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WO1993018065A1 - Physiologically active proteinaceous substance - Google Patents

Physiologically active proteinaceous substance Download PDF

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Publication number
WO1993018065A1
WO1993018065A1 PCT/JP1993/000294 JP9300294W WO9318065A1 WO 1993018065 A1 WO1993018065 A1 WO 1993018065A1 JP 9300294 W JP9300294 W JP 9300294W WO 9318065 A1 WO9318065 A1 WO 9318065A1
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WO
WIPO (PCT)
Prior art keywords
physiologically active
gly
active substance
chromatography
proteinaceous
Prior art date
Application number
PCT/JP1993/000294
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French (fr)
Japanese (ja)
Inventor
Hajime Nawata
Fumio Umeda
Teruaki Yamauchi
Mitsunori Masakado
Original Assignee
Mochida Pharmaceutical Co., Ltd.
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Publication of WO1993018065A1 publication Critical patent/WO1993018065A1/en

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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention broth tag run Gin 1 2 (also known; prosulfuron evening cyclin (Pros ta cy c 1 in) , hereinafter referred to as PG 1 2) Novel proteinaceous raw physiologically active substance having a production stimulating activity, their preparation and
  • the present invention relates to a pharmaceutical composition containing the proteinaceous physiologically active substance.
  • novel proteinaceous physiologically active substance having an activity to promote the production of PG 1 2 from vascular endothelial cells.
  • Prostaglandin pros tagl and ins PG was published in 1930 by Kurzrock et al. [Pro S0c Exp. Biol. Med. 268, 1930], and was reported as a uterine muscle contractile active substance present in semen. Since then, basic research on prostaglandins has developed rapidly. Prostaglandin is a general term for biologically active substances based on prostanoic acid contained in human and animal tissues and organs.A series of chemicals called arachidonic acid cascades starting from arachidonic acid in cells. The reaction produces several prostaglandins with different chemical structures. It is.
  • prostaglandin plays an important role as a vasoactive substance for vascular smooth muscle [Pathophysiology, Vol. 2, p. 792, 1983], and thromboxane, an active prostanoid derived from platelets and blood vessel walls.
  • TXA 2 TXA 2
  • PGI 2 potent platelet aggregation inhibitory action and vascular relaxing action, and antagonism to create the TXA 2 platelet derived are involved in the maintenance of Homeosutashisu in vivo [Pre Tissue Jar Fungus, Narob Pharmacol. (Br. J. Parmac.), Vol.
  • the decrease in production may be attributable to the decrease in PG12 production stimulating activity present in the blood above [Metabolism] (Me t ab o 1 ism) 38, 837, 1989, Ha emo stasis 16, 447, 1986 and Diabetis Research and Clinical Practice (Diab. Res. Cl in. Prac. 3), p. 243, 1987].
  • PGI 2 As described above having a platelet aggregation inhibitory action, smooth muscle relaxing action, such as a blood vessel contact and bronchi besides that, have a gastric acid secretion inhibiting action and the like. Based on these physiological effects, it is conceivable to develop PGI 2 as a pharmaceutical product, but PGI 2 is a very chemically unstable substance with a neutral water of 37 and a half-life of 5 minutes. Absent. On the other hand, while maintaining the natural PG I 2-like activity, chemically stable PG 1 2 class ⁇ the anticoagulants, attempts to cane developed as pharmaceuticals such as a vasodilator has been made. However, it may be more desirable for the living body to make PGI 2 unstable and short.
  • the living body must always be ready to respond to PGI 2 that is produced promptly in an emergency, and by providing a large amount of stable PGI 2 analogs, the PGI 2 It is possible that responsiveness to PGI 2 may be reduced, making it impossible to respond to PGI 2 in an emergency. Indeed, pretreatment of prostaglandin E! (PGEa) and stable PGI 2 analogs due Examples of cells naturally occur to c AMP rise response to PG 1 2 no longer occurs has been reported [Prostaglandins (Pros tag 1 an nd ins.) 19, 2 pages, 1980].
  • PSA is present in blood, but its active substance (PSF) is It has not been identified and identification is desired.
  • PSF stimulates PG 12 production from endothelial cells, inhibiting platelet aggregation having the PG 1 2 by increasing the PG 12 concentration in the blood, smooth muscle relaxing action, to express gastric acid secretion inhibiting action, etc. it can.
  • PSF hemolytic uremic syndrome
  • thrombotic thrombocytopenic purpura peripheral artery occlusion
  • cardiac ischemia cerebral ischemia
  • atherosclerosis cerebral obstruction
  • hyperlipidemia diabetes, heart failure, angina It has the potential to be used in the treatment of sickness, ischemic heart disease, depressive heart disease, choroidal circulation disorder, bronchial disease, gastric ulcer, pregnancy and eclampsia. Disclosure of the invention
  • An object of the present invention is to provide a novel protein bioactive substance by purifying and isolating a substance that stimulates the production of PGI 2 from vascular endothelial cells in view of the above situation. Another object of the present invention is to provide a method for producing the same. It is a further object of the present invention to provide a pharmaceutical composition for the above-mentioned diseases based on the action of the proteinaceous physiologically active substance to stimulate PG12 production.
  • the present inventors have conducted intensive studies on PG12 stimulating activity present in the blood and in the culture supernatant of human cell lines in order to achieve the above-mentioned object, and found that the culture supernatant of normal human diploid fibroblasts It was confirmed that a substance having a PG12 production stimulating activity was present at a high concentration. Next, we succeeded in isolating and purifying a novel proteinaceous physiologically active substance having PGI 2 ⁇ stimulating activity from the culture supernatant, and determined its amino acid sequence.
  • the present invention provides a novel proteinaceous physiologically active substance having the following features.
  • the molecular weight is 30 to 36 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions.
  • the present invention uses at least one of the following steps (a) to (c) from a raw material containing the novel proteinaceous physiologically active substance, preferably a culture supernatant of normal human diploid fibroblasts.
  • a raw material containing the novel proteinaceous physiologically active substance preferably a culture supernatant of normal human diploid fibroblasts.
  • the present invention also provides a novel pharmaceutical composition for treating the following diseases, which comprises the novel proteinaceous physiologically active substance as an active ingredient.
  • FIG. 1 is a graph showing an elution pattern obtained by DEAE-5 PW anion exchange chromatography.
  • FIG. 2 is a graph showing an elution pattern obtained by heparin-5 PW affinity chromatography.
  • FIG. 3 is a graph showing the development pattern of protein-, gel-filtration and ram chromatography.
  • FIG. 4 is a schematic diagram showing an experimental result by SDS-PAGE. BEST MODE FOR CARRYING OUT THE INVENTION
  • the proteinaceous physiologically active substance of the present invention is a novel substance having at least one amino acid sequence represented by any of the following formulas (1) to (II).
  • amino acid sequence portion of any one of the above formulas (I) to (III) may be any portion of the proteinaceous physiologically active substance of the present invention.
  • the amino acid sequence portions of the above formulas (I) to (III) may be adjacent to each other or may contain an arbitrary amino acid sequence between them.
  • novel proteinaceous physiologically active substance has a molecular weight of 30 to 36 kDa in non-reducing conditions in sodium dodecyl sulfate sodium acrylamide gel electrophoresis (SDS-PAGE).
  • novel proteinaceous bioactive substances of the present invention have activity which act on vascular endothelial cells, stimulate the production of prostaglandin 1 2 (PG 1 2) from the cells. The method for measuring this activity will be described later.
  • the novel proteinaceous physiologically active substance of the present invention may be of any origin, but is preferably of human origin, more preferably of normal human diploid fibroblasts. Those produced in the culture supernatant of diploid fibroblasts are preferred. For example, it can be obtained by culturing normal human diploid fibroblasts or the like in an appropriate growth medium, collecting a culture solution having a PG production stimulating activity, and purifying a culture supernatant obtained. For culturing normal human diploid cells, a commonly used medium can be used, but Dulbecco's modified Eagle medium, RPMI164 medium, Eagle's minimum essential medium or ham medium is particularly preferable.
  • serum-free medium because the substance of the present invention is purified and isolated from the culture supernatant. It is desirable that the culture supernatant after completion of the culture is concentrated by, for example, a filter method or an ultrafiltration method.
  • the raw material containing the novel proteinaceous physiologically active substance of the present invention obtained as described above is purified by using at least one of the following steps.
  • the following steps (a), (b) and (c) ) May be performed according to a known protocol.
  • the order may be arbitrary, and may be repeated as necessary.
  • operations such as concentration, dialysis, etc. may be added between or before and after one step and the other, depending on the further, for example, chromatofocusing, adsorption chromatography, water chromatography, reverse phase Combination with other chromatography methods such as chromatography, thin-layer chromatography, partition chromatography, and other protein purification methods such as isoelectric focusing, polyacrylamide electrophoresis, and salting Is also good.
  • the collected fraction may be further subjected to reversed-phase high-performance liquid chromatography for the purpose of isolation, purification, purity confirmation, and the like.
  • appropriate elution conditions or developing conditions, eluate or developing solution may be selected in consideration of the properties of each chromatography carrier, target substance, contained contaminants, and the like.
  • the elution may be linear gradient elution or stepwise gradient elution.
  • the novel proteinaceous physiologically active substance of the present invention can be isolated from the culture supernatant preferably by the following method.
  • Culture supernatant of normal human diploid fibroblasts is subjected to anion exchange chromatography, for example, DEAE-5PW (manufactured by Tosoh I), onoQ (manufactured by Pharmacia) or Q-Sepharose column (manufactured by Pharmacia) And elute by sodium chloride concentration gradient method.
  • the PG 1 2 Stimulating activity of the eluted each fractions were measured to obtain fractions which are active. The measurement of the PGI 2 production stimulating activity will be described later.
  • this fraction is adsorbed to affinity chromatography, for example, heparin-15PW (manufactured by Tosoh Corporation) or the like, and eluted by a sodium chloride concentration gradient method.
  • the PGI 2 production stimulating activity is measured in the same manner, and the active fraction is collected. Next, this fraction is subjected to gel filtration chromatography, for example, Protein One Pack (Millipore Limited Waters Chromatography Division), Sephacryl S-100HR (Pharmacia) or TSK Gel G3000 SW XL column (Tosoichisha). Ltd.) and the like developed using, by Rukoto measuring the PG 1 2 production stimulating activity can be obtained biologically active fraction. This fraction is then subjected to affinity chromatography, for example, insulin-like growth factor (Insulin-like growth factor).
  • affinity chromatography for example, insulin-like growth factor (Insulin-like growth factor).
  • IGF e-like growth factor
  • the PGI 2 production stimulating activity can be measured by the following method.
  • vascular endothelial cells collected from the thoracic aorta membrane by a detachment method were subcultured in Dulbecco's modified medium containing 10% fetal serum, and cultured until they reached a saturated cell density. After adding the measurement sample and incubating for 60 minutes, the stable metabolite of PGI 2 in the supernatant is
  • O - Ke Bok - r F la (hereinafter, 6 - keto - PGF described as 1 alpha) can be obtained PG 1 2 production amount by the ⁇ a constant!.
  • a commercially available kit for measuring 6-keto PGF1 ⁇ may be used.
  • the amino acid sequence of the novel proteinaceous bioactive substances of the present invention with purified PG 1 2 production stimulating activity it may be used the following method.
  • the substance of the present invention can be directly analyzed by the sequencer, or peptide fragments obtained by enzymatic cleavage of trypsin, lysyl endopeptidase, etc. can be analyzed.
  • the sequence of each amino acid can be determined.
  • the novel proteinaceous physiologically active substance of the present invention is characterized by inhibiting platelet aggregation of PG I 2
  • the present invention provides a pharmaceutical composition containing the proteinaceous physiologically active substance of the present invention as at least one active ingredient for the following various diseases because it has an action, a smooth muscle relaxing action, a gastric acid secretion inhibitory action and the like.
  • Hemolytic uremic syndrome Hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, peripheral arterial occlusion, cardiac ischemia, cerebral ischemia, arteriosclerosis, cerebral obstruction, hyperlipidemia, diabetes, heart failure, angina, ischemic heart disease, depression Hematologic heart disease, choroidal circulation disorder, bronchial disease, gastric ulcer, pregnancy eclampsia.
  • the dosage of the novel proteinaceous physiologically active substance of the present invention may vary depending on the patient's sex, age, body weight, disease type and its pathological condition, depending on the dosage form, but the effective dosage is 1 ng. 22 mg / kg, preferably 10 ng to 200; / g / kg.
  • the pharmaceutical form of the pharmaceutical composition of the present invention may be any as long as it can supply an effective amount to the disease lesion, and examples thereof include tablets, powders, powders, capsules, ointments, powders, and injections. Etc. are exemplified.
  • the pharmaceutical composition of the present invention contains a commonly used pharmaceutical mixture such as an excipient, a stabilizer ⁇ ) or a solubilizing agent, as long as the pharmacological properties are not impaired. May be.
  • Specific examples include Ringer's solution, phosphate buffer, human serum albumin, hydrolyzed gelatin, sucrose, dextran, polyethylene glycol, etc., which are appropriately selected and used depending on the drug form.
  • a culture supernatant was prepared and purified by the following method to obtain a novel proteinaceous physiologically active substance of the present invention.
  • Normal human diploid fibroblasts were prepared in 4.8 ⁇ 10 4 cells ZmL in Dulbecco's modified Eagle's medium containing 15% fetal bovine serum. 3 L of this cell suspension was inoculated in a rotary culture vessel, and 5% CO 2 -95% air 37. C. Three days after the cell implantation, the culture solution was replaced with 3 L of fresh Dulbecco's modified Eagle's medium containing 15% fetal calf serum, and the culture was continued for 2 days.
  • the culture solution was removed, and the cells were washed with a Ca2 + -and Mg2 + -free Dulbecco's monophosphate physiological saline solution, and 3 L of Dulbecco's modified Eagle's medium without phenol red was added. And cultured for 2 days. After recovering the culture, the cells were filtered through a 2.5 / zm filter (CN cartridge, 30 inch, manufactured by Millipore) to remove cell debris.
  • CN cartridge 2.5 / zm filter
  • a two-stage concentration using a hollow fiber module (Molsep fiber FS-106 kDa cut-off, manufactured by Daicel Chemical Industries, Ltd.) and a filtration filter (Omega mini-set, manufactured by Fuji Filter Industries, Ltd.) with a molecular weight cutoff of 10 kDa. It concentrated by operation.
  • This concentrated solution is used in a dialysis tube (stained with a molecular weight cutoff of 3.5 kDa
  • the solution was dialyzed against 2 OmM Tris-HCl buffer (pH 7.8) for 4 to 1 minute using the following method. After further dialysis with the same buffer at 4 e C for 8 hours, the mixture was sequentially filtered through 1.2 ⁇ m filter Yuichi (DF Assembly, Nippon Pall) and 0.22 zm filter-one system (Koining). did.
  • Culture supernatant of normal human diploid fibroblasts prepared according to (1) was pre-equilibrated with 2 OmM Tris-monohydrochloride buffer (PH7.8). DEAE-5 PW (Tosoichi) Anion exchange chromatography The mixture was adsorbed on a column, eluted with a linear gradient method of 0 to 1.0 M sodium chloride prepared using a 2 OmM Tris-monohydrochloride buffer (pH 7.8), and the absorbance was simultaneously measured at 280 nm.
  • the active fraction obtained in step 1 was dialyzed against 10 mM phosphate buffer (pH 7.4) and equilibrated with 1 OmM phosphate buffer (pH 7.4).
  • Heparin (HEPAR IN) -5PW E. Adsorbed on an affinity column and eluted with a linear gradient of 0 to 0 M sodium chloride prepared using 1 OmM phosphate buffer (pH 7.4). The absorbance was simultaneously measured at nm.
  • the eluted PGI 2 production stimulation activity of each off Rakushiyon measures, the fractions with the eluted PG I 2 production stimulating activity at sodium ⁇ beam chloride concentration of 450 to 500 mM were pooled (FIG. 2) o
  • the active fraction obtained in step 2 was concentrated with Centricon-10 (manufactured by Amicon), and then pre-equilibrated with 10 mM phosphate buffer (pH 7.4). It was developed using two connected gel filtration columns from Millipore Limited Waters Chromatography Division, and absorbance was simultaneously measured at 280 nm. Was measured PG 1 2 Stimulating activity of each fraction, activity was observed around a molecular weight 3 OKD a (FIG.
  • Fig. 3 o
  • the numbers in Fig. 3 indicate molecular weight (kDa), Vt indicates the total volume of the filler, and Vo indicates the excluded volume.
  • the active fraction obtained in step 3 was applied to an IGF-affinity column equilibrated with 1 OmM phosphate buffer (pH 7.4).
  • IGF-affinity column a pressure-resistant ram in which recombinant IGF-I was bound as a ligand to AFFEPREP 10 (manufactured by Nippon Bio-Rad Laboratories) was used for this experiment. After adsorption, the sample was eluted with 0.5 M acetic acid. Where was measured PG 1 2 Stimulating activity, PG 1 2 surname stimulating activity was found in non-adsorbed surface min.
  • the molecular weight of the novel proteinaceous physiologically active substance of the present invention obtained in step 5 of Example 1 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions. A single band was observed at a molecular weight of 33 kDa (Fig. 4).
  • Figure 4 is a schematic diagram of a reference photograph after SDS-PAGE electrophoresis.
  • lines A and C are bands of a standard sample having a known molecular weight
  • line B is a band of the novel proteinaceous bioactive substance of the present invention.
  • the numbers represent the molecular weight (kDa) of the standard sample.
  • the novel proteinaceous physiologically active substance of the present invention obtained in step 5 of Example 1 was digested with trypsin to form a peptide fragment.
  • Formula (I) Formula (I)
  • Vascular endothelial cells were collected from the thoracic aorta intima by an isolation method. The obtained vascular endothelial cells were then placed in a Dulbecco's modified Eagle's medium containing 10% OU / mL penicillin and 100 gZmL streptomycin containing 10% fetal calf serum, at 37 ° C under 5% CO 2 -95%. The cells were subcultured in C. The medium was changed twice a week, and after passage 5-10, vascular endothelial cells were treated with 0.05% trypsin.
  • vascular endothelial cells were transferred to a 24-well culture dish containing 1 mL of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, and cultured to 5 ⁇ 10 4 cells Z well.
  • Dulbecco's modified Eagle medium 500 containing the following various samples, the mixture was incubated at 37 ° C for 60 minutes.
  • the PG12 production stimulating activity of the proteinaceous physiologically active substance of the present invention increased in a concentration-dependent manner.
  • Example 4 A toxicity test in mice was carried out using the proteinaceous physiologically active substance of the present invention obtained in Example 1. That is, the proteinaceous physiologically active substance obtained in Example 1 was intravenously administered once daily for one week to five male and female 6-week-old ICR mice once a day. Administration period The general characteristics of each animal were observed throughout. The highest dose group in this study
  • Example 6 10 Omg of the proteinaceous physiologically active substance of the present invention obtained in Example 1 was dissolved in 10 OmL of a physiological saline solution containing 1 Omg / mL of hydrolyzed gelatin, and a filter having a pore size of 0.22 m (Mirex GV Filtration and sterilization were carried out using Millipore Corporation. This was aseptically dispensed in 2 mL aliquots into glass vials, sealed and sealed for injection lysate. (Example 6)
  • Example 2 10 Omg of the proteinaceous physiologically active substance of the present invention obtained in Example 1 was dissolved in 10 OmL of 1 OmM PBS (pH 7.4) containing 10 Oiiig / mL of human serum albumin, and the pore size was 0.22 m. And sterilized by filtration using a filter (Mirex GV Millipore). This was aseptically dispensed into glass vials in 3 mL portions, freeze-dried and sealed to give a freeze-dried preparation for injection. Availability of S ⁇
  • the novel proteinaceous bioactive substances of the present invention acts on vascular endothelial cells has the effect of stimulating the PG 1 2 of flavor from the cells. Therefore, based on the inhibitory action of PG12 on platelet aggregation, smooth muscle relaxation, gastric acid secretion, etc., hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, peripheral arterial occlusion, cardiac ischemia, cerebral ischemia, arteriosclerosis, brain It can be expected to be used as a therapeutic drug for obstruction, hyperlipidemia, diabetes, heart failure, angina pectoris, ischemic heart disease, depressive heart disease, choroidal circulatory disorder, bronchial disease, gastric ulcer, pregnancy eclampsia, etc. .

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Abstract

A novel, physiologically active proteinaceous substance having at least one amino acid sequence represented by any of formulae (I) to (III) and a molecular weight of 30-36 kDa as determined by SDS-PAGE under a non reducing condition. It has an activity of acting upon hemangioendothelial cells to stimulate the production of prostaglandin I2 (PGI2) therefrom, thus being useful as a therapeutic pharmaceutical composition.

Description

明 細 書 発明の名称  Description Name of Invention
蛋白性生理活性物質 技術分野  Technical field of proteinaceous bioactive substances
本発明は、 ブロスタグランジン 12 (別名；プロス夕サイクリン （Pros t a cy c 1 i n)、 以下 PG 12 と記す）産生刺激活性を有する新規な蛋白性生 理活性物質、 その製造方法および該蛋白性生理活性物質を含有する医薬組成物に 関する。 詳しくは血管内皮細胞に作用し、 血管内皮細胞からの PG 12 の産生を 促進させる活性を有する新規な蛋白性生理活性物質に関する。 背景技術 The present invention broth tag run Gin 1 2 (also known; prosulfuron evening cyclin (Pros ta cy c 1 in) , hereinafter referred to as PG 1 2) Novel proteinaceous raw physiologically active substance having a production stimulating activity, their preparation and The present invention relates to a pharmaceutical composition containing the proteinaceous physiologically active substance. For more acts on vascular endothelial cells, novel proteinaceous physiologically active substance having an activity to promote the production of PG 1 2 from vascular endothelial cells. Background art
プロスタグランジン （pros tagl and ins PG)は 1930年にク ルツロックら [プロシーデイング ォブ ザ ソサイエティ フォー ェクスぺ リメンタル バイオロジー アンド メディスン （Pro S 0 c Exp. Bi o l. Med. ) , 28卷、 268頁、 1930年] により、 精液中に存在 する子宮筋収縮活性物質として発見報告された。 その後プロスタグランジンに関 する基礎的研究は急速な発展をとげた。 プロスタグランジンはヒト、 動物の 組織、 臓器に含まれるプロスタン酸を基本構造とする生理活性物質の総称で あり、 細胞内でァラキドン酸を出発物質としてァラキドン酸カスケ一ドと呼ばれ る一連の化学反応により化学構造の異なる数種のプロスタグランジンが生合成さ れる。 それらは内分泌系、 神経系、 炎症、 免疫系その ft ^体の恒常性の維持に関 与する重要な活性物質として注目されており、 炎症、 血栓形成、 末梢循環、 動脈硬化、 老化などの諸機構に広汎に関与する [臨床科学、 17巻、 958頁、 1981年] と考えられている。 さらに、 プロスタグランジンは血管平滑筋に対 する血管作動性物質として重要な役割をはたしており [病態生理、 2巻、 792 頁、 1983年]、 血小板や血管壁由来の活性型プロスタノィドであるトロンボ キサン A2 (TXA2 ) [プロシーデイング ォブ ザ ナショナル ァカ デミ一 ォブ サイエンス ォブ ザ ユナイテッド ステイツ ォブ ァメリ 力 （Proc. Nat l. Acad. S c i. USA) , 72巻、 2994頁、 1975年] および PG 12 [ネイチヤー （Na tur e)、 263巻、 663 頁、 197 &年] が、 血小板と血管系の生理と病態における臨床的意義において 注巨されている。 これらプロスタグランジンの中で、 とりわけ PGI2 は強力な 血小板凝集抑制作用と血管弛緩作用を呈し、 血小板由来の TXA2 と拮抗的に作 用し生体内のホメォスターシスの維持に関与している [プリティッシュ ジャー 菌ナル ォブ ファーマコロジ一 （Br. J. P a rma c. ) 、 76巻、 3頁、 1982年] 。 正常の血管内皮細胞は、 物理的、 化学的刺激が加わると、 主として PGI2 を生成し、 血小板の活性化を抑制するのみならず自ら血管 壁トーヌスを調節して局所循環の恒常性を維持する機構が作動している。 すなわ ち、 血栓症や動脈硬化症などの血管障害の発症には TXA2 および PGI2 の産 生不均衡とりわけ PGI 2 の産生低下が関与している [プリティッシュ ジャー ナル ォブ ファーマコロジー （B r. J. Ph a rma ） 、 76巻、 3頁、 1982年] ことが知られている。 1978年マックインタイヤーら [ネ イチヤー （Na t u r e)、 271巻、 549頁、 1978年] は流血中に血管 壁での PGI2 産生を刺激する因子の存在を認めており、 本因子は熱に不安定な 高分子物質であることを報告している。 その後レムッジらにより溶血性尿毒 症症候群で本因子が低下していることが報告されて以来 [ランセッ ト (L a n c e t ) 2巻、 871頁、 1 978年] 、 血栓性血小板減少性紫斑 病 [ランセット （Lan c e t) 2巻、 748頁、 1 979年] 、 鎌状赤血 球性貧血 [ブリティッシュ ジャーナル ォブ へマトロジ一 （Br. J. Ha ema ΐ o 1. ) 48巻、 545頁、 1981年] 、 急性心筋梗塞 [コロナ リ一 （C 0 r 0 n a r y) 2巻、 49頁、 1985年] 、 糖尿病等の血栓性 ·動 脈硬化性疾患で本因子の低下がその病因と密接に関与していることが明らかにさ れている。 とくに、 糖尿病性血管障害の発症 '進展因子としての血小板凝集亢進 機序については、 血小板由来 TXA2 の産生亢進 [トロンボシス リサーチ (Th r omb. R e s . ) 19巻、 211頁、 1980年、 ジャーナル ォブ ラボラトリ一 アンド クリニカル メディスン （J. Lab. C l i n. Me d. ) 97巻、 87頁、 1 981年] の他に、 血管由来の PG I 2 産生 低下が糖尿病患者や実験糖尿病動物について報告されている [ランセッ ト (Lance t) 1巻、 325頁、 1979年、 ランセット （Lance t) 2 巻、 1365頁、 1979年、 ザ 二ユウ イングランド ジャーナル ォブ メディスン （N. Engl. J. Me d. ) 300巻、 366頁、 1979 年およびライフ サイエンス （L i f e Sc i. ) 23巻、 351頁、Prostaglandin (pros tagl and ins PG) was published in 1930 by Kurzrock et al. [Pro S0c Exp. Biol. Med. 268, 1930], and was reported as a uterine muscle contractile active substance present in semen. Since then, basic research on prostaglandins has developed rapidly. Prostaglandin is a general term for biologically active substances based on prostanoic acid contained in human and animal tissues and organs.A series of chemicals called arachidonic acid cascades starting from arachidonic acid in cells. The reaction produces several prostaglandins with different chemical structures. It is. They have been attracting attention as important active substances involved in the maintenance of homeostasis of the endocrine system, nervous system, inflammation, immune system and the ft ^ body, and have been used in various fields such as inflammation, thrombus formation, peripheral circulation, arteriosclerosis, and aging. It is considered to be widely involved in the organization [Clinical Science, 17, 958, 1981]. Furthermore, prostaglandin plays an important role as a vasoactive substance for vascular smooth muscle [Pathophysiology, Vol. 2, p. 792, 1983], and thromboxane, an active prostanoid derived from platelets and blood vessel walls. A 2 (TXA 2 ) [Proceding of the National Academia Science of the United States of America (Proc. Natl. Acad. Sci. USA), Vol. 72, p. 2994, 1975] and PG 12 [Naturé, 263, 663, 197 & years] have been overwhelmed by their clinical significance in platelet and vascular physiology and pathology. Of these prostaglandins, especially PGI 2 exhibits a potent platelet aggregation inhibitory action and vascular relaxing action, and antagonism to create the TXA 2 platelet derived are involved in the maintenance of Homeosutashisu in vivo [Pre Tissue Jar Fungus, Narob Pharmacol. (Br. J. Parmac.), Vol. 76, p. 3, 1982]. Normal vascular endothelial cells mainly produce PGI 2 when subjected to physical and chemical stimuli, and not only suppress platelet activation, but also regulate vascular wall tonus to maintain local circulation homeostasis. The mechanism is working. Sunawa Chi, thrombosis or onset is decreased production of TXA 2 and production imbalance especially PGI 2 of PGI 2 in vascular disorders such as arteriosclerosis are involved [pre tissue journal O Bed Pharmacology (B r. J. Pharma), 76, 3 pages, 1982]. In 1978, Mac Internia et al. Nature, 271: 549, 1978] found that blood bleeding had a factor that stimulated PGI 2 production in the blood vessel wall, and this factor was a heat-labile macromolecular substance. Have reported that. Since Remuzzi et al. Reported that the factor is reduced in hemolytic uremic syndrome [Lancet, Volume 2, p. 871, 1978], thrombotic thrombocytopenic purpura [Lancet (Lancet) 2, 748, 1979], Sickle cell anemia [Br. J. Haema o 1.] 48, 545, 1981 ], Acute myocardial infarction [C 0 r0 nary 2, Vol. 49, p. 1985], Reduction of this factor in thrombotic and arteriosclerotic diseases such as diabetes is closely related to its etiology. It has been clarified. In particular, for the platelet aggregation enhancement mechanism as onset 'progression factors of diabetic angiopathy, enhanced production of platelet-derived TXA 2 [Toronboshisu Research (Th r omb. R es. ) 19 vol, 211 pp., 1980, Journal Oblin Laboratory and Clinical Medicine (J. Lab. Clin. Med.) 97, 87, 1981], as well as decreased vascular PGI 2 production in diabetic patients and experimental diabetic animals. Reported [Lancet, Volume 1, p. 325, 1979, Lancet, Volume 2, p. 1,365, 1979, The Niu England Journal of Medicine (N. Engl. J. Me.) d.) 300, 366, 1979 and Life Science (Life Sc i.) 23, 351,
1978年] 。 しかも、 この産生低下は、 上 の血中に 存在する PG 12 産生 刺激活性の低下が一因を担っている可能性が彔唆されている [メタボリズム (Me t ab o 1 i sm) 38巻、 837頁、 1989年、 へモ ス タ シ ス (Ha emo s t a s i s) 16巻、 447頁、 1 986年およびダイァ ベティス リサーチ アンド クリニカル プラクティス （D i ab. Re s. Cl in. P r a c t. ) 3巻、 243頁、 1987年] 。 1978]. In addition, it has been suggested that the decrease in production may be attributable to the decrease in PG12 production stimulating activity present in the blood above [Metabolism] (Me t ab o 1 ism) 38, 837, 1989, Ha emo stasis 16, 447, 1986 and Diabetis Research and Clinical Practice (Diab. Res. Cl in. Prac. 3), p. 243, 1987].
上述のごとく P G 12 は血小板凝集抑制作用を有するが、 それ以外にも血管お よび気管支などの平滑筋弛緩作用、 胃酸分泌抑制作用等を有する。 これらの生理 作用に基づき PGI2 を医薬用品として開発することが考えられるが、 PGI2 は 37で中性の水で半減期 5分と極めて化学的に不安定な物質であり、 実用 化に至ってない。 一方、 天然の PG I2 様作用を保ちながら、 化学的に安定 な P G 12類緣体を血液凝固阻止剤、 血管拡張剤等の医薬品として開発しょうと する試みがなされている。 しかし、 生体にとっては、 PGI2 が不安定で短 である方がむしろ望ましい場合も考えられる。 すなわち、 緊急の事態に至って速 やかに生成される PGI 2 に対して、 生体は常に反応できる態勢にならなければ ならず、 安定な PGI2類縁体を多量に与えることによって、 細胞の PGI2 に 対する応答性が低下し、 緊急の場合に PGI2 に反応できなくなる可能性も考え られる。 実際、 プロスタグランジン E! (PGEa )や安定な PGI2類縁体の 前処理が原因で、 P G 12 に対して当然起こるべき c AMP上昇反応が生じなく なった細胞の例が報告されている [プロスタグランジンズ（Pros tag 1 a nd i ns. ) 19巻、 2頁、 1980年] 。 While PG 1 2 as described above having a platelet aggregation inhibitory action, smooth muscle relaxing action, such as a blood vessel contact and bronchi besides that, have a gastric acid secretion inhibiting action and the like. Based on these physiological effects, it is conceivable to develop PGI 2 as a pharmaceutical product, but PGI 2 is a very chemically unstable substance with a neutral water of 37 and a half-life of 5 minutes. Absent. On the other hand, while maintaining the natural PG I 2-like activity, chemically stable PG 1 2 class緣体the anticoagulants, attempts to cane developed as pharmaceuticals such as a vasodilator has been made. However, it may be more desirable for the living body to make PGI 2 unstable and short. In other words, the living body must always be ready to respond to PGI 2 that is produced promptly in an emergency, and by providing a large amount of stable PGI 2 analogs, the PGI 2 It is possible that responsiveness to PGI 2 may be reduced, making it impossible to respond to PGI 2 in an emergency. Indeed, pretreatment of prostaglandin E! (PGEa) and stable PGI 2 analogs due Examples of cells naturally occur to c AMP rise response to PG 1 2 no longer occurs has been reported [Prostaglandins (Pros tag 1 an nd ins.) 19, 2 pages, 1980].
従って、 PGI 2 の有する上述の作用を期待するのであれば、 化学的に安定 な類縁体を用いるよりも、 必要なときに必要な濃度の PG 12 を必要な部位 に産生させてやることが生体にとってより好ましいと考えられる。 生体内で の PG I 2 代謝は 2つの因子により制御されており、 その 1つは PG I 2 安 定化因子で近年アポリポプロティン A— 1 (ΑροΑ— 1) であることが同 定された [ザ ジャーナル ォブ クリニカル インべスティゲージヨン （J. C l i n. I nve s t. ) 82巻、 803頁、 1 988年】 。 他の 1つは PG 12 産生刺激因子 (Prostacyclin Production Stimulating Factor, PSF)で ある。 上述のごとくすでに血中には PG 12 產生刺激活性 (Pros t acyc 1 i n St imu l at ing Ac t iv i ty. PSA)が存在すること が報告されているが、 その活性本体（PSF)は同定されておらず同定が望まれ ている。 PSFは血管内皮細胞からの PG 12 産生を刺激し、 血中の PG 12 濃 度を高めることにより P G 12 の有する血小板凝集抑制作用、 平滑筋弛緩作用、 胃酸分泌抑制作用等を発現することができる。 このような作用に基づき P S Fは 溶血性尿毒症症候群、 血栓性血小板減少性紫斑病、 末梢動脈閉塞、 心虚血、 脳虚血、 動脈硬化、 脳閉塞、 高脂血症、 糖尿病、 心不全、 狭心症、 虚血性心 疾患、 うつ血性心疾患、 脈絡膜循環障害、 気管支疾患、 胃潰瘪、 妊娠子癇等の治 療に用いうる可能性を有している。 発明の開示 Therefore, if you expect the effect described above with a PGI 2, that'll chemically than with stable analogs were produced to the site in need of PG 1 2 concentration when needed It is considered more preferable for the living body. In vivo PG I 2 metabolism is regulated by two factors, one of which is the PG I 2 stabilizing factor, which was recently identified as apolipoprotein A-1 (ΑροΑ—1) [The Journal Bu Clinical Investigation Y. (J. Clin. Invest., 82, 803, 1988). The other is Prostacyclin Production Stimulating Factor (PSF). As described above, it has already been reported that PG 12 proliferative stimulating activity (Prostacy 1 in Stimulating Activity. PSA) is present in blood, but its active substance (PSF) is It has not been identified and identification is desired. PSF stimulates PG 12 production from endothelial cells, inhibiting platelet aggregation having the PG 1 2 by increasing the PG 12 concentration in the blood, smooth muscle relaxing action, to express gastric acid secretion inhibiting action, etc. it can. Based on these effects, PSF is considered to be hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, peripheral artery occlusion, cardiac ischemia, cerebral ischemia, atherosclerosis, cerebral obstruction, hyperlipidemia, diabetes, heart failure, angina It has the potential to be used in the treatment of sickness, ischemic heart disease, depressive heart disease, choroidal circulation disorder, bronchial disease, gastric ulcer, pregnancy and eclampsia. Disclosure of the invention
本発明の目的は、 上述のような状況を鑑み、 血管内皮細胞からの PGI 2 の産 生を刺激する物質を精製単離し、 新規な蛋白性生理活性物質を提供することであ る。 また、 本発明はその製造方法を提供することである。 さらに、 本発明の目的 は、 該蛋白性生理活性物質の PG 12 產生を刺激するという作用に基づいた上述 の疾患に対する医薬組成物を提供することにある。 本発明者らは上述の目的を達成するために、 血中およびヒト細胞株培養上清中 に存在する PG 12 刺激活性について鋭意研究を重ねた結果、 正常ヒトニ倍 体線維 胞の培養上清中に PG 12産生刺激活性を有する物質が高濃度に存在 することを確認した。 次いで、 この培養上清より PGI 2 ^^刺激活性を有する 新規な蛋白性生理活性物質を単離精製することに成功し、 さらにアミノ酸配列の を決定した。 An object of the present invention is to provide a novel protein bioactive substance by purifying and isolating a substance that stimulates the production of PGI 2 from vascular endothelial cells in view of the above situation. Another object of the present invention is to provide a method for producing the same. It is a further object of the present invention to provide a pharmaceutical composition for the above-mentioned diseases based on the action of the proteinaceous physiologically active substance to stimulate PG12 production. The present inventors have conducted intensive studies on PG12 stimulating activity present in the blood and in the culture supernatant of human cell lines in order to achieve the above-mentioned object, and found that the culture supernatant of normal human diploid fibroblasts It was confirmed that a substance having a PG12 production stimulating activity was present at a high concentration. Next, we succeeded in isolating and purifying a novel proteinaceous physiologically active substance having PGI 2 ^^ stimulating activity from the culture supernatant, and determined its amino acid sequence.
すなわち、 本発明は、 下記の特徵を有する新規な蛋白性生理活性物質を提供す 。  That is, the present invention provides a novel proteinaceous physiologically active substance having the following features.
(1)下記式 (I)ないし（III) いずれかで表されるアミノ酸配列を、 少なく とも 1つ有している。  (1) It has at least one amino acid sequence represented by any of the following formulas (I) to (III).
式（I) Formula (I)
lie Thr Val Val Asp Ala Leu His Glu lie Pro Val Lys'Lys Gly 1 5 10 15 lie Thr Val Val Asp Ala Leu His Glu lie Pro Val Lys'Lys Gly 1 5 10 15
Glu GLy Ala Glu Leu Glu GLy Ala Glu Leu
20  20
式（ω Equation (ω
Gly His Tyr Gly Val Gin Arg Gly His Tyr Gly Val Gin Arg
式 (ΠΙ) Expression (ΠΙ)
Thr Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala lie Gin Thr 1 5 10 15 Thr Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala lie Gin Thr 1 5 10 15
Arg Arg
16  16
(2)非還元条件下におけるドデシル硫酸ナトリウム一ポリアクリルアミ ドゲ ル電気泳動 （SDS— PAGE) において分子量が 30〜36kDaである。  (2) The molecular weight is 30 to 36 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions.
(3)血管内皮細胞に作用し、 該細胞からのプロスタグランジン I 2 (PG 12 ) の産生を刺激する活性を有する。 (3) acts on vascular endothelial cells, it has an activity of stimulating the production of prostaglandin I 2 (PG 1 2) from the cells.
また、 本発明は、 上記の新規な蛋白性生理活性物質を含む原料、 好ましくは正 常ヒトニ倍体線維芽細胞の培養上清から下記（a)〜（c) の工程の少なくとも 1つを用いて精製することを特徴とする該蛋白性生理活性物質の製造方法を提供 する。  In addition, the present invention uses at least one of the following steps (a) to (c) from a raw material containing the novel proteinaceous physiologically active substance, preferably a culture supernatant of normal human diploid fibroblasts. To provide a method for producing the proteinaceous physiologically active substance, characterized in that the substance is purified by purification.
(a) イオン交換クロマトグラフィー  (a) Ion exchange chromatography
(b)ァフィ二ティークロマトグラフィー  (b) Affinity chromatography
(c) ゲルろ過クロマトグラフィー  (c) Gel filtration chromatography
また、 本発明は上述の新規な蛋白性生理活性物質を有効成分として含有するこ とを特徴とする、 下記疾患に対する治療用の新規な医薬組成物を提供する。 溶血性尿毒症症候群、 血栓性血小板減少性紫斑病、 末梢動脈閉塞、 心虚血、 脳 虚血、 動脈硬化、 脳閉塞、 高脂血症、 糖尿病、 心不全、 狭心症、 虚血性心疾患、 うつ血性心疾患、 脈絡膜循環障害、 気管支疾患、 胃潰瘍、 妊娠子癤。 図面の簡単な説明 The present invention also provides a novel pharmaceutical composition for treating the following diseases, which comprises the novel proteinaceous physiologically active substance as an active ingredient. Hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, peripheral arterial occlusion, cardiac ischemia, cerebral ischemia, arteriosclerosis, cerebral obstruction, hyperlipidemia, diabetes, heart failure, angina, ischemic heart disease, depression Bloody heart disease, choroidal circulation disorder, bronchial disease, gastric ulcer, pregnant child. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 D EAE— 5 PW陰イオン交換クロマトグラフィーによる溶出パター ンを示すグラフである。  FIG. 1 is a graph showing an elution pattern obtained by DEAE-5 PW anion exchange chromatography.
図 2は、 へパリンー 5 PWァフィ二ティークロマトグラフィーによる溶出 パターンを示すグラフである。  FIG. 2 is a graph showing an elution pattern obtained by heparin-5 PW affinity chromatography.
図 3は、 プロテインーノ、 'ックゲルろ過力ラムクロマトグラフィ一による展開パ ターンを示すグラフである  FIG. 3 is a graph showing the development pattern of protein-, gel-filtration and ram chromatography.
図 4は、 S D S— PAGEによる実験結果を示す模式図である。 発明を実施するための最良の形態  FIG. 4 is a schematic diagram showing an experimental result by SDS-PAGE. BEST MODE FOR CARRYING OUT THE INVENTION
以下本発明を詳細に説明する。  Hereinafter, the present invention will be described in detail.
本発明の蛋白性生理活性物質は、 下記式 ( 1 ) ないし （ΙΙΓ)いずれかで表され るァミノ酸配列を、 少なくとも 1つ有している新規な物質である。  The proteinaceous physiologically active substance of the present invention is a novel substance having at least one amino acid sequence represented by any of the following formulas (1) to (II).
式（I ) Formula (I)
lie Thr Val Val Asp Ala Leu His Glu lie Pro Val Lys Lys Gly  lie Thr Val Val Asp Ala Leu His Glu lie Pro Val Lys Lys Gly
10 15  10 15
Glu Gly Ala Glu Leu  Glu Gly Ala Glu Leu
20  20
式（II) Formula (II)
Gly His Tyr Gly .Val Gin Arg 式 (ΠΙ) Gly His Tyr Gly .Val Gin Arg Expression (ΠΙ)
Thr Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala lie Gin Thr 1 5 10 15 Thr Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala lie Gin Thr 1 5 10 15
Arg Arg
16  16
上記式（I ) ないし（III) いずれかのアミノ酸配列の部分は本発明の蛋白性生 理活性物質の任意の部分でよい。 上記式 ( I ) ないし（II I) のアミノ酸配列部分 は互いに隣接していても、 それらの間に任意のァミノ酸配列が含まれていてもよ い 0  The amino acid sequence portion of any one of the above formulas (I) to (III) may be any portion of the proteinaceous physiologically active substance of the present invention. The amino acid sequence portions of the above formulas (I) to (III) may be adjacent to each other or may contain an arbitrary amino acid sequence between them.
この新規な蛋白性生理活性物質は、 非還元条件下におけるドデシル硫酸ナトリ ゥムーボリアクリルアミ ドゲル電気泳動 (S D S - PAG E) において分子量は 3 0〜3 6 kD aである。  The novel proteinaceous physiologically active substance has a molecular weight of 30 to 36 kDa in non-reducing conditions in sodium dodecyl sulfate sodium acrylamide gel electrophoresis (SDS-PAGE).
また、 本発明の新規な蛋白性生理活性物質は、 血管内皮細胞に作用し、 該細胞 からのプロスタグランジン 1 2 (P G 1 2 ) の産生を刺激する活性を有する。 こ の活性の測定方法は後述する。 The novel proteinaceous bioactive substances of the present invention have activity which act on vascular endothelial cells, stimulate the production of prostaglandin 1 2 (PG 1 2) from the cells. The method for measuring this activity will be described later.
次に本発明の新規な蛋白性生理活性物質の製造方法について説明する。  Next, a method for producing the novel proteinaceous physiologically active substance of the present invention will be described.
本発明の新規な蛋白性生理活性物質は、 いかなる起源のものでも良いが、 ヒト 由来のものが好ましく、 さらに好ましくは、 正常ヒト二倍体線維芽細胞由来のも のであり、 特に、 この正常ヒト二倍体線維芽細胞の培養上清中に産生されるもの が好ましい。 例えば、 適当な生育媒体中で正常ヒト二倍体線維芽細胞等を培 養し、 P G 産生刺激活性を有する培養液を回収して得られる培養上清を精製 することにより、 得ることができる。 正常ヒトニ倍体線維 胞の培養は、 通常用いられる培地を用いることができ るが、 特にダルベッコ改変イーグル培地、 R PM I 1 6 4 0培地、 イーグル最少 必須培地またはハム培地等が好ましい。 また、 血清を添加してもしなくてもよい が、 培養上清より本発明の物質を精製し、 単離することから、 無血清培地を用い た方が望ましい。 培養終了後の培養上清は、 例えばフィルタ一法または限外ろ過 法等の方法により濃縮した方が望ましい。 The novel proteinaceous physiologically active substance of the present invention may be of any origin, but is preferably of human origin, more preferably of normal human diploid fibroblasts. Those produced in the culture supernatant of diploid fibroblasts are preferred. For example, it can be obtained by culturing normal human diploid fibroblasts or the like in an appropriate growth medium, collecting a culture solution having a PG production stimulating activity, and purifying a culture supernatant obtained. For culturing normal human diploid cells, a commonly used medium can be used, but Dulbecco's modified Eagle medium, RPMI164 medium, Eagle's minimum essential medium or ham medium is particularly preferable. Although serum may or may not be added, it is preferable to use a serum-free medium because the substance of the present invention is purified and isolated from the culture supernatant. It is desirable that the culture supernatant after completion of the culture is concentrated by, for example, a filter method or an ultrafiltration method.
上述のようにして得られた本発明の新規な蛋白性生理活性物質を含む原料を下 記工程の少なくとも 1つを用いて精製するが、 下記工程 （a ) 、 （b ) およ び（c ) の各々は、 公知のプロトコールに従って行えばよい。  The raw material containing the novel proteinaceous physiologically active substance of the present invention obtained as described above is purified by using at least one of the following steps. The following steps (a), (b) and (c) ) May be performed according to a known protocol.
(a) イオン交換クロマトグラフィー  (a) Ion exchange chromatography
(b) ァフィ二ティーク口マトグラフィー  (b) Affinity mouth chromatography
( c) ゲルろ過クロマトグラフィー '  (c) Gel filtration chromatography ''
また、 その順序は任意でよく、 必要に応じて繰り返してもよい。 さらに、 —つの工程と他の工程との間または前後に、 濃縮、 透析等の操作を加えても よく、 さらに に応じて、 例えば、 クロマトフォーカシング、 吸着クロマトグ ラフィ一、 驟水性クロマトグラフィー、 逆相クロマトグラフィー、 薄層クロマト グラフィ一、 分配クロマトグラフィー等の他のクロマトグラフィーや、 等電点電 気泳動法、 ポリアクリルアミ ド電気泳動法、 塩折法等の他の蛋白質の精製方法と 組み合わせてもよい。  In addition, the order may be arbitrary, and may be repeated as necessary. In addition, — operations such as concentration, dialysis, etc. may be added between or before and after one step and the other, depending on the further, for example, chromatofocusing, adsorption chromatography, water chromatography, reverse phase Combination with other chromatography methods such as chromatography, thin-layer chromatography, partition chromatography, and other protein purification methods such as isoelectric focusing, polyacrylamide electrophoresis, and salting Is also good.
また、 工程（a：) 〜（c) の後、 単離精製、 純度確認等の目的等のため、 さら に、 収集画分を逆相高速液体クロマトグラフィ一にかけてもよい。  After the steps (a :) to (c), the collected fraction may be further subjected to reversed-phase high-performance liquid chromatography for the purpose of isolation, purification, purity confirmation, and the like.
上記工程（a) 、 (b) および（c ) は各々、 低圧液体クロマトグラフィーで あっても、 高圧液体クロマトグラフィー （高性能液体クロマトグラフィー、The above steps (a), (b) and (c) are each performed by low pressure liquid chromatography. High-performance liquid chromatography (high-performance liquid chromatography,
HPLC)であってもよい。 HPLC).
各クロマトグラフィ一における溶出または展開方法は、 各クロマトグラフィー 担体、 目的物質、 含有される夾雑物質の物性等を考慮して、 適当な溶出条件また は展開条件、 溶出液または展開液を選択すればよい。 溶出に際しては、 直線濃度 勾配溶出であっても、 段階的濃度勾配溶出であってもよい。  For the elution or developing method in each chromatography, appropriate elution conditions or developing conditions, eluate or developing solution may be selected in consideration of the properties of each chromatography carrier, target substance, contained contaminants, and the like. . The elution may be linear gradient elution or stepwise gradient elution.
上記工程 (a)〜（c)を用いて、 好ましくは下記の方法により培養上清から 本発明の新規な蛋白性生理活性物質を単離することができる。  Using the above steps (a) to (c), the novel proteinaceous physiologically active substance of the present invention can be isolated from the culture supernatant preferably by the following method.
正常ヒトニ倍体線維芽細胞の培養上清を陰ィォン交換ク口マトグラフィー、 例 えば DEAE— 5PW (東ソ一社製）、 onoQ (フアルマシア社製） または Q—セファロ一スカラム （フアルマシア社製） 等に吸着させ、 塩化ナトリウムの 濃度勾配法にて溶出する。 溶出した各フラクションの PG 12 産生刺激活性 を測定し、 活性のある画分を得る。 PGI2 産生刺激活性の測定については後 述する。 さらにこの画分をァフィ二ティークロマトグラフィー、 例えばへパリン 一 5 PW (東ソ一社製） 等に吸着させ、 塩化ナトリゥムの 濃度勾配法にて溶 出する。 PGI2 産生刺激活性を同様に測定し、 活性のある画分を集める。 次にこの画分をゲルろ過クロマトグラフィー、 例えばプロテイン一パック （日 本ミリポア リミテッド ウォーターズ クロマトグラフィー事業部） 、 セファ クリル S— 100HR (フアルマシア社製） または TSKゲル G 3000 SWXLカラム （東ソ一社製） 等を用いて展開し、 PG 12 産生刺激活性を測定す ることにより、 生物学的活性画分を得ることができる。 さらにこの画分をァフィ 二ティークロマトグラフィー、 例えばインシユリン様増殖因子 （Insu l i n e— l ike growth f ac tor, I GF) — Iまたは IIをリガンド として結合させた IGF—ァフィ二ティ一カラム、 または、 小麦胚芽凝集素をリ ガンドとして結合させた WGA—ァフィ二ティーカラムにかけることにより、 PG 12産生刺激活性を示す新規な蛋白性生理活性物質を単離精製できる。 各クロマトグラフィーにおける目的とする本発明の蛋白性生理活性物質の画分 は、 溶出した各フラクションの PG 12 産生刺激活性を測定し、 活性のある面分 を集めることにより得ることができる。 Culture supernatant of normal human diploid fibroblasts is subjected to anion exchange chromatography, for example, DEAE-5PW (manufactured by Tosoh I), onoQ (manufactured by Pharmacia) or Q-Sepharose column (manufactured by Pharmacia) And elute by sodium chloride concentration gradient method. The PG 1 2 Stimulating activity of the eluted each fractions were measured to obtain fractions which are active. The measurement of the PGI 2 production stimulating activity will be described later. Further, this fraction is adsorbed to affinity chromatography, for example, heparin-15PW (manufactured by Tosoh Corporation) or the like, and eluted by a sodium chloride concentration gradient method. The PGI 2 production stimulating activity is measured in the same manner, and the active fraction is collected. Next, this fraction is subjected to gel filtration chromatography, for example, Protein One Pack (Millipore Limited Waters Chromatography Division), Sephacryl S-100HR (Pharmacia) or TSK Gel G3000 SW XL column (Tosoichisha). Ltd.) and the like developed using, by Rukoto measuring the PG 1 2 production stimulating activity can be obtained biologically active fraction. This fraction is then subjected to affinity chromatography, for example, insulin-like growth factor (Insulin-like growth factor). e-like growth factor (IGF) — IGF-affinity column with I or II bound as ligand or WGA-affinity column with wheat germ agglutinin bound as ligand by subjecting it is isolated and purified a novel proteinaceous bioactive substances exhibiting PG 1 2 stimulating activity. Fractions of proteinaceous bioactive substances of the invention intended in each chromatography, the PG 1 2 Stimulating activity of the eluted each fractions were measured, it can be obtained by collecting the surface region which is active.
PGI2産生刺激活性の測定は、 以下の方法により実施できる。 すなわちゥシ 胸部大動脈內膜より剝離法にて採取した血管内皮細胞を 10 %ゥシ胎児血清含有 ダルべッコ改変ィ一グル培地で継代培養し、 飽和細胞密度に達するまで培養した 後、 測定試料を添加し 60分間インキュベーション後、 上清中の PGI2 の安定 代謝産物である、 The PGI 2 production stimulating activity can be measured by the following method. In other words, vascular endothelial cells collected from the thoracic aorta membrane by a detachment method were subcultured in Dulbecco's modified medium containing 10% fetal serum, and cultured until they reached a saturated cell density. After adding the measurement sample and incubating for 60 minutes, the stable metabolite of PGI 2 in the supernatant is
Ό —ケ 卜 — r F la (以下、 6 -ケト- PGF 1 αと 記す） を !ί定することにより P G 12 産生量を求めることができる。 この測定に は市販の 6—ケトー PGF 1 α測定用キットを用いてもよい。 O - Ke Bok - r F la (hereinafter, 6 - keto - PGF described as 1 alpha) can be obtained PG 1 2 production amount by the ί a constant!. For this measurement, a commercially available kit for measuring 6-keto PGF1α may be used.
精製された PG 12産生刺激活性を有する本発明の新規な蛋白性生理活性物質 のアミノ酸配列を決定するために、 以下の方法を用いることができる。 例えば、 自動気相シークェンサ一を使用する場合は、 直接本発明の物質を該シークェ ンサ一で分析しても、 または、 トリプシン、 リジルエンドべプチダ一ゼ等の酵素 切断により得られるぺプチド断片を分析してもよく、 それぞれのァミノ酸配列を 決定することができる。 To determine the amino acid sequence of the novel proteinaceous bioactive substances of the present invention with purified PG 1 2 production stimulating activity, it may be used the following method. For example, when an automatic gas-phase sequencer is used, the substance of the present invention can be directly analyzed by the sequencer, or peptide fragments obtained by enzymatic cleavage of trypsin, lysyl endopeptidase, etc. can be analyzed. Alternatively, the sequence of each amino acid can be determined.
本発明の新規な蛋白性生理活性物質は、 PG I2 の有する血小板凝集抑制 作用、 平滑筋弛緩作用、 胃酸分泌抑制作用等を有するので、 以下の各種疾患に対 して、 本発明の蛋白性生理活性物質を少なくとも 1つの有効成分として含有する 医薬組成物を提供する。 The novel proteinaceous physiologically active substance of the present invention is characterized by inhibiting platelet aggregation of PG I 2 The present invention provides a pharmaceutical composition containing the proteinaceous physiologically active substance of the present invention as at least one active ingredient for the following various diseases because it has an action, a smooth muscle relaxing action, a gastric acid secretion inhibitory action and the like.
溶血性尿毒症症候群、 血栓性血小板減少性紫斑病、 末梢動脈閉塞、 心虚血、 脳 虚血、 動脈硬化、 脳閉塞、 高脂血症、 糖尿病、 心不全、 狭心症、 虚血性心疾患、 うつ血性心疾患、 脈絡膜循環障害、 気管支疾患、 胃潰瘍、 妊娠子癇。  Hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, peripheral arterial occlusion, cardiac ischemia, cerebral ischemia, arteriosclerosis, cerebral obstruction, hyperlipidemia, diabetes, heart failure, angina, ischemic heart disease, depression Hematologic heart disease, choroidal circulation disorder, bronchial disease, gastric ulcer, pregnancy eclampsia.
本発明の新規な蛋白性生理活性物質の投与量は、 患者の性別、 年齢、 体重、 疾 患の種類やその病態あるレ、は投与剤型により適宜変動するが、 有効投与量は ,1 n g〜2 mg/kg、 好ましくは 1 0 n g〜2 0 0；/ g/kgである。  The dosage of the novel proteinaceous physiologically active substance of the present invention may vary depending on the patient's sex, age, body weight, disease type and its pathological condition, depending on the dosage form, but the effective dosage is 1 ng. 22 mg / kg, preferably 10 ng to 200; / g / kg.
本発明の医薬組成物の医薬形態は、 その疾患病巣に有効量を供給できるもので あればいかなるものでもよく、 例えば、 錠剤、 粉末剤、 散剤、 カプセル剤、 軟膏 剤、 粉霧剤あるいは注射剤等が例示される。 また、 本発明の医薬組成物は、 その 薬理学的特性を損なわない限り、 一般的に使用される製剤学的混合物である賦型 剤、 安定化剤^)るいは溶解補助剤等を含有していてもよい。 具体例として、 リンガ一液、 燐酸緩衝液、 ヒト血清アルブミン、 水解ゼラチン、 蔗糖、 デキスト ラン、 ポリエチレングリコール等があり、 薬剤形態により、 適宜選択使用さ れる。 実施例  The pharmaceutical form of the pharmaceutical composition of the present invention may be any as long as it can supply an effective amount to the disease lesion, and examples thereof include tablets, powders, powders, capsules, ointments, powders, and injections. Etc. are exemplified. In addition, the pharmaceutical composition of the present invention contains a commonly used pharmaceutical mixture such as an excipient, a stabilizer ^) or a solubilizing agent, as long as the pharmacological properties are not impaired. May be. Specific examples include Ringer's solution, phosphate buffer, human serum albumin, hydrolyzed gelatin, sucrose, dextran, polyethylene glycol, etc., which are appropriately selected and used depending on the drug form. Example
以下に本発明を実施例をもってより具体的に示すが、 これは本発明の実施態様 の一つの例示であり、 本発明はこれに限定されるものではない。 なお、 実施例中 の学術用語、 H各号等は特に断らなレ、限り当該技術分野で一般的に使用されている ものに従った。 また、 蛋白質精製に関わる基本的操作は、 日本生化学会編Hereinafter, the present invention will be described more specifically with reference to examples. However, this is one example of an embodiment of the present invention, and the present invention is not limited thereto. In addition, the technical terms, H items, and the like in the examples are not particularly limited, and are generally used in the technical field. Followed the ones. Also, basic operations related to protein purification are described in
" 新生化学実験講座第 1巻 蛋白質 I 分離，精製，性質" 東京化学同人 1990年を参考として実施した。 各種機器、 試薬の使用法は各々附属の使用 説明書に従った。 "Shinsei Kagaku Kenkyusho Vol.1 Protein I Isolation, Purification, and Properties" The usage of various instruments and reagents was in accordance with the attached instruction manual.
(実施例 1 )  (Example 1)
下記の方法で、 培養上清を作製し、 精製し、 本発明の新規な蛋白性生理活性物 質を得た。  A culture supernatant was prepared and purified by the following method to obtain a novel proteinaceous physiologically active substance of the present invention.
なお、 PG 12 産生刺激活性の測定は実施例 3に示す後述の方法で行った。 ( 1 ) 正常ヒトニ倍体線維芽細胞の培養上清の作製 The measurement of the PG 1 2 production stimulating activity was carried out by the method described below in Examples 3. (1) Preparation of culture supernatant of normal human diploid fibroblasts
正常ヒトニ倍体線維芽細胞を 15 %ゥシ胎児血清含有ダルべッコ改変イーグル 培地にて 4. 8X 104 細胞 ZmLに調製した。 この細胞懸濁液 3 Lを回転培 養容器に植え込み、 5%炭酸ガス- 95%空気下、 37。Cにて培養した。 細胞植 え込み 3日後に培養液を新鮮な 15 %ゥシ胎児血清含有ダルべッコ改変ィー グル培地 3 Lに交換し、 さらに培養を 2日間継続した。 次いで、 培養液を除 去し、 Ca2+、 Mg2+不含ダルベッコ一リン酸生理食塩溶液を用いて細胞を 洗浄した後、 フヱノールレツド不含のダルベッコ改変イーグル培地 3 Lを添 加し、 37て、 2日間培養した。 培養液を回収後、 2. 5/zmのフィルター (CNカートリッジ 30インチ、 ミリポア社製） にてろ過し細胞片を除去 した。 次いでホロ一ファイバーモジュール（モルセップファイバー FS— 10 6 kDa カツトオフ、 ダイセル化学工業社製） 、 分画分子量 10 kDaの限タ ろ過カセット （オメガミニセット、 富士フィルター工業社製） による 2段階の濃 縮操作にて濃縮した。 この濃縮液を分画分子量 3. 5 kDaの透析チューブ（ス ぺクトラム社製） を用いて、 2 OmMトリスー塩酸緩衝液 （pH 7. 8) で 4 、 1晚透析した。 同緩衝液でさらに 4 eC、 8時間透析後、 1. 2〃 mフィル 夕一（ディエフエイ アセンブリ、 日本ポール社製） 、 0. 22 zmフィルタ一 システム （コ一二ング社製） で順次ろ過した。 Normal human diploid fibroblasts were prepared in 4.8 × 10 4 cells ZmL in Dulbecco's modified Eagle's medium containing 15% fetal bovine serum. 3 L of this cell suspension was inoculated in a rotary culture vessel, and 5% CO 2 -95% air 37. C. Three days after the cell implantation, the culture solution was replaced with 3 L of fresh Dulbecco's modified Eagle's medium containing 15% fetal calf serum, and the culture was continued for 2 days. Next, the culture solution was removed, and the cells were washed with a Ca2 + -and Mg2 + -free Dulbecco's monophosphate physiological saline solution, and 3 L of Dulbecco's modified Eagle's medium without phenol red was added. And cultured for 2 days. After recovering the culture, the cells were filtered through a 2.5 / zm filter (CN cartridge, 30 inch, manufactured by Millipore) to remove cell debris. Next, a two-stage concentration using a hollow fiber module (Molsep fiber FS-106 kDa cut-off, manufactured by Daicel Chemical Industries, Ltd.) and a filtration filter (Omega mini-set, manufactured by Fuji Filter Industries, Ltd.) with a molecular weight cutoff of 10 kDa. It concentrated by operation. This concentrated solution is used in a dialysis tube (stained with a molecular weight cutoff of 3.5 kDa The solution was dialyzed against 2 OmM Tris-HCl buffer (pH 7.8) for 4 to 1 minute using the following method. After further dialysis with the same buffer at 4 e C for 8 hours, the mixture was sequentially filtered through 1.2〃m filter Yuichi (DF Assembly, Nippon Pall) and 0.22 zm filter-one system (Koining). did.
(2) P G 12 産生刺激活性因子の精製 (2) PG 1 2 production purification stimulating activity factor
工程 1  Process 1
( 1 ) に従って作製した正常ヒトニ倍体線維芽細胞の培養上清を予め 2 OmM トリス一塩酸緩衝液 （PH7. 8) で平衡化した DEAE— 5 P.W (東ソ一 社製） 陰イオン交換クロマトカラムに吸着させ、 2 OmMトリス一塩酸緩衝 液（pH7. 8)を用いて作製した 0〜1. 0Mの塩化ナトリウムの直線濃度勾 配法にて溶出させ、 280 nmにおいて吸光度を同時に測定した。 溶出した各フ ラクシヨンの P G 12 産生刺激活性を上述の方法に従って測定し、 50〜 150 mMの塩化ナトリウ厶濃度で溶出された PG I 2 産生刺激活性を有する画分 をプールした （図 1)。 Culture supernatant of normal human diploid fibroblasts prepared according to (1) was pre-equilibrated with 2 OmM Tris-monohydrochloride buffer (PH7.8). DEAE-5 PW (Tosoichi) Anion exchange chromatography The mixture was adsorbed on a column, eluted with a linear gradient method of 0 to 1.0 M sodium chloride prepared using a 2 OmM Tris-monohydrochloride buffer (pH 7.8), and the absorbance was simultaneously measured at 280 nm. The eluted PG 1 2 Stimulating activity of each off Rakushiyon measured according to the method described above, the fractions with the eluted PG I 2 production stimulating activity at sodium chloride厶濃degree of 50 to 0.99 mM were pooled (Figure 1) .
工程 2  Process 2
工程 1で得られた活性画分を 10 mMリン酸緩衝液 (pH7. 4)で透析後、 1 OmMリン酸緩衝液 (pH7. 4)で平衡化したへパリン （HEPAR IN) -5PW (東ソ一社製） ァフィ二ティーカラムに吸着させ、 1 OmMリン酸緩衝 液（pH7. 4)を用いて作製した 0〜し 0Mの塩化ナトリウムの直線濃度勾 配法にて溶出させ、 28 O.nmにおいて吸光度を同時に測定した。 溶出した各フ ラクシヨンの PGI2 産生刺激活性を測定し、 450〜 500 mMの塩化ナトリ ゥム濃度で溶出された PG I 2 産生刺激活性を有する画分をプールした （図 2) o The active fraction obtained in step 1 was dialyzed against 10 mM phosphate buffer (pH 7.4) and equilibrated with 1 OmM phosphate buffer (pH 7.4). Heparin (HEPAR IN) -5PW (E. Adsorbed on an affinity column and eluted with a linear gradient of 0 to 0 M sodium chloride prepared using 1 OmM phosphate buffer (pH 7.4). The absorbance was simultaneously measured at nm. The eluted PGI 2 production stimulation activity of each off Rakushiyon measures, the fractions with the eluted PG I 2 production stimulating activity at sodium © beam chloride concentration of 450 to 500 mM were pooled (FIG. 2) o
工程 3  Process 3
工程 2で得られた活性画分をセントリコンー 10 (アミコン社製） にて濃 縮後、 予め 10 mMリン酸緩衝液 (pH7. 4)で平衡化したプロティンーパッ ク （PROTE IN— PAK) 300 (日本ミリポア リミテッド ウォーター ズクロマトグラフィ一事業部） のゲルろ過カラムを 2本連結したものを用いて展 開し、 280 nmにおいて吸光度を同時に測定した。 各フラクションの PG 12 産生刺激活性を測定したところ、 活性は分子量 3 OkD a付近に認められた （図The active fraction obtained in step 2 was concentrated with Centricon-10 (manufactured by Amicon), and then pre-equilibrated with 10 mM phosphate buffer (pH 7.4). It was developed using two connected gel filtration columns from Millipore Limited Waters Chromatography Division, and absorbance was simultaneously measured at 280 nm. Was measured PG 1 2 Stimulating activity of each fraction, activity was observed around a molecular weight 3 OKD a (FIG.
3) o 図 3中の数字は分子量（kDa)、 Vtは充填剤の総体積、 Voは排除体 積を表す。 3) o The numbers in Fig. 3 indicate molecular weight (kDa), Vt indicates the total volume of the filler, and Vo indicates the excluded volume.
工程 4  Process 4
工程 3で得られた活性画分を 1 OmMリン酸緩衝液 (pH7. 4)で平衡化し た I GF—ァフィ二ティーカラムにかけた。 I GF—ァフィ二ティーカラムは、 ァフイブレップ 10 (日本バイオラッドラボラトリース社製） に組換 IGF— I をリガンドとして結合させた耐圧力ラムを作製し、 本実験に用いた。 サンプルを 吸着後、 0. 5 M酢酸で溶出した。 PG 12 産生刺激活性を測定たところ、 PG 12姓刺激活性は、 非吸着面分に認められた。 The active fraction obtained in step 3 was applied to an IGF-affinity column equilibrated with 1 OmM phosphate buffer (pH 7.4). For the IGF-affinity column, a pressure-resistant ram in which recombinant IGF-I was bound as a ligand to AFFEPREP 10 (manufactured by Nippon Bio-Rad Laboratories) was used for this experiment. After adsorption, the sample was eluted with 0.5 M acetic acid. Where was measured PG 1 2 Stimulating activity, PG 1 2 surname stimulating activity was found in non-adsorbed surface min.
工程 5 '  Step 5 '
工程 4で得られた活性画分を 0 · 1 %トリフルォロ酢酸含有 10 %ァセトニト リノレ水溶液で平衡化した C 4 逆相 HP LCカラム （ウォーターズ社製） にァプラ ィした後、 溶出液 0. 1%トリフルォロ酢酸/ァセトニトリル溶液を用いて作製 した 10〜60%ァセトニトリルの直線濃度勾配法にて溶出した。 その結果、 本 発明の新規な蛋白性生理活性物質は単一なピークとして溶出された。 ， (実施例 2 ) After Apura I to the active fraction obtained in step 4 was equilibrated with 0 - 1% Torifuruoro acetate containing 10% Asetonito Rinore solution C 4 reverse-phase HP LC column (Waters), eluent 0.1% Elution was performed by a linear gradient method of 10-60% acetonitrile prepared using a trifluoroacetic acid / acetonitrile solution. As a result, the book The novel proteinaceous bioactive substance of the invention eluted as a single peak. , (Example 2)
正常ヒトニ倍体線維芽細胞由来の PG 12 産生刺激因子の物性の測定  Measurement of physical properties of PG12 production stimulating factor derived from normal human diploid fibroblasts
( 1 ) 分子量の測定  (1) Measurement of molecular weight
実施例 1の工程 5で得られた本発明の新規な蛋白性生理活性物質の分子量を、 非還元条件下におけるドデシル硫酸ナトリウム一ポリアクリルアミ ドゲル電気泳 動 （SDS— PAGE)で求めたところ、 分子量 33 kD aに単一のバンドとし て認められた （図 4)。 図 4は SDS— PAGEの泳動後の参考写真の模式図で ある。 図 4中、 ライン Aおよび Cは既知分子量の標準試料のバンドであり、 ライ ン Bは本発明の新規な蛋白性生理活性物質のバンドである。 また、 数字は標準試 料の分子量 (kDa)を表す。  The molecular weight of the novel proteinaceous physiologically active substance of the present invention obtained in step 5 of Example 1 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions. A single band was observed at a molecular weight of 33 kDa (Fig. 4). Figure 4 is a schematic diagram of a reference photograph after SDS-PAGE electrophoresis. In FIG. 4, lines A and C are bands of a standard sample having a known molecular weight, and line B is a band of the novel proteinaceous bioactive substance of the present invention. The numbers represent the molecular weight (kDa) of the standard sample.
(2) アミノ酸配列の解析  (2) amino acid sequence analysis
実施例 1の工程 5で得られた本発明の新規な蛋白性生理活性物質を、 トリプシ ンで消化しペプチドフラグメント化した。 このサンプルを 0. 1%トリフルォロ 酢酸水溶液で平衡化した C8 逆相 HPLCカラム （セパレーシヨンズグループ社 製） にかけた後、 0. 08 %トリフルォロ酢酸 Zァセトニトリル溶液を用いて作 製した 0〜80%ァセトニトリルの直線濃度勾配法にて溶出させ、 得られたフラ グメントペプチドについて自動気相シークェンサ一（アプライドバイォシステム ズ 477A- 12 OA)を用い、 エドマン分解により内部のアミノ酸配列、 下記 式（I)〜(ΙΠ) を決定した。 式（I ) The novel proteinaceous physiologically active substance of the present invention obtained in step 5 of Example 1 was digested with trypsin to form a peptide fragment. After applying the sample to 0. C 8 reversed phase HPLC column equilibrated with 1% Torifuruoro aqueous acetic acid (Se Palais Shiyonzu Group Inc.), 0.08% Torifuruoro acetic Z Asetonitoriru solution 0-80% was made created using Acetonitrile was eluted by the linear gradient method, and the obtained fragment peptide was subjected to Edman degradation using an automated gas-phase sequencer (Applied Biosystems 477A-12OA) to decompose the internal amino acid sequence into the following formula (I) ~ (ΙΠ) was determined. Formula (I)
He Thr Val Val Asp Ala Leu His GIu lie Pro Val Lys Lys Gly 1 5 10 15 He Thr Val Val Asp Ala Leu His GIu lie Pro Val Lys Lys Gly 1 5 10 15
Glu Gly Ala Glu Leu Glu Gly Ala Glu Leu
20  20
式（II) Formula (II)
Gly His Tyr Gly Val Gin Arg 式 (III)  Gly His Tyr Gly Val Gin Arg Formula (III)
Thr Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala He Gin Thr 1 5 10 15 Thr Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala He Gin Thr 1 5 10 15
' Arg ' 'Arg'
16  16
(実施例 3 )  (Example 3)
P G 1 2 刺激活性の測定 Measurements of PG 1 2 stimulating activity
血管内皮細胞はゥシ胸部大動脈内膜より剝離法にて採取した。 次いで得られた 血管内皮細胞を 1 0 %ゥシ胎児血清を含む 1 0 O U/m Lペニシリンおよび 1 0 0 gZmLストレプトマイシン含有ダルベッコ改変イーグル培地中、 5 % 炭酸ガス一 9 5 % 下、 3 7 °Cにて継代培養した。 培地は週 2回交換し、 5〜 1 0代継代後、 血管内皮細胞を 0. 0 5 %トリプシン処理した。  Vascular endothelial cells were collected from the thoracic aorta intima by an isolation method. The obtained vascular endothelial cells were then placed in a Dulbecco's modified Eagle's medium containing 10% OU / mL penicillin and 100 gZmL streptomycin containing 10% fetal calf serum, at 37 ° C under 5% CO 2 -95%. The cells were subcultured in C. The medium was changed twice a week, and after passage 5-10, vascular endothelial cells were treated with 0.05% trypsin.
次いで、 1 0 %ゥシ胎児血清含有ダルベッコ改変イーグル培地 l mLの入った 2 4穴培養皿に血管内皮細胞を移し、 5 X 1 0 4 細胞 Z穴まで培養した。 1 0 % 以下の各種試料含有ダルベッコ改変イーグル培地 500 を添加後、 37°Cで 60分間インキュベーションした。 Next, the vascular endothelial cells were transferred to a 24-well culture dish containing 1 mL of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, and cultured to 5 × 10 4 cells Z well. Ten % After adding Dulbecco's modified Eagle medium 500 containing the following various samples, the mixture was incubated at 37 ° C for 60 minutes.
培養上清からの 6—ケトー PGF 1 αの抽出と精製はジヤッフェらの方法 [ザ ジャーナル ォブ クリニカル インべスティゲーシヨン （J. Cl in. I nve s t. ) 52巻、 398頁、 1973年] を改良して行った。 培養上清 lmLに 0. 1N塩酸 1. 5mLを加え、 5 mL酢酸ェチルで 2度抽出した。 窒 素ガス下で酢酸ェチルを蒸発させ、 エタノ一ルに再溶解させた。 アツセィに使用 するまで一 20でで保存した。 4 (TCでエタノールを蒸発させ 0. 1Mリン酸緩 衝液（pH7. 2、 1M塩化ナトリウム、 1 %ゼラチン含有） に溶解させた。  Extraction and purification of 6-keto PGF 1α from the culture supernatant was performed according to the method of Jaffe et al. [J. Clin. Investigation. 52, 398, 1973 Year]. 1.5 mL of 0.1N hydrochloric acid was added to 1 mL of the culture supernatant, and extracted twice with 5 mL of ethyl acetate. The ethyl acetate was evaporated under nitrogen gas and redissolved in ethanol. Stored at 1-20 for use in Atsushi. 4 (The ethanol was evaporated with TC and dissolved in 0.1 M phosphate buffer (pH 7.2, 1 M sodium chloride, containing 1% gelatin).
6- [ 3H]ケト PGF 1 αと抗 6—ケトー PGF 1 α抗体を用いたニューィ ングランドヌクレア一 （N ew Eng l and Nu c l e a r)社製の 6—ケトー PGF 1 α測定ラジオィムノアッセィキットを用い、 添付マニュ アルに従い 6—ケトー PGF 1 αの濃度を測定した。 PG I2 產生量は、 1 時間、 104 細胞当たりの &ーケトー PGF 1 α産生量（pgZl 04 細胞 Z時 間） によって、 求めた。 6- [3 H] keto PGF 1 alpha and anti-6-Keto PGF 1 alpha Nyui down ground Nuclear one of antibodies using (N ew Eng l and Nu clear ) manufactured 6 Keto PGF 1 alpha measurements Rajioimuno Using an assay kit, the concentration of 6-keto PGF1α was measured according to the attached manual. PG I 2產生amount is 1 hour, the & Keto PGF 1 alpha production per 104 cells (pgZl 0 4 during the time of cell Z), was determined.
本発明の蛋白性生理活性物質の示す PG 12 産生刺激活性は濃度依存的に増加 した。  The PG12 production stimulating activity of the proteinaceous physiologically active substance of the present invention increased in a concentration-dependent manner.
(実施例 4 ) 実施例 1で得られた本発明の蛋白性生理活性物質を用いてマウスにおける毒性 試験を実施した。 すなわち、 6週齢の I CRマウス雌雄各々 5匹に対して実施例 1で得られた蛋白性生理活性物質を 1日 1回 1週間連日静脈内投与した。 投与期 間中を通して各動物個体の一般性状を観察した。 本試験では最高投与量群である(Example 4) A toxicity test in mice was carried out using the proteinaceous physiologically active substance of the present invention obtained in Example 1. That is, the proteinaceous physiologically active substance obtained in Example 1 was intravenously administered once daily for one week to five male and female 6-week-old ICR mice once a day. Administration period The general characteristics of each animal were observed throughout. The highest dose group in this study
1 Omg/kg群でもいずれの動物においても死亡例は観察されず、 一般性状におい ても変化はみられなかった。 No deaths were observed in any of the animals in the 1 Omg / kg group, and there was no change in general characteristics.
(実施例 5 )  (Example 5)
注射用溶液剤の調製 Preparation of solution for injection
実施例 1で得られた本発明の蛋白性生理活性物質 10 Omgを 1 Omg/mLの水 解ゼラチンを含有する生理食塩液 1 0 OmLに溶解し、 ポアサイズ 0. 22 m のフィルター （マイレックス GV ミリポア社製） を用いてろ過減菌した。 これ を無菌的に 2 m Lずつガラスパイアルに分注後密栓し、 注射用溶翻とした。 (実施例 6 )  10 Omg of the proteinaceous physiologically active substance of the present invention obtained in Example 1 was dissolved in 10 OmL of a physiological saline solution containing 1 Omg / mL of hydrolyzed gelatin, and a filter having a pore size of 0.22 m (Mirex GV Filtration and sterilization were carried out using Millipore Corporation. This was aseptically dispensed in 2 mL aliquots into glass vials, sealed and sealed for injection lysate. (Example 6)
注射用凍結乾燥剤の調製  Preparation of lyophilized preparation for injection
実施例 1で得られた本発明の蛋白性生理活性物質 1 0 Omgを 10 Oiiig/mLの ヒト血清アルブミンを含有する 1 OmM PBS (pH7. 4) 10 OmLに溶 解し、 ポアサイズ 0. 22 mのフィルター （マイレックス GV ミリポア 社製） を用いてろ過滅菌した。 これを無菌的に 3 mLずつガラスバイアルに 分注、 凍結乾燥後密栓し、 注射用凍結乾燥剤とした。 産 S±の利用可能性  10 Omg of the proteinaceous physiologically active substance of the present invention obtained in Example 1 was dissolved in 10 OmL of 1 OmM PBS (pH 7.4) containing 10 Oiiig / mL of human serum albumin, and the pore size was 0.22 m. And sterilized by filtration using a filter (Mirex GV Millipore). This was aseptically dispensed into glass vials in 3 mL portions, freeze-dried and sealed to give a freeze-dried preparation for injection. Availability of S ±
本発明の新規な蛋白性生理活性物質は血管内皮細胞に作用し、 該細胞から の PG 12 の趣を刺激する作用を有する。 従って PG 12 の有する血小板凝集 抑制作用、 平滑筋弛緩作用、 胃酸分泌抑制作用等に基づき溶血性尿毒症症候群、 血栓性血小板減少性紫斑病、 末梢動脈閉塞、 心虚血、 脳虚血、 動脈硬化、 脳 閉塞、 高脂血症、 糖尿病、 心不全、 狭心症、 虚血性心疾患、 うつ血性心疾患、 脈 絡膜循環障害、 気管支疾患、 胃潰瘍、 妊娠子癇等の治療用医薬用途としての応用 が期待できる。 The novel proteinaceous bioactive substances of the present invention acts on vascular endothelial cells has the effect of stimulating the PG 1 2 of flavor from the cells. Therefore, based on the inhibitory action of PG12 on platelet aggregation, smooth muscle relaxation, gastric acid secretion, etc., hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, peripheral arterial occlusion, cardiac ischemia, cerebral ischemia, arteriosclerosis, brain It can be expected to be used as a therapeutic drug for obstruction, hyperlipidemia, diabetes, heart failure, angina pectoris, ischemic heart disease, depressive heart disease, choroidal circulatory disorder, bronchial disease, gastric ulcer, pregnancy eclampsia, etc. .
配列表 Sequence listing
配列番号 ： 1  SEQ ID NO: 1
配列の長さ： 2 0  Array length: 2 0
配列の型 ：アミノ酸  Sequence type: Amino acid
トポロジー：直鎖状  Topology: linear
配列の種類：ぺプチド  Sequence type: peptide
配列  Array
lie Thr Val Val Asp Ala Leu His Glu [le Pro Val Lys Lys Gly 1 5 10 15 Glu Gly Ala Glu Leu  lie Thr Val Val Asp Ala Leu His Glu (le Pro Val Lys Lys Gly 1 5 10 15 Glu Gly Ala Glu Leu
20 配列番号 ： 2  20 SEQ ID NO: 2
配列の長さ： 7  Array Length: 7
配列の型 ：アミノ酸  Sequence type: Amino acid
トポロジー：直鎖状  Topology: linear
配列の種類：ぺプチド  Sequence type: peptide
配列  Array
Gly His Tyr Gly Val Gin Arg 配列番号 ： 3 Gly His Tyr Gly Val Gin Arg SEQ ID NO: 3
配列の長さ： 1 6 Array Length: 1 6
配列の型 ：アミノ酸 Sequence type: Amino acid
トポロジー：直鎖状  Topology: linear
配列の種類：ぺプチド Sequence type: peptide
配列 Array
Thr Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala l ie Gin Thr 1 5 10 15 Thr Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala lie Gin Thr 1 5 10 15
Arg Arg

Claims

請求の範囲 The scope of the claims
1. 下記の特徴を有する蛋白性生理活性の物質、  1. a proteinaceous bioactive substance having the following characteristics:
(1)下記式（I)ないし σπ) レ、ずれかで表されるアミノ酸配列を、 少なくと も 1つ有する、  (1) having at least one amino acid sequence represented by the following formula (I) or σπ)
式（I) Formula (I)
lie Thr Val Val Asp Ala Leu His Glu lie Pro Val Lys Lys Gly 1 5 10 15 lie Thr Val Val Asp Ala Leu His Glu lie Pro Val Lys Lys Gly 1 5 10 15
Glu Gly Ala Glu Leu Glu Gly Ala Glu Leu
20  20
式（II) Formula (II)
Gly His Tyr Gly Val Gin Arg  Gly His Tyr Gly Val Gin Arg
1 5  1 5
式 (I I I) Equation (I I I)
Thr Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala lie Gin Thr 1 5 10 15 Thr Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala lie Gin Thr 1 5 10 15
Arg Arg
16  16
(2)非還元条件下におけるドデシル硫酸ナトリウム一ポリアクリルァミドゲル 電気泳動 (SDS-PAGE)において分子量が 30〜36kDaである、 (3)血管内皮細胞に作用し、 該細胞からのプロスタグラ ンジン I 2 (PG 12 ) の を刺激する活性を有する。. (2) sodium dodecyl sulfate-polyacrylamide gel under non-reducing conditions has a molecular weight of 30 to 36 kDa in electrophoresis (SDS-PAGE). (3) acting on vascular endothelial cells, prostaglandin I from the cells It has an activity to stimulate 2 (PG 1 2) of. .
2. 請求項 1に記載の蛋白性生理活性物質を含む原料を、 下記（a)〜（c)の 工程の少なくとも 1つを用いて精製することを特徴とする製造方法。 2. A raw material containing the proteinaceous physiologically active substance according to claim 1 is prepared using the following (a) to (c): Purification using at least one of the steps.
( a ) イオン交換クロマトグラフィー  (a) Ion exchange chromatography
(b ) ァフィ二ティ一クロマトグラフィー  (b) affinity chromatography
( c ) ゲルろ過クロマトグラフィー  (c) Gel filtration chromatography
3. 請求項 1に記載の蛋白性生理活性物質を有効成分として含有することを特徵 とする、 下記疾患のいずれか 1つの疾患に対する治療用医薬組成物、 3. A pharmaceutical composition for treating any one of the following diseases, which comprises the proteinaceous physiologically active substance according to claim 1 as an active ingredient:
溶血性尿毒症症候群、 血栓性血小板減少性紫斑病、 末梢動脈閉塞、 心虚血、 脳 虚血、 動脈硬化、 脳閉塞、 高脂血症、 糖尿病、 心不全、 狭心症、 虚血性心疾患、 うつ血性心疾患、 脈絡膜循環障害、 気管支疾患、 胃潰瘍、 妊娠子癇。  Hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, peripheral arterial occlusion, cardiac ischemia, cerebral ischemia, arteriosclerosis, cerebral obstruction, hyperlipidemia, diabetes, heart failure, angina, ischemic heart disease, depression Hematologic heart disease, choroidal circulation disorder, bronchial disease, gastric ulcer, pregnancy eclampsia.
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