JPH04198195A - Method for stabilizing cpb-i and pharmaceutical composition - Google Patents

Abstract

PURPOSE:To stabilize CPB-I (calphobindin I) useful as an antiblood coagulant, a therapeutic agent for dermatopathy and keratopathy, etc., so as not to cause inactivation with operation such as freezing, melting or dissolving by adding a basic amino acid to the CPB-I or recombinant CPB-I. CONSTITUTION:CPB-I or recombinant CPB-I is dissolved in 10mM phosphate buffer solution (pH 7.4) and one or more of basic amino acids such as lysine, arginine and ornithine as a stabilizer are added to the resultant solution. Thereby, the stabilized CPB-I or recombinant CPB-I and its pharmaceutical composition capable of retaining its activity even by performing separating and purifying operation such as freezing or melting without deteriorating the activity even by dissolving in an aqueous medium, etc., preparing a pharmaceutical and preserving the pharmaceutical for a long period are obtained.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a method and a pharmaceutical composition for stabilizing CPB-1 or recombinant CPB-4 useful as an anticoagulant and a therapeutic agent for skin and corneal diseases. The present invention relates to a stabilization method useful for these operations such as separation and purification, freeze-drying, and a pharmaceutical composition with excellent storage safety.

[Problems to be solved by conventional technology and invention] CPB
-I is widely distributed in tissues and secretions in living organisms, including the human placenta, and is widely distributed in Chem, Pharma, Bul
1, 38° 1957-1960, 1990), and in cells, it is a substance that exists in the cytoplasm and has physiological activities such as anticoagulant effects.

CPB-I can be extracted from human or animal organs (JP-A-62-174023), and the thus obtained [:PB-1] has the following properties.

■ Molecular weight (SO3-polyacrylamide gel electrophoresis method, reduced state) 34,000±2.000 ■ Isoelectric focusing column electrophoresis method using isoelectric focusing ampholite) 4.7±0.1 ■ Stability (I) ) Deactivated by heat treatment at 50℃ for 30 minutes (b)p! (Stable at 4-10 (c) Stable in plasma at 37°C for 30 minutes ■ Effect on blood coagulation system (a) Prolongs calcium recirculation clotting time (b) Prolongs prothrombin time (c) Activated partial thromboplastin time Extension■ Amino acid analysis Amino acid analysis reveals the presence of aspartic acid, threonine, serine, glutamic acid, proline, chrysine, alanine, each cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine.

Furthermore, CPB-r can be expressed in Escherichia coli, yeast, etc. by genetic recombination technology using a gene fragment encoding human or animal [:PB-1].
20095 Publication). CPB-I obtained through genetic recombination (hereinafter referred to as "recombinant CPB-I J") has the following amino acid sequence.

Ala-GI n-Va I-Leu-Arg-GIy
-Thr-Val-Thr-Asp-Phe-Pro-
G 1y-Phe-^5p-Glu-^rg-Ala-
Asp-^1a-G Iu-Thr-Leu-A r
g-Ly s-A la-Met-Ly s-G 1
y-Leu-G 1y-Th r-Asp-G 1u-
G 1u-3e r-I 1e-Leu-Th r-
Leu-Leu-Thr-3e r-A r g-3e
r-Asn-A 1 a-61n-Ar g-G In
-G 1 u-11e-3er-A 1a-A 1a-
Phe-Lys-T hr-Leu-Phe-G
1y-Ar g-Asp-Leu-Leu-A s
p-^s p-Le u-Lys-5er-G 1 u
-Lau-T hr-G 1 y-Lys-Ph e-
G 1 u-Lys-Leu-11e-VaiAI a
-Leu-Met-Lys-Pro-3er-Arg-
eu-Tyr-Asp-^1a-Tyr-Glu-L
eu-Lys-His-Ala-Leu-Lys-Gl
y-Ala-Gly-Thr-^5n-Glu-Lys
-Val-Leu-Thr-G Iu-I 1e-11
e-A la-3er-Arg-Thr-Pro-G
1u-G lu-Leu-Arg-A la-I 1e
-Lys-G In-Va I-Tyr-G 1u-G
1u-G 1u-Tyr-Gly-3er-3er-
Leu-Glu-Asp-85p-Va1-Va1-G
ly-Asl)-Thr-3er-Gly-Tyr-T
yr-Gln-8rg-Met-Leu-Val-Va
l-Leu-Leu-GIn-Ala-Asn-Arg
-Asp-Pro-Asp-81a=Gly-11e-
Asp-G 1u-A 1a-G In-Va 1-G
1u-G I n-Asp-A 1a-G 1 n-
^1a-Leu-Phe-GIn-AIa-GIy-G
Iu-Leu-Lys-Trp-Gly-Thr-A
sp-GI u-G l u-Lys-Phe-11
e-Thr-11e-Phe-G 1y-Th r-A
rg-3er-Va 1-3er-Hi 5-Leu
-A rg-L ys-V a l-P h e-^s
p-Lys-Tyr-Met-Thr-T Ie-3e
r-GIy-Phe-GIn-I 1e-G 1 u-
G 1 u-Thr-11e-Asp-Arg-G I
u-Th r-3er-G Iy-Asn-Leu-G
lu-Gln-Leu-Leu-Leu-Ala-Va
l-Val-Lys-3er-I 1e-Ar g-3
er-I 1e-Pro-A 1a-Tyr-Leu-
A 1a-G 1 u-Thr-Leu-Tyr-
Tyr-A 1 a-Met-L y s-G 1y-
A 1a-G 1y-Th r-A 5p-Asp-H
i 5-Thr-Leu-I 1e-Ar g-Va
1-Met-Va 1-3er-A1-3er-Ar
1u-I 1e-Asp-Leu-Phe-Asn-1
1e-Arg-Lys-G lu-Phe-Arg-L
ys-Asn-Phe-AIa-Thr-3er-Le
u-Tyr-3er-Met-I 1e-Lys-Gl
y-Asp-Thr-3er-GIy-Asp-Tyr
-Lys-Lys-A1 a-Leu-Leu-Leu
-Lau-Cys-Gly-G1 u-Asp-Asp
Furthermore, recombinant CPB-I can be produced using yeast other than E. coli. In this case, the amino acid sequence will be an acetyl group bonded to alanine at the amino terminal.

Such CPB-1 or recombinant CPB-I can be made into a number of dosage forms such as a liquid, a gelling agent, an ointment, an eye drop, and an injection.

However, it is necessary to carry out separation and purification, freeze-drying, etc. in the process of formulation, and after making the formulation as described above, it is necessary to store it for a long period of time. The disadvantage was that it deteriorated.

Therefore, a method for stabilizing CPB-1 or recombinant CPB-1 and a stable pharmaceutical composition have been desired.

[Means to solve the problem]

The inventors of the present invention have conducted extensive research in view of the above-mentioned circumstances, and have found that C.
By adding basic amino acids to PB-1 or recombinant CPB-1, these can be stabilized.
We have completed the present invention by discovering that the activity does not decrease even after performing operations such as separation and purification and freeze-drying, or even when it is formulated into a formulation and stored for a long period of time.

That is, the present invention provides CPB-I or recombinant CP
C characterized by adding a basic amino acid to B-I
PB-I or recombinant Method for stabilizing CPB-I and CPB-I or recombinant CPB-1 or recombinant characterized by containing CPB-1 and a basic amino acid ["Preparation composition in which FB-1 is stabilized" It is something that provides something.

Examples of the basic amino acids used in the present invention include:
Examples include lysine, arginine, ornithine, and when preparing an injection preparation, L-lysine or L-arginine is preferable. The basic amino acid content is CPB-I or recombinant CPB-I (hereinafter referred to as rCPB-1, etc.)
It is preferable to use about 0.6 to 20 μm per ml of the solution containing the following. Note that the amount of the stabilizer added to the powder may be such that when the powder is dissolved, the stabilizer has the above-mentioned solution concentration. If the amount is less than 0.6 .mu., it is difficult to achieve stabilization, and even if the amount exceeds 20 .mu., no significant improvement in the effect can be expected.

The method of adding the basic amino acid is not particularly limited, and CPB-
A method in which basic amino acids are added directly to a solution such as I, a method in which the basic amino acid is dissolved in water or an appropriate buffer solution beforehand, and a method in which basic amino acid powder is mixed with CPB-1, etc. and then added and dissolved in water, etc. The method may be selected as appropriate. Also,
The timing of addition is not particularly limited, and may be in the separation and purification process of CPB-1 etc. or in the formulation process.

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 at 0 to 30°C, particularly 0 to 10°C, in the case of a solution. Also,
When storing the same solution in a frozen state, keep it below 0℃, especially at -2℃.
The temperature is preferably 0°C or lower.

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

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

〔Effect of the invention〕

According to the method of the present invention, even if CPB-1 etc. undergoes separation/purification such as freezing/thawing, dissolving in an aqueous medium, and formulation process, CPB-1, etc.
Activities such as PB-I are maintained. Further, the pharmaceutical composition of the present invention does not decrease CPB-I activity even after long-term storage, and is useful as a pharmaceutical.

〔Example〕

Example 1 CPB dissolved in 10mM phosphate buffer (pH 7,4)
-16,76■/- aspartic acid as a stabilizer,
5 each of glycine or lysine per yn1
100 μl of the solution containing mg was frozen at -40°C and thawed at room temperature. After repeating this operation six times, analysis was performed using the following measurement method. The results are shown in Table 1.

Determination of CPB-1 polymer by HPLC
．． 10mM containing 14M NaCl! J acid buffer (
Tosoh HPLC gel filtration applicable column TSK gel G3000SWxt (pH 7, 4) equilibrated with pH 7, 4)
20 μl of the sample solution diluted to 1 mg/mf of the same buffer was injected into the tube. Separation from the polymer was performed at a flow rate of 1 mf/a+in, and the absorbance of the sample eluted from the column was measured at a wavelength of 280 nm, and based on the area value, C'PB-
The ratio of I and its polymer was calculated.

Table 1 with blank spaces below Example 2 C dissolved in 10+r+M phosphate buffer (pH 7,4)
Add 1 mjl of L-lysine hydrochloride as a stabilizer to PB-16,76■/-! A 100 μm solution containing 0.04 to 20 mg per sample was frozen at -40° C. and then thawed at room temperature.

After repeating this operation five times, analysis was conducted in the same manner as in Example 1. The results are shown in Table 2.

Table 2 with blank space below Example 3 CPB dissolved in 10mM phosphate buffer (pH 7,4)
The anti-coagulant activity of a solution prepared by adding 10 mg of L-IJ gin hydrochloride as a stabilizer to -16,76 cm/barrel was measured by the following method. The results are shown in Table 3.

Determination of anti-coagulant activity 0.5% bovine serum albumin and 0.15M NaCl
5 in Lis-HCl buffer (pH 7,4) containing 20mM
and 10ttg/rrd! 100μ of sample solution diluted to
Oblastin manufactured by Mochida Pharmaceutical Co., Ltd. diluted with 30rnMCaC12 solution in J2 0.5mg/rnf! 100 μm of was added and incubated at 37° C. for 3 minutes. Furthermore, 37
Add 200μβ control plasma manufactured by Oates dissolved in 2mf of physiological saline at
The time taken for plasma to coagulate was measured using B-600.

Table 3 that's all

Claims (1)

[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, characterized by containing CPB-I or recombinant CPB-I and a basic amino acid. 3. The stabilization method according to claim 1, wherein the basic amino acid is one or more selected from lysine, arginine, and ornithine. 4. The pharmaceutical composition according to claim 2, wherein the basic amino acid is one or more selected from lysine, arginine, and ornithine.