JP2916947B2 - Method for stabilizing CPB-I and formulation composition - Google Patents

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for stabilizing CPB-I or recombinant CPB-I, which is useful as an anticoagulant and a therapeutic agent for skin and corneal diseases, and a pharmaceutical composition thereof. In detail, the present invention relates to 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.

[Prior art and problems to be solved by the invention]

CPB-I is widely distributed in tissues and secretions in the body, including the human placenta (Chem. Pharma. Bull, 38, 1957-196).
0, 1990), and within cells it is a substance present in the cytoplasm that has physiological activity such as anticoagulant properties.

CPB-I can be extracted from human or animal organs (Japanese Patent Application Laid-Open No. 62-174023), and the CPB-I thus obtained has the following properties:

Molecular weight (SDS-polyacrylamide gel electrophoresis, reduced state): 34,000 ± 2,000 Isoelectric point (isoelectric focusing column electrophoresis using ampholyte): 4.7 ± 0.1 Stability (a) Inactivated by heating at 50°C for 30 minutes (b) Stable at pH 4-10 (c) Stable in plasma at 37°C for 30 minutes Actions on the blood coagulation system (a) Prolongs recalcification clotting time (b) Prolongs prothrombin time (c) Prolongs activated partial thromboplastin time Amino acid analysis Amino acid analysis showed the following: aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, 1/
The presence of 2 cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine and arginine is observed.

CPB-I can also be produced by recombinant gene technology in Escherichia coli,
The CPB-1 gene obtained by gene recombination can be expressed in yeast or the like (JP Patent Publication 2009-20095).
I (hereinafter referred to as "recombinant CPB-I")
has the following amino acid sequence:

Recombinant CPB-I can also be produced using yeast in addition to E. coli. In this case, the amino acid sequence has an acetyl group bound to the amino-terminal alanine.

Such CPB-I or recombinant CPB-I can be made into various dosage forms such as liquids, gels, ointments, eye drops, and injections.

However, in the process of formulation, separation, purification, freeze-drying, etc. are required, and after the above-mentioned formulation is made,
It has the disadvantage that it must be stored for a long period of time, during which time CPB-I undergoes polymerization, etc., resulting in a decrease in its activity.

Therefore, there has been a demand for a method for stabilizing CPB-I or recombinant CPB-I and a stable pharmaceutical composition.

[Means for solving the problems]

As a result of intensive research conducted by the present inventors in light of the above-mentioned circumstances,
The present inventors have discovered that the addition of a basic amino acid to CPB-I or recombinant CPB-I can stabilize the compound, and that its activity does not decrease even when the compound is subjected to procedures such as separation and purification and lyophilization, or when the compound is formulated and stored for a long period of time, thereby completing the present invention.

That is, the present invention relates to a method for producing CPB-I or recombinant CPB-
CPB characterized by the addition of I and a basic amino acid
Method for stabilizing CPB-I or recombinant CPB-I and
CPB-I or recombinant CPB-I, characterized by containing a basic amino acid
-I provides a stabilized pharmaceutical composition.

Examples of basic amino acids used in the present invention include lysine, arginine, ornithine, etc., and L-lysine or L-arginine is preferred for injectable preparations. The amount of basic amino acid is preferably about 0.6 to 20 mg per ml of solution containing CPB-I or recombinant CPB-I (hereinafter referred to as "CPB-I, etc."). The amount of stabilizer added to powders is:
The above solution concentration should be achieved when the powder is dissolved. If the amount is less than 0.6 mg, stabilization is difficult, and if the amount exceeds 20 mg, little improvement in effect can be expected.

The method of adding the basic amino acid is not particularly limited.
A method of adding a basic amino acid directly to a solution of CPB-I or the like, a method of dissolving the basic amino acid in water or an appropriate buffer solution in advance and adding the solution, a method of mixing a powder of a basic amino acid with CPB-I or the like and adding/dissolving the mixture in water or the like, etc., etc., may be appropriately selected. The timing of addition is not particularly limited, and may be any of the following: a separation and purification step of CPB-I or the like,
The process may be any of the formulation processes.

The stabilized composition of the present invention, in which a basic amino acid is added to CPB-I or the like, is preferably separated and purified, formulated, or stored in the form of a solution at 0 to 30° C., particularly at 0 to 10° C. When the solution is stored in a frozen state, it is preferably stored at 0° C. or below, particularly at −20° C. or below.

The composition of the present invention also contains glucose, glucosamine, xylose, sucrose, dextran T-40,
Sugars such as mannitol, albumin, etc. may be added.

The stabilized preparation composition containing CPB-I or the like of the present invention can be in various dosage forms depending on the application. For example, when used as an anticoagulant, it can be in the form of an aqueous injection, etc.
When used as a therapeutic agent for skin and corneal diseases, the composition may be in the form of a liquid, gel, cream, ointment, eye drops, eye ointment, or the like.

[Effects of the Invention]

According to the method of the present invention, CPB-I and the like can be obtained without any loss of CP even after separation and purification steps such as freezing and thawing, dissolving in an aqueous medium, and preparation steps.
Furthermore, the preparation composition of the present invention does not exhibit a decrease in the activity of CPB-I even after long-term storage, and is therefore useful as a pharmaceutical.

[Example]

Example 1 6.76 mg/mL of CPB-I dissolved in 10 mM phosphate buffer (pH 7.4)
100μl of a solution containing 5mg of aspartic acid, glycine or lysine per ml as a stabilizer was added to −4
The samples were frozen at 0°C and thawed at room temperature. This procedure was repeated six times, and then the samples were analyzed using the following measurement method. The results are shown in Table 1.
As shown in.

Measurement of CPB-I polymer by HPLC The polymer was first dissolved in 10 mM phosphate buffer (pH 7.
4) Equilibrated HPLC gel filtration column TSK g (Tosoh Corporation)
20 μl of sample solution diluted with 1 mg/ml of the same buffer was injected into an el G3000SW _XL (7.8×300 mm). Separation from the polymer was performed at a flow rate of 1 ml/min, and the absorbance of the sample eluted from the column was measured at a wavelength of 280 nm, and the ratio of CPB-I to its polymer was calculated from the area value.

Example 2 6.76 mg/mL of CPB-I dissolved in 10 mM phosphate buffer (pH 7.4)
0.04 ml of L-lysine hydrochloride as a stabilizer
100 μl of solution containing 20 mg of the compound was frozen at −40° C. and then thawed at room temperature. This procedure was repeated five times, after which analysis was carried out in the same manner as in Example 1. The results are shown in Table 2.

Example 3 6.76 mg/mL of CPB-I dissolved in 10 mM phosphate buffer (pH 7.4)
10 mg of L-lysine hydrochloride per ml as a stabilizer
The anticoagulant activity of the solution to which the above was added was measured by the following method. The results are shown in Table 3.

Measurement of anticoagulant activity 20 mM saline containing 0.5% bovine serum albumin and 0.15 M NaCl
100 μl of sample solution diluted to 5 and 10 μg/ml with Tris-HCl buffer (pH 7.4) was added with 100 μl of 0.5 mg/ml of Lyoplastin (Mochida Pharmaceutical Co., Ltd.) diluted with ₃₀ mM CaCl2 solution and incubated at 37°C for 3 h.
The plate was preincubated for 1 min at 37°C in physiological saline.
200 μl of Ortho control plasma dissolved in 2 ml was added and the time until the plasma coagulated was measured using Elma Optical's Coregtech TE-600.

───────────────────────────────────────────────────────── Continued from the front page (72) Inventor: Satoshi Adachi 3190 Shinyama, Tsukure, Kikuyo-machi, Kikuchi-gun, Kumamoto Prefecture, Japan 202 (56) References: JP 63-165328 (JP, A) (58) Field of investigation (Int.Cl. ⁶ , DB name): C07K 14/47 A61K 38/17 A61L 47/18

Claims (4)

(57) [Claims] 1. A method for stabilizing CPB-I or recombinant CPB-I, which comprises adding a basic amino acid to CPB-I or recombinant CPB-I. 2. A pharmaceutical composition in which CPB-I or recombinant CPB-I is stabilized, comprising CPB-I or recombinant CPB-I and a basic amino acid. 3. The method for stabilization according to claim 1, wherein the basic amino acid is one or more selected from the group consisting of lysine, arginine and ornithine. 4. The pharmaceutical composition according to claim 2, wherein the basic amino acid is one or more selected from the group consisting of lysine, arginine and ornithine.