JPS6043116B2 - Urokinase derivative - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel urokinase derivative, and more specifically, a water-soluble urokinase-dextran conjugate has been developed in which urokinase as a urokinase derivative is chemically linked with activated dextran. However, since this urokinase derivative has various novel and superior oxygen properties and biological properties compared to urokinase itself, the aim is to provide this urokinase derivative as a medicine.

Urokinase, which is a fibrin and thrombus dissolving element, 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.

When urokinase is administered to a living body, it quickly disappears from the blood both as a protein and as an enzyme, and its half-life in the blood is only 1 to 2 minutes. Furthermore, it is known that the oxygen activity of administered urokinase is affected by urokinase inhibitors in the blood, and thrombolytic ability does not develop unless the dose exceeds a certain threshold. Such blood dynamics of urokinase inevitably leads to large-dose administration in order to obtain the administration effect.
It is understood that this has led to today's high dose therapy.

The inventors have long been researching how to maintain the efficacy of urokinase in the blood and make it less susceptible to the effects of inhibitory factors in the blood, and as a result of repeated experiments and studies, a water-soluble urokinase-dextran conjugate has been found to be effective against urokinase. can improve various disadvantages observed when administered alone;
The present inventors have discovered that the oxygen activity of urokinase and urokinase can be sufficiently and sustainably exerted, and based on these new findings, the present invention has been completed. Various attempts have been made to immobilize urokinase by binding, embedding, or adsorbing it to some types of carriers, but all of them have used water-insoluble synthetic resins, collagen fibers, polymeric natural compounds, etc. as carriers. These are in the form of water-insoluble urokinase, and these were developed for use in medical devices such as artificial blood vessels and dialysis circuits, and are not used as medicines like urokinase administered alone.

A substance similar to the present invention is a urokinase-heparin complex (Japanese Unexamined Patent Publication No. 56888/1988). This takes advantage of the property of the polyanionic body hebalin to non-specifically adsorb proteins, and separates hevalin from urokinase.
It is introduced into the purification process, and urokinase is coprecipitated and recovered in the form of a hebalin complex. When the complex obtained in this way is dissolved in water, it dissociates again into urokinase and hebalin, and when administered to a living body, the two behave separately in the blood. This results in no change and does not exhibit the effect that the substance of the present invention brings. The urokinase derivative according to the present invention is a water-soluble urokinase-dextran conjugate, which has excellent properties in terms of stability against heat or inhibitors and sustainability of enzyme activity. The urokinase used in the present invention may be derived from either human urine or tissue kidney culture as long as it has been purified as a medicine, and can be arbitrarily selected from the molecular weight range of 25,000 to 60,000. Dextran is also not particularly limited in origin, and has a molecular weight of 1000 to 1000, which can be used as a pharmaceutical.
You can arbitrarily choose from a range of 2 million. The composition ratio of urokinase and dextran is 1 m9 of dextran to 1 m9 of dextran. 100 to 100,001 U (international units) (For international units, see Pharmaceutical Research (3), 29
5-308, 1974). The method for producing the conjugate is to first activate dextran, and then use urokinase and activated dextran either directly or via a chain molecule having 1 to 3 carbon atoms. and combine them. For activation of dextran, known methods can be used, such as covalent bonding method (J●B
lOl. Chem. 25l, lO8l, l976),
Cleavage method of aldohexopyranose ring of dextran (P
rOe. Natl. Acad. Sci. USA. 73,
2l28, 1976), -bifunctional compound bonding method (P
rOe. Natl. Acad. Sci. USA. 73,
2l28, 1976), etc.

In the covalent bonding method, cyanbromide is added at a ratio of 0.05 to 1 m9 to 1 mg of dextran, the pH thereof is adjusted to about 9 to 11.5, and reaction is carried out in about 2 to 1 powder at room temperature. After the reaction is complete, excess halide is removed, and the resulting activated dextran is brought into contact with urokinase. The reaction formulas for these activation and binding are as follows. The method for cleaving the aldohexopyranose ring uses sodium periodate as an oxidizing agent, and approximately 0.01 to 1
Add 0.2 mg of oxidizing agent and carry out treatment for 5-2 hours.

After the reaction is complete, excess additives are removed and the resulting activated dextran is brought into contact with urokinase. These reaction formulas are as follows. In the bifunctional compound bonding method, for example, dextran is activated with cyanobromide, this activated dextran is bonded with a bifunctional compound such as diaminoethane to produce a chain molecule having 1 to 3 carbon atoms, and then bromoacetylbromide is bonded to the activated dextran. After binding, contact with urokinase.

These activation and binding reaction formulas are as follows. The binding reaction between urokinase and activated dextran reduces the pH to 7.
．． Adjust to 2-11, 0.5-10c at a temperature of 3-25℃
Make contact between tf and tf.

After the reaction is completed, unbound substances are separated and the desired urokinase◆dextran-conjugated substance is recovered. Known gel filtration methods, molecular sieve separation methods, ion exchange methods, etc. can be used for the recovery method, but when fractionated using the gel filtration method, the urokinase-dextran bound product and unbound dextran and urokinase are very clearly separated. Because they behave differently, the target bound product can be easily recovered. The recovered urokinase-dextran conjugate is subjected to sterilization filtration and heat treatment, and then dispensed and freeze-dried to obtain a urokinase-dextran conjugate preparation. The properties of the urokinase-dextran conjugate thus obtained are such that it is easily soluble in water and physiological saline.

The urokinase activity per TLg of dextran varies depending on the reaction conditions, but it is determined by the human fibrin standard plate method (B.B.
．． A. ? , 278-282, 1975) and is in the range of 100 to 10,000 PU (Ploeg unit unit). (For Ploug units, see Pharmaceutical Research (3)
, 295-308, 197). The binding ratio of urokinase and dextran can be arbitrarily selected by changing the reaction amounts and reaction conditions of both. The substrate specificity of this substance was determined based on the titer (Ploeg units) measured using human fibrin and standard plate method.
K for nemet11y1ester (TLME)
Inetics are as shown in Table 1. In other words, the ester resolution per PU is Vmax (×101μ
MOITLME/Min/PU) is 3 to 4 times higher for the urokinase dextran conjugate than for the original urokinase alone. Furthermore, when comparing the Michaelis constant Km (MM), which indicates substrate affinity, it is 0.79 for urokinase alone, whereas it increases to 0.97 for the urokinase-dextran combination, and the two are completely different Michaelis constants. It turns out that it has a constant. When examining the PH stability, thermal stability, and stability against urokinase inhibitors in the blood of the urokinase-dextran conjugate,
Regarding pH, urokinase complex is relatively stable over a pH range of 3 to 10, but urokinase-dextran conjugate is even more stable, with no decrease in potency observed over a wide range of pH 2 to 11. Nakatsuta.

Regarding thermal stability, urokinase is the most stable at pH8.
．． When heated at 60°C in O2, as shown in Figure 1, urokinase alone was safely inactivated after 2 hours of heating, whereas the urokinase-dextran combination was inactivated even after 1 hour of heating. In addition, it showed a 100% activity residual rate and was found to be extremely stable against heat.
Next, we investigated how urokinase activity is inhibited using a human blood urokinase inhibitor purified from human placenta (Japanese Patent Publication No. 46-21464). The test method is to adjust the urokinase-dextran combination to 751 U/Mt and the urokinase alone to 1501 U/Mt, add equal amounts of various urokinase inhibitor solutions, mix, and heat at 3rC for 3 minutes. Then, 0.02 mt was taken and the urokinase titer was measured using the human fibrin standard plate method. The test results are shown in Figure 2. Looking at the amount of 100% inhibition, urokinase alone has an inhibitory factor of 50
1 U/ml (units are based on the method of Kawano et al. in Japanese Patent Publication No. 46-21464), whereas it was almost completely inhibited.
2001 U/m for urokinase-dextran conjugate
It was found that this conjugate was extremely resistant to the action of urokinase inhibitors in the blood. This suggests that this conjugate is resistant to the effects of urokinase inhibitors in the blood. Also, urokinase
The dextran conjugate was measured by starch gel electrophoresis at 0.0%.
17M, Tris-H2BO4 buffer, PH8.8, l
When analyzed under the conditions of mA/C7n, lm at 4°C, it showed almost the same migration pattern as that of urokinase alone, and it was observed that there was almost no difference in the electrical properties between the two. The thrombolytic ability of urokinase-dextran conjugate and urokinase alone was compared using the Chandra loop method.

This test method uses 1ml of fresh, normal human citrated blood.
was placed in a plastic tube with an inner diameter of 3TW1 and a length of 270 mm, and after adding 0.1 ml of 3.8% calcium chloride dihydrate saline to the blood, a tube with an inner diameter of 5 Tn was placed at both ends of the tube. Tt, length 15? Join the silicone tubes to make a loop. This tube was immediately placed on a rotary plate designed to rotate once per minute at an angle of 80° from the horizontal plane, and rotated three times in a thermostatic chamber at 3rC. As a result, an artificial thrombus with a length of 13w1 is generated 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 creating such an artificial thrombus in the blood in the loop, add 0.1 mL of urokinase solutions of various concentrations to adjust the final concentration of urokinase in the blood to 1 to 20 PU/ml, and then The artificial thrombus is lysed by continuing rotation at a hill rotation/min for exactly 4 hours at °C. After the rotation is complete, remove the artificial thrombus inside the tube and add fan sample solution (1).
After fixing with one solution, the weight of the thrombus was weighed, and the thrombolysis rate was calculated using the following formula. As a control, the same amount of physiological saline was added instead of the urokinase solution, and after rotating in the same manner for 4 hours, the same treatment was performed and the weight of the thrombus was measured. The test results are shown in Figure 3, and urokinase alone showed no thrombolytic ability at concentrations below 10 PU/ml, but the urokinase-dextran conjugate showed no thrombolytic activity at concentrations below 10 PU/ml.
Even in a low concentration range of PU/ml or less, remarkable thrombolytic ability was brought about.

This suggests that this conjugate can be expected to have excellent effects as a thrombolytic agent. Next, we conducted an experiment on the maintenance effect of the urokinase-dextran conjugate in the blood using beagle dogs (male, weight 12-15k9) and compared it with administration of urokinase alone. The test method is Chloramine T method (B
iOehemJ. ? , 114, 1963), 100,000 units of urokinase was dissolved in 0.5 ml of phosphate buffer, 25 l/MCi of Nal was added, and 1% chloramine-TO. After adding 5 ml and reacting at room temperature for 2 minutes, the reaction mixture was passed through a Sephadex G-25 column to separate 1251-urokinase and free NaIl25. The 1251-urokinase thus obtained was divided into two parts, one was used as a sample for animal experiments, and the other was reacted with activated dextran with a molecular weight of 500,000.
A conjugate of 1-urokinase and dextran was obtained and used as a sample for animal experiments. 2 x 1C each of 1251-urokinase dextran conjugate and 1251-urokinase
f'Cpm was administered intravenously to vehicle dogs, and the respective blood concentrations were determined every other time.

The experimental results are shown in Table 2 and Figure 4. Compared to urokinase alone, this conjugate has a half-life in the blood that is 5.1 times longer from the first decay curve and 5.1 times longer from the second decay curve. is 4.4
It was found that the binding of urokinase with dextran had a remarkable effect on maintaining blood circulation. In order to investigate the acute toxicity test by intravenous administration into the tail vein of Wistar rats, 500,000 IU of this conjugate, urokinase, was injected intravenously per 1 kg of body weight, and general symptoms were observed and body weight was measured for 7 days after administration. The patient's weight increased steadily and no abnormal findings were observed, and no abnormalities were found in the results of the physical examination and histological examination.

The dosage and administration method for clinical use of the urokinase-dextran conjugate according to the present invention is 50 to 3000
Dissolve this product (Japanese Pharmacopoeia) in 0.5 to 5 ml of distilled water for injection, and administer intravenously, intravenously, intravenously, subconjunctivally or bulbally, depending on age, symptoms, and progress. Used after injection.

The urokinase-dextran conjugate according to the present invention is extremely stable in the blood, does not dissociate or decompose easily, is extremely stable against heat, and is not susceptible to the effects of urokinase inhibitors in the blood. Urokinase alone has no thrombolytic effect; even low-titer administration produces thrombolytic effects, and when administered in vivo, the blood retention time is significantly extended, and toxicity is almost undetectable. It has the effect of providing extremely useful medicines that have never existed before.

Example 1 Dextran T-10, T-40, T-70, T-50
0 or T-2000 (manufactured by Pharmacia, Sweden)
Weigh out 20 m9 of either of these, add 10 m1 of 0.1M sodium carbonate to it, and dissolve.

60 mg of bromcyan in solid or solution form is added to this dextran solution, and the mixture is stirred at room temperature for 5 minutes. During this time, the pH of the reaction solution is maintained at 11.0 using a hemorrhoidal sodium hydroxide solution, and the activation reaction of dextran is carried out. After stirring for 5 minutes, the reaction was stopped by lowering the pH to 8.5 with hemorrhoidal hydrochloric acid solution.

Among these, the reaction product is dialysis or Sephadex G-2
Gel filtration is performed using the column No. 5 to remove unreacted bromide and the like to obtain an activated dextran solution. Next, urokinase 50yj[U is added to the activated dextran solution and dissolved, and the binding reaction is carried out overnight at room temperature with gentle stirring.

When the reaction is complete, transfer the reaction mixture to Sephadex G-200.
gel filtration through a column to separate the urokinase-dextran conjugate and unreacted urokinase. Note that instead of gel filtration, molecular sieving may be performed using a membrane filter with a bore size of 100,000 to 200,000. The urokinase-dextran combination obtained in this way is collected, sterilized and filtered using a millibore filter, the urokinase titer is measured, and the amount to be dispensed is determined. A urokinase-dextran conjugate preparation is obtained by drying. This formulation possessed various properties of the urokinase-dextran conjugate described above. Example 2 Dextran 1y was dissolved in 9ml of distilled water, 1mt of 10% sodium periodate was added thereto, and the mixture was allowed to stand overnight in a dark place at 4°C to carry out an activation reaction.

The activation reaction is stopped by adding 3 ml of 3% sodium bisulfite, dialyzed against distilled water overnight, and then freeze-dried to obtain activated dextran powder. Add 100 mg of activated dextran powder to 100,000 IU of urokinase solution and adjust the pH to 9.
．． The binding reaction is carried out by allowing the mixture to stand at 4° C. for about 4 hours. Thereafter, in the same manner as in Example 1, free, unreacted urokinase is removed by molecular sieving, the urokinase-dextran conjugate is sterilized, and the dispensed product is freeze-dried to form a preparation. This formulation retained the properties of the urokinase-dextran conjugate described above. Example 3 Add and dissolve 40ml of distilled water to 1y of dextran, add 150ml of Promium to this and add 1.5ml of cyanide methane.
Add the solution dissolved in it dropwise and stir thoroughly.

During this time, add 1N sodium hydroxide to PHLO. 2-10.
Keep it at 5. After 5 minutes from the start of the reaction, P was added with concentrated hydrochloric acid.
The pH was lowered to 2.2, diaminoethane 27rL1 was added to raise the pH to 9.5, and the pH was kept at 9.5 and allowed to stand overnight at 4°C. This is followed by dialysis against distilled water and freeze-drying to obtain aminoethylaminodextran. Then change this to 0
．． Dissolved in 25ml of 1M phosphate buffer (PH7.O),
Add 1 ml of bromoacetyl bromide dropwise and leave it at 1N for about 2 hours.
pH7 with sodium hydroxide. After maintaining the temperature at O, it is dialyzed against distilled water and freeze-dried to obtain activated dextran powder. 0.4 m of 0.1M carbonate buffer (PH9.5)
1, add 20,000 IU of urokinase, and let stand at about 5°C for 5 minutes to perform a binding reaction. The reaction mixture was then gel-filtered through a Sephadex G-200 column.
A urokinase-dextran conjugate is obtained. FIG. 5 shows a gel filtration pattern using a Sephadex G-200 column, with urokinase and dextran patterns added as controls. The preparations thus obtained possessed various properties of the urokinase-dextran conjugate described above.

[Brief explanation of the drawing]

Figure 1 is a diagram showing thermal stability, Figure 2 is a diagram showing stability against inhibitory factors, Figure 3 is a diagram showing thrombolysis rate, and Figure 4 is a diagram showing blood maintenance effect. , FIG. 5 is a diagram showing the pattern of gel permeation.

Claims (1)

[Scope of Claims] 1. A urokinase derivative consisting of a water-soluble urokinase-dextran conjugate in which urokinase is chemically bonded to dextran. 2. The urokinase derivative according to claim 1, wherein the dextran has a molecular weight of 10 to 2 million. 3. The urokinase derivative according to claim 1, wherein the urokinase is derived from urinary or cultured kidney tissue. 4. The urokinase derivative according to claim 1, wherein the composition ratio is 100 to 10,000 IU of urokinase per 1 mg of dextran.