JPH07121869B2 - Inhibitors of inflammatory edema promotion - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an agent for suppressing inflammatory edema promotion.

[0002]

2. Description of the Related Art Various plant-derived protease inhibitors have been reported. These inhibitors have specificity.For example, maize trypsin inhibitor and pumpkin trypsin inhibitor show inhibitory activity against trypsin and active Hageman factor (Thromb.
Res. 20 , 149, 1980, etc.), among the soybean trypsin inhibitors, Kunitz-type trypsin inhibitor is known to have an inhibitory effect on trypsin, plasma kallikrein, activated factor X and the like. In addition, when inflammatory edema is promoted, bradykinin activity is expressed and pain and vascular permeability are increased, but a scheme in which Hageman factor and kallikrein are involved in bradykinin activity expression has been proposed.

However, the relationship between protease inhibitors and inflammation remains at the stage of research in vitro, and there are very few reports demonstrating the effect in vivo.

On the other hand, ascites is often accumulated in patients with gastric cancer, renal cancer, ovarian cancer, liver cancer and the like, and pleural effusion is often accumulated in patients with lung cancer. Tumor metastases within the thoracic cavity, resulting in similar pooling. When ascites accumulates, it causes a decrease in plasma protein, which causes the patient to lose physical strength and other exacerbations.
If the therapeutic effect is reduced, the need for high-volume plasma transfusion, and the accumulation of pleural effusion cause respiratory failure, which in turn accelerates the introduction of the disease epidemics in the patient, which has serious therapeutic consequences for cancer.

[0005]

The present inventor has conducted various studies on the relationship between inflammation suppression and protease inhibitors, and as a result, soybean Kunitz-type trypsin inhibitor, especially its derivative, is effective in suppressing inflammatory edema in vivo. It was found to have a remarkable effect. Also,
Similar mechanism of vascular permeability increase in inflammation and vascular permeability increase in solid tumor (similar vascular permeability increase and plasma component leakage in solid tumor and inflammatory site)
Among the various studies focusing on, the inhibitor and its derivative have an effect on the suppression of cancerous thoracic / ascites retention,
That is, the inhibitor strongly inhibits vascular permeability in cancer tissues, and by consecutive administration of the inhibitor in the system of ascites cancer (Meth A, Sarcoma 180), the ascitic fluid is significantly suppressed, and tumor-bearing mice The present invention has been completed based on the finding that it will be a drug useful for treating cancer, by discovering that it will have a life-prolonging effect.

[0006]

[Means for Solving the Problems] That is, one of the present inventions is a cancerous thoracic / ascites retention inhibitor and an inflammatory edema acceleration inhibitor containing a soybean Kunitz-type trypsin inhibitor or a derivative thereof as an active ingredient. And the other one is
A soybean Kunitz-type trypsin inhibitor or a derivative thereof as an active ingredient.

Soybean trypsin inhibitor includes Kunitz-type trypsin inhibitor having a molecular weight of about 20,000 and Bowman-Birk having a molecular weight of about 6,000 to 8,000.
k) Type trypsin inhibitors are classified into two major categories. Their basic properties are Methods in Enzymology 19 , 853 (197
0) and "New viewpoints of protein research (mainly chemical research)", page 1738 (1982) (Kyoritsu Shuppan), among which Kunitz (Kunits) is a representative example.
z) Trypsin inhibitor and Bowman ─ Burk (Bowma
The structure of the (n-Birk) inhibitor has already been determined.
(Eur. J. Biochem., 32,417 (1973), J. Biochem. 74, 697
(1973)), and therefore their chemical and biological methods (
Synthesis by cell culture, gene recombination, etc.) is also possible today.

The agent of the present invention is soybean Kunitz
-Type trypsin inhibitor (hereinafter referred to as KTI) is used as an active ingredient, but KTI is derived from soybean, and is a substance that retains KTI-specific activity by limiting decomposition of soybean, and these chemical and biological methods described above. And those obtained by synthesizing

As a method for preparing KTI derived from soybean, some specific methods have been already proposed, and any method may be adopted, but generally, soybean, defatted soybean, soybean whey, etc. are used as raw materials. Then, from these, extraction with an aqueous medium or polar organic solvent (such as ethanol, acetone, etc.), membrane separation, isoelectric focusing, concentration by salting out, etc. It is possible to obtain a further purified product by means of ion exchange, physical or chemical adsorption means, etc., and its activity and purity can also be determined by a known method, for example, the casein digestion method of Kunitz [notation in this specification] Was measured by this method, and the amount that inhibits 1 mg of trypsin "Type-XI" (7500 to 9000 BAEE Unit / mg of active protein) manufactured by Sigma was set as 1 Unit], or by SDS-containing polyacrylamide gel electrophoresis etc. Measurement Rukoto can.

The amount of the active ingredient varies depending on the degree of purification, administration method and residual activity, but is 1.5 to 2.5 Unit /
Converted to the weight of active KTI protein, it is appropriate that the content is approximately 1 to 3000 mg / 60 kg body weight for intraperitoneal administration and 1 to 300 mg / 60 kg body weight for intravenous injection. .

Derivatives reduce the antigenicity of KTI, which is a protein, increase the stability against degrading substances (SH-protease, etc.) (improve in vivo half-life), or impart hydrophobicity to the amphipathic substances. It can be used for one or more purposes such as increasing sex. Such derivatives include KTI and various modified products such as styrene maleic anhydride copolymer (SMA) and its partial esterified product, polyethylene glycol, vinyl ether copolymer, pyran, and West German Patent Publication No. 315541. And the like, and other low molecular weight modified products and the like, and the method of binding with these is known, but one or more of the above objects can be achieved. Any method may be used as long as it is available. For example, when the modified product is SMA or its partial esterified product, a method of directly reacting with the N-terminal amino group of KTI or the ε-amino group of lysine residue in a neutral or alkaline solution to form a peptide bond. Was reported by Maeda et al., The inventor of the present invention (Journal o
f Medicinal Chemistry, 28, 455-461, (1985)).

The substance and its derivatives can be administered locally, intravenously, transarterally, transmucosally, transdermally, orally and intraperitoneally.

That is, for example, as a water-soluble drug for intravenous injection, KTI can be used as it is, or can be dissolved in physiological saline, 5% glucose solution, or other water-soluble injection to be used. It can also be used as a lipiodol preparation as an oil solution of KTI.
It is also possible to emulsify an aqueous solution in which is dissolved with oils and fats such as olive oil, medium chain fatty acid ester, linoleic acid ester, soybean oil and tung oil using an emulsifier, and to use it in the form of liposome.

KTI or its derivative can be used in combination with an agent having an autologous cancer action. Neocartinostane (NCS), Smunx
[SMANCS: Styrene-maleic acid (SMA) copolymer bound to neocarzinostanne (NCS) via amino group]
, Anti-cancer antibiotics such as mitomycin (trade name), 5-F
u (trade name) and other antimetabolites, Picibanil (trade name)
An immunostimulant such as the above, or other anticancer drug can be used, and examples of the combination mode with KTI include a method of processing into a single preparation or a method of individually administering monosodium. .

[0015]

[Action] KTI has an effect of suppressing inflammatory edema promotion and an effect of suppressing cancerous ascites and pleural effusion, resulting in a life prolonging effect. KTI further enhances the anti-cancer effect of the agent when used in combination with the agent having an autologous cancer effect. The reason for these effects is not entirely clear, but kallikrein-which is thought to be involved in the case of inflammatory edema-
KTI effectively blocks the bradykinin system in vivo, suppresses the development of pain and increased vascular permeability due to the expression of bradykinin, and the presence of a similar system in increasing the vascular permeability of cancer tissues causes vascular permeability. Sex, that is, the leakage of plasma proteins and the like to the tissue is suppressed, the storage of pleural effusion and ascites is suppressed, and the plasma component-nutrition supply to the tissue is blocked, and the direct action of the anticancer agent is suppressed. It is estimated that it will increase the Moreover, there is a possibility that the action of promoting the growth of cancer cells by protease is partially suppressed. further,
Since the molecular weight of KTI is about 20,000 (derivatives are higher than this), there is almost no leakage to normal tissues where vascular permeability is not enhanced, and selective permeation / action to tissues where vascular permeability is enhanced to some extent. It is also presumed that it contributes to and suppresses the growth of edema and cancer tissue. In addition, exudate (leakage) in the local area of inflammation
Is recovered from the lymph, but it is considered that the exuded polymer does not recover in the tumor tissue because it does not have a lymphatic system and remains there for a long period of time.This property is also considered to be useful for selective action on the affected area. To be

Although the cutoff of nutritional supply to cancer tissue is similar to the idea of suppressing nutritional supply by physical embolization therapy, physical embolization often has a harmful effect of destroying even normal tissues, as compared with the present invention. The drug does not cause such side effects.

[0017]

EXAMPLES Examples of the present invention will be described below, but the examples are for the purpose of explanation and are not intended to limit the spirit of the invention. Example 1 (Preparation example of KTI) 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 was added to 1 volume, and the mixture was stirred for about 1 hour. To the supernatant obtained by centrifugation, 1.5 volume of acetone was added, and the mixture was further stirred for about 1 hour. The resulting precipitate fraction was dialyzed against water. 1 for this dialysate
Add / 50 volume of 0.5M sodium phosphate buffer (pH 7.0),
The pH was adjusted to 7.0, the solution was passed through a DEAE-cellulose ion exchange column to be adsorbed on the resin, and then the eluate having a linear salt concentration gradient of 0 to 0.4 M was fractionated by a fraction collector to obtain a BBI trypsin inhibitor. Or KTI
Each of the rich fractions was further salted out and concentrated (for BBI trypsin inhibitor, further purified by CM cellulose ion exchange resin), and each concentrated and purified product was subjected to isoelectric point precipitation and freeze-dried to obtain KTI evaluation product and BBI. A type trypsin inhibitor was obtained. The purity of each product was 95% or higher in the protein by SDS-containing polyacrylamide gel electrophoresis. The specific activity was 1.97 Unit / mg protein in the former and 3.37 Unit / mg protein in the latter. Example 2 (suppression of ascites accumulation and life prolonging effect) Sa was intraperitoneally administered to female ddY-mice (7 mice per group) at 8 weeks of age.
rcoma 180 suspension was transplanted to 500,000 cells / mouse,
From that day, KT obtained in the above preparation example against 1 cc of physiological saline
A water-soluble drug in which 3 mg of I or BBI type trypsin inhibitor was dissolved or physiological saline which was not dissolved was intraperitoneally administered for 1 day per animal for 1 or 4 days for 4 or 14 consecutive days. Let An untreated case without any Sarcoma 180 transplantation was also conducted in parallel.

The results of body weight measurement during this period are shown in FIG. 1 (administered for 14 days) and FIG. 2 (administered for 4 days). From the results shown in both figures and the macroscopic observation, the inhibitory effect on weight gain by KTI (the inhibitory effect on ascites retention) was clearly observed, while there was almost no difference from the untreated group.
The inhibitory effect of BBI-type trypsin inhibitor was hardly observed.

Further, the average survival days and the like in the above-mentioned administration examples are shown in the following table, and a life prolonging effect was recognized.

[0020]

[Table 1] Example 3 (Preparation example of derivative bound with partially butylated SMA) 300 mg of KTI prepared by the method of Example 1 was added to 30 cc of 0.5 M
After dissolving in sodium hydrogen carbonate solution, 150 mg of partially butylated SMA (average molecular weight of about 2000) was added and stirred at 25 ° C for 90 minutes, and then a glycine solution was added to stop the reaction. The residual activity of this product is 36.3%, and the TNBS method (protein, nucleic acid, enzyme,
18 (13), 1153-1159, (1973)), the number of modified amino groups was 1.94 / molecule. Furthermore, it is dialyzed against ammonium hydrogen carbonate solution, freeze-dried and SMA of KTI.
A derivative (KTI-SMA) modified with was obtained. Example 4 (Inhibitor of inflammatory edema acceleration) 1% prepared by dissolving the modified KTI-SMA obtained in Example 3 in physiological saline as a test drug and mixing it with the test drug or physiological saline. Carrageenan solution (KTI-SMA content
5 mg / cc, or 15 mg / cc) of SD male rats (average body weight 1
50g ± 5g, 5 animals per group), 0.1cc / site was administered to induce inflammation, and the volume of the foot was measured over time to examine the inhibitory effect on carrageenin-induced paw edema. It was as shown in the figure.

As shown in the figure, a remarkable inflammatory paw edema inhibitory effect was observed in the KTI-SMA 15 mg / cc administration group 3 hours after the induction of inflammation.

The inhibitory effect of using the unmodified KTI 20 mg / cc obtained in Example 1 in place of KTI-SMA was KTI-SMA.
The effect was comparable to that of the 5 mg / cc administration group. Example 5 (Effect of anticancer drug enhancing action) Sarcoma 180 suspension was transplanted into the abdominal cavity of female ddY-mice (10 mice per group) at 8 weeks of age at a dose of 500,000 cells / mouse, and physiology was started from that day. Obtained in the above Preparation Example against saline
KTI, neocarzinostatin (NCS) or a mixture of both was administered once daily for 7 days continuously, and a group to which only physiological saline was administered was used as a control. During the experimental period, they were allowed to eat and drink freely.

The changes in dose and survival rate (%) are shown in the following table.

[0024]

[Table 2] As shown by the results in the above table, it was found that KTI not only has a life-prolonging effect by itself, but also has a synergistic effect of enhancing the effect of an anticancer drug (NCS).

[0025]

Industrial Applicability As described above, the agent containing KTI or a derivative thereof as an active ingredient has an inhibitory effect on inflammatory edema, an inhibitory effect on the accumulation of cancerous pleural and ascitic fluid, and an effect on enhancing the anticancer effect. Suppression of inflammatory edema suppresses the occurrence or enhancement of pain due to edema, and by the suppression of pleural and ascites accumulation, adverse effects due to the accumulation, for example, decrease in physical strength of the patient, decrease in therapeutic effect,
It has the effect of prolonging the life by, for example, preventing or suppressing a large amount of plasma transfusion, respiratory failure, introduction of a bad epidemic, and enhancing the therapeutic effect.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between administered drug and weight gain (≈ascites storage amount) in Example 2.

FIG. 2 is a graph showing the relationship between the administered drug and weight gain (≈retention volume of ascites) in Example 2.

FIG. 3 is a graph showing a volume change of foot edema in Example 4.

[Explanation of symbols]

In Fig. 1, (1) (△) .... Saline administration group (Sarcoma 180 tumor-bearing control), (2) (◇) .... BBI trypsin inhibitor administration group, (3) (□). .. KTI administration group, (4) (○)
(Non-cancer control), (Open arrow) .... Date of cancer cell transplant, (
↓) .... Date of drug administration. In Fig. 2, (1) (○) .... Saline administration group (Sarcoma 180 tumor-bearing control), (2) (△) .... BBI trypsin inhibitor administration group, (3) (□) .. .. KTI administration group,
(Folded broken line) ... untreated group (non-cancer control), (open arrow) ... cancer cell transplantation date, (↓) ... drug administration date. Figure 3
Medium, (1) (○) .... Saline administration group, (2) (△) .... KTI-
SMA 5 mg / cc administration group, (3) (□) .... KTI-SMA 15 mg / cc administration group.

Claims (1)

[Claims] 1. An inflammatory edema acceleration inhibitor, which comprises a soybean Kunitz type trypsin inhibitor or a derivative thereof as an active ingredient.