JPH08782B2 - Anti-inflammatory agent - Google Patents

Description

TECHNICAL FIELD The present invention relates to an anti-inflammatory agent. More specifically, it relates to an anti-inflammatory agent comprising human-derived antithrombin-III (hereinafter simply referred to as antithrombin-III) as an active ingredient.

[Prior art]

Antithrombin-III is a kind of glycoprotein belonging to α ₂ globulin existing in plasma, and its molecular weight is 65,000.
It has a protease inhibitory activity and strongly inhibits the thrombin coagulation activity.

Further, it has not only an inhibitory effect on thrombin but also an inhibitory effect on other coagulation factors such as activated factor X and activated factor IX. In addition, it has been reported that it has an inhibitory effect on plasmin and trypsin.

It is generally known that these inhibitory effects proceed more rapidly in the presence of heparin.

Antithrombin-III having such pharmacological action
Is used for the purpose of correcting hypercoagulability, specifically, diffuse angiopathy (DIC).

[Problems to be solved by the invention]

The present inventors have conducted various studies with the intention of using the antithrombin-III for other pharmaceutical applications.

Therefore, the present inventors have found that various types of inflammation are associated with an increase in the activity of proteolytic enzymes such as plasmin and trypsin.

Therefore, when research was repeated focusing on the antiproteolytic enzyme action of antithrombin-III, antithrombin-III
It was found that III has an anti-inflammatory effect.

An object of the present invention is to provide a drug that utilizes the above-mentioned action of antithrombin-III.

[Means for solving problems]

The present invention was completed based on the above new findings, and is an anti-inflammatory agent characterized by comprising human-derived antithrombin-III as an active ingredient.

The antithrombin-III used in the present invention is of human origin and is not particularly limited as long as it is purified to such an extent that it can be used as a medicine, and for example, human whole blood, plasma, serum or coagulation. It can be purified from the serum or the like that is squeezed from the collected blood.

As a starting material for the preparation of antithrombin-III, for example, fraction IV-1 obtained by the cold corn ethanol method is used.

Examples of the method for purifying antithrombin-III include the methods disclosed in JP-A-48-35017 and JP-B-59-7693.

The anti-inflammatory agent of the present invention is antithrombin-III alone,
The antithrombin-III is used in combination with heparin, the antithrombin-III / heparin complex and the like are used.

It should be noted that antithrombin-III and heparin originally have an affinity, and the antithrombin-III / heparin complex itself is known. The complex can be easily produced by, for example, mixing antithrombin-III and heparin in the same amount in the presence of a solvent.

The heparin may be any that has been purified to the extent that it can be used as a medicine, and it is sufficient to use the one currently clinically used (listed in the 11th revised pharmacopoeia).

The amount of heparin in the case of combined use of antithrombin-III and heparin is usually antithrombin-II.
40 to 160%, preferably 60 to 140%, especially 80
~ 120% (optimally both units).

Antithrombin-III, which is an active ingredient of the anti-inflammatory agent of the present invention, has an excellent anti-inflammatory effect on mammals such as humans, dogs, cows, horses, mice and rats. Specifically, for example, hepatitis, pancreatitis in the above animals,
It has an excellent anti-inflammatory action against nephritis, arthritis and the like.

The anti-inflammatory agent of the present invention is usually orally or parenterally administered and prepared as a formulation depending on each administration route, but usually as a freeze-dried product with antithrombin-III alone or with other additives. It is preferable to prepare a preparation which is dissolved before use. Such a preparation is diluted with distilled water for injection to prepare an antithrombin-III solution of about 1 to 10% W / V, and more preferably adjusted to a physiologically isotonic salt concentration and then intravenously administered. To be done.

The dose may be appropriately selected depending on symptoms, body weight, sex, animal species, etc.
~ 1000 units.

In the present specification, the titer of antithrombin-III is prepared in advance by measuring the extent of prolongation of coagulation time when fibrin is added to the reaction mixture of thrombin and the sample at 28 ° C. for 5 minutes. The titer was calculated from the calibration curve. The unit at this time was expressed as a unit of 100 / ml for convenience of the antithrombin activity of the supernatant liquid obtained by subjecting normal human plasma to heat treatment at 56 ° C. for 3 minutes and defibrinating.

[Examples and experimental examples]

Experimental Example 1 Anti-inflammatory effect on carrageenin edema in rats Carrageenin was administered to the hind paws of 150 to 200 g rats (5 animals per group), and at the same time, 100 units / k of antithrombin-III.
g or the same volume of physiological saline solution (control group) was administered through the tail vein, and the volume of edema was measured every hour.

As a result, in the control group, the volume immediately after carrageenin administration was 10%.
At 3 hours after carrageenin administration, 0% increased edema to 170%, but in the antithrombin-III administration group, it increased to 135%, confirming that antithrombin-III has a significant anti-inflammatory effect. It was

Further, 100 units of the same amount of antithrombin-III was previously mixed with 100 units of heparin to form an antithrombin-III / heparin complex, and then the complex was administered in the same manner as above, and antithrombin-III. Edema was observed in each group that received heparin in the same unit after
Only 8% and 125% increase, it was found that these groups also had an anti-inflammatory effect.

In addition, after the antithrombin-III alone group, the antithrombin-III / heparin complex pair administration group and the antithrombin-III administration heparin administration group,
The effect tended to be slightly strong in the group, but it was not significant.

Experimental Example 2 A freeze-dried preparation of antithrombin-III was dissolved in distilled water for injection, 40000 units / kg equivalent amount was administered to 5 mice through the tail vein, and observation was continued for 7 days. I couldn't do it. In addition, 5000 units / kg of the same solution in rabbits
After administration, it was observed for 24 hours, but no abnormality in body temperature was observed.

Example 1 10 kg of a paste of fraction IV-1 obtained by the cold alcohol fractionation method of corn was suspended in 100 physiological saline, barium sulfate was added thereto at 5 W / V%, and the mixture was stirred at room temperature for 30 minutes. , A small amount of prothrombin was adsorbed and removed by barium sulfate. Adjust the supernatant to pH 6.5, add polyethylene glycol # 4000 to 13 W / V%, remove the precipitate by centrifugation, and add polyethylene glycol # 4000 to 30 W / V%. In addition, the resulting precipitate was collected by centrifugation. Approximately 20 this cold saline solution
Was injected into a column of heparin sepharose prepared in advance with physiological saline to adsorb antithrombin-III on the column. This column was washed with a 0.4 M sodium chloride solution to remove impure proteins, and then a 2.0 M sodium chloride solution was applied to the column to collect the eluted portion.

To this aqueous solution of antithrombin-III, sodium citrate was added at a concentration of 0.6M, and the pH was adjusted to 7.8.
Heat treatment for 0 hours, then dialyzing against 0.9% sodium chloride solution overnight while concentrating to obtain a 1 W / V% aqueous solution of antithrombin-III, and filter or centrifuge if necessary. It was a clear liquid.

To this 1 W / V% aqueous solution of antithrombin-III, 2 W / V% of mannitol and 0.2 W / V% of sodium citrate were added, diluted with a small amount of cold distilled water so that the content of sodium chloride was 0.5%, and 1N hydroxylated. After the pH was adjusted to 7.6 with sodium, the bacteria was filtered through a sterilized Millipore filter, dispensed at 500 units, and lyophilized to give a dry preparation.

Example 2 In 1 vial, antithrombin-III 500 units Mannitol 200 mg Sodium chloride 50 mg Sodium citrate 52 mg A freeze-dried product was dissolved in 10 ml of distilled water at the time of use to prepare an intravenous preparation.

Example 3 Effect on Acute Deoxycholic Acid Pancreatitis (Dog) A mixed-breed dog (body: 8 dogs) weighing 10-18 kg was subjected to laparotomy under pentobarbital anesthesia, and 30% deoxycholic acid (DCA) 0.2 ml / from the large pancreatic duct. kg was injected to create acute hemorrhagic necrotizing pancreatitis. Immediately after DCA administration, the following samples were administered in the femoral vein at the following doses respectively, and the survival rate up to 24 hours,
Blood pancreatic secretory trypsin inhibitor (PSTI, dog PSTI,
The method of Kitahara et al. Using RIA) and the amount of fibrinogen (thrombin time method) were measured. Note that lactated Ringer's solution was continuously infused at 100 to 120 ml / kg for 6 hours after preparation of pancreatitis.

Sample / Dose (1) Antithrombin-III 100 units / ml 1 ml / kg (2) Antithrombin-III 100 units Heparin was mixed in advance with 100 units to form an antithrombin-III / heparin complex. Later, the complex was treated as antithrombin-III at 100 units / kg (complex administration group) (3) Antithrombin-III at 100 units / ml at 1 ml / kg and heparin at 100 units / ml at 1 ml / kg (combination) Administration group) (4) Physiological saline solution 2 ml / kg As described above, antithrombin-III, antithrombin-III / heparin complex, and antithrombin-III combined with heparin were found to be effective against pancreatitis. There was no difference between these 3 treatment groups.

Example 4 Effect on Masugi Hepatitis (Rabbit) The renal cortex of a Japanese white-blooded male white rabbit weighing 2.5 to 3.0 kg was taken out, and its homogenate (20% suspension) was used as a rabbit kidney antigen in 10 ml duck (2.5 to 3.0 kg). ) Was intraperitoneally administered and immunized. Antigen administration was performed 15 times at 7-day intervals to obtain anti-rabbit renal antiserum. This duck antiserum was intravenously administered to male white rabbits weighing 2.0 to 2.5 kg at 2 ml / kg to produce Masugi nephritis. 5 days after administration of antiserum, urinary protein (colorimetric method) was 300 mg / dl
The above animals (150 mg / dl or less in normal animals) were selected as nephritic animals (8 birds per group), and the sample was administered for 11 days or less consecutively from that day.

Specimen / Dose (1) Antithrombin-III 100 units / ml administered intravenously at 1 ml / kg (2) Antithrombin-III Antithrombin-III formed by mixing 100 units of heparin in advance
100 units of heparin complex as antithrombin-III
/ kg intravenous administration (3) Antithrombin-III 100 units / ml 1 ml / kg intravenous administration, then heparin 100 units / ml 1 ml / kg intravenous administration (4) Physiological saline 1 ml / kg intravenous administration antiserum The life and death of the rabbits were observed from 5 days to 16 days after the administration, and urinary protein and BUN (diacetyl monooxime method) were measured after 16 days.

As described above, antithrombin-III, antithrombin-III / heparin complex, and antithrombin-III and heparin combined use were found to be effective against nephritis (Masugi nephritis). There was no difference between these 3 treatment groups.

Example 5 0.1 ml of Freund's complete adjuvant (6.0 mg / ml liquid paraffin for injection) was injected subcutaneously into the footpad of the right hind leg of Sprague-Dawley female rats weighing 180 to 220 g (6 rats per group), and from that day The following samples were administered on consecutive days for 18 days.

Specimen / Dose (1) Antithrombin-III 100 units / ml intravenously administered at 1 ml / kg (2) Antithrombin-III 100 units plus 100 heparin
Antithrombin-III / heparin complex formed by mixing units as antithrombin-III is 100 units / kg
Intravenous administration (3) Antithrombin-III 100 units / ml 1 ml / kg intravenously followed by heparin 100 units / ml 1 ml / kg intravenously (4) Physiological saline 1 ml / kg intravenously.

The volume of the right and left hind limbs was measured every other day until 18 days after the adjuvant administration.

Swelling of the adjuvant-treated foot (right hind leg), ie (first
(Secondary inflammation) increased linearly up to 6 days after treatment, and stopped increasing thereafter. However, from day 12 on, the inflammation increased again.
Swelling (secondary inflammation) of the untreated foot (left hind limb) appeared from the 12th day and increased linearly thereafter. The effect was determined from the swelling of the treated paw after 12 days and the tumor of the untreated paw after 18 days (Table 3). As a result, each antithrombin-III administration suppressed the primary inflammation of the treated foot to about 77 to 84% of the physiological saline administration, and the secondary inflammation of the untreated foot to about 82 to 85%. It was

As described above, it was found that antithrombin-III, antithrombin-III / heparin complex, and antithrombin-III and heparin combined use were effective for the adjuvant arthritis by the preliminary administration method.

 There was no difference between these 3 treatment groups.

Claims (3)

[Claims] 1. An anti-inflammatory agent comprising human-derived antithrombin-III as an active ingredient. 2. The anti-inflammatory agent according to claim (1), wherein the human-derived antithrombin-III is a complex of human-derived antithrombin-III and heparin. 3. The anti-inflammatory agent according to claim 1, which further comprises heparin.