JPH01316400A - Modified polypeptide - Google Patents
Modified polypeptideInfo
- Publication number
- JPH01316400A JPH01316400A JP1079748A JP7974889A JPH01316400A JP H01316400 A JPH01316400 A JP H01316400A JP 1079748 A JP1079748 A JP 1079748A JP 7974889 A JP7974889 A JP 7974889A JP H01316400 A JPH01316400 A JP H01316400A
- Authority
- JP
- Japan
- Prior art keywords
- formula
- derivative
- positive integer
- chemically modified
- activity
- 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.)
- Granted
Links
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Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- 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、ヒト頚粒球コロニー刺激因子（以下ｈＧ−Ｃ
３Ｆと略記する）活性を有するポリペプチドの分子中の
少なくとも１個のアミノ基を化学修飾して得られる化学
修Ｍ５ｈＧ−Ｃ３Ｆポリペブチドおよびその製造法に関
する。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to human cervical granulocyte colony stimulating factor (hG-C).
The present invention relates to a chemically modified M5hG-C3F polypeptide obtained by chemically modifying at least one amino group in the molecule of a polypeptide having activity (abbreviated as 3F), and a method for producing the same.
ｈ Ｇ−ＣＳ Ｆは、造血幹細胞を増殖・分化させ種々
の血球を形成させる際に必須なポリペプチドの１種で、
主として頚粒球、なかでも好中球の増加を促進する効果
をもつ。好中球は生体の感染防御において重要な役割を
担っているが寿命が短く、前駆細胞の恒常的な増殖・分
化によって常に補給されていなければならない。近年広
く行われている増殖性腫瘍に対する治療は、同時に好中
球前駆細胞の増殖を阻止するため、担癌患者の感染防御
機能を低下させるという重篤副作用をもつ。ｈＧ−Ｃ５
Ｆは、好中球の増加を促進することにより、この副作用
を軽減し、他方、感染症を予防・治療する効果を持つも
のと期待される。さらに、ｈＧ−Ｃ８Ｆには、白血病細
胞株をｉｎ ｖｉｔｒｏにおいて分化させる活性があり
、白血病に対する治療薬となる可能性もあるものとみら
れる。本発明の化学修飾ｈＧ−Ｃ５Ｆポリペプチドは、
既知のｈＧ−Ｃ５ＦよりすぐれたｈＧ−Ｃ３Ｆ活性をを
しており、医薬品としての利用が期待される。h G-CSF is a type of polypeptide essential for the proliferation and differentiation of hematopoietic stem cells to form various blood cells.
It mainly has the effect of promoting an increase in cervical granulocytes, especially neutrophils. Neutrophils play an important role in the defense of living organisms against infection, but they have a short lifespan and must be constantly replenished through the constant proliferation and differentiation of progenitor cells. Treatments for proliferative tumors that have been widely used in recent years also inhibit the proliferation of neutrophil progenitor cells, which has the serious side effect of reducing the infection defense function of cancer-bearing patients. hG-C5
F is expected to reduce this side effect by promoting the increase in neutrophils, and on the other hand, to have the effect of preventing and treating infectious diseases. Furthermore, hG-C8F has the activity of differentiating leukemia cell lines in vitro, and appears to have the potential to serve as a therapeutic agent for leukemia. The chemically modified hG-C5F polypeptide of the present invention is
It has hG-C3F activity superior to that of known hG-C5F, and is expected to be used as a pharmaceutical.
従来の技術
近年急速に発展してきた組換えＤ Ｎ Ａ技術により、
血球の増殖・分化に係わるタンパク質性の因子の遺伝子
が次々と単離されてきた。それらの因子は、微生物や動
物細胞を利用して遺伝子工学の手法で生産されている。Conventional technologyWith recombinant DNA technology, which has developed rapidly in recent years,
Genes for protein factors involved in the proliferation and differentiation of blood cells have been isolated one after another. These factors are produced using genetic engineering techniques using microorganisms and animal cells.
ｈＧ−Ｃ３Ｆは、長田らがヒト扁平上皮癌細胞株ＣＨＵ
−ｎよりｃＤＮＡを単離してその塩基配列を決定し、Ｃ
Ｏ５細胞での発現を報告している〔長田ら：ネイチ＋
−（Ｎａｔｕｒｅ） ３１９． ４１５ （１９８６）
］。hG-C3F was derived from the human squamous cell carcinoma cell line CHU by Nagata et al.
Isolate cDNA from -n, determine its base sequence, and
reported expression in O5 cells [Nagata et al.: Neichi+
-(Nature) 319. 415 (1986)
].
また、ススープＳｏｕｚａ）らはヒト膀胱癌細胞株５６
３７よりｃＤＮＡを単離してその塩基配列を決定し、犬
ｌｌ１３菌での発現を報告している〔スープら：サイエ
ンス （Ｓｃｉｅｎｃｅ） ２３２． ６１ （１９
８６）］。In addition, Souza et al.
cDNA was isolated from 37, its nucleotide sequence was determined, and its expression in canine 113 bacteria was reported [Suup et al.: Science 232. 61 (19
86)].
このようにして得られたｈＧ−Ｃ３Ｆは、生体内に投与
した場合、効果の持続には連続投与が必要であり、投与
を中止すると効果はすみやかに消失するという報告があ
る〔ジャーナル・オブ・エックスヘリメンタル・メディ
スン（Ｊ、 ＥＸＩＴ、 １ｃｅｄ、）１６５、９４１
−９４８ （１９８７） 〕。これは、ｈＧ−Ｃ５Ｆの
血中における持続性が低いためと考えられる。There is a report that when hG-C3F obtained in this way is administered in vivo, continuous administration is required to maintain the effect, and that the effect disappears quickly when administration is discontinued [Journal of Exhermental Medicine (J, EXIT, 1ced,) 165, 941
-948 (1987)]. This is thought to be due to the low persistence of hG-C5F in the blood.
また、アスパラギナーゼ［Ｉｎａｄａ、 Ｙ、、 ｅ
ｔ ａｌ、。In addition, asparaginase [Inada, Y, e
tal,.
トレンズ・イン・バイオテクノロジー（Ｔｒｅｎｄｓ
ｉｎＢｉｏｔｅｃｈｎｏｌｏｇｙ）、 ４．６８−７３
． （１９８６）） 、 アルギナーゼ［５ａｖｏ
ｃａ、 Ｋ、Ｖ、、 ｅｔ ａｌ、、 バイオケミ
力・エト・バイオフィジカ・アクタ （Ｂｉｏｃｈｅｍ
ｉｃａ ｅｔＢｉｏｐｈｙｓｉｃａ Ａｃｔａ＞、
５７８． ４７−５３． （１９７９）：ｌ 、
バトロキソビンｌ：ＮｉｓｈＮ１５ｈｉ、 ＩＩ、、
ｅｔ ａｔ、、 ライフ・サイエンス （Ｌｉｆ
ｅ ５ｃｉｅｎｃｅ）、 ３３． １４６７−１４７
３゜（１９８３）１などの酵素類について、ポリエチレ
ングリコールで化学修飾を行うことにより血中持続性の
向上、抗原性の減弱がδ忍められている。Trends in Biotechnology
inBiotechnology), 4.68-73
．． (1986)), arginase [5avo
ca, K, V,, et al.
ica et Biophysica Acta>,
578. 47-53. (1979):l,
Batroxobin I: NishN15hi, II,
et at,, Life Science (Lif
e5science), 33. 1467-147
Chemical modification of enzymes such as 3° (1983) 1 with polyethylene glycol has been shown to improve blood persistence and reduce antigenicity.
一般にタンパク質を医薬として用いる場合、凍結乾燥剤
として供給されることが多い。ところが、凍結乾燥時あ
るいは凍結乾燥後に温度、湿度、酸素、紫外線等の外的
因子により影響を受け、会合、重合あるいは酸化などの
物理的、化学的変化を受け、活性の低下を招く場合があ
る。いくつかの添加剤による凍結乾燥時の安定化が試み
られている（ｈＧ−Ｃ３Ｆについては、例えば特開昭６
３−１４６８２６、特開昭６：３−１４６８２９等に添
加剤の例がある）が、実用上更に安定化された製剤が望
まれている。Generally, when proteins are used as medicines, they are often supplied as lyophilized agents. However, during or after freeze-drying, it may be affected by external factors such as temperature, humidity, oxygen, and ultraviolet light, and may undergo physical and chemical changes such as association, polymerization, or oxidation, leading to a decrease in activity. . Stabilization during freeze-drying using several additives has been attempted (for hG-C3F, for example,
3-146826, JP-A-6:3-146829, etc.), but for practical purposes, a more stabilized formulation is desired.
発明が解決しようとする課題
医薬品としてｈＧ−Ｃ３Ｆを用いる場合、生体内に投与
されたｈＧ−Ｃ３Ｆが、その活性を保持したまま、血中
での安定性が高く、高い持続性を有し、さらにその抗原
性が減弱されたものであることが望ましい。また、ｈＧ
−Ｃ３Ｆを医薬として用いる場合、通常凍結乾燥製剤と
して供給されるので、凍結乾燥時ならびに凍結乾燥後に
安定性を向上させることは有用である。しかしながら、
そのような性質を有するｈＧ−Ｃ５Ｆおよびその製造法
は開発されていない。Problems to be Solved by the Invention When using hG-C3F as a pharmaceutical, hG-C3F administered in vivo retains its activity, has high stability in blood, and has high persistence; Furthermore, it is desirable that the antigenicity thereof be attenuated. Also, hG
When -C3F is used as a medicine, it is usually supplied as a lyophilized preparation, so it is useful to improve its stability during and after lyophilization. however,
hG-C5F having such properties and its production method have not been developed.
課題を解決するための手段
本発明者は、ｈＧ−Ｃ３Ｆ活性を有するポリペプチドの
分子中の少なくとも１個のアミノ基を化学修飾すること
により、未修飾のｈ （、−ＣＳ Ｆに比して、血中に
おける持続性を向上させることができることおよび凍結
乾燥時ならびに凍結乾煙後において安定性を向上させる
ことができることを見出し、本発明を完成した。Means for Solving the Problems The present inventors chemically modified at least one amino group in the molecule of a polypeptide having hG-C3F activity, thereby increasing the completed the present invention by discovering that persistence in blood can be improved and stability can be improved during freeze-drying and after freeze-drying.
以下に本発明の詳細な説明する。The present invention will be explained in detail below.
本発明は、ｈＧ−Ｃ５Ｆ活性を有するポリペプチドの分
子中の少なくとも１個のアミノ基に式ＲＴ（ＯＣＨ，Ｃ
Ｈ２−）−、Ｘ−Ｒ２−（Ｔ）（式中Ｒ１はアルキル基
またはアルカノイル基、一
〔式中Ｒ３はＯＨ，ＣＩ！、Ｏ（ＣＨ＝ＣＨ２０）ｎ
ＲＩ（式中Ｒ，，ｎは前記と同意義を表す）を表し、
Ｙは存在しないか、またはＺ （ＣＨ２）ｐＣＯ（式
中Ｚは０．ＳまたはＮＨを表し、ｐは任意に変わりうる
正の整数を表す）を表す〕または（ＣＯ）ｍ （ＣＨ
２） ｔ ＣＯ（式中、ｍは０または１、ｌは任意に変
わりつる正の整数を表す）を表す）で表される基を結合
してなる修飾ポリペプチドを提供する。The present invention provides that at least one amino group in the molecule of a polypeptide having hG-C5F activity has the formula RT (OCH, C
H2-)-, X-R2-(T) (wherein R1 is an alkyl group or an alkanoyl group,
RI (in the formula, R,, n represents the same meaning as above),
Y is absent or represents Z (CH2)pCO (wherein Z represents 0.S or NH, and p represents a positive integer that can vary arbitrarily)] or (CO)m (CH
2) Provides a modified polypeptide in which a group represented by t CO (in the formula, m represents 0 or 1, and l represents an arbitrarily variable positive integer) is bonded.
本発明に使用するｈ（、−Ｃ３Ｆ活性を有するポリペプ
チドは、第１表に示されるアミノ酸配列を有するポリペ
プチドや、第２表に代表されるような、第１表のアミノ
酸配列中で少なくとも１個のアミノ酸が他種のアミノ酸
に置換されたポリペプチドまたはＮ末端アミノ酸が１−
１１個欠失したポリペプチドでｈＧ−Ｃ３Ｆ活性を有す
るものであればよい。さらに特開昭６３−２９９ 、特
公表６３−５００６３６に記載のｈ Ｇ−ＣＳ Ｆｍ導
体も用いることができる。The polypeptides having h(, -C3F activity) used in the present invention include polypeptides having the amino acid sequences shown in Table 1, and polypeptides having at least one of the amino acid sequences shown in Table 1, such as those represented by Table 2. A polypeptide in which one amino acid is substituted with another type of amino acid or the N-terminal amino acid is 1-
Any polypeptide with 11 deletions and hG-C3F activity may be used. Further, the hG-CS Fm conductor described in Japanese Patent Application Laid-open No. 63-299 and Japanese Patent Publication No. 63-500636 can also be used.
第 １ 表 ＡｌａＧｌｎＰｒ。Chapter 1 Table AlaGlnPr.
（Ｘ＝Ｈまたは！Ｊｅｔ）
本発明に用いられる化学修飾基に関し、Ｒ１で示される
末端酸素の保護基としてのアルキル基、アルカノイル基
としては、具体的にはアルキル基は０１〜ｌ（メチル、
エチル、プロピルなど）のもの、アルカノイル基はＣ１
〜５．のもの（ホルミル。(X=H or !Jet) Regarding the chemical modification group used in the present invention, the alkyl group or alkanoyl group as a protecting group for the terminal oxygen represented by R1 is specifically an alkyl group of 01 to 1 (methyl,
(ethyl, propyl, etc.), alkanoyl groups are C1
~5. (formyl).
アセチル、プロピオニルなど）があげられる。acetyl, propionyl, etc.).
ｎで表される正の整数は、５００以下である。The positive integer represented by n is 500 or less.
とりわけ７〜２３０が好ましい。Especially preferred is 7-230.
βで表される正の整数は、１００以下であり、好ましく
は、０〜６である。ｐは表される正の整数は１〜１８、
好ましくは１〜６である。本発明の化学修飾基の分子量
は、３０，０００以下であり、好ましくは３００〜１０
，０００の範囲内にあるものである。The positive integer represented by β is 100 or less, preferably 0-6. p is a positive integer from 1 to 18,
Preferably it is 1-6. The molecular weight of the chemical modification group of the present invention is 30,000 or less, preferably 300 to 10
,000.
本発明の化学修飾ｈＧ−Ｃ３Ｆは、例えばｈｃＣβ
（Ｒ，、ｎ、Ｘ、Ｒ，は前記と同意義である）で示され
るハロゲン化物との縮合あるいはｈ Ｇ−ＣＳ Ｆと、
Ｒｒ−（ＯＣＨ，ＣＨ２）、、−Ｘ−（Ｃｏ）、（ＣＨ
，）７！−ＣＯＯＨ（Ｉｎ）（Ｒ，、ｎ、Ｘ、ｍ、１．
は前記と同意義である）で示されるカルボン酸との縮合
あるし）はｈ Ｇ−ＣＳ Ｆと
（式中Ｒ１，ｎ、 Ｘ、 Ｒ３、Ｚ、 ｐは前記
と同意義である）で示されるカルボン酸との縮合により
製造される。The chemically modified hG-C3F of the present invention is, for example, condensed with a halide represented by hcCβ (R, , n, X, R, are the same as defined above) or with hG-CSF,
Rr-(OCH,CH2), -X-(Co), (CH
,)7! -COOH(In)(R,, n, X, m, 1.
is the same meaning as above)) is a condensation with a carboxylic acid represented by h G-CSF (wherein R1, n, It is produced by condensation with carboxylic acid.
式（Ｕ）のハロゲン化物は、Ｒ３−（○ＣＨ２ＣＨ−）
ｎ−ＸＨ（Ｒ２，ｎ、Ｘは前記と同意義である）と塩
化シアヌルとの縮合で得られる〔例えば、！Ｊａｔｓｕ
ｓｈｉｍａ。The halide of formula (U) is R3-(○CH2CH-)
Obtained by condensation of n-XH (R2, n, and X have the same meanings as above) and cyanuric chloride [for example,! Jatsu
Shima.
Ａ、、 ｅｔ ａｌ、、ケミストリー・レターズ（Ｃ
ｈｅｍｉｓｔｒｙ。A, et al, Chemistry Letters (C
hemistry.
Ｌｅｔｔｅｒｓ）、 ７７３−７７６、 （１９８０
）、＾ｂｕｃｈｏｗｓｋｉ、 Ａ、、ｅｔａｌ、、
ジャーナル・オブ・バイオロジカル・ケミストリー（Ｊ
、 Ｂｉｏｌ、Ｃｈｅｍ、）、 ２５２． （１
２） ３５７８−３５８１ （１９７７） ］。このハ
ロゲン化物は反応性を有するので、ｈＧ−Ｃ３Ｆ活性を
有するポリペプチドと直接反応させることができる。Letters), 773-776, (1980
), ^buchowski, A,, etal,,
Journal of Biological Chemistry (J
, Biol, Chem, ), 252. (1
2) 3578-3581 (1977)]. Since this halide has reactivity, it can be directly reacted with a polypeptide having hG-C3F activity.
式（■）のカルボン酸は、Ｒ，−（ＯＣ８２ＣＨ，）、
、−ＸＨ（Ｒ，、ｎ、Ｘは前記と同意義である）とアル
カンジカルボン酸のカルボキシル基の脱水縮合によりカ
ルボキシル基を導入するか、へロゲル化モノカルボン酸
によりカルボキシル基を導入するか、あるいは末端の水
酸基を酸化し、カルボキシル基に変換することにより得
られる。このカルボン酸は反応性を有さないので、活性
化して用いる必要がある。カルボン酸の活性化法として
は、例えばＮ−ヒドロキシコハク酸イミド、Ｎ−ヒドロ
キシフタル酸イミド、ｌ−ヒドロキシベンゾトリアゾー
ル、ｐ−ニトロフェノールなどとの活性エステルとする
方法、イソブチルクロロホルメート、エチルクロロホル
メートとの混合酸無水物とする方法、または塩化チオニ
ルなどのハロゲン化剤を作用させて、酸ハロゲン化物と
する方法〔いずれも「ペプチド合成」 （泉屋信夫ほか
著、丸善刊）参照〕などがあげられる。The carboxylic acid of formula (■) is R, -(OC82CH,),
, introducing a carboxyl group by dehydration condensation of the carboxyl group of an alkanedicarboxylic acid with -XH (R, , n, and X have the same meanings as above), or introducing a carboxyl group by using a hero gelling monocarboxylic acid, Alternatively, it can be obtained by oxidizing the terminal hydroxyl group and converting it into a carboxyl group. Since this carboxylic acid has no reactivity, it is necessary to activate it before use. Methods for activating carboxylic acids include, for example, methods for forming active esters with N-hydroxysuccinimide, N-hydroxyphthalimide, l-hydroxybenzotriazole, p-nitrophenol, etc., isobutyl chloroformate, ethyl chloroformate, etc. A method of forming a mixed acid anhydride with a formate, or a method of forming an acid halide by reacting with a halogenating agent such as thionyl chloride [see "Peptide Synthesis" (written by Nobuo Izumiya et al., published by Maruzen)], etc. can be given.
前述の式（ｒＶ）のカルボン酸は式（ＩＩ）の塩化物と
Ｒ２（Ｃｉ（２）ｐ Ｃ０２Ｈ（ＺＳｐは前記と同義
である）との縮合により得られる。この式（ｒＶ）のカ
ルボン酸は式（ＩＩＩ）のカルボン酸と同様に、活性化
して用いる。The above-mentioned carboxylic acid of formula (rV) can be obtained by condensation of the chloride of formula (II) and R2(Ci(2)pC02H (ZSp has the same meaning as above).This carboxylic acid of formula (rV) is activated and used in the same manner as the carboxylic acid of formula (III).
これらのハロゲン化物あるいはカルボン酸活性化物を、
ｈＧ−Ｃ３Ｆ活性を有するポリペプチドの分子中に存在
するアミノ基の２〜１００倍量（モル比）加え、４〜３
７℃、好ましくは４〜１０℃、ｐＨ７〜１０で１時間〜
２日間好ましくは１〜２４時間反応させることにより目
的とする化学修飾ｈ Ｇ−ＣＳ Ｆを製造することがで
きる。These halides or carboxylic acid activated products,
Add 2 to 100 times the amount (molar ratio) of amino groups present in the molecule of a polypeptide having hG-C3F activity, and add 4 to 3
1 hour at 7°C, preferably 4-10°C, pH 7-10
The desired chemically modified hG-CSF can be produced by reacting for 2 days, preferably 1 to 24 hours.
化学修飾ｈ Ｇ−ＣＳ Ｆまたは化学修飾ｈ Ｇ−Ｃ３
Ｆｉ導体において、式■のハロゲン化物との反応物を■
型、弐■のカルボン酸との反応物を■型、弐■のカルボ
ン酸との反応物を■型と、それぞれ称する。Chemical modification h G-CS F or chemical modification h G-C3
In the Fi conductor, the reaction product with the halide of the formula ■ is
The reaction product of type 2 and carboxylic acid is called type 2, and the reaction product of type 2 and carboxylic acid is called type 2, respectively.
化学修飾の程度は遊離アミノ基の減少債をトリニトロベ
ンゼンスルホン酸で定量することにより、あるいは、ド
デシル硫酸ナトリウム−ポリアクリルアミドゲル電気泳
動法を用いて、化学！飾ｈＧ−Ｃ３Ｆの移動度の変化を
追うことにより確認される。The degree of chemical modification can be determined by quantifying the decrease in free amino groups using trinitrobenzene sulfonic acid or by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This is confirmed by tracking changes in the mobility of decorated hG-C3F.
化学修飾ｈＧ−Ｃ５Ｆおよび化学修飾ｈＧ−Ｃ３Ｆ誘導
体は水または適当な緩衝液に溶解した液剤または凍結乾
煙剤として医薬用途に供される。Chemically modified hG-C5F and chemically modified hG-C3F derivatives are provided for pharmaceutical use as a solution dissolved in water or an appropriate buffer or as a freeze-dried agent.
凍結乾燥の条件はとくに限定しないが、通常は一５０℃
以下で１〜５時間凍結し、棚温−２０℃〜０℃、真空度
５０〜１５０ｍＴｏｒｒで２４〜４８時間乾燥し、つい
で側温ｌＯ〜３０℃、真空度５ （１〜１００ｍＴｏｒ
ｒで１６〜２４時間乾燥し、凍結乾燥品を得る。Freeze-drying conditions are not particularly limited, but usually -50℃
Freeze for 1 to 5 hours below, dry for 24 to 48 hours at a shelf temperature of -20°C to 0°C and a degree of vacuum of 50 to 150 mTorr, then dry at a side temperature of 10 to 30°C and a degree of vacuum of 5 (1 to 100 mTorr).
Dry at r for 16-24 hours to obtain a lyophilized product.
液剤はそのまま無菌濾過して注射剤として用い凍結乾煙
剤は水または適当な緩衝液に溶解した後無菌濾過して注
射剤として使用する。The liquid preparation is directly sterile-filtered and used as an injection; the freeze-dried smoke preparation is dissolved in water or an appropriate buffer, and then sterile-filtered and used as an injection.
なお、本発明において使用するｈＧ−Ｃ３Ｆは：通常の
各種製薬担体、賦形剤、希釈剤、安定化剤あるいは吸着
防止剤などを含むことができる。The hG-C3F used in the present invention may contain various conventional pharmaceutical carriers, excipients, diluents, stabilizers, and anti-adsorption agents.
本誘導体の投与量は、その対象となる疾患および患者の
病状にあわせて決められるが、通常の成人−人当り０．
１〜５００μｇ１好ましくは０．５〜２００μｇの化学
修飾ｈＧ−Ｃ３Ｆおよび化学修飾ｈＧ−Ｃ５Ｆ誘導体含
を製剤を１週間当り１〜７回投与する。The dosage of this derivative is determined according to the target disease and the patient's condition, but it is 0.000 mg per normal adult.
The formulation containing 1 to 500 μg, preferably 0.5 to 200 μg of chemically modified hG-C3F and chemically modified hG-C5F derivatives is administered 1 to 7 times per week.
化学修飾ｈＧ−Ｃ３Ｆおよび化学修飾ｈＧ−Ｃ３Ｆ誘導
体にはポリエチレングリコール誘導体が一分子〜三分子
結合する。したがって、本修飾ｈＧ−Ｃ５Ｆならびに修
飾ｈＧ−Ｃ３Ｆ誘導体は一分子結合体〜三分子結合体の
混合物で用いるか、あるいは−分子結合体〜三分子結合
体をそれぞれ分離して用いる。One to three molecules of polyethylene glycol derivative are bound to chemically modified hG-C3F and chemically modified hG-C3F derivatives. Therefore, the present modified hG-C5F and modified hG-C3F derivatives can be used as a mixture of monomolecular conjugates to trimolecular conjugates, or used separately as monomolecular conjugates to trimolecular conjugates, respectively.
本発明における蛋白質量の測定は以下に記載する実験方
法によって測定する。The amount of protein in the present invention is measured by the experimental method described below.
実験方法ｌ。Experimental method l.
オ・エッチ・ローリ−の方法［Ｌｏｗｒｙ、 Ｏ，Ｈ，
。Lowry, O, H,
.
ｅｔ ａｌ、、ジャーナル・オブ・バイオロジカル・
ケミストリー （Ｊ、 Ｒｉａｌ、 Ｃｈｅｍ、）
、 １９３． ２６５ （１９５１＞１により行う。et al., Journal of Biological Sciences
Chemistry (J, Rial, Chem,)
, 193. 265 (performed by 1951>1).
実験方法２゜
レムリの方法〔口、に、（４Ｃ＋＋＋ｍｌ ｉ ：ネイ
チ＋ −（Ｎａｔｕｒｅ）２２７、６８０ （１９７０
） 〕により〕５ＤＳ−ポリアクリルアミドゲル電気泳
を行った後、クロマトスキャナー＜Ｃ５−９３０島津製
作所）で測定する。Experimental method 2゜Laemmli's method [mouth, ni, (4C+++ml i:Nature) 227, 680 (1970
)] After performing 5DS-polyacrylamide gel electrophoresis, measurement is performed using a chromato scanner <C5-930 Shimadzu Corporation).
本発明におけるＧ−Ｃ３Ｆ活性の測定は以下のように行
った。８〜１２週令のＣ３Ｈ／Ｈｅ雄マウス（静岡実験
動物協同組合）の大腿骨より骨髄細胞を無菌的にとり出
し、牛胎児血清（ＦＢＳ）を１０％添加したａ −！Ｊ
ｉｎｉｍｕｍ Ｅｓ５ｅｎｔｉａｌ Ｍｅｄｉｕｍ（Ｆ
ｌｏｗ Ｌａｂｏｒａｔｏｒｉｅｓ、以下Ｕ −Ｍ Ｅ
Ｍ培地と略す）に懸濁した。この細胞（約５Ｘ１０’
個）！ｇ４液１．５ｍｌをナイロン・ウール（Ｎｙｌｏ
ｎ ｗｏｏｌ） （和光純薬、Ｎｙｌｏｎ Ｆｉｂｅｒ
１４６−０４２３１＞をつめたカラム（０，３ｇ）に
浸漬し、５％ＣＯ。インキュベーター内にて３７℃９０
分間反応させた。次いで予め３７℃に加温したα−ＭＥ
Ｍ培地をカラムに流し、溶出してくるナイロン・ウール
非吸着性の骨髄細胞を得た。この細胞をα−ＭＥＭ培地
で一回洗浄し、所定の濃度に調製した。Measurement of G-C3F activity in the present invention was performed as follows. Bone marrow cells were aseptically removed from the femurs of 8-12 week old C3H/He male mice (Shizuoka Experimental Animal Cooperative Association), and 10% fetal bovine serum (FBS) was added to a-! J
inimum Es5nential Medium (F
low Laboratories, hereinafter referred to as U-ME
(abbreviated as M medium). This cell (approximately 5X10'
Individual)! Transfer 1.5 ml of g4 solution to nylon wool (Nylo
n wool) (Wako Pure Chemical, Nylon Fiber
146-04231> (0.3 g) and 5% CO. 37℃90 in incubator
Allowed to react for minutes. Next, α-ME preheated to 37°C
M medium was poured into the column, and eluted bone marrow cells that were not adsorbed to nylon wool were obtained. The cells were washed once with α-MEM medium and adjusted to a predetermined concentration.
次いで、間部らの方法［０ｋａｂｅ Ｔ、 ｅｔ
ａｔ、。Then, the method of Mabe et al.
at.
キ丁ンサーｅリサーチ （Ｃａｎｃｅｒ Ｒｅ５ｅａｒ
ｃｈ） ４４゜４５０３−４５０６ （１９８６）］
に準じて骨髄造血幹細胞コロニー形成能を測定した。す
なわち、α−ＭＥＭ培地０．２ｍｌ、 ＦＢＳ ０
．４ｍｌおよび２段階希釈した各サンプル０．２ｍｌの
混液に、上記の方法で調製した骨髄細胞（２Ｘ１０ｇ個
／ｍ１）の０．２ｍｌを混和した。これに４２℃に保温
した０、６％寒天（Ｄｉｆｃｏ。Cancer Re5ear
ch) 44°4503-4506 (1986)]
Bone marrow hematopoietic stem cell colony forming ability was measured according to . That is, 0.2 ml of α-MEM medium, FBS 0
．． 0.2 ml of bone marrow cells (2×10 g cells/ml) prepared by the above method was mixed with a mixture of 4 ml and 0.2 ml of each two-step diluted sample. This was supplemented with 0.6% agar (Difco) kept at 42°C.
Ａｇａｒ ｐｕｒｉｆｉｅｄ ： ０５６０−０１）溶
液を等’７１ （１，０ｍｌ）混和し、その０．５ｍｌ
を２４穴マルチデイツシユ（Ｎｕｎｃ社製、＄ １４３
９８２）に播種した（５Ｘ１０’個／ｗｅｌｌ、 ｎ
＝３） 、 ５％ＣＯ２インキュベーター中で３７℃
７日間培養し、４０個以上の細胞からなるコロニーの数
を顕微鏡（Ｏ１ｙｍｐｕｓ社製、×４０）で計数した。Agar purified: 0560-01) solution (1,0 ml) was mixed, and 0.5 ml of it was mixed.
24-hole multi-date (manufactured by Nunc, $143)
982) (5 x 10' pieces/well, n
=3), 37°C in a 5% CO2 incubator
After culturing for 7 days, the number of colonies consisting of 40 or more cells was counted using a microscope (manufactured by Olympus, ×40).
コロニー計数後、注意深くスライドグラス上にとり出し
、アセトン・ホルマリン混液で３０秒間固定後、Ｋｕｂ
ｏＬａらの方法［Ｋｕｂｏｔａ Ｋ、。After counting the colonies, carefully take them out onto a slide glass, fix with acetone/formalin mixture for 30 seconds, and then
The method of oLa et al. [Kubota K,.
ｅｔ ａｌ、、エックスペリメンタル・ヘマトロジイ
（Ｅｘｐ、）Ｉｅｍａｔｏｌｏｇｙ） ８．３３９−３
４４ （１９８０）］でエニステラー２重染色を施し、
各コロニーの同定を行った。et al, Experimental Hematology (Exp,) Iematology) 8.339-3
44 (1980)] and subjected to Ennisteller double staining.
Each colony was identified.
各サンプルの力価は、コロニー形成試験の２段階稀釈に
於ける計数結果から以下のように算出した。スタンダー
ドとして用いたインタクトＧ−ＣＳＦのコロニー形成の
最大値の２値を与える活性を５０単位と定義し、これに
各サンプルの稀釈率および単位ｍ１当りの活性に換算す
るため、２０を乗じて力価（単位）とした。比活性は、
単位蛋白質（ｍｇ）当りの力価（単位／ｍｇ）で表示し
た。The titer of each sample was calculated as follows from the counting results in two stage dilutions of the colony formation test. The activity that gives the maximum binary value of colony formation of intact G-CSF used as a standard is defined as 50 units, and in order to convert this to the dilution rate of each sample and the activity per unit ml, multiply it by 20 and calculate the power. value (unit). The specific activity is
It was expressed as titer (unit/mg) per unit protein (mg).
以下に実施例、参考例、実験例を示す。Examples, reference examples, and experimental examples are shown below.
実施例１゜
第１表のアミノ酸配列を有するｈＧ−Ｃ５Ｆ１８６ μ
ｇ／ｍｌを含む０．ＩＭホウ酸緩衝液（ｐＨ１０）３ｍ
ｌに後述の参考例１で得られたクロル化物５６ｍｇを添
加し、攪拌しながら４℃で２４時間反応させた。Example 1 hG-C5F186μ having the amino acid sequence shown in Table 1
0.g/ml. IM borate buffer (pH 10) 3m
56 mg of the chloride obtained in Reference Example 1 described later was added to the mixture, and the mixture was reacted at 4° C. for 24 hours with stirring.
限外濾過（分画分子量３万）により、未反応のりＤＪＩ
、化物を除去後、Ｍ Ｍ Ｃ−ＰａｃｋΔＭ−３１２０
ＤＳ（栗田工業社製）を用いるアセトニ） ＩＪル０−
７０％の直線匂配による逆相ＨＰＬＣを行った。化学修
飾ｈＧ−Ｃ３Ｆポリペプチドは、アセトニトリル約５０
％の溶出分画に溶出された（収量３０μｇ、収率５％）
。得られた化学修飾ｈＧ−Ｃ３Ｆポリペプチドは、ｈＧ
−Ｃ３ＦＩ分子に対し、クロル化物１分子結合している
ことが、５ＤＳ−ポリアクリルアミドゲル電気泳動で確
Ｊ忍された。By ultrafiltration (molecular weight cut off: 30,000), unreacted glue DJI
, after removing compounds, MMC-PackΔM-3120
Acetonitrile using DS (manufactured by Kurita Kogyo Co., Ltd.) IJ Le 0-
Reversed phase HPLC with 70% linear gradient was performed. Chemically modified hG-C3F polypeptides can be prepared using acetonitrile at approximately 50%
% elution fraction (yield 30 μg, yield 5%)
. The obtained chemically modified hG-C3F polypeptide
- It was confirmed by 5DS-polyacrylamide gel electrophoresis that one molecule of chloride was bound to the -C3FI molecule.
純度は９０％以上であった。The purity was over 90%.
実施例２゜
第１表のアミノ酸配列を有するｈＧ−Ｃ５Ｆ５７０ ｑ
／ｍｌを含む５０ｍＭリン酸緩衝液（ｐＨ７，２）５０
ｍｌに、後述の参考例２で得られた活性エステル２４０
ｍｇを添加し、攪拌しながら４℃で６時間反応させた。Example 2 hG-C5F570q having the amino acid sequence shown in Table 1
50mM phosphate buffer (pH 7,2) containing /ml
ml of active ester obtained in Reference Example 2 described below.
mg was added thereto, and the mixture was reacted at 4° C. for 6 hours with stirring.
１０ｍＭＩＪス塩酸緩衝液−０．７Ｍ硫酸アンモニウム
（ｐＨ８，０）５０ｍｌを加えた後、ｌＱｍＭトリス・
塩酸−〇、３５Ｍ硫酸アンモニウム（ｐＨ８，０）で充
たされたブチル−トヨパール６５０Ｍ（東ソーり（２，
２ｃｍｘ２６ｃｍ＞に１００ｍ１／ｈｒの流速で通塔し
た。次いで１０ｍＭ）ＩＪス・塩酸−０，３５Ｍ硫酸ア
ンモニウム（ｐＨ８，０）１００ｍｌを１００ｍ１／ｈ
ｒの流速で通塔して洗浄後、１０ｍ Ｍ ト’）ス・塩
酸（ｐＨ８，０＞系に直線匂配で０．３５Ｍ硫酸アンモ
ニウムから０Ｍ硫酸アンモニウムを総１ｔ４００ｍｌか
けて加え、１００ｍｌ／ｈｒの流速で溶出を行った。目
的物は、硫酸アンモニウム５０ｍＭから１３０ｍＭで溶
出された。溶出画分１３０ｍ１を限外濾過〔分子量分画
１万；膜ＹＭ１０ （アミコン社製）〕シ、７ ｍｌま
でａ縮した。After adding 50 ml of 10mM JS hydrochloric acid buffer-0.7M ammonium sulfate (pH 8,0), 1QmM Tris.
Butyl-Toyopearl 650M (Tosori (2,
2 cm x 26 cm> at a flow rate of 100 ml/hr. Then, 100ml of 10mM) IJ-hydrochloric acid-0.35M ammonium sulfate (pH 8.0) was added at 100ml/h.
After washing by passing through the column at a flow rate of r, a total of 1t400ml of 0.35M ammonium sulfate to 0M ammonium sulfate was added to the 10mM t')-hydrochloric acid (pH 8,0> system) using a linear gradient, and the mixture was washed at a flow rate of 100ml/hr. Elution was performed. The target product was eluted from 50 to 130 mM ammonium sulfate. 130 ml of the eluate fraction was subjected to ultrafiltration [molecular weight fraction 10,000; membrane YM10 (manufactured by Amicon)] and concentrated to 7 ml.
次いで、ａ縮液を１０ｍＭ’Ｊン酸緩衝液−生理食塩水
（ｐＨ７，２） （ＰＢＳ）で充たされたセファクリ
ルＳ−２００（ファルマシア社ｍ） （２，８ｃｍＸ
７Ｑｃｍ）に１２０ ｍｌ／ｈｒの流速で通塔した。次
いで、同流速でＰＢＳを通塔した。Next, the acondensate was transferred to Sephacryl S-200 (Pharmacia) (2,8 cm
7 Qcm) at a flow rate of 120 ml/hr. Then, PBS was passed through the column at the same flow rate.
化学修飾ｈＧ−Ｃ３Ｆポリペプチドはポリエチレングリ
コール誘導体三分子結合体（Ｔ「１体）がＰＢＳを流し
始めて１５０ｍ＆から１６０ｔｒｌｆ！あたりに溶出さ
れたく収ｌ ２ ｍｇ 収率７％）。次いで、ポリエ
チレングリコール誘導体二分子結合体（Ｄｉ体）が１６
５Ｍから１８５−あたりに溶出された（収５４１．５
ｍｇ 収＝１！５％）。次いで、ポリエチレングリコ
ール誘導体−分子結合体（！、ｌ ｏ ｎ ｏ体）が１
９０ｍｆ＋から２１０ｍ１あたりに溶出された（収量４
、５ ｍｇ 収率１６％）。得られたポリペプチドは
＋１１ｏｎｏ体ではｈＧ−Ｃ５Ｆ−分子に対し、ポリエ
チレングリコール誘導体のカルボン酸が一分子結合して
おり、Ｄｉ体では二分子結合しており、Ｔｒｉ体では三
分子結合していることが５ＤＳ−ポリアクリルアミドゲ
ル電気泳動で確認された。それぞれの純度は９０％以上
であった。The chemically modified hG-C3F polypeptide was obtained by eluating the polyethylene glycol derivative trimolecular conjugate (T'1 body) from 150 m to 160 trlf! (yield 2 mg, yield 7%) after starting to flow PBS. The bimolecular conjugate (Di form) is 16
It was eluted from 5M to around 185- (yield 541.5
mg yield = 1!5%). Next, the polyethylene glycol derivative-molecule conjugate (!, l o no form) is 1
It was eluted from 90mf+ to around 210ml (yield 4
, 5 mg yield 16%). In the obtained polypeptide, one molecule of carboxylic acid of a polyethylene glycol derivative is bound to the hG-C5F- molecule in the +11ono form, two molecules in the Di form, and three molecules in the Tri form. This was confirmed by 5DS-polyacrylamide gel electrophoresis. The purity of each was 90% or more.
実施例３゜
後述の参考例３で得られたｈＧ−ＣＳＦ誘導体（５７０
μｇ／ｍｌ）を含む０．１Ｍホウ酸緩１軒液（ｐＨ９＞
ｌｏｍｌに、後述の参考例２で得られた活性エステル５
４ｍｇを添加し、４℃で１０時間反応させた。Example 3 hG-CSF derivative (570
0.1M boric acid solution (pH 9>
loml, active ester 5 obtained in Reference Example 2 described below.
4 mg was added and reacted at 4°C for 10 hours.
限外濾過膜ＹＭ３０（アミコン社製）を用いて、未反応
の活性エステルおよびその分解物を除去後、間膜を用い
て１０ ｍＭ ） ＩＪス・塩酸緩衝液（ｐＨ８）に内
液の交換を行った。残液を、１０ｍＭ）リス・塩酸緩衝
液（ｐＨ８，０）で充たされたＤＥＡＥ−トヨパール６
５０Ｍ（東ソー１） （１，７ａｍ ｘ ４．４Ｃ［ｌ
ｌ）にｌｏｍｌ／ｈｒの流速で通塔した。次いでｌＯｍ
Ｍ）リス・塩酸緩衝液（ｐＨ８）２０ｍｌを５ｍｌ／ｈ
ｒの流速で通塔して洗浄後、１０ｍＭ）リス・塩酸（ｐ
Ｈ８）の緩（ｌ？液系に、直線匂配で０ＭＮａＣｆから
０．４Ｍ ＮａＣｊ！を総量１００ｍ１かけて加え、
５ｍｌ／ｈｒの流速で溶出を行った。化学修飾ｈＧ−Ｃ
ｓｐポリペプチドはＮａＣｌ１１００〜１２０ｍＭで溶
出された（収量ｏ、ａ５ｍｇ、収率１５％）。得られた
ポリペプチドは、ｈＧ−Ｃ５Ｆ誘導体１分子に対し、ポ
リエチレングリコール誘導体のカルボン酸１分子結合し
ていることが、５ＤＳ−ポリアクリルアミドゲル電気泳
動で確認された。純度は９０％以上であった。After removing unreacted active esters and their decomposition products using an ultrafiltration membrane YM30 (manufactured by Amicon), the inner solution was exchanged with a 10 mM IJ-hydrochloric acid buffer (pH 8) using an interlayer membrane. went. The remaining solution was added to DEAE-Toyopearl 6 filled with 10mM) Lis-HCl buffer (pH 8,0).
50M (Tosoh 1) (1.7am x 4.4C[l
1) was passed through the column at a flow rate of loml/hr. Then lOm
M) 20ml of Lis-HCl buffer (pH 8) at 5ml/h
After washing by passing through the column at a flow rate of r, 10mM)
Add 0M NaCf to 0.4M NaCj! to the slow liquid system of H8) in a linear manner over a total amount of 100ml.
Elution was performed at a flow rate of 5 ml/hr. Chemically modified hG-C
The sp polypeptide was eluted at 1100-120 mM NaCl (yield o, a5 mg, 15% yield). It was confirmed by 5DS-polyacrylamide gel electrophoresis that in the obtained polypeptide, one molecule of the carboxylic acid of the polyethylene glycol derivative was bound to one molecule of the hG-C5F derivative. The purity was over 90%.
実施例４゜
参考例３で得られたｈＧ−Ｃ３Ｆ誘導体（５７０μｇ
／ ｒｒｄｌ ）を含む５０ｍＭリン酸緩衝液（ｐＨ７
，２）５０−に、参考例２で得られた活性エステル３０
０ｍｇを添加し、４℃で６時間反応させた。Example 4 hG-C3F derivative obtained in Reference Example 3 (570 μg
/rrdl) in 50mM phosphate buffer (pH 7)
, 2) 50-, active ester 30 obtained in Reference Example 2
0 mg was added and reacted at 4°C for 6 hours.
１０ｍＭ）リス塩酸緩衝液−０，７Ｍ硫酸アンモニウム
（ｐＨ８，０）５０−を加えた後１０ｍＭ）リス塩酸緩
衝液−０，３５Ｍ硫酸アンモニウム（ｐＨ８，０）で充
たされたブチル−トヨバール６５０Ｍ（東ソー製）
（２，２ｃｍＸ ２６ｃｍ）に１００ Ｉｎ１／ｈｒの
流速で通塔した。次いで１０ｍＭ）リス塩酸緩衝液−０
，３５Ｍ硫酸アンモニウム（ｐＨ８，０）１００ｍｌを
１００ｍ＆／ｈｒの流速で通塔して洗浄後、１０ｍＭ）
リス塩酸緩衝液（ｐＨ８，０）に直線勾配で０．３５Ｍ
硫酸アンモニウムから０Ｍ硫酸アンモニウムを総量４゛
００−かけて加え、１００ｍ＆／ｈｒの流速で溶出を行
った。目的物は、硫酸アンモニウム５０ｍＭから１５０
ｍＭで溶出された。溶出画分１５０ｍ１２を限外濾過〔
分子量分画１万：ＹＭｌｏ （アミコン社製）〕シ、１
０ｍ１２まで濃縮した。Butyl-Toyovar 650M (manufactured by Tosoh) filled with 10mM) Lis-HCl buffer - 0,7M ammonium sulfate (pH 8,0) 50-10mM) Lis-HCl buffer - 0,35M ammonium sulfate (pH 8,0) )
(2.2 cm x 26 cm) was passed through the column at a flow rate of 100 In1/hr. Then 10mM) Lis-HCl buffer-0
, 100 ml of 35 M ammonium sulfate (pH 8,0) was passed through the column at a flow rate of 100 m&/hr, and then washed, 10 mM)
0.35M in a linear gradient to Lis-HCl buffer (pH 8,0)
From ammonium sulfate to 0M ammonium sulfate was added over a total amount of 400 ml, and elution was performed at a flow rate of 100 m/hr. The target substance is ammonium sulfate from 50mM to 150mM.
Eluted at mM. Ultrafiltrate 150ml of the eluate fraction [
Molecular weight fraction 10,000: YMlo (manufactured by Amicon), 1
It was concentrated to 0ml.
ついで、濃縮液をＰＢＳで充たされたセファクリルＳ−
２００（ファルマシアＭ） （２，８ｃｍｘ ７０ｃ
Ｉ１１）に１２０ｍｆｆ／ｈｒの流速で通塔した。次い
で、同流速でＰＢＳを通塔した。化学修飾ｈＧ−Ｃ５Ｆ
ポリペプチドはポリエチレングリコール誘導体三方子結
合体（Ｔｒｉ体）がＰＢＳを流し始めて１５０−から１
６０−あたりに溶出されたく収量１、５ ｍｇ 収率
５％）。次いで、ポリエチレングリコール誘導体二分子
結合体（Ｄｉ体）が１６５−から１８５ｍ１！あたりに
溶出された（収量３ｍｇ 収率１１％）。次いで、ポ
リエチレングリコール誘導体−分子結合体（Ｍ ｏ ｎ
ｏ体）が１９０ｍ１から２１０−あたりに溶出された
（収量４ｍｇ 収率１４％）。The concentrate was then poured into a Sephacryl S-
200 (Pharmacia M) (2.8cmx 70c
I11) at a flow rate of 120 mff/hr. Then, PBS was passed through the column at the same flow rate. Chemically modified hG-C5F
The polypeptide is a polyethylene glycol derivative trigonal conjugate (Tri body) that changes from 150 to 1 when PBS starts flowing.
The sample was eluted around 60-mg (yield: 1.5 mg, yield: 5%). Next, the polyethylene glycol derivative bimolecular conjugate (Di form) is 165 to 185 m1! (yield: 3 mg, yield: 11%). Next, a polyethylene glycol derivative-molecule conjugate (M on
o form) was eluted from 190 ml to around 210-ml (yield: 4 mg, yield: 14%).
得られたポリペプチドはｈｌｏｎｏ体ではｈＧ−Ｃ３Ｆ
−分子に対し、ポリエチレングリコール誘導体のカルボ
ン酸が一分子結合しており、０１体では二分子結合して
おり、Ｔｒｉ体では３分子結合していることが５ＤＳ−
ボアクリルアミドゲル電気泳動で確認された。それぞれ
の純度は９０％以上であった。The obtained polypeptide is hG-C3F in hlono form.
-One molecule of the carboxylic acid of the polyethylene glycol derivative is bound to the molecule, two molecules are bound in the 01 form, and three molecules are bound in the Tri form.5DS-
Confirmed by boreacrylamide gel electrophoresis. The purity of each was 90% or more.
実施例５゜
参考例３で得られたｈＧ−Ｃ３Ｆ誘導体（３００ｕｇ／
ｍｌ ）を含む５０ｍＭリン酸緩衝液（ｐＨ７，２）
１００ｍｌに、参考例４で得られた活性エステル８００
ｍｇを添加し、４℃で２４時間反応させた。Example 5 hG-C3F derivative obtained in Reference Example 3 (300ug/
50mM phosphate buffer (pH 7,2) containing
800 mL of active ester obtained in Reference Example 4 to 100 ml.
mg was added and reacted at 4°C for 24 hours.
１０ ｍＭ ） ’Ｊス塩酸緩衝液−０．７Ｍ硫酸アン
モニウム（ｐＨ８，０）ｌｏｏｍ＆を加えた後、１０ｍ
１トリス塩酸緩衝液−０，３５Ｍ硫酸アンモニウム（ｐ
Ｈ８，０）で充たされたブチル−トヨパール６５０Ｍ（
東ソー製）（２，２ｃｍＸ２６ｃｍ）に１００ｍ１／ｈ
ｒの流速で通塔した。次いで１０ｍＭトリス塩酸緩衝液
−０，３５Ｍ硫酸アンモニウム（ｐＨ８，０）１００−
を１００ｄ／ｈｒの流速で通塔して洗浄後、ｌ ＯｍＭ
） ’Ｊス塩酸緩衝液（ｐＨ８，０）系に直線勾配で
０．３５Ｍ硫酸硫酸アンモニウム０Ｍ硫酸アンモニウム
を＆ｅ量４００＋ｎ＋２かけて加え、１００ｄ／ｈｒの
流速で溶出を行った。目的物は、硫酸アンモニウムＯｍ
Ｍから２５０ｍＭで溶出された。溶出画分２５０−を限
外−過〔分子量分画１万：ＹＭｌｏ（アミコン社製）〕
シ、ＩＯ−まで濃縮した。ついで、ＰＢＳで充たされた
セファクリルＳ−２００（ファルアシア製） （５，６
ｃｍＸ４０ｃｍ＞に１６０ｍ１２／ｈｒの流速で通塔し
た。次いで、同流速でＰＢＳを通塔した。化学修飾ｈＧ
−Ｃ３Ｆポリペプチドはポリエチレングリコール誘導体
三方子結合体（Ｔ「１体）がＰＢＳを流し始めて３６０
−から４００ｍ１あたりに溶出された（収量２．１ ｆ
ｆ１ｇ収率７％）。次いで、ポリエチレングリコール誘
導体二分子結合体（Ｄｉ体）が４２０ｍ１から４５０ｍ
１あたりに溶出された（収１１．５ ｍｇ収率５％）。10mM) 'JS hydrochloric acid buffer-0.7M ammonium sulfate (pH 8,0) room
1 Tris-HCl buffer - 0,35M ammonium sulfate (p
Butyl-Toyopearl 650M (H8,0) filled with
Tosoh) (2.2cm x 26cm) 100m1/h
It passed through the tower at a flow rate of r. Then 10mM Tris-HCl buffer - 0,35M ammonium sulfate (pH 8,0) 100-
After washing by passing through the column at a flow rate of 100 d/hr, l OmM
) 0.35M ammonium sulfate sulfate and 0M ammonium sulfate were added in a linear gradient to a JS hydrochloric acid buffer (pH 8,0) system in an amount of 400+n+2, and elution was performed at a flow rate of 100 d/hr. The target is ammonium sulfate Om
It was eluted at 250mM from M. The elution fraction 250- was ultrafiltered [molecular weight fraction 10,000: YMlo (manufactured by Amicon)]
The mixture was concentrated to 10-10%. Then, Sephacryl S-200 (manufactured by Falcia) filled with PBS (5,6
cm×40 cm> at a flow rate of 160 m12/hr. Then, PBS was passed through the column at the same flow rate. chemically modified hG
-C3F polypeptide is a polyethylene glycol derivative three-way conjugate (T "1 body") after starting to flow PBS.
- eluted around 400 ml (yield 2.1 f
f1g yield 7%). Next, the polyethylene glycol derivative bimolecular conjugate (Di form) was added from 420 ml to 450 ml.
1 (yield: 11.5 mg, yield: 5%).
次いで、ポリエチレングリコール誘導体−分子結合体（
Ｍｏｎｏ体）が５００ｍ１から５３０ｍ１あたりに溶出
された（収５１１．５ ｍｇ 収量５％）。Next, a polyethylene glycol derivative-molecule conjugate (
Mono body) was eluted from 500 ml to 530 ml (yield: 511.5 mg, yield: 5%).
得られたポリペプチドは！、ｔ ｏ ｎ ｏ体ではｈ（
、−Ｃ５Ｆ−分子に対し、ポリエチレングリコール誘導
体のカルボン酸が一分子結合しており、Ｄｉ体では三方
子結合しており、Ｔｒｉ体では三方子結合していること
が５ＤＳ−ポリアクリルアミドゲル電気泳動で確認され
た。それぞれの純度は９０％以上であった。The obtained polypeptide is! , h(
, 5DS-polyacrylamide gel electrophoresis shows that one molecule of the carboxylic acid of the polyethylene glycol derivative is bound to the -C5F- molecule, which is trigonal in the Di form and triangular in the Tri form. It was confirmed. The purity of each was 90% or more.
実施例６゜
化学修飾ｈＣ，Ｃ３Ｆ凍結乾燥標品の製造およびその保
存安定性
実施例２と同様の方法で、ｈ Ｇ−ＣＳ Ｆと活性エス
テルとを反応させ、限外−過膜を用いて未反応の活性エ
ステルおよびその分解物を除去した。Example 6 Production of chemically modified hC,C3F freeze-dried specimen and its storage stability In the same manner as in Example 2, hG-CSF and an active ester were reacted, and an ultrafiltration membrane was used to react the hG-CSF with an active ester. Unreacted active ester and its decomposition products were removed.
間膜を用いて、ＩＭ塩化ナトリウム含有５０ｍＭリン酸
緩衝液（ｐ Ｈ７，２＞で内液の交換を行った。The internal fluid was exchanged with IM sodium chloride-containing 50 mM phosphate buffer (pH 7,2>) using the intermembrane.
得られた修飾ｈＧ−Ｃ３Ｆ２００μｇ／ｍｌ含有溶液を
凍結乾燥し凍結乾燥剤を製造した。対照とじてｈＧ−Ｃ
３Ｆとポリエチレングリコールとの混液の凍結乾怪を同
様に行い凍結乾燥剤を製造した。The obtained solution containing 200 μg/ml of modified hG-C3F was freeze-dried to produce a freeze-dried agent. hG-C as control
A lyophilizer was produced by similarly freeze-drying a mixture of 3F and polyethylene glycol.
それぞれの凍結乾燥剤を６５℃で放置後、経時的に溶解
し、ｈＧ−Ｃ５Ｆの残存活性を前述の活性測定法に従い
測定した。結果は第３表に示す。After each freeze-dried agent was left at 65°C, it was dissolved over time, and the residual activity of hG-C5F was measured according to the activity measurement method described above. The results are shown in Table 3.
残存活性（％）は、凍結乾煙前の初期活性に対する相対
割合であり、以下の式で定義される。Residual activity (%) is a relative proportion to the initial activity before freeze-drying and is defined by the following formula.
凍結乾爆条件：未修＃ｈＧ−Ｃ３Ｆ溶液、修飾ｈＧ−Ｃ
３Ｆ溶液とも以下の条件で行う。ｈｃ−Ｃ３Ｆ溶液をガ
ラスバイヤルに入れ、−５０℃以下で２時間凍結する。Freeze-drying conditions: unmodified #hG-C3F solution, modified hG-C
The 3F solution is also used under the following conditions. Place the hc-C3F solution into a glass vial and freeze at below -50°C for 2 hours.
次いで、棚温−２０℃下真空度１００ Ｔｏｒｒで２４
時間乾燥する。次いで、２０℃下真空度８０ｍＴｏｒｒ
で２４時間乾燥し、凍結乾迷品を得る。Next, at a shelf temperature of -20°C and a vacuum degree of 100 Torr,
Dry for an hour. Next, at 20°C, the vacuum level was 80 mTorr.
Dry for 24 hours to obtain a freeze-dried product.
（５０ｍＭリン酸緩衝液、 ｐＨ７，２，ＬＭ ＮａＣ
ｊり（１） ｈ Ｇ −ＣＳ Ｆ １部当り２．５部
（重量比）（２） ｈ Ｇ −ＣＳ Ｆ １部当り
５部 （重量比）実施例７゜
化学修飾ｈｃ−Ｃ３Ｆ、ｔＭ導体の凍結乾燥条件の製造
およびその保存安定性
実施例３と同様の方法で、ｈＧ−Ｃ３Ｆ誘導体と活性エ
ステルとを反応させ、限外濾過膜を用いて未反応の活性
エステルおよびその分解物を除去した。間膜を用いて、
１Ｍ塩化す） ＩＪウム含有、１） Ｏｍ Ｍ リン緩
衝液（ｐＨ７，２）で内液の交換を行った。得られた修
飾ｈＧ−Ｃ３Ｆ誘導体２００μｇ／ｍ＆含有溶液を凍結
乾燥し、凍結乾燥剤を製造した。また、対照としてｈＧ
−Ｃ３Ｆ誘導体およびポリエチレングリコール添加溶液
の凍結乾燥を同様に行い凍結乾燥剤を製通した。それぞ
れ凍結乾燥剤を３７℃で７日間放置し、経時的に溶液中
のｈＧ−Ｃ５Ｆの残存活性を前述の活性測定法に従い測
定した。結果は第４表に示す。表中残存活性（％）およ
び凍結乾燥条件は実施例５と同じである。(50mM phosphate buffer, pH 7.2, LM NaC
(1) h G -CS F 2.5 parts per part (weight ratio) (2) h G -CS F per part
5 parts (weight ratio) Example 7 Chemically modified hc-C3F, Freeze-drying conditions for tM conductor Production and its storage stability In the same manner as in Example 3, an hG-C3F derivative and an active ester were reacted, Unreacted active ester and its decomposition products were removed using an ultrafiltration membrane. Using the mesentery,
The internal solution was exchanged with 1) OmM phosphorus buffer (pH 7,2) containing 1M chloride). The obtained modified hG-C3F derivative 200 μg/m&containing solution was freeze-dried to produce a freeze-dried agent. In addition, as a control, hG
The solution containing the -C3F derivative and polyethylene glycol was similarly freeze-dried to obtain a freeze-dried agent. Each lyophilizate was left at 37° C. for 7 days, and the residual activity of hG-C5F in the solution was measured over time according to the activity measurement method described above. The results are shown in Table 4. The residual activity (%) and freeze-drying conditions in the table are the same as in Example 5.
（５０ｍＭリン酸緩衝液、 ｐａｌ ７．２ 、 １！
、ｌ Ｎａ１Ｊ）（１） ｈＧ−Ｃ３Ｆ１部当り２．
５部（重量比）（２） ｈ Ｇ −ＣＳ Ｆ １部当
り ５部 （重量比）参考例１゜
２．４−ビス（０−メトキシポリエチレングリコール）
−６−クロル−８−トリアジンの製造１０ｇの無水炭酸
ナトリウムを含む１００ｍ１の無水トルエンに平均分子
１４０００のモノメトキシポリエチレングリコール（日
本油脂社製）２０ｇを溶解し、１１０℃で３０分間加熱
した後、塩化シアヌル５００ｍｇを加え、２４時間、１
１０℃で加熱した。反応残留物を戸去し、石油エーテル
３００ｍ１を加えて、沈澱を生じさせ、その沈澱を数回
石油エーテルで洗浄し、２．４−ビス（０−メトキシポ
リエチレングリコール）−６−クロル−５−）リアジン
を１０ｇ取得した（収率５０％）。(50mM phosphate buffer, pal 7.2, 1!
, l Na1J) (1) 2.0% per part of hG-C3F.
5 parts (weight ratio) (2) h G -CS F 5 parts per part (weight ratio) Reference example 1゜2.4-bis(0-methoxypolyethylene glycol)
Production of -6-chloro-8-triazine 20 g of monomethoxy polyethylene glycol (manufactured by NOF Corporation) with an average molecular weight of 14,000 was dissolved in 100 ml of anhydrous toluene containing 10 g of anhydrous sodium carbonate, and after heating at 110°C for 30 minutes, Add 500mg of cyanuric chloride and leave for 24 hours.
Heated at 10°C. The reaction residue was removed, 300 ml of petroleum ether was added to form a precipitate, and the precipitate was washed several times with petroleum ether to give 2,4-bis(0-methoxypolyethylene glycol)-6-chloro-5- ) 10 g of riazine was obtained (yield 50%).
参考例２゜
モノメトキシポリエチレングリコールサクンニルーＮ−
ヒドロキシサクシンイミドエステルの合成
充分脱水した平均分子ｆｆ１５０００のモノメトキシポ
リエチレングリコール（Ｌｉｎｉｏｎ Ｃａｒｂｉｄｅ
社製）２０ｇ、無水コハク酸２ｇを無水トルエン５０ｍ
１に加え、１５０℃に加熱し５時間還流した。トルエン
を減圧下留去した残渣に塩化メチレン３０ｍ１添加し、
完全可溶化後、無水エチルエーテル４００−を加えて、
沈澱を生じさせた。この沈澱物を塩化メチレン：エチル
エーテル系（１：３容量比）で再結晶を行い、サクシニ
ル化モノメトキシポリエチレングリコール１０ｇ （収
率約５０％）を得た。このサクシニル化物３．３ｇとＮ
−ヒドロキシコハク酸イミド１００ｍｇを無水塩化メチ
レン５ｍｌに可溶化し、水冷下ジシクロへキシルカルボ
ジイミド（ＤＣＣ＞２００ｍｇを添加後、室温下２０時
間攪拌した。生じたジシクロへキシルウレア（ＤＣＵ＞
を戸別し、ｐ液に エチルエーテルを加えて沈澱を生じ
させた。得られた沈澱物を塩化メチレン：エチルエーテ
ル系で再結晶を行い、モノメトキシポリエチレングリコ
ール−サクシニル−Ｎ−ヒドロキシサクシンイミドエス
テル２．５ｇ（収率７２％）を得た。Reference example 2゜Monomethoxypolyethylene glycol sacunniru N-
Synthesis of hydroxysuccinimide ester Thoroughly dehydrated monomethoxypolyethylene glycol (Linion Carbide) with an average molecular weight of 15,000
) 20g, 2g of succinic anhydride, 50ml of anhydrous toluene
1 and heated to 150°C and refluxed for 5 hours. Toluene was distilled off under reduced pressure, and 30 ml of methylene chloride was added to the residue.
After complete solubilization, add 400% of anhydrous ethyl ether,
A precipitate formed. This precipitate was recrystallized from a methylene chloride:ethyl ether system (1:3 volume ratio) to obtain 10 g (yield: about 50%) of succinylated monomethoxypolyethylene glycol. 3.3 g of this succinylated product and N
- 100 mg of hydroxysuccinimide was solubilized in 5 ml of anhydrous methylene chloride, and after adding dicyclohexylcarbodiimide (DCC>200 mg) under water cooling, the mixture was stirred at room temperature for 20 hours.The resulting dicyclohexylurea (DCU>
The p-solution was distributed from house to house, and ethyl ether was added to the p-liquid to form a precipitate. The obtained precipitate was recrystallized from a methylene chloride:ethyl ether system to obtain 2.5 g (yield: 72%) of monomethoxypolyethylene glycol-succinyl-N-hydroxysuccinimide ester.
参考例３゜
第１表に示したアミノ酸配列を有するｈＧ−Ｃ５Ｆの１
１目のスレオニンをアラニンに、３番目のロイシンをス
レオニンに、４番目のグリシンをチロシンに、５番目の
プロリンをアルギニンに、１７７番目シスチンをセリン
にそれぞれ置換したｈＧ−Ｃ３Ｆ誘導体を以下のように
して得た。Reference Example 3゜1 of hG-C5F having the amino acid sequence shown in Table 1
The hG-C3F derivative in which the 1st threonine is replaced with alanine, the 3rd leucine with threonine, the 4th glycine with tyrosine, the 5th proline with arginine, and the 177th cystine with serine is prepared as follows. I got it.
上記のｈＧ−Ｃ３Ｆ誘導体をコードするＤＮＡを含むプ
ラスミドｐｃ ｆ ＢＤ２８を保有する大腸菌’ｖＶ３
１１０５ｔｒＡ株（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ
［１ＣｆＢ口２８ＦＥＲｊｌ ＢＰ−１４７９）をＬ
Ｇ培地〔バタトトリプトン１０ｇ、酵母エキス５ｇ、Ｎ
ａＣｊ！ ５ｇ、グルコース１ｇを水１１に溶かし、
ＮａＯＨにてｐＨを７，０とする。〕で３７℃、１８時
間培養し、この培養液５ｍｌを２５μｇ ／ ｍ ｌの
トリプトファンと５０ｇｇ／ｍｌのアンピシリンを含む
ＭＣＧ培地（Ｎａ２８Ｐ０．０．６％、ＫＨ，Ｐｏ、０
．３％。Escherichia coli 'vV3 carrying plasmid pc f BD28 containing DNA encoding the above hG-C3F derivative
1105trA strain (Escherichia coli
[1CfB port 28FERjl BP-1479) to L
G medium [Batato tryptone 10g, yeast extract 5g, N
aCj! Dissolve 5g of glucose and 1g of glucose in 11 parts of water,
Adjust the pH to 7.0 with NaOH. ] at 37°C for 18 hours, and 5 ml of this culture was added to MCG medium (Na28P 0.0.6%, KH, Po, 0
．． 3%.
ＮａＣＡ ０．５％、カザミノ酸０．５％、Ｍｇ５Ｏ
＜１ ｍＭ、 ビタミンＢ＋ ４μｇ／ｍ！、ｐＨ
７，２） １００ｍｌに接種し、３０℃で４〜８時間
培養後、トリプトファンの誘導物質である３β−インド
ールアクリル酸（３β−１ｎｄｏｌｅａｃｒｙｌｉｃ
ａｃｉｄ、以下ＩＡＡと略す）を１０■／ｍｌ加え、さ
らに２〜１２時間培養を続けた。培養液を８．００ Ｏ
ｒｐｍ 、 １０分間遠心して集菌し、３０ ｍＭ
Ｎ ａ Ｃｊ！ 、 ３０ ｍＭトリス・塩酸緩衝
液（ｐ Ｈ７，’ ５ ）で洗浄した。洗浄菌体を上記
緩衝液３０ｍ１に懸濁し、０℃で超音波破砕（ＢＲＡＮ
ＳＯＮ ５ＯＮＩＣＰＯＩ髪ＩＥＲＣＯＭＰＡＮＹ社５
ＯＮＩＰＩＥＲＣＥＬＬ ＤＩＳＲＵＰＴ［ｌＲ２０
０，０ＵＴＰＵＴ ［：０ＮＴＲＣＩＬ２． １０
分間処理）した。これを９．００Ｏｒρｍ、３０分間
遠心して菌体残渣を得た。この菌体残渣からマーストン
らの方法ＣＦ、Ａ０口０Ｍａｒｓｔｏｎら：バイオ・チ
クノロシイ （ＢＩＯ／ＴＥＣＩＩＮＯＬＯＧＹ）
２．８００ （１９８４）］によりｈＧ−Ｃ５Ｆ誘導体
を抽出・精製・可溶化・再生した。NaCA 0.5%, casamino acid 0.5%, Mg5O
<1mM, Vitamin B+ 4μg/m! , pH
7,2) After inoculating 100 ml and culturing at 30°C for 4 to 8 hours, inoculate with 3β-indoleacrylic acid (3β-1ndoleacrylic acid), which is a tryptophan inducer.
Acid (hereinafter abbreviated as IAA) was added at 10 μ/ml, and the culture was continued for an additional 2 to 12 hours. Culture solution at 8.00 O
rpm, centrifuged for 10 minutes to collect bacteria, and diluted with 30 mM
N a Cj! , and washed with 30 mM Tris/HCl buffer (pH 7,'5). The washed bacterial cells were suspended in 30 ml of the above buffer solution and disrupted by ultrasonication (BRAN) at 0°C.
SON 5ONICPOI hair IER COMPANY company 5
ONIPIERCELL DISRUPT[lR20
0,0UTPUT [:0NTRCIL2. 10
(processed for 1 minute). This was centrifuged at 9.00 Orrpm for 30 minutes to obtain bacterial cell residue. From this bacterial cell residue, the method CF of Marston et al.
2.800 (1984)], the hG-C5F derivative was extracted, purified, solubilized, and regenerated.
参考例４゜
２．４−ビス（０−メトキシポリエチレングリコール）
−６−（３−カルボキシブチルアミノ）−Ｓ−）リア
ジン（ＩＶａ）のＮ−ヒドロキシサクシンイミドエステ
ル（ｒＶ ｂ ）の製造参考例１で得られた塩化物５０
０ＩＩ１ｇを無水テトラヒドロフラン９ｍ１２に溶解し
た。一方、ｒ−アミノ酪酸ｌｏｎｇ、）リエチルアミン
２８μｇを無水ジメチルアミド１ｍｌに溶解した液に上
記溶液を添加後、室温下１６時間攪拌した。次いで、減
圧乾固の後、塩化メチレン３０ｍｆ、１０ｍＭ’）ン酸
緩衝液（ｐＨ１０）１５ｍ＆を加え分配した。上層を２
ＮＨＣ１でｐＨ１にした後、塩化メチレン３０ｍ１を加
え、再び分配した。下層を分画し、無水硫酸す） ＩＪ
ウムで乾燥後、戸別し、減圧濃縮を行い、上記カルボン
酸（ＩＶａ） １５０ｍｇ （収率３０％）を（ｌた
。次いで乾燥したカルボン酸（ｒＶａ） １５０ｍｇと
Ｎ−ヒドロキシサクシンイミド３ｍｇを堵水塩化メチレ
ン１ｍｆ！に可溶化し、水冷下ジシクロヘキ。Reference example 4゜2.4-bis(0-methoxypolyethylene glycol)
Production of N-hydroxysuccinimide ester (rV b ) of -6-(3-carboxybutylamino)-S-)riazine (IVa) Chloride obtained in Reference Example 1 50
1 g of OII was dissolved in 9 ml of anhydrous tetrahydrofuran. On the other hand, the above solution was added to a solution in which 28 μg of r-aminobutyric acid (long) ethylamine was dissolved in 1 ml of anhydrous dimethylamide, and then stirred at room temperature for 16 hours. Next, after drying under reduced pressure, 30mf of methylene chloride, 15mF of 10mM' acidic acid buffer (pH 10) was added and distributed. 2 upper layers
After adjusting the pH to 1 with NHC1, 30 ml of methylene chloride was added and partitioned again. Fractionate the lower layer and add sulfuric anhydride) IJ
150 mg (yield 30%) of the above carboxylic acid (IVa) was obtained by drying the carboxylic acid (rVa) and 3 mg of N-hydroxysuccinimide in diluted water. Solubilize in 1mf! of methylene chloride and dicyclohexane under water cooling.
ジルカルボジイミド（ＤＣＣ）６ｍｇを添加後、室温下
１２時間攪拌した。生じたジシクロへキシルウレア（Ｄ
ＣＵ）を戸別し、ｐ液にエチルエーテルを加えて、沈澱
を生じさせた。次いで、沈澱をρ別後、減圧乾燥し、目
的のエステル（ｒＶｂ）１００ｍｇ（収率６７％）を得
た。After adding 6 mg of dicarbodiimide (DCC), the mixture was stirred at room temperature for 12 hours. The resulting dicyclohexylurea (D
CU) was taken from house to house, and ethyl ether was added to the p solution to form a precipitate. Next, the precipitate was separated by ρ and dried under reduced pressure to obtain 100 mg (yield: 67%) of the desired ester (rVb).
実験例１゜
化学修飾ｈＧ−Ｃ５Ｆ誘導体の比活性及びマウス白血病
細ＭＩＮＦＳ６０の増殖促進活性実施例３と同様の方法
で、ｈＧ−Ｃ３Ｆ！Ｓ導体と活性エステルを反応させ、
限外ｐ過膜を用いて未反応の活性エステルおよびその分
解物を除去した。同腹を用いて、ＰＢＳに内液の交換を
行い、得られた残液中の化学修飾ｈＧ−Ｃ３Ｆ誘導体の
Ｇ−Ｃ３Ｆ活性及びＮＦＳ６０細抱に対する増殖促進活
性［Ｋ、Ｌ、Ｈｏｌｍｅｓ、 ｅｔ ａｌ、、 プ
ロシーディング・オブ・ザ・ナショナル・アカテ′ミイ
・オブ・サイエンス （Ｐｒｏｃ、 Ｎａｔｌ、Ａｃａ
ｄ、 Ｓｃｉ、 ｌｌ５Ａ ）８２、６６８７ （１
９８５） ］を測定し、結果を第５表に示した。Experimental Example 1 Specific activity of chemically modified hG-C5F derivative and growth promoting activity of mouse leukemia cells MINFS60 In the same manner as in Example 3, hG-C3F! React the S conductor with the active ester,
Unreacted active ester and its decomposition products were removed using an ultrapolar membrane. Using the same litter, the internal fluid was exchanged with PBS, and the chemically modified hG-C3F derivative in the resulting residual fluid was tested for G-C3F activity and proliferation-promoting activity for NFS60 binding [K, L, Holmes, et al. , Proceedings of the National Academy of Sciences
d, Sci, ll5A) 82, 6687 (1
985) ] and the results are shown in Table 5.
第５表
ｈＧ−ＣＳＦ誘導体 １００％ １０
０％化学修飾ｈＧ−Ｃ３Ｆ誘導体は、マウス骨髄幹細胞
に対してあきらかにＣ５Ｆ活性を保持していた。さらに
、Ｇ−Ｃ５Ｆ依存性の増殖を示すＮＦＳ６０細胞に対し
ても増殖促進活性を有していた。Table 5 hG-CSF derivatives 100% 10
The 0% chemically modified hG-C3F derivative clearly retained C5F activity against mouse bone marrow stem cells. Furthermore, it also had proliferation-promoting activity against NFS60 cells, which exhibit G-C5F-dependent proliferation.
実験例２゜
白血球（頚粒球）増加効果
実験例１で用いた化学修飾ｈＧ−Ｃ３Ｆ誘導体をＣ３Ｈ
／Ｈｅ７ウス（雄、ｎ＝３）の皮下に単回または一日一
回６日間投与し、以下経時的に採血して末梢血中の白血
球（’ｖＶＢＣ）を計数した。Experimental Example 2゜White blood cell (cervical granulocytes) increasing effect The chemically modified hG-C3F derivative used in Experimental Example 1 was
/He7 mice (male, n=3) were subcutaneously administered once or once a day for 6 days, and blood was collected over time to count white blood cells ('vVBC) in the peripheral blood.
その結果を第６表（単回投与）及び第７表（連日投与）
に示した。The results are shown in Table 6 (single administration) and Table 7 (daily administration).
It was shown to.
第６表 単回投与（皮下）時の白血球（Ｗ Ｂ Ｃ）散
策７表 ６日間連日投与（皮下）時の白血球（ＷＢＣ）
敗ｈ（ｉ−［：ＳＦ誘導体 １ ７９．３ ！１５．
５ ｇ５．１ ９１．２ ７９．１ １１Ｂ、８ｈＧ
−［：ＳＦ誘導体＋０ １６３．０ ２２１．５ ２２
０．：ｌ ２８９．３ ２７３．０ ２Ｂ４．０単回
投与では、投与後８時間をピークとする白血球数の増加
が認められたが、その後ｈＧ−Ｃ３Ｆ誘導体では４８時
間でほぼ正常レベルに低下したのに対して、化学修飾ｈ
Ｇ−Ｃ３Ｆ誘導体では４８時間においても有意に高値の
白血球増加の持続効果を示した。Table 6 White blood cells (WBC) after single administration (subcutaneous) Table 7 White blood cells (WBC) after daily administration (subcutaneous) for 6 days
Defeat h(i-[:SF derivative 1 79.3 !15.
5 g5.1 91.2 79.1 11B, 8hG
-[: SF derivative +0 163.0 221.5 22
0. :l 289.3 273.0 With a single dose of 2B4.0, an increase in the number of white blood cells was observed that peaked at 8 hours after administration, but after that, with the hG-C3F derivative, the number decreased to almost normal levels within 48 hours. For chemical modification h
The G-C3F derivative showed a sustained effect of increasing leukocytes at a significantly high level even after 48 hours.
連日投与では、特に低用量投与群において、化学修飾ｈ
ＧＣ５Ｆ誘導体はｈＧ−Ｃ３Ｆ誘導体に比べてあきらか
に有意な白血球増加効果を示した。With daily administration, especially in the low dose group, chemical modification h
The GC5F derivative clearly showed a significant leukocyte increasing effect compared to the hG-C3F derivative.
実験例３゜
血中濃度の推移
実験例１で用いた化学修飾ｈＧ−Ｃ３Ｆ誘導体をＣ３Ｈ
／ Ｈｅ ７ウス（雄、ｎ＝３）の皮下に単回または連
日６回投与し、以下経時的に採血し血漿中のＧ−Ｃ５Ｆ
ａ度を測定した。その結果を第８表（単回投与）及び第
９表（連日投与）に示した。また、一部の実験では静脈
内に単回投与（第１０表）した実験も行った。Experimental Example 3゜Change in Blood Concentration The chemically modified hG-C3F derivative used in Experimental Example 1 was
/ He 7 mice (male, n = 3) were subcutaneously administered once or 6 times daily, and blood was collected over time to determine G-C5F in plasma.
The a degree was measured. The results are shown in Table 8 (single administration) and Table 9 (daily administration). In some experiments, a single dose of intravenous administration (Table 10) was also conducted.
第８表 単回投与（皮下投与）
誘導体１０２４８．３７７２．７２７４４．５ ２１４
．０ １６３Ｊ ４９．７ ６．２注）ａ） ｈ
Ｇ−Ｃ３Ｆ蛋白として同一重量を投与した。Table 8 Single dose (subcutaneous administration) Derivative 10248.3772.72744.5 214
．． 0 163J 49.7 6.2 Note) a) h
The same weight was administered as G-C3F protein.
ｂ） ＮＦＳ６０細胞の増殖促進活性（Ｈａｌｆ ｍ
ａｘ＝＝５０１１＞より算出した。b) Proliferation promoting activity of NFS60 cells (Half m
Calculated from ax==5011>.
第９表 連日投与（皮下投与）
４゜８ ２．２ ＮＴ、 −リ −−１４，２
１１Ｊ ＮＴ、 ４．７ ２．２ ２．２注
ａ）ｈＧ−Ｃ３Ｆ蛋白として同一重量を投与した。Table 9 Daily administration (subcutaneous administration) 4゜8 2.2 NT, -Re -14,2
11J NT, 4.7 2.2 2.2 Note a) The same weight was administered as hG-C3F protein.
ｂ）上役、投与後１時間での血中濃度
下段、投与後２４時間での血中濃度
ｃ）ＮＦＳ６０細胞の増殖促進活性（ｌｌａｌｆ ｍａ
ｘ−５０ｕ）より見出した。b) Superior, lower blood concentration 1 hour after administration, blood concentration 24 hours after administration c) NFS60 cell proliferation promoting activity (llalf ma
Found out from x-50u).
ｄ） ＮＴ、 （Ｎｏｔ Ｔｅ５ｔｅｄ）ｅ）＝（検
出限界以下）
第１Ｏ表 単回投与（静脈内投与）
誘導体 １０ １３０７ １：１５６ ９０１
６３１Ｊ ５６３ ３５５．８誘導体
注 ａ）ｈＧ−Ｃ３Ｆ蛋白として同一重量を投与した。d) NT, (Not Te5ted) e) = (below detection limit) Table 1O Single dose (intravenous administration) Derivative 10 1307 1:156 901
631J 563 355.8 Derivative Note a) The same weight was administered as hG-C3F protein.
ｂ） ＮＦＳ６０細抱の増殖促進活性（ｌｌａｌｆ
ｍａｘ＝５０ｕ）より算出した。b) Proliferation-promoting activity of NFS60 (llalf
max=50u).
単回投与（皮下）の場合、ｈＧ−Ｃ３Ｆ誘導体では１時
間をピークとして以下急速に血中濃度が低下したのに対
し、化学修＃ｈＧ−Ｃ５Ｆｌｉｌ導体では５〜７時間に
かけて徐々に血中濃度が上がり２４時間でも比較的高値
を維持した（第８表）。In the case of a single administration (subcutaneously), the blood concentration of the hG-C3F derivative peaked at 1 hour and then rapidly decreased, whereas the blood concentration of the chemically modified hG-C5Flil conductor gradually decreased over 5 to 7 hours. rose and remained relatively high for 24 hours (Table 8).
一方、連日投与の実験では、１時間後血中濃度はいずれ
もｈＧ−Ｃ３Ｆ誘導体の方が高値であったが、２４時間
では低値であり、３日目以降は検出できなかった。これ
に対して、化学修飾ｈＧ−Ｃ３Ｆ、Ｉ導体では２４時間
後でも検出可能であり、かつｈＧ−Ｃ５Ｆ誘導体よりも
高値であった。On the other hand, in the experiment of daily administration, the hG-C3F derivative had a higher blood concentration after 1 hour, but it was lower after 24 hours, and could not be detected after the 3rd day. In contrast, the chemically modified hG-C3F, I conductor was detectable even after 24 hours, and the value was higher than that of the hG-C5F derivative.
一方、静脈内投与の場合は、第１０表に示したように化
学修飾ｈＧ−Ｃ３ＦＸＮ導体の方があきらかに高い血中
濃度を示した。On the other hand, in the case of intravenous administration, as shown in Table 10, the chemically modified hG-C3FXN conductor showed a clearly higher blood concentration.
実験例化
化学修＃ｊｈＧ−Ｃ３Ｆおよび化学修飾ｈｃ−ｃＳＦ誘
導体の比活性及びマウス白血病細胞ＮＦＳ６０の増殖促
進活性
（１）実施例２で得られた化学修飾ｈ（、−Ｃ３Ｆ（■
型）を用い、実験例１と同様の方法で比活性及び増殖促
進活性を測定した。結果を第１１表に示す。Experimental Examples Specific activities of chemically modified hG-C3F and chemically modified hc-cSF derivatives and growth-promoting activity of murine leukemia cells NFS60 (1) Chemically modified h(, -C3F (■) obtained in Example 2
The specific activity and proliferation promoting activity were measured using the same method as in Experimental Example 1 using the following method. The results are shown in Table 11.
第 １１ 表
（２）実施例４で得られた化学修飾ｈ Ｇ −ＣＳ Ｆ
誘導体（■型）および実施例５で得られた化学修飾ｈＧ
−Ｃ３Ｆ誘導体（■型）を用い、実験例１と同様の方法
で比活性及び増殖促進活性を測定した。Table 11 (2) Chemical modification obtained in Example 4 hG-CSF
Derivative (■ type) and chemically modified hG obtained in Example 5
-C3F derivative (■ type) was used to measure specific activity and growth promoting activity in the same manner as in Experimental Example 1.
結果を第１２表に示す。The results are shown in Table 12.
第１２表
： 未修飾体 １００％ １００％
ｌ実験例５
白血球（顆粒球）増加効果
（１）実施例２で得られた化学修飾ｈＧ−Ｃ３Ｆ（■型
）（２，５μｇ／匹）をＢＡＬＢ／ Ｃマウス （雄３
匹、対照群は４匹）の皮下に単回投与し、以下経時的に
採血して末梢血中の白血球（ＷＢＣ）数を係数した。そ
の結果を第１３表に示す。Table 12: Unmodified form 100% 100%
l Experimental Example 5 White blood cell (granulocyte) increasing effect (1) Chemically modified hG-C3F (■ type) obtained in Example 2 (2.5 μg/mouse) was administered to BALB/C mice (3 male
The drug was subcutaneously administered once to 4 mice in the control group), and blood was collected over time to calculate the number of white blood cells (WBC) in the peripheral blood. The results are shown in Table 13.
第１３表 単回投与（皮下）時の白血球（ＷＢＣ）数（
２）実施例４でｉ５られた化学修飾ｈＧ−Ｃ５Ｆ誘導体
（１１１型）および実施例５で（）られた化学修飾ｈＧ
−Ｃ３Ｆ誘導体（■型）を２．５μｇ／匹用い上記（１
）と同様にして白血球（ＷＢＣ）数を計数した。Table 13 White blood cell (WBC) count after single administration (subcutaneous) (
2) Chemically modified hG-C5F derivative (type 111) obtained in Example 4 and chemically modified hG obtained in Example 5
-C3F derivative (■ type) was used at 2.5 μg/mouse and the above (1
) The number of white blood cells (WBC) was counted.
その結果を第１４表に示す。The results are shown in Table 14.
第１４表 単回投与（皮下）時の白血球（＋−１［Ｉ
Ｃ）敗発明の効果
本発明の化学修飾ｈＧ−Ｃ３Ｆ及び化学修飾ｈＧ−Ｃ５
Ｆ誘導体は、末梢白血球（顆粒球）の増多効果が増大さ
れ、血中での安定性及び持続性が向上しており、凍結乾
員製剤として安定性が向上しているので、治験薬として
有利に使用できる。Table 14 Leukocytes (+-1[I
C) Effect of the defeated invention Chemically modified hG-C3F and chemically modified hG-C5 of the present invention
The F derivative has an increased effect on increasing peripheral leukocytes (granulocytes), has improved stability and persistence in the blood, and has improved stability as a freeze-dried preparation, so it can be used as an investigational drug. Can be used to advantage.
Claims (4)
プチドの分子中の少なくとも１個のアミノ基に式（ I
）で表される基を結合してなる修飾ポリペプチド。 式▲数式、化学式、表等があります▼（ I ） 式中Ｒ＿１はアルキル基またはアルカノイル基、ｎは任
意に変わりうる正の整数、ＸはＯ、ＮＨまたはＳ、Ｒ＿
２は▲数式、化学式、表等があります▼ 〔式中Ｒ＿３はＯＨ、Ｃｌ、Ｏ−（ＣＨ２ＣＨ＿２Ｏ）
＿ｎ−Ｒ＿１（式中Ｒ＿１、ｎは前記と同意義を表す）
を表し、Ｙは存在しないか、またはＺ−（ＣＨ＿２）＿
ｐＣＯ（式中ＺはＯ、ＳまたはＮＨを表し、式中ｐは任
意に変わりうる正の整数を表す）を表す〕または（ＣＯ
）＿ｍ−（ＣＨ＿２）＿ｌＣＯ（式中、ｍは０または１
、ｌは任意に変わりうる正の整数を表す）を表す。(1) At least one amino group in the molecule of a polypeptide having human granulocyte colony stimulating factor activity has the formula (I
) A modified polypeptide formed by bonding a group represented by: Formula▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) In the formula, R_1 is an alkyl group or an alkanoyl group, n is a positive integer that can be changed arbitrarily, and X is O, NH or S, R_
2 has ▲ mathematical formulas, chemical formulas, tables, etc. ▼ [In the formula, R_3 is OH, Cl, O-(CH2CH_2O)
__n-R_1 (in the formula, R_1, n represents the same meaning as above)
and Y is absent or Z-(CH_2)_
pCO (in the formula, Z represents O, S or NH, and p represents a positive integer that can vary arbitrarily)] or (CO
)_m-(CH_2)_lCO (where m is 0 or 1
, l represents a positive integer that can change arbitrarily).
乾燥標品。(2) A lyophilized preparation containing the modified polypeptide according to claim 1.
胞増殖促進剤。(3) A white blood cell proliferation promoter comprising the modified polypeptide according to claim 1.
Ｏ、ＳまたはＮＨを表し、ｐは任意に変わりうる正の整
数を表す）(4) New carboxylic acid represented by the following formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, R_1, n and X represent the same meanings as above, Z represents O, S or NH, and p is optional (represents a positive integer that can change to )
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1079748A JPH0796558B2 (en) | 1988-03-31 | 1989-03-30 | Modified polypeptide |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63-80088 | 1988-03-31 | ||
| JP8008888 | 1988-03-31 | ||
| JP1079748A JPH0796558B2 (en) | 1988-03-31 | 1989-03-30 | Modified polypeptide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01316400A true JPH01316400A (en) | 1989-12-21 |
| JPH0796558B2 JPH0796558B2 (en) | 1995-10-18 |
Family
ID=26420744
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1079748A Expired - Lifetime JPH0796558B2 (en) | 1988-03-31 | 1989-03-30 | Modified polypeptide |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0796558B2 (en) |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990006952A1 (en) * | 1988-12-22 | 1990-06-28 | Kirin-Amgen, Inc. | Chemically modified granulocyte colony stimulating factor |
| WO1991018630A1 (en) | 1990-05-30 | 1991-12-12 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Polyether-substituted tumor agents |
| JPH04217693A (en) * | 1990-10-23 | 1992-08-07 | Fuji Photo Film Co Ltd | Peptide-containing polyethylene glycol derivative and its use |
| JPH04504801A (en) * | 1989-04-21 | 1992-08-27 | ジェネティックス・インスチチュート・インコーポレーテッド | Cysteine-added polypeptide variants and chemical modifications thereof |
| US5336782A (en) * | 1991-04-24 | 1994-08-09 | Kuraray Co., Ltd. | Long chain carboxylic acid imide ester |
| US5342940A (en) * | 1989-05-27 | 1994-08-30 | Sumitomo Pharmaceuticals Company, Limited | Polyethylene glycol derivatives, process for preparing the same |
| WO1995023165A1 (en) * | 1994-02-23 | 1995-08-31 | Kyowa Hakko Kogyo Co., Ltd. | Platelet growth accelerator |
| WO1996034016A1 (en) * | 1995-04-26 | 1996-10-31 | Kyowa Hakko Kogyo Co., Ltd. | Novel polypeptides |
| WO1998055500A1 (en) * | 1997-06-06 | 1998-12-10 | Kyowa Hakko Kogyo Co., Ltd. | Chemically modified polypeptides |
| WO2005053730A1 (en) * | 2003-12-05 | 2005-06-16 | Kirin Beer Kabushiki Kaisha | Therapeutic agent for terminal heart failure |
| JP2015532264A (en) * | 2012-09-25 | 2015-11-09 | ジェンザイム・コーポレーション | Peptide-bound morpholino antisense oligonucleotides for the treatment of myotonic dystrophy |
| JP2016037488A (en) * | 2014-08-11 | 2016-03-22 | 日本化薬株式会社 | CHIMERIC PROTEIN HAVING TGFβ INHIBITING FUNCTION |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57192435A (en) * | 1981-05-20 | 1982-11-26 | Toyobo Co Ltd | Modified polypeptide |
| JPS62115280A (en) * | 1985-07-05 | 1987-05-26 | Takeda Chem Ind Ltd | Modifying enzyme, production and use thereof |
| JPS6360938A (en) * | 1986-09-02 | 1988-03-17 | Meiji Milk Prod Co Ltd | Plasminogen activator of modified tissue type and production thereof |
| JPS63146828A (en) * | 1986-07-18 | 1988-06-18 | Chugai Pharmaceut Co Ltd | Stable granulocyte colony stimulating factor-containing preparation |
-
1989
- 1989-03-30 JP JP1079748A patent/JPH0796558B2/en not_active Expired - Lifetime
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57192435A (en) * | 1981-05-20 | 1982-11-26 | Toyobo Co Ltd | Modified polypeptide |
| JPS62115280A (en) * | 1985-07-05 | 1987-05-26 | Takeda Chem Ind Ltd | Modifying enzyme, production and use thereof |
| JPS63146828A (en) * | 1986-07-18 | 1988-06-18 | Chugai Pharmaceut Co Ltd | Stable granulocyte colony stimulating factor-containing preparation |
| JPS6360938A (en) * | 1986-09-02 | 1988-03-17 | Meiji Milk Prod Co Ltd | Plasminogen activator of modified tissue type and production thereof |
Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990006952A1 (en) * | 1988-12-22 | 1990-06-28 | Kirin-Amgen, Inc. | Chemically modified granulocyte colony stimulating factor |
| JPH04504801A (en) * | 1989-04-21 | 1992-08-27 | ジェネティックス・インスチチュート・インコーポレーテッド | Cysteine-added polypeptide variants and chemical modifications thereof |
| US5342940A (en) * | 1989-05-27 | 1994-08-30 | Sumitomo Pharmaceuticals Company, Limited | Polyethylene glycol derivatives, process for preparing the same |
| WO1991018630A1 (en) | 1990-05-30 | 1991-12-12 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Polyether-substituted tumor agents |
| JPH04217693A (en) * | 1990-10-23 | 1992-08-07 | Fuji Photo Film Co Ltd | Peptide-containing polyethylene glycol derivative and its use |
| USRE36359E (en) * | 1991-04-24 | 1999-10-26 | Kuraray Co., Ltd. | Long chain carboxylic acid imide ester |
| US5336782A (en) * | 1991-04-24 | 1994-08-09 | Kuraray Co., Ltd. | Long chain carboxylic acid imide ester |
| US5414089A (en) * | 1991-04-24 | 1995-05-09 | Kuraray Co., Ltd. | Long chain carboxylic acid imide ester |
| KR100403688B1 (en) * | 1994-02-23 | 2004-02-05 | 교와 핫꼬 고교 가부시끼가이샤 | Thrombocytopenia and pharmaceutical compositions comprising the same |
| WO1995023165A1 (en) * | 1994-02-23 | 1995-08-31 | Kyowa Hakko Kogyo Co., Ltd. | Platelet growth accelerator |
| WO1996034016A1 (en) * | 1995-04-26 | 1996-10-31 | Kyowa Hakko Kogyo Co., Ltd. | Novel polypeptides |
| WO1998055500A1 (en) * | 1997-06-06 | 1998-12-10 | Kyowa Hakko Kogyo Co., Ltd. | Chemically modified polypeptides |
| US6583267B2 (en) | 1997-06-06 | 2003-06-24 | Kyowa Hakko Kogyo Co., Ltd. | Chemically modified polypeptides |
| US7547675B2 (en) | 1997-06-06 | 2009-06-16 | Kyowa Hakko Kirin Co., Ltd. | Chemically modified polypeptides |
| WO2005053730A1 (en) * | 2003-12-05 | 2005-06-16 | Kirin Beer Kabushiki Kaisha | Therapeutic agent for terminal heart failure |
| JP2015532264A (en) * | 2012-09-25 | 2015-11-09 | ジェンザイム・コーポレーション | Peptide-bound morpholino antisense oligonucleotides for the treatment of myotonic dystrophy |
| US10111962B2 (en) | 2012-09-25 | 2018-10-30 | Genzyme Corporation | Peptide-linked morpholino antisense oligonucleotides for treatment of myotonic dystrophy |
| JP2019006808A (en) * | 2012-09-25 | 2019-01-17 | ジェンザイム・コーポレーション | Peptide-linked morpholino antisense oligonucleotides for treatment of myotonic dystrophy |
| JP2016037488A (en) * | 2014-08-11 | 2016-03-22 | 日本化薬株式会社 | CHIMERIC PROTEIN HAVING TGFβ INHIBITING FUNCTION |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0796558B2 (en) | 1995-10-18 |
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