-04 | 上海交通大学 | A superoxide dismutase-based nanotransdermal delivery system and its preparation method |
-
1991
- 1991-05-30 JP JP3155575A patent/JPH0785717B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0698766A (en) | 1994-04-12 |
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