JP2916948B2 - Method for stabilizing CPB-I and its pharmaceutical composition - Google Patents

Description

The present invention relates to a method for stabilizing CPB-I or recombinant CPB-I which is useful as an anticoagulant or a therapeutic agent for skin / corneal diseases, and a pharmaceutical composition. The separation and purification of these,
The present invention relates to a stabilization method useful for performing operations such as freeze-drying and a pharmaceutical composition excellent in storage safety.

[Problems to be solved by conventional technology and invention]

CPB-I is widely distributed in in vivo tissues and secretions, including human placenta (Chem. Pharma. Bull, 38, 1957-196).
0,1990), and a substance having a physiological activity such as an anticoagulant effect existing in the cytoplasm within a cell.

CPB-I can be extracted from human or animal organs (JP-A-62-174023). The CPB-I thus obtained has the following properties.

Molecular weight (SDS-polyacrylamide gel electrophoresis, reduced state) 34,000 ± 2,000 Isoelectric point (isoelectric point column electrophoresis using ampholite) 4.7 ± 0.1 Stability (a) Loss by heating at 50 ° C for 30 minutes Active (b) Stable at pH 4-10 (c) Stable at 37 ° C, 30 minutes in plasma Effect on blood coagulation system (a) Extend calcium recoagulation time (b) Extend prothrombin time (c) Activated partial thromboplastin Extend time Amino acid analysis Amino acid analysis shows aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, 1 /
2 The presence of cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine and arginine is observed.

In addition, CPB-I is obtained by using a gene fragment encoding human or animal CPB-I,
It can be expressed in yeast and the like (JP-A-64-20095). CPB- thus obtained by genetic recombination
I (hereinafter referred to as "recombinant CPB-I")
Has the following amino acid sequence:

Recombinant CPB-I can also be produced using yeast other than E. coli. In this case, an amino acid sequence in which an acetyl group is bonded to alanine at the amino terminus is obtained.

Such CPB-I or recombinant CPB-I can be made into various dosage forms such as a solution, a gelling agent, an ointment, an ophthalmic solution, and an injection.

However, it is necessary to separate and purify, freeze-dry, etc. during the formulation process, and after the formulation as described above,
It has to be stored for a long period of time, and at this time, there is a drawback that the activity of the CPB-I is reduced due to polymerization or the like.

Therefore, a method for stabilizing CPB-I or recombinant CPB-I and a stable pharmaceutical composition have been desired.

[Means for solving the problem]

The present inventors have conducted intensive research in view of the above situation,
Sugars can be added to CPB-I or recombinant CPB-I to stabilize them, and even if they are subjected to operations such as separation / purification / lyophilization, or even if they are formulated and stored for a long period of time, they remain active. The present invention has been completed by finding that the value does not decrease.

That is, the present invention relates to CPB-I or recombinant CPB-
A method for stabilizing CPB-I or recombinant CPB-I, characterized by adding a saccharide to I, and a method characterized by containing CPB-I or recombinant CPB-I and a saccharide.
The present invention relates to a pharmaceutical composition in which PB-I or recombinant CPB-I is stabilized.

Examples of the saccharide used in the present invention include glucose, glucosamine, xylose, saccharose, dextran T-40 and the like. In the case of an injectable preparation, glucose is preferred. The amount of the saccharide is preferably about 0.4 to 20 mg per 1 ml of a solution containing CPB-I or recombinant CPB-I (hereinafter referred to as “CPB-I or the like”). The amount may be adjusted so that the above-mentioned solution concentration is obtained when the powder is dissolved.If the amount of the saccharide is less than 0.4 mg, it is difficult to stabilize, and even if it is more than 20 mg, the effect is not so good. Improvement cannot be expected.

The method of adding the saccharide is not particularly limited, a method of directly adding the saccharide to a solution such as CPB-I, a method of previously dissolving the saccharide in water or an appropriate buffer, and adding the saccharide powder to CPB-I or the like. And the method of adding and dissolving in water or the like may be appropriately selected. The timing of addition is not particularly limited, and CPB-
It may be any of the separation / purification step such as I and the preparation step.

In the case of a solution, the stabilized composition of the present invention in which a saccharide is added to CPB-I or the like is preferably separated and purified at 0 to 30 ° C, particularly preferably at 0 to 10 ° C, and is stored or formulated. When the solution is stored in a frozen state, the temperature is preferably 0 ° C. or lower, particularly -20 ° C. or lower.

The stabilized preparation composition containing CPB-I etc. of the present invention can be made into various dosage forms depending on the use. For example, when used as an anticoagulant, in the form of an aqueous injection or the like, and when used as a therapeutic agent for skin and corneal diseases, liquids, gels,
It can be in the form of a cream, ointment, ophthalmic solution, eye ointment, or the like.

〔The invention's effect〕

According to the method of the present invention, CPB-I and the like are frozen and thawed, separated and purified by dissolution in an aqueous medium, etc.
Activities such as BI are retained. In addition, the pharmaceutical composition of the present invention does not decrease the activity of CPB-I even when stored for a long time, and is useful as a pharmaceutical.

〔Example〕

Example 1 CPB-I 6.76 mg / dissolved in 10 mM phosphate buffer (pH 7.4)
glucose, glucosamine hydrochloride, as stabilizer in ml
100 μl of a solution containing 5 mg each of xylose, soccerose and dextran T-40 per ml was frozen at −40 ° C. and thawed at room temperature. After repeating this operation six times, analysis was performed by the following measurement method. Table 1 shows the results.

Determination of CPB-I polymer by HPLC 10 mM phosphate buffer (pH 7.
Tosoh HPLC gel filtration column TSK g equilibrated in 4)
20 μ of a sample solution diluted to 1 mg / ml in the same buffer as above was injected into el G3000SW _XL (7.8 × 300 mm). The polymer was separated at a flow rate of 1 ml / min, the absorbance of the sample eluted from the column was measured at a wavelength of 280 nm, and the ratio between CPB-I and the polymer was calculated from the area value.

Example 2 CPB-I 6.76 mg / dissolved in 10 mM phosphate buffer (pH 7.4)
0.04-20mg of glucose per ml as stabilizer in ml
100 μm of the added solution was frozen at −40 ° C. and then thawed at room temperature. After repeating this operation five times, analysis was performed in the same manner as in Example 1. Table 2 shows the results.

Example 3 CPB-I 6.76 mg / dissolved in 10 mM phosphate buffer (pH 7.4)
The anticoagulant activity of a solution obtained by adding 10 mg per ml of glucose as a stabilizer to ml was measured by the following method. Table 3 shows the results.

Measurement of anticoagulant activity 20 mM containing 0.5% bovine serum albumin and 0.15 M NaC1
100 μl of a sample solution diluted to 5 and 10 μg / ml with Tris-HCl buffer (pH 7.4) was added with 100 μl of lipoplastin 0.5% / ml from Mochida Pharmaceutical Co., Ltd. diluted with 30 mM CaCl ₂ solution.
Preincubated for minutes. In addition, saline at 37 ° C
200 μm of Ortho control plasma dissolved in 2 ml was added, and the time until the plasma coagulated was measured using a Coagtech TE-600 manufactured by Elma Optical.

──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Satoshi Adachi 3190-202, Tsukurei, Kikuyo-cho, Kikuchi-gun, Kumamoto Prefecture (56) References JP-A-63-165328 (JP, A) (58) Fields investigated ( Int.Cl. ⁶ , DB name) C07K 14/47 A61K 38/17 A61K 47/26 A61K 47/36

Claims (4)

(57) [Claims] 1. A method for stabilizing CPB-I or recombinant CPB-I, comprising adding a saccharide to CPB-I or recombinant CPB-I. 2. A pharmaceutical composition containing stabilized CPB-I or recombinant CPB-I, comprising CPB-I or recombinant CPB-I and a saccharide. 3. The method according to claim 1, wherein the saccharide is one or more selected from glucose, glucosamine, xylose, saccharose and dextran. 4. The pharmaceutical composition according to claim 2, wherein the saccharide is one or more selected from glucose, glucosamine, xylose, saccharose and dextran.