JPH0143553B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel urokinase derivative and a method for producing the same. Urokinase, which is a fibrin and thrombus dissolving enzyme, is widely used in the treatment of various thrombosis and embolic diseases, as well as in combination therapy with anticancer drugs, and has excellent clinical effects. However, when urokinase is administered to a living body, it quickly disappears from the blood both as a protein and as an enzymatic activity, and its half-life in the blood is only 1 to 2 minutes. Furthermore, it is known that the enzymatic activity of administered urokinase is affected by urokinase inhibitors in the blood, and thrombolytic ability does not develop unless an amount exceeding a certain threshold is administered. It is understood that such blood dynamics of urokinase inevitably leads to large-dose administration in order to obtain sufficient effects, leading to today's high-dose therapy. The inventors have long researched ways to maintain the efficacy of urokinase in the blood and make it less susceptible to inhibitory factors in the blood.
As a result of repeated experiments and studies, we have created water-soluble urokinase/monosaccharides, urokinase/monosaccharide derivatives, and urokinase/disaccharide conjugates, and these conjugates can improve various drawbacks seen when urokinase is administered alone. The inventors have discovered that the enzymatic activity of urokinase can be sufficiently and sustainably exerted, and have completed the present invention by focusing on its usefulness as a medicine. That is, the present invention provides a water-soluble urokinase-saccharide conjugate formed by directly or indirectly covalently bonding urokinase and at least one sugar selected from monosaccharides, derivatives thereof, and disaccharides, and urokinase and monosaccharides. This invention relates to a method for producing a urokinase-saccharide conjugate by reacting activated monosaccharides, activated monosaccharide derivatives, and activated disaccharides with one selected from activated monosaccharides, and optionally subjecting them to a reduction reaction. The urokinase used in the present invention may be derived from either human urine or kidney tissue culture as long as it has been purified as a medicine. Furthermore, the human-derived urokinase gene is introduced into Escherichia coli using genetic engineering techniques, and after culturing, Urokinase produced by E. coli may be used, and it is preferable to use one with a molecular weight in the range of 25,000 to 60,000. Examples of monosaccharides used in the present invention include glucose, galactose, fructose, etc., and examples of monosaccharide derivatives include uronic acids such as glucuronic acid and galactyuronic acid, glucosamine, and N-acylglucosamine (e.g., N-acetylglucosamine). glucosamine), 2-amino-
Amino sugars and acylated amino sugars such as 2-deoxine-D-glucose, methyl-6-bromo-6
Examples include glycosides such as -deoxy-glycoside, and further examples of disaccharides include lactose and maltose. The urokinase/saccharide of the present invention is produced by reacting urokinase with a monosaccharide as it is, or by reacting it with an activated saccharide obtained by activating the above saccharide. Here, activated saccharide is one in which the hydroxyl group of saccharide is activated so that it can react with the amino in the urokinase molecule. More specifically, the urokinase-saccharide conjugate is produced as follows. (1) (Method 1) A method that utilizes the reactivity of monosaccharides to directly bind to urokinase. This method is applicable only to monosaccharides;
This reaction utilizes an aldehyde group or a keto group of a monosaccharide as a functional group, and the formula for this reaction is as follows, taking the case of using glucose as the monosaccharide as an example. This method is carried out by reducing a compound obtained by reacting a monosaccharide with urokinase. In the reaction between urokinase and monosaccharide, monosaccharide is added to 1 to 10 parts by weight of urokinase.
Preferably, 10 to 3000 parts by weight are reacted.
Usually, monosaccharides are dissolved in water, and urokinase is dissolved in a 0.1-1M buffer (for example, phosphate buffer, pH 6-8) for the reaction. Reduction of the reaction product thus formed is usually carried out with sodium cyanoborate hydride. This reaction is usually carried out by adding a urokinase buffer and a reducing agent to an aqueous monosaccharide solution and stirring the mixture at 1 to 10°C for 10 to 30 hours. (2) A method of binding activated monosaccharides, their derivatives, or disaccharides to urokinase. This method is broadly divided into an aldehyde method and an alkylation method. These two methods will be described below. (Second method) Aldehyde conversion method After oxidizing monosaccharides and their derivatives or disaccharide hydroxyl groups to aldehyde groups and activating them,
This is a method for producing urokinase derivatives by reacting with urokinase and further reducing it. This is shown as a schematic reaction formula as follows. As the oxidizing agent used in the oxidation step in this reaction, periodic acid or the like is preferably used. The oxidizing agent is based on 180 parts by weight of sugars,
It is usually used in a proportion of 20 to 240 parts by weight, and
The oxidation is usually carried out for 10 to 60 minutes at room temperature in the dark. The activated saccharide thus obtained is reacted with urokinase. This reaction usually involves first neutralizing the activated saccharide obtained in the above reaction with a 1-4M alkali (e.g., sodium hydroxide), and then
This is carried out by adding 10 parts by weight of urokinase at about 0.9°C. The product thus obtained is treated with a reducing agent (preferably a cyanoborohydride metal salt).
0.6 to 2 parts by weight),
It can lead to urokinase-saccharide conjugate. As a solvent in the reduction treatment, for example, a 0.1-1M buffer (e.g. phosphate buffer) PH6
~8 is used. Further, the reduction is carried out by stirring at about 4°C for 15 to 20 hours. (Third Method) Alkylation Method This method is schematically illustrated as follows. (In the formula, X and X' each represent a halogen atom (e.g., prom, chloro), and n represents an integer of 1 to 6.) It is added in a proportion of 0.1 to 0.3 parts by weight to form an activated saccharide, and then treated with a difunctional compound [for example, a compound such as diaminoethane or diaminohexane ()], and then combined with a compound such as bromoacetyl bromide (). let The activated saccharide thus obtained is brought into contact with urokinase to obtain a urokinase derivative. The resulting reaction generally has a pH of 7.2
-11 and contact at a temperature of 3 to 25°C for 12 to 48 hours. The urokinase-saccharide conjugate thus obtained can be separated and purified by known separation and purification methods, such as gel filtration, molecular sieving, and ion exchange. The recovered urokinase/saccharide conjugate can be made into a urokinase/saccharide conjugate preparation, that is, a thrombolytic agent, by, for example, performing sterilization and heat treatment, dispensing it, and freeze-drying it. . The properties of the urokinase/saccharide conjugates thus obtained, ie, urokinase/monosaccharides, urokinase/monosaccharide derivatives, and urokinase/disaccharide bonds, are as follows. Easily soluble in water and saline. The molar ratio of urokinase and each sugar is 1:5 ~
It is 1:1000. PH stability, thermostability, resistance to protease inhibitors in plasma, persistence in blood, and acute toxicity are as shown in the experimental examples below. The urokinase/saccharide conjugates used in the following experimental examples are urokinase/monosaccharide conjugates of urokinase/glucose and urokinase/galactose produced by the second method (aldehyde method). As a derivative conjugate, a conjugate of urokinase/glucosamine and urokinase/N-acetylglucosamine produced by the third method [alkylation method, using diaminoethane as the compound ()] was used as a second urokinase/disaccharide conjugate. A conjugate of urokinase/lactose and urokinase/maltose produced by the aldehyde method (aldehyde method) was used. Experimental Example 1 PH Stability: Urokinase itself is relatively stable over a PH range of 3 to 10, but the various urokinase-saccharide conjugates mentioned above are even more stable, ranging from PH 2 to 11.
No decrease in titer was observed over a wide range. Experimental example 2 Thermal stability: 60℃ at pH8.0, where urokinase is most stable
When heated, urokinase alone was completely inactivated after 2 hours of heating, whereas the above-mentioned urokinase/saccharide conjugate still showed 100% residual activity even after 10 hours of heating. It was found to be very stable to heat. Experimental Example 3 Resistance to plasma protease inhibitors: Using human plasma, we tested the resistance of urokinase, the above-mentioned urokinase/monosaccharide, urokinase/monosaccharide derivatives, and urokinase/disaccharide conjugates to plasma protease inhibitors. Examined. The test method is as follows. That is, the activity is 100IU
(International units)/ml (For international units, see Pharmaceutical Research (3), 295-308 (1974)) A solution of urokinase alone or each of the above-mentioned conjugates diluted to
Mix 15μ of 18% human plasma albumin with 225μ, then add human plasma to 20μ of this mixture.
After mixing 80μ and incubating for 1 hour at 37℃, human fibrin standard plate method (BBA, 24 ,
278-282, 1975), and the residual urokinase activity was calculated based on the urokinase activity before incubation with human plasma as 100%. The test results are shown in Figure 1, and the residual activity of urokinase alone was 1.5%.
In contrast, the residual activities of the conjugates of urokinase-glucose, urokinase-galactose, urokinase-glucosamine, urokinase-N-acetylglucosamine, urokinase-lactose, and urokinase-maltose are 8%, 7%, and 6%, respectively. They were 7%, 8%, and 9%.
From this, it was confirmed that the conjugate of the present invention is resistant to the effects of plasma protease inhibitors. Experimental Example 4 Thrombolytic ability test: Using the Chandra loop method, the thrombolytic ability of urokinase or the various conjugates listed above was comparatively investigated. In this test method, 1 ml of fresh, normal human citrated blood is placed in a plastic tube with an inner diameter of 3 mm and a length of 270 mm, and 0.1 ml of 3.8% calcium chloride dihydrate solution is added to the blood.
Join silicon tubes with an inner diameter of 5 mm and a length of 15 mm to both ends of the tube to form a loop. This tube was immediately placed on a rotary plate designed to rotate 12 times per minute at an angle of 80° from the horizontal plane, and rotated for 20 minutes in a thermostatic chamber at 37°C. This creates an artificial thrombus approximately 13 mm in length at the tip of the blood column. This artificial thrombus is said to have a histological image that is very similar to a mixed thrombus formed in the human body in histopathological terms. After removing the loop from this device, remove one end of the looped polyvinyl tube from the silicone tube, and add 0.1% of the sample solution or physiological saline.
ml was put into the tube, the tube was made into a loop again, and placed on a rotating device to continue rotating. Four hours after starting thrombolysis in the first loop, the first loop was removed from the rotating device, the thrombus in the tube was homogenized in 10 ml of distilled water, and centrifuged (3600 rpm, 5 ml). The absorbance of the supernatant was measured at 540 nm.
The second and subsequent loops were extracted following the first loop and processed in the same manner as before. In addition, the thrombolysis rate (Y) of the sample solution addition group was determined according to the following formula. Y=(1-A/Ac)×100(%) However, A: 540 nm for the loop to which the sample solution was added
Absorbance Ac; for the loop with saline added
Average value of absorbance at 540 nm The results of the thrombolytic test using the Chandra loop method for urokinase alone and the combination of urokinase/glucose, urokinase/galactose, urokinase/glucosamine, urokinase/N-acetylglucosamine, urokinase/lactose, and urokinase/maltose are as follows: , as shown in FIG. The horizontal axis of the graph in Figure 2 is the sample liquid.
The final titer when 0.1 ml was injected into the loop is shown, and the titer is the value measured by the fibrin plate method. From this graph, it can be seen that the thrombolytic curves of the various conjugates mentioned above are shifted to the left side of the curves of urokinase itself, indicating that the thrombolytic ability of the conjugates of the present invention is stronger than that of urokinase itself. It will therefore be understood that these conjugates of the present invention have excellent thrombolytic activity and are useful as thrombolytic agents. Experimental Example 5 Blood Persistence Test: Using beagle dogs (male, weight 12-15 kg), the effects of maintaining each of the above-mentioned urokinase-saccharide conjugates in the blood were tested and compared with administration of urokinase alone. The labeling method is the chloramine T method (Biochem J. 89 , 114,
(1963), dissolve 100,000 units of urokinase in 0.5 ml of phosphate buffer, add 1 ml of 1 mCi/ml Na ¹²⁵ I to 0.1 ml of this urokinase aqueous solution, add 0.1 ml of 0.05% chloramine-T, and let the mixture cool to room temperature. te2
After reacting for a minute, 0.1% sodium bisulfite
The reaction was stopped by adding 0.1 ml, and the reaction mixture was passed through a Sephadex G-25 column to separate ¹²⁵ I-urokinase and free NaI ¹²⁵ . ^{The 125} I-urokinase thus obtained was divided into two parts, one was used as a sample for animal experiments, and the other was reacted with a monosaccharide, a derivative thereof, or a disaccharide of the activated conjugate having a molecular weight of 500,000. ^{A 125} I-urokinase-saccharide conjugate was obtained and used as a sample for animal experiments. Each ¹²⁵ I-urokinase/saccharide conjugate and ¹²⁵ I
- 2×10 ⁶ cpm of each urokinase was administered intravenously to beagle dogs, and the blood concentration of each was determined every 10 minutes. The experimental results are shown in Table 1, and compared to ¹²⁵ I-urokinase alone, the blood half-life is shorter in the first and second decay curves.
¹²⁵ I-Urokinase glucose conjugate: 5.3 times and 4.7 times, ¹²⁵ I-Urokinase galactose conjugate: 5.3 times and 4.4 times, ¹²⁵ I-Urokinase glucosamine conjugate: 5.9 times and 5 times, ¹²⁵ I -Urokinase/N-acetylglucosamine conjugate
5.3 times and 4.9 times, ¹²⁵ I-urokinase lactose conjugate 5.3 times and 4 times, ¹²⁵ I-urokinase conjugate
For the maltose conjugates, the increase was 5.9 times and 4.4 times, respectively, indicating that these urokinase conjugates had a significant blood maintenance effect.

【table】

[Table] Experimental Example 6 Acute toxicity test of urokinase-saccharide conjugate: To investigate the acute toxicity test by intravenous administration of urokinase to Wistar rats, 500,000 IU of urokinase of each of the above urokinase conjugates was intravenously injected per 1 kg of body weight. When general symptoms were observed and body weight was measured for 7 days after administration, the body weight increased steadily and no abnormal findings were observed.
No abnormalities were found in the results of autopsy and histological examination. The dosage and administration method for clinical use of the urokinase-saccharide conjugate according to the present invention are, for example, as follows. That is, dissolve 50 to 30,000 IU of this product in 0.5 to 5 ml of distilled water for injection in the Japanese Pharmacopoeia, and
Administer the drug by intravenous injection, intravenous drip, subconjunctival or retrobulbar injection, adjusting the dosage as appropriate depending on the symptoms and progress. The urokinase-saccharide conjugate according to the present invention is extremely stable in blood and does not easily dissociate or decompose.
It is extremely stable against heat, is not susceptible to the effects of urokinase inhibitors in the blood, and produces thrombolytic effects even when administered at a low titer, while urokinase alone does not exhibit thrombolytic effects, making it possible to administer it in vivo. It has excellent properties such as a significantly extended blood residence time and almost no detectable toxicity, and has the effect of providing an extremely useful medicine that has never existed before. Examples 1 to 4 Weigh out 180 mg of glucose, galactose, fructose or lactose, and add 3 ml of distilled water to this.
Add and dissolve, containing 6 mg of urokinase.
2ml of 0.1M phosphate buffer (PH7) and 1ml of the same buffer
A solution of 2 mg of sodium cyanoborohydride was added to the solution, and the mixture was stirred at 4°C for 24 hours. After stirring, dialysis is performed using the above buffer. The obtained material was sterilized using a Millipore filter,
After measuring the urokinase titer and determining the amount to dispense, divide the specified amount into small portions and freeze-dry them to obtain a molar ratio of 1:
A conjugate of 58 urokinase glucose, urokinase galactose in a molar ratio of 1:32, urokinase fructose in a molar ratio of 1:46, and urokinase-lactose in a molar ratio of 1:36 is obtained. These had various properties of the urokinase-monosaccharide conjugate described above. Examples 5-10 Glucose, galactose, glucosamine, N
- Weigh out 1 g of acetylglucosamine, lactose, or maltose, add 20 ml of distilled water to dissolve it, add 1.18 g of sodium periodate dissolved in 5 ml of distilled water, and store in the dark at room temperature. The mixture was stirred for 30 minutes. After stirring, neutralize with 4M sodium hydroxide solution and then add 10 mg of urokinase.
4 ml of an aqueous solution containing is added and stirred at 4°C for 18 hours. After stirring, sterilization was performed using a Millipore filter, the urokinase titer was measured and the amount to be dispensed was determined, and the specified amount was divided into small portions and lyophilized to obtain urokinase/glucose at a molar ratio of 1:96. , urokinase/galactose in a molar ratio of 1:70, urokinase/glucosamine in a molar ratio of 1:46, urokinase/N-acetylglucosamine in a molar ratio of 1:66, urokinase/N-acetylglucosamine in a molar ratio of 1:160.
A conjugate of lactose and urokinase/maltose in a molar ratio of 1:140 is obtained. These had the properties of the conjugates of the invention described above. Example 11 1 g of methyl-6-bromo-6-deoxy-D-glucoside was added to 0.1 M phosphate buffer (PH10) (10 ml)
Add the same buffer as above containing 10 mg of urokinase and dilute with 10 mM sodium hydroxide.
Adjust the pH to 10 and stir at 4°C for 24 hours. After stirring,
Dialysis is performed using the above buffer. Obtained molar ratio 1:
62 urokinase methyl-6-deoxy-D-
The glucoside conjugates had the various properties mentioned above. Example 12, 13 Either glucuronic acid or galacturonic acid
Dissolve g and dicyclohexylcarbodiimide in 0.1M phosphate buffer (PH4.5), and adjust the pH to 4.5 with 1MHCl.
Then, add 0.1M phosphate buffer (PH4.5) containing 10mg of urokinase and allow to react for 24 hours. As described in the previous example, urokinase and urokinase in a molar ratio of 1:76 were obtained by dialysis, sterilization, and freeze-drying.
Glucuronic acid, a conjugate of urokinase and galacturonic acid in a molar ratio of 1:83, had the various properties described above. Examples 14 and 15 1 g of either 2-amino-2-deoxy-D-glucose or 2-amino-2-deoxy-D-galactose and dicyclohexylcarbodiimide were dissolved in 0.1 M phosphate buffer (PH4.5), 1M
Adjust the pH to 4.5 with hydrochloric acid, add 0.1M phosphate buffer (PH4.5) containing 10 mg of urokinase, and allow to react for 24 hours. After the reaction is complete, urokinase 2 with a molar ratio of 1:62 is obtained by dialysis, sterilization, and freeze-drying.
-Amino-2-deoxy-D-glucose or a conjugate of urokinase and 2-amino-2-deoxy-D-galactose in a molar ratio of 1:50 had the various properties mentioned above. Examples 16-21 Glucose, galactose, glucosamine, N
-Add 49ml of distilled water to 1g of acetylglucosamine, lactose, or maltose and dissolve.
Next, 3.2 g of triphenylphosphine and 2.2 g of N-bromosuccinimide were added to 1.5 ml of tetrahydrofuran.
Add the solution dissolved in it dropwise and stir thoroughly. During this time
Maintain pH 10.2-10.5 with 1M sodium hydroxide. 5 minutes after the start of the reaction, adjust the pH to 2.2 with concentrated hydrochloric acid.
Add 2 ml of diaminoethane to raise the pH to 9.5, keep it at 9.5, and leave it at 4°C overnight. Thereafter, unreacted substances are removed by partition chromatography using a flash column, followed by freeze-drying to obtain the above-mentioned aminoethylaminated compound. Next, dissolve this in 25 ml of 0.1M phosphate buffer (PH7.0), add 1 ml of bromoacetyl bromide dropwise, and
After maintaining the pH at 7.0 with 1N sodium hydroxide for a period of time, unreacted substances were removed by partition chromatography using a flash column, and the activated compound powder was obtained by lyophilization. Dissolve 50mg of it in 0.4ml of 0.1M carbonate buffer (PH9.5) and add urokinase 2.
Add 10,000 IU and leave to stand at about 5°C for 50 hours to perform a binding reaction. After completion of the reaction, dialysis, sterilization, and lyophilization were performed to obtain urokinase/glucose at a molar ratio of 1:130, urokinase/galactose at a molar ratio of 1:150, urokinase/glucosamine at a molar ratio of 1:116, and urokinase/glucosamine at a molar ratio of 1:120. A preparation of a conjugate of urokinase/N-acetylglucosamine, urokinase/lactose in a molar ratio of 1:260, and urokinase/maltose in a molar ratio of 1:310 was obtained. These formulations had the various properties mentioned above. The characteristics of each product obtained in each of the above examples are as shown in Table 2, and in Table 2, "UK"
means "urokinase".

【table】

【table】 [Brief explanation of drawings]

FIG. 1 shows resistance to plasma protease inhibitors, and FIG. 2 shows thrombolytic curves of urokinase and various urokinase conjugates.
1 to 7 in FIG. 2 are curves for the following combinations. 1... Urokinase, 2... Urokinase glucose, 3... Urokinase galactose,
4...Urokinase/N-acetylglucosamine, 5...Urokinase/glucosamine, 6...
Urokinase maltose, 7... Urokinase lactose.

Claims (1)

[Claims] 1. A water-soluble urokinase obtained by directly or indirectly covalently bonding urokinase and a type of sugar selected from monosaccharides, derivatives thereof, and disaccharides.
Sugar conjugates. 2. The urokinase-saccharide conjugate according to claim 1, wherein the saccharide is bound to 1 mole of urokinase at a ratio of 5 to 1000 moles. 3. A water-soluble urokinase-saccharide bond characterized by reacting urokinase with one selected from monosaccharides, activated monosaccharides, activated monosaccharide derivatives, and activated disaccharides, and subjecting it to a reduction reaction as necessary. How things are manufactured.