KR100458996B1 - Cerebral function improving agent - Google Patents

Abstract

The present invention provides a brain function improving agent containing a compound represented by the following Chemical Formula 1 as an active ingredient, wherein the compound is composed of 2-10 molecules when R ₁ represents a hydroxyl group and R ₂ and R ₃ represent a hydrogen atom It may be an oligomer:

[Formula 1]

[Wherein, R ₂ represents a hydrogen atom if R ₁ is a hydroxyl group; Or R ₁ and R ₂ are bonded together to form an oxo group; R ₃ represents a hydrogen atom, an alkali metal or a monovalent, divalent or _trivalent alcohol residue.

It suppresses hydrocephalus, protects brain function, activates damaged brain energy metabolism and reduces cerebral infarction.

Description

CEREBRAL FUNCTION IMPROVING AGENT}

The present invention relates to a brain function improving agent, and more particularly to a brain function improving agent that acts by inhibiting hydrocephalus, protecting brain function, activating brain metabolism or reducing the degree of cerebral infarction.

Recently, many people suffer from brain injury due to cerebrovascular disease or traffic accidents. This background is often associated with impaired hydrocephalus and brain metabolism. This trend is probably due not only to dietary changes with increased fat intake, but also to the increased morbidity of cerebrovascular disease due to an increase in the elderly population.

Hydrocephalus, which impairs brain function, is defined as an increase in the volume of the brain by increasing the water content of the brain parenchyma. Hydrocephalus not only causes excessive metabolism and dysfunction of nerve cells, but also increases intracranial pressure, leading to disruption of brain circulation and metabolism. In addition, this change can lead to cerebral hernia formation, which further exacerbates hydrocephalus, sometimes with fatal consequences.

Hydrocephalus can be classified into two etiological types, angioedema and cytotoxic species.

Vascular edema: Vascular edema occurs as a result of the functional disorder of the blood-brain barrier of cerebral capillaries, due to the interstitial retention of serum components that escape from blood vessels into brain tissue. This type of species is typically associated with scoliosis, brain tumors, brain abscesses or cerebral hemorrhages and intracranial hematoma.

2. Cytotoxic species: Cytotoxic species are caused by cell membrane injury caused by lactate accumulation when energy metabolism is impaired in brain parenchyma. This type of species is typically associated with cerebral ischemia, hypoxemia, and lactic acidosis.

Current therapeutic approaches against this background include:

(1) Intravenous hypertonic fluid: Hypertonic fluid increases blood osmotic pressure to relieve hydrocephalus by aspirating interstitial fluid into blood vessels. However, repulsion is observed because stopping the hypertonic infusion decreases the blood osmotic pressure again.

(2) Corticosteroids: Corticosteroids are used in the treatment of vascular species associated with brain tumors or brain abscesses because they enhance the function of the blood-brain barrier, stabilize cell membranes, and inhibit inflammatory reactions and cerebrospinal fluid production. However, there are reports that corticosteroids exacerbate ischemic neuronal disorders.

(3) Antioxidants. Antioxidants relieve hydrocephalus by scavenging free radicals produced by ischemia or cytotoxicity.

(4) Calcium channel blockers: Calcium channel blockers are expected to inhibit the delayed neuronal cell death caused by the influx of calcium into brain neurons in the presence of several species. However, calcium channel blockers have vasodilating effects, which can increase local blood flow and worsen hydrocephalus.

(5) Barbiturate: Barbiturate protects the brain against ischemia because it inhibits further lactate accumulation and acidosis by reducing brain metabolic activity (brain oxygen and glucose consumption). However, barbiturate exerts a beneficial effect at doses that cause respiratory depression and cardiovascular toxicity. Therefore, careful use of breathing and cardiac care is required during use.

Therefore, there is a need for the development of drugs that are effective in the prevention and treatment of hydrocephalus, and are free of potential repulsion or other toxicity.

The inventors have discovered that β-hydroxybutyric acid, its salts and esters thereof, which have not been previously evaluated in their effect on brain function, inhibits hydrocephalus by activating brain metabolism. The compound was also found to activate brain metabolism to protect brain mitochondria, thereby reducing the extent of cerebral infarction caused by ischemia.

Accordingly, the present invention provides a brain function improving agent that functions specifically by inhibiting hydrocephalus, protecting brain function, activating brain metabolism or reducing the degree of cerebral infarction.

The present invention provides a brain function improving agent containing a compound represented by the following formula (1) as an active ingredient:

[Wherein, R ₂ represents a hydrogen atom if R ₁ is a hydroxyl group; Or R ₁ and R ₂ are bonded together to form an oxo group; R ₃ represents a hydrogen atom, an alkali metal or a monovalent, divalent or _trivalent alcohol residue.

If R ₁ represents a hydroxyl group and R ₂ and R ₃ represent a hydrogen atom, the compound may be an oligomer consisting of 2-10 molecules.

In other words, an object of the present invention is to provide a brain function improving agent containing a compound of formula (1), more specifically esters of β-hydroxybutyric acid, β-hydroxybutyric acid sodium and / or β-hydroxybutyric acid It is to provide a brain function improving agent containing.

The compound of formula 1, that is, the active ingredient of the brain function improving agent according to the present invention acts by inhibiting hydrocephalus, protecting brain function, activating brain metabolism or reducing the degree of cerebral infarction.

Accordingly, it is an object of the present invention to provide an hydrocephalus inhibitor, a brain function protectant, a brain metabolic active agent, and an infarction reducer containing a compound of formula 1 as an active ingredient.

Β-hydroxybutyric acid, a representative type of the compound of formula 1, is a ketone (eg acetone and acetoacetic acid) produced by fatty acid degradation of the liver. Since β-hydroxybutyric acid is detected in the urine of diabetic patients and the blood concentration of the compound increases during fasting, a study of metabolic activity in obese patients by Owen et al. [J. Clin. Invest. 46 (10), 1589-1595 (1967)], it has been considered that β-hydroxybutyric acid is the end product of metabolism and is unnecessary to the living body. The findings point to the use of β-hydroxybutyric acid as an energy source instead of glucose in the brains of obese patients starved for 5-6 weeks.

β-hydroxybutyric acid is a water soluble and low molecular weight compound (M.W.104). 1 g of β-hydroxybutyric acid has an energy of 3.8 kcal. During normal metabolism, the blood concentration of β-hydroxybutyric acid is only 0-3 mg / dl and the compound is not used again but is excreted in urine and released through the breath into the air. However, during hunger or fasting, blood levels rise above 20-30 mg / dl and are used again. Β-hydroxybutyric acid can be replaced as an energy source instead of glucose during hunger or fasting. It is metabolized and used by important organs and tissues (ie heart, kidney, brain and muscle) except the liver.

Japanese Patent Application Laid-Open No. 201746/1983 discloses that salts of 3-hydroxybutyric acid and compositions containing them protect myocardial metabolism. In addition, Fujii et al. [Jpn. J. Acute Med., 4 (5), 484 (1933)] reported that intravenous administration of β-hydroxybutyric acid after acute cerebral injury results in increased ketone concentrations in cerebrospinal fluid and decreased lactate concentrations.

However, there have been no studies showing that the compound of formula 1 improves brain function by improving impaired brain metabolism, inhibiting hydrocephalus, protecting brain function or reducing the extent of cerebral infarction.

We have found that intravenous pretreatment with β-hydroxybutyric acid prolongs the survival time of animals intravenously administered a lethal dose of potassium cyanide (KCN).

KCN may be cytotoxic by inhibiting mitochondrial cytochrome oxidase, reducing oxygen supply to the electron transport system and blocking ATP production. This effect of KCN can only be antagonized by promoting brain glucose uptake, activating the mitochondrial succinate oxidase system and increasing cerebral blood flow. We observed that death induced by KCN is delayed by continuous intravenous infusion of β-hydroxybutyric acid. The blood concentration of β-hydroxybutyric acid is most likely increased during continuous infusion, and β-hydroxybutyric acid may exhibit this effect by promoting brain ATP production and accumulation. That is, continuous injection of β-hydroxybutyric acid increases brain mitochondria's resistance to oxygen deficiency subsequently induced by KCN lethal dose, thereby prolonging survival mostly.

The inventors also found that simultaneous intravenous administration of β-hydroxybutyric acid prevents the development of hydrocephalus in animals with cerebral ischemia caused by ligation of the left and right common carotid arteries. In this study, animals treated with β-hydroxybutyric acid maintain high levels of ATP in the brain. Based on this finding, β-hydroxybutyric acid can inhibit hydrocephalus by the following mechanisms: it can be metabolized into the TCA circuit to promote ATP production or to inhibit ATP consumption. It can maintain the high activity of Na ⁺ / K ⁺ -ATPase, which can then actively disperse the retained sodium and water out of the cell.

In addition, the inventors have found that administration of β-hydroxybutyric acid during ligation of the middle cerebral artery and subsequent reperfusion significantly reduces the extent of cerebral infarction due to acute ischemia caused by arterial ligation. β-hydroxybutyric acid may exhibit this effect by protecting brain mitochondria against ischemia and improving brain metabolism.

The present invention provides a brain function improving agent containing a compound of formula 1 as an active ingredient, wherein R ₂ represents a hydrogen atom if R ₁ is a hydroxyl group; And R ₁ and R ₂ are bonded together to form an oxo group; R ₃ represents a hydrogen atom, an alkali metal (eg sodium, potassium and lithium) or a monovalent, divalent or trihydric alcohol residue. Examples of alcohol residues include C ₁ -C ₁₂ monohydric alcohols such as methanol, ethanol and butanol, dihydric alcohols such as ethylene glycol, 1,3-butanediol and 2-butene-1,4-diol, trihydric alcohols such as glycerin and tartaric acid And acids such as succinic acid. The compound may be an oligomer composed of 2-10 molecules when R ₁ represents a hydroxyl group and R ₂ and R ₃ represent a hydrogen atom.

Examples of the compound of formula 1 include esters of acetoacetic acid, β-hydroxybutyric acid, β-hydroxybutyric acid sodium and β-hydroxybutyric acid. Esters include methanol, ethanol and glycerin (mono-, di- and tri-esters).

The brain function improving agent of the present invention can be used for the treatment of cerebral injury, brain tumor or cerebral hemorrhage, intracranial hematoma or terminal cerebral ischemia, and hemangiomas associated with cytotoxicity due to cerebral ischemia, severe cerebral injury or hypoxia.

The brain function improving agent of the present invention may be administered by intravenous infusion at a constant rate, preferably in parenteral fluid, but may also be administered via enteral nutrition or intravenous injection. This agent is preferably used in the form of an aqueous solution.

In general, although the optimal concentration of this drug depends on the weight of the patient, it can be added to the parenteral solution at a concentration of 5-500 mM and administered at a rate of 1-2 ml / kg / hour. The concentration of this agent in the parenteral solution may preferably be 10-300 mM, more preferably 20-100 mM.

In general, the duration of administration of this agent also depends on the patient's initial condition and response, but may preferably be administered for 1-7 days.

Brain function improving effect of the compound of Formula 1 will be described through the examples.

A): hydrocephalus inhibitory effect:

Example 1 Relationship Between Dose and Effect of Sodium β-Hydroxybutyrate

The inhibitory effect of sodium β-hydroxybutyrate on hydrocephalus induced by ligation and cleavage of the left and right common carotid arteries was studied in male Wister rats (weight 158.8 ± 1.2 g). Sodium β-hydroxybutyrate solution is prepared at concentrations of 2.5, 5.0, 10, 20, 40, 80, 160 and 320 mM and administered at a rate of 10 ml / kg / hour for 5 hours. Thus, the doses of β-hydroxybutyrate sodium are 3.1, 6.3, 12.5, 25, 50, 100, 200 and 400 mg / kg / hour, respectively.

Rats are operated under anesthesia maintained by induction with ether and inhalation of a mixture of isoflurane / nitrous oxide / oxygen (0.5: 70: 30). Make a midline incision in the neck and carefully cut out the connective tissue without injuring the vagus nerve, exposing the left and right carotid arteries.

For drug administration, the cannula is inserted into the right outer jugular vein and the other end of the cannula is pulled out through the back of the animal. The common carotid artery is then ligated at two points (head and heart) and cut between the two ligations. The incised skin is sutured and each rat is immediately given either intravenous infusion (10 ml / kg / hour for 5 hours) using either an physiological saline solution or one of the β-hydroxybutyrate solutions using an infusion pump.

In sham-manipulated rats, the left and right common carotid arteries are exposed in the same manner and the incised skin is closed without arterial ligation and amputation.

When dosing is complete, each rat is gushed and all brains are immediately isolated. After removing the cerebellum and training, the wet weight of the cerebrum is measured. The cerebrum is then dried at 105 ° C. for 24 hours and the dry weight is measured. The water content of the brain is calculated according to the following formula: Brain water content (%) = (Cerebral Wet Weight-Cerebral Dry Weight) / Cerebral Wet Weight x 100.

As shown in FIG. 1, brain water content is related to the dose of β-hydroxybutyrate.

(In FIG. 1, BHBNa represents sodium β-hydroxybutyrate and BHBMe represents methyl β-hydroxybutyrate.)

The brain moisture content of mock-engineered rats was 79.56 ± 0.05%. The brain water content of rats receiving physiological saline after ligation of the left and right common carotid arteries is much higher than that of the mock-engineered group, indicating the expression of hydrocephalus.

In all groups treated with sodium β-hydroxybutyrate, the brain water content was lower than that in the physiological saline group and this difference was statistically significant (p <0.01) at doses above 5 mM. These results indicate that β-hydroxybutyrate is effective for the prevention and treatment of hydrocephalus.

Example 2 Relationship Between Dose and Effect of Methyl β-Hydroxybutyrate

Using the same procedure as described above, the β-hydroxybutyrate methyl is administered to the rats at a dose of 25 mg / kg / hour for 5 hours. In addition, the results are shown in Table 1.

Example 3 :

A parenteral liquid is prepared according to the appropriate sequence using a specific amount of the following ingredients:

2.42 g of methyl β-hydroxybutyrate

Sodium chloride 2.05 g

Potassium Chloride 1.47 g

Glucose 50.00 g

These ingredients are dissolved in about 950 ml of water and the pH is adjusted to 7.0 with sodium hydroxide. Subsequently, water is added to the solution to make 1,000 ml uniformly. The solution thus prepared is filtered through a filter having a pore size of 0.45 μm. The filtrate is filled into 500 ml glass vials and autoclaved to give a precipitate-free, colorless parenteral.

Using this solution, the effect of methyl β-hydroxybutyrate on hydrocephalus is assessed in the same animal model as detailed in Example 1. All rats treated with this solution had lower cerebral water content than the normal saline treated group, indicating that β-hydroxybutyrate methyl inhibited the development of hydrocephalus.

B): protect brain function

Example 4

(Materials and methods)

1. Test Animal

6-9 Main male wister rats (Japan S.L.C.Ltd.) Are used. Animals are maintained at 23 ± 3 ° C. and 55 ± 15% humidity for 12 hours daily (7: 00-19: 00), providing light. Animals are fed an unlimited supply of pellet food (MF, Oriental Yeast) and tap water. The number of animals in one group is 6-8.

2. Test and Control Drugs

1) Test drug

β-hydroxybutyrate sodium (purity: 99.8%)

Lot samples used in the study showed no change in quality at the end of the study. It was confirmed that the preparation solution was stable for at least one week and that the solution was prepared at an appropriate concentration.

2) Reference drug

(1) Physiological Saline JP

Manufacturer: Otsuga Pharmaceutical Factory

(2) Grenol (trade name)

 Composition (amount for 100 ml):

   10 g of concentrated glycerin

   5 g of fructose

   0.9 g of sodium chloride

Manufacturer: Shimizu Pharmaceutical Co., Ltd.

3. Administration:

The doses of β-hydroxybutyrate sodium tested are 50 and 100 mg / kg / hour (solutions are prepared at 80 mM and 160 mM, respectively), whereas grenolen is the recommended clinical dose (5 mg / kg / hour). To supply. In all groups, the solution is administered at a rate of 5 mg / kg / hour for 1 hour through the cannula in the right external jugular vein. Another group was given 3 days of sodium β-hydroxybutyrate at a dose of 100 mg / kg / hour to study the effect of dosing period on the effect of the test drug.

4. Order

Rats are operated under anesthesia maintained by induction with ether and inhalation of a mixture of isoflurane / nitrous oxide / oxygen (0.5: 70: 30). Insert the cannula into the right outer jugular vein and fix the other end of the cannula to the muscles of the back. Through the cannula, potassium cyanide (KCN: 3 mg / kg) is administered immediately after administration of the specific amount of the test or control drug. Survival time is defined as the time from the administration of potassium cyanide to the onset of respiratory arrest.

5. Statistical Analysis

Data are expressed as mean ± standard error (S.E.). Military differences are tested for significance, using a one-way ANOVA. If significant differences are found between groups, Dunnett's multiple comparison test should be conducted. The significance level is set to p <0.05.

(result)

FIG. 2 shows the relationship between the dose of β-hydroxybutyrate sodium and the survival time, and FIG. 3 shows the relationship between the administration period and the survival time.

Survival time was 50 or 100 mg / kg / hour for 1 hour, compared with the group treated with β-hydroxybutyrate sodium (physiological saline) (58.8 ± 2.4 and 58.8 ± 1.2 seconds vs. 44.6 ± 1.6 seconds; 2) It is greatly extended. Although Greno treated rats also survive longer than the saline feed group, the difference is not statistically significant (52.8 ± 2.3 seconds vs. 44.6 ± 1.6; FIG. 2).

Survival time of rats treated with β-hydroxybutyrate at 100 mg / kg / hour for 3 hours was not significantly different from that of rats treated for 1 hour (61.0 ± 1.6 seconds versus 58.2 ± 2.2 seconds; 3).

Potassium cyanide is thought to express its cytotoxicity by inhibiting mitochondrial cytochrome oxidase inhibition of oxygen delivery to the electron transport system and blocking ATP production. It has been reported that anti-oxygen deficiency mechanisms may include the promotion of brain glucose capture, the activation of the mitochondrial succinate oxidase system and the promotion of increased brain blood flow. Sodium β-hydroxybutyrate significantly inhibits the increase in brain water content caused by left and right carotid artery ligation (BLCN) for 6 hours at the test dose in this study. At the same dose level, this compound significantly prolongs the survival time after administration of potassium cyanide. In contrast, the grenol-treated group does not show a significant increase in survival time compared to the physiological saline-treated group. Based on this finding, the isotonic solution of β-hydroxybutyric acid can inhibit the increase in brain water content of the BLCL-induced hydrocephalus model by a mechanism different from the activation of brain metabolism and the anti-edema effect of Gregol.

C) Activation of Brain Metabolism:

Example 5

(Materials and methods)

1. Test Animal

Male Wister rats purchased from S.L.C.Ltd., Japan, are used after acclimation for one week. Animals are maintained at 23 ± 2 ° C. and 55 ± 10% humidity for 12 hours daily (7: 00-19: 00), providing light. Animals are fed an unlimited supply of pellet food (MF, Oriental Yeast) and tap water. The average body weight of the animals was 172.1 ± 6.7 g at the time of study.

2. Test and Control Drugs

1) Test drug

β-hydroxybutyrate (BHBNa)

BHBNa was stable for 5 months when stored at room temperature. The samples of the lot used in the study did not change in quality upon testing after study completion. It was confirmed that the preparation solution was stable for at least one week and that the solution was prepared at an appropriate concentration.

2) Reference Drug

Physiological Saline JP

Manufacturer: Otsuga Pharmaceutical Factory

3. Experimental Design

The dose of BHBNa tested is 25 mg / kg / hour. The test group is treated with 20 mM BHBNa solution and the control group is fed with saline solution. Both groups are administered at a rate of 10 mg / kg / hour for 5 hours.

4. Test Procedure

Rats are operated under anesthesia maintained by induction with ether and inhalation of a mixture of isoflurane / nitrous oxide / oxygen (0.5: 70: 30). Make an incision at the neck. Carefully cut off the connective tissue without injuring the vagus nerve and expose the left and right common carotid arteries. Insert the cannula into the right outer jugular vein and withdraw the other end of the cannula through the back. Each exposed common carotid artery is ligated at two points (head and heart) and cut between the two ligations. The incised skin is sutured and each rat is immediately infused intravenously with 10 ml / kg / hour for 5 hours using a test drug or physiological saline infusion pump. In mock-engineered rats, the left and right common carotid arteries are exposed in the same manner and the incised skin is closed without arterial ligation and amputation. When dosing is complete, each animal is killed by a guillotine and the whole brain is immediately isolated. After removing the cerebellum and training, the wet weight of the cerebrum is measured. The cerebrum is then dried at 105 ° C. for 24 hours and the dry weight is measured. The water content of the brain is calculated according to the following formula: Brain water content (%) = (Cerebral Wet Weight-Cerebral Dry Weight) / Cerebral Wet Weight x 100.

After weighing, the dry cerebrum is dissolved in 0.6 N-HNO ₃ (1 ml / 100 g cerebrum) and wet burned at 125 ° C. until a clear colorless product is obtained. Purified water is added to the combustion product to make exactly 1 ml, and the solution is analyzed for Na ⁺ and K ⁺ using a flame photometer (FLAME-30C: Nihonbunko Medical). Rats were used as the nine groups in the study.

5. Statistical Analysis

Data are expressed as mean ± standard error (S.E.). The differences between groups were tested for significance using unidirectional ANOVA followed by Dunnett's multiple comparison test. The significance level is set to p <0.05.

(result)

Data on the amount of cerebral water and brain electrolyte in rats ligated with left and right common carotid arteries, with or without BHBNa, are shown in Table 1 and FIGS. 4-6.

Brain fluid and electrolyte levels in the brain County (n = 9) Cerebral fluid rate (%) Na ⁺ volume (mEq / kg dry weight) K ⁺ volume (mEq / kg dry weight) Mock-operation 79.32 ± 0.16 ^{＊＊ °} 224.08 ± 5.93 ^{＊＊ °} 458.31 ± 13.44 ^{＊＊ °} Saline solution 81.96 ± 0.21 ^° 304.39 ± 12.83 ^° 405.99 ± 20.96 ^° BHBNa 79.50 ± 0.09 ^{＊＊ °} 269.74 ± 15.98 ^{＊＊ °} 420.42 ± 5.76 ^°

^＊＊ P <0.01 in preparation for physiological saline group

Left and right total carotid artery ligation (BLCL) significantly increased cerebrospinal fluid and Na ⁺ content (p <0.01) in physiological saline group as well as significantly reduced brain K ⁺ content (p <0.01) compared to mock-manipulated group. Let's do it. Rats receiving BHBNa continuous injection show no significant difference in brain K and Na ⁺ levels (p <0.01), although the brain's K ⁺ content does not differ significantly compared to normal saline-treated rats.

Example 6

(Materials and methods)

1. Test Animal

Male Wister rats (S.L.C.Ltd., Japan) are used for the study. Animals are maintained at 23 ± 3 ° C. and 55 ± 15% humidity for 12 hours daily (7: 00-19: 00), providing light. Animals are fed an unlimited supply of pellet food (MF, Oriental Yeast) and tap water. Animal weight ranges from 161.7 to 249.0 g at the time of study.

2. Test and Control Drugs

1) Test drug

β-hydroxybutyrate (BHBNa)

2) Reference Drug

Glyceol (trade name)

  Composition (amount for 100 ml):

     10 g of concentrated glycerin

     5 g of fructose

     0.9 g of sodium chloride

Distributor: Suga Pharmaceutical Company

3) excipients

(1) excipient 1

Physiological Saline JP

Distributor: Otsuga Pharmaceutical Factory

(2) excipient 2

Distilled Water JP

Distributor: Otsuga Pharmaceutical Company

3. Preparation of Dosage Solution

Accurately weigh 0.2 g of BHBNa, dissolve in distilled water for injection and bring the final volume to 10 ml. To 9 ml of this solution, physiological saline is added to make 30 ml. The resulting 0.6% BHBNa solution is filtered and sterilized through DISMIC-25CS (0.2 μM, ADVANTEC Toyo) before use.

4. Preparation of Cerebral Ischemia Model

Rats are operated under anesthesia maintained by induction with ether and inhalation of a mixture of isoflurane / nitrous oxide / oxygen (0.5: 70: 30). Make a midline incision in the neck and carefully cut out the connective tissue without injuring the vagus nerve, exposing the left and right carotid arteries.

For drug administration, the cannula is inserted into the right outer jugular vein and the other end of the cannula is pulled out through the animal. The common carotid artery is then ligated at two points (head and heart) and cut between the two ligations. The incised skin is sutured and each rat is immediately infused intravenously with 5 ml / kg / hour of saline, 0.6% BHBNa solution or glycerol using an infusion pump. In sham-operated rats, the left and right common carotid arteries are exposed in the same manner and the incised skin is closed without arterial ligation and amputation.

After 3 hours of cerebral ischemia, microwaves (5 kW) were irradiated to the head of each rat for 2 seconds using a microwave irradiator (TMW-6402C, Toshiba) to produce all enzyme reactions and intermediate metabolites in brain tissue. Terminate the decomposition. The cerebrum is separated from the cerebellum and soft water and placed in a pre-weighed test tube containing 3 ml of ice-cold 10% trichloroacetic acid (TCA). Weigh the test tube again. The cerebrum is then homogenized in TCA in an ice cold bath using a homogenizer (Physcotron, Nichion). Homogenates are centrifuged at 4 ° C. at 3,000 rpm for 15 minutes and the supernatant is collected. The pellet is homogenized again in 2 ml of 10% TCA and the homogenate is centrifuged under the same conditions. The supernatant thus obtained is thoroughly mixed with the previous supernatant by stirring to use the mixture as a sample solution. Divide the sample solution into two fractions equally and add 3 ml of saturated ether to one fraction. The mixture is stirred and centrifuged at 3,000 rpm for 15 minutes at 4 ° C., then the ether layer (supernatant) is removed. This extraction process is carried out three times, and the aqueous phase thus obtained is analyzed to determine the amount of ATP. Determine the lactate content using another fraction of the sample solution. The weight of the cerebrum is obtained from the difference from the measured in vitro weight.

Brain ATP levels are determined by enzyme method (Luciferin-Luciferase method) using Lumet LB9507 (EG & G BERTHOLD). The data obtained are expressed in μmol / wet weight g.

Brain lactate level is determined by enzyme method (Lactate dehydrogenase method) using Determiner LA (Kyowa Medics). The data obtained are expressed in μmol / wet weight g.

5. Statistical Analysis

Data are expressed as mean ± standard error (S.E.). Military vehicles are tested for significance using unidirectional ANOVA. If there are significant differences, Scheffe's multiple comparison test is performed. The significance level is set to p <0.05.

(result)

Data on brain ATP and lactate levels are shown in FIGS. 7 and 8, respectively.

1. Effect of BHBNa on Brain ATP and Lactate Levels in Normal Rats

Continuous infusion of BHBNa for 3 hours did not show a significant effect on brain ATP or lactate levels in normal rats without BLCL.

2. Effects of BHBNa and Glycerol on Brain ATP and Lactate Levels in Rats with BLCL

1) ATP level in the brain

In physiological saline-treated rats, ATP levels in the brain after 3 hours BLCL are significantly reduced (0.52 ± 0.10 vs 1.85 ± 0.13 μmol / wet weight g) compared to mock-manipulated rats. Rats fed BHBNa at a dose of 30 mg / kg / hour during BLCL showed significantly lower ATP levels in the brain compared to the physiological saline group, its final concentration (1.56 ± 0.10 μmol / wet weight g). Is similar to the concentration of mock-engineered rats. Continuous infusion of glycerol during BLCL did not cause a significant difference in brain ATP levels compared to the physiological saline group (1.00 ± 0.04 vs. 0.52 ± 0.10 μmol / wet weight g).

2) Lactate levels in the brain

In physiological saline-treated rats, the lactate concentration in the brain after 3 hours of BLCL is significantly increased (11.39 ± 0.85 vs. 0.61 ± 0.12 μmol / wet weight g) compared to mock-manipulated rats. Rats fed BHBNa at a dose of 30 mg / kg / hour during BLCL showed significantly lower lactate levels in the brain compared to the physiological saline group, with a final concentration of 1.36 ± 0.17 μmol / wet g. . In addition, rats fed glycerol during BLCL showed a significantly smaller increase in brain lactate (7.16 ± 0.54 vs 11.39 ± 0.85 μmol / wet weight g) compared to the physiological saline group, but the final value was still simulated-manipulated. Significantly higher than the group.

D): Cerebral infarction effect

Example 7

(Materials and methods)

1. Test Animal

Eight major male wister rats (Charles, Charles River, Japan) are used for the study. Animals are maintained at 23 ± 3 ° C. and 55 ± 15% humidity for 12 hours daily (7: 00-19: 00), providing light. Animals are fed an unlimited supply of pellet food (MF, Oriental Yeast) and tap water. Animal weight ranges from 270.6 to 338.9 g at the time of study.

2. Test and Control Drugs

1) Test drug

β-hydroxybutyrate (BHBNa)

2) excipients

(1) excipient 1

Physiological Saline JP

Distributor: Otsuga Pharmaceutical Company

(2) excipient 2

Distilled Water JP

Distributor: Otsuga Pharmaceutical Company

3. Preparation of Test Solution

A BHBNa solution is prepared just before use.

Accurately weigh 0.2 g of BHBNa, dissolve in distilled water for injection and bring the final volume to 10 ml. To 9 ml of this solution, physiological saline is added to make 30 ml. The resulting 0.6% BHBNa solution is filtered and sterilized through DISMIC-25CS (0.2 μM, ADVANTEC Toyo) before use.

4. Preparation of Occlusion / Reperfusion Model of Middle Cerebral Artery

1) Occlusion model of the middle cerebral artery

Under ether anesthesia, the midline is dissected. Make an incision to the right carotid artery while being careful not to hurt the vagus nerve. At the branch circumference, the carotid and external carotid arteries are separated from the surrounding connective and adipose tissue. The laryngeal and thyroid arteries originating in the external carotid artery are cut out, ligated twice with 6-0 silk and then cut. The external carotid artery is then ligation and cut twice with 5-0 silk. In addition, the right internal carotid artery is cut out of the surrounding connective tissue to expose the pterygium. The external carotid artery is dissected and a 21 mm embolizing thread is inserted through the incision into the internal carotid artery. The tip of the thread enters the forearm cerebral artery through the start of the middle cerebral artery, about 1-2 mm, and the middle cerebral artery is blocked by the central portion of the thread.

In mock-manipulated rats, the incised skin is closed without inserting any color chamber into the internal carotid artery. The rats are then left to the time they are supposed to kill.

2) Reperfusion Model

The middle cerebral artery is occluded with the chromosomal chamber for 2 hours and then the thread is removed under ether anesthesia for reperfusion.

5. Administration of Test and Control Drugs

Immediately after insertion of the color chamber, the rats are continuously injected intravenously with femoral vein via cannula the saline solution or 0.6% BHBNa solution the day before. The infusion pump is used at a rate of 5 ml / kg / hour and continued until the time scheduled for killing (24 hours after reperfusion).

6. Preparation of Brain Specimens

Each rat is killed 24 hours after reperfusion. The cerebrum is separated and cut using a brain slicer (MUROMACHI KIKAI) into 2-mm coronal sections from the 7 mm point behind the forebrain edge to the atrial trunk (the cerebrum). Brain sections are stained by soaking in 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC) in physiological saline for 30 minutes to detect normal mitochondria. The stained sections are fixed with 10% neutral buffered formalin.

7. Evaluation of Brain Sections

TTC-stained coronal sections of the cerebrum are scanned using a film scanner and the data is processed with image analysis software (NIH Image). The percentage of infarcted area (death of the mitochondria) for each brain section is calculated from the total area of the unstained portion over the entire area of the section.

(result)

The percentage of obtained infarcting area is shown in Table 2.

Infarct data (n = 4) Infarction (%) S.E. BHBNa 18.99 ± 1.59 Saline solution 35.18 ± 2.69

The average percentage of infarcts in rats receiving physiological saline over the 2-hour middle cerebral artery occlusion and subsequent 24-hour reperfusion periods was 35.18 ± 2.69%. Infarcts are found in the middle cerebral artery region (temporal lobe), the anterior cerebral artery region (parietal lobe), and in the periphery of the cerebral hemisphere. This finding suggests that after occlusion of the middle cerebral artery, infarction can progress not only in the central ischemic area but also in the peripheral areas (parietal lobe and striatum).

In contrast, continuous infusion of BHBNa (30 mg / kg / hour) immediately after arterial occlusion reduced the average infarct percentage to 18.99 ± 1.59%. Most infarcts are localized to the central ischemic zone and rarely progress to the periphery.

Example 8 Toxicity Study

(1) BHBNa is administered orally or intravenously to a single female dose of 2000 mg / kg to female rats or SD rats. No abnormalities were found and the rat survived.

(2) BHBNa is repeatedly orally or intravenously administered to male Wisteria or SD rats for 2 weeks. Rats administered 500 mg / kg-2000 mg / kg survived without finding any abnormalities.

Example 9 : Formulations

A parenteral liquid is prepared according to the proper sequence using a specific amount of the following ingredients:

16.0 g of β-hydroxybutyric acid monoglycerate

3.15 g sodium chloride

0.30 g of potassium chloride

Each component is dissolved in about 950 ml of water and the pH is adjusted to 7.0 with hydrochloric acid or sodium hydroxide. Then further water is added to make exactly 1,000 ml. The solution thus prepared was filtered with a membrane filter of 0.20 mu m, the filtrate was filled in 500 ml of glass vessel, and then autoclaved to give a colorless parenteral without precipitation.

Example 10 : Other Formulations

Using a specific amount of the ingredients listed in Table 3, a colorless parenteral liquid without precipitation is obtained in the order described above.

The abbreviations used in Table 3 are as follows:

BHB = β-hydroxybutyric acid;

BHBNa = β-hydroxybutyrate;

BHBMe = β-hydroxybutyric acid methyl; And

BHB-oligo (3 mer) = oligomer (trimer) of β-hydroxybutyric acid

List of ingredients and quantities (unit: g / ℓ) BHB BHBNa BHBMe BHB-oligo (trimer) NaCl KCl glucose (One) 31.2 - - - 2.05 1.47 50 (2) 2.42 - - - 2.05 1.47 50 (3) 15.6 - - - 7.02 1.47 50 (4) 2.42 - - - 7.02 1.47 50 (5) - 16.4 - - 0.585 - 50 (6) - 16.4 - - - 1.47 50 (7) - - 36.3 - 2.05 1.47 50 (8) - - 18.15 - 7.02 - 50 (9) - - 2.42 - 7.02 1.47 50 10 - - - 46.5 2.05 1.47 50 (11) - - - 6.2 2.05 1.47 50 (12) - - - 46.5 7.02 1.47 50 (13) - - - 6.2 7.02 1.47 50

As described above, the brain function improving agent of the present invention containing the compound of formula 1 as an active ingredient can inhibit hydrocephalus, protect brain function, improve impaired brain metabolism, and reduce the degree of cerebral infarction. Therefore, this drug will be very effective in improving brain function.

The figure on the back of the present specification shows the results of animal experiments.

1 shows the relationship between the β-hydroxybutyrate or methyl β-hydroxybutyrate and the water content of the brain.

2 shows the relationship between the β-hydroxybutyrate sodium and the survival time.

Figure 3 shows the relationship between the β-hydroxybutyrate sodium administration period and survival time.

4 shows the effect of β-hydroxybutyrate on the water content of the brain.

5 shows the effect of sodium β-hydroxybutyrate on the Na ⁺ content of the brain.

6 shows the effect of β-hydroxybutyrate on the K ⁺ content of the brain.

7 shows the effect of sodium β-hydroxybutyrate on the ATP content of the brain.

8 shows the effect of sodium β-hydroxybutyrate on lactate content in the brain.

Claims (6)

A pharmaceutical composition for improving brain function for treating impaired brain metabolism, which contains a compound represented by the following formula (1) as an active ingredient, wherein the compound may be an oligomer of 2-10 β-hydroxybutyrate molecules: [Formula 1] [Wherein, R ₁ is a hydroxyl group and R ₂ represents a hydrogen atom, or R ₁ and R ₂ are bonded together to form an oxo group; R ₃ represents a hydrogen atom, an alkali metal, or a monovalent, divalent or trivalent alcohol residue. A compound according to claim 1, wherein the compound of formula 1 is β-hydroxybutyrate, β-hydroxybutyrate, oligomer of 2-10 β-hydroxybutyrate molecules, β-hydroxybutyrate and methanol, ethanol, butanol, A pharmaceutical composition for improving brain function selected from esters with ethylene glycol, 1,3-butanediol, 2-butene-1,4-diol, glycerin, tartaric acid or succinic acid. A pharmaceutical composition for inhibiting hydrocephalus, comprising as an active ingredient a compound of formula 1 according to claim 1. A pharmaceutical composition for protecting brain function, comprising the compound of formula 1 described in claim 1 as an active ingredient. A pharmaceutical composition for activating brain metabolism containing the compound of formula 1 as described in claim 1 as an active ingredient. A pharmaceutical composition for reducing cerebral infarction containing the compound of formula 1 according to claim 1 as an active ingredient.