JPH09100298A - Trypsin inhibitor derivative having kallikrein inhibiting action - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an acylgelatinized trypsin inhibitor derivative useful as an agent for treating and preventing septicemia, pancreatitis or shock diseases and having kallikrein inhibiting action. SOLUTION: This acylgelatinized trypsin inhibitor derivative is obtained by fragmenting gelatin comprising a collagen or a soluble collagen or a substance obtained by irreversibly converting a collagen to water-soluble derivative, fractionating the fragment, purifying a part having low antigenicity, further, acylating the gelatin fraction and further converting the acylated material to an acylgelatin derivative increased in water soluble property. The inhibitor hardly has rejection symptom and has long half value period in blood and can be used as an agent for treating and preventing septicemia, pancreatitis or shock diseases.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention provides an acyl-gelatinized trypsin inhibitor derivative having a kallikrein inhibitory activity, which is promising as a therapeutic and preventive agent for sepsis, pancreatitis, or shock symptoms.

[0002]

2. Description of the Related Art Sepsis is caused by infectious lesions in the living body.
Bacteria and toxins produced by them are produced continuously, especially proteases, which flow into the blood and activate bradykinin-producing enzymes such as Hageman factor and prekallikrein in the blood, resulting in severe systemic symptoms. It is a disease (C
linica Chimica Acta, 185, 3
57-368, 1989, Journal Biolo
musical Chemistry, 264, 10589
10594,1989). Kallikrein acts on kininogen to produce a large amount of quinine (bradykinin), resulting in a so-called shock state with decreased blood pressure, oliguria, respiratory failure, consciousness disorder, and often death. . Kinins are also involved in the dissemination of toxins from other infected organs such as bacteria to other organs and the diffusion of toxins. Thus, regarding the mechanism of falling into sepsis, the excess or abnormal biological reaction to infection is the main factor, various chemical mediator networks, in particular, the production of kinin by kallikrein is involved, and after cell damage, It is known to be associated with multiple organ failure at a high rate.

[0003] On the other hand, acute pancreatitis is caused by reflux of duodenal fluid into the pancreatic duct, activation and release of trypsin by the surfactant activity of bile, and maturation process of cell endoplasmic reticulum or lysosome by hyperstimulation of cerulein. Due to the disorder, dissociation of proenzyme and lysosomal enzymes such as cathepsin B does not proceed, trypsinogen is activated, and a series of abnormal activation of digestive enzymes is said to occur. Trypsin also
The above-mentioned prekallikrein is activated to kallikrein, resulting in the production of a large amount of quinine, blood pressure lowering, pain, and in severe cases of acute pancreatitis, often with shock at the early stage, organ damage is inevitable, but at that time blood Clot progression, and in severe cases, acute intravascular coagulation (DI
The finding of C) is seen, and in actual human symptoms, it is often an incurable disease designated by the Ministry of Health and Welfare, which often accompanies lung disease.
Regarding the mechanism of falling into this acute pancreatitis, the main cause is the endotoxemia associated with blood translocation of proteolytic enzymes from the pancreas or infection, and the first step is the overproduction of quinine and nitric oxide by them, and further infection. A hypercoagulable state of blood or a shock symptom appears, and then in the second stage, the release of a neutral protease represented by neutrophil elastase occurs from neutrophils due to the state generated in the first stage. However, both endotoxin and elastase ultimately produce kallikrein, which in turn activates (produces) quinine.
Then, quinine also produces nitric oxide, which causes hypotension and shock symptoms. This is believed to lead to organ failure. At this time, it is kallikrein that holds an important key, and suppressing it suppresses quinine production and improves the condition.

As described above, due to the activation of kallikrein and the production of quinine, septicemia and acute pancreatitis cause an increase in vascular permeability, a vasodilatory action, a decrease in blood pressure, endotoxemia, and a hypercoagulable state. It is a disease in which a disorder, organ failure, shock symptom, or the like develops, and early prevention of these shock symptom, etc. is regarded as an important treatment that greatly affects the prognosis. From the mechanism of pathogenesis, administration of proteolytic enzyme inhibitors in sepsis acute phase and severe acute pancreatitis is considered to be an appropriate treatment method.However, at present, few drugs have sufficient therapeutic effect and severe acute The mortality rate of pancreatitis is extremely poor at 30 to 50% or more.

[0005]

DISCLOSURE OF THE INVENTION The present inventors have found that in these diseases, activation of plasma kallikrein, which is caused by the release of a large amount of proteolytic enzyme into blood, causes an increase in vascular permeability and vascular permeability. It is thought that production of a large amount of kinin, which is a physiologically active substance such as dilation and hypotension, is closely related, and the causative bacteria causing sepsis such as Pseudomonas aeruginosa, Serratia, Streptomyces, and Porphyromonas. That the increase in vascular permeability, the decrease in blood pressure, the shock, and the like caused by the above-mentioned or proteolytic enzymes derived from them may be improved or prevented by uniformly inhibiting the kallikrein-kinin system (Infecti
on and Immunity 48, 747-75
3, 1985, J. Bioactive and Co
compatible Polymers 3,27-4
3, 1988, Recent Progress on
Kinin, 362-369, 1992, FEMS
Microbiology Letter, 114, 10
9-114, 1993) and a soybean Klinitz-type trypsin inhibitor, which is one of the inhibitors of the kallikrein-kinin system, shows excellent effects in application to sepsis, acute pancreatitis, or shock symptoms associated therewith. (J. Pharm. Sci. 78 (3) 219-22.
2, 1989).

However, soybean Klinitz-type trypsin inhibitor is an enzyme having a very strong inhibitory activity (Ki value, < 10 ⁻¹⁰ M) of blood kallikrein activity, and temporarily shows an excellent effect in sepsis and acute pancreatitis. Though
The half-life and effect time in vivo are short, and because soybean Krinitz-type trypsin inhibitor itself is a foreign protein to the body, increasing the dose or repeated administration assuming a short half-life will cause fever, rash, and allergies. However, there is a problem that the foreign body rejection reaction of the living body becomes an obstacle.

[0007]

[Means for Solving the Problems] As a result of intensive studies to solve the problems of the soybean Klinitz-type trypsin inhibitor, the present inventors have found that collagen, preferably soluble collagen or collagen is irreversibly water-soluble. Gelatin, which is a derivative, is preferably fragmented, fractionated, and purified from the low-antigenic portion of the purified gelatin, and a derivative obtained by binding soybean Klinitz-type trypsin inhibitor is effective. Is desired to be acylated to obtain an acyl gelatin derivative having increased water solubility, that is, succinyl gelatinized soybean Kunitz trypsin inhibitor (S
It was found that the acyl-gelatinized soybean Kunitz-type trypsin inhibitor derivative of the present invention represented by ac-gel-SBTI) has less foreign body rejection in vivo and its effect is dramatically increased and lasts. ,
The present invention has been completed.

The acyl-gelatinized soybean Kunitz-type trypsin inhibitor derivative used in the present invention is a gelatin, preferably a fragmented gelatin having a molecular weight of about 20,000 to 100,000, such as succinic acid, malic acid, malonic acid or the like. The anhydrides thereof are preferably neutral to weakly alkaline, more preferably pH 7.0 to 9.5, and the free amino groups of gelatin are almost completely blocked, preferably 95% or more, and intermolecular cross-linking between gelatins is performed. Prevent. The acylated gelatin derivative is separated from unreacted organic acid by dialysis or the like. This and soybean Kunitz-type trypsin inhibitor are reacted with a condensing agent such as 1-ethyl-3- [3-dimethylaminopropyl] -carbadiimide (EDCI), preferably under acidic conditions, to obtain an acyl gelatin condensation soybean A Kunitz type trypsin inhibitor derivative, that is, a succinyl gelatinized soybean Kunitz type trypsin inhibitor, a maloyl gelatinized soybean Kunitz type trypsin inhibitor, a malonyl gelatinized soybean Kunitz type trypsin inhibitor and the like can be obtained. Isolation of these products from the reaction solution can be performed by a general method, for example, a method of separating with a column or the like.

[0009]

INDUSTRIAL APPLICABILITY The acyl-gelatinized soybean Kunitz type trypsin inhibitor derivative of the present invention has sepsis, acute pancreatitis,
In the treatment of shock symptoms, etc., the effect of diminishing, preventing or maintaining it dramatically increased and continued.

The present invention will be specifically described below, but the examples are for the purpose of explanation, and are not intended to limit the present invention.

[0011]

Example 1 (Preparation Example of Soybean Kunitz Type Trypsin Inhibitor) Soybean whey obtained in the process of producing isolated soybean protein from low-denaturation defatted soybean was concentrated, and this concentrate (crude protein content 5.
5%) 0.5 volume of acetone cooled to 0 ° C. or lower is added to 1 volume, and the mixture is stirred for about 1 hour. To the supernatant obtained by centrifugation, 1.5 volume of acetone is further added to 0 volume. ~ 4 ° C
The precipitate fraction obtained by centrifuging and stirring for about 1 hour at
It was dialyzed against water. 1/50 volume of 0.5 in this dialysate
Add M1 sodium phosphate buffer (pH 7.0) to pH
Was adjusted to 7.0 and passed through a DEAE-cellulose ion exchange column to adsorb to the resin, and then the salt concentration was adjusted to 0.
The eluate, which had a linear gradient of ˜0.4 M, was fractionated by a fraction collector and Bowman-
Fractions rich in Burk trypsin inhibitor or Kunitz trypsin inhibitor were obtained. This is salted out and concentrated, and further, the Bowman-Birk type trypsin inhibitor is further purified by a CM cellulose ion exchange resin, and ammonium sulfate is added to each concentrated and refined product so as to have a saturation of 0.8, followed by freezing after isoelectric focusing. After drying, a Kunitz-type trypsin inhibitor preparation and a Bowman-Birk-type trypsin inhibitor preparation were obtained. The purity of each preparation was 95% or more in protein by SDS-containing polyacrylamide gel electrophoresis. The specific activity of the former was 1.97 Units / mg protein, and the latter was 3.3 Units / mg protein.

[0012]

Example 2 (Preparation Example of Succinyl Gelatinized Soybean Kunitz Type Trypsin Inhibitor Derivative) Fragmentation into a molecular weight of 20,000 to 100,000 and fractionation were carried out to obtain an excess of about 100-fold molar amount of excess succinic anhydride with respect to gelatin. The reaction was carried out under conditions of pH 8.3 and succinylation. This almost completely blocked the free amino acids of gelatin. This suppresses intermolecular cross-linking between gelatin molecules. In this process, the modification rate of the amino group was estimated to be 98% by the trinitrobenzene sulfonate (TNBS) method. This succinyl gelatin was purified by a conventional method (dialysis, gel chromatography) and then freeze-dried. Soybean Kunitz-type trypsin inhibitor (SBTI) 50 mg obtained by the method of Example 1 and succinyl gelatin 160 m
g in 6 ml of water and 0.1M hydrochloric acid to pH 4.75
Then, 60 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added, and the mixture was reacted at 4 ° C. for 5 hours with stirring. After completion of the reaction, dialyzed with 0.05M potassium phosphate buffer (pH 7.0), and further gel filtration using a Cellulofine GCL-300-m (3.5 × 70 cm, manufactured by Chisso KK) column at a flow rate of 60 ml / hr. Was performed, and 5 ml each was collected. Succinyl gelatinized soybean Kunitz-type trypsin inhibitor (Suc-ge
For 1-SBTI), a fraction having ultraviolet absorption and antitrypsin activity was collected. Amicon-8050
(YM-30 membrane) was used for concentration and dialysis with deionized water and freeze-drying.

Succinyl gelatin (Suc-gel),
Soybean Kunitz trypsin inhibitor (SBTI),
Table 1 shows the physical properties of succinyl-gelatinized soybean Kunitz-type trypsin inhibitor (Suc-gel-SBTI). Further, gelatin fragment (gel), succinyl gelatin (Suc-gel), soybean Kunitz-type trypsin inhibitor (SBTI), succinyl-gelatinized soybean Kunitz-type trypsin inhibitor (Suc-ge).
1-SBTI) for each 1 mg / ml concentration of ultraviolet absorption spectrum is shown in FIG. 1, and each infrared absorption spectrum is shown in FIG.
As shown in FIG.

[0014]

[Table 1]

[0015]

FIG.

[0016]

FIG. 2

[0017]

FIG. 3

[0018]

FIG. 4

[0019]

FIG. 5

[0020]

Example 3 (Improvement effect on trypsin-induced lethal shock mouse survival rate) Male ddY strain mice are injected with 25 mg / kg trypsin through the tail vein, and the survival rate after 2 hours is calculated. Soybean Kunitz trypsin inhibitor (SBT
I), succinyl gelatinized soybean Kunitz type trypsin inhibitor (Suc-gel-SBTI), and physiological saline were administered 420 to 1 minute before the administration of trypsin. As shown in Table 2, in the control group, all the animals died 2 hours after the injection of trypsin from the tail vein, whereas the survival rate was obviously increased by the administration of the soybean Kunitz-type trypsin inhibitor. Furthermore, when the succinyl-gelatinized soybean Kunitz-type trypsin inhibitor was administered, the survival rate of 75% was still obtained even 240 minutes before the administration when the administration of the soybean-Kunitz-type trypsin inhibitor was not effective. Furthermore, 50% even after 420 minutes
The succinyl-gelatinized soybean Kunitz-type trypsin inhibitor was superior to that of succinyl-gelatinized soybean.

[0021]

[Table 2]

[0022]

Example 4 (Inhibitory Effect on Pseudomonas aeruginosa-derived Elastase-Induced Blood Pressure Reduction) Male Wistar rats are anesthetized with sodium pentobarbital, a catheter is inserted and fixed in the carotid artery, and the arterial blood pressure is monitored with an electric sphygmomanometer. Insert a catheter for general administration of aorto-venous drug, and use Pseudomonas aeruginosa-derived elastase (P
E) Injecting 0.8 mg / kg shows a marked decrease in systemic blood pressure and causes shock symptoms. If soybean Kunitz-type trypsin inhibitor was administered to this,
The systemic blood pressure drop by E was completely inhibited, showing a protective effect against shock (Fig. 6). Furthermore, succinyl gelatinized soybean Kunitz-type trypsin inhibitor (Suc
-Gel-SBTI) was shown to have a stronger and longer-lasting suppression of lowering of blood pressure as compared to the case where only soybean Kunitz trypsin inhibitor (SBTI) was administered (Fig. 7).

[0023]

FIG. 6

[0024]

FIG. 7

[0025]

[Example 5] (Inhibitory effect on blood kinin (bradykinin) release increasing action induced by Pseudomonas aeruginosa-derived elastase) The amount of blood kinin (bradykinin when carried out in the same manner as in Example 4 using male Wistar rats Amount: BK) Enzyme antibody method (Dainippon Pharmaceutical Merkit M, Bradykinin assay kit)
As a result of the measurement by the method, a clear increase in blood kinin amount was observed after PE administration. The increase in quinine was completely suppressed 1 minute after administration of soybean Kunitz trypsin inhibitor (SBTI) or succinyl gelatinized soybean Kunitz trypsin inhibitor (Suc-gel-SBTI). After 180 minutes, the soybean Kunitz-type trypsin inhibitor administration does not already have an inhibitory effect, but administration of the succinyl-gelatinized soybean Kunitz-type trypsin inhibitor still shows sufficient inhibition, and the effect is long-term (8 hours or more). ) It was shown to be persistent and significantly improved (Fig. 8).

[0026]

FIG. 8

[0027]

[Example 6] (Pharmacokinetics of succinyl gelatinized soybean Kunitz type trypsin inhibitor (Suc-gel-SBTI)) Soybean Kunitz type trypsin inhibitor (SBTI) and succinyl gelatinized soybean Kunitz type trypsin inhibitor (Suc-gel-SBTI) were used. After attachment to the DTPA chelating agent, it was radiolabeled with ⁵¹ CrCl and the labeled proteins were separated using a Sephadex G-25 column. ⁵¹ Cr-labeled soybean Kunitz-type trypsin inhibitor and succinyl-gelatinized soybean Kunitz-type trypsin inhibitor (about 50,000 γpm) were tail vein-administered to ddY mice, and the blood radioactivity was measured by a gamma counter over time to halve the blood. The period was calculated. Compared to the blood half-life AUC of soybean Kunitz-type trypsin inhibitor, that of succinyl-gelatinized soybean Kunitz-type trypsin inhibitor is 6 times as long as half-life and 32 at AUC.
Each was 5-fold enhanced, and it was shown that derivatization to a succinyl-gelatinized soybean Kunitz-type trypsin inhibitor prolongs the action time (Table 3, FIG. 9).

[0028]

[Table 3]

[Brief description of the drawings]

FIG. 1 shows gelatin fragments (g
e), succinyl gelatin (Suc-el), soybean Kunitz-type trypsin inhibitor (SBTI), and succinyl-gelatinized soybean Kunitz-type trypsin inhibitor (Suc-gel-SBTI) at a concentration of 1 mg / ml, respectively.

FIG. 2 is an infrared absorption spectrum of succinyl gelatin (Suc-gel).

FIG. 3 is an infrared absorption spectrum of succinyl-gelatinized soybean Kunitz-type trypsin inhibitor (Suc-gel-SBTI).

Fig. 4 Soybean Kunitz trypsin inhibitor (SB
It is an infrared absorption spectrum of TI).

FIG. 5 is an infrared absorption spectrum of a gelatin fragment (gel).

[Fig. 6] Pseudomonas aeruginosa-derived elastase (P
E) Soybean Kunitz-type trypsin inhibitor (SBT)
I) Suppression of decrease in blood pressure in a group to which PE was administered after administration was recorded by a sphygmomanometer.

FIG. 7 is a graph showing the degree of decrease in blood pressure by PE administration in Example 4 over time (after 1 minute, 120 minutes, and 180 minutes), soybean Kunitz-type trypsin inhibitor (SBTI) and succinyl gelatinization. It is shown by comparing the effect of soybean Kunitz type trypsin inhibitor (Suc-gel-SBTI).

FIG. 8: Soybean Kunitz-type trypsin inhibitor (SBTI) and succinyl-gelatinized soybean Kunitz-type trypsin inhibitor (Suc-gel-SB) for blood kinin amount and its blood kinin production by PE administration in Example 5.
The effect of TI) is shown at 1 minute and 180 minutes after administration.

FIG. 9: ⁵¹ Cr-labeled soybean Kunitz trypsin inhibitor and succinyl gelatinized soybean Kunitz trypsin inhibitor according to Example 6 (about 50,000).
(γpm) was administered to the ddY mouse by tail vein, and the radioactivity in blood was measured with a gamma counter over time, and the results of comparison of changes in blood concentration are shown.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Hironori Oide 1-3-40 Nishiotsuka, Matsubara-shi, Osaka Fujimoto Pharmaceutical Co., Ltd. (72) Inventor Shiro Yoneda 1-3-40 Nishi-Otsuka, Matsubara-shi, Osaka Fujimoto Pharmaceutical Co., Ltd. (72) Inventor Chikako Kinoshita 1-3-40 Nishiotsuka, Matsubara-shi, Osaka Prefecture Fujimoto Pharmaceutical Co., Ltd.

Claims (9)

[Claims] 1. An acyl-gelatinized soybean Kunitz-type trypsin inhibitor derivative having a kallikrein inhibitory action. 2. The derivative according to claim 1, wherein the acyl group is selected from the group consisting of a succinyl group, a malonyl group, a maloyl group and an aconyl group. 3. A therapeutic and prophylactic agent for sepsis or pancreatitis, which comprises the derivative according to claim 1 or 2. 4. A therapeutic and prophylactic agent for shock symptoms, which comprises the derivative according to claim 1 or 2. 5. The therapeutic and prophylactic agent according to claim 4, wherein the shock symptom is caused by sepsis or acute pancreatitis. 6. The method according to claim 6, which is obtained by combining an acyl group derived from an organic acid with gelatin to obtain an acyl gelatin, and then combining it with a soybean Kunitz-type trypsin inhibitor in the presence of a condensing agent. The derivative according to 1. 7. A succinyl gelatin obtained by combining succinyl derived from succinic acid and gelatin, and then using 1-ethyl-3- [3-dimethylaminopropyl] as a condensing agent.
-Succinyl-gelatinized soybean Kunitz-type trypsin inhibitor, which is obtained by using carbodiimide and combining with a soybean Kunitz-type trypsin inhibitor 8. The method according to claim 1, which comprises a step of binding an acyl group derived from an organic acid to gelatin to obtain an acyl gelatin, and then binding the soybean Kunitz-type trypsin inhibitor in the presence of a condensing agent. Method for producing derivative 9. The acyl group is a succinyl group derived from succinic acid, the condensing agent is 1-ethyl-3- [3-dimethylaminopropyl] -carbadiimide, and the resulting derivative is succinyl gelatinized soybean Kunitz type. 9. The method according to claim 8, which is a trypsin inhibitor.