JPH0249708B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a novel fibrin adsorbent protein-urokinase complex. The thrombolytic enzyme urokinase is widely used clinically as a therapeutic agent for thrombosis, but urokinase does not have adsorption properties to fibrin, which is a protein component of blood clots, and it cannot be administered intravenously. Since the urokinase that has been used is rapidly metabolized and excreted, the therapeutic effect as expected from its in vitro activity has not been achieved.
For this reason, large doses of urokinase are being administered in Europe and the United States, but in this case, it causes systemic activation of plasminogen and has significant side effects such as bleeding tendency due to fibrinogen degradation. In addition, bloodshed contains large amounts of plasmin inhibitors such as α _{2 -Plasmin} Inhibitor and _{α 2 -Macroglobulin} , which activate (generate) plasmin of urokinase. It prevents the thrombolytic effect of From this point of view, the present inventors believe that if it is possible to give urokinase fibrin-binding properties by binding a protein that is adsorbed to fibrin to urokinase, urokinase will be adsorbed to the thrombus, which is the therapeutic objective, and will be metabolized and excreted. It is expected that fibrin will be less susceptible to the effects of the above-mentioned plasmin inhibitors, increasing the therapeutic effect, and that plasminogen will be activated locally at the site of thrombus. Based on the idea that urokinase can be expected to reduce side effects such as bleeding tendency due to its ability to bind to fibrin, intensive research has been conducted with the aim of obtaining urokinase that is adsorbable to fibrin. As a result, plasmin-derived heavy chain (hereinafter referred to as ``plasmin'') was found to be a fibrin-adsorbing protein.
The complex obtained by binding this to urokinase via a specific protein binding reagent has fibrin adsorption ability and the desired urokinase activity, and is a novel thrombolytic agent. It was found to be effective as The present invention was completed based on this new knowledge. That is, the present invention combines fibrin-adsorbing protein and urokinase with the general formula [In the formula, A and B each represent a lower alkylene group,
l and m each represent 0 or 1. ] This relates to a method for producing a fibrin-adsorbing protein-urokinase complex, which is characterized by carrying out the reaction in the presence of a protein-binding reagent represented by the following. The complex obtained by the method of the present invention acts selectively (locally) on the thrombus site using a smaller amount as a thrombolytic agent than urokinase alone,
Metabolism and excretion are delayed, there is little influence from plasmin inhibitors, sufficient thrombolytic action can be sustained, and there are few side effects such as systemic bleeding tendency, making it very effective. The present invention establishes a new method for easily and efficiently obtaining a new complex useful as such a thrombolytic agent, and will greatly contribute to the pharmaceutical industry and other fields. In the method of the present invention, the lower alkylene groups represented by A and B in the compound represented by the general formula [] used as a protein binding reagent include methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, 2
- C1-C6 alkylene groups such as methyl and trimethylene groups can be exemplified. The compound represented by the general formula [] includes known or new compounds, and includes, for example, the literature [T.Kitagawa et al., J.Biochem. (Tokyo)]
79, 233 (1976)]. Furthermore, plasmin HC as a fibrin-adsorbing protein used in the present invention is described, for example, in the literature [Eur.J.
Biochem., 57 , 441 (1975)], etc., and has a mercapto group obtained by partially reducing plasmin. Furthermore, in the present invention, the urokinase is not particularly limited, and various known ones such as low molecular weight urokinase and high molecular weight urokinase can be used.
-Urokinase, LMW-UK, average molecular weight 32000;
High Molecular Weight―Urokinase, HMW
-UK, average molecular weight 54000, Biochemistry, 5 ,
2160 (1966)] etc. can be used. The binding reaction between the fibrin-adsorbing protein and urokinase of the present invention is preferably carried out, for example, in an aqueous solution, physiological saline, or a normal buffer solution with a pH of 4 to 10.
The reaction is carried out in a buffer solution having a pH of 6 to 8, at a temperature of 0 to 40°C, preferably around room temperature, and preferably in a nitrogen stream. The reaction is usually completed in about 1 to 24 hours, preferably 2 to 3 hours. As the buffer solution used above, any of various known buffer solutions that do not contain ammonia or amino groups can be used. In the above binding reaction, the proportions of fibrin-adsorbing protein, urokinase, and the protein-binding reagent represented by the general formula [] are determined as appropriate, but usually fibrin-adsorbing protein is used in an amount of 0.3 to 4 times the molar amount of urokinase. degree, preferably about 0.5~
It is preferable to use about 2 times the molar amount, and about 1 to 50 times the molar amount of the protein binding reagent, preferably about 3 to 10 times the molar amount. Thus, the complex produced by the production method of the present invention mainly contains urokinase in a proportion of 1 to 3 moles, preferably approximately 1 mole, per mole of fibrin-adsorbing protein.
After the completion of the binding reaction, it can be easily isolated and purified by conventional methods such as dialysis, gel filtration, fractional precipitation, and affinity chromatography. Moreover, it can be preserved by the usual freeze-drying method. The complex obtained as described above has fibrin adsorption ability and urokinase activity, and is effective as a thrombolytic agent in the treatment of thrombosis. When the complex obtained by the production method of the present invention is used as a thrombolytic agent, it is made into a pharmaceutical composition together with a conventional pharmaceutical carrier. The dosage unit form of the thrombolytic agent can be selected from various forms depending on the therapeutic purpose, but it is usually used as an injection. The injection is sterilized in a conventional manner,
Preferably it is isotonic with blood. Examples of diluents used when forming into injections include various commonly used diluents in this field, such as water and physiological saline. In this case, the thrombolytic agent may contain a sufficient amount of salt, glucose, or glycerin to prepare an isotonic solution, and may also contain conventional solubilizing agents, buffers,
Add soothing agents, preservatives, etc., as well as coloring agents, if necessary.
Preservatives, fragrances, flavors, sweeteners, etc. and other pharmaceutical agents may also be included in the thrombolytic agent. The amount of the complex of the present invention to be contained in the thrombolytic agent is not particularly limited and is appropriately selected from a wide range, but is usually about 0.01 to 30% by weight in the total pharmaceutical composition.
It is preferable to set the amount to be contained. The above-mentioned thrombolytic agents are administered by various methods depending on the form, but are usually administered intravenously as an injection alone or mixed with a replacement fluid such as glucose or amino acid infusion. The dosage is appropriately selected depending on the purpose of use, symptoms, etc., but it is usually about 1,000 to 500,000 units per day as the active ingredient, fibrin-adsorbing protein-urokinase complex.
Kg, which may be administered in 2 to 4 doses. Below, examples of the production and preparation of the fibrin-adsorbing protein and urokinase used in the present invention are listed as reference examples, followed by examples of producing the complexes of the present invention as examples, and further pharmacological test examples of the complexes obtained in the examples are listed. . The methods for measuring various activities and testing for other physical properties in each example are as follows. ΓUrokinase activity measurement Synthetic substrate method; urokinase sample at 0.15M
- Dilute to an appropriate concentration with an aqueous solution containing NaCl and 5 g/polyethylene glycol (PEG6000, Wako Pure Chemical Industries, Ltd.), and 0.05M Tris-HCl buffer (PH = 8.4) containing 0.1M NaCl per 100μ.
After adding 800μ and heating at 37℃ for 1 minute, the substrate S-2444 (Pyro Glu-Gly-Arg-p-
Add 100 μM of Nitroanilide (manufactured by Daiichi Kagaku Yakuhin Co., Ltd.) to the above buffer solution, and heat at 37°C.
Incubate for 2 minutes. 50% acetic acid aqueous solution
Stop the reaction by adding 100μ and measure the absorbance at 405nm. The activity of the sample (expressed as international units) is calculated from the absorbance obtained in the same manner using standard urokinase. Fibrin plate method: Add 0.3% bovine fibrinogen (manufactured by Poviet) to 6 ml of 0.05M veronal buffer (PH = 8.0) containing 0.1M-NaCl, final 0.02M.
of CaCl ₂ and a final concentration of about 3 NIH units/ml bovine thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.), stirred, and spread on a shear plate (inner diameter 8.5 cm) to prepare a fibrin plate. 0.1% rabbit serum albumin (manufactured by Sigma)
10μ of the urokinase sample dissolved in the above buffer solution was spotted on the above fibrin plate, and after incubation at 37°C for 16 hours, the diameter of the dissolution circle was measured. Quantification of ΓSH group After degassing 100μ of the sample, 1 ml of 0.2M-Tris-HCl buffer (PH = 8.2) substituted with degassed _N2 was added to it, and then dissolved in degassed methanol.
Add 100μ of 0.01M-5,5'-dithiobis(2-nitrobenzoic acid), stir, and leave at room temperature for about 30 minutes, then measure the absorbance at 412 nm. Calculate the amount of SH groups in the sample using 2-mercaptoethanol as a standard. Quantification of Γm-maleimidobenzoyl group (MB group) After degassing 100μ of the sample, add 25μ of an aqueous solution of 2-mercaptoethanol (35 nanomoles) and incubate at 37°C for 20 minutes. Above above
In the same manner as the determination of SH groups, the amount of residual 2-mercaptoethanol is measured and the amount of MB groups in the sample is converted. ΓSDS polyacrylamide gel electrophoresis (SDS-
PAGE) Mix equal amounts of the sample with 0.02M Tris-HCl buffer (PH = 8.0) containing 40% glycerol and 2% SDS, heat at 100°C for 2 minutes, and then create a 12.5% separation gel and a 4% concentration gel. using Laemeri (UK
[Nature (London), 227 ,
680 (1970)] and gradient gel PAA4/30 manufactured by Pharmamia. After electrophoresis, stain with Commercy Brilliant Blue (CBB), and calculate the molecular weight using a standard protein (Molecular Weight Kit, manufactured by Pharmamia). Reference example 1 Production of plasmin HC Method of EERickli et al. [Eur.J.
Biochem., 59 , 441 (1975)]. That is, plasminogen purified from human blood according to the method of BAK Chibber et al. [Methods in Enzymology, 34 , 424 (1974)].
Dissolve 150 mg in 0.05 M Tris-HCl buffer (PH = 7.8) containing 0.1 M NaCl and 25% glycerol, add approximately 3000 units of urokinase (manufactured by Nippon Chemical Research), and incubate at room temperature for 8 hours. . Further, 1500 units of the same urokinase are added and incubated at room temperature for 16 hours. This solution was purged with nitrogen, and 2-mercaptoethanol was added to a final concentration of 0.1M.
Leave for a minute. Thereafter, the lysine-sepharose serum was cooled on ice and sufficiently equilibrated with a 0.01M sodium phosphate buffer (PH = 7.2) containing 0.01M EDTA and 0.001M 2-mercaptoethanol, which had been previously replaced with nitrogen. 5 cmφ Concentrate under nitrogen pressure using a membrane (manufactured by Amicon). Add the above buffer solution excluding 2-mercaptoethanol to this and concentrate again. Repeat this operation several times to obtain about 60 mg of plasmin HC. This was replaced with nitrogen and stored at -80°C. This one is 1
Approximately 3 moles of SH groups were detected per mole. Example 1 LMW-UK (manufactured by Nippon Chemical Research) was concentrated using Amicon PM-10 and then dialyzed against 0.01M sodium phosphate buffer (PH=7.0).
6.7 mmol of m-maleimidobenzoyl N-hydroxysuccinimide ester (general formula [], m
- position substitution, l=m=0, manufactured by Pierce, below
Add a dimethylformamide (DMF) solution of MBS (referred to as MBS) dropwise with stirring for 30 min at room temperature.
Stir for 1 minute. This was applied to a Sephadex G-25 fine column (manufactured by Pharmacia, 1.5 cmφ x 20 cm) equilibrated with a degassed 0.1M sodium phosphate buffer (PH=6.0), and developed with the same buffer. The absorbance peak fraction at 280 nm was collected and LMW-UK0.9 with MB group attached was collected.
Get mmol. In this product, an average of 2.7 moles of MB groups were bonded to 1 mole of LMW-UK. Plasmin obtained by the same procedure as Reference Example 1
HC123mg and MB group-containing LMW obtained above
Mix 29.5 mg of UK, purify the mixture thoroughly with nitrogen, and stir at room temperature for 3 hours. Add 2-mercaptoethanol to a final concentration of 1mM and stir for 10 minutes, then add N-ethylmaleimide to a final concentration of 2mM and stir at room temperature for 20 minutes. This was equilibrated with a 0.1M sodium phosphate buffer containing 0.4M NaCl (Sephadex G-25 fine column (manufactured by Pharmacia);
Benzamidine-CH-Sepharose (method of L. Holmberg et al. [Biochemica et Biophysica Acta, 445 , 215
(1976)]) column (2.5 cm
After washing with 900ml of the same buffer,
0.1M acetic acid aqueous solution containing 0.1M-NaCl and
Elute with 6M-urea aqueous solution, combine the absorbance peak fractions at 280nm, and gel-filter through a Sephadex G-25 column (5cmφ x 25cm) equilibrated with 0.1M ammonium bicarbonate aqueous solution.The absorbance peak obtained The fraction was applied to a lysine-Sepharose column (1.6 cmφ x 14 cm) equilibrated with a 0.1 M aqueous ammonium bicarbonate solution, washed with 400 ml of the same solution, then with 100 ml of a 0.2 M 6-aminohexanoic acid aqueous solution, and then washed with a 2 M aqueous solution of 6-aminohexanoic acid. Elute with KSCN aqueous solution. After desalting the absorbance peak fraction of the eluate, it was concentrated using Amicon PM-10 to obtain 22.3 mg of the plasmin HC-LMW-UK complex of the present invention [hereinafter referred to as "complex A"]. The obtained complex A was confirmed as a band with a molecular weight of 80,000 to 90,000 on SDS-PAGE, and it was confirmed that the LMW-
Approximately 1 mole of plasmin HC is bound to 1 mole of UK. Example 2 As a protein binding reagent in Example 1 above
In place of MBS, each of the following compounds is used to obtain a plasmin HC-LMW-UK complex in the same manner. <Protein binding reagent> Γ1-[4-(2,5-dioxo-1-pyrrolidinyloxycarbonyl)phenyl]-2,5-
Dihydropyrrole-2,5-dione [general formula [], p-position substitution, m=l=0] Γ1-{4-[4-(2,5-dioxo-1-pyrrolidinyloxycarbonyl)butyl] phenyl}-2,5-dihydropyrrole-2,5-dione [general formula [], p-position substitution, (A) _l = (-CH ₂ )- ₄ ,
m=0] Γ1-[4-(2,5-dioxo-1-pyrrolidinyloxycarbonyl)benzyl]-2,5-
Dihydropyrrole-2,5-dione [general formula], p-position substitution, (B) _n = -CH ₂ -,
l=0] The obtained complexes are sequentially referred to as "complex B,""complexC," and "complex D." Each such complex is
Both were confirmed as 80,000 to 90,000 bands on SDS-PAGE, and the molar ratio of LMW-UK and plasmin HC was about 1:1. <Pharmacological tests> (1) Fibrinolytic activity test The dissolving activity of the complex obtained in Example 1 was measured by the fibrin plate method. The results are shown in Figure 1. In FIG. 1, the horizontal axis shows the urokinase activity (unit/ml) measured by the synthetic substrate method, and the vertical axis shows the diameter (mm) of the lysis circle measured by this method. Further, in FIG. 1, line 1 indicates the composite A obtained in Example 1 of the present invention, and line 2 indicates LMW-UK (control). From the figure, it is clear that at 10 units/ml, Complex A of the present invention retains fibrinolytic activity to the same extent as the control LMW-UK, and at lower concentrations it exhibits higher lytic activity than the control. It is. (2) Fibrin adsorption ability test The fibrin binding ability of Complex A obtained in Example 1 was tested by the following method. In other words, fibrin
Monomer-Sepharose 6B (Hahne (DL)
[Thrombosis Research,
2, 137 (1973)] containing 0.135 M NaCl.
0.005M sodium phosphate buffer (PH=7.4)
After equilibration with, complex A dissolved in the above buffer solution,
Apply HNW-UK, plasminogen or plasmin HC (2-3 mg each) and wash with 30 ml of the above buffer. Elution was performed with the above buffer containing 10mM-6-aminohexanoic acid, and plasminogen and plasmin HC were determined from the absorption of the elution fraction at 280 nm, and complex A and
HMW-UK determines the recovery rate for each elution fraction from the activity measured by the synthetic substrate method of urokinase activity in the elution fraction, and uses this as the fibrin adsorption capacity. As a result of the above test, the complex A of the present invention has a fibrin adsorption ability that is about 1/2 that of plasminogen and about 1/4 that of plasmin HC, and has a fibrin adsorption ability that is about 10 times stronger than that of urokinase. showed that. (3) Therapeutic effect on experimental pulmonary embolism in rabbits This test was conducted according to the method of O. Matsuo et al. [Nature, 291 , 590 (1981)]. That is, an artificial thrombus is prepared from fresh human blood mixed with ¹²⁵ I-fibrinogen, and is injected into a rabbit jugular vein under anesthesia to create a pulmonary embolism model. Complex A obtained in Example 1, HMW-UK as a control, or physiological saline was continuously injected into the ear vein over a period of 6 hours, and blood was collected over time to measure its radioactivity. Calculate the percentage from the total radioactivity. After 9 hours, the lungs are removed, the remaining thrombus is collected, and the lysis rate of the administered thrombus is determined from its radioactivity. Furthermore, the recovery rate of radioactivity in the lungs and urine is calculated. The total radioactivity recovery rate is the sum of radioactivity in blood, urine, lungs, and recovered thrombus.
It was calculated as a percentage of the radioactivity of the administered artificial thrombus. The results are shown in Table 1. [Table] As is clear from Table 1 above, HMW―
UK50,000 units/body administration was no different from physiological saline alone, but in the group administered 50,000 units of Complex A, radioactivity in the blood was significantly higher throughout the period, and radioactivity in the urine was significantly higher. The quantity is also increasing. Furthermore, the thrombolytic rate calculated from the radioactivity of the collected thrombi increased to about 10%, indicating a significantly high thrombolytic effect. In addition, no bleeding tendency was observed in the group administered with complex A.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the fibrinolytic activity of complex A obtained according to the present invention.

Claims (1)

[Claims] 1. Fibrin-adsorbing protein and urokinase,
general formula [In the formula, A and B each represent a lower alkylene group,
l and m each represent 0 or 1. ] A method for producing a fibrin-adsorbing protein-urokinase complex, which comprises reacting in the presence of a protein-binding reagent represented by: