JPH036790B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel urokinase derivative, a method for producing the same, and a thrombolytic agent. More specifically, the present invention relates to a water-soluble urokinase-starch conjugate, a method for producing the same, and a thrombolytic agent containing the conjugate as an active ingredient. 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 a water-soluble urokinase-starch conjugate and found that this conjugate can improve various drawbacks seen when urokinase is administered alone, and that the enzymatic activity of urokinase can be maintained sufficiently and sustainably. The present invention was completed by focusing on its usefulness as a medicine. That is, the present invention provides a compound having a molecular weight of 90,000 to 500,000, a molar ratio of urokinase to starch of 1:1 to 1:10, and urokinase and starch having -CH ₂ NH- or

A water-soluble urokinase-starch conjugate bonded via [formula] (wherein n represents an integer of 1 to 6), the above-mentioned water-soluble urokinase-starch obtained by reacting urokinase with a reactive derivative of starch The present invention relates to a method for producing a conjugate, and a thrombolytic agent containing the above-mentioned water-soluble urokinase-starch conjugate as an active ingredient. 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 also be used. Moreover, it is preferable to use one having a molecular weight in the range of 25,000 to 60,000. The starch used in the present invention includes starch derivatives such as hydroxyethyl starch (HES), and usually has a molecular weight of 40,000 to 400,000. In the following description, the urokinase-starch conjugate used in experiments and examples other than those used includes the conjugate of such starch derivatives. The urokinase-starch conjugate of the present invention may be one in which activated starch (for example, starch hydroxyl group is activated to aldehyde) and urokinase are directly bound together, or activated starch and urokinase may be combined in another form. It may also be indirectly bonded via a group. Activated starch is one in which the hydroxyl group in the starch molecule is activated so that it can react with the amino group in the urokinase molecule, for example,

【formula】

Examples include compounds represented by the following formula. Compound () is produced, for example, by oxidizing starch with periodic acid or the like. Compound () is obtained by reacting starch and cyanbromide. These activation methods are described in J.Biol.Chem. 251 , 1081 (1976), Proc.
Natl. Acad. Sci. USA 73 , 2128 (1976), etc. The method for producing the urokinase-starch conjugate of the present invention is, for example, as follows. In this reaction, starch is first oxidized with an oxidizing agent (for example, sodium periodate). At this time, add 10 to 40 parts of oxidizing agent to 100 parts by weight of starch.
Parts by weight are used, and the reaction time is 10 to 60 minutes, and the reaction is carried out at room temperature and in the dark with stirring. The activated starch thus obtained is usually neutralized with 3 to 5 M sodium hydroxide after the reaction is completed.
Dialyze against water and freeze-dry. The degree of oxidation of the activated starch thus obtained is between 10 and 50%. The reaction between urokinase and activated starch is usually performed using 20 to 20 parts of activated starch per 100 parts by weight of urokinase.
This is done by reacting 2000 parts by weight.
At this time, urokinase is subjected to the reaction as an aqueous solution, and activated starch is subjected to the reaction as a phosphate buffer (PH 6 to 8) solution. The target product can be obtained by reducing the compound thus obtained, but in this case,
As the reducing agent, a borohydride metal salt is used, and it is particularly preferable to reduce the borohydride metal salt with a cyanoborohydride metal salt before the reduction with borohydride. Main alkali metal salts (eg, sodium salts) are exemplified as preferred. The reduction is usually carried out by stirring at 0 to 30°C, preferably 4°C for 6 to 12 hours. [In the formula, X and X' each represent a halogen atom (for example, chlor), and n represents an integer of 1 to 6. ] In the reaction between starch and cyanogen bromide, the former 100
The latter is used in an amount of 10 to 100 parts by weight. The activated starch is bound by the action of a difunctional compound (eg diaminoethane, diaminohexane) and then a halogen acetyl halide (eg bromoacetyl bromide). Next, this activated starch and urokinase are brought into contact to obtain a urokinase-starch conjugate. This binding reaction was carried out at a temperature of 12 to 48°C at a temperature of 3 to 25°C, with a pH of 7.2 to 11.
This is done by contacting for a period of time. The urokinase-starch conjugate thus obtained can be recovered by known gel filtration methods, molecular sieve separation methods, ion exchange methods, etc. However, when fractionated by gel filtration, the urokinase-starch conjugate and unbound starch and Since urokinase behaves with very distinct differences, it is easy to recover the target conjugate. The recovered urokinase-starch conjugate is subjected to sterilization, heat treatment, etc., and then dispensed and freeze-dried to obtain a thrombolytic agent containing the urokinase-starch conjugate. The properties of the urokinase-starch conjugate thus obtained are that it is easily dissolved in water and physiological saline, and the molar ratio of urokinase and starch is 1:1.
~1:10. In addition, when examining the PH stability, heat stability, and stability against plasma protease inhibitors of the urokinase-starch conjugate, it was found that urokinase itself is relatively stable over a PH range of 3 to 10; - The starch conjugate was more stable, and no decrease in potency was observed over a wide range of pH 2 to 11. Regarding heating stability, when heating at 60℃ at PH8.0, where urokinase is most stable,
While urokinase alone was safely inactivated after 2 hours of heating, the urokinase-starch combination still showed 100% residual activity even after 10 hours of heating, making it extremely stable against heat. This was recognized. The urokinase-starch conjugate according to the present invention has a thrombolytic effect and is therefore useful as a thrombolytic agent, and is administered parenterally, for example, as an injection. Specifically, for example, 50-30000IU
Dissolve this product in 0.5 to 5 ml of distilled water for injections in the Japanese Pharmacopoeia, and administer by intravenous injection, intravenous drip, intravenous drip injection, subconjunctival or retrobulbar injection, adjusting the dosage as appropriate according to age, symptoms, and course. use The urokinase-starch conjugate according to the present invention is extremely stable in the blood, does not easily dissociate or decompose, is extremely stable against heat, and is not susceptible to the effects of urokinase inhibitors in the blood. , Urokinase alone does not exhibit thrombolytic activity; even low-activity administration produces thrombolytic effects; when administered in vivo, blood retention time is significantly extended, and toxicity is almost undetectable. However, it has the effect of providing extremely useful medicines that have never existed before. Hereinafter, the present invention will be explained in more detail with reference to Examples and Experimental Examples. Example 1 Using human plasma, the resistance of urokinase or urokinase-starch conjugate and urokinase-HES conjugate to plasma protease inhibitors was investigated. The test method is that the activity is 100 IU (international unit)/ml (for international units, please refer to Pharmaceutical Research (3),
295-308, 1974) and 225 μ each of urokinase alone, urokinase-starch conjugate, or urokinase-HES conjugate were mixed with 15 μ of 18% human plasma albumin, and then 20 μ of this mixture was mixed with 80 μ of human plasma. Mix and heat at 37℃.
After incubation for an hour, urokinase activity was measured using the human fibrin standard plate method (BBA, 24 , 278-282, 1975), and the urokinase activity before incubation with human plasma was taken as 100%, and the residual urokinase activity was calculated. . The test results are shown in Figure 1, and the residual activity of urokinase alone was 1.5%, while the residual activity of urokinase alone was 1.5%.
The rates were 28% and 25% for starch conjugate and urokinase/HES conjugate, respectively. From this, it was confirmed that the present conjugate was resistant to the effects of plasma protease inhibitors. Experimental Example 2 Urokinase-starch conjugate and urokinase-HES conjugate were analyzed using high-performance liquid chromatography (LC-3A: Shimadzu Corporation).
Someno et al. [J. Chromat., 188 , 185-92 (1980)]
It was analyzed according to the method of TSK- as carrier
Using Gel3000SW (manufactured by Toyo Soda), 0.2M of PH3
When the phosphate buffer solution was developed at a flow rate of 1.2 ml/min, the high performance liquid chromatogram shown in Figure 2 was obtained.
As a result, the urokinase-starch conjugate and the urokinase-HES conjugate were eluted on the polymer side as expected. Experimental Example 3 The thrombolytic ability of urokinase/starch conjugate, urokinase/HES conjugate, and urokinase alone was compared using the Chandra loop method. This test method involves placing 1 ml of fresh, normal human citrate blood into a plastic tube with an inner diameter of 3 mm and a length of 270 mm, adding 0.1 ml of 3.8% calcium chloride dihydrate solution to the blood, and then inserting 1 ml of fresh, normal human citrate blood into a plastic tube with an inner diameter of 3 mm and a length of 270 mm. A silicone tube with an inner diameter of 5 mm and a length of 15 mm is joined to 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 placed in a constant temperature room at 37°C.
Rotate for 20 minutes. 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 from a histopathological viewpoint. 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 Figure 3 shows the results of the thrombolysis test using the Chandler loop method for urokinase alone, urokinase/starch conjugate, and urokinase/HES conjugate. The horizontal axis of the graph in FIG. 3 indicates the final titer when 0.1 ml of the sample solution was injected into the loop, and the titer is the value measured by the fibrin plate method. From this figure, the thrombolytic curves of urokinase-starch conjugate and urokinase-HES conjugate are
Both of them are shifted to the left side of the same curve of urokinase alone, indicating that the thrombolytic ability is stronger than that of urokinase alone. Experimental Example 4 Next, using a beagle dog (male, weight 12-15 kg), we tested urokinase-starch conjugate and urokinase-starch.
The maintenance effect of HES conjugate in the blood was tested and compared with administration of urokinase alone. Urokinase was labeled with ¹²⁵ I by the chloramine T method (Biochem J. 89 , 114, 1963). That is, urokinase
After dissolving 100,000 units in 0.5 ml of phosphate buffer, add 1 mCi/ml Na ¹²⁵ I to 0.1 ml of this urokinase aqueous solution.
Add 0.1ml of
was added and allowed to react for 5 minutes at room temperature, then 0.1 ml of 0.1% sodium bisulfite was added to stop the reaction, and the reaction mixture was passed through a Sephadex G-25 column to separate ¹²⁵ I-urokinase and free NaI ¹²⁵ . did. ^{The 125} I-urokinase thus obtained was
One was used as a sample for animal experiments, and the others were each made of activated starch or HES with a molecular weight of 400,000.
¹²⁵ I-urokinase/starch conjugate and ¹²⁵ I-urokinase/HES conjugate were obtained and used as animal experiment samples. 2× each of ¹²⁵ I-urokinase starch conjugate, ¹²⁵ I-urokinase HES conjugate, and ¹²⁵ I-urokinase
10 ⁶ cpm was administered intravenously to beagle dogs, and the respective blood concentrations were determined every 10 minutes. The experimental results are the first
As shown in the table, the blood half-lives of urokinase-starch conjugate and urokinase-HES conjugate are 5.6 times and 5.5 times higher than that of urokinase alone, respectively, from the first decay curve, and from the second decay curve. The results showed that urokinase was 4.7-fold and 4.6-fold longer, respectively, indicating that binding urokinase to starch or its derivatives had a significant effect on maintaining it in the blood.

[Table] Experimental Example 5 To investigate acute toxicity test by intravenous administration to Wistar rats, 500,000 IU of urokinase, a conjugate of urokinase and starch, was intravenously injected per 1 kg of body weight, and general symptoms were observed for 7 days after administration. When the body weight was measured, the body weight increased steadily and no abnormal findings were observed, and autopsy and histological examination revealed no abnormalities at all. Example 1 1 g of starch was weighed out, 20 ml of distilled water was added thereto to dissolve it, 356 mg of sodium periodate dissolved in 5 ml of distilled water was added, and the mixture was stirred at room temperature in the dark for 30 minutes. After stirring, the mixture was neutralized with 4M sodium hydroxide solution and thoroughly dialyzed against water. After dialysis, lyophilize. Then urokinase 10mg (100
28 mg (3.5 molar equivalents) of the above activated starch dissolved in 0.1 M phosphate buffer (PH7) was added to 4 ml of an aqueous solution containing 10,000 IU), and further 1.22 mg of sodium cyanoborohydride was added. at 4℃
Stir for 18 hours. After stirring, 4.3 mg of sodium borohydride dissolved in 0.1 M phosphate buffer (PH7) is added, and the mixture is stirred at 4°C for 18 hours. After stirring, 3 to 6 mg of sodium borohydride dissolved in the above buffer solution is added, and the mixture is stirred at 4°C for 6 to 12 hours. After stirring, dialyze against the above buffer. The resulting reaction mixture is gel-filtered through a Sephadex G-200 column to separate urokinase, the above-mentioned starch conjugate, and unreacted urokinase. The conjugate obtained by fractionation is collected, sterilized using a Millipore filter, the urokinase titer is measured, and the amount to be dispensed is determined. A thrombolytic agent containing a starch conjugate was obtained. This conjugate had the properties of urokinase starch described above. Example 2 A urokinase/HES conjugate was obtained by using HES (molecular weight: 40,000) in place of the starch in Example 1 and carrying out the same treatment as in Example 1. Moreover, the obtained urokinase/HES conjugate had the above-mentioned properties. Example 3 Weigh out 1 g of starch (molecular weight: 40,000), add 49 ml of distilled water to dissolve it, and add cyanogen bromide to it.
Add dropwise a solution of 150 mg dissolved in 1.5 ml of cyanide methane and stir thoroughly. During this time, maintain the pH at 10.2 to 10.5 with 1M sodium hydroxide. 5 from the start of the reaction
After 4 minutes, lower the pH to 2.2 with concentrated hydrochloric acid, add 2 ml of diaminoethane and raise the pH to 9.5, and keep at 9.5.
Leave at ℃ overnight. This is followed by dialysis against distilled water and freeze-drying to obtain aminoethylamino starch. Next, add this to 0.1M phosphate buffer (PH7.0)
Dissolve in 25 ml, add 1 ml of bromoacetyl bromide dropwise, maintain the pH at 7.0 with 1M sodium hydroxide for about 2 hours, dialyze against distilled water, and freeze-dry to obtain activated compound powder. 50mg of it was added to 0.1M carbonate buffer (PH
9.5) Dissolve in 0.4ml of urokinase 5mg (500,000
IU) and left to stand at about 5°C for 50 hours to carry out the binding reaction. The reaction mixture was then transferred to Cephadex G.
-200 column and gel filtration to obtain urokinase-starch conjugate. This conjugate had the properties described above. Example 4 A urokinase/HES conjugate was obtained by using HES (molecular weight: 40,000) in place of the starch in Example 3 and carrying out the same treatment as in Example 3. Moreover, this urokinase/HES conjugate had the above-mentioned properties. Example 5 1 g of starch (molecular weight: 40,000) was reacted with 150 mg of cyanogen bromide in the same manner as in Example 3, 2 ml of diaminohexane was added, and the mixture was treated under the same conditions to obtain aminohexylamino starch. Example 3
Treated with bromoacetyl bromide in the same manner as N-
Bromoacetylaminohexylamino starch was obtained. Dissolve 50mg of this in 1ml of the above buffer solution,
5 mg (50,000 IU) of urokinase was added and reacted at about 5°C for 50 hours. The reaction mixture was subjected to gel filtration fractionation in the same manner as in Example 2. The resulting conjugate had the properties described above. Example 6 Using HES (molecular weight: 40,000) in place of the starch in Example 5, the same treatment was carried out to obtain a urokinase/HES conjugate. This urokinase
The HES conjugate had the properties described above. In addition, the characteristics of the products in Examples 1 to 6 are as follows.

【table】 [Brief explanation of the drawing]

Figure 1 shows resistance to plasma protease inhibitors; Figure 2 shows resistance to urokinase inhibitors;
Figure 2 shows urokinase-starch conjugate, Figure 2.3
are high performance liquid chromatograms of urokinase/HES conjugate, and the solid line is 280n
It is the absorbance at m, and the broken line shows the refractive index. Figure 3 shows urokinase, urokinase-starch conjugate, and urokinase-HES
Figure 3 shows the thrombolytic curve of the conjugate.

Claims (1)

[Scope of Claims] 1. The molecular weight is 90,000 to 500,000, the molar ratio of urokinase and starch is 1:1 to 1:10, and the urokinase and starch are -CH ₂ NH- or [Formula] (Formula (n is an integer of 1 to 6). 2. By reacting urokinase and a reactive derivative of starch, the molecular weight is 90,000 to 500,000 and the molar ratio of urokinase to starch is 1:1 to 1:10,
Urokinase and starch are bonded via -CH ₂ NH- or [Formula] (in the formula, n represents an integer of 1 to 6) to obtain a water-soluble urokinase-starch conjugate. A method for producing a water-soluble urokinase-starch conjugate. 3. The water-soluble urokinase according to claim 2, wherein the starch has a molecular weight of 40,000 to 400,000.
Method for producing starch conjugates. 4 The molecular weight is 90,000 to 500,000, the molar ratio of urokinase and starch is 1:1 to 1:10, and urokinase and starch are -CH ₂ NH- or [formula] (where n is 1 to A thrombolytic agent containing a water-soluble urokinase-starch conjugate as an active ingredient, which is formed by bonding via an integer of 6).