JPH029900A - Modified erythropoetine - Google Patents
Modified erythropoetineInfo
- Publication number
- JPH029900A JPH029900A JP8978088A JP8978088A JPH029900A JP H029900 A JPH029900 A JP H029900A JP 8978088 A JP8978088 A JP 8978088A JP 8978088 A JP8978088 A JP 8978088A JP H029900 A JPH029900 A JP H029900A
- Authority
- JP
- Japan
- Prior art keywords
- epo
- modified
- erythropoietin
- present
- cells
- 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
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical class [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 claims abstract description 47
- 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 claims abstract description 19
- 230000000694 effects Effects 0.000 abstract description 19
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 5
- 230000003394 haemopoietic effect Effects 0.000 abstract description 5
- 229940088597 hormone Drugs 0.000 abstract description 5
- 239000005556 hormone Substances 0.000 abstract description 5
- GAZRNXIMWKZADY-UHFFFAOYSA-N 3,5-dimethylpyrazole-1-carboximidamide Chemical compound CC=1C=C(C)N(C(N)=N)N=1 GAZRNXIMWKZADY-UHFFFAOYSA-N 0.000 abstract description 4
- 102000003951 Erythropoietin Human genes 0.000 abstract description 4
- 108090000394 Erythropoietin Proteins 0.000 abstract description 4
- 229940105423 erythropoietin Drugs 0.000 abstract description 4
- 208000007502 anemia Diseases 0.000 abstract description 3
- RMAHPRNLQIRHIJ-UHFFFAOYSA-N methyl carbamimidate Chemical compound COC(N)=N RMAHPRNLQIRHIJ-UHFFFAOYSA-N 0.000 abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 3
- 230000006798 recombination Effects 0.000 abstract description 2
- 241000699802 Cricetulus griseus Species 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 230000002611 ovarian Effects 0.000 abstract 1
- 108010092408 Eosinophil Peroxidase Proteins 0.000 description 25
- 102100031939 Erythropoietin Human genes 0.000 description 25
- 238000007745 plasma electrolytic oxidation reaction Methods 0.000 description 25
- QUOGESRFPZDMMT-YFKPBYRVSA-N L-homoarginine Chemical group OC(=O)[C@@H](N)CCCCNC(N)=N QUOGESRFPZDMMT-YFKPBYRVSA-N 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 9
- 235000018977 lysine Nutrition 0.000 description 9
- 238000007385 chemical modification Methods 0.000 description 8
- 241000700159 Rattus Species 0.000 description 7
- 235000001014 amino acid Nutrition 0.000 description 7
- 150000001413 amino acids Chemical class 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 5
- 210000002798 bone marrow cell Anatomy 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 239000004472 Lysine Substances 0.000 description 4
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 4
- 108020004511 Recombinant DNA Proteins 0.000 description 4
- 238000010348 incorporation Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- QUOGESRFPZDMMT-UHFFFAOYSA-N L-Homoarginine Natural products OC(=O)C(N)CCCCNC(N)=N QUOGESRFPZDMMT-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 231100000673 dose–response relationship Toxicity 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 241000282693 Cercopithecidae Species 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000987586 Homo sapiens Eosinophil peroxidase Proteins 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 102000044890 human EPO Human genes 0.000 description 2
- 238000000099 in vitro assay Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 102000035118 modified proteins Human genes 0.000 description 2
- 108091005573 modified proteins Proteins 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- KZMAWJRXKGLWGS-UHFFFAOYSA-N 2-chloro-n-[4-(4-methoxyphenyl)-1,3-thiazol-2-yl]-n-(3-methoxypropyl)acetamide Chemical compound S1C(N(C(=O)CCl)CCCOC)=NC(C=2C=CC(OC)=CC=2)=C1 KZMAWJRXKGLWGS-UHFFFAOYSA-N 0.000 description 1
- 208000032467 Aplastic anaemia Diseases 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 101000929799 Homo sapiens Acyl-CoA-binding protein Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- YRKCREAYFQTBPV-UHFFFAOYSA-N acetylacetone Chemical compound CC(=O)CC(C)=O YRKCREAYFQTBPV-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- HAMNKKUPIHEESI-UHFFFAOYSA-N aminoguanidine Chemical compound NNC(N)=N HAMNKKUPIHEESI-UHFFFAOYSA-N 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 210000000267 erythroid cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229960002413 ferric citrate Drugs 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- NPFOYSMITVOQOS-UHFFFAOYSA-K iron(III) citrate Chemical compound [Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NPFOYSMITVOQOS-UHFFFAOYSA-K 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 210000003924 normoblast Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000003760 tallow Substances 0.000 description 1
- -1 that is Chemical group 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
［産業上の利用分野〕
本発明は、少なくとも一つのリジン残基がグアニジル化
によりホモアルギニン残基に変換された修飾エリスロボ
エチンに関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to modified erythroboetin in which at least one lysine residue has been converted to a homoarginine residue by guanidylation.
［従来の技術］
エリスロポエチン（以下、ＥＰＯという。）は、主に腎
臓で産生・分泌される造血ホルモンの一種であり、赤芽
球前駆体細胞に作用して該細胞を赤血球に分化させる機
能を有する、分子１約３４．０００の糖タンパク質であ
る。[Prior Art] Erythropoietin (hereinafter referred to as EPO) is a type of hematopoietic hormone mainly produced and secreted by the kidneys, and has the function of acting on erythroblast precursor cells and causing them to differentiate into red blood cells. Molecule 1 is a glycoprotein with approximately 34,000 molecules.
動物に於いて、貧血症の治療に代表されるエリスロボエ
チン治療に有用な物質であることが、判明している。It has been found that erythroboetin is a useful substance in the treatment of anemia in animals.
天然由来のＥＰＯとしては、例えば、再生不良性貧血思
考の尿から精製したものが知られているが（ミャケ他、
ジャーナル・オブ・バイオロジカル・ケミストリー、第
２５２巻、第１５号、 ５５５８−５５６４頁、 １９
７７年）、この方法では収率が低くその供給量には限界
があり、工業的大１生産には適さないものである。そこ
で、組換えＤＮＡ技術を利用した生産性の高いＥＰＯ生
産方法が提案されている（特表昭６１−５０１６２７号
公報）。しかしながら、ＥＰＯは糖鎖部分を失うとイン
・ビボ活性がなくなってしまうために、この組換えＤＮ
Ａ生産方法では動物細胞を使うことが望ましく、この場
合、動物細胞培養は経済性の点で高価につく。Naturally derived EPO is known, for example, to be purified from the urine of patients with aplastic anemia (Myake et al.
Journal of Biological Chemistry, Volume 252, No. 15, Pages 5558-5564, 19
(1977), this method has a low yield and a limited supply, making it unsuitable for industrial large-scale production. Therefore, a highly productive EPO production method using recombinant DNA technology has been proposed (Japanese Patent Publication No. 501,627/1983). However, if EPO loses its sugar chain moiety, it loses its in vivo activity, so this recombinant DNA
In production method A, it is desirable to use animal cells, and in this case, animal cell culture is expensive in terms of economy.
このように、従来のＥＰＯ生産方法はコストが非常に高
くつき、その経済性に問題があった。As described above, the cost of the conventional EPO production method is very high, and there is a problem in its economic efficiency.
ところで、従来から、化学修飾は、主に酵素活性の構造
的な解明の手段として使用されてきた方法である。しか
しながら、酵素を含めたタンパク質に対して化学修飾を
施したことによって、これらタンパク質の比活性が高ま
ったという報告はこれまであまりなされていない。それ
どころか、むしろ、化学修飾に伴って、修飾タンパク質
の活性が著しく低下するか又は実質的に失活してしまう
例が多い。リジン残基のグアニジル化に伴って修飾タン
パク質が不活性化した例として、Ｄ、Ｈ，Ｇｌ＋ｃｋ、
Ｅ、Ａ、Ｂａｒｎａｒｄ、 Ｂｉｏｃｈｉｍ、Ｂｉｏ
ｐｈｙｓ、Ａｃｔａ、 ２１４．３２６（１９７０）及
びＰｌａｐｐ、Ｂ、Ｖ、、 Ｈｏｏｖａ、Ｓ、、 Ｓｔ
ｅ＋ｎ、Ｗ、Ｈ。By the way, chemical modification has traditionally been a method used mainly as a means for structurally elucidating enzyme activity. However, there have been few reports to date that chemical modification of proteins, including enzymes, increases the specific activity of these proteins. On the contrary, in many cases, the activity of the modified protein is significantly reduced or substantially inactivated due to chemical modification. Examples of modified proteins inactivated by guanidylation of lysine residues include D, H, Gl+ck,
E, A, Barnard, Biochim, Bio
phys, Acta, 214.326 (1970) and Plapp, B.V., Hoova, S., St.
e+n, W, H.
Ｊ、Ｂｉｏｌｏｇｉｃａｌ Ｃｈｅｍｉｓｔｒｙ ２６
４． （４） ９３９ （１９７１）を挙げることがで
きる。J, Biological Chemistry 26
4. (4) 939 (1971).
また、前記特表昭６１−５０１６２７号公報には、ＥＰ
Ｏポリペプチド類縁体もしくは誘導体が開示されている
が、本発明の修飾ＥＰＯのように化学修飾によってリジ
ン残基がグアニジル化されているものに関する具体的な
記載はなく、又それを示唆乃至教示する記載も見当たら
ない。In addition, in the above-mentioned Japanese Patent Publication No. 61-501627, EP
O polypeptide analogs or derivatives have been disclosed, but there is no specific description of those in which lysine residues are guanidylated by chemical modification like the modified EPO of the present invention, nor is there any suggestion or teaching thereof. I can't find any description.
［発明が解決しようとする課題］
本発明は、化学修飾によって比活性の高い修飾ＥＰＯを
得ることを目的としており、その結果、より少量のＥＰ
Ｏで所望の効果を達成し、ひいて（よＥＰＯ生産の最終
的なコストを低減することを目的とするものである。[Problems to be Solved by the Invention] The present invention aims to obtain modified EPO with high specific activity through chemical modification, and as a result, a smaller amount of EP
The aim is to achieve the desired effect with O and thus reduce the final cost of EPO production.
［課題を解決する為の手段］
上記課題を解決する為に、本発明は、少なくとも一つの
リジン残基がグアニジル化されている修飾エリスロボエ
チンを提供するものである。[Means for Solving the Problems] In order to solve the above problems, the present invention provides modified erythroboetin in which at least one lysine residue is guanidylated.
本発明で使用し得るＥＰＯは、ヒト及びサル等の哺乳動
物に於いて天然に産生されるものの他、前記特表昭６１
−５０１６２７号公報に開示の組換えＥＰＯに代表され
る組換えＤＮＡ技術によって得られ天然ＥＰＯの生物学
的活性を有するポリペプチド、及び同じく天然ＥＰＯの
生物学的活性を有するそれらの類縁体をも意味する。EPO that can be used in the present invention includes those that are naturally produced in mammals such as humans and monkeys, as well as those that are naturally produced in mammals such as humans and monkeys.
Polypeptides having the biological activity of natural EPO obtained by recombinant DNA technology, such as the recombinant EPO disclosed in Publication No. 501627, and their analogues having the biological activity of natural EPO as well. means.
本発明で使用するＥＰＯは、好ましくはヒト型ＥＰＯで
あり、更に好ましくは組換えＤＮＡ技術によって得られ
るヒト型ＥＰＯである。The EPO used in the present invention is preferably human EPO, more preferably human EPO obtained by recombinant DNA technology.
グアニジル化されるリジン残基数は４以上が好ましく、
更に好ましくは７以上である。The number of lysine residues to be guanidylated is preferably 4 or more,
More preferably, it is 7 or more.
本発明に於けるグアニジル化は公知の化学修飾方法によ
りリジン残基のε−アミノ基をグアニジル化するもので
あり、例えばＯ−メチルイソ尿素又は１−グアニル−３
，５−ジメチルピラゾールを修飾試薬として用いて実施
することができる。The guanidylation in the present invention involves guanidylating the ε-amino group of a lysine residue by a known chemical modification method, such as O-methylisourea or 1-guanyl-3
, 5-dimethylpyrazole as the modifying reagent.
反応条件は一般的に、Ｉ）Ｈ９−１１，４℃前後、数時
間〜数日間である。尚、この化学修飾反応に際して、使
用したＥＰＯのほぼ全量を回収することができ、また、
その安定性に関しても実質的に何等の影響も受けない。The reaction conditions are generally I) H9-11, around 4°C, for several hours to several days. In addition, during this chemical modification reaction, almost the entire amount of EPO used can be recovered, and
Its stability is also virtually unaffected.
本発明のグアニジル化によって得られる修飾ＥＰＯ中の
修飾リジン残基、即ち、ホモアルギニン残基の個数は、
得られた修１ＥＰＯを酸分解の侵アミノ酸分析にかけ、
夫々のアミノ酸溶出ピークから常法に従い計算して得ら
れる平均値である。The number of modified lysine residues, that is, homoarginine residues, in the modified EPO obtained by the guanidylation of the present invention is:
The obtained Shu1EPO was subjected to invasive amino acid analysis by acid decomposition,
This is an average value calculated from each amino acid elution peak according to a conventional method.
従って、当業者には自明のことであるが、本発明の修ｆ
！１ｌｉＥＰｏに於いて個々のポリペプチド分子が有す
るホモアルギニン残基の個数には、該平均値を中心とし
た一定の偏差が存在するものである。Therefore, it will be obvious to those skilled in the art that the modification of the present invention
! In 1liEPo, the number of homoarginine residues that each polypeptide molecule has has a certain deviation around the average value.
［作 用］
本発明の修ｆｌＥＰｏはラット骨髄細胞を用いたｎ ｖ
ｉｔｒｏ ａｓｓａｙに於いて、未修飾ＥＰＯに比べて
鉄の取込み量が著しく増大する。従って、本発明の修飾
ＥＰＯはより高いＥＰＯ比活性（造血ホルモンとしての
作用）を示すことができるものである。[Effect] The modified flEPo of the present invention can be used for nv using rat bone marrow cells.
In itro assay, iron uptake is significantly increased compared to unmodified EPO. Therefore, the modified EPO of the present invention can exhibit higher EPO specific activity (action as a hematopoietic hormone).
因みに、実施例１及び２で用いたヒトＥＰ○のリジン残
基をカルバミル化又はトリニトロベンゾイル化してリジ
ン残基の電荷を消失させた場合には該ＥＰＯ活性は消失
する。更には、アミジン化によって正電荷を維持した場
合でもその活性に何の変化も認められない。Incidentally, when the lysine residue of human EP○ used in Examples 1 and 2 is carbamylated or trinitrobenzoylated to eliminate the charge on the lysine residue, the EPO activity disappears. Furthermore, even when the positive charge is maintained by amidination, no change is observed in its activity.
以下、実施例に基づいて本発明を更に詳しく説明する。Hereinafter, the present invention will be explained in more detail based on Examples.
実施例
チャイニーズハムスター卵巣細胞を用いて遺伝子組み換
えによって得られたヒト型組み換えエリスロポエチン（
特表昭６１−５０１６２７号）を化学修飾した。Example Human recombinant erythropoietin (obtained by genetic recombination using Chinese hamster ovary cells)
Japanese Patent Publication No. 61-501627) was chemically modified.
［実施例１］
ＥＰＯ２００ｕｉを、０．５Ｍ Ｏ−メチルイソ尿素（
Ｈａ叶でｐＨを１０．３に調整したもの）４０ｇに溶解
し、４°Ｃで０〜１４４時間反応させた。反応終了後、
水に対して透析して試薬を除き、本発明の修ｆｆ１ｊＥ
ＰＯを得た。これを６８ ＨＣｆ！中で１１０℃、２４
時間の加水分解を行ない、アミノ酸自動分析計（日立８
３５及び日立８５００ ）を用いてリジン、ホモアルギ
ニン数を測定した。−例として、日立８５００を使用し
て７２時間反応させた試料を分析した際のクロマトグラ
ムを第１図に示す。標準的な分析条件を基本として、分
析サイクルを３０分から３５分に延長したところ、ホモ
アルギニンは、アルギニンの約５分俊に溶出された。[Example 1] 200ui of EPO was mixed with 0.5M O-methylisourea (
The pH was adjusted to 10.3 with Ha leaves) and the mixture was dissolved in 40 g of the mixture and reacted at 4°C for 0 to 144 hours. After the reaction is complete,
Dialyze against water to remove reagents and prepare the modified ff1jE of the present invention.
Got PO. This is 68 HCf! Inside 110℃, 24
Amino acid automatic analyzer (Hitachi 8)
35 and Hitachi 8500) to measure the number of lysine and homoarginine. - As an example, FIG. 1 shows a chromatogram obtained when a sample reacted for 72 hours was analyzed using a Hitachi 8500. When the analysis cycle was extended from 30 minutes to 35 minutes based on standard analysis conditions, homoarginine was eluted approximately 5 minutes earlier than arginine.
尚、標準的な分析条件とは以下の文献に記載のものであ
る。Note that the standard analysis conditions are those described in the following literature.
（以下余白）
＃ ２６２２カラム（４，６ｓ ＩＤＸ ６０順）を用
いてクエン酸緩衝液を使用
日立テクニカルデータ ＬＣ８２。(Left below) #2622 column (4,6s IDX 60 order) using citrate buffer Hitachi Technical Data LC82.
日立Ｓ Ｉ ニュース、 Ｖｏｌ ２９（４） １８
−２３（１９８６）１’−１８５００日立高速アミノ酸
分析計の開発」、及び、
日立評論 ６９．３３３−３３８ （１９８７）「粒形
３ミクロンイオン交換樹脂を用いる高速アミノ酸分析計
の開発」。Hitachi SI News, Vol 29(4) 18
-23 (1986) 1'-18500 Development of Hitachi High-speed Amino Acid Analyzer" and Hitachi Review 69.333-338 (1987) "Development of High-speed Amino Acid Analyzer Using Particle Size 3 Micron Ion Exchange Resin."
反応時間と、得られたＩｆｓＥＰｏ中の未修飾リジン数
及びホモアルギニン数を表１に示した。いずれの場合も
、未修飾リジン数とホモアルギニン数の和は８残基（本
実施例で使用した原料ＥＰＯ１分子中のリジン数）であ
った。１４４時間の反応で８残基中７．３残基のリジン
がＩ飾された修飾ＥＰ○が得られた。Table 1 shows the reaction time and the number of unmodified lysines and homoarginine in the obtained IfsEPo. In either case, the sum of the number of unmodified lysines and the number of homoarginines was 8 residues (the number of lysines in one molecule of raw material EPO used in this example). After 144 hours of reaction, modified EP○ in which 7.3 of the 8 residues of lysine were decorated with I was obtained.
また、ＤＨ９，６において同様の実験を行なった結果を
表２に示した。４０８時間の反応で８残基中４．９残基
のリジンが修飾されたＥＰＯが得られた。Further, Table 2 shows the results of similar experiments conducted on DH9 and DH6. After 408 hours of reaction, EPO in which 4.9 of the 8 residues were modified with lysine was obtained.
表１
表２
し実施例２］
表
アミノグアニジンと２，４−ペンタジオンをＲ，Ａ、Ｂ
Ｂａｎｎａｒｄ他の方法（Ｃｏｎ、 Ｊ、 Ｃｈｅｍ、
Ｖｏｌ ３６．１５４１（１９５Ｂ） ）に従って５
０％エタノール中で還流し、１−グアニル−３，５−ジ
メチルピラゾールを得た。Table 1 Table 2 Example 2 Table Aminoguanidine and 2,4-pentadione are R, A, B
The method of Bannard et al. (Con, J. Chem.
Vol 36.1541 (195B)) 5
The mixture was refluxed in 0% ethanol to obtain 1-guanyl-3,5-dimethylpyrazole.
実施例１で用いたものと同じ［Ｐ０１■を、０．３５Ｍ
１−グアニル−３，５−ジメチルピラゾール（ＮａＯＨ
でｐＨを９５に調整したもの） ３００ ［に溶解し、
４℃でＯ〜４８時間反応させた。反応終了後、４倍邑の
０．０２Ｎ ＨＣｌ）を加え、１ｍＨｔｌα１次いで水
に対して透析して本発明の修飾ＥＰＯを得た。反応残基
数の測定は、実施例１と同様に行なった。Same as that used in Example 1 [P01■, 0.35M
1-guanyl-3,5-dimethylpyrazole (NaOH
pH adjusted to 95) 300 [dissolved in
The reaction was carried out at 4° C. for 0 to 48 hours. After the reaction was completed, 4 times the volume of 0.02N HCl) was added, and the mixture was dialyzed against water for 1 mHtlα1 to obtain the modified EPO of the present invention. The number of reactive residues was measured in the same manner as in Example 1.
反応時間と未修飾リジン数及びホモアルギニン数を表３
に示した。４８時間の反応で８残基中５，８残基のリジ
ンが修飾された修飾ＥＰＯが得られた。Table 3 shows the reaction time, number of unmodified lysines, and number of homoarginines.
It was shown to. After 48 hours of reaction, modified EPO in which 5 and 8 of the 8 residues were modified with lysine was obtained.
［実施例３］
実施例１および２で得られたｖｉ篩ＥＰＯの生物学的活
性を測定するためにｉｎ ＶｉｔｒＯａｓｓａｙを行な
った。方法は、ラットの骨髄細胞への５９Ｆｅの取込み
法を用いた。[Example 3] In VitrOassay was performed to measure the biological activity of the vi sieve EPO obtained in Examples 1 and 2. The method used was the incorporation of 59Fe into rat bone marrow cells.
ラットより調製した骨髄細胞（０，５〜１．０×１０６
ｃｅｌｌｓ／ｃｕｌｔｕｒｅ ｗｅｌｌ）を上記修１１
１ＥＰＯ含有試料（修ｆｌｉＥ ＰＯをａ −ｎ＋ｅｄ
ｉｕｍに溶解したもの）存在下、２０％牛脂児血清入り
のα−ａ＋ｅｄｉｕｍ中で２０時間インキュベートした
。次に５９Ｆｅでラベルしたラット血清（５９Ｆｅクエ
ン酸第二鉄溶液を０．２μ偏／ＣＩＪＩｔｔｌｒ８ ｗ
ｅｌｌとなるようにａ −ｍｅｄｉｕｍで希釈し、ラッ
ト血清に加えたもの）を添加し、さらに２０時間インキ
ュベートした。その後細胞を遠心によって集め、洗浄し
、細胞に取込まれた５９Ｆｅ量をγ−カウンターで測定
した。該試料中のＥＰＯｍはアミノ酸分析値より求めた
。Bone marrow cells prepared from rats (0.5-1.0 x 106
cells/culture well) above in Modification 11.
1 EPO-containing sample (modified EPO a -n+ed
The cells were incubated for 20 hours in α-a+edium containing 20% tallow serum. Next, 59Fe-labeled rat serum (59Fe ferric citrate solution with 0.2μ bias/CIJIttlr8 w
(diluted with a-medium so as to give a total volume of 100 ml, added to rat serum) was added, and further incubated for 20 hours. Thereafter, the cells were collected by centrifugation, washed, and the amount of 59Fe incorporated into the cells was measured using a γ-counter. EPOm in the sample was determined from amino acid analysis values.
実施例１および２で得られた修飾ＥＰＯ存在下における
ラット骨髄細胞の５９Ｆｅ取込みｌを表４に示した。い
ずれの修飾ＥＰＯも、コントロール（未修ＩＥＰＯ）に
比べて５９Ｆｅ取込み量が太きくなっていた。また修飾
リジン数（ホモアルギニン数）が増えるにつれて５９Ｆ
ｅ取込み量も増加していた。ここで、未修ｆｌＥＰＯと
は実施例１及び２の化学修飾反応に於いて、夫々修飾試
薬を除い尚、この未修飾ＥＰ○は１ｎｔａＣｔなＥＰＯ
と同等の活性を保持している。Table 4 shows the 59Fe uptake of rat bone marrow cells in the presence of the modified EPO obtained in Examples 1 and 2. All of the modified EPOs had a larger amount of 59Fe incorporated than the control (unmodified IEPO). In addition, as the number of modified lysines (the number of homoarginines) increases, 59F
The amount of e-uptake was also increasing. Here, unmodified flEPO is obtained by removing the modifying reagent in the chemical modification reactions of Examples 1 and 2, and this unmodified EP○ is 1ntaCt EPO.
It maintains an activity equivalent to that of
このａｓｓａｙの結果は、本発明修飾ＥＰＯの造血ホル
モンとしてのＥＰＯ比活性が、未修ｆ！ｉＥ ＰＯより
大きいことを示している。つまり、本発明修ＭＥＰＯは
、骨髄細胞中での赤血球系細胞の増殖・分化をより強く
促進したものといえる。The results of this assay showed that the EPO specific activity as a hematopoietic hormone of the modified EPO of the present invention was unmodified f! It shows that it is larger than iE PO. In other words, it can be said that the modified MEPO of the present invention more strongly promotes the proliferation and differentiation of erythroid cells in bone marrow cells.
尚、本発明の修１１ＥＰＯは、４℃で１力月保存した侵
でも活性の低下はみられず、その安定性も充分に実用的
なものであった。Incidentally, the Xi-11EPO of the present invention showed no decrease in activity even after being stored at 4° C. for one month, and its stability was sufficiently practical.
た以外は同条件下で処理して得られたものをいう。It refers to the product obtained by processing under the same conditions except for the above conditions.
表 ４
（実施例１）
５９Ｆｅの取込み（ＣＰＭ）
［実施例４］
実施例２で得られたホモアルギニン残基数５．８の修飾
ＥＰＯについて、実施例３で行なった １ｎＶｉｔｒＯ
ａｓｓａｙにおける用量応答曲線を得た。結果を図２に
示した。ラット骨髄ｆａ胞（ＲＢＨＣ） ６．２５ｘ１
０ ！ｌ！ｉｌに、５９Ｆｅ最人取込み世の半量が取
込まれる時の試料中のＥＰ○は、未修飾Ｅ Ｐ Ｏ１，
５８Ｘ １０’ｎｍｏｌに対して修飾ＥＰＯでは５．９
６ｘ １０−１０−６ｎ テあった。即ち、修飾ＥＰＯ
の、一定量の細胞に対するあるレベルまでの増殖・分化
作用は、未修飾ＥＰＯよりより低濃度であられれたとい
える。Table 4 (Example 1) Incorporation of 59Fe (CPM) [Example 4] Incorporation of 59Fe (CPM) [Example 4] Incorporation of 1nVitrO performed in Example 3 on the modified EPO with 5.8 homoarginine residues obtained in Example 2
A dose response curve in the assay was obtained. The results are shown in Figure 2. Rat bone marrow follicle (RBHC) 6.25x1
0! l! When half of the maximum amount of 59Fe is incorporated into il, EP○ in the sample is unmodified EP O1,
58X 10'nmol versus 5.9 for modified EPO
There were 6x 10-10-6n te. That is, modified EPO
It can be said that the proliferation and differentiation effect of EPO to a certain level on a certain amount of cells was achieved at a lower concentration than that of unmodified EPO.
また用量応答曲線の直線領域での直線の傾きが両者で異
なることから、修１！１５ＥＰｏは未修ｆｌｌｉＥＰＯ
とは作用機作が異なることが考えられる。例えば修＃Ｅ
ＰＯは未修飾ＥＰＯより細胞に対する結合のアフィニテ
ィーが大きいことが考えられる。In addition, since the slope of the straight line in the linear region of the dose-response curve is different between the two,
It is thought that the mechanism of action is different. For example, repair #E
It is believed that PO has a greater affinity for binding to cells than unmodified EPO.
［効 果コ
以上の記載から明らかなように、本発明の修飾Ｅｌ”’
Ｏは、造血ホルモンとしてのＥＰＯ活性に関して未修ｆ
ｌｉ［ＰＯと比較してその比活性がより高くなったもの
である。その結果、貧血症等の治療薬として使用する場
合により少量で所望の効果を達成し１杯、経済性・安全
性等の見地から優れたものといえよう。[Effects] As is clear from the above description, the modified El"' of the present invention
O is untrained regarding EPO activity as a hematopoietic hormone.
li[It has a higher specific activity compared to PO. As a result, when used as a therapeutic agent for anemia, etc., the desired effect can be achieved with just a small amount, and it can be said to be excellent from the viewpoints of economy, safety, etc.
第１図はアミノ酸自動分析計から得られた本発明１１Ｆ
Ｅｌ）Ｏの酸分解物のクロ？トゲラムを示す。
第２図は本発明修飾ＥＰＯ（○）とコントロールＥＰＯ
（・）のｉｎ ＶｉｔｒＯａｓｓａｙに於ける用量応答
曲線を示す。Figure 1 shows the present invention 11F obtained from an automatic amino acid analyzer.
El) Chlorine of the acid decomposition product of O? Shows Togerum. Figure 2 shows modified EPO of the present invention (○) and control EPO.
(•) shows a dose-response curve in in VitrOassay.
Claims (1)
ている修飾エリスロポエチン。(1) Modified erythropoietin in which at least one lysine residue is guanidylated.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8978088A JPH029900A (en) | 1988-04-12 | 1988-04-12 | Modified erythropoetine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8978088A JPH029900A (en) | 1988-04-12 | 1988-04-12 | Modified erythropoetine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH029900A true JPH029900A (en) | 1990-01-12 |
Family
ID=13980193
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP8978088A Pending JPH029900A (en) | 1988-04-12 | 1988-04-12 | Modified erythropoetine |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH029900A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002032957A1 (en) * | 2000-10-16 | 2002-04-25 | Chugai Seiyaku Kabushiki Kaisha | Peg-modified erythropoietin |
| JP2013166775A (en) * | 2000-12-29 | 2013-08-29 | Kenneth S Warren Inst Inc | Protection, restoration and enhancement of erythropoietin-responsive cell, tissue and organ |
-
1988
- 1988-04-12 JP JP8978088A patent/JPH029900A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002032957A1 (en) * | 2000-10-16 | 2002-04-25 | Chugai Seiyaku Kabushiki Kaisha | Peg-modified erythropoietin |
| US8022191B2 (en) | 2000-10-16 | 2011-09-20 | Chugai Seiyaku Kabushiki Kaisha | PEG-conjugated erythropoietin |
| JP5170931B2 (en) * | 2000-10-16 | 2013-03-27 | 中外製薬株式会社 | PEG-modified erythropoietin |
| JP2013166775A (en) * | 2000-12-29 | 2013-08-29 | Kenneth S Warren Inst Inc | Protection, restoration and enhancement of erythropoietin-responsive cell, tissue and organ |
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