WO1983004179A1

WO1983004179A1 - Prophylactic and/or therapeutic agent for infestation

Info

Publication number: WO1983004179A1
Application number: PCT/JP1982/000187
Authority: WO
Inventors: Haruo Ohnishi; Hiroshi Kosuzume; Yoshikazu Ashida; Yasuo Suzuki; Ei Mochida
Original assignee: Mochida Pharmaceutical Co., Ltd.
Priority date: 1982-05-22
Filing date: 1982-05-22
Publication date: 1983-12-08
Also published as: AU8451782A

Abstract

A prophylactic and/or therapeutic agent for infestation into a living body due to exposure to radiation or various surgical operations, which contains trypsin inhibitor present in human urine, and a prophylactic and/or therapeutic method for various infestations, which comprises administering the agent orally or parenterally.

Description

Description Title of invention

Invasive prevention and Z or therapeutic agents

Technical field .

TECHNICAL FIELD The present invention relates to an invasive prevention and / or Z or therapeutic agent, which is an active ingredient of a trypsin inhibitor in urine.

Background art

Organisms have the function of keeping the internal environment constant with respect to the external environment that is constantly changing. This is called the homeostasis of the living body (homeostasis), which is a stimulus that causes a change in the external environment or the external environment that causes a certain degree of change in the internal environment, that is, a biological reaction. Invasive conditions are defined as the pathological conditions caused by these stimuli, including various factors such as radiation exposure, bleeding, trauma, trauma, and infection. However, surgery involving factors such as pain, wounding, infection, starvation or anesthesia is one of the most typical invasions.

Many of the biological reactions to various invasions are reversible, but if the magnitude of the invasion is excessive or if the patient has various comorbidities, the organism can withstand the invasion. It is a well-known fact that function is permanently impaired, leading to delayed recovery or complications unless proper treatment is given.

In particular, during surgery, disturbances in body protein metabolism may occur, Adverse opportunistic conditions such as opportunistic infection or rapid growth or metastasis of residual tumors due to decreased immunity, development of postoperative ulcers, and wound infection in surgical wounds are often observed. Occasionally, these causes are associated with acute renal failure, severe infection, and even death, which is not uncommon.

Many of the production reactions to invasions such as radiation exposure and surgery are persistent, sometimes as long as several months, and can result in severe blood pressure due to transient sudden circulatory disturbances. This is different from a so-called shock with a decline.

It is also believed that local organs are most susceptible to shock, as the shock is a systemic response, whereas most invasions elicit a local response first.

Despite the fact that the invasion is clearly different from a shock, no drug has been developed to treat the invasion. In other words, current treatments for various invasions include nutritional management in the sense of energy supplementation for postoperative hyperglycaemia, lipid and protein catabolism, or symptomatic treatment such as adaptation of antibiotics for postoperative infection. It has only been done and has not always had a sufficient effect. As an example, increased nutrition immediately after gastrectomy is not possible with any current nutritional management method, and nitrogen supplementation for protein catabolism is not possible. No known therapeutic agent that improves overall deterioration, etc.

In this way, we can prevent various types of invasion typified by surgery.

OMPI Or, the development of safe drugs to treat is an enhancement of recent remarkable surgical treatments, and the importance of pre- and post-operative patient isolation is being recognized more and more. Is strongly desired by many people.

Disclosure of the invention

The present inventor 5 has studied over many years on metabolic abnormalities and functional deterioration caused by surgical invasion typified by surgery, and consequently found that trypsin inhibitor present in urine was obtained.

(Hereinafter referred to as UTI) excretion was found to vary during surgery depending on the degree of invasion.

Therefore, the present inventors conducted further research and found that, as shown in the experimental examples described later, this UTI was exposed to various kinds of abnormalities due to radiation exposure or surgical invasion, that is, a decrease in immune function. Have been found to improve overall resistance to infection and swelling, and to improve protein metabolism and renal dysfunction, etc., which has been used as an anti-shock agent. U

Aprotinin and gabexate mesylate (hereinafter called gabexate mesi late, which are referred to as gabexate) which have a trypsin inhibitory action like TI, are exposed to radiation or It had no effect on these symptoms caused by surgical invasion. This indicates that various anti-invasive effects during the week indicate that various disease states resulting from radiation exposure or surgical invasion are different from those at the time of the shock. It does not appear to be common to busin preparations, but indicates that it is a unique effect of UTI. The present invention has been completed based on the above findings.

OMPI It has been achieved.

'' UTI, an active ingredient of the invasive and / or therapeutic agent of the present invention, is a substance widely present in the urine of mammals, and its properties vary slightly depending on the type of animal from which it is derived. (Carls son et al., Enzynie, 18: 176, 1976). The human UTI has a distribution of molecular weight "170,000-700,000", and each of them has a trypsin inhibitory activity (Proksh, Journal of Propertory and Clinical Medin Shin (J. Lab. Clin. Med.) 79 949 1 197 2), for example, the method of Sumi et al. [Journal or Pie Ochemi Tree (J. Biochem., Pp. 83, 141, pp. 1978), which means that human urine is reduced to S and arginine sef. After passing through a column, the column is eluted with 2% ammonia water containing 0.2 M sodium chloride, and then eluted with a Sephadex G—200 column according to a conventional method. Then, gel chromatography is performed to obtain a trypsin S harmful substance fraction.Urine trypsin purified in this manner is purified. The inhibitor is an acidic glycoprotein having a molecular weight of 67000, an isoelectric point of 2 to 3, and containing 5 to 12% of neutral sugar.

Next, the effectiveness and toxicity of the UTI of the present invention will be described based on experimental examples. EXPERIMENTAL EXAMPLE 1 Effect on Immune Function Decrease by Radiation Exposure Takeya et al. : Journal of General Microbiology (vol. 100, pp. 373, 197). That is, 10 groups of IGR male mice (body weight: 15 to 179) were irradiated with X-ray 450 Lentogen (Soflon Soken) to examine the effects on various immunity. . UTI 20 mgZ or aprotinin 2 O mgZkg, which is widely used as a shock remedy, or gabexetol 20 mgZkg twice a day for 3 days did .

1) Macrofuge function

Three days after irradiation, peritoneal exudate cells were collected and analyzed by the method of Stuart et al. [Hundbook of expe- rimentary medicine. The phagocytic activity of sheep erythrocytes was measured according to rimenta (immunology) 2nd edition 24. M1973 years], and the results are shown in Table 1.

UTI improved macrophage function, which was reduced by radiation exposure, but aprotinin and gabexet were ineffective.

1 0 ο Sheep red blood cell phagocytic cell count z Total cell count Untreated group 18.5 ± 2 3 Control group 3.6 ± 2 1

UTI 20 mgZ kgx 6 times group 9.8 ± 1 9 * aprotinin 20 mgZ kgx 6 times group 4.0 ± 12 Gabexet 20 mg / kgx 6 times group '― 3.-8 ± twenty five

P <0.0 5

2) Humoral immunity

In mice after irradiation 3 days to篛注1 0 ⁸ sheep erythrocytes, the spleen cells after 4 days were collected, the method [I Takeno B di one (I mmunology) Cunningham (cunninghara) 1 4 Volume 5 9 9 pp [1968]], the number of plaque-forming cells was counted, and the results are shown in Table 2.

UTI improved humoral immunity, which was reduced by radiation exposure, but aprotinin and gabexetate were ineffective.

Table 2

Number of plaque forming cells Z 1 O ⁶ Number of spleen cells Untreated group 3 5 1 ± 2 8

Control. Group 8 3 ± 1 9

U T I 20 rag / kgx 6 times group 1 4 2 ± 16 *

Aprotinin 20 mg / kg x 6 groups 9 5 ± 2 9

Gabexet 20 mg / kg x 6 groups 8 8 ± 1 1

* P <0. 0 5

3) Cellular immunity

Three days after irradiation, Natsuume et al.

I [Vol.5, 225, pp. 1975]. That is,

A 7% picric chloride solution was applied to the abdomen to sensitize, and 7th, a 1% picric chloride solution was applied to the earlobe, and the thickness was measured 48 hours later. It is shown in the table.

UTI improved cellular immunity reduced by radiation exposure, but aprotinin and gabexate were ineffective

OMPI Table 3

Ear swelling (m 丄 untreated group 1 9 1 ± 6

Control group 1 6 3 ± 7

U T I 20 mg kgx 6 groups 1 8 0 ± 9

Aprotinin 20 mg / kg x 6 groups 1 60 ± 5

Gabexet 20 mg kg x 6 groups 1 6 2 ± 6 Experimental Example 1 2 Effect on postoperative immunological decline

Sasaki way {

It was carried out according to the journal of Tsutsuga University of Medicine 95 5 6 3 6 7 That is, 10 dd Y male mice (body weight of 30 to 329) of 1 group each were laparotomized 2 cm along the median line under pentobarbital anesthesia, and the inside of the abdominal cavity was excised for 3 minutes. After stirring with a stick, the limb was restrained for 2 hours while the abdomen was open, and the abdomen was closed to examine the effect on various immunity. In addition, UTI 20 mgZ kg, aprotinin 20 nig / kg or gabexetol 20 mgZ kg was administered 2 hours immediately after laparotomy using a continuous infusion device (truth A-I type). It was intravenously injected.

1) Mac mouth fusing function

Peritoneal exudate cells were collected after the laparotomy 1 and the phagocytic activity of sheep erythrocytes was measured in the same manner as in Experimental Example 11 and the results are shown in Table 4.

UTI improved macrophage function reduced by laparotomy invasion, but no aprotinin and gabexate Table 4 which was effective

1 ο ο χ Sheep erythrocyte phagocytic cell count Z Total cell count Untreated group 1 7.4 Sat 1 5

Control group 8.5 people 1

U Τ ί 20 mg / kg group 15.8 ± 25 *

Aprotinin 20 mgZ kg group 9.3 ± 22

Gaudexate 20 mgZ kg group 9.0 ± 3 2

* P <0. 0 5

2) Humoral immunity

1 0 ⁸ sheep red blood cells by intravenous injection to mice of open surgery "! Day after

(4) Collect the spleen cells after the above and plaque the same as in Experimental Example 11

The number of formed cells was counted, and the results are shown in Table 5.

UTI has reduced humoral immunity due to invasive laparotomy

Improved, but aprotinin and gabexetet were ineffective

O

One O PI ~. Table 5

Plastic chromatography click forming cell number Z 1 0 ⁶ spleen cell number

Untreated, group 3 17 ± 20

Control group 1 9 7 ± 1 6

U T I 20 rngX kg group 2 8 6 ± 1 8 **

Ablotinin 20 nig / kg group 2 1 2 ± 1 7

Gabexet 2 „0 mgX kg group 2 0 5 ± 3 1

** p <0.0

3) Cystic immunity

One day after laparotomy, thickening of the earlobe as in Experimental Example 1

Was measured, and the results are shown in Table 6.

UTI has reduced cellular immunity due to invasive laparotomy

Improved, but aprotinin and gabexet were ineffective

I got it. Table 6

Ear swelling (UL Π1)

Untreated group 2 1 0 ± 8

Control group 1 8 1 ± 5

U Τ Γ 20 mgZ kg group 2 0 0 ± 9

Aprotinin 20 mgZ kg group 1 8 5 ± 6

Gabexet 2-0 mg / kg group 1 8 3 ± 7

Ο ΡΙ Experimental Example 1 3 Effect on postoperative infection

Experimental Example 1 and 2 A group of 10 ddY male mice (body weight 30-32 9) were inoculated laparotomy-invasively for 2 hours and 4 hours.

The limb was restrained. Proteus Bulgaris (P.S.

Vulgaris GN 5298) 10 ⁵ Z animals were infected intraperitoneally and the abdomen was closed, and the survival rate 7 days later was calculated. Note that

UTI 20 mgZ kg, aprotinin 20 mgZ kg or gabexate 20 mgz kg were intravenously administered continuously for 2 hours immediately after laparotomy. The results are shown in Table 7.

U T 改善 improved decreased survival due to postoperative infection

Aprotinin and gabexet were ineffective.

Table 7

Survival rate% untreated group 0 0 control group 0

*

U T I 20 mgZ kg group 6 0

Aprotinin 20 mg / kg group 0

Gabexet 20 mgZ kg group 0

P <0.0 5

Experimental Example 1 4 Effect on tumor growth after surgery

As in Experimental Example 1 and 2, a group of 10 ddY male mice (body weight: 30-32 g.) Were invaded by laparotomy, restrained the limbs for 2 hours, closed the abdomen, and axilla Subcutaneous tumor cells (S

O PI

, coma- 1 8 0) were implanted with 1 0 ^7. One week after tumor implantation, the tumor was excised and weighed. UTI 20 mgZ kg, aprotinin 2 O mgZkg or gabexet 2 O mg

/ kg twice a day from the day of laparotomy for 3 days. The results are shown in Table 8.

UTI inhibited the growth of subcutaneous tumors in the axilla, but aprotinin and gabexate were ineffective. Table 8

Tumor weight (mg)

Control group 8 5 0 ± 6 5

U T I 20 rag / kgx 6 times group 5 3 3 ± 7 4 **

Aprotinin 20 fiig / kgx 6 groups 7 9 3 ± 8 1

Gabexet 20 mgZ kgx 6 times group 8 0 8 ± 9 4

** p 0.0 0 Experimental example 1 5 Effects on postoperative protein metabolism and renal function abnormalities

10 ddY male mice per group as in Experimental Example 1

(Body weight 30-329) was subjected to laparotomy surgery, the limb was restrained for 2 hours, and then the abdomen was closed to reduce protein catabolism and renal function.

Effects were investigated. In addition, UTI 2 mgZ kg, aprotinin 2 mgZ kg or gabexantol 2 nig / kg was intravenously infused for 2 hours immediately after laparotomy using a continuous intravenous infusion device.

O PI 1) Protein catabolism

Two hours after laparotomy, blood is collected by cardiac puncture, and blood urea nitrogen (BUN) is measured using a ceriotti method [Clinical Chemistry] 1 7, p. Pp. 1971] according to the diacetyl monoxime method, and the results are shown in Table 9.

UTI improved protein catabolism associated with the invasion of laparotomy, but aprotinin and gabexet were ineffective. Table 9

B U

(mgXdl) Untreated group 24.3 ± 0.8 Control group 32.0 ± 1.6

U T I 2 mgZ kg group 27.5 ± 0.1 aprotinin 2 mg / kg group 3 1.5 ± 2.1 Gabex-to 2 mgZ kg group 30.8 ± 2.6

* p <0. 0 5

2) Smart function

Two hours after laparotomy, phenol sulfone phthalein (PSP) was intravenously injected after 30 minutes, and the amount of PSP remaining in the blood 30 minutes later was measured by the method of Ecker et al. Toxicology and Applied Pharmacology 3 4 1 7 8 1 9 75 years], and the results are shown in Table 10.

UTI improved renal function decreased by invasive surgery, but aprotinin and gabexet were ineffective.

0 Table

PSP elimination test (g Ζ Βΐί blood)-untreated group 4.

Control group 37.5 ± 3

1 **

U Τ I 2 mgZ kg group 2 3, 2 Sat 1

Aprotinin 2 fog, kg group 34.8 ± 4 2 Gabexe-total 2 mg group 32.6 ± 5 4

** P <0.0 Experimental example 1 6 Acute toxicity

UTI4Q was intravenously or intraperitoneally administered to 10 male ddY mice (body weight: 20-229) per group, and the symptoms were observed for one week. The change in body weight during the observation period was similar to that in the saline-administered group, and no deaths were observed. In addition, no abnormalities were found in the autopsy findings after one week.

As is evident from the above experimental examples, UTI is not useful for living organisms, such as metabolic abnormalities caused by bombardment or invasion of various laparotomy operations, decreased renal function such as anuria, or decreased immune function. Action to reduce and improve various undesired symptoms Remarkably strong, the dose that exerts these effects is a safe dose due to the acute toxicity. Aprotinin or gabexet, which is widely used clinically as a shock treatment, is not suitable for these symptoms caused by radiation exposure or surgical invasion. No effect was shown.

Therefore, UTI can be a very useful drug for the prevention and / or treatment of invasiveness that occurs frequently during radiation exposure and during various surgical procedures.

BEST MODE FOR CARRYING OUT THE INVENTION

The therapeutic dose of UTI of the present invention is 1-1000 mg, preferably 420 mg per day, but can be increased or decreased depending on the condition of the patient or the use. .

The UTI of the present invention is usually administered as an injection, but it can also be used as an oral administration, an oral preparation, or a vaginal or rectal preparation. Injectables can be lyophilized preparations that are dissolved after use, and capsules, tablets, granules-powders or oral liquid preparations can be absorbed but not absorbed. It is preferable to use ribosome inclusions to promote the treatment. <Also, suppositories are appropriate for vaginal and rectal preparations.

In preparing these preparations, it is possible to use conventional methods together with pharmaceutical carriers or excipients for resentment. Next, the method for obtaining UTI will be described with reference to examples, but the method for obtaining UTI is not limited to these examples, and a general protein purification method may be used. Is, of course.

O PI Example 1 1. Proksh method (Journal of Clinical Medicine) <J. Lab. CI in. Med.) 7 9 4 9 1 p. 1972). Immediately after evacuation, healthy adult urine was dialyzed against deionized water, adjusted to pH 7.8 with 1N sodium hydroxide solution, and then adjusted to 0.05 M tris. Adsorbed to DEAE cellulosic column (20 x 80 cm) fully equilibrated with hydrochloric acid buffer (PH7.8) After washing with the same buffer 40, 0.3 M The adsorbed UTI was eluted using the same buffer solution containing sodium, and then heat treatment was performed at 60 ° C for 20 minutes to inactivate contaminating proteases. The crude UTI thus obtained was equilibrated with 0.02 M glycine-monohydrochloride buffer (PH 3.4). The column was adsorbed on a DEAE cellulosic column (8 x 60 cm), and the column was washed successively with 10 £ of the same buffer and then with 10 of the same buffer containing 0.2 M sodium chloride. After, 0.4 M salt UTI was eluted with perimeter buffer containing sodium 8. The eluate was subjected to ultra-concentration, and then Sephadex G-1 prepared so as to be free of pyrogens. Using a column (100 x 95 cm) filled with 0, fractionation was performed using physiological saline as a developing solvent. Method [Methods in Enzymo Iogy] 19 9 8 4 4 1 9 70 years], and the amount of periribulin (300 000 N

F U Zmg, Miles) 100%

The specific activity when the amount of I is 1 U is 2500 UZ mg.The formulation of this invasive prevention and / or therapeutic agent is shown below as an example. It is not limited to only.

Example 1-2 Lyophilized Injection

4 g of UTI is dissolved in 200 m of physiological saline and filtered aseptically using a filter. Example 1 3 tablets by filling the sterilized solution into sterilized glass containers 1 m at a time to obtain a lyophilized formulation

U T I 40 K g

Lactose 32 Kg

Potato starch 15 Kg

Polyvinyl alcohol 0 15 Kg

Magnesium stearate 0 15 K g

The above components are weighed, and UTI lactose and potato starch are uniformly mixed. An aqueous solution of polyvinyl alcohol is added to this mixture, and granules are prepared by a wet granulation method. The granules are dried, mixed with magnesium stearate, compressed and compressed into tablets weighing 100 mg, and then enteric-coated to give enteric-coated tablets.

OMPI

, Example 1 4 Ribosome Inclusion Body

Egg yolk lecithin, cholesterol and diacetyl phosphate were mixed in a molar ratio of 7: 2: 1, and the resulting mixture was added to the mixture.

It was dissolved in a 2.5 ^ cross-mouthed form, and a thin film was prepared on a Frasco wall. This film was mixed with a phosphate buffer 25 containing 100 g of UTI to prepare a dispersion. After the ultrasonic treatment, centrifugation is performed at 110,000, and the obtained precipitate is turbidized in 3 πιΰ physiological saline. After sterilization, a liposome encapsulated preparation containing UTI is obtained.

Claims

The scope of the claims

1. Invasive prevention and / or Z or therapeutic agent containing tribsine inhibitor in human urine as an active ingredient

2. The invasive prophylactic and / or therapeutic agent according to claim 1, wherein the human urinary trypsin inhibitor is an acidic glycoprotein having the following properties.

a) Molecular weight 6 0 0 0 0 to 7 0 0 0 0

b》 isoelectric point 2-3

c) Neutral sugar content 5-12%

3. The invasive prophylactic and / or Z or therapeutic agent according to claim 1 or 2, comprising an active ingredient and a liquid or solid medium.

4. The infringement according to any one of claims 1 to 3, wherein the dose of "1" of the active ingredient is "1 to 100 rag!" ! Prevention and Z or therapeutic agents

5. The invasive preventive and / or therapeutic agent according to claim 4, wherein the daily dose of the active ingredient is 4 to 200 nig.

6. The therapeutic agent according to any one of claims 1 to 5, wherein the dosage form is a single dosage form selected from the group consisting of injections, pills, inhalants, and suppositories. Invasive prophylactic and / or therapeutic agents according to item 1.

7. The invasive prophylactic and / or therapeutic agent according to any one of claims 1 to 6, wherein the disease to be treated is surgical invasion.

8. The scope of the disease to be treated or the invasion due to radiation exposure The invasive prevention and / or Z or therapeutic agent according to any one of paragraphs 1 to

9. Prevention and / or treatment of invasion comprising administering a therapeutically effective amount of a human urinary trypsin inhibitor.

10. The invasive prophylaxis and Z or treatment method according to claim 9, wherein the human urinary tribsin inhibitor is an acidic glycoprotein having the following properties.

a) Molecular weight 6 0 0 0 0 0 0 0 0

b> isoelectric point 2-3

c) Neutral sugar content 5 to 1 2 96

11 1. The scope of the simulant consisting of the active ingredient and a liquid or solid medium described in paragraph 9 or 10

Z or treatment method

1 2. Claims in which a therapeutically effective amount of a trypsin inhibitor in human urine is administered from one or more administration routes selected from the group consisting of injection, oral, inhalation, rectal and vaginal. Invasion prevention and / or Z or treatment method as described in any one of paragraphs 9 to 11

13.1 Therapeutically effective amount per day;] The method of invasion prevention and Z or treatment according to any one of paragraphs 9 to 12 of the range of 1000 mg

14. The invasive prevention and / or treatment method according to claim 13, wherein the therapeutically effective amount per day is 420 mg.

1 5. Claim 9 where the disease to be treated is surgical invasion

O PI Invasion prevention and / or Z or treatment method according to any one of paragraphs 1 to 14

16. The scope of the claim wherein the disease to be treated is an invasion due to radiation exposure.