JPS6331446B2 - - Google Patents
Info
- Publication number
- JPS6331446B2 JPS6331446B2 JP57191820A JP19182082A JPS6331446B2 JP S6331446 B2 JPS6331446 B2 JP S6331446B2 JP 57191820 A JP57191820 A JP 57191820A JP 19182082 A JP19182082 A JP 19182082A JP S6331446 B2 JPS6331446 B2 JP S6331446B2
- Authority
- JP
- Japan
- Prior art keywords
- urease
- solution
- freeze
- activity
- nac
- 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
Links
- 108010046334 Urease Proteins 0.000 claims description 28
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 239000003381 stabilizer Substances 0.000 claims description 6
- 150000002016 disaccharides Chemical class 0.000 claims description 3
- 229940071106 ethylenediaminetetraacetate Drugs 0.000 claims description 2
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 claims 1
- 229960004308 acetylcysteine Drugs 0.000 claims 1
- 230000000694 effects Effects 0.000 description 16
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 12
- 239000000243 solution Substances 0.000 description 10
- 238000004108 freeze drying Methods 0.000 description 8
- 238000004090 dissolution Methods 0.000 description 7
- 108010024636 Glutathione Proteins 0.000 description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 6
- 229930006000 Sucrose Natural products 0.000 description 6
- 229960003180 glutathione Drugs 0.000 description 6
- 239000005720 sucrose Substances 0.000 description 6
- -1 thiol compounds Chemical class 0.000 description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 6
- 239000000203 mixture Substances 0.000 description 5
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 4
- 235000018417 cysteine Nutrition 0.000 description 4
- 239000004372 Polyvinyl alcohol Substances 0.000 description 3
- 229920002451 polyvinyl alcohol Polymers 0.000 description 3
- 229910000160 potassium phosphate Inorganic materials 0.000 description 3
- 235000011009 potassium phosphates Nutrition 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- ABBQHOQBGMUPJH-UHFFFAOYSA-M Sodium salicylate Chemical compound [Na+].OC1=CC=CC=C1C([O-])=O ABBQHOQBGMUPJH-UHFFFAOYSA-M 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- XEYBHCRIKKKOSS-UHFFFAOYSA-N disodium;azanylidyneoxidanium;iron(2+);pentacyanide Chemical compound [Na+].[Na+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].[O+]#N XEYBHCRIKKKOSS-UHFFFAOYSA-N 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 229940083618 sodium nitroprusside Drugs 0.000 description 2
- 229960004025 sodium salicylate Drugs 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 239000007992 BES buffer Substances 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
Landscapes
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
本発明は、安定化したウレアーゼ製剤に関する
ものである。
ウレアーゼは特異的に尿素をアンモニアと二酸
化炭素に分解する酵素として、これまで広く利用
されてきたが、近年、特に、腎機能疾患の重要な
指標のひとつである生体成分中の尿素の測定に多
く使われ、その重要性が高まつている。
しかし、高度に精製されたウレアーゼは、非常
に不安定であり、これまで種々の安定化の試みが
なされているが、いづれも満足できる結果は得ら
れていない。例えば、安定剤としてグルタチオ
ン、エチレンジアミンテトラ酢酸塩（以下、
EDTAと略す）及びクエン酸塩の一定重量比に
よる混合物を含有する調製物（特開昭52−11748
号）あるいは、チオール化合物、キレート試薬、
及び有機二塩基酸またはその塩の混合物を含有す
るウレアーゼ組成分（特開昭57−138389号）など
が提案されているが十分な効果は上がつていな
い。
本発明者らは、鋭意、研究した結果、Ｎ−アセ
チルステイン（以下、NACと略す）EDTA、お
よびシユークロース等の二糖類を加えることによ
り、従来の安定化法に比し、著しく安定なウレア
ーゼ製剤が得られることを見出し、本発明を完成
した。
本発明の特徴のひとつは、ウレアーゼの安定化
剤として、シユークロース、ラクトース等の二糖
類を用いることにある。これによつて後述の試験
例２に示すように37℃に２ケ月保存後も溶解直後
のウレアーゼ活性についてはチオール化合物の有
無あるいは種類に関係なくほゞ100％のウレアー
ゼ残存活性率を示す安定な製剤が得られた。
又、一般にチオール化合物が、ウレアーゼの安
定化に効果があることは古くから知られており、
なかでもグルタチオンがよく使われているが、
（特開昭52−117488号特開昭57−138389号）本発
明者らの詳細な検討の結果、試験例２に示すよう
に、NACが最もすぐれていることが判明した。
本発明における安定なウレアーゼ製剤とは、例
えば、緩衝液に安定化剤を加えて溶解し、PHを調
整（望ましくはPH6.5）した後、ウレアーゼを溶
解した溶液を凍結乾燥して得られるものである。
次に、実施例について説明するが本発明はこれ
によつて限定されるものではない。
実施例 １
リン酸カリウム 50ｍＭ
EDTA 100ｍＭ
NAC 20ｍＭ
シユークロース ２ｇ／dl
ラクトース ２ｇ／dl
以上の溶液のPHを6.5に調整した後ウレアーゼ
90u／mlを加え凍結乾燥して安定なウレアーゼ製
剤を得る。
実施例 ２
リン酸カリウム 25ｍＭ
EDTA 25ｍＭ
NAC ５ｍＭ
シユークロース ２ｇ／dl
以上の溶液のPHを6.5に調整した後ウレアーゼ
22.5u／mlを加え凍結乾燥して安定なウレアーゼ
製剤を得る。
次に試験例により本発明の効果について説明す
る。
試験例 １
リン酸カリウム10ｍＭ、NAC4ｍＭ、EDTA40
ｍＭの溶液のPHを6.5に調整後、ウレアーゼを
40u／mlとなるように加え、更に、表１に示す安
定化剤(1)シユークロース、(2)フイコール（フアル
マシア社製品名）、(3)KClをそれぞれ２ｇ／dlと
なるよう加え、凍結乾燥後そのウレアーゼ活性を
凍結乾燥直後および一定期間（95日）37℃に保存
したものについて測定した結果を表１に示す。
こゝで溶解には緩衝液20ｍＭ BESにサリチル
酸ナトリウム２％、ニトロプルシツドナトリウム
0.15％、ポリビニルアルコール0.4％を添加した
溶液を用いた。数値はそれぞれ凍結乾燥前のウレ
アーゼ活性を100とした場合の残存活性率を表わ
す。
表１から安定化剤としてシユークロースが最も
すぐれていることが分る。
The present invention relates to stabilized urease formulations. Urease has been widely used as an enzyme that specifically decomposes urea into ammonia and carbon dioxide, but in recent years it has been used extensively for measuring urea in biological components, which is one of the important indicators of renal function disease. used and its importance is increasing. However, highly purified urease is extremely unstable, and although various attempts have been made to stabilize it, no satisfactory results have been obtained. For example, as a stabilizer, glutathione, ethylenediaminetetraacetate (hereinafter referred to as
A preparation containing a mixture of EDTA (abbreviated as EDTA) and citrate in a fixed weight ratio (JP-A-11748
No.) or thiol compounds, chelating reagents,
Although urease compositions containing a mixture of organic dibasic acids and organic dibasic acids or their salts (Japanese Patent Laid-open No. 138389/1983) have been proposed, sufficient effects have not been achieved. As a result of extensive research, the present inventors have found that by adding N-acetylsteine (hereinafter abbreviated as NAC), EDTA, and disaccharides such as sucrose, the urease preparation is significantly more stable than conventional stabilization methods. The present invention was completed based on the discovery that the following can be obtained. One of the features of the present invention is the use of disaccharides such as sucrose and lactose as urease stabilizers. As a result, as shown in Test Example 2 below, even after storage at 37°C for 2 months, the urease activity immediately after dissolution is stable, showing almost 100% residual urease activity regardless of the presence or absence of thiol compounds or the type of thiol compound. A formulation was obtained. In addition, it has been known for a long time that thiol compounds are generally effective in stabilizing urease.
Among them, glutathione is often used,
(JP-A-52-117488 and JP-A-57-138389) As a result of detailed study by the present inventors, as shown in Test Example 2, NAC was found to be the most excellent. The stable urease preparation in the present invention is, for example, one obtained by adding and dissolving a stabilizer in a buffer solution, adjusting the pH (preferably to PH 6.5), and then freeze-drying the solution in which urease is dissolved. It is. Next, examples will be described, but the present invention is not limited thereto. Example 1 Potassium phosphate 50mM EDTA 100mM NAC 20mM Sucrose 2g/dl Lactose 2g/dl After adjusting the pH of the solution to 6.5, urease
Add 90u/ml and lyophilize to obtain a stable urease preparation. Example 2 Potassium phosphate 25mM EDTA 25mM NAC 5mM Sucrose After adjusting the pH of a solution with 2g/dl or more to 6.5, urease
Add 22.5u/ml and lyophilize to obtain a stable urease preparation. Next, the effects of the present invention will be explained using test examples. Test example 1 Potassium phosphate 10mM, NAC4mM, EDTA40
After adjusting the pH of the mM solution to 6.5, add urease.
In addition, stabilizers shown in Table 1 (1) Seuucrose, (2) Ficoll (product name of Pharmacia), and (3) KCl were added to a concentration of 2 g/dl each, and freeze-dried. The urease activity was then measured immediately after freeze-drying and on those stored at 37°C for a certain period (95 days). Table 1 shows the results.
For dissolution, add 2% sodium salicylate and sodium nitroprusside in 20mM BES buffer.
A solution containing 0.15% polyvinyl alcohol and 0.4% polyvinyl alcohol was used. Each numerical value represents the residual activity rate when the urease activity before freeze-drying is set as 100. It can be seen from Table 1 that sucrose is the best stabilizer.
【表】
試験例 ２
実施例２のウレアーゼ製剤および比較のためチ
オール化合物として実施例２のNACの替りにグ
ルタチオン、システインをそれぞれ同量（５ｍ
Ｍ）用いた場合及び無添加の場合について試験例
１と同様ウレアーゼ活性を測定した結果を表２に
示す。こゝで溶解には緩衝液20ｍＭ HEPESに
サリチル酸ナトリウム1.6％、ニトロプルシツド
ナトリウム0.12％、ポリビニルアルコール0.4％
を添加した溶液（PH6.8）を用いた。
数値はそれぞれ凍結乾燥前のウレアーゼ活性を
100とした場合の残存活性率を表わす[Table] Test Example 2 The same amount of glutathione and cysteine (5 m
Table 2 shows the results of measuring the urease activity in the same manner as in Test Example 1 when M) was used and when it was not added. For dissolution, add 20mM HEPES buffer, 1.6% sodium salicylate, 0.12% sodium nitroprusside, and 0.4% polyvinyl alcohol.
A solution (PH6.8) was used. Each value represents the urease activity before freeze-drying.
Represents the residual activity rate when set to 100
【表】
この表から明らかなように溶解直後のウレアー
ゼ活性はいずれもほゞ100％であるがこれはシユ
ークロース、EDTAの効果によるものと考えら
れる。これに反し37℃で２ケ月保存したものは溶
解後の溶液でのウレアーゼの安定性が添加するチ
オール化合物によつて著しく差があり、グルタチ
オン、システインの場合はNACに較べ劣化して
いる。すなわちNACの場合は溶液として30℃７
日保存後の残存活性率が凍結乾燥直後のものでは
69.4％、凍結乾燥後37℃で63日保存したものでは
67.4％とその低下がほとんど変らない。グルタチ
オンの場合は前者は69.3％、後者は15.1％と著し
く低下が大きい。またシステインの場合は前者は
18.6％、後者は10.3％と双方とも残存活性率は低
い。
このことはNAC添加のものは凍結乾燥直後と、
37℃、63日保存後とで品質的に変化がないことを
示し、グルタチオンの場合は37℃、63日保存中に
品質が劣化したとを示している。システインの場
合は溶液保存中の劣化が大きく凍結乾燥品の保存
中の変化は問題にならない。
試験例 ３
実施例１のウレアーゼ製剤を37℃で保存した場
合のウレアーゼ活性の安定性を測定した結果を表
３に示す。
ここで溶解液には試験例２と同様のものを用
い、数値の表示も同様とする。[Table] As is clear from this table, the urease activity immediately after dissolution was almost 100% in all cases, which is thought to be due to the effects of sucrose and EDTA. On the other hand, when stored at 37°C for 2 months, the stability of urease in the solution after dissolution differed markedly depending on the thiol compound added, and in the case of glutathione and cysteine, it deteriorated compared to NAC. In other words, in the case of NAC, the temperature is 30℃7 as a solution.
The residual activity after storage for 1 day is not the same as that immediately after freeze-drying.
69.4%, when stored at 37℃ for 63 days after freeze-drying
The decline remains almost unchanged at 67.4%. In the case of glutathione, the former was 69.3% and the latter 15.1%, which was a significant decrease. In the case of cysteine, the former is
The residual activity rate of both is low at 18.6% and 10.3% for the latter. This means that the NAC-added product is immediately after freeze-drying.
It shows that there is no change in quality after storage at 37°C for 63 days, and in the case of glutathione, the quality deteriorates during storage at 37°C for 63 days. In the case of cysteine, the deterioration is large during solution storage, and changes during storage of lyophilized products are not a problem. Test Example 3 Table 3 shows the results of measuring the stability of urease activity when the urease preparation of Example 1 was stored at 37°C. Here, the same dissolving solution as in Test Example 2 is used, and the numerical values are displayed in the same manner.
【表】
この表３から本発明のウレアーゼ製剤は37℃、
約６ケ月保存後においてもなお溶解直後のウレア
ーゼ活性は勿論、溶解後の保存性についても、凍
結乾燥直後とほとんど品質に変わりがなく非常に
安定である。[Table] From this Table 3, the urease preparation of the present invention is
Even after storage for about 6 months, the urease activity immediately after dissolution as well as the storage stability after dissolution are very stable with almost no difference in quality compared to immediately after lyophilization.
Claims (1)
テイン、エチレンジアミンテトラ酢酸塩、および
二糖類を含有することを特徴とする安定なウレア
ーゼ製剤。1. A stable urease preparation containing N-acetylcysteine, ethylenediaminetetraacetate, and a disaccharide as urease stabilizers.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57191820A JPS5982318A (en) | 1982-11-02 | 1982-11-02 | Stable pharmaceutical preparation of urease |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57191820A JPS5982318A (en) | 1982-11-02 | 1982-11-02 | Stable pharmaceutical preparation of urease |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5982318A JPS5982318A (en) | 1984-05-12 |
| JPS6331446B2 true JPS6331446B2 (en) | 1988-06-23 |
Family
ID=16281062
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP57191820A Granted JPS5982318A (en) | 1982-11-02 | 1982-11-02 | Stable pharmaceutical preparation of urease |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5982318A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20200040323A1 (en) * | 2018-07-31 | 2020-02-06 | Fresenius Medical Care Holdings, Inc. | Urease Purification And Purified Urease Products Thereof And Sorbent Cartridges, Systems And Methods Using The Same |
-
1982
- 1982-11-02 JP JP57191820A patent/JPS5982318A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5982318A (en) | 1984-05-12 |
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