JPH0615477B2 - Anti-infective agent - Google Patents
Anti-infective agentInfo
- Publication number
- JPH0615477B2 JPH0615477B2 JP60023777A JP2377785A JPH0615477B2 JP H0615477 B2 JPH0615477 B2 JP H0615477B2 JP 60023777 A JP60023777 A JP 60023777A JP 2377785 A JP2377785 A JP 2377785A JP H0615477 B2 JPH0615477 B2 JP H0615477B2
- Authority
- JP
- Japan
- Prior art keywords
- human
- csf
- infection
- human csf
- molecular weight
- 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
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/53—Colony-stimulating factor [CSF]
- C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- General Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Animal Behavior & Ethology (AREA)
- Oncology (AREA)
- Public Health (AREA)
- Communicable Diseases (AREA)
- Immunology (AREA)
- Toxicology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Description
【発明の詳細な説明】 本発明はヒトコロニー刺激因子（以下ＣＳＦと略す）を
有効成分とする感染防禦剤に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an infection control agent comprising a human colony stimulating factor (hereinafter abbreviated as CSF) as an active ingredient.
ＣＳＦはヒトまたは動物の骨髄細胞の顆粒球系幹細胞に
作用して、単球マクロファージおよび好中球への***増
殖と分化とを誘導することが知られている（例えば、Me
tcalf.Ｄ．等 Experimental Haematology １ 185(1
973)を参照）。CSF is known to act on granulocyte stem cells of human or animal bone marrow cells to induce mitotic proliferation and differentiation into monocyte macrophages and neutrophils (eg Me.
tcalf.D. Etc. Experimental Haematology 1 185 (1
973)).
ヒトＣＳＦに関してはこれまでヒト正常組織由来のＣＳ
Ｆやヒト腫瘍細胞由来のＣＳＦについて多数報告されて
いる（例えば、Stanley 等Fed.Proc.３５ 2272(197
5)，Burgess等Blood 49 573(1977)，Shah 等 Blood
50 811(1977)，Fojo 等 Biochem，17 3109(197
8)，Okabe 等 Cancer Res 38 3910(1978)，Asano
等Blood 49 845(1977)，Golde 等 Blood ５７
1321(1981)，Wu 等 Ｊ．Biol. Chem 254 6226(197
9)，Dipersio等 Blood 56 717(1980) などを参
照。）。Regarding human CSF, CS derived from normal human tissue has been used so far.
Many reports have been made on F and CSF derived from human tumor cells (eg, Stanley et al. Fed. Proc. 35 2272 (197).
5), Burgess et al. Blood 49 573 (1977), Shah et al. Blood
50 811 (1977), Fojo et al. Biochem, 17 3109 (197)
8), Okabe et al. Cancer Res 38 3910 (1978), Asano
Etc Blood 49 845 (1977), Golde etc Blood 57
1321 (1981), Wu et al. Biol. Chem 254 6226 (197
9), Dipersio et al. Blood 56 717 (1980), etc. ).
しかしながらこれ等のヒトＣＳＦは完全に純化されたも
のではなく従ってヒトＣＳＦの医薬としての有用性また
は有効性については未だ不明のままである。However, these human CSFs are not completely purified, and therefore the usefulness or efficacy of human CSF as a drug remains unclear.
近年、感染症領域に於ける化学療法剤の顕著な発達は広
範囲なスペクトルを有し、かつ強力な殺菌力を有する抗
菌剤の登場を促す一方で従来の特定の病原体を有する強
毒物質産生菌からヒトの正常細菌叢を構成するグラム陰
性菌などへの原因菌の菌交交現象を招いており、臨床的
には日和見感染など新たな問題を引き起こしている。さ
らに、強力な抗生物質の使用により減少した細菌類にか
わって、最近は Candida などのカビ類が患者体内で
増殖するため臨床的に重要な問題となっているが、抗真
菌剤は一般的に極めて副作用が強く、その使用は大幅に
制限されている。これ等の感染症の治療には従来の化療
法剤の効果に加え、宿主の防禦機能を賦活するような薬
剤の使用が望まれているが現在まで有効な薬剤は見出さ
れていない。In recent years, the remarkable development of chemotherapeutic agents in the field of infectious diseases has broad spectrum, and has promoted the emergence of antibacterial agents having strong bactericidal activity, while conventional bacteria producing strong poisonous substances with specific pathogens. Cause a crossing phenomenon of causative bacteria such as Gram-negative bacteria that compose normal human flora in humans, and clinically cause new problems such as opportunistic infections. In addition, fungi such as Candida have recently become a clinically important problem because of the growth of molds such as Candida in place of the bacteria reduced by the use of powerful antibiotics. It has very strong side effects and its use is severely restricted. For the treatment of these infectious diseases, in addition to the effects of conventional chemotherapeutic agents, it is desired to use a drug that activates the defense function of the host, but no effective drug has been found to date.
細菌感染の初期には宿主のもつ防禦機能のうち白血球の
貧食殺菌作用が最も強く影響すると考えられている事か
ら好中球やマクロファージの増殖、分化成熟を高めるこ
とにより宿主の感染防禦能を亢進することできれば有効
な感染治療薬となり得る可能性が示唆される。In the early stage of bacterial infection, it is considered that the phagocytic bactericidal action of leukocytes has the strongest effect on the defense function of the host, and therefore the host's infection defense ability can be improved by enhancing the proliferation and differentiation / maturation of neutrophils and macrophages. If it can be enhanced, it may be an effective therapeutic drug for infection.
従って純粋なヒトＣＳＦの大量生産の研究が治発に行わ
れているが現在まで成功したものはない。このような状
況に於いて本発明者等は口腔底癌患者の腫瘍細胞から極
めて高いＣＳＦ産生能を有し、かつ良好な増殖能を示す
細胞株を樹立し（ＣＮＣＭ受託番号「１−３１５」）、
この細胞株の培養上清からヒト好中球のコロニー形成促
進活性を示す純粋なヒトＣＳＦの単離に初めて成功した
（特願昭５９−１５３２７３号）。Therefore, studies on mass production of pure human CSF have been under way, but none have been successful to date. In such a situation, the present inventors established a cell line having extremely high CSF-producing ability and good proliferating ability from tumor cells of patients with oral floor cancer (CNCM Accession No. “1-315”). ),
For the first time, we have succeeded in isolating pure human CSF exhibiting human neutrophil colony-promoting activity from the culture supernatant of this cell line (Japanese Patent Application No. 59-153273).
本発明者等は感染動物モデルを用いてこのヒトＣＳＦの
感染防禦効果を調べたところ、ヒトＣＳＦが ｉｎ vi
voに於いて顕著な好中球の成熟能を有し、従って感染防
禦効果を示したことから感染症治療薬として有効である
ことを見出し、本発明を完成した。The present inventors have investigated the anti-infective effect of this human CSF using an infected animal model, and found that human CSF was found to be in vitro.
The present invention was found to be effective as a therapeutic agent for infectious diseases because it has a remarkable neutrophil maturation ability in vo and therefore has an effect of preventing infection, and thus completed the present invention.
本発明は新規な感染防禦剤の提供に係るものである。The present invention relates to the provision of a novel infection control agent.
すなわち、本発明はヒトＣＳＦを有効成分とする感染防
禦剤である。That is, the present invention is an infection control agent containing human CSF as an active ingredient.
本発明の有効成分であるヒトＣＳＦは前述の特許出願に
開示された方法に従って得ることができる他、例えばＣ
ＳＦ産生細胞と、該細胞と区別可能な性質を有し且つ自
己増殖を有する悪性腫瘍細胞とを細胞融合して得られる
ハイブリドーマをマイトジエンの存在または非存在下で
培養することによって得るか、またはヒトＣＳＦをコー
ドする遺伝子を用いて組換体ＤＮＡを作成し、これを適
当な宿主細胞（例えば大腸菌、酵母菌、チャイニーズハ
ムスターの卵巣細胞等）で発現させることによって得る
こともできる。Human CSF, which is the active ingredient of the present invention, can be obtained according to the method disclosed in the above-mentioned patent application.
Obtained by culturing a hybridoma obtained by cell fusion of a SF-producing cell and a malignant tumor cell having a self-proliferating property that is distinguishable from the cell, by culturing in the presence or absence of mitogen, or human It can also be obtained by preparing a recombinant DNA using a gene encoding CSF and expressing it in an appropriate host cell (eg, Escherichia coli, yeast, Chinese hamster ovary cell, etc.).
これ等の方法で得たヒトＣＳＦは全て本発明に含まれ
る。All human CSFs obtained by these methods are included in the present invention.
得られたヒトＣＳＦ含有液は必要により公知の手段でさ
らに精製、濃縮した後凍結保存とするかまたは凍結乾
燥、真空乾燥等の手段により水分を除去して保存するこ
とができる。また所望によりヒトＣＳＦを適当な緩衝液
に溶解した後、ミリポアフィルター等で無菌ろ過して注
射剤とすることもできる。The obtained human CSF-containing solution may be further purified and concentrated by known means, if necessary, and then frozen or stored, or may be stored after removing water by means such as freeze-drying and vacuum-drying. If desired, human CSF may be dissolved in an appropriate buffer solution, and then sterile filtered with a Millipore filter or the like to prepare an injection.
さらに本発明の感染防禦剤はヒトまたは動物医薬用に適
した医薬製剤としての形態をとるために必要な製薬担体
や賦形剤を、さらには安定化剤、吸着防止剤を含むこと
ができる。Furthermore, the infection control agent of the present invention may contain a pharmaceutical carrier or excipient necessary for taking a form as a pharmaceutical preparation suitable for human or veterinary medicine, and further, a stabilizer and an adsorption inhibitor.
本発明の感染防禦剤に含まれるヒトＣＳＦの投与量、投
与回数は対象の疾患患者の病状を配慮して決めることが
できるが、通常成人一人当たり０．１〜500μg好ましく
は５〜100μgのヒトＣＳＦを有する製剤を１週間に１〜
７回投与することができる。しかし本発明はヒトＣＳＦ
の含有量によって限定されるものではない。The dose of human CSF contained in the anti-infective agent of the present invention, the number of administrations can be determined in consideration of the medical condition of the target disease patient, but usually 0.1 to 500 μg per adult, preferably 5 to 100 μg of human. 1 to 1 weekly preparations with CSF
It can be administered 7 times. However, the present invention
Is not limited by the content of.
本発明のヒトＣＳＦを有効成分とする感染防禦剤が使用
され得る対象感染菌としては種々の菌を挙げることがで
き、特に限定されるものではないが、なかでもスタフィ
ロコッカス属，ストレプトコッカス属などのグラム陽性
球菌，エシェリシア属，セラチア属，クレブシェラ属な
どの腸内細菌やヘモフィルスなどを含むグラム陰性通性
嫌気性菌，シュードモナス属，アルカリゲネス属などの
グラム陰性好気性菌，バクテロイデスなどのグラム陰性
嫌気性菌，およびキャンディダ属，アスペルギス属など
の真菌等による単独感染又はいくつかの菌による複合感
染に対して高い感染防禦効果が得られる。Examples of the target infectious bacteria for which the anti-infective agent containing human CSF of the present invention as an active ingredient can be used include various bacteria and are not particularly limited. Among them, Staphylococcus spp., Streptococcus spp. Gram-positive cocci, Gram-negative facultative anaerobes such as Escherichia, Serratia, Klebsiella and other Haemophilus, Gram-negative aerobes such as Pseudomonas and Alcaligenes, and Gram-negative anaerobes such as Bacteroides A high infection control effect can be obtained against sexually transmitted bacteria and single infections with fungi such as Candida and Aspergillus or combined infections with several fungi.
＜参考例１＞アッセイ法 ヒトＣＳＦによるコロニー形成促進活性（以下ＣＳＡと
略す）は以下の方法によって測定された。<Reference Example 1> Assay method The colony formation promoting activity by human CSF (hereinafter abbreviated as CSA) was measured by the following method.
ウマ血清０．４ml，被検検体０．１ml，C3H/He（メス）
マウスの骨髄細胞浮遊液０．１ml（０．５〜１×105有
核細胞），寒天を0.75％含む改変McCoy's5A培養液０．
４mlを混合し直径３５ｍｍの組織培養用プラスティック
ディッシュに入れて固まらせたのち、３７℃，５％炭酸
ガス／９５％空気，１００％湿度の条件下にて５日間培
養し、形成されたコロニー数（５０個以上の細胞からな
る集落を１コロニーとする）を数え、１個のコロニーを
形成する活性を１単位(Unit)としてＣＳＡを求めた。Horse serum 0.4 ml, test sample 0.1 ml, C3H / He (female)
0.1 ml of mouse bone marrow cell suspension (0.5 to 1 × 10 5 nucleated cells), modified McCoy's 5A culture medium containing 0.75% agar.
After mixing 4 ml and putting it in a plastic dish for tissue culture with a diameter of 35 mm to solidify, it was cultured for 5 days at 37 ° C., 5% carbon dioxide / 95% air, 100% humidity, and the number of colonies formed (A colony consisting of 50 or more cells was defined as one colony), and the activity for forming one colony was defined as 1 unit (Unit) to determine CSA.
＜参考例２＞ヒトＣＳＦの単離 ヒトＣＳＦ産生細胞（ＣＮＣＭ受託番号「Ｉ−３１
５」）が完全に密に増殖した１５０ｃm2の培養フラスコ
２本より細胞を回収し、これをウシ胎児血清を１０％含
有するＦ−１０培養液500mlに浮遊させたのち、１５８
０cm2のガラス製ローラーボトル（Belco社製）に移し、
０．５ｒ．ｐ．ｍ．の速度で回転培養を行った。細胞が
ローラーボトルの内壁に完全に密に増殖した時点で培養
液を血清を含まないＲＰＭＩ１６４０に交換し、４日間
培養したのち培養上清を回収し、ウシ胎児血清を１０％
含有するＦ−１０を加えて培養を続行する。３日間培養
したのち再び血清を含まないＲＰＭＩ１６４０に液替を
行い、４日後に培養上清を回収した。以下同様の操作を
くり返すことにより、毎週１ボトルより５００ｍｌずつ
の血清を含まない培養上清が得られ、しかもこの方法に
よりかなり長期間にわたって細胞を維持し、培養上清を
回収することが可能であった。<Reference Example 2> Isolation of human CSF Human CSF-producing cells (CNCM Accession No. "I-31
5 ") were collected from two 150 cm 2 culture flasks in which they were completely densely grown, and the cells were suspended in 500 ml of F-10 culture medium containing 10% fetal bovine serum.
Transfer to a 0 cm 2 glass roller bottle (Belco),
0.5r. p. m. The rotation culture was performed at the speed of. When the cells were completely and densely grown on the inner wall of the roller bottle, the culture solution was exchanged with serum-free RPMI1640, and the cells were cultured for 4 days, and then the culture supernatant was collected and 10% of fetal bovine serum was collected.
F-10 containing is added and culture is continued. After culturing for 3 days, the serum-free RPMI1640 was changed again, and after 4 days, the culture supernatant was collected. By repeating the same procedure below, 500 ml of serum-free culture supernatant can be obtained from 1 bottle each week, and this method allows the cells to be maintained for a considerably long time and the culture supernatant can be recovered. Met.
得られた培養上清5を１バッチとし、これに０．０１
％ツイーン２０を添加後Hollow Fiber DC-4 およびA
micon ＰＭ−１０（アミコン社製）を用いた限外ろ過
法により約１０００倍に濃縮したのち、これを以下の順
序で精製した。直径４．６cm，長さ90cmのUltrogel
AcA 54カラム（ＬＫＢ社製）を用い、０．１５ Ｍ N
aClおよび０．０１％ツィーン２０（半井化学社製）を
含む０．０１ Ｍ トリス塩酸緩衝液（pH７．４）を用
いて前記濃縮した培養上清５mlを流速約５０ｍｌ／時間
でゲルろ過した。尚カラムはあらかじめウシ血清アルブ
ミン（分子量：６７０００），オボアルブミン（分子
量：４５０００），チトクロームＣ（分子量：1240０）
にてキャリブレーションを行った。。ゲルろ過終了後各
フラクションより０．１mlずつ採取し、１０倍に希釈し
た後、前述したＣＳＡの測定方法により活性を示す画分
を調べた。この結果、先ずＶｅ＝400〜700ml画分がマク
ロファージ優位のＣＳＡを示し、Ｖｅ＝800〜1200mlの
画分が好中球優位のＣＳＡを示すことがわかったので、
後者の画分を集めＰＭ−１０（アミコン社製）を用いる
限外ろ過器によって約５ｍｌに濃縮した。The obtained culture supernatant 5 was treated as 1 batch, and 0.01
% After adding Tween 20, Hollow Fiber DC-4 and A
After being concentrated about 1000 times by the ultrafiltration method using micon PM-10 (manufactured by Amicon), it was purified in the following order. Ultrogel with a diameter of 4.6 cm and a length of 90 cm
AcA 54 column (manufactured by LKB) using 0.15 MN
5 ml of the above concentrated culture supernatant was subjected to gel filtration at a flow rate of about 50 ml / hour using 0.01 M Tris-HCl buffer (pH 7.4) containing aCl and 0.01% Tween 20 (manufactured by Hanai Chemical Co., Ltd.). The column was previously bovine serum albumin (molecular weight: 67,000), ovalbumin (molecular weight: 45000), cytochrome C (molecular weight: 12400).
Was calibrated at. . After the completion of gel filtration, 0.1 ml was collected from each fraction, diluted 10-fold, and the fractions showing activity were examined by the above-mentioned CSA measurement method. As a result, it was first found that the Ve = 400 to 700 ml fraction showed macrophage-dominated CSA, and the Ve = 800 to 1200 ml fraction showed neutrophil-dominated CSA.
The latter fractions were collected and concentrated to about 5 ml by an ultrafilter using PM-10 (manufactured by Amicon).
上記濃縮画分にｎ−プロパノール（東京化成社製，ア
ミノ酸配列決定用）を３０％含む０．１％トリフルオロ
酢酸水溶液を添加し、氷中に１５分程度放置したのち、
１５，０００ｒ．ｐ．ｍ． １０分の遠心により沈殿を
除去した。次いで先のｎ−プロパノールおよびトリフル
オロ酢酸を含む水溶液で平衡化した μ Bondapak Ｃ
１８カラム（Waters 社製，セミ分取用，８ｍｍ×３０
ｃｍ）に吸着後、３０〜６０％の直線濃度勾配のｎ−プ
ロパノールを含む０．１％トリフルオロ酢酸水溶液で順
次溶出した。高速液体クロマト装置は日立６８５−５０
型を、検出は日立６３８−４１型検出器（いずれも日立
製作所社製）を用い、２２０ｎｍと２８０ｎｍの吸収を
同時に測定した。溶出後、各画分より１０μｌを分取し
１００倍希釈したのち、活性を示す画分を調べた。この
結果、ｎ−プロパノール４０％にて溶出されるピークに
好中球優位のＣＳＡが認められたので、このピークを集
め再度同じ条件で再クロマトを行い上記と同様にしてＣ
ＳＡを調べたところ、やはりｎ−プロパノール４０％の
位置のピークに好中球優位のＣＳＡが認められたので、
このピークを集め（４フラクション＝４ｍｌ）凍結乾燥
した。After adding 0.1% trifluoroacetic acid aqueous solution containing 30% of n-propanol (manufactured by Tokyo Kasei Co., Ltd. for amino acid sequence determination) to the above concentrated fraction and leaving it in ice for about 15 minutes,
15,000 r. p. m. The precipitate was removed by centrifugation for 10 minutes. Then, μ Bondapak C equilibrated with the above aqueous solution containing n-propanol and trifluoroacetic acid
18 columns (Waters, semi-preparative, 8mm x 30)
cm), followed by sequential elution with a 0.1% trifluoroacetic acid aqueous solution containing 30 to 60% linear concentration gradient of n-propanol. High performance liquid chromatograph is Hitachi 685-50
The detection was performed using a Hitachi 638-41 type detector (both manufactured by Hitachi, Ltd.), and absorptions at 220 nm and 280 nm were simultaneously measured. After elution, 10 μl of each fraction was collected and diluted 100-fold, and the fraction showing activity was examined. As a result, neutrophil-dominated CSA was observed in the peak eluted with 40% of n-propanol, so this peak was collected and re-chromatographed again under the same conditions to perform C chromatography in the same manner as above.
When SA was examined, neutrophil-dominated CSA was observed at the peak at the position of n-propanol 40%.
This peak was collected (4 fractions = 4 ml) and freeze-dried.
上記凍結乾燥粉末をｎ−プロパノールを４０％含む
０．１％トリフルオロ酢酸水溶液２００μｌに溶解し、
ＴＳＫ−Ｇ３０００ＳＷカラム（東洋曹達社製，２．５
mm×60cm）を用いた高速液体クロマトグラフィーにかけ
た。溶出は同水溶液により０．４ml／分の流速で行い、
フラクションコレクターＦＲＡＣ−１００（ファルマシ
ア社製）により０．４mlずつ分取した。分取した各画分
についてＣＳＡを前記と同様にして調べた結果、保持時
間が３７〜３８分の画分（分子量約２万に相当）に好中
球優位のＣＳＡが認められたので、この画分を回収し、
更に分析用μBondapak Ｃ１８カラム（４．６mm×30c
m）による精製を施したのち、メインピークを回収し凍
結乾燥した。The lyophilized powder was dissolved in 200 μl of 0.1% trifluoroacetic acid aqueous solution containing 40% of n-propanol,
TSK-G3000SW column (Toyo Soda Co., Ltd., 2.5
mm × 60 cm) and subjected to high performance liquid chromatography. Elution is performed with the same aqueous solution at a flow rate of 0.4 ml / min.
A fraction collector FRAC-100 (Pharmacia) was used to collect 0.4 ml each. As a result of examining CSA for each of the collected fractions in the same manner as described above, neutrophil-dominated CSA was found in the fraction having a retention time of 37 to 38 minutes (equivalent to a molecular weight of about 20,000). Collect the fractions,
Furthermore, μBondapak C18 column for analysis (4.6 mm × 30c
m), the main peak was recovered and freeze-dried.
このようにして得られたヒトＣＳＦは以下の理化学的性
質を有していた。The human CSF thus obtained had the following physicochemical properties.
Ｉ）分子量：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による測定で約19,000±1,000。I) Molecular weight: about 19,000 ± 1,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
II）等電点：以下の表−１に示す三つの等電点（Ａ，
Ｂ，Ｃ）のうち少なくとも１つを有する。II) Isoelectric points: Three isoelectric points (A,
At least one of B and C).
III）紫外部吸収：２８０ｎｍに極大吸収を有し、２５
０ｎｍに極小値をもつ。 III) Ultraviolet absorption: maximum absorption at 280 nm, 25
It has a minimum value at 0 nm.
IV）Ｎ末端から２１残基目迄のアミノ酸配列が次の如く
である。IV) The amino acid sequence from the N-terminal to the 21st residue is as follows.
Ｖ）アミノ酸組成：明細書中の表−IIに示すアミノ酸組
成を有する。 V) Amino acid composition: It has the amino acid composition shown in Table-II in the specification.
＜参考例３＞本発明のＣＳＦの等電点 測定フラットベッド型等電点電気泳動装置ＦＢＥ−３０
００（ファルマシア社製）を用いた pH４〜６．５のPharmalyte （ファルマシア社製）及び
４Ｍ尿素を含むポリアクリルアミドゲル（Ｔ＝５％，Ｃ
＝３％， 115mm×230mm）にて３０Ｗ定電力（最大電圧
２０００Ｖ）で２時間泳動を行ったのち、３０％メタノ
ール／１０％トリクロル酢酸／３５％スルホサリチル酸
により固定し、次いでクマーシーブリリアントブルーＲ
−２５０染色を行った。等電点マーカーとしてLow pI
Kit pH２．５〜６．５（ファルマシア社製）を用い
た。 Reference Example 3 Isoelectric Point Measurement of CSF of the Present Invention Flat Bed Type Isoelectric Focusing Device FBE-30
Polyacrylamide gel (T = 5%, C) containing Pharmalyte (manufactured by Pharmacia) having a pH of 4 to 6.5 using 00 (manufactured by Pharmacia) and 4M urea.
= 3%, 115 mm x 230 mm), and electrophoresed at 30 W constant power (maximum voltage 2000 V) for 2 hours, then fixed with 30% methanol / 10% trichloroacetic acid / 35% sulfosalicylic acid, and then Coomassie Brilliant Blue R
-250 staining was performed. Low pI as isoelectric point marker
Kit pH 2.5-6.5 (Pharmacia) was used.
pH４〜６．５の間で分離を検討したところpI＝5.73，6.
03，6.37の３本のバンドが認められた。このうち pI＝
5.73 および 6.03 の２本のバンドが主たる成分であ
った。また尿素の不存下において同様にして等電点を測
定したところ、pI＝5.52，5.80 および 6.13の３本の
バンドが認められた。When separation was examined between pH 4 and 6.5, pI = 5.73, 6.
Three bands, 03 and 6.37, were recognized. Of these, pI ＝
Two bands of 5.73 and 6.03 were the main components. When isoelectric points were measured in the same manner in the absence of urea, three bands with pI = 5.52, 5.80 and 6.13 were recognized.
上記等電点測定を１０回繰り返し行った結果、その値は
表−Ｉに示すとおりであり等電点Ａ，Ｂ，Ｃは互いにほ
ぼ０．３ずつの相違が見られた。As a result of repeating the isoelectric point measurement 10 times, the values are as shown in Table-I, and the isoelectric points A, B, and C were different from each other by about 0.3.
＜参考例４＞本発明のＣＳＦの分子量の測定 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（ＳＤＳ−ＰＡＧＥ）により行った。電気泳動装
置はGE-2/4（ファルマシア社製）、ゲルはＴ＝１５％、
Ｃ＝２．６％のポリアクリルアミドスラブゲル（７０ｍ
ｍ×７０ｍｍ×３ｍｍ）および濃縮ゲル（Ｔ＝３％、Ｃ
＝２０％）を用いた。試料はあらかじめ、0.64 Ｍ２−
メルカプトエタノールにドデシル硫酸ナトリウムを濃度
が２％になるように加えた溶液中で３分間煮沸後、尿素
を４Ｍになるように添加することにより変性させておい
たものを２μｇを用いた。１２０Ｖで３時間泳動したの
ちゲルを取り出し、メタノール：酢酸：水（４：１：
５）にて固定し、銀染色（Silver Stain ： パイオ
ラッド社製）によりバンドを検出した。分子量マーカー
としてウシ血清アルブミン（ＢＳＡ，分子量67,000）、
オボアルブミン（ＯＶＡ，45,000）、チトクロームＣ
（Cyt.Ｃ，12,000）、インスリン（Ins.Ａ鎖：3,300，
Ｂ鎖：2,400）を同様に処理して用いた。この結果、分
子量約18,000の単一バンドが認められた。<Reference Example 4> Measurement of molecular weight of CSF of the present invention It was carried out by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresis device is GE-2 / 4 (Pharmacia), gel is T = 15%,
C = 2.6% polyacrylamide slab gel (70 m
m × 70 mm × 3 mm) and concentrated gel (T = 3%, C
= 20%) was used. The sample is 0.64 M2-
After boiling in a solution prepared by adding sodium dodecylsulfate to mercaptoethanol to a concentration of 2% for 3 minutes and then adding urea to a concentration of 4M, 2 μg of the product was used. After running at 120 V for 3 hours, the gel was taken out, and methanol: acetic acid: water (4: 1:
After fixing with 5), a band was detected by silver staining (Silver Stain: manufactured by Piolad). Bovine serum albumin (BSA, molecular weight 67,000) as a molecular weight marker,
Ovalbumin (OVA, 45,000), Cytochrome C
(Cyt.C, 12,000), insulin (Ins. A chain: 3,300,
B chain: 2,400) was similarly treated and used. As a result, a single band with a molecular weight of about 18,000 was observed.
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（ＳＤＳ−ＰＡＧＥ）により行った。電気泳動装
置はPROTEANTM16cm（バイオラツド社製）、ゲルはＴ＝
１５％、Ｃ＝２．６％のポリアクリルアミドスラブゲル
（140mm×160mm×１．５mm）および濃縮ゲル（Ｔ＝３
％，Ｃ＝２０％）を用いた。試料はあらかじめ0.64 Ｍ
２＝メルカプトエタノールにドデシル硫酸ナトリウムを
濃度が２％になるように加えた溶液中で３分間煮沸し変
性させておいたものを４μｇ用いた。３０ｍＡ定電流で
４時間泳動したのちゲルを取り出し、0.25％クーマシー
プリリアントブルーＲ２５０（シグマ社製）による染色
にてバンドを検出した。分子量マーカーとしてホスホリ
ラーゼＢ（Phosphorylass Ｂ，分子量92,500）、ウシ
血清ア清アルブミン（ＢＳＡ，67,000）オボアルブミン
（ＯＶＡ，45,000）、カルボニックアンヒドラーゼ（Ca
rbonic Anhydrase，31,000）、ソイビーントリプシン
インヒビター（Soybean Trypsin Inhibitor，21,50
0）、リゾチーム（Lysozyme，14,400）を同様に処理し
て用いた。この結果、分子量約19,000の単一バンドが認
められた。It was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresis device is PROTEAN ™ 16cm (manufactured by Bio-Rad), gel is T =
15%, C = 2.6% polyacrylamide slab gel (140 mm × 160 mm × 1.5 mm) and concentrated gel (T = 3
%, C = 20%) was used. Sample is 0.64 M in advance
2 = 4 µg of a solution obtained by boiling and denaturing a solution of sodium dodecyl sulfate added to mercaptoethanol at a concentration of 2% for 3 minutes. After running for 4 hours at a constant current of 30 mA, the gel was taken out and the band was detected by staining with 0.25% Coomassie Primitive Blue R250 (manufactured by Sigma). Phosphorylase B (Phosphorylass B, molecular weight 92,500), bovine serum albumin (BSA, 67,000) ovalbumin (OVA, 45,000), carbonic anhydrase (Ca) as molecular weight markers
rbonic Anhydrase, 31,000), Soybean Trypsin Inhibitor, 21,50
0) and lysozyme (Lysozyme, 14,400) were similarly treated and used. As a result, a single band with a molecular weight of about 19,000 was observed.
以上およびの結果から、本発明のＣＳＦの分子量
はＳＤＳ−ＰＡＧＥによる測定で19,000±1,000である
と考えられる。」 ＜参考例５＞本発明のヒトＣＳＦのアミノ酸組成試料を
常法により加水分解し、その蛋白部分のアミノ酸組成を
日立８３５アミノ酸自動分析装置（日立製作所社製）を
用いて特殊アミノ酸分析法により分析した。この結果を
表−IIに示した。尚、加水分解条件は次の如くである。From the above results and above, it is considered that the molecular weight of CSF of the present invention is 19,000 ± 1,000 as measured by SDS-PAGE. <Reference Example 5> An amino acid composition sample of human CSF of the present invention was hydrolyzed by a conventional method, and the amino acid composition of the protein portion was analyzed by a special amino acid analysis method using a Hitachi 835 amino acid automatic analyzer (manufactured by Hitachi, Ltd.). analyzed. The results are shown in Table-II. The hydrolysis conditions are as follows.
6N ＨＣｌ，１１０℃，２４時間，真空中 4N メタンスルホン酸＋０．２％３−（２−アミノエ
チル）インドール，１１０℃，２４時間，４８時間，７
２時間，真空中 試料は、４０％ｎ−プロパノールと０．１％トリフルオ
ロ酢酸を含む溶液（１．５ml）に溶かした後、各々０．
１mlをとり、乾燥窒素ガスにより乾燥させた後、又は
の試薬を加えて真空封管し、加水分解に供した。6N HCl, 110 ° C, 24 hours, 4N methanesulfonic acid + 0.2% 3- (2-aminoethyl) indole in vacuum, 110 ° C, 24 hours, 48 hours, 7
2 hours in vacuum The sample was dissolved in a solution containing 40% n-propanol and 0.1% trifluoroacetic acid (1.5 ml), and then dissolved in water.
After taking 1 ml and drying with dry nitrogen gas, or the reagent of (1) was added and the tube was sealed in a vacuum and subjected to hydrolysis.
表中、実測値はの２４時間値との２４，４８，７２
時間値の合計４回の平均値である。但しThr，Ser，1/2C
ys，Met，Val，IleおよびTrpは以下の方法で算出した。
（生化学実験講座、タンパク質化学II（東京化学同人
出版）を参照） ・Thr，Ser，1/2Cys，Metはの２４，４８，７２時間
値の経時変化をとり、零時間に補外。In the table, the measured value is 24, 48, 72 with the value of 24 hours.
It is an average of four time values in total. However, Thr, Ser, 1 / 2C
ys, Met, Val, Ile and Trp were calculated by the following method.
(Biochemistry Laboratory, Protein Chemistry II (Tokyo Kagaku Dojin)
(Published)) ・ Thr, Ser, 1 / 2Cys, and Met change over time at 24, 48, and 72 hours and are extrapolated to zero hours.
・Val，Ileはの７２時間値。・ Val and Ile are 72-hour values.
・Trpはの２４，４８，７２時間値の平均値。・ Trp is the average of 24, 48 and 72 hours.
表中のアミノ酸残基数はLeuを３３個と仮定して算出し
た予測値である。一般に上記の如き補正が必要なアミノ
酸は加水分解時に一部又はかなりの部分が破壊される
か、あるいは加水分解を受け難いものであり、さらにPr
oは発色率が低い等のことから、それ等のアミノ酸の実
測値(nmol)、従ってそれから算出される残基数は実際よ
りも低い値を示す傾向がある（例えば前述の生化学実験
講座を参照）。The number of amino acid residues in the table is a predicted value calculated assuming 33 Leu. In general, amino acids that require correction as described above are either partially or substantially destroyed during hydrolysis, or are less susceptible to hydrolysis.
Since o has a low coloring rate, etc., the measured values (nmol) of those amino acids, and therefore the number of residues calculated from them tend to show lower values than they actually are (for example reference).
＜実施例＞ヒトＣＳＦの感染防禦効果 １．Pseudomonas aeruginosa 感染に対する防禦効果 ８〜９週令（体重35.3±1.38 ｇ）のＩＣＲ系マウス
（雄）にエンドキサン（シオノギ社製、商品名）２００
ｍｇ／ｋｇを腹腔内投与した後３群に分け、その２群に
ヒトＣＳＦ（25000μ/マウス又は５００００μ／マウ
ス）を含む溶媒（１％プロパノール、５％(W/V)マウス
血清アルブミン）を、そして別の１群には溶媒のみを、
それぞれ２４時間毎に０．１mlずつ４回皮下投与した。
４回目の投与後３時間して各々の群にシュードモナス
アエルギノーザ(Pseudomonasaeruginosa)GNB−139
（３．９×105ＣＦＵ／マウス）を皮下投与して感染さ
せた。感染後２１時間してさらにもう一度ヒトＣＳＦ
（２５０００μ／マウス又は５００００μ／マウス）を
含む溶媒又は溶媒のみをそれぞれ対応する群に皮下投与
した。<Example> Infection prevention effect of human CSF 1. Protective effect against Pseudomonas aeruginosa infection 8 to 9 week-old (body weight 35.3 ± 1.38 g) ICR mice (male) Endoxan (Shionogi, trade name) 200
After intraperitoneal administration of mg / kg, it was divided into 3 groups, and the solvent (1% propanol, 5% (W / V) mouse serum albumin) containing human CSF (25000 μ / mouse or 50,000 μ / mouse) was divided into 2 groups, And another one group only solvent,
Each 24 hours, 0.1 ml was subcutaneously administered 4 times.
Pseudomonas was given to each group 3 hours after the 4th administration.
Aeruginosa (Pseudomonas aeruginosa) GNB-139
(3.9 × 10 5 CFU / mouse) was subcutaneously administered to infect. Human CSF 21 hours after infection
A solvent containing (25,000 μ / mouse or 50,000 μ / mouse) or only the solvent was subcutaneously administered to the corresponding group.
感染後１０日目までの生存マウス数により感染防禦効果
を調べた（表−III）。The effect of preventing infection was examined by the number of surviving mice up to 10 days after infection (Table-III).
（菌液の調製） ハートインフュージョン寒天平板（Difco社製、商品
名）を用いて３７℃で一夜Pseudomonas aeruginosa
ＧＮＢ−１３９を振とう培養する。培養液を生理食塩水
に懸濁させて調製した。(Preparation of Bacterial Solution) Pseudomonas aeruginosa was used overnight at 37 ° C. using a heart infusion agar plate (Difco, trade name).
GNB-139 is cultured by shaking. The culture solution was prepared by suspending it in physiological saline.
２．Candida感染に対する防禦効果 ８週令（体重40.5±1.60 ｇ）のＩＣＲ系マウス（雄）
にエンドキサン（シオノギ社製：商品名）２００ｍｇ／
ｋｇを腹腔内投与した後２群に分け１群にヒトＣＳＦ
（50000ｕ／マウス）を含む溶媒（１％プロパノール、
１０％(W/V)同種マウス血清）を、別の１群には溶媒の
みをそれぞれ２４時間毎に０．１mlずつ４回皮下投与し
た。４回目の投与後４時間して各々の群にキヤンディダ
アルビカンス（Candida albicans）Ｕ−５０−１（白
血病患者分離株、東北大細菌学教室分与）５．６×105
ＣＦＵ／マウスを静脈内投与して感染させた。感染後１
０日目までの生存マウス数により感染防禦効果を調べた
（表−IV）。2. Anti-protective effect against Candida infection 8-week-old (body weight 40.5 ± 1.60 g) ICR mouse (male)
Endoxan (Shionogi: trade name) 200 mg /
After intraperitoneal administration of kg, human CSF was divided into 2 groups and 1 group
(50000u / mouse) containing solvent (1% propanol,
(10% (W / V) allogeneic mouse serum) was subcutaneously administered to another group 4 times by 0.1 ml of the solvent alone every 24 hours. 4 hours after the 4th administration, Candida albicans U-50-1 (leukemia patient isolate, Tohoku University Bacteriology Department) 5.6 × 10 5
CFU / mouse was administered intravenously to infect. After infection 1
The infection control effect was examined by the number of surviving mice up to day 0 (Table-IV).
（菌液の調製） サブロー液体培地（２％デキストロース（純正化学社
製）１０％トリプトケースペプトン（ＢＢＬ社製：商品
名）、５％酵母抽出物（Difco社製：商品名）、pH５．
６）を用いて３７℃で一夜Candida albicans Ｕ−５
０−１を振とう培養する。培養液を生理食塩水で２回洗
浄した後、生理食塩水に懸濁させて調製した。(Preparation of bacterial solution) Sabouraud liquid medium (2% dextrose (manufactured by Junsei Kagaku) 10% tryptocase peptone (manufactured by BBL: trade name), 5% yeast extract (manufactured by Difco: trade name), pH5.
6) with Candida albicans U-5 at 37 ° C. overnight
Incubate 0-1 with shaking. The culture solution was washed twice with physiological saline and then suspended in physiological saline to prepare.
表−III，IVに示される如く本発明のヒトＣＳＦは顕著
な感染防禦効果を有することが認められた。 As shown in Tables III and IV, it was confirmed that the human CSF of the present invention has a remarkable anti-infective effect.
以下に実施例を挙げて本発明を具体的に説明するが、本
発明はこれ等に限定されるものではない。The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto.
実施例１ 参考例２の方法によって得られたヒトＣＳＦ含有フラク
ションを無菌処理した後−２０℃で凍結された凍結物を
用いて注射剤とした。Example 1 The human CSF-containing fraction obtained by the method of Reference Example 2 was subjected to aseptic treatment, and a frozen product frozen at -20 ° C was used to prepare an injection.
実施例２ 参考例２の方法によって得られたヒトＣＳＦを無菌操作
で１０ｍｌバイアル瓶に５ｍｌ充填し、−２０℃で凍結
乾燥後ゴム栓にて施栓した凍結乾燥物を用いて注射剤と
した。Example 2 Human CSF obtained by the method of Reference Example 2 was aseptically filled into a 10 ml vial in an amount of 5 ml, and the freeze-dried product was freeze-dried at −20 ° C. and capped with a rubber stopper to give an injection.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 松本 雅彦 東京都豊島区高田３丁目41番８号 中外製 薬株式会社内 (56)参考文献 特開 昭58−59924（ＪＰ，Ａ) 特開 昭59−210027（ＪＰ，Ａ) 特開 昭61−227526（ＪＰ，Ａ) ─────────────────────────────────────────────────── --- Continuation of the front page (72) Inventor Masahiko Matsumoto 3-41-8 Takada, Toshima-ku, Tokyo Inside Chugai Pharmaceutical Co., Ltd. (56) References JP-A-58-59924 (JP, A) JP-A-SHO 59-210027 (JP, A) JP-A-61-227526 (JP, A)
Claims (4)
Ｆを有効成分とする感染防禦剤。 〔理化学的性質〕 (i) 分子量：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による測定で１９０００±１００
０。 (ii) 等電点：以下の表−Ｉに示す三つの等電点（Ａ、
Ｂ、Ｃ）のうち少なくとも１つを有する。 (iii) 紫外部吸収：２８０ｎｍに極大吸収を有し、２
５０ｎｍに極小値をもつ。1. A human G-CS having the following physicochemical properties.
Anti-infective agent containing F as an active ingredient. [Physical and chemical properties] (i) Molecular weight: sodium dodecyl sulfate-polyacrylamide gel electrophoresis 19000 ± 100
0. (ii) Isoelectric points: Three isoelectric points (A,
At least one of B and C). (iii) Ultraviolet absorption: maximum absorption at 280 nm, 2
It has a minimum value at 50 nm.
範囲第１項の表−ＩのＡである特許請求の範囲第１項記
載の感染防禦剤。2. The infection control agent according to claim 1, wherein the isoelectric point pl of human G-CSF is A in Table I of claim 1.
範囲第１項の表−ＩのＢである特許請求の範囲第１項記
載の感染防禦剤。3. The infection control agent according to claim 1, wherein the isoelectric point pl of human G-CSF is B in Table-I of claim 1.
範囲第１項の表−ＩのＣである特許請求の範囲第１項記
載の感染防禦剤。4. The infection control agent according to claim 1, wherein the isoelectric point pl of human G-CSF is C in Table-I of claim 1.
Priority Applications (8)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60023777A JPH0615477B2 (en) | 1985-02-08 | 1985-02-08 | Anti-infective agent |
| PCT/JP1986/000053 WO1986004506A1 (en) | 1985-02-08 | 1986-02-07 | Infection-protective agent containing human granulocyte colony-stimulating factor as effective ingredient |
| EP86901138A EP0215126B1 (en) | 1985-02-08 | 1986-02-07 | Human granulocyte colony stimulating factor |
| AT86901138T ATE65798T1 (en) | 1985-02-08 | 1986-02-07 | HUMAN GRANULOXCYTE COLONY STIMULATION FACTOR. |
| DE8686901138T DE3680613D1 (en) | 1985-02-08 | 1986-02-07 | HUMAN GRANULOXCYT COLONY STIMULATION FACTOR. |
| PCT/JP1986/000052 WO1986004605A1 (en) | 1985-02-08 | 1986-02-07 | Human granulocyte colony stimulating factor |
| HK662/93A HK66293A (en) | 1985-02-08 | 1993-07-08 | Human granulocyte colony stimulating factor |
| BG98614A BG61925B2 (en) | 1985-02-08 | 1994-02-28 | Human granulocytic colonies stimulation factor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60023777A JPH0615477B2 (en) | 1985-02-08 | 1985-02-08 | Anti-infective agent |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS61186327A JPS61186327A (en) | 1986-08-20 |
| JPH0615477B2 true JPH0615477B2 (en) | 1994-03-02 |
Family
ID=12119764
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP60023777A Expired - Lifetime JPH0615477B2 (en) | 1985-02-08 | 1985-02-08 | Anti-infective agent |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JPH0615477B2 (en) |
| WO (1) | WO1986004506A1 (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5532341A (en) * | 1985-03-28 | 1996-07-02 | Sloan-Kettering Institute For Cancer Research | Human pluripotent hematopoietic colony stimulating factor |
| US4810643A (en) * | 1985-08-23 | 1989-03-07 | Kirin- Amgen Inc. | Production of pluripotent granulocyte colony-stimulating factor |
| US6004548A (en) | 1985-08-23 | 1999-12-21 | Amgen, Inc. | Analogs of pluripotent granulocyte colony-stimulating factor |
| JPH0618778B2 (en) * | 1985-10-04 | 1994-03-16 | 中外製薬株式会社 | Leukopenia treatment |
| JPH0618781B2 (en) * | 1986-10-18 | 1994-03-16 | 中外製薬株式会社 | Infectious disease treatment agent |
| FR2607344B1 (en) * | 1986-11-21 | 1994-04-29 | Nexo Distribution | DEVICE FOR PROCESSING AN AUDIO FREQUENCY ELECTRIC SIGNAL |
| CA1329119C (en) * | 1988-03-29 | 1994-05-03 | Milton David Goldenberg | Cytotoxic therapy |
| WO1989010932A1 (en) * | 1988-05-13 | 1989-11-16 | Amgen Inc. | Compositions and method for treating or preventing infections in animals |
| US6979442B1 (en) | 1998-08-17 | 2005-12-27 | Pfizer Inc. | Stabilized protein compositions |
| JP5680534B2 (en) | 2008-07-23 | 2015-03-04 | イーライ リリー アンド カンパニー | Modified bovine G-CSF polypeptides and their use |
| AR083006A1 (en) | 2010-09-23 | 2013-01-23 | Lilly Co Eli | FORMULATIONS FOR THE STIMULATING FACTOR OF COLONIES OF GRANULOCITS (G-CSF) BOVINE AND VARIANTS OF THE SAME |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6030655B2 (en) * | 1981-10-03 | 1985-07-17 | 科学技術庁 放射線医学総合研究所長 | Method for producing CSF using CSF-producing tumor transplantation method |
| JPS59210027A (en) * | 1983-05-14 | 1984-11-28 | Kagaku Gijutsucho Hoshasen Igaku Sogo Kenkyusho | Production of cerebrospinal fluid (csf) |
-
1985
- 1985-02-08 JP JP60023777A patent/JPH0615477B2/en not_active Expired - Lifetime
-
1986
- 1986-02-07 WO PCT/JP1986/000053 patent/WO1986004506A1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| WO1986004506A1 (en) | 1986-08-14 |
| JPS61186327A (en) | 1986-08-20 |
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| EXPY | Cancellation because of completion of term |