JP2861655B2 - Liquid preparation containing human urinary trypsin inhibitor and method for producing the same - Google Patents
Liquid preparation containing human urinary trypsin inhibitor and method for producing the sameInfo
- Publication number
- JP2861655B2 JP2861655B2 JP4203211A JP20321192A JP2861655B2 JP 2861655 B2 JP2861655 B2 JP 2861655B2 JP 4203211 A JP4203211 A JP 4203211A JP 20321192 A JP20321192 A JP 20321192A JP 2861655 B2 JP2861655 B2 JP 2861655B2
- Authority
- JP
- Japan
- Prior art keywords
- liquid preparation
- trypsin inhibitor
- human urinary
- urinary trypsin
- producing
- 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 - Fee Related
Links
Landscapes
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
【０００１】[0001]
【産業上の利用分野】本発明はヒト尿性トリプシンイン
ヒビターの液状製剤及びその製造方法に関する。The present invention relates to a liquid preparation of a human urinary trypsin inhibitor and a method for producing the same.
【０００２】[0002]
【従来の技術】ヒト尿性トリプシンインヒビター（ヒト
尿由来トリプシンインヒビター、以下ＨＵＴＩ）は、Ｐ
ｒｏｋｓｃｈらによりヒト尿より初めてｉｎｔａｃｔな
形で分離・精製された（Ｊ．Ｌａｂ．Ｃｌｉｎ．Ｍｅ
ｄ．，７９，４９１，（１９７２））。同分子はシアル
酸及び中性ヘキソースを数十％含む、分子量約６７ｋｄ
（ゲル濾過分析）、等電点２〜３の糖タンパク質である
（Ｊ．Ｌａｂ．Ｃｌｉｎ．Ｍｅｄ．，７９，４９１，
（１９７２）、日泌尿会誌、７４，１６２７，（１９８
３）、Ｐｒｏｔｅｉｎａｓｅ Ｉｎｈｉｂｉｔｏｒｓ，
１２，３８９，（１９８６）、Ｂｉｏｃｈｉｍ．Ｂｉｏ
ｐｈｙｓ．Ｒｅｓ．Ｃｏｍｍｕｎ．，１０９，１２４
７，（１９８２））。2. Description of the Related Art Human urinary trypsin inhibitor (human urine-derived trypsin inhibitor, hereinafter referred to as HITI) is a P urinary trypsin inhibitor.
were first isolated and purified from human urine in an intact form by Roksch et al. (J. Lab. Clin. Me.)
d. , 79, 491, (1972)). The molecule contains sialic acid and neutral hexose at tens of percent and has a molecular weight of about 67 kd.
(Gel filtration analysis), which is a glycoprotein having an isoelectric point of 2-3 (J. Lab. Clin. Med., 79, 491,
(1972), Journal of the Japan Urinary Association, 74, 1627, (198
3), Proteinase Inhibitors,
12, 389, (1986), Biochim. Bio
phys. Res. Commun. , 109,124
7, (1982)).
【０００３】このＨＵＴＩは、膵酵素のトリプシン及び
キモトリプシンを特に強く阻害するが、ＡＴ−III やＦ
ＯＹと異なり凝固・線溶系の酵素を全く或は弱くしか阻
害しない（日薬理会誌、８１，２３５，（１９８
３））。また、ライソゾーム膜の安定化効果を持つこと
からライソゾーム酵素の産生を抑制したり、心筋抑制因
子の産生抑制作用を有すると言われている（麻酔、３
３，１３７，（１９８４））。[0003] This HITI particularly strongly inhibits the pancreatic enzymes trypsin and chymotrypsin.
Unlike OY, it inhibits coagulation / fibrinolytic enzymes at all or only weakly (Jpn Pharmaceutical Association, 81, 235, (198)
3)). In addition, it has a stabilizing effect on the lysosomal membrane, and is therefore said to suppress the production of lysosomal enzymes and to inhibit the production of myocardial depressants.
3, 137, (1984)).
【０００４】以上の活性・作用を有することから、急性
膵炎や急性循環不全への有用性が期待され、持田製薬
（株）により初めて製剤化され、１９８５年に商品名ミ
ラクリッドとして発売されるに到った（持田製薬
（株）、ミラクリッド添付文書（１９８５）） 本製剤は、臨床的に急性膵炎及び手術時やエンドトキシ
ンショック時に対処する重篤な副作用の見られない製剤
として使われており、近年の売上は１００億円に達して
いる。[0004] Having the above activities and effects, it is expected to be useful for acute pancreatitis and acute circulatory insufficiency. It was first formulated by Mochida Pharmaceutical Co., Ltd. and launched in 1985 under the trade name Miracrid. (Mochida Pharmaceutical Co., Ltd., Miracled package insert (1985)) This formulation has been clinically used as a formulation without acute side effects to cope with acute pancreatitis and surgery or endotoxin shock. Has reached 10 billion yen.
【０００５】ところで、ＨＵＴＩを製剤化するに当た
り、液状製剤とすることが可能である。この際、添加剤
として、塩化ナトリウム、リン酸塩、リン脂質、マンニ
トール、アルブミン、ゼラチン等を用いることが従来よ
り知られている。（特開昭５５−１６０７２４号、同５
８−２２５０２６号、同６３−２６７７３０号）。[0005] By the way, when huti is formulated, it can be made into a liquid formulation. At this time, it has been conventionally known to use sodium chloride, phosphate, phospholipid, mannitol, albumin, gelatin and the like as additives. (JP-A-55-160724, 5
Nos. 8-225026 and 63-267730).
【０００６】[0006]
【発明が解決しようとする課題】本発明の目的は、保存
時の安定性に優れたトリプシンインヒビター含有液状製
剤を提供することにある。SUMMARY OF THE INVENTION An object of the present invention is to provide a liquid preparation containing a trypsin inhibitor which has excellent storage stability.
【０００７】[0007]
【問題を解決するための手段】本発明者らはＨＵＴＩの
液状製剤について鋭意研究を行い、従来より公知の製剤
に比べて保存時の安定性に優れた製剤を調製できること
を見出して本発明を完成した。即ち、本発明は、イオン
強度が０．１Ｍ以下であり、ｐＨが７以下であることを
特徴とするヒト尿性トリプシンインヒビター含有液状製
剤である。また、本発明は、ヒト尿性トリプシンインヒ
ビター含有溶液をｐＨ５〜７で６０〜１００℃に加熱後
に、その温度を維持しつつ、ｐＨを２〜５に下げて１〜
３０分間加熱処理して得られるヒト尿性トリプシンイン
ヒビター含有液状製剤である。Means for Solving the Problems The present inventors have conducted intensive studies on a liquid formulation of HITI and found that a formulation having excellent storage stability can be prepared as compared with a conventionally known formulation, and found the present invention. completed. That is, the present invention is, ion
Strength is at 0.1M or less, a human urinary trypsin inhibitor-containing liquid formulation having a pH and wherein 7 or less der Rukoto. In addition, the present invention relates to a method of heating a human urinary trypsin inhibitor-containing solution at a pH of 5 to 7 and heating the solution to 60 to 100 ° C.
It is a human urinary trypsin inhibitor-containing liquid preparation obtained by heat treatment for 30 minutes.
【０００８】また、本発明は、ヒト尿性トリプシンイン
ヒビター含有溶液をｐＨ５〜７で６０〜１００℃に加熱
後に、その温度を維持しつつ、ｐＨを２〜５に下げて１
〜３０分間加熱処理することからなる前記液状製剤の製
造方法である。 （１）ＨＵＴＩ 本発明のＨＵＴＩは、尿由来、細胞培養由来、遺伝子工
学由来のいずれにも限定されない。また、医薬品として
適用可能な程度まで精製されておればよい。[0008] The present invention also relates to a method of heating a human urinary trypsin inhibitor-containing solution at a pH of 5 to 7 at 60 to 100 ° C, and maintaining the temperature while lowering the pH to 2 to 5 to 1
A method for producing the liquid preparation, which comprises heat-treating for up to 30 minutes. (1) huti The huti of the present invention is not limited to those derived from urine, cell culture, or genetic engineering. Further, it may be purified to the extent that it can be applied as a pharmaceutical.
【０００９】ＨＵＴＩの精製法としては陰イオン交換体
処理、限外濾過、ゲル濾過、アフィニティクロマト、無
機吸着剤処理、塩析、等電点沈殿法、キトサン処理、不
溶性トリプシン処理等が知られている（特開昭５１−５
１５７９、同５１−１１８８１０、同５１−１２３８１
０、同５５−１６０７２４、同５６−９９４２７、同５
７−１４０７２８、同６０−２６０５１８、同６１−３
７７３６等）。As a method for purifying HITI, anion exchanger treatment, ultrafiltration, gel filtration, affinity chromatography, inorganic adsorbent treatment, salting out, isoelectric precipitation, chitosan treatment, insoluble trypsin treatment and the like are known. (Japanese Patent Laid-Open Publication
1579, 51-118810, 51-12381
0, 55-160724, 56-99427, 5
7-140728, 60-260518, 61-3
7736).
【００１０】また、金属キレート樹脂処理、疎水性担体
処理、加熱処理等を組み合わせてもよい。こうして得ら
れるＨＵＴＩは分子量６万〜７万程度（好ましくは６７
０００）、等電点は２〜３程度、比活性は２０００〜３
５００単位／ｍｇ蛋白程度である。 （２）液状製剤 本発明の液状製剤におけるＨＵＴＩの濃度は、１０００
〜５００００単位／ｍＬ程度が例示される。本発明の特
徴は低イオン強度、ｐＨ７以下である。低イオン強度と
は、ｐＨ調整等を行ったあとの液状製剤のイオン強度
が、塩化ナトリウム濃度に換算して０．１Ｍ（モル／
Ｌ）以下である。好ましくは０．０５Ｍ以下、さらに好
ましくは、０．０１Ｍ以下である。ｐＨは３〜７が例示
されるが、好ましくは３〜６程度である。ただし、酸性
蛋白質分解酵素が共存する場合はｐＨ４〜６程度、共存
しない場合はｐＨ３〜６程度が好都合である。該ｐＨ調
整等に使用される物質としては、リン酸塩、塩化ナトリ
ウム、グリシン−塩酸等が挙げられるが、ＨＵＴＩの調
製過程で既に上記条件が満足されているならば、特にな
にも添加しなくともよい。尚、本発明において酸性蛋白
質分解酵素とは以下の性質を有するものを指す。 （ｉ）酸性（ｐＨ２〜４付近）でＨＵＴＩを分解する。 （ｉｉ）アスパラギン酸プロテアーゼの阻害剤であるペ
プスタチンまたはアミノペプチダーゼの阻害剤であるベ
スタチンによって阻害される。 （ｉｉｉ）１００℃３分間またはｐＨ２〜４程度での６
０℃、３０分間の加温処理により不活化される。Further, a metal chelate resin treatment, a hydrophobic carrier treatment, a heat treatment and the like may be combined. The huti thus obtained has a molecular weight of about 60,000 to 70,000 (preferably 67 to 70,000).
000), isoelectric point is about 2-3, specific activity is 2000-3
About 500 units / mg protein. (2) Liquid preparation The concentration of huti in the liquid preparation of the present invention is 1000
For example, about 50,000 units / mL. The features of the present invention are low ionic strength and pH 7 or less. Low ionic strength is the ionic strength of the liquid preparation after pH adjustment, etc.
Is 0.1 M (mol / mol)
L) The following is true. Preferably it is 0.05M or less, more preferably 0.01M or less. The pH is, for example, 3-7, but preferably about 3-6. However, the pH is preferably about 4 to 6 when the acidic protease is present, and about 3 to 6 when the acidic protease is not present. Substances used for the pH adjustment and the like include phosphate, sodium chloride, glycine-hydrochloric acid, and the like. It is not necessary. In the present invention, the acid protease refers to one having the following properties. (I) Decompose huti under acidic conditions (around pH 2-4). (Ii) Inhibited by pepstatin, an inhibitor of aspartic protease, or bestatin, an inhibitor of aminopeptidase. (Iii) 6 at 100 ° C. for 3 minutes or pH of about 2 to 4
Inactivated by heating at 0 ° C. for 30 minutes.
【００１１】また、当該酵素が共存しないようなＨＵＴ
Ｉの製法としては、 当該酵素の阻害剤を固定化した担体を用いて、酵素を
吸着除去する方法。 弱酸性ｐＨ（例えば、ｐＨ５〜７程度）で６０〜１０
０℃に加温後に、ｐＨを下げて（例えば、ｐＨ２〜５程
度）、１〜３０分程度加温処理することにより、酵素を
不活化する方法等を用いることができる。[0011] Further, a HUT in which the enzyme does not coexist
The method for producing I is a method in which an enzyme is adsorbed and removed using a carrier on which an inhibitor of the enzyme is immobilized. 60 to 10 at a weak acidic pH (for example, about pH 5 to 7)
After heating to 0 ° C., a method of inactivating the enzyme by lowering the pH (for example, about pH 2 to 5) and performing a heating treatment for about 1 to 30 minutes can be used.
【００１２】本発明の液状製剤には公知の添加剤、二糖
類（例、ショ糖、マルトース）あるいは糖アルコール
（例、マンニトール、ソルビトール）を、例えば０．０
１〜１ｍｇ／ｍＬ程度用いてもよい。ＨＵＴＩ含有溶液
に必要に応じて添加剤を添加後、通常の方法により、除
菌濾過、分注、加熱処理等の操作を施して、液状製剤と
して供与できる。本製剤は注射剤、点眼剤、点鼻剤等と
して、患者に投与することができる。The liquid preparation of the present invention contains known additives, disaccharides (eg, sucrose, maltose) or sugar alcohols (eg, mannitol, sorbitol), for example, 0.0
About 1 to 1 mg / mL may be used. After adding additives as needed to the huti-containing solution, the solution can be provided as a liquid preparation by subjecting it to operations such as sterilization filtration, dispensing, and heat treatment by a usual method. This preparation can be administered to patients as injections, eye drops, nasal drops and the like.
【００１３】[0013]
【実施例】本発明をより詳細に説明するために実施例お
よび参考例を挙げるが、本発明はこれらによって、なん
ら限定されるものではない。 実施例１ ＨＵＴＩ（比活性 ２５００Ｕ／ｍｇ蛋白）１０ｍｇを
精製水１ｍＬ（ミリリットル）に溶解し、ｐＨを６に調
整して、本発明の液状製剤を得た。The present invention will be described in more detail with reference to Examples and Reference Examples, which should not be construed as limiting the invention thereto. Example 1 10 mg of HITI (specific activity 2500 U / mg protein) was dissolved in 1 mL (milliliter) of purified water, and the pH was adjusted to 6 to obtain a liquid preparation of the present invention.
【００１４】実施例２ ＨＵＴＩ（比活性 ２５００Ｕ／ｍｇ蛋白）１０ｍｇを
０．０５Ｍリン酸緩衝液（ｐＨ６）１ｍＬに溶解し、本
発明の液状製剤を得た。 実施例３ ＨＵＴＩ（比活性 ２５００Ｕ／ｍｇ蛋白）１０ｍｇを
精製水１ｍＬに溶解し、１００℃に加温した。温度を保
ちつつ、ｐＨ３〜４に調整して３分間置き、その後に温
度を下げることにより、本発明の液状製剤を得た。Example 2 10 mg of HITI (specific activity 2500 U / mg protein) was dissolved in 1 mL of 0.05 M phosphate buffer (pH 6) to obtain a liquid preparation of the present invention. Example 3 10 mg of HITI (specific activity 2500 U / mg protein) was dissolved in 1 mL of purified water, and heated to 100 ° C. While maintaining the temperature, the pH was adjusted to 3 to 4 and the mixture was kept for 3 minutes, and then the temperature was lowered to obtain the liquid preparation of the present invention.
【００１５】実施例４ ＨＵＴＩ（比活性 ２５００Ｕ／ｍｇ蛋白）１０ｍｇを
０．１５Ｍリン酸緩衝液（ｐＨ７）１ｍＬに溶解した後
に、ペプスタチンを固定化したセファロース６Ｂカラム
にアプライし、その非吸着画分を回収した。さらにｐＨ
を４に調整して、本発明の液状製剤を得た。Example 4 After dissolving 10 mg of HITI (specific activity 2500 U / mg protein) in 1 mL of 0.15 M phosphate buffer (pH 7), the solution was applied to a Sepharose 6B column on which pepstatin was immobilized, and the non-adsorbed fraction was obtained. Was recovered. Further pH
Was adjusted to 4 to obtain a liquid preparation of the present invention.
【００１６】実施例５ ＨＵＴＩ（比活性 ２５００Ｕ／ｍｇ蛋白）１０ｍｇを
精製水１ｍＬに溶解し、６０℃に加温した。温度を保ち
つつ、ｐＨ３〜４に調整して３０分間置き、その後に温
度を下げることにより、本発明の液状製剤を得た。 実施例６ ＨＵＴＩ（比活性 ２５００Ｕ／ｍｇ蛋白）１０ｍｇを
精製水１ｍＬに溶解し、６０℃で１０時間加熱処理後
に、温度を保ちつつ、グリシン−塩酸緩衝液でｐＨ３〜
４に調整して３０分間置き、その後に温度を下げること
により、本発明の液状製剤を得た。Example 5 10 mg of HITI (specific activity 2500 U / mg protein) was dissolved in 1 mL of purified water and heated to 60 ° C. While maintaining the temperature, the pH was adjusted to 3 to 4 and the mixture was placed for 30 minutes, and then the temperature was lowered to obtain the liquid preparation of the present invention. Example 6 10 mg of HITI (specific activity 2500 U / mg protein) was dissolved in 1 mL of purified water, and after a heat treatment at 60 ° C. for 10 hours, the pH was adjusted to 3 to 3 with a glycine-hydrochloric acid buffer while maintaining the temperature.
4, and the temperature was lowered for 30 minutes, and then the liquid preparation of the present invention was obtained.
【００１７】以下に実験例を示す。イオン強度について
は、それぞれの液状製剤のイオン強度を塩化ナトリウム
濃度に換算してモル濃度（Ｍ）で表した。 実験例１ 実施例１で調製した液状製剤を用いて、ｐＨ６、イオン
強度０〜１Ｍの条件で６０℃、７日間保存し、その効果
を確認した。ＨＵＴＩの凝集による高分子体およびＨＵ
ＴＩの分解による低分子体の各々の含有率はゲル濾過法
により測定した。結果を表１に示す。 An experimental example will be described below. About ionic strength
The sodium chloride of the ionic strength of each liquid formulation
The concentration was converted to a molar concentration (M). Experimental Example 1 The liquid preparation prepared in Example 1 was stored at 60 ° C. for 7 days under conditions of pH 6 and ionic strength of 0 to 1 M, and the effect was confirmed. Polymers and HU by aggregation of huti
The content of each of the low molecular weight substances due to the decomposition of TI was measured by a gel filtration method. Table 1 shows the results.
【００１８】[0018]
【表１】 [Table 1]
【００１９】実験例２ 実施例１で調製した液状製剤を用いて、イオン強度０．
０１、ｐＨ１〜１０の条件下で６０℃１０時間保存し、
その効果を確認した。ＨＵＴＩの高分子体および低分子
体の各々の含有率はゲル濾過法により測定した。ＨＵＴ
Ｉ活性はカッセルらの方法（メソッド・イン・エンザイ
モロジー、１９巻、８４４頁、１９７０年）に準じて測
定した。結果を表２に示す。EXPERIMENTAL EXAMPLE 2 Using the liquid preparation prepared in Example 1, an ionic strength of 0.
01, stored at 60 ° C. for 10 hours under conditions of pH 1 to 10,
The effect was confirmed. The content of each of the high molecular weight and low molecular weight HITI was measured by a gel filtration method. HUT
I activity was measured according to the method of Kassel et al. (Method in Enzymology, 19, 844, 1970). Table 2 shows the results.
【００２０】[0020]
【表２】 [Table 2]
【００２１】実験例３ 実施例３で調製した液状製剤を用いて、ｐＨ５、イオン
強度０〜１Ｍの条件で６０℃、６日間保存し、その効果
を確認した。ＨＵＴＩの凝集による高分子体および低分
子体の各々の含有率はゲル濾過法により測定した。結果
を表３に示す。Experimental Example 3 The liquid preparation prepared in Example 3 was stored at 60 ° C. for 6 days under conditions of pH 5 and ionic strength of 0 to 1 M, and the effect was confirmed. The content of each of the high molecular weight substance and the low molecular weight substance due to the aggregation of HITI was measured by a gel filtration method. Table 3 shows the results.
【００２２】[0022]
【表３】 [Table 3]
【００２３】実験例４ 実施例３および５で調製した液状製剤中の、酸性蛋白質
分解酵素が不活化されたかどうかを確認した。その効果
は、液状製剤をｐＨ２．５で３７℃１５分間処理して生
じたＨＵＴＩの低分子体の生成度から判断した。結果を
表４に示す。Experimental Example 4 It was confirmed whether or not the acidic protease was inactivated in the liquid preparations prepared in Examples 3 and 5. The effect was determined based on the degree of generation of HITI low molecular weights produced by treating the liquid preparation at pH 2.5 at 37 ° C. for 15 minutes. Table 4 shows the results.
【００２４】[0024]
【表４】 [Table 4]
【００２５】実験例５ 実施例６で得た液状製剤をイオン強度０．０１Ｍ、ｐＨ
４〜５．５に調整した上で４０℃で２月間保存し、安定
性を確認した。また、安定化剤としてソルビトールを
０．２ｍｇ／ｍＬ添加した場合の効果も確認した。ＨＵ
ＴＩの高分子体および低分子体の含有率、ＨＵＴＩ活性
は、実験例１または２に準じて測定した。結果を表５に
示す。Experimental Example 5 The liquid preparation obtained in Example 6 was subjected to ionic strength 0.01 M, pH
After adjusting to 4-5.5, it was stored at 40 ° C. for 2 months to confirm the stability. The effect of adding 0.2 mg / mL of sorbitol as a stabilizer was also confirmed. HU
The contents of the high molecular weight and low molecular weight TI and the HITI activity were measured according to Experimental Example 1 or 2. Table 5 shows the results.
【００２６】[0026]
【表５】 [Table 5]
【００２７】参考例 人尿を特開昭６２−９３２３８の方法に準じて調製した
ものを本実施例に使用されるＨＵＴＩの出発原料とし
た。出発原料４２０ｍＬをｐＨ６．４に調整後、０．１
Ｍリン酸緩衝液（ｐＨ６．４）で平衡化したＱＡＥ−ア
ガロースゲル（商品名Ｑ−セファロース ファストフロ
ー、ファルマシア社）にアプライした。その後、０．５
Ｍ塩化ナトリウム加０．１Ｍリン酸緩衝液（ｐＨ６．
４）で溶出しＨＵＴＩ含有画分を回収した。Reference Example Human urine prepared according to the method of JP-A-62-93238 was used as a starting material of HITI used in this example. After adjusting 420 mL of the starting material to pH 6.4, 0.1
The mixture was applied to a QAE-agarose gel (trade name: Q-Sepharose Fast Flow, Pharmacia) equilibrated with M phosphate buffer (pH 6.4). Then 0.5
M sodium chloride and 0.1 M phosphate buffer (pH 6.
The fraction eluted in 4) was collected.
【００２８】溶出画分８００ｍＬをｐＨ８に調整後、
０．５Ｍ塩化ナトリウム加０．１Ｍリン酸緩衝液（ｐＨ
８）で平衡化したＣｕ2+キレートアガロースゲル（商品
名キレートセファロース ファスト フロー、 ファル
マシア社）にアプライし、非吸着画分を回収した。非吸
着画分２６０ｍＬに酢酸緩衝液（ｐＨ５）を加えて、最
終５０ｍＭに調整し、６０℃、１０時間加熱処理した。
さらに２Ｍ硫酸アンモニウムを添加して濃度０．８Ｍに
調整後、０．８Ｍ硫酸アンモニウムで平衡化したフェニ
ル−アガロースゲル（商品名フェニル セファロース４
Ｂ、ファルマシア社）にアプライし、非吸着画分を回収
した。After adjusting 800 mL of the eluted fraction to pH 8,
0.5 M sodium chloride and 0.1 M phosphate buffer (pH
The mixture was applied to a Cu 2+ chelate agarose gel (trade name: Chelate Sepharose Fast Flow, Pharmacia) equilibrated in 8), and a non-adsorbed fraction was collected. An acetate buffer (pH 5) was added to 260 mL of the non-adsorbed fraction to adjust the final concentration to 50 mM, and the mixture was heated at 60 ° C. for 10 hours.
Further, after adding 2 M ammonium sulfate to adjust the concentration to 0.8 M, a phenyl-agarose gel (trade name: Phenyl Sepharose 4) equilibrated with 0.8 M ammonium sulfate was used.
B, Pharmacia) and the non-adsorbed fraction was collected.
【００２９】非吸着画分６１０ｍＬにＥＤＴＡを１ｍＭ
となるように添加後に限外濾過膜（商品名ＵＦメンブラ
ン、フィルトロン社、分子量１万以下の画分をカット）
で処理し、濃縮画分を回収した。濃縮画分２８ｍＬを
０．１５Ｍ塩化ナトリウム加５０ｍＭ酢酸緩衝液（ｐＨ
６．２）で平衡化したポリアクリルアミドゲル（商品名
セファクリルＳ−２００ＨＰ、ファルマシア社）にアプ
ライし、同緩衝液で溶出しＨＵＴＩ画分を回収した。最
後に精製水で透析した。1 mM EDTA was added to 610 mL of the non-adsorbed fraction.
Ultrafiltration membrane (trade name: UF membrane, Filtron Co., cut off fractions with molecular weight of 10,000 or less)
And concentrated fractions were collected. 28 mL of the concentrated fraction was added to 50 mM acetate buffer (pH 5
The solution was applied to a polyacrylamide gel (trade name: Sephacryl S-200HP, Pharmacia) equilibrated in 6.2) and eluted with the same buffer to collect a HITI fraction. Finally, it was dialyzed against purified water.
【００３０】[0030]
【発明の効果】本発明によれば、ＨＵＴＩの長期保存時
の安定性が改善される。特に、重合による高分子体およ
び分解による低分子体の生成を抑えた製剤を調製するこ
とができる。According to the present invention, the stability of HITI during long-term storage is improved. In particular, it is possible to prepare a preparation in which the production of high molecular weight by polymerization and low molecular weight by decomposition is suppressed.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 広瀬 正明 大阪府枚方市招提大谷２丁目25番１号 株式会社ミドリ十字 中央研究所内 (72)発明者 真崎 厚司 大阪府枚方市招提大谷２丁目25番１号 株式会社ミドリ十字 中央研究所内 (56)参考文献 特開 昭61−37736（ＪＰ，Ａ) (58)調査した分野(Int.Cl.6，ＤＢ名) A61K 38/55 - 38/57 ＣＡ（ＳＴＮ) ＲＥＧＩＳＴＲＹ（ＳＴＮ)────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Masaaki Hirose 2- 25-1, Invitation Otani, Hirakata-shi, Osaka Midori Cross Co., Ltd. Central Research Laboratory (72) Inventor Atsushi Masaki 2-25, Invitation Otani, Hirakata-shi, Osaka No. 1 Midori Cross Co., Ltd. Central Research Laboratory (56) References JP-A-61-37736 (JP, A) (58) Fields investigated (Int. Cl. 6 , DB name) A61K 38/55-38/57 CA (STN) REGISTRY (STN)
Claims (5)
が７以下であることを特徴とするヒト尿性トリプシンイ
ンヒビター含有液状製剤。Claims: 1. An ionic strength of 0.1 M or less,
Is a human urinary trypsin inhibitor-containing liquid preparation, wherein is less than or equal to 7.
項１記載の液状製剤。2. The liquid preparation according to claim 1, which has an ionic strength of 0.05 M or less.
００単位／ｍｇ蛋白である請求項１または２に記載の液
状製剤。3. Specific activity is 2000 units / mg protein to 35.
3. The liquid preparation according to claim 1, wherein the liquid preparation is 00 units / mg protein.
液をｐＨ５〜７で６０〜１００℃に加熱後に、その温度
を維持しつつ、ｐＨを２〜５に下げて１〜３０分間加熱
処理して得られる請求項１記載の液状製剤。4. A solution obtained by heating a human urinary trypsin inhibitor-containing solution at 60 to 100 ° C. at a pH of 5 to 7 and lowering the pH to 2 to 5 while maintaining the temperature for 1 to 30 minutes. The liquid preparation according to claim 1 .
液をｐＨ５〜７で６０〜１００℃に加熱後に、その温度
を維持しつつ、ｐＨを２〜５に下げて１〜３０分間加熱
処理することからなる請求項１記載の液状製剤の製造方
法。5. A method comprising heating a human urinary trypsin inhibitor-containing solution at a pH of 5 to 7 to 60 to 100 ° C., lowering the pH to 2 to 5 while maintaining the temperature, and performing a heat treatment for 1 to 30 minutes. A method for producing the liquid preparation according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4203211A JP2861655B2 (en) | 1992-07-08 | 1992-07-08 | Liquid preparation containing human urinary trypsin inhibitor and method for producing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4203211A JP2861655B2 (en) | 1992-07-08 | 1992-07-08 | Liquid preparation containing human urinary trypsin inhibitor and method for producing the same |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP10026198A Division JPH10182482A (en) | 1998-02-06 | 1998-02-06 | Liquid preparation containing human urinary trypsin inhibitor and its production |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0625011A JPH0625011A (en) | 1994-02-01 |
| JP2861655B2 true JP2861655B2 (en) | 1999-02-24 |
Family
ID=16470308
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP4203211A Expired - Fee Related JP2861655B2 (en) | 1992-07-08 | 1992-07-08 | Liquid preparation containing human urinary trypsin inhibitor and method for producing the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2861655B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2660484B2 (en) * | 1994-04-12 | 1997-10-08 | 株式会社ミドリ十字 | Liquid preparation containing human urinary trypsin inhibitor |
| US6355243B1 (en) * | 1999-11-13 | 2002-03-12 | Bayer Corporation | Method of thrombolysis by local delivery of active plasmin |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6137736A (en) * | 1984-07-31 | 1986-02-22 | Nippon Chem Res Kk | Preparation of human urine trypsin inhibitor drug |
-
1992
- 1992-07-08 JP JP4203211A patent/JP2861655B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0625011A (en) | 1994-02-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0100982B1 (en) | Novel purified plasminogen activator, process for its production and thrombolytic composition containing it | |
| JP2003535574A (en) | Method for preparing reversibly inert acidified plasmin composition | |
| EP0037078A2 (en) | Process for heat treatment of aqueous solution containing human blood coagulation factor XIII | |
| JPS62289182A (en) | Novel plasminogen activated substance | |
| JPS6160049B2 (en) | ||
| EP0155852B1 (en) | Thrombin-binding substance and process for its production | |
| US6280729B1 (en) | Preparation of factor IX | |
| JP2861655B2 (en) | Liquid preparation containing human urinary trypsin inhibitor and method for producing the same | |
| JP2002533473A (en) | Pharmaceutical formulations containing hyaluronan degrading enzymes of microbial origin | |
| JPS6360938A (en) | Plasminogen activator of modified tissue type and production thereof | |
| US6051223A (en) | Method of improving solubility of tissue plasminogen activator | |
| JP2722143B2 (en) | Lyophilized formulation containing trypsin inhibitor | |
| JP2660484B2 (en) | Liquid preparation containing human urinary trypsin inhibitor | |
| JPH10182482A (en) | Liquid preparation containing human urinary trypsin inhibitor and its production | |
| JPS59118717A (en) | Plasminogen activator | |
| JP2545231B2 (en) | Method for suppressing molecular weight reduction of urokinase by heating | |
| CN109952374A (en) | Technique for the plasminogen purifying since human plasma | |
| US20020031518A1 (en) | Plasminogen fragment having activity to inhibit tumor metastasis and growth and process for preparing same technical field | |
| JP2722140B2 (en) | Method for producing human urinary trypsin inhibitor | |
| JP2714817B2 (en) | Method for producing urokinase precursor | |
| JPS60248621A (en) | Method of stabilizing single stranded tissue plasminogen activator | |
| JPS6140392B2 (en) | ||
| JPS588835B2 (en) | Heat stabilization method for urokinase | |
| JPH0977680A (en) | Haptoglobin-containing liquid preparation | |
| JPH0329390B2 (en) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| LAPS | Cancellation because of no payment of annual fees |