KR101932247B1 - Composition containing deoxycholate or salt thereof and stabilization method of enzyme - Google Patents

Abstract

The present invention relates to an enzyme; And a deoxycholic acid or a salt thereof, and a method of adding a deoxycholic acid or a salt thereof to an enzyme to prepare a composition. According to the enzyme composition of the present invention and the method of stabilizing the enzyme, the period of maintaining the activity at room temperature as well as the refrigeration after the reconstitution of the lyophilized preparation and the lyophilized preparation with the injection water is prolonged. It is possible to maintain.

Description

TECHNICAL FIELD The present invention relates to a composition comprising deoxycholinic acid or a salt thereof and a method for stabilizing an enzyme,

The present invention relates to a composition comprising deoxycholic acid or a salt thereof and a method of stabilizing the enzyme, and more particularly, to an enzyme composition which maintains stability in freeze-dried preparations and liquid preparations by containing deoxycholic acid or a salt thereof, and And a method for stabilizing an enzyme using the same.

Hyaluronidase regulates the binding gap through hydrolysis of hyaluronic acid which links cell binding in human connective tissues, promotes absorption of drug into human connective tissues, and reabsorption of body fluids exiting into connective tissue (Carbohydrase) that promotes the metabolism of carbohydrates. Chronic pain treatment (neuroprotection) and postoperative swelling and bruising.

Enzymatically-containing proteins can lose biological activity as a result of chemical instability, such as physiological instability (e.g., denaturation, formation of aggregates, etc.) and hydrolysis, oxidation, and deamidation. The stability of the protein is further influenced by factors such as pH, temperature, wall, and number of freeze-thaw cycles. To ensure stability, therapeutic protein preparations are typically fed into a protein solution containing lyophilized protein, which is dissolved just prior to use in a separately packaged aqueous diluent, or an additive to improve stability. For example, additives such as free amino acids useful for formulating protein solutions (e.g., leucine, tryptophan, serine, arginine, and histidine) have been proposed. Some protein preparations currently available in the market contain proteins as stabilizers. For example, human serum albumin or purified gelatin is used to inhibit chemical and physical changes in protein solutions. However, the addition of these proteins involves a complex process to eliminate virus contamination. Freeze drying is another method used to ensure stability; This process increases the manufacturing cost and involves the increased risk of improper administration since the lyophilized protein must be dissolved just prior to use. In addition, the purpose of lyophilization is to remove water from the formulation, since harmful physical and chemical reactions often occur in the aqueous phase. That is, freeze-drying is carried out because stability is lowered in an aqueous liquid phase. However, the freeze-dried state has a disadvantage in that there is the above-described problem and limitation in use.

Patent Registration No. 10-0330540 Patent Registration No. 10-1084412 Patent Application No. 10-2011-7004838 Patent Application No. 10-2006-7006765 Patent Application No. 10-2005-7017941 Patent Application No. 10-2011-7024336

In order to solve the above-mentioned problems, the present invention provides a method for stabilizing an enzyme having excellent stability by maintaining deoxycholic acid or a salt thereof in an enzyme in which the enzyme activity is maintained before lyophilization and after lyophilization, And the like.

Disclosure of the Invention In order to solve the above-mentioned problems, And deoxycholic acid or a salt thereof.

In one embodiment of the present invention, the enzyme may be hyaluronidase.

In one embodiment of the present invention, the content of the enzyme may be 0.01 to 50% by weight based on the total weight of the composition.

In one embodiment of the present invention, the amount of deoxycholic acid or salt thereof may be 10 to 90% by weight based on the total weight of the composition.

In one embodiment of the present invention, the composition may be a lyophilized formulation.

In one embodiment of the present invention, the composition may be a formulation of a solution to be lyophilized or a reconstituted solution form prepared from a lyophilized composition and a diluent.

In one embodiment of the present invention, the deoxycholic acid or its salt may be sodium deoxycholinate.

In one embodiment of the present invention, the composition may be stable at a temperature of 20-70 < 0 > C and a relative humidity of 10-90%.

In one embodiment of the present invention, the enzyme of the solution may be maintained at a temperature of 20 to 70 DEG C and at a relative humidity of 10 to 90%.

The present invention also provides a method of stabilizing an enzyme comprising adding deoxycholic acid or a salt thereof to the enzyme.

In one embodiment of the invention, the stabilization method comprises: after the composition is prepared, lyophilizing the prepared composition; As shown in FIG.

In one embodiment of the present invention, the stabilization method may further comprise, after lyophilization, dissolving the lyophilized composition in a diluent to prepare a solution.

According to the enzyme composition of the present invention and the method of stabilizing the enzyme, the period of maintaining the activity at room temperature as well as the refrigeration after the reconstitution of the lyophilized preparation and the lyophilized preparation with the injection water is prolonged. And it is possible to improve the stability of the enzyme upon packaging the composition and reduce the amount of the composition adsorbed on the vial.

1 is a graph comparing the stability of hyaluronidase of a composition according to an embodiment of the present invention.
2 is a graph comparing the stability of hyaluronidase after storage of the composition according to an embodiment of the present invention at a temperature of 25 ° C and a relative humidity of 60%.
3 is a graph comparing the stability of hyaluronidase after storage of the composition according to an embodiment of the present invention at a temperature of 40 ° C and a relative humidity of 75%.

Hereinafter, preferred embodiments of the present invention will be described in detail in order to facilitate the present invention by those skilled in the art.

The present invention relates to an enzyme; And deoxycholic acid or a salt thereof.

In the present invention, the enzyme is not particularly limited, but may be a carbohydrase. The carbohydrate enzyme may be, for example, at least one selected from amylase, chondroitinase, and hyaluronidase. The enzyme may preferably be hyaluronidase.

The content of the enzyme may be 0.01 to 50% by weight, preferably 0.02 to 20% by weight, based on the total weight of the composition. Alternatively, the enzyme may contain 1 to 3,000 IU of enzyme per vial. When the content of the enzyme is less than 0.01% by weight, the decomposition efficiency of the enzyme is insignificant. For example, the effect of degrading hyaluronic acid in the body, which is the main purpose of hyaluronidase, may become insignificant. In addition, if it exceeds 50% by weight, an allergic reaction may occur due to excessive injection.

In the present invention, the deoxycholic acid or its salt is not particularly limited, but may be any one of deoxycholic acid, rusodooxycholic acid, chenodeoxycholic acid and salts thereof derived from bile acid. It may preferably be sodium deoxycholate.

The amount of the deoxycholic acid or its salt may be 10 to 90% by weight, preferably 30 to 90% by weight, based on the total weight of the composition. Or 0.0067 to 200 mg per vial. When the deoxycholic acid salt is less than 10% by weight in the combination with the enzyme, the storage stability of the enzyme is the lowest level at which the storage stability is guaranteed, and when it exceeds 90% by weight, the storage stability is the highest level.

In the present invention, the composition may be a lyophilized formulation. Such lyophilized formulations include enzymes; And deoxycholic acid or a salt thereof is a lyophilized solid preparation.

Further, in the present invention, the composition may be a formulation of a solution to be lyophilized or a reconstituted solution prepared from a lyophilized composition and a diluent. That is, it is a solution before freeze-drying containing a solvent in addition to an enzyme and deoxycholic acid or a salt thereof, or a liquid preparation which is reconstituted by dissolving a lyophilized solid preparation in a diluent. The diluent may be water for injection.

The concentration of the enzyme in the liquid preparation is preferably 1 to 3,000 IU / mL, more preferably 15 to 1,500 IU / mL, and most preferably 15 IU / mL. If the enzyme is less than 1 IU / mL, the enzyme, for example hyaluronidase, which is the major component of hyaluronidase, can not be degraded. If it is more than 3,000 IU / mL, allergic reaction may occur due to overdose.

When the composition containing the enzyme is a lyophilized solid preparation, the stability with respect to the activity of the enzyme is excellent. When the composition containing the enzyme is a liquid preparation, the activity stability of the enzyme is reduced as compared with the solid preparation, but the composition according to the present invention maintains the activity stability of the enzyme even in the liquid preparation. Specifically, the composition according to the present invention maintains excellent stability at a temperature of 20 to 70 캜 and a relative humidity of 10 to 90%, regardless of the formulation of the solid preparation or the liquid preparation. More specifically, it may be a temperature of 25 to 45 캜, a temperature of 30 to 40 캜, and may be a relative humidity of 50 to 90% and 65 to 75%. That is, the enzyme activity of the conventional enzyme composition is lost or decreased in an environment of high temperature and high humidity, while the present invention has excellent stability under high temperature and high humidity environment. This can be confirmed in Test Example 2 to be described later.

The present invention also provides a method of stabilizing an enzyme using a composition prepared by adding deoxycholic acid or a salt thereof to an enzyme. Hereinafter, a method of stabilizing the enzyme of the present invention will be specifically described.

First, the composition is prepared by adding deoxycholic acid or a salt thereof to the enzyme. The enzyme is preferably present in an amount of 0.01 to 50% by weight, based on the total weight of the composition, of the composition, and the deoxycholic acid or its salt is preferably added in an amount of 10 to 90% by weight based on the total weight of the composition .

The enzyme and deoxycholic acid or its salt used in the present invention are as described above. The addition of deoxycholic acid or its salt to the enzyme makes it possible to maintain the stability of the enzyme when the composition is in a solid or liquid phase.

Then, the prepared composition is freeze-dried. Lyophilization can be by any method known in the art.

The lyophilized composition is then dissolved in a diluent to form a solution. When the composition according to the present invention is formed into a liquid preparation, the enzyme activity is still maintained. The activity of such an enzyme is stably maintained at a temperature of 20 to 70 DEG C and a relative humidity of 10 to 90%.

The composition containing deoxycholic acid or a salt thereof of the present invention can be improved in utilization because the activity of the enzyme is maintained for a long time and the stability is excellent even in a liquid form preparation. In addition, the composition according to the enzyme stabilization method of the present invention can improve the stability of the enzyme in the preparation of the composition and prevent the reduction of the activity by adsorption of the glass bial introduced at the time of storage, thereby improving the storage stability.

The present invention will be described in more detail through the following examples. However, the examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

[Examples 1 to 3]

A composition containing hyaluronidase (represented by Hdase) and sodium deoxycholate (represented by NaDC) was prepared by the composition as shown in Table 1 below and lyophilized to prepare a lyophilized preparation. Each lyophilized formulation contains Hyaluronidase at 1,500 IU per vial. Syringing water was added to the lyophilized preparation to prepare solutions each having a concentration of 15 IU / mL.

ingredient content Example 1 Example 2 Example 3 Hyaluronidase  1,500 IU  1,500 IU  1,500 IU Lactose baggage 13.3 mg 13.3 mg 13.3 mg Sodium deoxycholate 10 mg 50 mg 100 mg Injection water 735 mg 735 mg 735 mg

[Comparative Examples 1 to 3]

The solution was prepared in the same manner as in Example 1 except that sodium lauryl sulfate was used instead of sodium deoxycholate in Example 1, as shown in Table 2 below.

ingredient content Comparative Example 1 Comparative Example 2 Comparative Example 3 Hyaluronidase  1,500 IU  1,500 IU  1,500 IU Lactose baggage 13.3 mg 13.3 mg 13.3 mg Sodium lauryl sulfate 10 mg 50 mg 100 mg Injection water 735 mg 735 mg 735 mg

[Comparative Example 4]

Was prepared in the same manner as in Example 1 but a solution was prepared with the composition shown in Table 3 below, that is, the sodium deoxycholate of Example 1 was not added.

ingredient content Hyaluronidase 1,500 IU Lactose baggage 13.3 mg Injection water 735 mg

[Test Example 1] Evaluation of stability immediately after manufacture

The stability of the solutions prepared in Examples 1 to 3 and Comparative Examples 1 to 3 was evaluated immediately after preparation. Stability was measured by assessing the residual activity by the potency test of Hyaluronidase. The potency test was carried out with reference to the USP Hyaluronidase Quantitative Test. The same composition as in Example 1, but without sodium deoxycholate was used as a control group, and the same composition as in Comparative Example 1, but without sodium deoxycholinate was used as a comparative control. After stopping the addition of the reaction stop solution prepared from horse serum, the absorbance was measured at 640 nm. The hyaluronidase content (%) was deduced from the absorbance.

The results are shown in Table 4 and FIG.

Comparative control (% by weight) Comparative Example 1 Comparative Example 2 Comparative Example 3 102.4 91.7 -0.4 -3.2 Control group Example 1 Example 2 Example 3 102.7 106.8 101.4 103.0

[Test Example 2] Evaluation of the stability and the content of sodium deoxycholate with time

The solutions prepared in Examples 1 to 3 and Comparative Example 4 were stored at a temperature of 25 ° C and a relative humidity of 60%, respectively, at a temperature of 40 ° C and a relative humidity of 75%. The residual activity of the hyaluronidase was evaluated by a potency test at the primary. The potency test was carried out with reference to the USP Hyaluronidase Quantitative Test. The diluted solution containing hydrolyzed gelatin was added to the hyaluronic acid, and the test substance was added thereto, followed by reaction at 37 ° C for 30 minutes. The reaction suspension prepared from horse serum was added and the absorbance was measured at 640 nm after 30 minutes. The hyaluronidase content (%) was deduced from the absorbance.

The results are shown in Tables 5 and 6 and Figs. 2 and 3, respectively.

Lt; RTI ID = 0.0 > 25 C < / RTI & (weight%) 0 days 5 days 10 days Comparative Example 4 99.8 49.8 23.2 Example 1 98.5 73.5 30.2 Example 2 100.0 77.7 29.2 Example 3 100.8 66.3 28.0

40 < 0 > C and a relative humidity of 75% (weight%) 0 days 5 days 10 days Comparative Example 4 99.8 28.7 3.8 Example 1 98.5 43.9 21.9 Example 2 100.0 51.4 27.6 Example 3 100.8 42.0 24.8

Claims (24)

A composition for stabilizing a lyophilized formulation of hyaluronidase containing deoxycholic acid or a salt thereof as an active ingredient. delete delete The method according to claim 1,
Wherein the content of hyaluronidase in the lyophilized formulation is 0.01 to 50% by weight based on the total weight of the lyophilized formulation. The method according to claim 1,
Wherein the deoxycholic acid or salt thereof is sodium deoxycholate. The method according to claim 1,
Wherein the content of deoxycholic acid or its salt is from 10 to 90% by weight based on the total weight of the lyophilized formulation. delete The method according to claim 1,
Wherein said composition is for the formulation of a solution to be lyophilized or for stabilization of reconstituted solution formulations prepared from a lyophilized composition and a diluent. 9. The method of claim 8,
Wherein the concentration of hyaluronidase in said solution is 1 to 3,000 IU / mL. The method according to claim 1,
Wherein said lyophilized formulation is stable at a temperature of 20-70 < 0 > C and a relative humidity of 10-90%. 9. The method of claim 8,
Wherein the solution of hyaluronidase maintains stability at a temperature of 20 to 70 DEG C and a relative humidity of 10 to 90%. Adding deoxycholic acid or a salt thereof to hyaluronidase to prepare a composition;
A method of stabilizing hyaluronidase comprising lyophilizing the prepared composition. delete delete 13. The method of claim 12,
Wherein the content of hyaluronidase is 0.01 to 50 wt% based on the total weight of the composition. 13. The method of claim 12,
Wherein said deoxycholic acid or its salt is sodium deoxycholinate. 13. The method of claim 12,
Wherein the deoxycholic acid or salt thereof is added in an amount of 10 to 90% by weight based on the total weight of the composition. delete 13. The method of claim 12,
The method of stabilization further comprises, after lyophilization, dissolving the lyophilized composition in a diluent to prepare a solution. 20. The method of claim 19,
Wherein the concentration of hyaluronidase in said solution is 1 to 3,000 IU / mL. 13. The method of claim 12,
Wherein the stabilization method maintains the stability of hyaluronidase at a temperature of 20 to 70 DEG C and a relative humidity of 10 to 90%. 20. The method according to any one of claims 12 and 19,
Wherein the composition has an effect of improving storage stability. 20. The method according to any one of claims 12 and 19,
Wherein the stability of hyaluronidase is increased when the composition is prepared. 20. The method according to any one of claims 12 and 19,
Wherein the composition is introduced into a glass vial during storage and the composition has the effect of preventing the reduction of the activity by adsorption to the glass vial.

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