JP2916947B2 - Method for stabilizing CPB-I and pharmaceutical composition - Google Patents
Method for stabilizing CPB-I and pharmaceutical compositionInfo
- Publication number
- JP2916947B2 JP2916947B2 JP2328286A JP32828690A JP2916947B2 JP 2916947 B2 JP2916947 B2 JP 2916947B2 JP 2328286 A JP2328286 A JP 2328286A JP 32828690 A JP32828690 A JP 32828690A JP 2916947 B2 JP2916947 B2 JP 2916947B2
- Authority
- JP
- Japan
- Prior art keywords
- cpb
- amino acid
- pharmaceutical composition
- recombinant
- stabilizing
- 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
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Medicinal Preparation (AREA)
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は抗血液凝固剤、皮膚・角膜疾患治療剤として
有用なCPB−Ｉ又はリコンビナントCPB−Ｉの安定化方法
及び製剤組成物に関し、詳細には、これらの分離精製、
凍結乾燥等の操作を行う際に有用な安定化方法及び保存
安全性に優れた製剤組成物に関する。The present invention relates to a method for stabilizing CPB-I or recombinant CPB-I which is useful as an anticoagulant or a therapeutic agent for skin / corneal diseases, and a pharmaceutical composition. The separation and purification of these,
The present invention relates to a stabilization method useful for performing operations such as freeze-drying and a pharmaceutical composition excellent in storage safety.
CPB−Ｉはヒト胎盤をはじめとする生体内の組織及び
分泌液に広く分布し（Chem.Pharma.Bull,38,1957〜196
0,1990）、また細胞内では細胞質に存在する抗血液凝固
作用等の生理活性を有する物質である。CPB-I is widely distributed in in vivo tissues and secretions, including human placenta (Chem. Pharma. Bull, 38, 1957-196).
0,1990), and a substance having a physiological activity such as an anticoagulant effect existing in the cytoplasm within a cell.
CPB−Ｉはヒトあるいは動物の臓器から抽出すること
ができ（特開昭62−174023号公報）、このようにして得
られたCPB−Ｉは以下の性質を有する。CPB-I can be extracted from human or animal organs (JP-A-62-174023). The CPB-I thus obtained has the following properties.
分子量（SDS−ポリアクリルアミドゲル電気泳動
法、還元状態） 34,000±2,000 等電点（アンフォライトを用いる等電点カラム電気
泳動法） 4.7±0.1 安定性 （イ）50℃、30分加熱処理で失活 （ロ）pH4〜10で安定 （ハ）血漿中37℃、30分で安定 血液凝固系に対する作用 （イ）カルシウム再加凝固時間を延長 （ロ）プロトロンビン時間を延長 （ハ）活性化部分トロンボプラスチン時間を延長 アミノ酸分析 アミノ酸分析で、アスパラギン酸、スレオニン、セリ
ン、グルタミン酸、プロリン、グリシン、アラニン、1/
2シスチン、バリン、メチオニン、イソロイシン、ロイ
シン、チロシン、フェニルアラニン、ヒスチジン、リジ
ン及びアルギニンの存在が認められる。Molecular weight (SDS-polyacrylamide gel electrophoresis, reduced state) 34,000 ± 2,000 Isoelectric point (isoelectric point column electrophoresis using ampholite) 4.7 ± 0.1 Stability (a) Loss by heating at 50 ° C for 30 minutes Active (b) Stable at pH 4-10 (c) Stable at 37 ° C, 30 minutes in plasma Effect on blood coagulation system (a) Extend calcium recoagulation time (b) Extend prothrombin time (c) Activated partial thromboplastin Extend time Amino acid analysis Amino acid analysis shows aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, 1 /
2 The presence of cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine and arginine is observed.
また、CPB−Ｉはヒト又は動物のCPB−Ｉをコードする
遺伝子断片を用いて、遺伝子組換え技術により大腸菌、
酵母等で発現させることができる（特開昭64−20095号
公報）。このように遺伝子組換えにより得られたCPB−
Ｉ（以下、これを「リコンビナントCPB−Ｉ」という）
は下記のアミノ酸配列を有する。In addition, CPB-I is obtained by using a gene fragment encoding human or animal CPB-I,
It can be expressed in yeast and the like (JP-A-64-20095). CPB- thus obtained by genetic recombination
I (hereinafter referred to as "recombinant CPB-I")
Has the following amino acid sequence:
また、リコンビナントCPB−Ｉは大腸菌以外にも酵母
を使用しても製造することができる。この場合、アミノ
末端のアラニンにアセチル基が結合したアミノ酸配列と
なる。 Recombinant CPB-I can also be produced using yeast other than E. coli. In this case, an amino acid sequence in which an acetyl group is bonded to alanine at the amino terminus is obtained.
このようなCPB−Ｉ又はリコンビナントCPB−Ｉは、液
剤、ゲル化剤、軟膏、点眼液、注射剤等種々の剤形とす
ることができる。Such CPB-I or recombinant CPB-I can be made into various dosage forms such as a solution, a gelling agent, an ointment, an ophthalmic solution, and an injection.
しかしながら、製剤化の過程で分離精製・凍結乾燥等
を行なう必要があり、また上記のような製剤とした後、
長期保存する必要があり、このときCPB−Ｉが重合等を
起こし、その活性が低下してしまうという欠点があっ
た。However, it is necessary to separate and purify, freeze-dry, etc. during the formulation process, and after the formulation as described above,
It has to be stored for a long period of time, and at this time, there is a drawback that the activity of the CPB-I is reduced due to polymerization or the like.
従って、CPB−Ｉ又はリコンビナントCPB−Ｉの安定化
方法及び安定な製剤組成物が望まれていた。Therefore, a method for stabilizing CPB-I or recombinant CPB-I and a stable pharmaceutical composition have been desired.
本発明者らは上記実状に鑑み鋭意研究を重ねた結果、
CPB−Ｉ又はリコンビナントCPB−Ｉに塩基性アミノ酸を
添加すれば、これらの安定化を図ることができ、分離精
製・凍結乾燥等の操作を行っても、またこれを製剤化し
て長期保存しても活性が低下しないことを見出し本発明
を完成した。The present inventors have conducted intensive research in view of the above situation,
By adding a basic amino acid to CPB-I or recombinant CPB-I, these can be stabilized, and even if an operation such as separation / purification / freeze-drying is performed, it can be formulated and stored for a long time. Found that the activity did not decrease, and completed the present invention.
すなわち本発明は、CPB−Ｉ又はリコンビナントCPB−
Ｉ及び塩基性アミノ酸を添加することを特徴とするCPB
−Ｉ又はリコンビナントCPB−Ｉの安定化方法及びCPB−
Ｉ又はリコンビナントCPB−Ｉ及び塩基性アミノ酸を含
有することを特徴とするCPB−Ｉ又はリコンビナントCPB
−Ｉが安定化された製剤組成物を提供するものである。That is, the present invention relates to CPB-I or recombinant CPB-
CPB characterized by adding I and a basic amino acid
-I or a method for stabilizing recombinant CPB-I and CPB-
CPB-I or recombinant CPB, characterized by containing I or recombinant CPB-I and a basic amino acid
-I provides a stabilized pharmaceutical composition.
本発明で用いられる塩基性アミノ酸としては、例え
ば、リジン、アルギニン、オルニチン等が挙げられる
が、注射用製剤とする場合はＬ−リジン又はＬ−アルギ
ニンが好ましい。塩基性アミノ酸の配合量はCPB−Ｉ又
はリコンビナントCPB−Ｉ（以下、「CPB−Ｉ等」とい
う）を含有する溶液1mlあたり0.6〜20mg程度が好まし
い。なお、粉末に添加する場合の安定化剤の添加量は、
当該粉末を溶解した際に、上記の溶液濃度になるように
すればよい。この配合量が0.6mg未満であると安定化が
図り難く、20mgを超えて配合してもあまり効果の向上は
望めない。The basic amino acid used in the present invention includes, for example, lysine, arginine, ornithine and the like. In the case of preparation for injection, L-lysine or L-arginine is preferable. The amount of the basic amino acid is preferably about 0.6 to 20 mg per ml of a solution containing CPB-I or recombinant CPB-I (hereinafter referred to as “CPB-I etc.”). In addition, the amount of the stabilizer to be added to the powder,
What is necessary is just to make it the said solution density | concentration when the said powder is melt | dissolved. If the amount is less than 0.6 mg, it is difficult to achieve stabilization, and if the amount exceeds 20 mg, the effect cannot be expected to be significantly improved.
塩基性アミノ酸の添加方法は特に限定されず、CPB−
Ｉ等の溶液に直接添加する方法、あらかじめ塩基性アミ
ノ酸を水又は適当な緩衝液に溶解して添加する方法、塩
基性アミノ酸の粉末をCPB−Ｉ等と混合せしめて水等に
添加・溶解する方法等、適宜選択すればよい。また、添
加時期も特に限定されず、CPB−Ｉ等の分離精製工程、
製剤工程のいずれであってもよい。The method for adding the basic amino acid is not particularly limited, and CPB-
I, a method of directly adding a basic amino acid to water or a suitable buffer solution, and a method of mixing a basic amino acid powder with CPB-I or the like and adding / dissolving it in water, etc. What is necessary is just to select a method etc. suitably. The timing of addition is not particularly limited, and a separation and purification step of CPB-I or the like,
Any of the formulation steps may be used.
CPB−Ｉ等に塩基性アミノ酸を添加した本発明の安定
化組成物は、溶液の場合０〜30℃、特に０〜10℃で分離
精製、製剤化又は保存することが好ましい。また、同溶
液を凍結状態で保存する場合は０℃以下、特に−20℃以
下とすることが好ましい。The stabilized composition of the present invention in which a basic amino acid is added to CPB-I or the like is preferably separated and purified, formulated or stored at 0 to 30 ° C, particularly 0 to 10 ° C in the case of a solution. When the solution is stored in a frozen state, the temperature is preferably 0 ° C. or lower, particularly -20 ° C. or lower.
また、本発明組成物には、グルコース、グルコサミ
ン、キシロース、サッカロース、デキストランＴ−40、
マンニトール等の糖類、アルブミン等を配合することが
できる。Further, the composition of the present invention includes glucose, glucosamine, xylose, saccharose, dextran T-40,
Sugars such as mannitol, albumin and the like can be added.
本発明のCPB−Ｉ等含有安定化製剤組成物は、用途に
応じて種々の剤形とすることができる。例えば、抗血液
凝固剤として用いる場合は、水性注射剤等の形態とし、
皮膚・角膜疾患治療剤として用いる場合は、液剤、ゲ
ル、クリーム、軟膏、点眼液、眼軟膏等の形態とするこ
とができる。The stabilized preparation composition containing CPB-I etc. of the present invention can be made into various dosage forms depending on the use. For example, when used as an anticoagulant, in the form of an aqueous injection or the like,
When used as a therapeutic agent for skin and corneal diseases, it can be in the form of a liquid, gel, cream, ointment, eye drop, ointment or the like.
本発明の方法によれば、CPB−Ｉ等を凍結・融解、水
性媒体への溶解等の分離・精製、製剤化工程を経てもCP
B−Ｉ等の活性が保持される。また本発明の製剤組成物
は長期保存してもCPB−Ｉの活性低下がなく、医薬品と
して有用である。According to the method of the present invention, CPB-I and the like are frozen and thawed, separated and purified by dissolution in an aqueous medium, etc.
Activities such as BI are retained. In addition, the pharmaceutical composition of the present invention does not decrease the activity of CPB-I even when stored for a long time, and is useful as a pharmaceutical.
実施例１ 10mMリン酸緩衝液（pH7.4）に溶解したCPB−I6.76mg/
mlに安定化剤としてアスパラギン酸、グリシンあるいは
リジンの夫々を1mlあたり各5mg加えた溶液100μを−4
0℃で凍結させ、室温で融解した。この操作を６回繰り
返した後下記測定方法により分析を行った。結果を表１
に示す。Example 1 CPB-I 6.76 mg / dissolved in 10 mM phosphate buffer (pH 7.4)
As a stabilizer, aspartic acid, glycine, or lysine was added as a stabilizer to each of 5 mg / ml of a 100 μl solution.
Frozen at 0 ° C. and thawed at room temperature. After repeating this operation six times, analysis was performed by the following measurement method. Table 1 shows the results
Shown in
HPLCによるCPB−Ｉの重合体の測定 あらかじめ0.14M NaC1を含む10mMリン酸緩衝液（pH7.
4）で平衡化した東ソー社製HPLCゲル濾過用カラムTSK g
el G3000SWXL（7.8×300mm）に同上緩衝液1mg/mlに希釈
した試料溶液20μを注入した。流速1ml/minで重合体
との分離を行い、カラムから溶出された試料の吸光度を
波長280nmで測定し、面積値よりCPB−Ｉとその重合体と
の割合を算出した。Determination of CPB-I polymer by HPLC 10 mM phosphate buffer (pH 7.
Tosoh HPLC gel filtration column TSK g equilibrated in 4)
20 μ of a sample solution diluted to 1 mg / ml in the same buffer as above was injected into el G3000SW XL (7.8 × 300 mm). The polymer was separated at a flow rate of 1 ml / min, the absorbance of the sample eluted from the column was measured at a wavelength of 280 nm, and the ratio between CPB-I and the polymer was calculated from the area value.
実施例２ 10mMリン酸緩衝液（pH7.4）に溶解したCPB−I6.76mg/
mlに安定化剤としてＬ−リジン塩酸塩を1mlあたり0.04
〜20mg加えた溶液100μを−40℃で凍結させた後室温
で融解した。この操作を５回繰り返した後実施例１と同
様にして分析を行った。結果を表２に示す。 Example 2 CPB-I 6.76 mg / dissolved in 10 mM phosphate buffer (pH 7.4)
L-lysine hydrochloride as a stabilizer in 0.04 ml / ml
100 μ of the solution containing 2020 mg was frozen at −40 ° C. and then thawed at room temperature. After repeating this operation five times, analysis was performed in the same manner as in Example 1. Table 2 shows the results.
実施例３ 10mMリン酸緩衝液（pH7.4）に溶解したCPB−I6.76mg/
mlに安定化剤としてＬ−リジン塩酸塩を1mlあたり10mg
を加えた溶液について下記方法により抗血液凝固活性の
測定を行った。結果を表３に示す。 Example 3 CPB-I 6.76 mg / dissolved in 10 mM phosphate buffer (pH 7.4)
10 mg / ml of L-lysine hydrochloride as a stabilizer
The anticoagulant activity of the solution to which was added was measured by the following method. Table 3 shows the results.
抗血液凝固活性の測定 0.5％ウシ血清アルブミン及び0.15M NaC1を含む20mM
トリス塩酸緩衝液（pH7.4）で５及び10μg/mlに希釈し
た試料溶液100μに30mM CaCl2溶液で希釈した持田製
薬社製リオプラスチン0.5mg/mlを100μ加え37℃で３
分間プレインキュベートした。更に、37℃で生理食塩水
2mlに溶解したオーソ社製コントロール血漿200μを加
えエルマ光学社製コアグテックTE−600を用いて血漿が
凝固するまでの時間を測定した。Measurement of anticoagulant activity 20 mM containing 0.5% bovine serum albumin and 0.15 M NaC1
100 μl of a sample solution diluted to 5 and 10 μg / ml with Tris-HCl buffer (pH 7.4) was added with 100 μl of lipoplastin 0.5% / ml from Mochida Pharmaceutical Co., Ltd. diluted with 30 mM CaCl 2 solution.
Preincubated for minutes. In addition, saline at 37 ° C
200 μm of Ortho control plasma dissolved in 2 ml was added, and the time until the plasma coagulated was measured using a Coagtech TE-600 manufactured by Elma Optical.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 足達 聡 熊本県菊池郡菊陽町津久礼字新山3190― 202 (56)参考文献 特開 昭63−165328（ＪＰ，Ａ) (58)調査した分野(Int.Cl.6，ＤＢ名) C07K 14/47 A61K 38/17 A61L 47/18 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Satoshi Adachi 3190-202 Tsuyama, Kikuyo-cho, Kikuchi-gun, Kumamoto Pref. Int.Cl. 6 , DB name) C07K 14/47 A61K 38/17 A61L 47/18
Claims (4)
基性アミノ酸を添加することを特徴とするCPB−Ｉ又は
リコンビナントCPB−Ｉの安定化方法。1. A method for stabilizing CPB-I or recombinant CPB-I, comprising adding a basic amino acid to CPB-I or recombinant CPB-I.
基性アミノ酸を含有することを特徴とするCPB−Ｉ又は
リコンビナントCPB−Ｉが安定化された製剤組成物。2. A pharmaceutical composition comprising stabilized CPB-I or recombinant CPB-I, comprising CPB-I or recombinant CPB-I and a basic amino acid.
オルニチンから選ばれる一種又は二種以上である請求項
１記載の安定化方法。3. The method according to claim 1, wherein the basic amino acid is one or more selected from lysine, arginine and ornithine.
オルニチンから選ばれる一種又は二種以上である請求項
２記載の製剤組成物。4. The pharmaceutical composition according to claim 2, wherein the basic amino acid is one or more selected from lysine, arginine and ornithine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2328286A JP2916947B2 (en) | 1990-11-28 | 1990-11-28 | Method for stabilizing CPB-I and pharmaceutical composition |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2328286A JP2916947B2 (en) | 1990-11-28 | 1990-11-28 | Method for stabilizing CPB-I and pharmaceutical composition |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH04198195A JPH04198195A (en) | 1992-07-17 |
| JP2916947B2 true JP2916947B2 (en) | 1999-07-05 |
Family
ID=18208533
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2328286A Expired - Fee Related JP2916947B2 (en) | 1990-11-28 | 1990-11-28 | Method for stabilizing CPB-I and pharmaceutical composition |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2916947B2 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1253932B1 (en) * | 2000-02-08 | 2005-04-27 | Allergan, Inc. | Botulinum toxin pharmaceutical compositions |
| EP1839670A3 (en) * | 2001-02-21 | 2009-11-11 | Alavita Pharmaceuticals, Inc. | Modified Annexin Proteins and their use against thrombosis |
| US7635676B2 (en) | 2001-02-21 | 2009-12-22 | Alavita Pharmaccuticals, Inc. | Modified annexin proteins and methods for their use in organ transplantation |
| US7635680B2 (en) | 2001-02-21 | 2009-12-22 | Alavita Pharmaceuticals, Inc. | Attenuation of reperfusion injury |
| US7645739B2 (en) | 2001-02-21 | 2010-01-12 | Alavita Pharmaceuticals, Inc. | Modified annexin compositions and methods of using same |
-
1990
- 1990-11-28 JP JP2328286A patent/JP2916947B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH04198195A (en) | 1992-07-17 |
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