JPH01221325A - Method for stabilizing tissue plasminogen activating factor - Google Patents

Abstract

PURPOSE:To obtain a stabilized tissue plasminogen activating factor (t-PA), excellent in solubility in redissolving at need with hardly any deterioration in activity in freeze-drying and preserving the factor and capable of stably maintaining even the single-stranded structure, by adding albumin as a stabilizer to the t-PA. CONSTITUTION:Albumin hydrolyzate, obtained by hydrolyzing albumin with a chemical reagent or enzyme and having preferably 3,000-60,000 average molecular weight is added to an aqueous solution in a process for purifying the t-PA or a pharmaceutical after the purification in an amount of 0.01-5mg based on 100,000 units t-PA.

Description

DETAILED DESCRIPTION OF THE INVENTION (1) Industrial field of application The present invention provides tissue plasminogen activator (hereinafter referred to as t) characterized in that an albumin decomposition product is added as a stabilizer.
- PA). L-
PA is useful as a medicine (thrombosis treatment drug) and a reagent for physiological activity research.

According to the present invention, stable formulations of t-PA pharmaceuticals, standards or reagents for assaying the potency of t-PA, and methods for producing them are provided.

(2) Conventional technology As a therapeutic agent for thrombosis, urokinase isolated and purified from human urine or tissue culture fluid of kidney cells has been widely used.

However, urokinase has a low affinity for fibrin, the main component of blood clots (and has the side effect of activating plasminogen in the circulating blood to produce plasmin, depleting coagulation factors in the blood and making bleeding more likely). has been pointed out.

On the other hand, t-PA has a high affinity for fibrin and has a weak effect of depleting blood coagulation factors in circulating blood, so it is attracting attention as a thrombus-specific drug with few side effects.

L-PA exists in various human tissues, and is produced by culturing normal cells derived from these tissues, established cells from normal cells, tumor cells, etc., or by culturing microorganisms or cells created by genetic recombination technology. There is.

The trace amount of t-PA that has accumulated in the culture solution can be removed by making full use of protein separation technology, combining dialysis, various chromatography, etc.
Through a long, multi-step purification process, it is highly purified to the extent that it can be used as a medicine.

The t-PA obtained in this way has two types: one in which the polypeptide chain is a single chain, one in which the polypeptide chain becomes two chains due to the action of a proteolytic enzyme during the culture and purification process, or a mixture of single and double chains. There are cases where

Since 2-terminal chain t-PA is produced by decomposition of natural 1-terminal chain t-PA, pharmaceuticals to be administered to the human body usually use natural 1-terminal chain t-PAO that exists in the body. is said to be preferable.

t-PA is unstable in an aqueous solution and tends to aggregate, and it is known that to prevent this, the ionic strength of the solution is increased [
Biokimi Power Eto Biochika Acta (Biochi
mica et Biophysica Acta) Vol. 580, pp. 140-153, 1979], and t-
When PA is handled as an aqueous solution, it tends to become insolubilized and adsorbed to the glass wall, so to prevent this, high concentration SCN
It is also known to add surfactants such as or Twin 80 [Journal of Biological Chemistry (
Journal of Biological Che
mistry) Vol. 256, pp. 7035-7041, 1
981].

As a method for stabilizing pharmaceutical preparations containing t-PA, the addition of albumin for the purpose of stabilizing the activity (Japanese Unexamined Patent Application Publication No. 1989-1999)
-17427, JP-A-58-65218) and addition of polyoxyethylene hydrogenated castor oil derivatives (JP-A-62
-429), the addition of lysine for the purpose of improving the solubility of t-PA (Japanese Patent Application Laid-Open No. 60-181028), and the addition of gelatin partial hydrolyzate combined with diisocyanate (Jikai, No. 62-59221). ), the addition of gelatin for the purpose of preventing the conversion of 1-end chain t-PA to 2-end chain t-PA (Japanese Patent Application Laid-Open No. 60-248621), etc. are known.

However, no excellent stabilization method has been known to date that can simultaneously stabilize the solubility and activity of a t-PA preparation and prevent the conversion from single strands to double strands.

(3) Problems to be Solved by the Invention t-PA is a fairly unstable substance in aqueous solutions and formulations. For example, as mentioned above, t-PA tends to aggregate in an aqueous solution, and high ionic strength is required to prevent this. In addition, adsorption to glass walls is strong, and to prevent this, it is necessary to add a surfactant such as Twin 80.

However, it is practically undesirable for pharmaceutical preparations to be administered into the blood to contain high concentrations of salts and surfactants.

The purpose of the present invention is to establish a method for preventing deactivation in the purification process of t-PA and a method for stabilizing a preparation, which is characterized by using an albumin decomposition product as a stabilizer, which has no concerns about side effects. That is.

(4) Means for Solving the Problems The present invention is directed to an albumin decomposition product (preferably with a molecular weight of 300
0 to 60,000) as a stabilizer.

The albumin decomposition product used in the present invention is produced by hydrolyzing albumin with a chemical reagent or an enzyme. Methods for producing albumin decomposition products include cleavage of methionyl bonds with bromosyanide, partial hydrolysis with acids, cleavage of acyl cysteine bonds with 2-nitro-5-thiocyanobenzoic acid, N-bromosuccinimide and 2-(2-nitrophenyl )-3-Methyl-3-rhyromoindole to cleave the tryptophanyl bond, or use an enzymatic reaction such as trypsin, chymotrypsin, thermolysin, pepsin, hapain, subtilisin elastase, etc. I can do it.

As the raw albumin, bovine albumin, human albumin, egg albumin, lactalbumin, etc. can be used, but when t-PA is used as a pharmaceutical, human albumin is most preferable.

These albumins can be easily converted into albumin decomposition products having an average molecular fi of 3,000 to 60,000 by subjecting them to the above-mentioned chemical or enzymatic reactions.

The albumin decomposition product is added to the aqueous solution in the t-PA purification process or to the preparation after purification, with or without fractionation as appropriate. The appropriate amount of the albumin decomposition product to be added is usually 0.01 to 5 cm per 10,000 units of t-PAIO.

The t-PA pharmaceutical preparation containing an albumin degradation product according to the present invention is usually formulated as a lyophilized preparation that dissolves before use, and has excellent solubility when redissolved before use, and has excellent solubility during lyophilization and storage. Almost no decrease in t-PA activity was observed, and the single-tree chain structure was stably maintained.

The albumin decomposition product used in the present invention can be produced, for example, by the method shown in the following reference example.

Reference Example (Production method for albumin decomposition product) 2 g of human serum albumin was dissolved in 20 mff of 6NH (J) and hydrolyzed at 60°C for 30 minutes. After the reaction, a 0.2 M trishydroxyaminomethane solution was cooled to 4°C. Sephadex G-100 column (5X140c++) dialyzed against
+), the gel is filtered and the average molecular weight is 3000°10
000, 20000.30000.40000 and 50
Six types of albumin decomposition product fractions ranging from 000 to 60,000 were obtained.

Example (L-PA lyophilized preparation) In 0.1 M phosphate buffer (pH 7.5), the albumin decomposition product obtained in the reference example (average molecular ff1loo00) and 1 bottle of L-PA were added at 10 mg/ml of the former. m7! , the latter 100
000IU/l117! 1 ml of the solution was aseptically filled into an ampoule and lyophilized to obtain a dry formulation of t-P A100OOOOI U (D stable fCtfau).

(5) Effects of the invention The present invention will be explained in detail below using test examples.

Test example 1 (stabilizing effect test of albumin decomposition product) 0,
1000 μl of 1-terminal t-PA in 1 M I77 acid buffer.
00 ([J/+l!), and the albumin decomposition products of various average molecular weights obtained in the above-mentioned reference examples were added as stabilizers at a concentration of lO/1I11. The residual activity and single chain content of t-PA after incubation for a certain period of time were measured.

For comparison, similar measurements were carried out with and without addition of human serum albumin and acid-treated gelatin (molecular size approximately 8000, manufactured by Nisopi Co., Ltd.) instead of the albumin decomposition product. The results of this test are shown in Table 1, and the albumin decomposition product has an average molecular weight of 13,000 to 6.
0000 showed clearly superior stabilizing effects compared to human serum albumin and acid-treated gelatin.

Table 1 Test Example 2 (Stabilizing effect test during lyophilization)
1 in sterile distilled water, and the turbidity (absorbance at 660 nm), residual t-PA activity, and single strand content were measured. For comparison, formulations using acid-treated gelatin and human serum albumin instead of albumin decomposition products as stabilizers and formulations without stabilizers were prepared and similar measurements were conducted.

The results of this test are shown in Table 2.

As is clear from the results in Table 2, in the preparations according to the present invention in which the albumin decomposition product is added as a stabilizer, there is almost no deterioration of the 1-end chain t-PA during the freeze-drying process, and when used, The solubility is also very good.

In the present invention, the activity and single chain content of t-PA were measured by the following method.

(Activity measurement method) 0.15% fibrinogen solution (veronal buffer,
70 mj? per ml of thrombin solution (IO
Fibrin plate (2
t-PA international standard product (WHO
) was measured as a standard product. All units are expressed in international units (IU) of t-PA.

(Measurement of single strand content) Add 10 μl of plasmin (10×7'nff1) to 100 μl of t-PA warm solution (1001 U/mj) and incubate at 37° C. for 30 minutes.

Thereafter, lOμl of aprotinin (100KIU/mj
After stirring well, add 1OOμl of synthetic substrate S.
-2288 solution (0.5mM solution) was added and heated to 37°C.
After incubating for 1 hour at 405nn+, the absorbance value (A
+) to 2-wavelength microplate photometer M T
Measured using P-22 (Corona IElectric). Furthermore, the absorbance value (Ao) at 405 nm is also measured at the same time when aprotinin is added before the addition of plasmin and the same operation is performed.

The single product t-PA content ff1 (x%) was calculated using the following formula.

However, a: 1 terminal chain t-PAI, 178 S-2288
Ice-melting activity (ΔA405/μg) t: N of t-PA used for measurement (μg) k: 1 main t
- The content of PA is 8%.

- value when measured using a sample.

a Patent applicant: Wakamoto Pharmaceutical Co., Ltd.

Claims (2)

[Claims] (1) A method for stabilizing tissue plasminogen activator, which comprises adding an albumin decomposition product as a stabilizer. (2) Albumin decomposition product has a molecular weight of 3000 to 60000
The method for stabilizing tissue plasminogen activator according to claim 1, characterized in that