JPH11171790A - Denaturation preventing agent for thrombomodulin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject agent useful for preventing the denaturation of thrombomodulin having excellent suppressing actions on the blood coagulation by including monosaccharides such as mannitol or disaccharides such as trehalose, lactose or sucrose as an active ingredient. SOLUTION: This denaturation preventing agent for thrombomodulin capable of preventing the denaturation of the thrombomodulin manifesting excellent suppressing actions on the blood coagulation is obtained by using monosaccharides such as mannitol or disaccharides which are any of trehalose, lactose and sucrose as an active ingredient, as necessary, adding an effective amount of one or more amino acids such as argninine, glutamic acid, proline, serine, glycine, histidine, asparagine, lysine, phenylalanine or valine and salts thereof as an active ingredient, further adding an isotonic agent, a buffer or the like as the third ingredient thereto, dissolving the ingredients in water, distilled water for parenteral injection or a suitable buffer, filling the resultant solution in an ampul or a vial and freeze-drying the filled solution.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a thrombomodulin denaturation inhibitor.

[0002]

2. Description of the Related Art Streptokinase, urokinase and tissue plasminogen activator are currently used as thrombolytic agents. Heparin and warfarin are used as anticoagulants. Further, aspirin, sulfinpyrazone, dipyridamole and the like are used as platelet aggregation inhibitors.

[0003] These thrombolytic agents, anticoagulants and platelet aggregation inhibitors are used separately or in combination, for example, for example, myocardial infarction, thrombosis, embolism, peripheral vascular occlusion, obstructive arteriosclerosis. , Intravascular Coagulation Syndrome (DI
C), for the treatment and prevention of diseases such as angina pectoris, transient cerebral ischemic attacks, and preeclampsia. However, these thrombolytics, anticoagulants and platelet aggregation inhibitors act only on a small part of the blood coagulation / fibrinolysis system, which consists of a very complex mechanism. Thus, there has been a demand for a drug that acts widely on the coagulation / fibrinolysis system of blood and has an excellent blood coagulation inhibitory action.

By the way, thrombomodulin (hereinafter referred to as TM)
Has an action to promote the activation of protein C by thrombin. Protein C is a vitamin K-dependent protein that plays an important role in the blood coagulation / fibrinolysis system and is activated by the action of thrombin. Activated protein C inactivates the activated coagulation factors V and VIII in vivo and is involved in the production of plasminogen activator having a thrombolytic action Is known [Koji Suzuki,
History of Medicine, Vol. 125, pp. 901 (1983)
Year)〕.

[0005] TM promotes the activation of protein C by thrombin to produce a large amount of active protein C having an anticoagulant effect and a thrombolytic effect. Therefore, TM greatly contributes to anticoagulation and thrombolysis in a living body. As mentioned above, TM
Has anti-coagulant action, platelet aggregation inhibitory action and thrombolytic action, for example, myocardial infarction, thrombosis, embolism, peripheral vascular occlusion, atherosclerosis obliterans, intravascular coagulation syndrome (DIC), angina It is expected to be used in the treatment and prevention of diseases such as infectious diseases, transient ischemic attacks, and toxemia of pregnancy.

[0006] Since the amount of TM produced from cells is small, it has not been produced on an industrial scale. However, it has become possible to easily obtain TM by using a genetically modified product (Yamamoto et al., JP-A-64-6219), and its development as a drug has been started.

[0007] In order to widely and stably supply TM as an anticoagulant or thrombolytic agent, it is an essential operation to freeze-dry it to prepare a formulation. However, the present inventors have revealed that when a TM-containing solution is freeze-dried, a small amount, but a part thereof, is polymerized by denaturation, and a multimer in which several TM molecules are associated is formed. In lyophilization of proteins, it is considered that water is partially dehydrated to a hydrated layer, and the environment is partially dehydrated, thereby destroying the structure retention mechanism of proteins and causing denaturation.

[0008] Since TM has been developed as a pharmaceutical, if it contains a denatured product, safety including its antigenicity becomes a problem. Under such circumstances, it is impossible to provide a TM composition that is safe as a therapeutic agent despite its anticoagulant action and thrombolytic action.

[0009]

As described above, it is not preferable to administer a TM composition containing a denatured substance to the human body, and when used as a therapeutic agent, a TM composition containing no denatured substance is desirable. For this reason, T is used to prevent polymerization due to denaturation of TM and to provide a safe and stable TM composition.
A modification inhibitor for M is required.

[0010]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, have found that the addition of monosaccharides or disaccharides can prevent denaturation of TM. They have found and completed the present invention. The present invention has been completed based on the above findings, and is a thrombomodulin denaturation inhibitor containing a monosaccharide or a disaccharide as an active ingredient.

In the present invention, thrombomodulin is defined as a substance having an action of promoting the activation of protein C by thrombin, and particularly preferably, soluble thrombomodulin is used.

First, the TM raw material used in the present invention may be prepared by a known method or an analogous method. Examples of such a method include the method of Yamamoto et al.
-6219, see Examples). That is,
A human-derived TM gene is prepared by a genetic engineering technique,
The Chinese hamster ovary cells incorporated and modified as necessary are cultured and purified to a high purity from the culture solution, and a soluble TM containing no cytoplasmic domain, for example, a molecular weight of 62,000 by a light scattering method. Soluble TM.

The TM raw material used in the present invention may have an appropriate sugar chain. The method for producing the TM raw material used in the present invention is not limited to these. That is, a raw material production method in which TM is produced or a tissue culture solution is extracted and purified from the tissue culture solution can be employed.

In general, a TM-containing solution containing salts such as an isotonicity agent (eg, sodium chloride) and a buffering agent (eg, sodium phosphate) may be prepared through an extraction and purification step. As a monosaccharide as an active ingredient of the denaturation inhibitor for TM of the present invention, mannitol is mentioned as a particularly preferred example. Preferred examples of the disaccharide include trehalose, lactose, and sucrose.

If necessary, one or more effective amounts selected from the group consisting of amino acids and salts thereof may be added to the denaturation inhibitor for thrombomodulin in the present invention. As this amino acid or its salts, for example, arginine, glutamic acid, proline,
One or more selected from the group consisting of serine, glycine, histidine, asparagine, lysine, phenylalanine and valine or salts thereof, preferably arginine or an acid addition salt thereof, glutamic acid or a base addition salt thereof, histidine or Amino acids such as acid addition salts thereof, lysine or acid addition salts thereof or salts thereof and proline as free form, serine, glycine,
Asparagine, phenylalanine, valine and the like.

Among the above additives, most preferred are arginine or an acid addition salt thereof, glutamic acid or a base addition salt thereof, proline and serine. In the case of an arginic acid addition salt, the acid that can be added is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include hydrochloric acid, citric acid, sulfuric acid, and the like, and substances functionally equivalent thereto. Can be. Similarly, in the case of the histidine acid addition salt, the acid that can be added is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include hydrochloric acid, citric acid, and sulfuric acid, and substances functionally equivalent thereto. Can be mentioned.

Similarly, in the case of a glutamic acid base addition salt, the base that can be added is not particularly limited as long as it is pharmaceutically acceptable. For example, sodium, potassium and the like, and those functionally equivalent thereto Substances can be mentioned. Similarly, in the case of a lysine acid addition salt, the acid that can be added is not particularly limited as long as it is pharmaceutically acceptable. For example, hydrochloric acid, citric acid, sulfuric acid, and the like, and substances functionally equivalent thereto Can be mentioned.

In the present invention, besides the above additives, the third
As a component, a tonicity agent, a buffering agent, and the like may be added, and these are not particularly affected, but the salts used for the tonicity agent or the buffering agent, particularly, the concentration of sodium chloride is high. This may harm the cake formation when freeze-drying using the present invention. Further, albumin, gelatin or the like may be appropriately added for the purpose of preventing adsorption of the cake forming aid or TM to the container.

When the denaturation inhibitor for TM of the present invention is used, the amount added as a monosaccharide or disaccharide is usually TM
0.01 to 1 mmol per 1 mg is exemplified. That is, it is preferable that the addition amount be 0.01 mmol or more. Also, more than 1 mmol may be added, but it can be selected from an economical point of view.

The method of adding the TM denaturing inhibitor of the present invention to TM is not particularly limited. For example, a method of directly adding the denaturation inhibitor to the TM-containing solution, or a method of adding the denaturation inhibitor to water, Examples thereof include a method of dissolving in distilled water for injection or an appropriate buffer, and adding the resultant to a TM or TM-containing solution.

When a preparation is prepared using the denaturation inhibitor of the present invention, for example, an ampoule or a vial may be used.
A solution containing 0.05 to 15 mg, preferably 0.1 to 5 mg of TM and the above-mentioned denaturation inhibitor per 1 ml of water, distilled water for injection, or a suitable buffer is added to, for example, 0.5 to
10 ml is filled, then freeze-dried by a conventional method to prepare a preparation for injection. As such an injection preparation, for example, 0.01 to 100 m for administration 1 to 3 times a day.
It may be obtained as a freeze-dried preparation containing g.

[0022]

Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples, but the present invention is not limited thereto. Example 1 1 mg of TM per 2 ml of distilled water for injection (T
Soluble TM having a molecular weight of about 62,000 obtained by culturing Chinese hamster ovary cells in which the M gene has been prepared and modified by a genetic engineering technique and having a sugar chain by light scattering; -6219) was prepared. Sodium glutamate 0.0
5 mmol was added. Further, 2 ml of the solution was dispensed into glass vials and freeze-dried. Lyophilization is pre-frozen at −40 ° C. for 18 hours, −40 to + 20 ° C.
Primary drying at a vacuum of 0.05 to 0.01 mmHg for 40 hours, then at 20 ° C. and a vacuum of 0.05 to 0.01 mmHg
For 6 hours.

Next, the lyophilized product was redissolved and subjected to size exclusion chromatography analysis to polymerize the TM.
Was determined. For analysis, inner diameter 7.5 mm, length 60
Using a column filled with a hydrophilic silica gel having an exclusion limit molecular weight of 500,000 in a stainless steel tube of 50 cm, a 50 mM sodium phosphate buffer solution (pH: 0.1 M) containing sodium sulfate was used.
7.0) was used as an eluent and detected at a wavelength of 210 nm.
The results are shown in Table 1.

[0024]

[Table 1]

Example 2 The procedure of Example 1 was repeated except that trehalose was used as the additive.

Example 3 The same procedure as in Example 1 was carried out except that lactose was used as an additive.

Example 4 The same operation as in Example 1 was carried out except that sucrose was used as an additive.

Example 5 As an additive, two kinds of arginine monohydrochloride and sucrose were used in combination, and the amount of each added was 0.05 mmol.
The procedure was performed in the same manner as in Example 1 except for the above.

Example 17 The above-mentioned Example 1 was repeated except that 0.05 mmol of arginine monohydrochloride and 10 mg of purified gelatin were used as additives.
The same was done.

Comparative Example 1 The procedure of Example 1 was repeated except that no additive was added. The results are shown in Table 2.

[0031]

[Table 2]

Comparative Examples 2 to 5 The additive was sodium chloride, and the amount of the additive was 0.01, 0.
The same procedure was performed as in Example 1 except that the amounts were 02, 0.05, and 0.1 mmol. The results are shown in Table 2.

[0033]

As is clear from the results of the above Examples and Comparative Examples, according to the present invention, TM is prevented from being polymerized by denaturation in the freeze-drying step, and TM containing no denatured product
It is possible to obtain a composition. As a result, T acts widely on the coagulation / fibrinolysis system and has an excellent blood coagulation inhibitory action.
M can be supplied as a safe therapeutic agent.

[Brief description of the drawings]

FIG. 1. TM for various amounts of arginine monohydrochloride added
FIG. 4 is a view showing a relationship between denatured product ratios.

Claims (4)

[Claims] 1. A denaturation inhibitor for thrombomodulin comprising a monosaccharide or a disaccharide as an active ingredient. 2. The disaccharide is trehalose, lactose,
The thrombomodulin denaturation inhibitor according to claim 1, which is any of sucrose. 3. The monosaccharide is mannitol.
3. The denaturation inhibitor for thrombomodulin according to item 1. 4. The denaturation inhibitor for thrombomodulin according to claim 1, wherein the thrombomodulin is soluble thrombomodulin.