JPS5922901A - Urokinase-starch composite, its manufacture, and thrombolytic agent - Google Patents

Abstract

PURPOSE:To obtain a novel water-soluble unokinase-starch composite with nontoxicity, having high thermal stability and stability in blood, useful as thrombolytic agent, by the reaction between urokinase and a reactive derivative from starch. CONSTITUTION:The objective composite can be obtained by the reaction between (A) urokinase (pref. with a molecular weight of 25,000-60,000) and (B) a reactive derivative from starch (e.g., an activated starch of formula I or II; pref. with a molecular weight of 40,000-400,000) in a molar ratio (B)/(A) of 1/1-10/1. For example, an activated starch prepared by the oxidation, using an oxidizing agent such as sodium periodate, of starch is made into a phosphoric acid buffer solution which is then brought to a reaction with an aqueous solution of urokinase, followed by the reduction of the resultant compound with, e.g., a hydrogenated boron metal salt, thus obtaining the objective composite.

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 lytic enzyme, is widely used in the treatment of thrombosis and embolic diseases, as well as in combination therapy with anticancer drugs, and has excellent clinical effects. However, urokinase administered to a living body
Both itself and its enzymatic activity disappear from the blood quickly, and the half-life of this substance 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 urokinase of a certain open chain level or higher is administered.

It is understood that such blood dynamics of urokinase result in today's high-dose therapy, which inevitably progresses to high-dose administration in order to obtain a sufficient effect. Invention Moss et al. have been researching ways 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, we have developed a water-soluble urokinase-starch conjugate. We have discovered that this conjugate can improve the various drawbacks seen when urokinase is administered alone, and that it can sufficiently and sustainably exert the enzymatic activity of urokinase, and we have confirmed its usefulness as a medicine. The present invention was completed by paying attention to this.

Specifically, the present invention relates to (1) a urokinase-starch conjugate, (2) a reactive derivative of urokinase and starch, and a thrombolytic agent.

The urokinase used in the present invention may be derived from 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 cultured. Urokinase produced by E. coli may also be used. Also the molecule fi250
The starch used in the present invention in the range of 00 to 60,000 is, for example, hydroxyethyl starch (HES
), etc., and those having a molecular weight of 40,000 to 400,000 are usually used. In the following description, the urokinase-starch conjugate used in experiments and examples other than those used includes conjugates 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 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, and includes, for example, a compound represented by the formula %. Compound ill is produced, for example, by oxidizing starch with periodic acid or the like. Compound +Ill is obtained by reacting starch with cyanbromide.

These activation methods are described in J. Biol.

Ohem, 251.1081 (1976), Pr.
oc, Natl.

Acad, Sci, USA 78.2128 (197
6 years) etc.

The method for producing the urokinase-starch conjugate of the present invention is, for example, as follows.

(First Method) Starch) In this reaction, starch is first oxidized with an oxidizing agent (for example, sodium periodate). At this time, starch 1
The oxidizing agent is used in an amount of 10 to 40 parts by weight per 00 parts by weight. 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 8-5M sodium hydroxide after the reaction is completed, dialyzed against water, and then freeze-dried. The degree of oxidation of the activated starch thus obtained is between 10 and 50%. The reaction between urokinase and activated starch is usually carried out using 20 to 20 parts of activated starch per 100 parts by weight of urokinase.
This is carried out 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 solution (pH 6 to 8). The target compound can be obtained by reducing the compound thus obtained. In this case, a metal borohydride salt is used as the reducing agent, and it is particularly preferable to reduce the metal cyanoborohydride before the reduction with the borohydride. Reduce with salt. The borohydride metal salt and the cyanoborohydride metal salt each include an alkali metal salt (for example, sodium salt).
are exemplified as preferred. The refund is usually 0
It is carried out by stirring at ~80°C, preferably 4°C, for 6 to 12 hours.

(Second method) (Urokinase) (Urokinase/starch conjugate)
[In the formula, x and x' are each a halogen atom (for example,
chlor, etc.), and n represents a # number from 1 to 6.] In the reaction between starch and cyanogen bromide, 10 to 10 parts by weight of the latter is used per 100 parts by weight of the former.

The activated starch is bound by the action of a difunctional compound (eg diaminoethane, diaminohexane) and then a halogen acetyl halide (eg promacetyl bromide). Next, this activated starch and urokinase are brought into contact to obtain a urokinase-starch conjugate. This binding reaction is

Adjust the pH to 7.2-11, and heat at a temperature of 3-25°C.
This is done by contacting for 48 hours.

The urokinase-starch conjugate thus obtained can be recovered by known gel filtration methods, single molecule sieving methods, ion exchange methods, etc., or if fractionated by gel filtration, the urokinase-starch conjugate and unbound starch can be recovered. Since urokinase and urokinase behave in a very distinct manner, the desired bound product can be easily recovered. The recovered urokinase
The starch conjugate is subjected to sterilization filtration and heat treatment, and then dispensed and freeze-dried to obtain a thrombolytic agent containing the urokinase-starch conjugate.

The urokinase-starch conjugate thus obtained is easily soluble in water and physiological saline, and the molar ratio of urokinase to starch is 1:1 to 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 has a pH of 3.
Although relatively stable over the range of ~IO, the urokinase starch conjugate is even more stable, with C1pH2 ~
No decrease in titer was observed over a wide range of 11. Regarding heating stability, when heating at 60°C at pH 8.0, where urokinase is most stable,
Urokinase alone was safely inactivated by heating for 2 hours, whereas urokinase-starch conjugate was heated for 10 hours.
It still shows 100% activity residual rate even after a long time,
It was found to be very stable to heat.

The urokinase-starch conjugate according to the present invention has a thrombolytic effect! Therefore, it is useful as a thrombolytic agent, for example, when administered parenterally as an injection. in particular,
For example, the urokinase O starch conjugate according to the invention is obtained by dissolving 50 to 30,000 IU of Motoichi in 0.5 to 5 of distilled water for injection in the Japanese Pharmacopoeia, and adding water as appropriate depending on age, symptoms, and progress. It is extremely stable in the blood and does not dissociate or degrade easily, is extremely stable against heat, is not susceptible to the effects of urokinase inhibitors in the blood, and does not exhibit thrombolytic activity when used alone. It has excellent properties such as producing thrombolytic effects even when administered with low activity, significantly prolonging blood retention time when administered in vivo, and having almost no detectable toxicity. It is effective in providing high-quality medicines. .

Hereinafter, the present invention will be explained in more detail with reference to Examples and Experimental Examples.

Example Human plasma was used to discuss the resistance of urokinase or urokinase-starch conjugate and urokinase-HES binding surface to plasma protease inhibitors. The test method is that the activity is 100 IU (International Unit)/*? (For international units, Pharmaceutical Research (8), 295-808.19
Urokinase alone, urokinase/starch conjugate, or urokinase/HES diluted to give (see 1974)
15 μl of 18% human plasma albumin in each 225 μl of binding
1 of human plasma, and then add 80 μl of human plasma to 20 μl of this mixture.
After mixing μl and incubating for 1 hour at 87°C, human fibrin standard plating method (B, B, A, 24.
278-282.19751, the urokinase activity before incubation with human plasma was taken as 100%, and the residual urokinase activity was calculated. The test results are as shown in FIG. 1, and while the residual activity of urokinase alone was 1.5%, it was 28% and 25% for the urokinase-starch conjugate and the urokinase-HE8 conjugate, respectively. From this, it was confirmed that this conjugate had resistance to all plasma protease inhibitors.

Example urokinase starch conjugate and urokinase-HE8
High performance liquid chromatography (L(!-
8A: 8crnen using Shimadzu Corporation).

(J, Ohromat, 188.185-92(1)
The analysis was performed according to the method of 980)). TSK- as carrier
Using Gelaooosw (manufactured by Toyo Soda), pI (3
When a 0°2M phosphate buffer solution was developed at a flow rate of 1.2 g//si, the high performance liquid chromatogram shown in FIG. 2 was obtained. As a result, the urokinase-starch conjugate and the urokinase-HES conjugate were eluted on the polymer side, as expected.

Example urokinase/starch conjugate and urokinase/HES
The thrombolytic ability of the conjugated conjugate rokinase alone was compared using the Chandra loop method.

This test method uses 1 ml of fresh, normal human citrated blood.
1 in a plastic tube with an inner diameter of 8 mm and a length of 270111 N, and after adding 0.1 tnl of 8.8% calcium chloride dihydrate saline solution to the blood, a plastic tube with an inner diameter of 5 mm and a length of 151 nm was placed at both ends of the tube. Join the silicone tubes to make a loop. This tube was immediately placed on a rotary plate designed to rotate 122 times per minute at an angle of 80°C from the horizontal plane, and rotated for 20 minutes in a thermostatic chamber at 87°C. As a result, an artificial thrombus of about 13 lengths 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 removing the loop from this device, remove one end of the looped polyvinyl duplex from the silicone tube, put the sample solution or physiological saline go, tysl into the tube, loop the tube again, and place it in the rotating device. I continued to do one rotation over the bridge.

4 hours after starting thrombolysis for the first loop, remove the first loop from the rotator and homoge the thrombus in the tube in 1011 distilled water.
centrifuge at 8600 rpm.

5 minutes), and the absorbance of the supernatant at 549 Nm was measured. The second and subsequent loops were extracted following the first loop and processed in the same manner as before.

The thrombolysis rate (Y) of the sample solution addition group was determined according to the following formula.

Y = (1-A/Ac)
Average value of absorbance at nm The results of the thrombolytic test using urokinase alone, urokinase-starch conjugate, and urokinase-HE8 conjugate chandler loop method are as follows:
As shown in the figure. The horizontal axis of the graph in FIG. 8 indicates the final titer when 0.1 vtl 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 the urokinase/starch conjugate and the urokinase/HES conjugate are both shifted to the left compared to the curve of urokinase alone, indicating that the thrombolytic ability is stronger than that of urokinase alone. I understand.

Example Next, urokinase/starch conjugate and urokinase/HES were prepared using Peagle dogs (1 male weighing 12-15 kg).
The retention effect in bound conjugates was studied and compared with administration of urokinase alone. Urokinase is produced using the Chlora ENT method (Eio
chem J, 89. 114゜1963). In other words, 100,000 units of urokinase is phosphate-relaxed? After dissolving in 0.5 ml of IJ H, add 1 mC1/1 t to this urokinase aqueous p0.1 tel.
Add 0.1 w/tl N a I and further 0.0
Add 5% chloramine-T O, 1yxl and incubate at room temperature for 5 minutes.
After reacting for minutes, 0.1% sodium bisulfite 0.1
The reaction was stopped by adding @/, and the reaction mixture was passed through a Sephadex G-25 column to separate and filter out -urokinase and free Na1125.

The ■-urokinase obtained in this way was divided into two parts, and 1
One was used as a sample for animal experiments, and the others each had a molecular weight of 40.
Reacted with activated starch or I[ES] 12
A 5I-urokinase starch conjugate and an eurokinase I (E8 conjugate) were obtained and used as samples for animal experiments.

125■-Urokinase starch conjugate and 125■-
Urokinase/HES conjugate and urokinase I (ES conjugate) were administered intravenously to pegle dogs at 2 x 10 cpm each, and the concentrations of urokinase/HES conjugate, urokinase I (ES conjugate) Compared to urokinase alone, the blood half-life was 5.6 times and 5.5 times, respectively, from the first decay curve, and 4.7 times and 4.6 times, respectively, from the second decay curve. extend,
It has been found that binding urokinase to starch or its derivatives has a significant effect on maintaining it in the blood.

Table 1 Examples To investigate acute toxicity tests by intravenous administration to Wistar rats, 500,000 IU of urokinase-starch conjugate was intravenously injected per 1 kq of body weight, and 7 days after administration.
When the general symptoms were observed and the body weight was measured over a period of days, the body weight increased steadily and no abnormal findings were observed.The results of autopsy and histological examination also showed no abnormalities.

Example starch 1y was weighed out, 20M/distilled water was added to dissolve it, and a solution of sodium periodate from 356 to 5m/t distilled water was added thereto, and the mixture was heated in the dark at room temperature for 30 minutes. Stirred. After stirring, the mixture was neutralized with 4M sodium hydroxide solution and thoroughly dialyzed against water.

After dialysis, freeze-dry. Next, urokinase 1 (1 (10
28 Mf/(8.5 molar equivalent) of the above activated starch was added to an aqueous solution containing 0.0000 IU). IM phosphoric acid
Add the solution dissolved in i solution (pH 7), add 1.221111/sodium cyanoborohydride to the solution,
Stir at °C for 18 hours. After stirring, add 4,3 of sodium borohydride to 0.1 M phosphate buffer (pH 7).
Add the solution dissolved in and stir at 4°C for 18 hours. After stirring, a solution of 3 to 6 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 with the above M tooth fluid. 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 bound substances obtained by fractionation are collected, sterilized through a Millipore filter, the amount of urokinase dissolved in water is determined, and the amount to be dispensed is determined.
A predetermined amount was divided into small portions and lyophilized to obtain urokinase den%. This conjugate has the above-mentioned properties of urokinase and dene. Example 2 The following treatment is carried out in the same manner as in Example 1, using IIBS (molecular weight 40,000) instead of the starch in Example 1. As a result, urokinase-HF; 5lf11 hardware was obtained.

Moreover, the obtained urokinase I (BS conjugate) had the above-mentioned characteristics.

Example 3 Starch (molecular weight 40,000) 1F was weighed out, 49*t of distilled water was added to it to dissolve it, and a solution of 150 cm of cyanogen bromide dissolved in 1.5 wt of cyanide methane was added dropwise to this. and stir thoroughly. During this time, adjust the pH with 1M sodium hydroxide.
Maintain at 1o, 2-1O15. After 5 minutes from the start of the reaction, the pH was lowered to 2.2 with concentrated hydrochloric acid, and diaminoethane'2
Add xl to raise the pH to 9.5, keep the pH at 9.5, and let stand overnight at 4°C. This is followed by dialysis against distilled water and freeze-drying to obtain aminoethylamino starch. Next, add this to 0.1M phosphate buffer (pH 7,0) for 25 minutes.
1 ml of bromacetyl bromide was added dropwise to the solution, and the mixture was kept at pH 7.0 with IM sodium hydroxide for about 2 hours, dialyzed against distilled water, and freeze-dried to obtain an activated compound powder. 50tly of 0.1M carbonate buffer (pH 9)
,5) Dissolve in 0.4g/,', urokinase 5~(
500,000 IU) and left to stand at about 5°C for 50 hours to perform a binding reaction.

Gel filtration is performed through a column to obtain a urokinase e-starch conjugate. This conjugate had the properties described above.

Example 4 HEM (molecular weight 4
Urokinase I (ES conjugate) was obtained by the following treatment in the same manner as in Example 8. Furthermore, this urokinase I (BS conjugate had the above-mentioned characteristics). .

Example 5 Starch (molecular weight: 40,000) IF was reacted with 150 My of cyanogen dibromide in the same manner as in Example 8, diaminohexane was added, and the mixture was treated under the same conditions to obtain aminohexylamino starch. This was treated with bromoacetyl bromide in the same manner as in Example 3 to obtain N-promoacetylaminohexylamino starch. Add this 504 to the above buffer 1.
ml, urokinase 51IIy (50,000 IU)
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 in i1.

Example 6 HEM (molecular weight 4
urokinase J (B).
An S-conjugate was obtained. This urokinase (ES) conjugate had the characteristics described above.

In addition, the characteristics of the products in Examples 1 to 6 are as follows.

Note) Composition ratio: activated starch/urokinase (, w/
w) Molar ratio: activated starch/urokinase (m017m
o l) Protein content: Protein content when the obtained urokinase conjugate is dissolved in 8 ml of water after lyophilization.pH: pH when the obtained urokinase derivative is dissolved in physiological saline.

[Brief explanation of drawings]

Figure 1 shows resistance to protease inhibitors in plasma; Figure 2 is an areal liquid chromatogram of urokinase alone, urokinase-starch conjugate, and urokinase-HE8 binding; Figure 8 shows urokinase and urokinase-starch conjugate. Figure 3 shows the binding surface plug lysis curve for urokinase and HE8 binding. Figure 1 -0-2550 Residual activity (/-) 2nd round %3 Figure 10 50 100 '500
1000 trials! Ill! + 1 in D 1 cokiner +゛4
/ [(t u” j ) Procedural amendment (method) % formula % 1. Indication of the case 1982 Patent Application No. 132790 3. Person making the amendment Relationship with the case Patent applicant name) Co., Ltd. Mitori Cross ＼ 4, Agent 〒541 7. Keyaki Mizubi drawing of a brief explanation of the drawing of the specification subject to amendment 8 Contents of amendment (1) On page 22 of the specification, lines 13 to 15, “ Figure 2 is a chromatogram.''[Figure 2 (1) shows urokinase, Figure 2 (2) shows urokinase-starch conjugate, and Figure 2 (3) shows urokinase-HES conjugate. The solid line is the absorbance at 280n*, and the broken line is the refractive index deposit. ” is corrected. (2) Correct Figure 2 as shown in the attached sheet. Figure 2↓ Molecular weight

Claims (1)

[Claims] (1) Water-soluble urokinase-starch conjugate. (2) A method for producing a water-soluble urokinase-starch conjugate, which comprises reacting urokinase with a reactive derivative of starch. (8) Water-soluble urokinase e according to claim (2), wherein the starch has a molecular weight of 40,000 to 400,000.
Method for producing starch conjugates. (4) 1 to 1 starch per mole of urokinase
Thrombolytic agent to form a water-soluble tumor according to claim (1), characterized in that the reaction is carried out at a ratio of 0 mol.