JPS5982318A - Stable pharmaceutical preparation of urease - Google Patents

Abstract

PURPOSE:The titled pharmaceutical preparation containing N-acetylcysteine, an ethylenediaminetetraacetate, and a disaccharide as a stabilizer for urease. CONSTITUTION:A stabilizer of N-acetylcysteine, an ethylene-diaminetetraacetate, and a disaccharide (e.g., sucrose, lactose, etc.) is dissolved in a buffer solution, and adjusted to 6.5 pH. Urease is dissolved in it, and the solution is lyophilized, to give a stable pharmaceutical preparation of urease. A large amount of urease is used especially in measurement of urea in organism components, one of important index of liver function diseases, and its importance has been raised. Highly purified urease is very unstable, however, the pharmaceutical preparation has urease activity corresponding to activity immediately after dissolution even after it is preserved at 37 deg.C for 6 months, it has quality almost equal to that immediately after lyophilization with respect to shelf stability after dissolution, and it is stable.

Description

[Detailed description of the invention] There is something to do.

Urease has been widely used as an enzyme that specifically decomposes urea into ammonia and carbon dioxide.
In recent years, it has become increasingly important, especially in the measurement of urea in biological components, which is one of the important indicators of renal function diseases.

However, urease purified at high altitudes is extremely unstable, and although various attempts have been made to stabilize it, no satisfactory results have been obtained. for example,
A preparation containing a mixture of glutathione, ethylenediaminetetraacetate (hereinafter abbreviated as EDTA) and citrate in a constant weight ratio as a stabilizer (JP-A-52-11)
No. 748) or thiol compounds, chelating reagents,
A urease composition containing a mixture of organic dibasic acid or a salt thereof (Japanese Unexamined Patent Publication No. 57-138389) has been proposed, but sufficient effects have not been achieved.

The present inventors have diligently researched N-acetylstine (hereinafter abbreviated as NAC) EDTA.

By adding trisaccharides such as and sucrose,
It was discovered that a significantly more stable urease preparation could be obtained compared to conventional stabilization methods, and the invention was completed.

One of the uninvented features is the use of disaccharides such as sucrose and lactose as urease stabilizers. It depends on the use. As a result, as shown in Test Example 2 below;
Even after storage at 7°C for 2 months, a stable preparation showing urease activity of 00% immediately after dissolution was obtained, regardless of the presence or absence of a thiol compound or the type of thiol compound.

In addition, it has been known for a long time that thiol compounds are generally less effective in stabilizing urease, and among them, glutathione is often used;
As a result of detailed study by the present inventors, as shown in Test Example 2, NAC was found to be the most excellent.

A stable lanase preparation in the present invention is, for example, prepared by adding a stabilizer to a buffer solution, dissolving it in a bath, adjusting the pH (desired pH 6.5), and then freeze-drying the solution in which lanase gold is dissolved. − is obtained.

Next, examples will be described, but the present invention is not limited thereto.

Example 1 Potassium phosphate 5QmM EDTA 100mMNAC20m
M Sucrose 2r/dz Lactose 21i'/dt After adjusting the pH of the solution to 6.5, 90 u of urease/gold was freeze-dried to obtain a stable urease preparation.

Example 2 Potassium phosphate 25mM EDTA 25 tnMNAo
5mM sucrose 217dt The total pH of the solution was adjusted to 6.5, and after fc urease 2
Stable urease preparation by adding 2゜5 u/m f and freeze-drying? obtain.

Next, the effects of the present invention will be explained using test examples.

Test example Potassium ecophosphate 10mM, NAC 4mM, E
After adjusting the pH of the DTA 40 tnM solution to 6.5, urease was added to a total of 40 u/-, and the stabilizers shown in Table 1 (1) Sucrose, (2) Ficoll (product name of Pharmacia) were added. , +3) Kα was added to each 2'if/dt, and the urease activity after freeze-drying was increased for a certain period of time (95 days) immediately after freeze-drying.
Table 1 shows the results of measurements on samples stored at 37°C. For dissolution, use buffer 20 mM BES, 2% sodium salicylate, nitroglucidonate l□')
A solution containing 0.15% of aluminum and 4% of polyvinyl alcohol O was used. Each numerical value represents the residual activity and percentage when the urease activity ligation before freeze-drying is 100%.

From Table 1, it can be seen that sucrose is the best stabilizer.

Test Example 2 R411 manufactured by Urease in Example 2 Test example for the case where the same amount (5ynM) of glutathione and stein grape were used instead of NAC of Example 2 as a thiol compound for comparison with J-paralysis, and the case where no additive was used. Table 2 shows the results of addition of warease activity as in 1. Dissolve with this buffer 20 tnM HEPES, 1.6% ditrium salicylate, 0.1 sodium nitrosorzodate.
2%.

A solution containing 0.4% of vinyl alcohol (pH
6, 8) was used.

The urease activity of each ton of numerical value before freeze-drying is 10
Residual activity when set to 04. As is clear from this table, the urease activity immediately after lysis is
, ioo%, which is sucrose, EDTA
This is thought to be due to the effect of On the other hand, at 37℃
When stored for several months, the stability of urease in the solution after dissolution varies markedly depending on the thiol compound added.
In the case of gluction and cysteine, they are degraded compared to 1-INA and CK. In other words, in the case of NAC, 30% as a solution
The residual activity rate after storage for 7 days at ℃ is 6 for the product immediately after freeze-drying.
9.4%, and when stored at 37°C for 63 days after freeze-drying, the decrease was almost unchanged to 67.4%.

In the case of glutathione, the former is 68.3% and the latter is 15.
This is a significant decrease of 1%. Further, in the case of cysteine, the residual activity rate of both is low, 18.6% for the former and 10.3% for the latter.

This means that for the NAC-added product, immediately after freeze-drying and at 37°C.
It shows that there is no change in quality after storage for 63 days, and in the case of glutathione, the quality deteriorates during storage for 63 days at 37°C. /stin case trj,,fB
Deterioration during liquid storage is a problem, but changes during storage of freeze-dried products are not a problem.

Test Example 3 Table 3 shows the results of measuring the stability of Vase activity when the Urease product Δ1ji of Example 1 was stored at 37°C.

Here, the same dissolving solution as in Test Example 2 was used, and the numerical values were displayed in the same manner.

Table 3 shows that the urease preparation of the present invention is very stable even after being stored at 37°C for about 6 months, with not only the urease activity immediately after dissolution, but also the storage stability after dissolution, with almost no change in quality compared to immediately after freeze-drying. And δri.

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Claims (1)

[Claims] N-acetylcysteine, ethylenediaminetetraacetate as a urease stabilizer. and a stable urease preparation characterized by containing a trisaccharide.