JPH0813751B2 - Brain function improving agent containing human urinary kininogenase as an active ingredient - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a brain function improving agent, particularly a brain function improving agent containing human urinary kininogenase as an active ingredient.

[0002]

2. Description of the Related Art In recent years, the average life span has tended to increase, and the incidence of Alzheimer's dementia has increased accordingly. This type of dementia is said to be caused by atrophy of cerebral nerve cells, but since its cause is unknown, no preventive or therapeutic method has been found. Furthermore, recently, a so-called modern illness, which is a mental illness that causes memory impairment, aphasia, apraxia, etc. due to stress and overwork due to a marked change in social environment, is also attracting attention, and cerebral infarction and cerebral hemorrhage. Brain dysfunction caused by is a problem. With respect to these cerebral dysfunctions, until now, there are almost no drugs whose efficacy has been sufficiently confirmed, and cerebral vasodilators such as cerebral vasodilators and avan are used depending on the symptoms. However, the development of effective drugs and early establishment of therapeutic methods are desired.

In view of the above situation, the inventors of the present invention have conventionally searched for substances having a brain function improving action, and as a result, the myo-inositol compound phytic acid and salts thereof have a function-retarding effect. It has been found to be effective against diseases, especially so-called "senile diseases" (Japanese Patent Laid-Open No. 1-3
05032 bulletin). In addition, it has been reported that a pyrazolopyridine compound also has a memory disorder-improving action (JP-A-2-
131424).

[0004] On the other hand, kininogenase brain function improving agent according to the present invention is to be used as an active ingredient is a bet Ripushi <br/> down-like enzyme international enzyme nomenclature committee has given the name of "Kininogenin", plasma It is a substance that exists in the inside and various tissues. Kininogenase in plasma has a molecular weight of about 80,000.
It is said that bradykinin is released, and kininogenase in tissues has a molecular weight of about 30,000 and releases kallidin. As described above, the kininogenase that is mainly biosynthesized in the liver and the tissue-derived kininogenase, which is present in high concentrations in the pancreas, submandibular gland and kidney, have different properties. have been referred to as the same thing in immunochemical those and those found in urine found in, not in the clear whether identical. Tissue-derived kininogenase decomposes kininogen in blood to produce quinine,
Through this, the arterioles are expanded to increase blood flow, and a blood pressure lowering action is expressed. Therefore, from the viewpoint of obtaining raw materials, those derived from porcine pancreas have hypertension, Meniere's syndrome and obstruction.
Of peripheral circulatory disorders in congenital thromboangiitis (Burger disease)
Improvement, concomitant symptoms in cerebral circulation disorder, and menopausal disorder
Has been used as an improvement purposes in pharmaceutical related circulatory disorders beauty chorioretinopathy, it is administered primarily orally in terms of antigenicity because it is a heterologous protein. However sucking
There is a problem with the profitability, and there is a question about the effectiveness. Human urine-derived quininogenase is considered to have low antigenicity unlike that derived from porcine pancreas, and thus has attracted attention in the past and various extraction and separation methods have been proposed (Japanese Patent Publication No. 46-
19067, Japanese Examined Patent Publication Nos. 1-41310 and 1-47997). However, it is unclear whether human urinary kininogenase itself is a single substance or not, and the standard has not been unified, and thus it has not been put to practical use as a medicinal product until now. Incidentally, no report has been made so far that kininogenase is effective as a brain function improving agent.

[0005]

In view of the above-mentioned conventional techniques, the problem to be solved by the present invention is to provide a novel agent for improving brain function, which enables prevention and treatment of various brain function disorders. There's something to do.

[0006]

Means and Actions for Solving the Problems The present inventors have
As described above, as a result of continuing diligently searching for a substance having a brain function improving action, the above-mentioned kininogenase, in particular, human urine-derived kininogenase has a strong cerebral dysfunction improving action and is safe to use. It was found to be expensive, and the present invention was completed.

The brain function-improving agent according to the present invention is extracted and purified from human urine, and has a major band of 41000 and a minor band of 31 in SDS-polyacrylamide gel electrophoresis.
It has a molecular weight in the range of 47,000-54000 according to the gel filtration method and has an isoelectric point of the stock solution of pI 3.92, 4.08 and 4.23 as the active ingredient.

Advantageously, the brain function-improving agent according to the present invention contains the above-mentioned human urinary kininogenase in an amount of about 0.15 PNA units per ml and 0.1-1% by weight of citric acid or a salt thereof.

The PNA unit showing the activity relating to human urinary kininogenase, which is the active ingredient of the brain function-improving agent according to the present invention, comprises HD-Val-Leu-Arg-p-nitroanilide as a substrate,
The above-mentioned human urinary kininogenase is allowed to act on this, and the liberated p-nitroanilide (PNA) is colorimetrically determined by measuring the absorbance at a wavelength of 405 nm, and at 37 ± 0.1 ° C for 1 minute.
The unit is 1 μmol of PNA produced. In the brain function-improving agent according to the present invention, citric acid or a salt thereof, for example, coexisting with sodium salt or potassium salt, quininogenase is not stable against heat or light,
The compounds used in the present invention are no exception, and the activity is decreased after 6 months when stored at room temperature.
This is because when light irradiation with an illuminance of 500 lux or more is observed, the activity is reduced in a relatively short time. Therefore,
In other words, when stored at low temperature in the dark, for example, in a brown ampoule (about -10 ° C), coexistence of citric acid or its salt as a stabilizer is not a requirement.

[0010]

[Production Examples, etc.] Next, the present invention will be described in more detail and specifically with reference to Production Examples, Formulation Examples, Activity Test Examples and Pharmacological Test Examples of the brain function improving agent according to the present invention. The brain function-improving agent according to the present invention is mainly intended as an intravenous injection from the viewpoint of suppression of decomposition, and further improvement of absorbability and bioavailability. Therefore, in the following, description will be made in connection with this.
It

Production Example 1 (stock solution) To 10 liters of healthy adult male urine, hydrochloric acid was added to adjust the pH to 5
After that, 100 g of chitosan was added and the mixture was stirred for 1 hour to adsorb quininogenase in urine to chitosan. After removing the adsorbent by filtration and washing it thoroughly with water, 0.2M
The extract was obtained by eluting quininogenase with 500 ml of Tris-HCl buffer (pH 9.0) and filtering. The extract was dialyzed with 0.1 M sodium hydrogen carbonate buffer (pH 9.0) containing 0.5 M sodium chloride. The obtained dialysate is aprotin-sepharose column
A urinary kininogenase stock solution (45 PNA units) was obtained by treating with (trypsin inhibitor) to adsorb quininogenase and then eluting with 0.1M phosphate buffer (pH 3.5) containing 0.5M sodium chloride.

The physicochemical properties of this stock solution were as follows: (1) Properties As a colorless to pale yellow clear liquid, it is odorless, has a slight salty taste, and has a pH of about 7 .

(2) Molecular weight (A) SDS-polyacrylamide gel electrophoresis a) Laemmli method A major band was found at a molecular weight of 41000, and a minor band was found at a position of 31000. Both bands were active. . (B) Gel filtration method a) Gel filtration method using Sephacryl S-200 A single peak was observed at a molecular weight of about 50,000, which coincided with the activity peak. b) Gel filtration method using Sephadex G-100 A single peak was observed at a molecular weight of about 47,000, which coincided with the activity peak. c) Using TSK-GEL (manufactured by Toyo Soda Co., Ltd.) at the position of standard molecular weight 5100-5500 (average: 5300 ± 2200) specified by high performance liquid chromatography.
Peak was observed, consistent with the peak of activity.

(3) Amino acid composition Considering the above-mentioned measured molecular weight values, the molecular weight is determined to be 50000.
Assuming that, the number of amino acid residues constituting the obtained kininogenase was 375, and its composition was as shown in Table 1 below. As is clear from this amino acid composition, the amino acids that make up this kininogenase are mostly basic and acidic. According to the Edman degradation method, the N-terminal amino acid sequence is Ile-Val-Gly,
This is identical to the primary structure of the N-terminal region that has already been revealed for human urinary kininogenase as well as the primary structure resulting from cDNA analysis of human kidney-derived kininogenase.

Table 1 A: Number of moles of amino acid in 1 mole of kininogenase
(Measured value), B: A value obtained by converting A into an integer, *: Half-cystine, nd: not detected

(4) Sugar composition The above quininogenase is an acidic glycoprotein substance, and its sugar composition is as shown in Table 2 below. It was confirmed that the N-glycoside type sugar chain and the 0-glycoside type sugar chain were bound.

Table 2

[0018] (5) treatment as measured by isoelectric point carriers am holin containing蔗sugar concentration gradient method, the isoelectric point of kininogenase stock pI 3.92,4.08
And 4.23.

[0019] (6) specific activity of standard activity of the kininogenase stock solution 1ml per 1.5 PNA units or more
Above (measured value is 2.4 PNA units or more), 1 mg protein
4.0 PNA units than the Examples of the per (measured value 5PNA Unit
It was ) .

[0020] (7) kininogenase active inhibitor standards above by aprotinin, its activity on more than 95% (measured value is 98% or more) is impaired inhibitory.

(8) Immunological properties When the immunological properties of the quininogenase stock solution were examined by the Ouchterlony method, a clear settling line was produced with the rabbit anti-human urinary quininogenase serum, but with the rabbit anti-porcine pancreatic kininogenase serum the settling line was obtained. It turned out that no.

(9) Enzymatic properties (A) Using natural kininogen as a substrate The effect of the above kininogenase on the quinine releasing ability was examined. First, when the effect of pH was investigated, free activity was observed in the range of pH 5-11 when human low molecular kininogen was used as a substrate and when dog low molecular kininogen was used as a substrate, and the maximum free activity was around pH 8.0. Met. On the other hand, when human macromolecule kininogen is used as a substrate, pH
Free activity was observed in the range of 5-12, and the maximum free activity was
The pH was around 8.5. Next, using various concentrations of canine low molecular weight kininogen, human low molecular weight kininogen and human high molecular weight kininogen as substrates, the kinin releasing ability of the above quinidinogenase was investigated, and the results of determining the enzyme kinetic constants of quinidinogenase are shown in Table 3 below. It was as shown in.

Table 3 Human L MWK: human low partial Koki Ninoge emissions (molecular weight: about 6
7000), human H MWK: human high molecular Kininoge emissions (molecular weight: about 12
0000), dog LMWK: canine low molecular weight kininogen (molecular weight: about 6
7000)

(B) When using a low molecular weight synthetic substrate The degradation activity of the above kininogenase against a low molecular weight synthetic substrate was examined in the same manner as in the above a), and the enzyme kinetic constants of the kininogenase were determined as follows. Table 4
It was as shown in.

Table 4 BAEE: Benzoyl-L-Arg ethyl ether hydrochloride, PNA: HD-Val-L-Leu-L-Arg-p-nitroanilide dihydrochloride, MCA: L-Pro-L-Phe-L-Arg-4- Methyl-coumaryl-7-amide

[0026] Production Example 2 (stabilized preparation of powder) Sodium citrate stock solution obtained in Production Example 1 the concentration of 1%
It was added to a, and treated at the 60 ° C. 10 hours,
The desired stabilized powder was then obtained by freeze-drying.

[0027] Production Example 3 (diluted ampoules formulation) Lyophilized Powder obtained in Production Example 2 to adjust the 1 ml per kininogenase to contain units 0.075 PNA unit or 0.15 PNA, 0.5 ml into ampoules under sterile, Dispensed to 1 ml and 2 ml, and sealed the ampoule. At the time of use, this ampoule is opened and a replacement fluid (for example, "Fish Salz", "Solita T
3 ”or“ Lactone gel solution “Fuso” ”etc. ) diluted to 100 ml and used for intravenous infusion. In this case, the required administration time is 30 minutes.

Pharmacological Test Example 1 (Effects on Cerebral Circulation and Metabolism in Normal Animals) (1) Vertebral Arterial Blood Flow The effect of kininogenase on canine vertebral artery blood flow was rapidly administered intraarterially and intravenously (normal manual operation). Injection speed)
And a more considered sustained dosing (infusion of over 30 minutes). 1.5 x 10 ^-4-5.in the vertebral artery of anesthetized dogs.
Rapid administration of quininogenase at 0 x 10 ^-2 PNA units / body increased the vertebral arterial blood flow in a dose-dependent manner, and the effect lasted for 1-2 minutes. Then 5.0 x into the brachial vein
10 ^-5 - 1.5 - 10 ^-1 PNA if the unit / kg to bolus and 5.0 x 10 ^-4 - 2.5 continuously administered x 10 ^-2 PNA units / kg
We were examined for the case to that. 1.5 x for rapid administration
10 ^-4 - 5.0 x 10 ^-3 increase in dose-dependent blood flow in the range of PNA units / kg but was observed, 5.0 x 10 ^-2 1 minute dosing blood flow in PNA units / kg or more administrations Later, it started to decrease. On the other hand, 1.5 x 10 ^-3 PNA for continuous administration
No increase in blood flow was observed at doses below the unit / kg.
increased throughout the bloodstream that administered in x 10 ^-3 PNA units / kg is maintained, it tends to reduce blood flow observed in closed of 5.0 x 10 ^-3 PNA units / kg in dose, 2.5 x 10 ^-2 PNA unit /
At kg, a clear decrease in blood flow was observed.

(2) Internal Carotid Blood Flow The effect of kininogenase on dog internal carotid blood flow was investigated by rapid and continuous administration into the artery and vein.
1.5 x 10 ^-4-5.0 x 10 in the internal carotid artery of anesthetized dogs
^When kininogenase was rapidly administered at ^-2 PNA unit / body, the internal carotid blood flow increased in a dose-dependent manner, and the effect lasted for about 1 minute. 5.0 x 10 ^-5-1.5 x 10 ^-1 PNA Single
Bolus dose position ^{/ kg, 5.0 x 10 -4 -} 2.5 x10 -2 PNA units /
Decreased blood flow was observed after continuous administration of kg, rapid administration of 1.5 x 10 ^-2 PNA unit / kg, and continuous administration of 5.0 x 10 ^-3 -2.5 x 10 ^-2 PNA unit / kg. It was

(3) Cerebral regional blood flow The effect of kininogenase on the regional brain (deep and cortical) of dogs was examined. Kininogenase a 5.0 x 10 intravenously anesthetized allowed dog ^-5 - 2.5 x 10 ^-3 PNA units / k
bolus dose or 2.5 x 10 ^-3-2.5 x 10 ^-2 PNA
It was continuously administered at a unit / kg for 30 minutes. Blood flow tended to increase with rapid administration, but 2.5 x 10 ^-3 with continuous administration
No change in blood flow was observed at -5.0 x 10 ^-3 PNA unit / kg, and a tendency for blood flow decrease was observed at doses of 1.0 x 10 ^-2 PNA unit / kg or higher.

(4) Brain buffy coat microcirculation 2.5 x 10 5 kininogenase was intravenously injected into anesthetized dogs.
^-3 or 5.0 x 10 ^-3 PNA Unit / kg in rapid dose, or
2.5 x 10 ^-3 PNA unit / kg was continuously administered for 30 minutes, and the diameter of cerebral pial arterioles was measured by photography. Vessel diameter is
Observations were made separately for less than 100 μm and more than 100 μm. Dilatation was observed in arterioles of any diameter with rapid administration, but 2.5 x 10 ^-3
No change in arteriolar diameter was apparent after continuous administration of PNA unit / kg.

(5) Cerebral Vascular Resistance Anesthetized dogs were intravenously injected with quininogenase at 1.5 × 10 5.
^-4-2.5 x 10 ^-3 PNA unit / kg bolus or 5.0
x10 ^-4 -2.5 x 10 ^-2 PNA unit / kg was continuously administered for 30 minutes. Rapid administration reduces cerebrovascular resistance in a dose-dependent manner,
At 2.5 x 10 ^-3 PNA units / kg, the effect lasted for about 15 minutes. On the other hand, 2.5 x 10 ^-3 PNA units for continuous administration
A decrease in cerebrovascular resistance was observed at doses of ≥ / kg and 2.5
At a dose of x 10 ^-2 PNA unit / kg, the effect was sustained even after the end of administration.

[0033] (6) rapidly administered kininogenase intravenously to dogs by cerebral arteriovenous oxygen hidden anesthesia, or continuously administered 30 minutes, arteriovenous oxygen hidden were taken Uwayahora venous bleeding and femoral arterial blood It was investigated. 1.5 x 10 ^-4-2.5 x 10 ^-3 PNA units / kg for rapid administration
Dose of 5.0 x 10 ^{-4-5.0 for} continuous administration
A dose of x 10 ^-3 PNA unit / kg tended to increase the cerebral arteriovenous oxygen range.

(7) Oxygen concentration in brain tissue Rapid administration of quininogenase intravenously to anesthetized dogs
^{(1.5 x 10 -4 - 2.5 x10} -3 PNA units / kg) and, or 30
Continuous administration for 5 minutes (5.0 x 10 ^-4-5.0 x 10 ^-3 PNA unit / kg)
Then, the oxygen concentration in the deep and cortical regions of the brain was measured.
In the case of rapid administration, the oxygen concentration tended to drop once and then rise immediately after the administration in both deep and cortical areas. On the other hand, in the case of continuous administration, both deep and cortical areas 5.0 x 10 ^-3 P
An increase in oxygen concentration was observed 10 minutes after the start of administration at NA units / kg or higher.

Pharmacological test example 2 (1) About the possibility of cerebral edema occurrence Normalized rabbits were divided into 3 groups, and in test groups 1 and 2, intravenous quininogenase was added to 2.5 x 10 ^-3 or 5.0 x 1
0 ^-3 PNA units / kg dose administered continuously for 30 minutes, and continuously administered saline control group, brains were removed immediately after completion of administration, moisture, Na ^+, K ⁺ and Cl ^- measuring the concentration Also, Na ⁺ / K ⁺
The ratio was calculated. As a result, no significant difference was observed between the two test groups and the control group, and therefore the possibility of cerebral edema development due to the administration of kininogenase was not recognized.

(2) Oxygen Uptake into Brain Tissue and Energy Metabolism Rabbits in a normal state are divided into 3 groups, and in test groups 1 and 2, intravenous quininogenase is added to 2.5 x 10 ^-3 or 5.0 x 1
The dose of 0 ^-3 PNA unit / kg was continuously administered for 30 minutes, and the control group was continuously administered with physiological saline. 30, 60 after completion of administration
And 90 minutes later, the brain was removed to collect oxygen and ATP,
Pyruvate, lactate, glucose and glucose-6-
The effect of quininogenase on oxygen uptake and metabolism of brain tissue was examined by measuring phosphate content.
As a result, it was found that quininogenase dose-dependently increased oxygen uptake in brain tissue. ATP,
Pyruvate, lactate, glucose and glucose-6-
Regarding the phosphate content, no significant difference was observed between the control group.

[0037] Pharmacological Test Example 3 (effect on hypoxia model) (1) and experimental methods weighing 3kg around the female New Zealand White rabbits experimental animals, pen Tubal Bitar Na anesthesia (20 mg / kg)
Then, the patient was immediately intubated into the trachea, and breathed by air control using an artificial respirator. Then tubocurarine chloride 0.5
After intravenously injecting mg / kg and performing muscle relaxation, it was transferred to a stereotaxic apparatus and the head muscle was peeled off along the midline of the parietal region, then 0.5 x 0.5 cm, posterior to the right front of the skull. At 1.0 x 0.5c
A bone window of m 3 was formed. Cortical EEG was measured by fixing a silver ball electrode to the anterior bone window, while local cortical blood flow and brain PO ₂ were measured using the posterior bone window. Heart rate is R on the ECG
Measured by triggering a wave. Incidentally, these parameters were sent to a polygraph, a cross thermocouple type tissue blood flow meter and a lecticorder and recorded. After stabilization of each parameter, insufflation from the ventilator was changed from air to 5% O _2, and at the same time, the drug described below was intravenously administered, and after 30 minutes, the insufflation was changed to air again for 10 minutes. The test continued.

(2) Dose and administration method of drug (A) Control group (n = 3) 3.3 ml of phosphate buffer (0.05 M, pH 7) was administered at a rate of 0.11 ml / min for 30 minutes. (B) Kininogenase 5.0 x 10 ^-3 PNA unit / kg administration group
(n = 3) 5.0 x 10 ^-2 PNA units / ml of quininogenase was diluted 10-fold with phosphate buffer (0.05M, pH 7), and 3.3 ml of this was diluted.
Administration at a rate of 0.11 ml / min for 30 minutes. (C) Kininogenase 2.5 x 10 ^-3 PNA unit / kg administration group
(n = 3) 2.5 x 10 ^-2 PNA units / ml of quininogenase was diluted 10-fold with phosphate buffer (0.05M, pH 7), and 3.3 ml of that was diluted.
Administration at a rate of 0.11 ml / min for 30 minutes. (D) Kininogenase 1.5 x 10 ^-3 PNA unit / kg administration group
(n = 3) 1.5 x 10 ^-2 PNA unit / ml of quininogenase was diluted 10-fold with phosphate buffer (0.05M, pH 7), and 3.3 ml of that was diluted.
Administration at a rate of 0.11 ml / min for 30 minutes. (E) Citicoline 100 mg / kg administration group (n = 3) 4 ml of nicoline injection solution (250 mg / 2 ml) was collected and 100 mg / kg
Diluted with phosphate buffer (0.05M, pH 7) to
3.3 ml was administered at a rate of 0.11 ml / min for 30 minutes. (F) Citicoline 10 mg / kg administration group (n = 3) 2 ml of nicoline injection solution (100 mg / 2 ml) was collected and 10 mg / kg
Diluted with phosphate buffer (0.05M, pH 7) to
3.3 ml was administered at a rate of 0.11 ml / min for 30 minutes. (G) Nicardipine 4 μg / kg administration group (n = 3) Nicardipine 10 mg was weighed, diluted to 4 μg / kg with physiological saline, and 0.1 ml / kg was administered over 20 seconds.

(3) Data processing method The rate of change was calculated for the heart rate, and the rate of change was calculated for the brain PO ₂ and regional blood flow in the brain. EEG analysis device (ATAC
-450, manufactured by Nihon Kohden Co., Ltd.), and the result was output to a printer and also output to XY as a graph.
FM 11 (manufactured by Fujitsu Limited) was used for the statistical test.
Student's t-test determined that there was a significant difference when the P value was 0.05 or less.

(4) Results (1) Effect on heart rate The control group was significantly decreased when compared with the untreated control group, but the kininogenase administration group and the citicoline administration group were not significantly different from the control group. There wasn't. On the other hand, the nicardipine administration group showed a significant decrease compared with the control group.

(B) Effect on brain cortex PO _{2 The} control group showed a significant decrease when compared with the untreated control group. That is, 5 minutes after the start of inhalation of 5% O _2,
There was a reduction of x 10 ^-8 PNA units / kg, whereas the untreated control group showed no reduction. In contrast, the quininogenase-administered group was 4.0 at a dose of 5.0 x 10 ^-3 PNA units / kg.
Kininogenase was found to cause PO in brain cortex in hypoxia, as it showed a decrease of 3.0 x 10 ^-8 PNA units / kg at doses of x 10 ^-8 PNA units / kg and 2.5 x 10 ^-3 PNA units / kg. It was found that the decrease of ₂ was significantly suppressed.
The quininogenase administration group of 1.25 x 10 ^-3 PNA unit / kg and the control drug citicoline and nicardipine administration group were almost the same as those of the control group, and the effect of suppressing PO ₂ decrease was not observed. This means that citicoline and nicardipine have no inhibitory effect on the decrease of oxygen in the brain cortex, and that the effective dose of kininogenase is 2.5.
x 10 ^-3 Indicates that PNA unit / kg or more.

(C) Effect on regional blood flow in cerebral cortex The control group increased significantly when compared with the untreated control group. That is, 5 minutes after the start of inhalation of 5% O ₂ , the control group was 26.5%.
Although there was an increase in μV, the untreated control group showed no increase. On the other hand, it was found that in the quininogenase administration group of 5.0 x 10 ^-3 PNA unit / kg, only an increase of 6.7 μV was shown and the increase in regional blood flow in the cerebral cortex was significantly suppressed. The control drugs citicoline and nicardipine were not found to have a blood flow increase suppressing effect.

[0043] (D) Effect control group on the cortical portion EEG, when compared to the untreated control group, high amplitude Nami Jo
It showed Ka傾 direction. In contrast, 5.0 x 10 ^-3 PNA units /
Low-amplitude fast waves in the kg and 2.5 x 10 ^-3 PNA units / kg quininogenase groups and 100 mg / kg citicoline group
Was shown. That is, quininogenase and citicoline are
By lowering the slow wave / fast wave ratio of the cortical brain waves that became high amplitude slow wave by inhalation of low oxygen air, it was found that the low amplitude fast wave was improved and the brain function was improved.

Pharmacological Test Example 4 (Effect on Cerebral Ischemia Animals) After bilateral vertebral arteries of normal rabbits were permanently ligated, both common carotid arteries were ligated for 40 minutes to leave the brain in an ischemic state. Then, the common carotid artery was reopened, and local blood flow in the cerebral cortex, electroencephalogram, etc. were observed over time. ATP, lactate and lipid peroxide concentrations in the cerebral cortex were measured 15 minutes after the opening of the common carotid artery. Quininogenase, citicoline and nicardipine are used as drugs, and 1.25 x 10 ^-3 , 2.5 x 10 ^-3 or 5.0 for quininogenase.
x 10 ^-3 PNA units / kg or 100 mg / k for citicoline
Infused continuously as an intravenous infusion from 15 minutes before common carotid artery ligation for 30 minutes, while for nicardipine 0.5 mg
It was given projected into the duodenum / a kg to 15 minutes before the common carotid artery ligation. Reopening of the common carotid artery markedly increased local blood flow in the cerebral cortex. Nicardipine tended to suppress the increase in blood flow, but quininogenase and citicoline did not. EEG was flattened by ischemia, but EEG activity was observed to reappear in the quininogenase administration group at 2.5 x 10 ^-3 PNA units / kg. On the other hand, reproduction of EEG activity was not observed in the citicoline and nicardipine administration groups. ATP of brain cortex and
When I attached to lactate, ATP was significantly increased by the administration of nicardipine. Lactate is 1.25 x 10 ^-3 PNA units / kg
Was significantly decreased by the administration of quininogenase and nicardipine . From the above, it is presumed that quininogenase has an action of suppressing the increase of lactate caused by the enhancement of anaerobic glycolysis due to ischemia and activating the flattened EEG activity.

Pharmacological test example 5 (cerebral circulation of pathological model animal
Effect on metabolism) A cerebral infarction model animal is prepared by injecting micro glass beads into the right cerebral hemisphere of anesthetized rabbits,
Local blood flow (cortical area), cerebral buffy coat microcirculation, cerebral blood flow distribution and PO ₂ , ATP in cerebral cortical area after drug administration
Lactate, pyruvate, glucose and glucose-6-
The phosphate concentration was measured. The measurement of the above biochemical substances in the brain was performed 60 minutes after the start of drug administration.

The results were as follows: (1) Cortical blood flow Kininogenase 2.5 x 10 ^-3 , 5 x 10 ^-3 or 1.25 x
When a continuous infusion of 10 ^-2 PNA unit / kg was given, a dose-dependent increase in cortical blood flow was observed on the ischemic side (glass beads) infused side, but on the non-ischemic side, Little increase was observed. On the other hand, as a control drug, nicardipine was administered into the duodenum (0.5 mg / kg) and citicoline was administered by continuous intravenous injection (10 mg / kg), but no increase in blood flow was observed on the ischemic side.

(2) Brain buffy coat microcirculation By the Cranial window method, the change in the diameter of arterioles in the buffy coat of the brain was examined under a microscope, and the blood vessel diameters were> 100, 50 <<100 and <50.
It was observed by dividing into μm. Dosage of kininogenase 5
x 10 ^-3 PNA Unit / kg or less 50 μm
Although the above-mentioned dilation of blood vessels is not remarkable, it is involved in substance metabolism.
Clearly extension was observed with respect to 50μm or less of the blood vessels are. When the dose was increased to 1.25 x 10 ^-2 PNA units / kg, clear dilation was observed for vessels 100 μm and above. On the other hand, the control drug, nicardipine, was 100μ
It dilates blood vessels of m or more, but almost no dilatation was observed in small blood vessels of 50 μm or less. This indicates that the vasodilatory action of kininogenase is more effective for thin blood vessels, and its microvasodilatory action kinetics is different from that of nicardipine, which is a typical Ca 2+ ion blocker.

(3) Cerebral blood flow distribution On the ischemic side caused by the injection of glass beads, a remarkable decrease in the blood flow distribution rate was observed in the gray matter region, but 5 × 10 5.
^The blood flow distribution was increased by the administration of ^-3 PNA unit / kg of quininogenase, and it was recovered to the same level as the non-injected side (normal side). No improvement in blood flow distribution was observed for the control drug nicardipine.

[0049] (4) PO _2, ATP in the ischemic side cerebral cortex by administering a kininogenase biochemicals such as 5 x 10 ^-3 PNA units / kg, Pi Rubin salt, glucose and glucose-6-phosphate It was found that the reduction of salt can be suppressed. The control drugs, citicoline and nicardipine, did not show the effect of suppressing the decrease of ATP.

Pharmacological Test Example 6 (Progress of Cerebral Infarction Area) Using the same model animal of cerebral infarction as in Pharmacological Test Example 4, the effect of the drug on the cerebral infarction area 24 hours after the infarction was examined, and the results are shown below. As shown in Table 5, quininogenase showed a significant inhibitory effect when compared with the control.

Table 5 *: 2.5 x 10 ^-3 PNA Unit / kg, continuous infusion for 30 minutes **: 100 mg / kg, continuous infusion for 30 minutes

Pharmacological Test Example 7 (Awakening Reaction of Cortical EEG) (1) Objective Similar to the pathological condition after cerebral hemorrhage by electrically cauterizing / destroying the internal capsule, which is a common site of human intracerebral hemorrhage, in rabbits. The model is prepared and the improving effect of quininogenase is examined. That is, as one of the causes of this internal destruction model, it is possible to partially impair the ascending reticular activation system, which is closely related to the arousal state of cortical EEG. Therefore, the effect of kininogenase on the changes in EEG arousal response to high-frequency stimulation of the midbrain reticulum is examined using the endothelium-disrupted rabbit as an experimental animal.

[0053] (2) was cannulated into the auricular vein of the experimental methods (A) to create a female New Zealand White rabbits contained destruction model (acute model) were anesthetized with pen Tubal Bitar Na 30 mg / kg IV, the back Fixed in position, cut the ventral neck and groin area and then perform a midline incision, expose the trachea in the neck area and cannulate, and expose the right femoral artery and vein in the groin area, A cannula for blood pressure measurement and blood collection was inserted and fixed in, and a cannula for intravenous drug injection was inserted and left in the vein. Then, the animal was fixed to a stereotaxic apparatus in a prone position, the scalp was incised after the head was shaved to expose the skull, and tubocurarine chloride 3 mg / body was intravenously immobilized to be used, and a ventilator was used. Artificial respiration with indoor air (15
-30ml / body / stroke, 30-40 stroke / min). Arterial blood P
CO ₂ was measured with a blood gas analyzer and the appropriate value (PCO
₂ : 35 ± 5 mmHg) was adjusted to the ventilator. Next, according to the brain chart, the inclusions on both sides (APO, L: 6, H
: 0) and left mesencephalic plexus (P: 8, L: 3, H: -2)
The electrodes were pierced so that the free ends of the bipolar bonded electrodes would reach each, and fixed with dental cement. Among these electrodes, both electrodes reaching the inner capsule are used for cautery destruction of the inner capsule, and the electrode reaching the midbrain reticular formation is used for EEG stimulation. For cortical EEG measurement, stainless steel screw-type electrodes were fixed at P: 3 and L: 7 sites. The electroencephalogram was unipolarly induced by the screw electrode and recorded on a polygraph and also on a magnetic tape by a data recorder. The electrocardiogram was recorded on lead II, the blood pressure was recorded via a pressure transducer, and the heart rate was recorded on a polygraph by triggering the R wave of the electrocardiogram with an instantaneous heart rate monitor. Destruction of the inner capsule was performed by applying a direct current of 60 V for 30 seconds through the inserted electrode and then again for 5 minutes. The observation of EEG arousal response was performed by stimulating the mesencephalic reticular body with high frequency by the inserted electrode (frequency of 100 Hz, voltage via electrical stimulator and isolator).
Square wave at 0.5-5.0V, interval 1msec.), Changes in activation threshold and duration of wakefulness reaction. After the measurement, saturated potassium chloride solution was intravenously injected for slaughter, the brain was quickly removed, fixed and stored in 10% formalin solution, and the electrode puncture site and damaged site were confirmed at a later date. .

(B) Method of judging cortical arousal reaction level The stimulating voltage of the midbrain reticular formation is increased at intervals of 0.5 V to 0.5 V, and the voltage at which the wakeful reaction first appears is used as a wakefulness reaction threshold, and the midbrain reticular formation is determined. The duration when the stimulus was applied at 4 V was defined as the awakening reaction duration. The threshold and duration of these wakefulness reactions were visually judged from the waveforms recorded on the recorder chart, and expressed as voltage value (V) and time (sec), respectively. Furthermore, at each time, the cortical EEG for about 2 minutes before the start of midbrain reticulum stimulation was recorded on a data recorder, and the data analysis device (ATAC-450 manufactured by Nihon Kohden Co., Ltd.) was used for A / D.
The power spectrum analysis was performed by performing the transform and the Fourier transform. This analysis was performed by dividing the power up to 0.496-30Hz into the following frequency bands. δ wave (0.496-3.968Hz), θ wave (3.968-8.432Hz), α wave (8.432-13.432Hz), β wave (13.432-30Hz). The power spectrum of the electroencephalogram is different for each time point. It was calculated as the ratio (%) of the power in the frequency band to the total ECoG power.

(C) Method for measuring regional cerebral blood flow. For the measurement of regional cerebral blood flow (rCBF), the tip of the hydrogen electrode was indwelled and fixed to the inner peripheral part (A: 4, L: 4, H: 3) and the cerebral cortex. Then
It carried out using the hydrogen clearance method. Measurement of regional cerebral blood flow by hydrogen clearance method was conducted in hydrogen inhaled using a set Ochiryu meter (Biomedical Sciences Inc. of RBF-2). The regional cerebral blood flow is calculated by A / D from the electrolytic tissue blood flow meter.
The calculation was performed by a computer through a converter or by a method of reading a chart of an electrolytic tissue blood flow meter and calculating by a personal computer. The local cerebral blood flow was measured only in the control group, and the EEG activity was not measured.

(D) Method for confirming disorder of blood-brain barrier In order to confirm whether or not a disorder of blood-brain barrier (BBB) has occurred, Evann's blue was intravenously injected immediately after the destruction of the internal capsule, and
One hour later, the brain was removed. In addition, this experiment was conducted only for the control group.

(E) Data processing method Threshold and duration of wakefulness reaction, δ wave, θ wave, α wave and
β wave and local cerebral blood flow are in units of V, sec,% and
It was expressed in ml / min / 100g, and heart rate and mean blood pressure were expressed as the rate of change (%) with respect to the value before internal capsule destruction. For the differences between the control and other groups in terms of the above parameters, see Stu
It was tested by dent'st-Test, and those with a risk rate of less than 5% were judged to be statistically significant.

(F) Administration dose (a) Control group (n = 7) PBS (phosphate buffer solution, 0.05M, pH 7) was intravenously administered at a rate of 0.11 ml / min for 30 minutes. (b) Sham surgery group (n = 6) (c) Kininogenase 1.25 x 10 ^-2 PNA unit / kg administration group
(n = 7) 1.5 x 10 ^-2 PNA unit / ml of quininogenase was diluted 10 times with phosphate buffer (0.05M, pH 7), and 3.3 ml of the diluted
Administration at a rate of 0.11 ml / min for 30 minutes. (d) Kininogenase 5.0 x 10 ^-3 PNA unit / kg administration group
(n = 6) 5.0 x 10 ^-2 PNA unit / ml of quininogenase was diluted 10-fold with phosphate buffer (0.05M, pH 7), and 3.3 ml of that was diluted.
Administration at a rate of 0.11 ml / min for 30 minutes. (e) Citicoline 100 mg / kg administration group (n = 6) 4 ml of nicoline injection solution (250 mg / 2 ml) was collected and 100 mg / kg
Diluted with phosphate buffer (0.05M, pH 7) to
3.3 ml was administered at a rate of 0.11 ml / min for 30 minutes.

(3) Results (A) Effect on cortical EEG awakening response (a) Awakening response threshold The awakening response threshold in the control group tended to be higher than that in the sham-operated group, but was recognized as a significant change. I couldn't do it. There was no obvious change in the alert threshold in the quininogenase and citicoline administration groups, and no significant difference was observed between the quininogenase and citicoline administration groups. (b) Arousal response duration The arousal response duration in the control group was significantly shorter than that in the sham operation group. In the 1.25 × 10 ^-2 PNA unit / kg quininogenase administration group, the duration of the arousal reaction was significantly prolonged compared to the control group at 60 minutes after the internal destruction, that is, at the end of continuous intravenous administration of the drug. However, there was a tendency to extend at other time points. on the other hand,
No significant prolongation or shortening of the duration of wakefulness was observed in the quininogenase administration group and citicoline administration group of 5.0 x 10 ^-3 PNA units / kg.

(B) Effect on each frequency component of cortical EEG The cortical EEG of the control group in about 2 minutes prior to evoking the arousal response of cortical EEG by electrical stimulation of the mesencephalic reticular formation is clearly less than that of the sham-operated group. A tendency to wavy was recognized. The ratio of the δ wave to the total power increased immediately after the internal destruction, and conversely, the θ wave decreased. As with the θ wave, the α and β waves, which are the fast wave components, tended to decrease due to the destruction of the inner capsule.

(C) Effects on regional cerebral blood flow, heart rate and mean blood pressure (a) Local cerebral blood flow Local cerebral blood flow in the acute phase of internal capsule disruption is significantly reduced around the internal capsule where the disruption was carried out, but Little effect on blood flow was observed. (b) Heart rate There was no significant difference in heart rate between the control group and the sham operation group. Regarding the kininogenase administration group, a transient but dose-dependent decrease in heart rate occurred. The heart rate was further decreased in the citicoline-administered group than in the quininogenase-administered group. (c) Mean blood pressure There was no significant difference in mean blood pressure between the control group and the sham-operated group. There was no significant difference in mean blood pressure between the citicoline-treated group and the control group, but it was significant 15 minutes after the start of treatment in the high-dose quininogenase (1.25 x 10 ^-2 PNA unit / kg) group. Blood pressure was decreased.

(D) Action on Blood-Brain Barrier Evans' blue leakage was observed around the internal-encapsulation destruction site in the blood-brain barrier during the acute phase of internal capsule destruction. Almost no leakage of Evans' blue was observed at the puncture site.

Pharmacological Test Example 8 (Effects on Neuropathy, Cerebral Circulation and EEG of Pathological Model Animal) A tube was inserted into the left external carotid artery of an anesthetized Japanese monkey,
A middle cerebral artery occlusion model was created by feeding a silicone plug (1 mmφ x 5 mm) into the left internal carotid artery via the tube. Kininogenase as a drug 2.5x10 ^-2 PNA units / 5 min after occlusion of the left middle cerebral artery
kg or citicoline 10 mg / kg was administered intravenously over 30 minutes. 24 from left middle cerebral artery occlusion by silicone plug
After a lapse of time, the control group exhibited clear neurological symptoms that were considered to be caused by disorders of the left hemisphere, including hemiplegia. Cerebral cortical blood flow was not significantly different from the sham-operated group in the right hemisphere (non-injured side), but decreased to about 1/3 in the left hemisphere. Furthermore, in the brains of the left and right frontal areas, parietal area and temporal area, α-waves and β-waves, which are fast-wave components in terms of activity, were compared with those in the sham-operated group.
It had fallen to 30%, and the patient had obvious brain dysfunction. In contrast, the kininogenase-administered group showed almost no neuropathy and the cerebral cortical blood flow tended to decrease in the right hemisphere (non-injured side). Showed a tendency to increase, and the left / right ratio was about 1. With regard to EEG activity, there was an increasing tendency in the fast wave component except for the right parietal area, and this tendency was remarkable in the left frontal cortex, which is considered to be the border area of ischemia. On the other hand, in the administration group of the control drug citicoline, the neurological symptom was improved as compared with the control group, but the cerebral cortical blood flow and the electroencephalogram activity were not improved, and therefore the brain function improving effect of the drug was clear. I couldn't say that.

Pharmacological Test Example 9 (Safety Confirmation Test for Healthy Subjects) Kininogenase 0.01, 0.0 for healthy adult male subjects
A single dose study of 2, 0.04, 0.075, 0.15 and 0.30 PNA units / body and a 3-day repeated dose study of 0.15 PNA units / body / day were conducted. There were 3 subjects in each treatment group, for a total of 21 subjects.
For example, the age was 21-37 years, the height was 160-183 cm, and the weight was 51-78 kg. 5% of Japanese drug administration of test drug
A predetermined amount of quininogenase was added to 100 ml of glucose solution to dissolve it, and the solution was intravenously infused through the great saphenous vein for 30 minutes. The evaluation was made by observing clinical symptoms such as subjective symptoms, blood pressure, heart rate, body temperature, respiration rate, and auscultation. As clinical tests, hematological tests, serum biochemical tests, fibrinolysis / coagulation system tests, urine tests, kininogenase antibody test (sensitized hemagglutination test), electrocardiogram and plasma pharmacokinetics were performed. Observation of clinical symptoms showed that the highest dose in a single dose study was 0.30 PNA units / body 3
A slight flushing sensation, which was probably due to the pharmacological action of kininogenase, was observed in about 2 minutes immediately after the start of administration in 2 of the 4 cases. Also, a single dose of 0.15,
The diastolic pressure tended to decrease during intravenous infusion in the 0.30 PNA unit administration group and the 0.15 PNA unit / day three-day repeated administration group, and the heart rate increased in the 0.075 PNA unit or more administration group. Although a tendency was observed, no other abnormal findings possibly attributable to the administration of quininogenase were observed. In the clinical examination, no change in the clinical examination value that was considered to be caused by the administration of kininogenase was observed in any of the administration groups. No antibody production was observed in the antibody test. Plasma concentrations of kininogenase were not detected in the 0.01 and 0.02 PNA unit dose groups, but were detected in 0.04 PNA unit doses and above. 0.04, 0.075, 0.15 and 0.30 PNA
A high linear relationship was observed between C _30min and AUC _{0-180min in} the unit dose group with the dose. The t _1/24 was about 170 minutes. It should be noted that in the repeated administration test for 3 days,
On the 3rd day, almost the same plasma concentration changes were observed, and there was almost no difference in the pharmacokinetic parameters. Therefore, it is considered that drug accumulation due to continuous injection does not occur.

Pharmacological Test Example 10 (Safety, Effective Dose, and Setting Time Required for Administration) Inpatients with various symptoms associated with cerebral infarction (cerebral thrombosis / cerebral embolism) and cerebral hemorrhage, one month or more after the attack. The kininogenase dose is divided into a 30-minute continuous intravenous infusion group (14 cases) and a 120-minute continuous intravenous infusion group (14 cases) for those who are judged to be in the chronic phase, and the dose is administered once a day according to the following procedure. The dose was gradually increased for 3 weeks. 1st week: 0.075 PNA unit / body, 2nd week: 0.15 PNA unit / body, 3rd week: 0.30 PNA unit / body. In the 30-minute continuous administration group, on the 5th day of the 1st week, one patient gave up treatment due to measles-like rash and hot flush, and the other one gave up because of palpitation, cold sensation, nausea and diarrhea. In the eyes, 1 patient had elevated blood pressure / hot flashes, 2 had facial redness, 1 had facial flushing, and 1 had headache / head weight / flushing. 7/26 example,
The expression rate was 25.0%. On the other hand, in the 120-minute continuous administration group, one patient died of diarrhea from the first week on the 6th day of the third week, and the other one had abandoned the administration due to a hot head sensation. Therefore, 2/14 cases of adverse drug reactions occurred and the incidence was 16.7%. Regarding effectiveness,
It was not clear in the 1st week, and the improvement of the symptoms came to be seen in the 2nd week, and there was no remarkable change in the improvement situation of the symptoms in the 3rd week. This is 0.15
The difference in the pharmacological effects between PNA unit / body administration and 0.30 PNA unit / body administration is considered to be small. In this respect,
There was no significant difference in safety between the 30-minute continuous administration group and the 120-minute continuous administration group, and the occurrence of side effects was third.
Considering the concentration at the time of weekly 0.30 PNA unit / body administration, the optimal dose of kininogenase was 0.15 PNA unit / body, and it was determined that continuous administration for 30 minutes by intravenous infusion was the optimal method. .

Pharmacological Test Example 11 (Clinical Test) Inpatients with cerebral infarction (cerebral thrombosis / cerebral embolism) and various symptoms associated with cerebral hemorrhage who had been ill for one month or more after an attack and were judged to be in the chronic stage. As a target, a clinical trial was conducted by consigning the facilities to various places. (1) Test method (A) Test drug (a) Placebo group: placebo 1.5 ml / body, (b) Low dose group: 0.075 PNA unit of quininogenase
(0.5 ml) + placebo 1.0 ml / body, (c) High dose group: 0.150 PNA units of quininogenase
(1.0 ml) + placebo 0.5 ml / body. (B) Method and period of administration The above-mentioned test drug and the vitamin B ₁ , B _6, and B ₁₂ complex for intravenous injection (for intravenous injection of vitamedin) were added to 200 ml of the replacement fluid to dilute, and the intravenous injection was performed for 30 minutes. did. The administration period was 2 weeks. (C) Concomitant drug As a general rule, during the administration period of the test drug, drugs that may affect the efficacy evaluation of the test drug, such as cerebral vasodilators and cerebral metabolism stimulants, should not be used in combination. However, if it is necessary, only a patient who has been receiving the brain metabolism stimulant Avan as a pretreatment drug from 1 month before the start of the study is to use 1 tablet once a day and 3 times a day during the study period. It was decided that it could be administered. (D) Analysis method For tactile data, t-test (comparison within group), analysis of variance
(Comparison between groups), Scheffe-type multiple comparison, Kruskal-Wallis H-test (hereinafter simply referred to as “H-test”) and Scheffe-type multiple comparison for ordinal data, and Χ ^2- for classification data. We decided to use the test. The significance level was set to 5% and 10% was also used as a reference.

(2) Investigators (A) Target The total number of patients who were the Investigators at the start of the study was 306, of which 3 were discharged from the hospital, and the date and time had not passed since the attack. However, the number of cases is 303. Of these, there are some that are not strictly appropriate, including violations of concomitant medications and discontinuation of administration due to the occurrence of side effects, etc. In the case of the placebo group (hereinafter referred to as “P group”), 8 cases were used.
There were 35 patients, 17 in the low dose group (hereinafter referred to as “L group”) and 10 in the high dose group (hereinafter referred to as “H group”). (B) Background factors of the subjects The background factors of 303 subjects analyzed were summarized and analyzed as shown in Table 6 below, and no significant difference was observed in any of them.

Table 6 (Part 1) Table 6 (Part 2) Table 6 (Part 3) In Table 6, *: 1 patient without CT scan (P group) was excluded from the aggregation, **: Cerebral vasodilators, brain metabolism stimulants (including Avan) were used, NS: P> 0.10.

(3) Test Results (Comprehensive Evaluation) (A) Overall Improvement Level The overall improvement level in the 1st week was 1.1% in P group, 7.9% in L group, and 5.4% in H group for improvement of moderate or higher. %,
Including mild improvement, P group 44.2%, L group 55.1%, H
The group was 55.9%. As a result of H test, there was no significant difference between these three groups. Overall improvement in week 2 was 12.9% in P group and 2 in L group, targeting moderate or higher improvement.
3.3% and 28.1% in H group, and even for mild improvement
65.6% in the P group, 75.6% in the L group, and 83.1% in the H group. As a result of H test, a significant difference was observed between these three groups (P <
0.01). As a result of multiple comparison, the H group was significantly superior to the P group (P <0.01).

(B) Safety Level In the first week, the percentage of patients judged to have no safety level was 92.1% in P group, 88.3% in L group, and 90.9% in H group. In week 2, P group 94.8%, L group 88.3%, H group
It was 89.1%. The final judgment (completely adopted case + partially adopted case) was P group 91.1%, L group 82.5%, and H group 86.9%. As a result of H test, there was no significant difference between the three groups at any evaluation time.

(C) Usefulness The usefulness in the 1st week was 2.0% in P group, 9.5% in L group, and 7.3% in H group in the reasonably useful or higher. 39.8%, L group 50.5%, H group 5
It was 5.2%. As a result of H test, there was some difference between these three groups (P <0.10). Result of multiple comparisons, H group was found <br/> that tend was excellent as compared with P group (P <0.10). The usefulness in the 2nd week was 12.9% in the P group and 21 in the L group, targeting more than usefulness.
8% and 30.3% in the H group, 61.3% in the P group, 70.1% in the L group, and 83.1% in the H group when targeting slightly more than useful. H
As a result of the test, a significant difference was found among these three groups (P
<0.001). As a result of multiple comparison, the H group was significantly superior to the P group (P <0.001).

(4) Overall improvement by symptom (A) Subjective symptom The improvement in the second week was 13.0% in the P group, 21.9% in the L group, and 21.2% in the H group, and was mild. For improvement, P group 58.0%, L group 62.5%, H group
It was 84.8%. As a result of H test, a significant difference was found between these three groups (P <0.01). As a result of multiple comparison, the H group was significantly superior to the P group (P <0.05).

(B) Psychiatric symptoms The improvement rate in the 2nd week was P group 11.8%, L group 17.1%, H group 21.8% for moderate or higher improvement, and P for mild improvement. 55.3%, L group 64.5%, H
The group was 75.6%. As a result of H test, a significant difference was found between these three groups (P <0.05). Result of multiple comparison, H
The group was significantly superior to the P group (P <0.05).

(C) The degree of improvement in the 2nd week of neurological symptoms was 2.2% in the P group, 7.0% in the L group, 9.0% in the H group for moderate or higher improvement, and P group for the mild improvement. 40.9%, L group 40.7%, H group 56.
It was 2%. As a result of H test, a significant difference was found between these three groups (P <0.05). As a result of multiple comparison, H group is P
It showed an excellent tendency compared with the group (P <0.10).

(D) Daily life movements The degree of improvement in the second week was 8.0% for the P group, 8.5% for the L group, and 9.5% for the H group for moderate or higher improvement, and P for the mild improvement. Group 40.9%, L group 48.8%, H group 54.
It was 8%. As a result of H test, there was no significant difference between these three groups.

(5) Improvement degree by symptom item (A) Subjective symptom As for the degree of improvement of each item of subjective symptom, there was no significant difference among the three groups as a result of H test. The number of symptoms was 10 or more, and the degree of improvement was relatively high (more than 20% with moderate improvement) Symptoms were vertigo (30.0%), shoulder / neck stiffness (25.0%), tinnitus. (23.1%), headache (21.7%) and dizziness (21.7%), all in the H group.

(B) Psychiatric symptoms As for the degree of improvement of each item of psychotic symptoms, a difference was found between the three groups in emotional incontinence and memory impairment as a result of H test.
(P <0.10). As a result of multiple comparison, for both items, the H group tended to be superior to the L group (P <0.10). There were 10 or more symptom cases, and the degree of improvement was relatively high.
(More than 20% with moderate improvement) Symptoms are depression (22.2%),
He was anxious and frustrated (20.0%), but both were in the H group.

(C) Neurological symptom The degree of improvement in each item of neurological symptom was H-tested, and a significant difference was observed among the three groups in dysphagia (P <0.05).
As a result of multiple comparison, the H group was significantly superior to the L group (P <0.05).

(D) Activities of daily living With respect to the degree of improvement of each item of activities of daily living, a significant difference was found among the three groups in the gait disturbance as a result of H test (P <0.0.
Five). As a result of multiple comparison, the H group was significantly superior to the L group (P <0.05).

(6) Stratified analysis As a result of the H test, a significant difference was observed among the three groups, or a tendency thereof was observed, and as a result of multiple comparison, a significant difference was observed between the groups or a tendency thereof was observed. The stratified items obtained were as follows, and in each case, the H group was superior. Male, cerebral infarction, severe case, mild case, lesion site / internal carotid system, disease duration, 3 months to less than 6 months and 6 months or more, initial case, recurrent case, complication / presence, pretreatment drug・ Yes, concomitant drug / no, avan combination / no and rehabilitation / yes.

(7) Side effects Side effects were observed in 10 patients (9.9%) in P group and 19 patients (18.4%) in L group.
%), 12 cases (12.1%) in the H group, and there was no significant difference between the 3 groups. Of the side effects, diarrhea was the most common, P group 1 patient,
There were 7 patients, 4 in L group and 2 in H group. Below, hypotension P group 1 case, L group 2 cases, H group 3 cases 6 cases in total, liver dysfunction P group 1 case, L group 2 cases, H group 1 case 4 cases in total,
Involuntary movements 3 in each group, 1 in dizziness P group, H
3 patients in 2 groups, nausea 2 patients in L group, 1 patient in H group 3 in total
It was in order of example. Other adverse reactions were sporadic in a total of 2 patients or less. The adverse reactions were evaluated as severe in 2 patients in the L group and 1 patient in the H group, for a total of 3 patients.

(8) Blood pressure and heart rate Within-group comparison before administration and week 1 and week 2 (corresponding t
-Test) showed a significant decrease in systolic blood pressure in the 1st week in the H group (P <0.05).
A similar tendency was seen between the H group and P group (P <0.1
0). There was a significant difference in the diastolic blood pressure in the 2nd week among the 3 groups (P <0.10), but there was no significant difference in the results of multiple comparison. Regarding the heart rate in the first week, there was a tendency of significant difference among the three groups (P <0.10), and as a result of multiple comparison, there was a tendency of significant difference between the L group and the H group.
(P <0.10).

(9) Clinical examination Within-group comparison before and after administration (paired t-test)
There were some items with significant differences in
In all cases, the fluctuations were mild, and there were no clinically significant items with significant differences.

[0084]

The present invention provides a brain function improving agent containing human urinary kininogenase as an active ingredient. The cerebral function improving agent according to the present invention is mainly administered intravenously, and improves various symptoms in the chronic stage due to cerebral infarction or cerebral hemorrhage, especially psychiatric symptoms, subjective symptoms, etc., relatively early. That is, it is generally used now,
This kind of drug is an oral agent, which requires administration for 8 weeks for clinical evaluation of treatment, but the brain function improving agent of the present invention can be evaluated by administration for 2 weeks.
The pharmacological effects are similar to those of oral administration for 8 weeks. The cerebral function-improving agent according to the present invention effectively acts and expands cerebral blood vessels, especially finer cerebral blood vessels to increase cerebral blood flow, but the blood flow increasing action is remarkable at the ischemic site, It is particularly suitable as an agent for improving cerebral dysfunction caused by cerebrovascular disorder because it has little effect on blood flow in non-ischemic regions.

Front page continuation (72) Inventor Tsunemasa, 35, Higashi Sotobori-cho, Higashi-ku, Nagoya, Aichi Sanwa Chemical Research Institute, Inc. Sanwa Chemical Laboratory (56) Reference JP-A-56-68618 (JP, A) JP-A-60-199827 (JP, A)

Claims (2)

[Claims] 1. SD extracted and purified from human urine
The major band was 4 in S-polyacrylamide gel electrophoresis.
1000 and the sub-band at 31000, and the gel filtration method showed a molecular weight in the range of 47000-54000, and the isoelectric point of the undiluted solution was pI 3.92, 4.08 and 4.23 in the brain containing human kininogenase as an active ingredient. Function improver. 2. A brain function-improving agent comprising the human urinary kininogenase according to claim 1 in an amount of about 0.15 PNA unit per 1 ml and containing citric acid or a salt thereof in an amount of 0.1 to 1% by weight. .