JPH11130801A - Production of low-molecular weight heparin - Google Patents
Production of low-molecular weight heparinInfo
- Publication number
- JPH11130801A JPH11130801A JP29696997A JP29696997A JPH11130801A JP H11130801 A JPH11130801 A JP H11130801A JP 29696997 A JP29696997 A JP 29696997A JP 29696997 A JP29696997 A JP 29696997A JP H11130801 A JPH11130801 A JP H11130801A
- Authority
- JP
- Japan
- Prior art keywords
- heparin
- low
- activity
- weight
- molecular weight
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、ヘパリンを解重合
して低分子ヘパリンを製造する方法に関する。The present invention relates to a method for producing low molecular weight heparin by depolymerizing heparin.
【０００２】[0002]
【従来の技術】ヘパリンは、汎発性血管内血液凝固症や
各種血栓性疾患の治療・予防や、血液透析や人工心肺な
どの体外循環時の血液凝固防止などに広く用いられてい
るが、一方では、出血傾向を助長し、血小板・脂質系に
対する影響も大きく、臨床上、使い易い医薬品ではな
い。近時、ヘパリンに亜硝酸処理・過酸化物処理やヘパ
リン分解酵素などの酵素処理によって、平均分子量５０
００前後の低分子ヘパリンが見出され、通常のヘパリン
に比べて優れた抗血栓効果を有しながら、出血の危険性
が極めて少ないことが報告されている。2. Description of the Related Art Heparin is widely used for the treatment and prevention of generalized intravascular coagulation and various thrombotic diseases, and for preventing blood coagulation during extracorporeal circulation such as hemodialysis and cardiopulmonary bypass. On the other hand, it promotes bleeding tendency and has a large effect on the platelet / lipid system, and is not a clinically easy-to-use drug. In recent years, heparin has been treated with nitrite, peroxide, or enzymatic treatment such as heparin-degrading enzyme to give an average molecular weight of 50.
A low molecular weight heparin of about 00 has been found, and it is reported that the risk of bleeding is extremely low while having an excellent antithrombotic effect as compared with normal heparin.
【０００３】[0003]
【発明が解決しようとする課題】ヘパリンから低分子ヘ
パリンを製造する方法には、分子量の差に基づくゲル濾
過法、亜硝酸や過酸化水素などを用いた化学的解重合
法、ヘパリン分解酵素などの特異酵素による酵素的解重
合法などがあるが、原料ヘパリンの製造条件、原材料に
よっては均質な低分子ヘパリン、特に抗トロンビン活性
の安定な低分子ヘパリンを得るのが困難であった。The method for producing low-molecular-weight heparin from heparin includes a gel filtration method based on a difference in molecular weight, a chemical depolymerization method using nitrous acid or hydrogen peroxide, a heparin-degrading enzyme, and the like. However, it is difficult to obtain a homogeneous low-molecular-weight heparin, particularly a low-molecular-weight heparin having a stable antithrombin activity, depending on the production conditions of the raw material heparin and the raw material.
【０００４】[0004]
【課題を解決するための手段】本発明は、ヘパリンを低
分子化する方法について鋭意検討した結果完成されたも
のであって、ヘパリン中の硫酸基置換ウロン酸の含有％
に応じて過酸化物の量を規制することによって、抗トロ
ンビン活性の安定な低分子ヘパリンを製造することがで
きることを知見した。即ち本発明は、ヘパリンを２価の
金属塩の存在下に過酸化物を用いて低分子化する方法で
あり、ヘパリン重量Ｚに対して次式で定められる過酸化
物重量Ｘの存在下に、６０〜８０℃で３〜８時間処理す
ることにより、抗第Ｘａ因子活性／抗トロンビン活性の
活性比が１．５〜３．０の低分子ヘパリンを得るもので
ある。Ｘ=Ｙ／100×Ｚ×Ｗ（式中、Ｙはヘパリン中の硫
酸基置換ウロン酸の含有％を、Ｗは1〜1／25を示す）DISCLOSURE OF THE INVENTION The present invention has been completed as a result of intensive studies on a method for reducing the molecular weight of heparin.
It has been found that by regulating the amount of peroxide according to the above, it is possible to produce a low-molecular-weight heparin having a stable antithrombin activity. That is, the present invention is a method for depolymerizing heparin using a peroxide in the presence of a divalent metal salt, wherein the heparin is dissolved in the presence of a peroxide weight X defined by the following formula with respect to the heparin weight Z. By treating at 60 to 80 ° C. for 3 to 8 hours, a low-molecular-weight heparin having an activity ratio of anti-factor Xa activity / antithrombin activity of 1.5 to 3.0 is obtained. X = Y / 100 × Z × W (wherein, Y represents the content of sulfated uronic acid in heparin, and W represents 1-1 / 25)
【０００５】[0005]
【発明の実施の形態】以下、本発明の実施の形態につい
て説明する。本発明における原料のヘパリンは、ウシ、
ブタなどいずれの動物由来のものでもよく、また、これ
らの動物の腸、肺などいずれの臓器由来のものでも用い
ることができる。ヘパリンは、ウロン酸とグルコサミン
が交互に１，４結合しており、ウロン酸の７０〜９０％
がイズロン酸で、残りがグルクロン酸であると考えられ
ている。Embodiments of the present invention will be described below. Heparin as a raw material in the present invention, bovine,
It may be derived from any animal such as pig, and may be derived from any organ such as intestine or lung of these animals. In heparin, uronic acid and glucosamine are alternately 1,4 bonded, and 70-90% of uronic acid
Is believed to be iduronic acid and the remainder to be glucuronic acid.
【０００６】本発明においては、このウロン酸のうち、
硫酸基置換のあるウロン酸の含有割合に応じて過酸化物
の使用量を規制することによって、望ましい品質の低分
子ヘパリンを安定的に製造することができる。なお、硫
酸基置換のあるウロン酸の含有割合は、例えばプロトン
核磁気共鳴分光（1ＨＮＭＲ）法によって測定し、算出
したものを使用する。In the present invention, among these uronic acids,
By regulating the amount of peroxide to be used in accordance with the content ratio of uronic acid having a sulfate group, low-molecular-weight heparin having desired quality can be stably produced. The content ratio of uronic acid having a sulfate group is measured and measured by, for example, proton nuclear magnetic resonance spectroscopy ( 1 HNMR) and used.
【０００７】過酸化物の使用量Ｘは、ヘパリン中の硫酸
基置換ウロン酸の含有％をＹ、ヘパリン重量をＺとする
と、Ｘ=Ｙ／100×Ｚ×Ｗで得られる量とするのがよい。
ここにＷは、1〜1／25、望ましくは4／5〜1／5である。
反応は水溶液中で行うのが望ましく、ヘパリン濃度とし
て１〜１０w/v％程度の範囲内で行うのがよい。必要に
応じ、反応液にアスコルビン酸等を適当量添加してもよ
い。反応温度は６０〜８０℃、望ましくは７０〜７５℃
にコントロールしながら行うのがよい。反応時間は３〜
８時間、望ましくは４〜６時間で終了するように行う。
また、反応液のｐＨは７〜８、望ましくは７．５〜７．
８に保ちながら行うのがよい。[0007] The amount X of peroxide used should be such that X = Y / 100 × Z × W, where Y is the content of sulfated uronic acid in heparin and Z is the weight of heparin. Good.
Here, W is 1 to 1/25, preferably 4/5 to 1/5.
The reaction is desirably performed in an aqueous solution, and is preferably performed in a heparin concentration range of about 1 to 10 w / v%. If necessary, an appropriate amount of ascorbic acid or the like may be added to the reaction solution. The reaction temperature is 60 to 80 ° C, preferably 70 to 75 ° C
It is good to control while controlling. Reaction time is 3 ~
The reaction is carried out in 8 hours, preferably 4 to 6 hours.
The pH of the reaction solution is 7 to 8, preferably 7.5 to 7.
It is better to keep it at 8.
【０００８】本発明における低分子ヘパリンの製造法で
は、２価の金属塩と過酸化物を用いる。ここに、２価の
金属塩としては、銅イオン、鉄イオン等の２価のイオン
を生成する、例えば酢酸銅、硫酸銅等の塩を使用する。
過酸化物としては、具体的には、過酸化水素等を使用す
る。このようにして得られる低分子ヘパリンの平均分子
量は、ＨＰＬＣ法で測定して、重量平均分子量として４
０００〜６５００である。In the method for producing low molecular weight heparin of the present invention, a divalent metal salt and a peroxide are used. Here, as the divalent metal salt, a salt which generates divalent ions such as copper ions and iron ions, for example, salts such as copper acetate and copper sulfate is used.
As the peroxide, specifically, hydrogen peroxide or the like is used. The average molecular weight of the low-molecular-weight heparin obtained in this way was measured by an HPLC method, and was found to be 4 as a weight average molecular weight.
000-6500.
【０００９】[0009]
【実施例】次に、本発明の実施例を挙げ、本発明の生じ
る作用効果を含めて具体的に説明する。Next, examples of the present invention will be described in detail, including the functions and effects produced by the present invention.
【００１０】（実施例１）硫酸基置換ウロン酸の含有割
合が７０％であるヘパリン３ｇを含む３w/v％塩化ナト
リウム、３w/v％酢酸ナトリウム三水和物混液２１０ｍ
Ｌに１．７５w/v％アスコルビン酸水溶液２０ｍＬ、
０．４５w/v％酢酸銅一水和物１０ｍＬを加えた。次に
３１w/v％過酸化水素水１．８ｍＬを加え、３０分間室
温で撹拌した後、さらに７０〜７５℃で８時間撹拌し
た。なお、この時反応混液中のｐＨが約７．８になるよ
うに１mol/L水酸化ナトリウムで調整した。(Example 1) 210 g of a 3 w / v% sodium chloride, 3 w / v% sodium acetate trihydrate mixed solution containing 3 g of heparin containing 70% of a sulfuric acid-substituted uronic acid
20 mL of 1.75 w / v% ascorbic acid aqueous solution to L
10 mL of 0.45 w / v% copper acetate monohydrate was added. Next, 1.8 mL of 31 w / v% hydrogen peroxide solution was added, and the mixture was stirred at room temperature for 30 minutes, and further stirred at 70 to 75 ° C. for 8 hours. At this time, the mixture was adjusted with 1 mol / L sodium hydroxide so that the pH in the reaction mixture was about 7.8.
【００１１】反応後、約１２０ｍＬに減圧濃縮（約１５
mmHg）し、エチレンジアミン四酢酸二ナトリウム(ＥＤ
ＴＡ)１．５ｇを加え、１mol/L水酸化ナトリウムでｐＨ
７に調整し、常法により、メタノールにより遠心分離し
た。メタノールによる遠心分離を２回繰り返し、最後に
得られた白色沈澱につき減圧乾燥（１５mmHg）し、平均
分子量５８００の低分子ヘパリン１．９ｇ（重量収率６
３％）を得た。この低分子ヘパリンについて、“４０℃
で６箇月保存後”における抗第Ｘａ因子活性／抗トロン
ビン活性の活性比は１．９と変わらなかった。なお、ｐ
Ｈの測定には「ｐＨ計“ＨＭ−６０Ｓ”（東亜電波工業
(株)製）」を使用し、遠心分離には「高速冷却遠心機
“ＣＲ２２Ｅ”（日立工機(株)製）」を使用した。After the reaction, the solution is concentrated under reduced pressure to about 120 mL (about 15 mL).
mmHg) and disodium ethylenediaminetetraacetate (ED
TA) 1.5 g and pH was adjusted with 1 mol / L sodium hydroxide.
7 and centrifuged with methanol in a conventional manner. The centrifugation with methanol was repeated twice, and the finally obtained white precipitate was dried under reduced pressure (15 mmHg), and 1.9 g of low-molecular-weight heparin having an average molecular weight of 5,800 (weight yield: 6).
3%). About this low molecular weight heparin, “40 ° C.
After storage for 6 months, the activity ratio of anti-factor Xa activity / antithrombin activity was unchanged at 1.9.
For the measurement of H, use the "HM-60S pH meter" (Toa Denpa Kogyo
And a high-speed cooling centrifuge “CR22E” (manufactured by Hitachi Koki Co., Ltd.).
【００１２】（実施例２〜５）試薬の量、反応温度、反
応時間を表１のように変化させ、前記実施例１と同様に
して、ヘパリンの解重合を行った。なお、ｐＨの測定及
び遠心分離には、実施例１と同じ機器を使用した。(Examples 2 to 5) Heparin was depolymerized in the same manner as in Example 1 except that the amounts of the reagents, the reaction temperature and the reaction time were changed as shown in Table 1. The same equipment as in Example 1 was used for pH measurement and centrifugation.
【００１３】[0013]
【表１】 [Table 1]
【００１４】[0014]
【発明の効果】本発明によれば、原料ヘパリンの製造条
件、原材料に関係なく均質な低分子ヘパリンを得ること
ができる。しかも、この低分子ヘパリンは、４０℃、６
箇月の保存に対しても安定であった。According to the present invention, homogeneous low-molecular-weight heparin can be obtained irrespective of the production conditions of the raw material heparin and the raw materials. Moreover, this low-molecular-weight heparin can be used at 40 ° C.
It was stable for months of storage.
Claims (1)
化物を用いて低分子化する方法において、ヘパリン重量
Ｚに対して次式で定められる過酸化物重量Ｘの存在下
に、６０〜８０℃で３〜８時間処理することを特徴とす
る、抗第Ｘａ因子活性／抗トロンビン活性の活性比が
１．５〜３．０の低分子ヘパリンの製造法。 Ｘ=Ｙ／100×Ｚ×Ｗ （式中、Ｙはヘパリン中の硫酸基置換ウロン酸の含有％
を、Ｗは1〜1／25を示す）1. A method for depolymerizing heparin using a peroxide in the presence of a divalent metal salt, comprising the steps of: A process for producing low-molecular-weight heparin having an activity ratio of anti-factor Xa activity / antithrombin activity of 1.5 to 3.0, which is performed at 60 to 80 ° C for 3 to 8 hours. X = Y / 100 × Z × W (wherein Y is the content of sulfated uronic acid in heparin in%
, W represents 1-1 / 25)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP29696997A JPH11130801A (en) | 1997-10-29 | 1997-10-29 | Production of low-molecular weight heparin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP29696997A JPH11130801A (en) | 1997-10-29 | 1997-10-29 | Production of low-molecular weight heparin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH11130801A true JPH11130801A (en) | 1999-05-18 |
Family
ID=17840555
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP29696997A Pending JPH11130801A (en) | 1997-10-29 | 1997-10-29 | Production of low-molecular weight heparin |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH11130801A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001240607A (en) * | 2000-02-29 | 2001-09-04 | Fuso Pharmaceutical Industries Ltd | Method for depolymerizing heparin, depolymerized heparin, derivative thereof and pharmaceutical composition |
| JP2002293804A (en) * | 2001-03-28 | 2002-10-09 | Itoham Foods Inc | Method for producing low molecular weight heparin |
| CN105461830A (en) * | 2016-01-08 | 2016-04-06 | 东营天东制药有限公司 | Method for removing copper ions through poly-chelate resin |
-
1997
- 1997-10-29 JP JP29696997A patent/JPH11130801A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001240607A (en) * | 2000-02-29 | 2001-09-04 | Fuso Pharmaceutical Industries Ltd | Method for depolymerizing heparin, depolymerized heparin, derivative thereof and pharmaceutical composition |
| JP2002293804A (en) * | 2001-03-28 | 2002-10-09 | Itoham Foods Inc | Method for producing low molecular weight heparin |
| CN105461830A (en) * | 2016-01-08 | 2016-04-06 | 东营天东制药有限公司 | Method for removing copper ions through poly-chelate resin |
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