WO1995019789A1

WO1995019789A1 - Motor-function disorder preventive or remedy

Info

Publication number: WO1995019789A1
Application number: PCT/JP1995/000057
Authority: WO
Inventors: Kenji Okajima; Yuji Taoka
Original assignee: The Green Cross Corporation
Priority date: 1994-01-21
Filing date: 1995-01-20
Publication date: 1995-07-27

Abstract

A motor-function disorder preventive or remedy, tissue trouble preventive or remedy, spinal ischemia preventive or remedy, and spinal injury preventive or remedy, each containing a human-origin antithrombin III as the active ingredient. The antithrombin III has the activity of ameliorating mammalian tissue troubles and motor-function disorders, especially those caused by spinal ischemia or spinal injury, for example, paralysis.

Description

Technical field

The present invention is effective for human-derived antithrombin-1π (hereinafter simply referred to as AT-m)!とする機機機機機機機機機機機機機^^

Light

Background art

AT- is a type of glycoprotein belonging to a2 globulin, and its amount is 65,000-68,000, indicating that it has protease inhibitory activity in the blood coagulation system. It strongly inhibits the clotting activity of thrombin.

In addition, it has a blocking effect on other coagulation factors, such as activated factor X and activated IX factor, as well as blocking against thrombin. In addition, it has been reported that there is a blocking effect on plasmin and trypsin.

These disruptions are known to progress more rapidly with ^ T of heparin.

AT-I [[, which has such pharmacological action, has been used for the purpose of correcting coagulation abnormalities 7¾1, and specifically for the treatment of generalized 'tt intubation coagulation ^! (D I C).

By the way, in parathoracic aneurysms, paraplegia caused by the spinal cord 報告 has been reported to occur on boats or '2.2 to 24%, and is attracting attention as a serious conclusion. Techniques such as temporary bypass have been reported as a method of protecting pharmacological aspects with dinsulin, steroids, etc., but the effect is not sufficient.

An object of the present invention is to provide a novel drug utilizing the unexplained action of AT-II. That is, it aims to use AT-II for a new medical use. Disclosure of the invention

Means for Solving the Problems The present inventors have conducted intensive studies to achieve the above object, and as a result, have found that the present invention has an effect of improving 改善 dysfunction and tissue disorder, and have achieved the present invention. That is, the present invention relates to a method for preventing agricultural dysfunction using AT-m:

And spinal injury prediction.

The AT-m used in the present invention is not particularly limited as long as it is derived from humans and is optionally purified to the extent possible. For example, AT-m from human whole blood, human serum, serum or It can be purified from serum or the like compressed from coagulated blood. The blood to be abolished is particularly preferable if it is negative for HBs¾H and anti-HIV antibody, and is less than twice as strong as GTP.

Starting materials for preparing ΑΤ—ΠΙ include, for example, fraction IV-1, fraction IV, supernatant I or supernatant Π + ΠΙ (Ε Ρ 5 8 4) Equalized.

Examples of the method for purifying AT- 、 include the methods disclosed in JP-A-48-35017 and JP-B-59-7693, and Kaihei 1-275600), anion exchanger treatment (Japanese Unexamined Patent Publication No. 2-47177), and the like.

In addition, AT-II is based on the cell culture method (for example, see Japanese Patent Application Laid-Open No. 57-500768), and the fetal culture (for example, see Japanese Patent Application Laid-Open No. 58-162,529). It may be prepared according to the publication. Also, a commercially available AT-III preparation (eg, trade name: Neuart Z Co., Ltd. Midori Cross, etc.) can be used.

AT-m, which is ¾ ^ of the present invention, has a protective action against spinal cord injury and spinal cord in mammals such as humans, dogs, dogs, dogs, mice, rats, etc. Ϋ¾ 改善 has the effect of improving dysfunction.

Here, 3 »dysfunction refers to a condition that causes abnormalities in taste function due to disorders of the central nervous system or peripheral nerves. For example, spinal cord disorders (eg, spinal cord injury, Specifically, for example, paraplegia, paralysis, hemiplegia, paraplegia and the like can be mentioned.

In addition, the thread job disorder refers to a failure or a functional disorder of each painter, for example, an obstacle of mi-woven. Therefore, AT-m is useful as an aza dysfunction disorder, preventive reversal of thread work disorder, 麵她 preventive cure U and circulatory wound preventive cure J, especially Mi function caused by 麵她 or spinal cord injury. Useful as prevention and Z or reversal of conditions involving at least one disorder or disorder. For example, AT-π has an improving effect on circulatory-induced threadwork disorder or S¾ function disorder (eg, paraplegia) that occurs as a complication in thoracic aneurysm surgery. AT-III also has the effect of improving complications associated with injuries, such as thrombosis and pulmonary thrombosis.

The preventive remedy ij of the present invention usually includes pharmacologically acceptable additives (eg, carriers, adjuvants, diluents, etc.), stabilizers and the like used in the adjuncts, as long as the object of the present invention is not violated. Alternatively, essential ingredients may be blended.

Examples of additives and stabilizers include sugars, for example, crafts such as glucose and fructose, Js such as sucrose, pulp li tang, maltose, sugar alcohols such as mannitol and sorbitol; citric acid, malic acid, tartaric acid, etc. Organic acids or salts thereof (for example, sodium salt, potassium salt, calcium salt); amino acids such as glycine, aspartic acid, and glutamic acid or salts thereof (for example, sodium salt); polyethylene glycol, polyp Surfactants such as ropylene glycol, its derivatives (eg, pull mouth nick), and polyoxyethylene sonolebitan fatty acid esters (eg, Tween); heparin, albumin and the like.

The preparation of the present invention is prepared by mixing AT AT with the above, and prepared in the form of powder, liquid, granules, tablets, capsules, syrups, tablets, etc., and administered orally or parenterally Is done. Preferably, it is a mode of intravenous administration.

This product is prepared by preparing AT-m as a 特に ¾ ^ product with a pharmacologically acceptable excipient, and dissolving it at the time of use into a fiber formulation or a liquid formulation. preferable. If necessary, such a preparation can be prepared as about 1 to 100 AT-DI single fe / ml sickle by using distilled water or sterile purified water, if necessary, more preferably a physiologically isotonic salt concentration and physiological Is adjusted to a preferable pH value (pH 6 to 8).

The dose may be selected according to the condition, body weight, animal species, etc. For human β¾Λ, AT-HI is usually 1 to 100 single fe kg body weight Z days, preferably 10 to 100 days. ~ 500 units kg body weight Z day is administered once or several times a day. For example, it is preferable to administer at a dose of 10 to 100 single feg kg body weight Z day for intravenous administration.

In the present specification, the titer of AT-IE corresponds to the amount of AT-m contained in 1 ml of normal human plasma. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, experimental examples and bell examples will be given in order to explain the present invention in detail, but the present invention is not limited thereto.

Experimental example 1

(1) Real method

Spinal cord R was obtained by opening the chest of a rat (body weight 300-400 g) and ligating the thoracic λ 胸 pulse (5th thoracic vertebral level) for 30 minutes. AT-1Π was given intravenously 30 minutes before ligation (250 single fe / kg body weight) or 15 minutes after ligation (500 single & / kg body weight), hind limbs »function, neutrophil accumulation in spinal cord and tissue Using the amount (measured as MP0 activity 12 hours after ligation) as a target, the preventive and therapeutic effects described in // function disorders and the pre-treatment effects in thread fiber disorders were examined. The impairment was determined 24 hours after the ligation procedure, and the degree of impairment was objectively evaluated using the following Talob (Tar 10 V) evaluation method.

Yell

0: voluntary JIl; none (so-called paraplegia), 1: joint movement can be recognized,

2: Inability to recognize any joint movement, 3: Walking and

4: Can be done freely.

(2) Results

Table 1 shows the dysfunction score and the percentage of paraplegia. AT-ΙΠ Insect (pre- and post-treatment) markedly improved. table 1

Number of rats Fiber conversion function C Complete paraplegia ratio Average (%)

(The score of each rattle '),

No treatment group 5 0.6 40%

(Ligation procedure) (0 0 1 1 1)

ΑΤ--Π pre-treatment group 4 1.5 0%

(1 1 2 2)

AT—user group 4 1. 5 25%

(0 1 2 3) In addition, the activity of myelin peroxidase (hereinafter referred to as M P0) in the tissue (β slide) was simulated with only thoracotomy (no ligation treatment, no AT-m administration group)

(Number of rats = 4) 13.8 ± 11.8 units Zg tissue, AT— Π non-administration group (ligation treatment) 92.4 ± 13.1 single fe / g tissue, AT— In the ΠΙΜ¾ group (number of rats = 4), it was 41.8 ± 11.2 single ii g thread. MPO is an index of the degree of local disability. In each rat group, the MP0 level in the damaged spine was measured 12 hours after spinal cord injury by an absorbance measurement method (wavelength: 460 nm). By administering AT-II, MPO was reduced to about 1Z2 and the degree of disability was reduced.

Experimental example 2

(1) mm

AT-m, heparin, ΑΤ- ΠΙ + heparin, dansyl-GlyG1yArg-chloromethylketone-treated FXa (hereinafter referred to as DEGR-Xa), etc. at the rate of 3 mg / kg body weight, respectively. Rats were intravenously administered, and 30 minutes later, the twelfth thoracic vertebra of each rat was compressed with 20 g of Wei to cause Mf injury.

無 -ΙΠ spinal cord injury-results were compared between non-administered and each-administered ϋ wound rats by comparing their functions, vascular permeability in Wf fiM, etc. The determination of the fiber failure was made 24 hours after the pressure was applied, and the failure was objectively dragoned using the Tar 1 ov basket method similar to that in Experimental Example 1.

(2) Results and discussion The ratio of ¾ fiber function and ¾ ^ paraplegia is as shown in Table 2, and the squamous dysfunction was significantly reduced by the pre-administration of the A τ_m stage insect.

Table 2 Number of drugs administered Number of rats 碰 General function (dots) Paraplegic rate No administration 17 0. 82 ± 0.70 35.3%

AT-1 7 1.46 ± 0.74 15.4¾

Heparin 7 0.50 ± 0.50 50%

AT— ΠΙ + heparin 7 0.6 ± 0.49 40 ¾

D EGR-X a 7 0.42 ± 0.49 57.1% In addition, the outflow in one tissue was reduced by about 40% by pre-administration of AT-II. A The protective effect of Τ—ΙΠ was offset by the combined use of heparin. Since heparin and DEG-Xa had no effect, it may be due to effects other than blood anticoagulant effect ο

Experiment 3

(1) Practical method

AT-ΠΙ was administered after injury: the effect of ^, ie, the therapeutic effect of AT-III administration was examined. First, spinal wound rats were prepared according to the method of Experimental Example 2. The experiments were performed in the following four groups, and their effects were compared. „Non-administration group, (DAT-HI pre-administration group (AT-ΠΙ administered intravenously 30 minutes before spinal cord injury at a dose of 250 / kg / body weight), ③ AT- ΠΙ body administration Group (AT-IE dose 250 mg, intravenously administered 30 minutes after spinal cord injury at Z kg body weight), ④ AT—Wm. Administration group (AT_m dose 500 Intravenous administration was performed 30 minutes after the spinal cord injury was given with a kg body weight.) The effect was confirmed and iffi was performed according to the method of Experimental Example 1.

(2) Results and discussion

Ϋ¾ ϋ¾ function and the rate of paraplegia are as shown in Table 3, and ϋ¾ dysfunction became prominent after administration of ΑΤ— ΙΠ reticulata (post). Table 3

Experiment 4

MP 0 was measured and confirmed in the same manner as in Experimental Example 1 for the behavior of MP 0 at the site of the injury when A τ -m was administered after the injury. First, spinal injured rats were prepared according to the method of Experimental Example 2. The experiment was performed in the following three groups, and the effects were compared. ① Normal rats 1 ^ »administration group (spine cord injury group), ③ AT—— post-treatment group (群 —Π administration dose 250 alone {intravenous administration 30 minutes after spinal cord injury with i kg body weight) . Table 4 shows the results. With AT-HI administration, MPO was reduced to about 12 and the damage was reduced.

Table 4

Experimental example 5

The acute toxicity (LDBO) did not differ by β in mice and rats, and was 15,000 single fekg body weight Ui for both intravenous and oral administration and 20000 single & kg body weight for subcutaneous administration. In monkeys (males), intravenous administration was 6000 units Zkg body weight J で ±.

Difficult case 1

Suspension of 10 kg of the fraction W-1 paste obtained by the corn cold alcohol fractionation method was suspended in 100 liters of physiological saline, and barium sulfate was added to 5 (w / v)%. Then, the mixture was stirred at room temperature for 30 minutes, and prothrombin was removed by adsorbing it on barium sulfate. Adjust the supernatant to pH 6.5 and use polyethylene glycol. # Add 400 000 to 13 (w / v)% and remove the resulting precipitate by centrifugation.Add polyethylene glycol # 40 to 30 (w / v)% And the resulting precipitate was collected by centrifugation. This precipitate is cooled and sanitary decoration J about 2

After dissolving in 0 l, heparin sepharose prepared in advance at physic: ft ^ z was introduced into a fiber-coated column, and AT-m was adsorbed to the column. The column was washed with 0.4 M sodium chloride to remove impure proteins, and then 2.0 M sodium chloride was passed through a force ram to collect the eluted portion.

Sodium citrate was added to the aqueous solution of AT-ΠΙ to a concentration of 0.6 M, the pH was adjusted to 7.8, and heat treatment was performed at 60 ° C for 10 hours, followed by 0.9% chloride. The sodium was subjected to fiber dialysis overnight while dialysis was performed to obtain 1- (wZv) XlYmL of AT-III, and filtered or centrifuged as necessary to obtain a transparent liquid.

To 1% (wZv)% water of this w-w, add 2% (w / v)% mannitol and 0.2% (wZv)% sodium citrate and add a small amount so that the sodium chloride becomes 0.5%. After diluting with cold distilled water and adjusting the pH to 7.6 with 1N sodium hydroxide, the solution is sterilized and filtered through a sterilized Millipore filter, and 500 units are dispensed. did.

Difficult case 2

In one vial,

AT— ΙΠ 500 unit

Mannitol 20 O mg

Shiojiri sodium 5 O mg

Sodium citrate 52 mg

The resulting condensate was dissolved in 2 Oml of distilled water for measurement at the time of use to prepare an static preparation. Production HJ: Available

AT-ΠΙ has the effect of improving dysfunction and threadwork disorder in mammals. In particular, spinal cord ^ ilX is effective against thread fiber disorder or »function disorder due to spinal cord injury, such as paraplegia Has an improving effect. Therefore, ΑΤ— ΙΠ is useful as 3 «/ function impairment prevention and return ij, Itoshiori Chapter P harm prevention and prevention and spinal cord injury prevention» J C that is 57

Claims

The scope of the claims

1. Preventive treatment of a disease state including at least one of a squamous dysfunction or a tissue disorder containing 1 m of human-derived antithrombin as a component.

2. The preventive transliteration according to claim 1, wherein the condition is attributable to a taste.

3. The preventive transliteration according to claim 1, wherein the condition is caused by spinal cord injury.

4. The preventive translation of a translational dysfunction according to any one of claims 1 to 3, wherein the condition is a fiber dysfunction.

5. The preventive transduction of a fiber disorder according to any one of claims 1 to 3, wherein the pathological condition is a silkworm disorder.

6. Spinal cord l preventive treatment with human-derived antithrombin 11 ビン as ¾ ^^.

7. Prevention of spinal cord injury with 1 m of human-derived antithrombin ^.