JPS631290B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing urokinase having a molecular weight of 54,000±10,000. Urokinase is an enzyme that exists in trace amounts in human urine, and has the function of activating plasminogen contained in serum to generate plasmin, which has fibrinolytic ability. Therefore, since urokinase is effective as an activator of the fibrinolytic system, it is isolated and purified from human urine or renal tissue culture fluid, and is widely used clinically for the treatment of various thromboses and in combination with anticancer drugs for cancer. As described above, urokinase has become extremely useful in the medical field, and the present inventors conducted research to further utilize this urokinase more efficiently in medical treatment. Urokinase is currently used as a drug with different molecular weights, and the currently known ones have a molecular weight of about 33,000±10,000.
54,000±10,000, etc., and is used as a preparation with a certain potency. The reason for the existence of enzymes with different molecular weights in urokinase is currently unknown, but it is possible that isoenzyme theory due to differences in the tissues that produce the enzyme, subunit formation due to the action of proteolytic enzymes present in urine, etc. It is thought that this may be due to decomposition and denaturation products during the recovery process. It is presumed that urokinases having such different molecular weights naturally differ in their physiological activities. However, in order to standardize the enzyme activity of conventional urokinase preparations, the commonly used test tube fibrin plate method, the two-step method (for pharmaceutical research (3))
295-308 (1974)), no sufficient suggestions or significant differences were found regarding this issue, and therefore, when administering as a medicine, standardization is carried out by adjusting the total force value in the test tube. Ta. In other words, regardless of the variation in molecular weight of urokinase, if the enzyme activity (potency) in a test tube was the same, it was assumed that the biological effectiveness would be the same. Against this background of the art, the diversity of molecular weights of urokinase was elucidated, and the therapeutic applications of animal experiments and clinical use were expanded. We discovered that there is a considerable difference between the enzyme activity in vitro and its dynamics in vivo. The present inventors focused on this problem and determined whether the enzyme activity in the test tube directly reflects the biological effectiveness in the living body, and whether urokinase with various molecular weights has the same biological effectiveness in the living body. We examined whether there were any other enzymatic differences based on differences in molecular weight. Surprisingly, the results showed that samples adjusted to the same titer based on the enzymatic activity measured by a two-step method in vitro were completely different in terms of thrombolytic ability in vivo and in vitro. It was found that there is a certain correlation between its molecular weight and thrombolytic effect. That is, it has been found that when the titer of urokinase is the same, the larger the molecular weight, the more pronounced the thrombolytic effect is, as determined by a two-stage test tube method. Furthermore, we found that urokinase with a large molecular weight has excellent storage stability.We labeled high-molecular urokinase (molecular weight 54,000) and low-molecular urokinase (molecular weight 33,000) with ^125I , and after intravenously injecting each into beagle dogs. As a result of determining the decay curve in the blood, it was found that the high molecular weight urokinase stays in the blood for a longer time, and its half-life in the blood is about twice that of the low molecular weight urokinase. The present invention has succeeded in developing a method for efficiently extracting and separating highly effective polymeric urokinase from raw materials. The present invention provides urokinase with a molecular weight characterized in that in each treatment step in the production of urokinase, the pH of at least the dialysis step for desalting is 7 to 12.
Concerning a method for producing 54,000±10,000 urokinase. The method for producing urokinase having a molecular weight of 54,000±10,000 is not particularly limited, except that at least the PH conditions in the dialysis step are as specified in the present invention. That is, after passing through the known treatment steps of separating, concentrating, and purifying urokinase from urine, a method of separating and recovering an enzyme of a certain molecular weight, such as fractionation by gel filtration, ion exchange column method, fractional precipitation method, etc. Urokinase can thus be isolated. Gel filtration is the simplest method for recovering urokinase of a certain molecular weight and provides good results. As the gel filter material, cross-linked dextran gel, polyacrylamide gel, agarose gel, etc. with a molecular weight of 1,000 to 150,000 are used. The gelling agent is retained in a column equilibrated with a low salt concentration buffer around pH 5.5 to 9.5, and an aqueous solution containing urokinase is developed on this column, resulting in a molecular weight of 54000±.
Collect 10,000 urokinase fractions. molecular weight
The method for recovering 54,000±10,000 urokinase is not limited to this method. The present inventor conducted research on improving the yield in the production method of urokinase with a molecular weight of 54,000±10,000, and found that it is effective to adjust the pH of the aqueous solution containing urokinase to 7 to 12 at least in the dialysis step for desalting. I found out. By maintaining these treatment conditions, the stability of urokinase is maintained, and the target urokinase with a molecular weight of 54,000±10,000 is efficiently recovered without decomposition of urokinase. Next, we will show Experiment 1 that suggests this. Experiment 1 Test on the relationship between urokinase activity residual rate, molecular weight distribution, and PH Urokinase adsorbed and recovered from human urine by silica gel was fractionated with ammonium sulfate, and the urokinase titer of the extract was adjusted to 9800 IU. The liquid is water, PH3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0,
11.0, 12.0 1M sodium acetate buffer or 1/15
against an external solution of M phosphate buffer for 40 hours at 4°C.
Dialysis experiments were performed by exchanging the external solution every 8 hours. Urokinase with a corresponding molecular weight was detected based on the residual activity rate after the dialysis and molecular sieving using gel filtration. The result is the residual activity rate in %
The molecular weight of urokinase was tested using a standard substance with a known molecular weight as a control, and the results are summarized in Table 1.

【table】

[Table] The present inventors have investigated the thrombolytic ability in vivo and in vitro, blood half-life after intravenous administration, stability, toxicity, and
Experiments regarding dosage and administration method were conducted. Next, we will explain these experimental methods and show the results. Experiment 2 Chandler's loop
Measurement of thrombolytic ability by method Purified urokinase with a molecular weight of approximately 33,000
±10000 (mostly 33000), 54000±10000 (mostly
54,000) and approximately 100,000 samples. In addition, molecules with a molecular weight of 100,000 exist only in extremely small amounts, and the molecular weight
It appears to be an aggregate of 54,000 urokinases and cannot be industrially recovered from urine. Dissolve each sample in physiological saline, and add the urokinase titer to 1 ml. Sample (1) 187.75 IU/ml, Sample (2) 37.5 IU/ml, Sample (3) 75.0 IU/ml, Sample (4) 150 IU. /ml, sample (5)
Prepare a solution containing 300 IU/ml and a control (saline). Here, the IU unit is a unit in which a test urokinase is applied to purified plasminogen, the secondary substrate fibrin is degraded by the generated plasmin, and the titer of urokinase is calculated from the dissolution time. It is an activity unit measured by a two-step test tube method (Pharmaceutical Research 5 (3) 295~
308, (1974)). In addition, the conventional proug unit (BB
A. 24 278-282, (1975)), 5000 prog units equal 6000 IU. To measure thrombus formation and lysis rate, 1ml of fresh blood supplemented with 3.8% citrate and 0.1ml of 0.25M calcium chloride solution was placed in a plastic tube with an inner diameter of 3mm (length 270mm).
ml, close both ends of the tube to make a loop,
Spin at 37 °C for 30 min at 12 revolutions per minute to form a clot. Inject 0.1 ml of each titer of urokinase solution or control into this loop, and repeat at 37°C with 12 revolutions per minute.
Rotated for 4 hours. After this, the remaining thrombus was removed and fixed overnight in Bouin's solution (prepared by mixing 75 ml of a saturated picric acid solution with 25 ml of a commercially available formalin solution and 5 ml of glacial acetic acid), and the IU titer was determined by measuring the thrombus weight. We investigated the correlation between , molecular weight, and thrombolytic ability. The thrombolytic ability of urokinase was determined by the weight ratio of the urokinase-added group thrombi to the average thrombus weight of physiological saline (control), and this is shown in Table 2 as the thrombolytic rate (%).

[Table] As a result, urokinase with a molecular weight of 54,000 maintains extremely excellent performance in terms of thrombolytic ability, but urokinase with a molecular weight of 33,000 has less than half the thrombolytic ability of high-molecular weight urokinase.
Although the testing of thrombolytic ability using the Chandler loop method is a test tube experiment system, it is a simple method recognized as the closest assay method to an in vivo system [JE xp.
Physiol. Vol. The results of Experiment 2 showed that although the titer was determined to be the same by conventional measurements such as test tube method, plate method, and two-stage method (Pharmaceutical research: mentioned above), when comparing the thrombolytic ability, the molecular weight was determined to be the same. It was surprising to find that the performance was completely different depending on the Experiment 3 Thrombolytic effect in vivo (rats) The in vivo fibrinolytic effect of urokinase was investigated using Y. Sasaki (Thrombosis) in rats.
Res. 9 , 513, 1976) and RNChakravart:
(Atherosclerosis 21 , 349, 1975). 125I-human fibrinogen was prepared by labeling human fibrinogen with ^125I by the method of Greenwood et al ^. (Biochen.J. 89 , 114, 1963),
200 IU of human thrombin was added to 4 ml of the 0.4% solution, mixed, and allowed to stand at room temperature for about 60 minutes to generate fibrin. After thoroughly absorbing the moisture from the fibrin using paper, it was thoroughly washed with physiological saline, and then the paper was cut into small pieces with scissors, and 4 ml of physiological saline was added thereto and thoroughly ground ^. I - A suspension of human fibrin particles was prepared. The fibrin particle size at this time was 5 to 10 μm. Fibrin contained in 1ml of suspension liquid is 12mg
wet weight, and the radioactivity of ¹²⁵ I is 2.93×
It was 10 ⁶ cpm. 1 ml of such fibrin suspension was administered through the tail vein of a Wistar rat (male, weight 250 g), and then the molecular weight was determined from the same site.
54000 or urokinase 30000IU with molecular weight 33000
was administered. Thereafter, 0.3 to 0.5 ml of blood was collected over time from the carotid artery of the rat into a test tube into which a drop of aprotinin solution (15,000 KIE/ml), an anti-plasmin agent, was added. After the blood was allowed to stand at room temperature for about 2 hours, 0.05 ml of serum was collected, and the amount of FDP (fibrin lysate) produced as a result of fibrin degradation was measured by radioactivity. Here, assuming that the radioactivity of the administered fibrin is 100%, the radioactivity value per ml of serum and the total circulating serum volume (31.1ml/Kg)
The blood production rate (%) of ¹²⁵ I-FDP at each time point was determined, and the results are summarized in Table 3.

【table】

[Table] This third table shows the polymer urokinase (molecular weight
54000) produces FDP earlier than that of low-molecular-weight products, indicating that the fibrin decomposition rate is higher and the amount produced is larger. Experiment 3 showed that polymer urokinase has a strong fibrin-dissolving effect even in vivo.
It was found to have high thrombolytic ability. Experiment 4 Differences in blood concentration attenuation of administered urokinase depending on molecular weight Urokinases with different molecular weights were each labeled with ¹²⁵ I according to the method of Greenwood et al. The attenuation of blood concentration after administration was compared. Urokinase is a polymer with a molecular weight of 54,000 (specific activity 100,000 IU/mg protein) and a molecular weight of 33,000.
using a low-molecular-weight compound (specific activity 160,000 IU/mg protein), respectively using the chloramine T method (Biochen.J. 89 ,
114, 1963) for ¹²⁵ I labeling. Their comparative radioactivity was 1.28×10 ⁷ cpm/mg protein for the former and 2.24×10 ⁷ cpm/mg protein for the latter.
Beagle dog (male, weight 12-15 kg) was used as the experimental animal.
Three animals were used in each group. Dosage for both high and low molecule urokinase
The dose was 1000 IU/Kg, which was administered through the ear vein.
After administration, 5 ml of blood was collected over time from the forelimb vein using a syringe containing 0.5 ml of 3.8% sodium citrate, then plasma was separated by centrifugation, and its radioactivity was measured using a γ-ray scintillation counter ( (manufactured by X Card). The results are as shown in the figure, and the decay curve H in the blood of high-molecular-weight urokinase is gentler than the decay curve L in the blood of low-molecular-weight urokinase. It was twice as hot. Experiment 5 Stability of molecular weight and enzyme activity The correlation between molecular weight and stability of enzyme activity was investigated using commercially available urokinase. Molecular weight 20000,
Urokinase gel filtration method of 33000, 54000, 100000 (Sephadex G-75, 70mM phosphate buffer PH)
8.0, 2.6 x 80cm), and each
It was prepared as a 10000IU/ml aqueous solution. This is 4
The samples were stored at -10°C and the changes in activity over time were examined, and the time required to reach 50% residual activity is shown in Table 5. As a result, it was found that urokinase with a molecular weight of 54,000 has about three times the stability as compared to low molecular weight urokinase.

[Table] Experiment 6 Toxicity test Five healthy, well-nourished white mice and guinea pigs each weighing approximately 20 g were used. Urokinase (15,000 IU), a polymer with a molecular weight of 54,000, was dissolved in 25 ml of distilled water for injection, and 0.5 ml of this aqueous solution was administered subcutaneously to a white mouse, and 5 ml was subcutaneously administered to a guinea pig. He didn't die. The dosage is 300 IU per mouse for white mice and 3000 IU per mouse for guinea pigs. The dosage and administration method for clinical use of the urokinase preparation obtained in the present invention is to dissolve 50 to 30,000 IU of this product in 0.5 to 5 ml of distilled water for injection in the Japanese Pharmacopoeia,
It is used by intravenous injection, intravenous drip injection, intravenous drip injection, subconjunctival or retrobulbar injection, adjusting the dosage as appropriate depending on the age, symptoms and course of the drug. Next, examples of the present invention will be shown. Example: Fresh urine collected from an adult male was adjusted to pH 7.2,
Part 5 is L-allerginin agarose (PH7.2
(equilibrated with 2% sodium chloride solution)
Flow down into a 50ml column to adsorb urokinase. After washing this column with 100 ml of 2% sodium chloride solution at pH 7.5, urokinase is eluted by flowing down 50 ml of 1M sodium chloride solution at pH 6.0. After concentrating the eluate to about 10 ml, a 2.6 x 80 cm Cephadex G-75 (manufactured by Pharmacia, PH8.0
After filling a column (equilibrated with 70mM phosphate buffer), gel filtration was performed using the same buffer as the one used for equilibration at a flow rate of 20ml/hr. Collect 0.5ml of the liquid,
An elution fraction with a molecular weight of 54,000±10,000 was collected. The recovered aqueous solution containing urokinase was analyzed using SDS-polyacrylamide gel electrophoresis and simple molecular weight analysis [Colcwickkaplan:
Method in Enzymeology
In Enzymology) 26 , 3, (1972)], it was confirmed as a single peak at a position corresponding to the molecular weight of the standard protein, approximately 54,000. This aqueous solution was dialyzed against physiological saline and then freeze-dried to obtain a sample of urokinase with a total amount of 17,000 IU.

[Brief explanation of the drawing]

The figure is a diagram showing the blood attenuation curve of ¹²⁵ I-specimen after intravenous injection into beagle dogs.

Claims (1)

[Claims] 1 In each treatment step in the production of urokinase, the pH of at least the dialysis step for desalting is 7.
Molecular weight characterized by ~12 54000±10000
The method for producing urokinase.