JPH0332356B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for improving the local affinity of thrombolytic proteins for diseases. This is a method for improving the local affinity of thrombolytic proteins for diseases, which is characterized by chemically bonding them to a thrombolytic protein. Thrombolytic agents such as urokinase are used not only as thrombolytic agents but also as anticancer agents.
By improving its affinity to the diseased local area, superior medicinal efficacy can be exerted with a small dose. In addition, thrombolytic proteins such as urokinase disappear quickly from the blood after in vivo administration.
Its half-life is a few minutes. Furthermore, since the administered thrombolytic protein such as urokinase is affected by inhibitors in the blood, it will not be effective unless administered in a certain amount or more. Therefore, large-dose administration is now becoming commonplace. Furthermore, thrombolytic proteins such as urokinase tend to lack thermal stability. Therefore, the first object of the present invention is to provide a method for improving the affinity of thrombolytic proteins to diseased sites (thrombus sites, cancerous sites). A second object of the present invention is to provide a method for improving the stability of thrombolytic proteins against heat and in blood. A third object of the present invention is to provide a stable thrombolytic protein and a method for producing the same. Other objects of the invention will become apparent from the description below. The present inventors have conducted various studies and found that blood clots can be improved by chemically bonding thrombolytic proteins to basic amino acids or their alkyl esters via polymeric polysaccharides. The present invention was completed by discovering that the stability of soluble proteins in blood and against heat is increased, and that their affinity to diseased areas is increased. Thrombolytic proteins used in the present invention include:
Examples include urokinase, streptokinase, tissue plasminogen activator (derived from melanoma cells, human kidney cells, etc.), and if these are highly purified, especially for medical use, There are no restrictions on its origin; for example, urokinase includes human urine,
Examples include those derived from tissue kidney culture, genetic recombination, etc. For example, in the case of urokinase, the molecular weight is generally preferably in the range of 25,000 to 60,000. The basic amino acids used in the present invention include:
Examples include arginine, lysine, histidine, etc., and arginine and lysine are preferably used.
The alkyl in the basic amino acid alkyl ester includes those having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, and isobutyl, with methyl being particularly preferred. Examples of the high molecular weight polysaccharide include dextran, chondroitin sulfate, starch, etc., and dextran is preferably used. The origin of the dextran used in the present invention is not particularly limited, and highly purified dextran, particularly for medical use, is preferably used.
Its preferred molecular weight is generally 10 to 2 million. The method of the present invention is carried out, for example, as follows. That is, a basic amino acid (for example, arginine, lysine, or an alkyl ester thereof) is reacted with a high molecular weight polysaccharide (for example, dextran), and then a thrombolytic protein is reacted. In the reaction with a basic amino acid or its alkyl ester, it is preferable to oxidize the hydroxyl group of the polymeric polysaccharide in advance to activate it to an aldehyde group, and the oxidizing agent at this time is usually sodium periodate. used. This reaction is usually carried out at room temperature, and the solvent is usually an aqueous solvent [preferably a phosphate buffer (especially
0.1M sodium phosphate] is used, the pH is about 4 to 8, preferably about 5 to 7, and the reaction time is about 2 to 10 hours, preferably 4 to 6 hours. Furthermore, the ratio of addition of the polymer polysaccharide and basic amino acid or its alkyl ester is approximately 10% of the polymer polysaccharide.
The ratio is such that the basic amino acid or its alkyl ester) is 0.1 to 50 μmol, particularly 1 to 30 μmol, per mg of basic amino acid or its alkyl ester. In this reaction, sodium cyanoborohydride, for example, is used as a reducing agent. The reaction product thus obtained is reacted with thrombolytic protein. In this reaction, sodium cyanoborohydride, for example, is used as a reducing agent. As a solvent, a phosphate buffer solution (usually pH 5 to 9, preferably about PH 6 to 8) is used, the reaction temperature is generally about 0 to 20°C, preferably about 1 to 10°C, and the reaction time is about 5 to 40 hours,
Preferably it is about 10 to 25 hours. The amount of thrombolytic protein (eg, urokinase) added is usually 100 IU to 100,000 IU per mg of polymeric polysaccharide. After the reaction, the terminally reacted aldehyde group is reduced to a hydroxyl group. As the reducing agent in this case, for example, sodium borohydride is used. An example of this reaction is schematically shown below. (In the formula, -NH-AA represents a basic amino acid or its alkyl ester residue. Here, the basic amino acid or its alkyl ester residue refers to a basic amino acid or its alkyl ester residue that is formed by removing one hydrogen atom from the amino group in the amino acid. It means a group formed. Note that the bonded product obtained by the method of the present invention is not limited to the above-mentioned model. For example, when a basic amino acid or its alkyl ester having multiple amino groups is used, The thrombolytic protein may be bound to the amino group that is not bound to the molecular polysaccharide, and in short, the three components mentioned above may be chemically bound.The binding ratio of the three components in the present invention is , preferably a basic amino acid or its alkyl ester per 1 mg of a polymeric polysaccharide, such as dextran,
For example, arginine (or its alkyl ester)
is 0.1-3 μmol, preferably 0.5-2.5 μmol, and thrombolytic proteins, such as urokinase, are 100-3 μmol.
The proportion corresponds to 100000 IU, preferably 5000 IU to 100000 IU. The conjugate produced according to the present invention is easily soluble in aqueous solvents (e.g., water, aqueous physiological salt solutions),
Also, human fibrin standard plate method (BBA, 24 ,
278, 1976) per mg of thrombolytic protein (e.g., urokinase).
It is about 5000-140000IU. The binding ratio of thrombolytic protein (eg, urokinase) and high molecular weight polysaccharide, such as dextran, is preferably such that 40 to 70% of the amino groups in the thrombolytic protein are modified. For arginine or its alkyl ester, the amino group can be quantified by R.Fields (Biochem.
J., 124 , 581, 1971), and also
In the case of lysine or its alkyl ester, it was measured by hydrolyzing the bound product in 6N hydrochloric acid at 110°C for 24 hours, followed by amino acid analysis.
The binding ratio was measured after removing thrombolytic protein and unreacted polymeric polysaccharide with DEAE (diethylaminoethyl)-Toyopearl 650M (manufactured by Toyo Soda). Experimental Example 1 The thermal stability of the conjugate obtained by the method of the present invention was investigated using urokinase as an example. When urokinase was heated at 60°C at pH 8.0, the most stable state, urokinase completely lost its activity after about 2 hours of heating, as shown in Figure 1, but the conjugate produced in Example 1 Approximately 90% activity was maintained even after heating for hours. Experimental Example 2 Using fresh human plasma, stability against thrombolytic protein inhibitors in blood was investigated. The test method is as follows. That is, 4 times the amount of plasma was added to urokinase diluted to 100 IU and 200 IU per ml or the conjugate obtained in Example 1 according to fibrinolytic activity using a fibrin plate, and the mixture was incubated at 37°C for 1 hour. After incubation, residual activity was measured by fibrin plate method. The results are shown in FIG. That is,
At 100 IU/ml, urokinase loses all activity, whereas the conjugate of Example 1 loses 10
~13%, at 200 IU/ml, urokinase is approximately 10
% of the remaining activity, whereas the conjugate of Example 1 retains about 20% of the remaining activity. Experimental Example 3 A comparison of thrombolytic ability between the conjugate of Example 1 and urokinase itself was performed in vivo using rabbits. ^Chloramine T method (Biochem.J.,
89, 114, 1963), fibrinogen (purified from rabbit blood) was labeled.
After mixing ¹²⁵ I-fibrinogen with rabbit blood, ^{a 125} I-thrombus was created using Chandler's apparatus. ¹²⁵ I- When circulating the blood clot by connecting the catheter inserted into the rabbit's femoral artery and femoral vein as shown in Figure 3, place the syringe into a syringe connected to the joint site with a rubber stopper ( ¹²⁵ I - The blood clot is suspended from a fishhook.) Then, the weight of the rabbit was accurately measured, and the fibrinolytic activity was 10 ⁵ IU of urokinase per 3 kg, or Example 1 was equivalent to the protein amount of this urokinase.
One hour after the start of blood circulation, the conjugate was administered from the three sides of the vein, and at 10, 20, 30, 45, 60,
At 90, 120, 150, and 180 minutes, blood was collected from the Sanho Kotsu on the artery side, and the radioactivity in the blood was measured using a γ-scintillation counter to determine the thrombus lysis rate.
The results are shown in Figure 4. From this result, it can be understood that the method of the present invention improves the thrombus lysis rate by more than twice. Example 1 Dextran T-10, T-40, T-70, T-
Weigh 1 g of either 500 or T-2000 (manufactured by Pharmacia) and dissolve it in 20 ml of distilled water. this
Sodium periodate after soaking in a 30℃ water bath
Add 1.078g as a solid. After reacting at 30℃ for 30 minutes, neutralize with 1N sodium hydroxide.
Oxidized dextran (activated dextran) was obtained by thorough dialysis against distilled water and freeze-drying. Add 13 μmol of arginine, lysine or their methyl esters to 0.2 ml of 0.1 M sodium phosphate (PH
6.1), add 9.3 mg of oxidized dextran and 13 μmol of sodium cyanoborohydride [6 mg of reagent dissolved in 1 ml of 0.1 M sodium phosphate (PH6.1)] to 0.8 ml of the same buffer. ,
React for 4-6 hours at room temperature. During this time, it is confirmed by silica gel thin layer chromatography that the amino acid or their ester is bound to dextran. After the reaction, cool at 5°C, dissolve in the same buffer solution, and add 0.1 μmol (1 to 2 μmol) of urokinase.
ml), followed by adding 26 μmol of sodium cyanoborohydride and reacting at 5° C. for 5 to 24 hours. After the reaction, 36 μmol of sodium borohydride (10 mg
(dissolved in 0.1M sodium phosphate (PH7.0)) and reacted at 5°C for 6 hours to completely reduce the aldehyde groups. After reduction, the mixture is thoroughly dialyzed against 0.1M sodium phosphate (PH7.0), sterilized through a Millipore filter, titer is measured, and lyophilized to obtain a urokinase conjugate. This conjugate had the characteristics of the urokinase/dextran/amino acid or methyl ester conjugate thereof described above.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the thermal stability improvement of the thrombolytic protein according to the method of the present invention, FIG. 2 is a diagram showing the stability of the thrombolytic protein according to the present invention against inhibitors in human blood, and FIG. The figure shows thrombolytic capacity test (in
Fig. 4 is a diagram showing the results of a thrombolytic ability test conducted using the experimental system shown in Fig. 3. 1: conjugate of Example 1 (using arginine methyl ester, dextran T-40), 2: conjugate of Example 1 (using arginine, dextran T-40), 3: urokinase itself, 4: femoral artery,
5: Femoral vein, 6: Catheter, 7: Three-sided tube, 8: Rubber tube, 9: ¹²⁵ I-thrombus, 1
0: Fishhook, 11: Syringe.

Claims (1)

[Scope of Claims] 1. A method for improving the local affinity of a thrombolytic protein for a disease, which comprises chemically bonding the thrombolytic protein to a basic amino acid or its alkyl ester via a polymeric polysaccharide. 2. The method according to claim 1, wherein the high molecular weight polysaccharide is dextran. 3. The method according to claim 2, wherein the dextran has a molecular weight of 10 million to 2 million. 4. The method according to any one of claims 1 to 2, wherein the basic amino acid is arginine or lysine. 5. According to claim 1, the basic amino acid or its alkyl ester is bound at a ratio of 0.1 to 3 μmol and the thrombolytic protein is bound to 1 mg of the polymeric polysaccharide at a ratio corresponding to 100 to 100,000 IU. Method. 6. The method according to claim 1, wherein the basic amino acid or its alkyl ester is bound to the polymeric polysaccharide, and then the thrombolytic protein is bound to the polymeric polysaccharide. 7. The method according to any one of claims 1 to 6, wherein the thrombolytic protein is urokinase.