JPH08169845A - Novel use of human-derived antithrombin-III - Google Patents

Abstract

PURPOSE: To obtain a preventive/therapeutic agent for motor dysfunction, tissue disorders or spinal disorders, containing, as active ingredient, human-derived antithrombin-III. CONSTITUTION: This preventive/therapeutic agent contains, as active ingredient, human-derived antithrombin-III (abbreviated as AT-III). The AT-III can be purified, for example, from the serum obtained by squeezing human whole blood, plasma, serum or coagulated blood, and the blood to be used therefor is esp. preferably negative to HBs antigens and anti-HIV antibodies and not more than a double level of the normal level GTP. The AT-III has improving effects on spinal disorders, tissue disorders due to spinal ischemia, etc., and motor dysfunction, also has spinal protective action and improving effect on complications involved in spinal disorders. The daily dose of this agent in terms of the AT-III is normally 1-1000 units/kg body weight devided in one to several portions for preventing/treating motor dysfunction, tissue disorders, spinal disorders or spinal ischemia. For intravenous administration, 10-100 units/kg body weight a day is preferable. As for the titer of the AT-III, one unit is equivalent to the AT-III level in 1ml of the plasma.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel drug utilizing the novel action of human-derived antithrombin-III (hereinafter simply referred to as AT-III). That is, a prophylactic / therapeutic agent for motor function disorders containing AT-III as an active ingredient, a prophylactic / therapeutic agent for tissue disorders containing AT-III as an active ingredient, a prophylactic / therapeutic agent for spinal cord disorders containing AT-III as an active ingredient, and AT-III are effective. Prophylactic / therapeutic agent for pathological conditions derived from myelopathy and AT
-III is a preventive and therapeutic agent for spinal cord ischemia containing III as an active ingredient.

[0002]

2. Description of the Related Art AT-III
Is a kind of glycoprotein belonging to α ₂ globulin existing in plasma, has a molecular weight of 65,000 to 68,000, has a protease inhibitory activity, and strongly inhibits the coagulation activity of thrombin. In addition to the inhibitory effect on thrombin, other coagulation factors such as activated factor X,
It also has an inhibitory effect on 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. AT-III, which has such a pharmacological action, corrects hypercoagulability, specifically, generalized angiopathy (DI).
It is used for the purpose of treating C).

By the way, paraplegia caused by spinal cord ischemia in thoracic aortic aneurysm surgery has been reported to occur at a frequency of 2.2 to 24%, and has been noted as a serious complication. As measures against this, devising surgical techniques such as temporary bypassing and pharmacological spinal cord protection with insulin and steroids have been reported, but the effect is not sufficient.

The object of the present invention is to provide a novel drug utilizing the heretofore unknown action of AT-III. That is, it is an object of the present invention to provide a new pharmaceutical use of AT-III.

[0005]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventors have found that AT-III has an effect of improving motor dysfunction, tissue damage, etc.
The present invention has been completed.

That is, the present invention relates to a prophylactic / therapeutic agent for motor dysfunction, a prophylactic / therapeutic agent for tissue disorders, and a prophylactic / therapeutic agent for spinal cord disorders containing AT-III as an active ingredient.

The AT-III used in the present invention is of human origin and is not particularly limited as long as it is purified to the extent that it can be used as a medicine. For example, human whole blood, plasma or serum. Alternatively, it can be purified from serum or the like that is compressed from coagulated blood. The blood used is especially negative for HBs antigen and anti-HIV antibody,
It is preferable that P is not more than twice the normal value.

As a starting material for preparing AT-III, for example, fraction IV-1 in the Cohn fractionation method of plasma,
Fraction IV, supernatant I or supernatant II + III (EP Publication 5510
84) and the like are used. As a method for purifying AT-III,
For example, JP-A-48-35017, JP-B-59-
The method disclosed in Japanese Patent No. 7693, hydrophobic chromatography (JP-A-1-275600), anion exchanger treatment (JP-A-2-4717) and the like are exemplified. Further, AT-III is a cell culture method (see, for example, JP-A-57-500768), a genetic engineering method (eg, JP-A-58-1625).
(See Japanese Patent Publication No. 29) and the like. Further, a commercially available AT-III preparation (for example, trade name: Neuart, manufactured by Midori Cross Co., Ltd.) can also be used.

[0009] The active ingredient of the present invention, AT-III, has the action and effect of preventive treatment of motor dysfunction and preventive treatment of tissue disorder in mammals such as humans, dogs, cows, horses, mice and rats. Here, motor dysfunction refers to a pathological condition that causes abnormal motor function due to disorders such as central nerves and peripheral nerves, and examples thereof include those caused by spinal cord disorders (for example, spinal cord injury, spinal cord ischemia, and spinal cord surgery). Specific examples include motor paralysis, complete paralysis, hemiplegia, and paraplegia. The tissue disorder means any disorder including injury and dysfunction of various organs, and examples thereof include disorders of spinal tissue. In particular, AT-III has a therapeutic and preventive effect on tissue damage or motor dysfunction (for example, paraplegia) and the like caused by spinal cord ischemia, which occurs as a complication in thoracic aortic aneurysm surgery.

Therefore, AT-III is useful as a preventive / therapeutic agent for motor dysfunction, a preventive / therapeutic agent for tissue disorders, a preventive / therapeutic agent for spinal cord ischemia and a preventive / therapeutic agent for spinal cord ischemia, and particularly spinal cord disorders (for example, spinal cord injury, spinal cord dysfunction). It is useful as a preventive and / or therapeutic agent for a pathological condition (for example, a pathological condition including at least one of motor dysfunction or tissue disorder) caused by blood.

In the preventive / therapeutic agent of the present invention, it is preferable to use AT-III alone as an active ingredient.

The preventive / therapeutic agent of the present invention is a pharmacologically acceptable additive (eg, carrier, excipient, diluent, etc.), stabilizer, stabilizer or the like which is usually used in pharmaceuticals unless it is against the object of the present invention. You may mix | blend the component required pharmaceutically. Examples of additives / stabilizers include sugars such as monosaccharides such as glucose and fructose, disaccharides such as sucrose, lactose and maltose, sugar alcohols such as mannitol and sorbitol; organic acids such as citric acid, malic acid and tartaric acid. Or a salt thereof (eg sodium salt, potassium salt, calcium salt etc.); an amino acid such as glycine, aspartic acid, glutamic acid or a salt thereof (eg sodium salt etc.); polyethylene glycol, polyoxyethylene-polyoxypropylene copolymer , Surfactants such as polyoxyethylene sorbitan fatty acid ester; heparin, albumin and the like.

The preparation of the present invention is prepared in the form of powder, liquid, granule, tablet, capsule, syrup, injection or the like by appropriately mixing AT-III and the above-mentioned components, and is orally or parenterally. Will be given to you. The preferred mode is intravenous administration. It is preferable that the present preparation be a preparation in which AT-III is used as a freeze-dried product together with a pharmacologically acceptable additive and dissolved at the time of use, or a liquid preparation. Such a formulation may be prepared by using distilled water for injection or sterilized purified water at the time of use at about 1 to 100 AT-II.
More preferably, as I unit / ml solution, physiologically isotonic salt concentration and physiologically preferable pH value (pH 6 to 8)
Adjusted to.

The dosage of the preparation of the present invention depends on symptoms, body weight, sex,
It may be appropriately selected depending on the animal species and the like, and in general, for human adults, it is usually 1 in both prevention and treatment of motor dysfunction, tissue damage, spinal cord injury, and spinal cord ischemia as AT-III. -1000 units / kg body weight / day, preferably 10-500 units / kg body weight / day, is administered in 1 to several divided doses per day. For example, 10 to 10 for intravenous administration
It is preferred to administer a dose of 0 units / kg body weight / day. In this specification, the titer of AT-III corresponds to the amount of AT-III contained in 1 ml of normal human plasma.

[0015]

[Experimental Examples / Examples] Experimental examples and examples will be given below to explain the present invention in detail, but the present invention is not limited thereto.

Experimental Example 1 (1) Experimental Method Antithrombin-III (hereinafter referred to as AT-III) was used as a test drug.
), Heparin, AT-III + heparin, Dansyl
-GlyGlyArg-Chlormethyket
One treated FXa (hereinafter referred to as DEGR-Xa) or the like was intravenously administered to the rats at a rate of 3 mg / kg body weight, and 30 minutes after that, the 12th thoracic vertebra of each rat was pressed with a weight of 20 g to prevent spinal cord injury. Gave. For each spinal cord injury rat to which various test drugs were administered, the hindlimb motor function, blood loss in spinal cord tissue, vascular permeability in spinal cord tissue, etc. were compared and examined to examine the protective effect of AT-III on spinal cord injury. The movement disorder was determined 24 hours after compression of the spinal cord, and the degree of the disorder was objectively evaluated using the following Tarlov evaluation method. -Evaluation- 0: No voluntary movement (so-called complete paraplegia), 1: Movement of joints can be recognized, 2: Movement of joints is good but unable to stand, 3: Standing and walking possible, 4: Jumping and sprinting possible .

(2) Results and Discussion Table 1 shows the proportions of hindlimb motor function and complete paraplegia. From this result, it was found that pre-administration of AT-III alone has a protective effect against spinal cord injury and significantly reduces motor dysfunction.

[0018]

[Table 1]

Further, the amount of bleeding in the spinal cord tissue was also reduced by about 40% by preadministration of AT-III alone. Such a spinal cord protective action of AT-III was offset by the combined use of heparin, and since heparin and DEGR-Xa had no effect, it is considered that the action mechanism is other than the blood anticoagulant action.

Experimental Example 2 (1) Experimental method The effect of administering AT-III after spinal cord injury, that is, the therapeutic effect of AT-III administration was examined. First,
According to the method of Experimental Example 1, a spinal cord injury rat was prepared. The experiment was conducted in the following 4 groups, and the effects were compared. No administration of study drug, pre-administration group of AT-III (Spinal cord injury 3
AT-III was intravenously administered at a dose of 250 units / kg body weight 0 minutes before, AT-III low administration group (at 30 minutes after the spinal cord injury, AT-III was administered at a dose of 2).
Intravenously administered at a rate of 50 units / kg body weight),
AT-III high dose group (30 minutes after the spinal cord injury,
T-III was intravenously administered at a dose of 500 units / kg body weight). In addition, the confirmation and evaluation of the effect were performed in Experimental Example 1
It was performed according to the method of.

(2) Results and Discussion The proportions of hindlimb motor function and complete paraplegia are shown in Table 2, and the motor dysfunction was significantly reduced by post-administration of AT-III alone.

[0022]

[Table 2]

Experimental Example 3 The behavior of myeloperoxidase (hereinafter referred to as MPO) in a damaged area when AT-III was administered after spinal cord injury was confirmed. MPO is an index of local degree of disability. First, a spinal cord injury rat was prepared according to the method of Experimental Example 1. The experiment was performed in the following three groups, and the effects were compared. Normal rat group, AT-III non-administered group (spinal cord injury group), AT-III post-administration group (AT-III was intravenously administered at a dose of 250 units / kg body weight 30 minutes after the spinal cord injury was given. thing). MPO in injured spinal cord tissue 24 hours after spinal cord injury for each group of rats
The amount was measured by an absorbance measuring method (wavelength 460 nm).
The results are shown in Table 3. By administration of AT-III, it was found that MPO was reduced to about 1/2 and the degree of injury of spinal cord tissue was reduced.

[0024]

[Table 3]

Experimental Example 4 (1) Experimental method Spinal cord ischemia was performed by opening the left thorax of a rat (body weight: 300 to 400 g) and ligating the thoracic aorta (fifth thoracic vertebra level) for 30 minutes. AT-III was intravenously administered to each rat 30 minutes before the spinal cord ischemia treatment (250 units / kg body weight) or 15 minutes after the spinal cord ischemia treatment (500 units / kg body weight), and hindlimb motor function, Using the amount of neutrophil accumulation in spinal cord tissues (measured as MPO activity 12 hours after ischemia) as an index, the protective effect of AT-III on motor dysfunction and the preventive and therapeutic effect on tissue damage were examined. The determination and evaluation of the movement disorder were performed 24 hours after spinal cord ischemia by the Tarlov evaluation method according to Experimental Example 1. Also,
MPO activity was measured by the absorbance measurement method (wavelength 460 nm).

(2) Results Table 4 shows the degree (score) of hindlimb motor dysfunction and the rate of complete paraplegia. In the AT-III alone administration group (before and after administration), motor dysfunction and paraplegia were significantly improved.

[0027]

[Table 4]

MPO activity in the spinal cord tissue (spinal cord at the lumbar spinal cord) was measured by a thoracotomy-only simulated surgery group (no ligation procedure, AT-
III non-administration group, n = 4) 13.8 ± 11.8 units / g
Tissue, 9 in AT-III non-administration group (ligation treatment, n = 4)
2.4 ± 13.1 units / g tissue, 41.6 ± 11.2 units / g tissue in the AT-III post-administration group (n = 4). MPO is an index of local degree of disability.
It was revealed that MPO was reduced to 1/2 or less by administration of T-III, and the degree of injury was reduced.

Experimental Example 5 There was no difference in acute toxicity (LD ₅₀ ) between male and female mice and rats, and AT-III 15000 was administered both intravenously and orally.
Unit / kg body weight or more, subcutaneously administered AT-III2000
It was 0 unit / kg body weight or more. In monkey (male), AT-III was 6000 units / kg body weight or more after intravenous administration.

Example 1 10 kg of the paste of fraction IV-1 obtained by the cold alcohol fractionation method of corn was suspended in 100 liters of physiological saline,
Barium sulfate was added at 5 (w / v)% and the mixture was stirred at room temperature for 30 minutes to remove a small amount of prothrombin by adsorbing it onto barium sulfate. PH of this supernatant
Adjusted to 6.5, polyethylene glycol # 4000 was added to 13 (w / v)%, the precipitate formed was removed by centrifugation, and polyethylene glycol # 400 was added.
0 was added to 30 (w / v)%, and the resulting precipitate was collected by centrifugation. This precipitate was dissolved in about 20 liters of cold physiological saline and injected into a column filled with heparin sepharose prepared in advance with physiological saline to obtain AT.
-III was adsorbed on the column. After washing this column with 0.4 M sodium chloride solution to remove impure proteins,
A 2.0 M sodium chloride solution was passed through the column to collect the eluted portion. Sodium citrate was added to this AT-III aqueous solution at a concentration of 0.6 M to adjust the pH to 7.8, and then heat treatment was carried out at 60 ° C. for 10 hours, and then to 0.9% sodium chloride solution overnight. The solution was concentrated while performing dialysis to obtain a 1 (w / v)% aqueous solution of AT-III, and filtered or centrifuged as necessary to obtain a transparent liquid.
2 (w / v)% mannitol and 0.2 (w / v) sodium citrate were added to a 1 (w / v)% aqueous solution of AT-III.
%, And dilute with a small amount of cold distilled water so that the sodium chloride content becomes 0.5%, and add 1N sodium hydroxide to adjust the pH.
After adjusting to 7.6, sterilized Millipore filter was used for sterilization filtration, and 500 units were dispensed and freeze-dried to obtain a dry preparation.

Example 2 A lyophilized product consisting of AT-III 500 units, mannitol 200 mg, sodium chloride 50 mg, sodium citrate 52 mg in one vial was dissolved in 20 ml of distilled water for injection at the time of use to prepare a preparation for intravenous injection.

[0032]

INDUSTRIAL APPLICABILITY AT-III has an action of improving locomotor dysfunction and tissue dysfunction in mammals, and is particularly effective for tissue dysfunction or locomotor dysfunction caused by spinal cord injury or spinal cord ischemia, such as paraplegia. Has an improving effect. Therefore, AT-III
Is useful as a preventive / therapeutic agent for motor dysfunction, a preventive / therapeutic agent for tissue disorders, a preventive / therapeutic agent for spinal cord injury and a preventive / therapeutic agent for spinal cord ischemia. Further, AT-III has an effect of improving spinal cord protection and motor dysfunction due to spinal cord injury (eg, spinal cord injury, spinal cord ischemia, etc.) or paraplegia, and complications associated with spinal cord injury (eg, venous thrombosis). , Pulmonary thrombosis, etc.). Therefore, AT-III is useful as a prophylactic / therapeutic agent for myelopathy and a prophylactic / therapeutic agent for pathological conditions (motor dysfunction, tissue disorder, etc.) derived from myelopathy.

Claims (7)

[Claims] 1. A preventive / therapeutic agent for motor dysfunction comprising human antithrombin-III as an active ingredient. 2. A preventive and / or therapeutic agent for tissue disorders, which comprises human-derived antithrombin-III as an active ingredient. 3. A prophylactic / therapeutic agent for spinal cord disorders containing human-derived antithrombin-III as an active ingredient. 4. A prophylactic / therapeutic agent for a pathological condition derived from myelopathy, which comprises human-derived antithrombin-III as an active ingredient. 5. The prophylactic / therapeutic agent according to claim 3, wherein the spinal cord injury is either spinal cord injury or spinal cord ischemia. 6. The preventive / therapeutic agent for the pathological condition derived from the spinal cord disorder according to claim 4 or 5, wherein the pathological condition derived from the spinal cord disorder is either motor dysfunction or tissue disorder. 7. A preventive and therapeutic agent for spinal cord ischemia containing human-derived antithrombin-III as an active ingredient.