WO2008110034A1 - 3-hydroxy fatty acid and its derivatives for improving of learning and/or memory of subjects - Google Patents

Abstract

The pharmaceutical composition for improving of learning and/or memory of subjects comprising 3-hydroxy fatty acid and its derivatives as active components, and the use of the compounds in the manufacture of medicaments for improving of learning and/or memory of subjects. escape response latency

Description

Technical field of ³ -hydroxy fatty acids and their derivatives for improving learning and/or memory ability of subjects

 The present invention relates to the use of hydroxy fatty acids and derivatives thereof for improving the learning and/or memory ability of a subject. The invention further relates to a pharmaceutical composition comprising the compound as an active ingredient. BACKGROUND OF THE INVENTION Knowledge of learning and memory

 Learning and memory are the necessary processes for animals to change their behavior or to generate new behaviors to adapt to the environment. Human learning and memory are the basis for understanding and transforming the objective world and participating in social practice activities. Physiologists say that learning mainly refers to the process by which the nervous system accepts external environmental information and affects its own behavior. Memory consists of two aspects: remembering and remembering: The mind refers to the brain extracting some useful information from the complicated information of the outside world to form the impression mark, and then selecting some information from the impression mark to form short-term memory, and finally from short-term memory. The selected part of the information forms a process such as long-term memory; the memory refers to the brain extracting relevant information experience stored in the brain according to the current external information, and generating some psychological behavior in combination with external information. The process of reaction or response.

 The types of learning can be divided into three types (Schunk, 1996).

 The first type of learning is non-joint learning, also known as simple learning. It does not require a clear connection between stimulation and response. Habituation and sensitization belong to this type of learning.

The two types of learning are joint learning, which includes classical conditioning and operational conditioning. Classical conditioning is the way animals learn to judge environmental relationships. For example, in Pavlov (1927), in animal experiments, light is given first and then animal meat is eaten. After several iterations, the animal responds to light and to the meat. The response is the same: light can also cause salivation. A typical example of operational conditioning: The experimenter placed a starved rat in a test box with a protruding pressure bar on one side of the wall. The rat pressed the rod by chance and then quickly got the food. The probability of the next pressure bar is higher than the spontaneity. This shows that the animal learns to use food to reward the reflection. After mastering this information, once the animal is hungry, it will go to the box to produce the corresponding Reacting to get food ( Ferster And Skinner, 1957).

 笫 Three kinds of learning are compound learning, and compound learning includes latent learning and alternative learning. Latent learning refers to the impact of potential experience on learning outcomes. For example, animals that have entered the labyrinth training environment, although not trained, are more likely to learn more than animals who have never entered the environment. Alternative learning refers to the ability of certain animals to mimic the actions of other animals, such as movements, sounds, etc., which are ubiquitous in higher animals and humans (Ormrod, 1999).

 The formation of memory consists of three processes of acquisition, consolidation and reproduction. Acquiring is the process of inputting signals into the brain through the sensory system. This process is susceptible to interference from external factors. Consolidation is the stage in which information is stored and stored in the brain. Long-term storage of information is always important to biological individuals. Meaning and often recurring information; Reproduction is the process of extracting information stored in the brain to reproduce it in consciousness (Han Taizhen, Wu Yumei, 1998).

 According to the length of the memory time, human memory can be divided into three stages. The first stage is sensory memory. When external stimuli appear, a certain amount of information is stored from the senses into the relative system. It is called sensory memory, also known as instantaneous memory, which lasts for several hundred seconds to one second (Wen Xuchu et al. , 1995).

The second stage is short-term memory, also known as first-level memory. Short-term memory is the result of further processing of words, numbers, words or other information based on sensory memory. The duration of short-term memory generally ranges from a few seconds to a minute (Miller, 19 ⁵ 6).

 The third stage is long-term memory, also known as long-term memory. It is the result of repeated processing by short-term memory. It lasts from a few minutes, a few days to a few years, and even a lifetime. Long-term memory is usually further divided into two types, namely, second-level memory and third-level memory. The second level of memory is a long-term memory stored with weak or slightly stronger memory traces that is easily forgotten. The third level of memory is a deep and deep memory in the mind, with strong memory traces, so that stored information can be called at any time (Schweickert, 1993). Enhance the development of memory drugs

On the basis of breakthroughs in the study of human genes, brain structures and neurochemicals by geneticists and neuroscientists, it is possible to artificially improve intelligence through a certain measure. At present, there are many "memory enhancing drugs" in the world that are in clinical trials. Some cognitive enhancers that improve memory have appeared on the market (Table 1). These cognitive enhancers are also called "smart drugs". , such as the central nervous system developed by Warner Lambert Inverse AchE inhibitor Tacrine (Hakansson, 1993); At the same time, some drugs for patients with memory impairment have been clinically proven to enhance the intelligence of healthy people. Many people take these drugs to improve the forgetfulness caused by aging. Such as galantamine (Sanda, 1995) and melamine phospholipids (Morris et al, 1998); Ritalin-like drugs for the treatment of mental illness can also be used to enhance certain regular brain functions, which can improve dysmotility Children's learning is also a similar effect for normal children (Jiao Pengtao et al. 2006). Table 1 Drugs that have been developed or are being developed to enhance memory

Table Note: AchE: (Acetylcholinesterase) University of Columbia Professor Eric Kandel is a pioneer in memory enhancer research. He found the mystery of memory through research on sea otters and won the Nobel Prize. (Kandel, 2001). He found that learning occurs in synapses between two neurons in several ways: when the CREB protein in the nerve cells (cAMP response) When the element binding protein, cAMP response element binding protein, is activated, the synapse is strengthened, and CREB protein also plays a role in memory formation in Drosophila and mice.

 Before a neuron naturally increases CREB, the NMDA (N-methyl-D-aspartate, N-methyl aspartate) channel on the nerve cell membrane must be opened, allowing positive ions to flow into the nerve cell through the NMDA channel, and then These positive ions trigger a cascade of cascades that cause CREB activation (Arumugam, et al 2005). Shimuzu et al. (2000) found that increasing the number of NMDA receptors in the hippocampus of mice resulted in better results in a spatial memory experiment. I have recalled related diseases and their treatment

 There are many diseases that can reduce the learning and memory ability of patients, resulting in memory impairment or decline, which has a great impact on people's EI life. These diseases include: various degenerative diseases of the brain (such as Alzhai, Alzheimer's disease, referred to as AD), traumatic brain injury and boxer dementia; sequelae of cerebrovascular disease such as subcortical arteriosclerotic encephalopathy, cerebral infarction and cerebral hemorrhage; sequelae of encephalitis; cerebral hypoxic sequelae such as carbon monoxide poisoning; Sexual encephalopathy (such as Wernicke encephalopathy (Reuler et al 1985)); alcoholism and biochemical disorders of the brain; mental illness. Such diseases can be collectively referred to as memory-related diseases.

 Among these diseases, Alzheimer's disease, which occurs in the elderly and pre-aged, is the most serious. The disease was first discovered by German neurologist Alois Alzheimer, a central nervous system degenerative disease characterized by progressive cognitive impairment and memory impairment (Muhamamad et al 1999), which increases with age. According to statistics, the incidence of AD in elderly people aged 65 or older is 4 to 12%, and the incidence rate among elderly people over 85 years old is 20 to 40% (Shi Anguo et al., 2000). Memory impairment or recession is a complex pathological process that causes abnormalities in the brain such as the frontal lobe, temporal lobe, hippocampus, thalamus, cingulate gyrus, diencephalon and midbrain reticular formation ( Andrew et al) 2005).

There is currently no effective way to treat memory disorders. Although lecithin foods, neurotransmitter precursors, Ritalin, dihydroergotamine vasodilators and other drugs have a certain effect, and also help the memory function training (Wilson et al l997), but still can not Achieve the desired results that people expect. With the improvement of people's living standards and the increase in life expectancy, the world's elderly population is increasing, and society is entering an aging society. Therefore, research on these diseases and their treatment is crucial. At the same time, people are learning any scientific knowledge. When you know, you can't live without it, and the biggest obstacle to learning is the poor memory. Enhance memory to quickly and accurately grasp the knowledge and skills you have learned, and to better understand and apply these knowledge and skills.

Brief description of 3-hydroxy fatty acid

 The structural units of polyhydroxyalkanoates (PHA) are diverse. It has been found that PHA has at least 125 different monomer structures, and new monomers are continuously discovered; carbon chain lengths are 4 - 14 The PHA building blocks between the carbons can also be chemically synthesized and the groups on the branches can be modified. Due to the different degradation methods of PHA, more kinds of monomer structures and derivatives can be obtained, which enrich the biological application of these products, in which 3-hydroxybutyric acid is part of the ketone body of bio-organic metabolites. Various aspects of physiological functions have been extensively studied (Chen and Wu, 2005).

 The ketone body is a product of fatty acid catabolism and is formed only in the liver. It includes 3-hydroxybutyric acid, acetoacetic acid and acetone, of which 3-hydroxybutyric acid is contained in a large amount. The liver contains an enzyme system that synthesizes a ketone body, so that it can form a ketone body, but lacks an enzyme that utilizes a ketone body, so that the ketone body cannot be oxidized, and the produced ketone body needs to be transported to the extrahepatic tissue for further oxidative decomposition by blood. The ketone body of the liver provides energy for extrahepatic tissues and plays an important role in ensuring the brain's energy supply during hypoglycemia to maintain its normal physiological function (Amiel et al, l l ).

 Using 3-hydroxybutyric acid (3HB) and other metabolic precursors as a combination of drugs can enhance myocardial efficiency and improve glucose utilization efficiency in the heart (Cross et al, 1995); it can also be diabetes and insulin resistant Patients provide energy (Rackley et al, 1981); these combination drugs also delay or prevent memory-related brain damage (Reger et al, 2004).

 3-hydroxyalkanoic acid (3HA) monomers and their derivatives have very special advantages:

 3HA monomer and its derivatives are colorless and transparent, with a slight aroma, can be made into most food additives, and also suitable for oral administration (Plecko et al, 2002);

 There is no metabolic enzyme of 3HA in the liver. All additives, which are normal diets, are not metabolized by the liver after being absorbed, but are transported directly to extrahepatic tissues that can utilize it, thus rapidly acting (Rasmussen et al, 1998);

They can directly cross the blood-brain barrier and reach the brain's nervous tissue directly, providing a camp Raise and delay the aging and death of nerve cells (Tieu et al, 2003)

 The present invention is based, in part, on the discovery that the 3-hydroxy fatty acids and derivatives thereof of the present invention are capable of significantly promoting the proliferation of nerve cells, delaying or inhibiting apoptosis and death of nerve cells, and increasing S phase and G2/ in the cell division cycle. The number of cells in the M phase; In the mouse test, the compound of the present invention significantly shortened the incubation period during the learning process, significantly enhanced the learning ability and spatial exploration ability of the mouse, and significantly enhanced the memory ability of the mouse.

 Accordingly, an aspect of the present invention provides a pharmaceutical composition for improving the learning and/or memory ability of a subject, which comprises the compound of the formula (I) as an active ingredient,

0

OH- CH _; C- OR,

(I)

 Or a pharmaceutically acceptable salt, solvate, hydrate, enantiomer, or prodrug thereof, wherein

a group consisting of an alkyl group of H, CC ₉ or a cycloalkyl group, an aryl group and a non-toxic metal ion;

R _{2 is} selected from the group consisting of H, C ₉ alkyl or cycloalkyl, alkoxy and aryl; and a pharmaceutically acceptable carrier.

 Without being bound by any particular theory, it is believed that when the compounds of the invention are amphiphilic molecules, they are advantageous for crossing the blood-brain barrier. When the compound of the present invention is a salt or an acid, the effect across the blood-brain barrier may be slightly inferior, but still has a good effect.

Therefore, the substituents in the compounds of the invention! ^ and R ₂ generally contain no more than nine carbon atoms. In a preferred embodiment of the invention, a group consisting of an alkyl group of H, C _r C ₇ and a non-toxic metal ion is selected; more preferably, an alkyl group of H, C ₅ and a non-toxic metal ion are selected. Further; preferably, a group consisting of an alkyl group of H, C _r C ₃ and a non-toxic metal ion is selected.

In another preferred embodiment of the invention, R _{2 is} selected from the group consisting of H, CC ₇ alkyl, CC ₇ alkoxy and aryl; more preferably, R _{2 is} selected from H, C ₅ alkane A group consisting of alkoxy groups of a group of CC ₅ ; more preferably, R _{2 is} selected from the group consisting of an alkyl group of H, C _r C ₃ and an alkoxy group of C ₃ .

Among the compounds of the present invention, preferred non-toxic metal ions are Na+, K+ and Ca2 ⁺ .

 Particularly preferred specific compounds of the invention are: methyl 3-hydroxybutyrate; ethyl 3-hydroxybutyrate; 3-hydroxybutyric acid; sodium D-3-hydroxybutyrate; sodium DL-3-hydroxybutyrate.

 Another aspect of the invention provides a method for increasing the learning and/or memory ability of a subject, comprising administering to the subject an effective amount of the above compound.

 A further aspect of the invention provides the use of a compound as described above for the manufacture of a medicament for increasing the learning and/or memory ability of a subject.

 According to a preferred embodiment of the invention, the compounds of the invention may be used to treat memory related diseases in a subject, including but not limited to: brain degenerative diseases (such as Alzheimer's disease), brain trauma and boxers Dementia; sequelae of cerebrovascular disease such as subcortical arteriosclerotic encephalopathy, cerebral infarction and cerebral hemorrhage; encephalitis sequelae; cerebral hypoxia sequelae; nutritional deficiency encephalopathy (eg Wernicke encephalopathy); alcoholism and biochemical metabolic disorders; mental illness . BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an infrared analysis of ethyl 3-hydroxybutyrate: Magna 750 Fourier transform near-infrared spectrometer, KBr compression, scanning spectrum area of 4 000 ~ 500cm - ¹ , scanning times of 20 times, resolution of 4 Cm- Figure 2 is a GC-MS analysis of 3-hydroxybutyrate ethyl ester: Shimadzu GC/MS (GC-MSQP5050A); temperature programmed (60 Ό for 5 min, 60 ~ 240, 2. C/min, 240 - 290 Ό , 23 ° C / min, 290 "C for 5 minutes); injection temperature 280 V, split ratio 20:1, carrier gas: He, 1 ml / min; ionization mode EI, ionization energy 70 eV, ion source temperature : 180 , Ion flow: 20 A, full scan acquisition mode (scan range (M/Z) 33 ~ 300 amu).

Figure 3 is an infrared analysis of methyl 3-hydroxybutyrate: Magna 750 Fourier transform near-infrared light transmitter, KBr compression, scanning spectrum area of 4 000 - 500 cm ¹ , scanning times 20 times, resolution For (:!^ ¹ .

Figure 4 is a bar graph showing the effect of 3-hydroxybutyrate, methyl 3-hydroxybutyrate and ethyl 3-hydroxybutyrate on the proliferation of rabbit cerebral cortical glial cells. (a): Each substance acts for 24 hours; (b): Each substance acts for 4 S hours. Figure 5 is a fluorescence micrograph showing the effect of ethyl 3-hydroxybutyrate on apoptosis and death of rabbit cerebral cortical glial cells: 37 细胞, 5% C0 ₂ , 2 % FBS DMEM medium Annexin-V-FITC Apoptosis Detection Kit Stained cell slides (Place 0.13-0.17 mm thick coverslips in 12-well plates, add ΙΟΟμΙ 1x10 ^s /ml cell suspension; cell adherent culture After 4 hours, add ⁹ 00 μl 2 % FBS DMEM medium for ²⁰ hours), and take a photo with a NIKON TE2000-E fluorescence microscope. ( _a ): control; (b): 0.004 g/1 ³ -hydroxybutyrate was applied to cells for 48 hours.

Figure 6 is a flow cytometry test showing the effect of ethyl 3-hydroxybutyrate on the glial cell cycle of rabbits. (a): control; (b): 0.005 _g / l ethyl 3-hydroxybutyrate was used to treat the cells.

 Figure 7 shows the changes in learning and memory behavior in mice in the MWM experiment. (a): The positioning navigation test measures the ability of mice to learn water maze; (b): The ability of spatial exploration experiments to measure spatial spatial memory in mice; (c): The memory retention of mice after 48 hours of spatial exploration test (d): Memory retention in mice was measured 45 days after the space exploration trial.

 Figure 8 shows the results of determination of crude fat content in the liver of mice.

 Figure 9 shows the results of measurement of serum cholesterol levels in mice.

 Figure 10 shows the number of nucleated cells in the femur bone marrow of mice.

 Figure 11 shows the identification of primary rat brain glial cells by immunohistochemistry.

 Figure 12 shows the effects of sodium D-3-hydroxybutyrate, sodium DL-3-hydroxybutyrate and methyl 3-hydroxybutanoate on apoptosis of murine glial cells by flow cytometry: negative control group ), D-3-hydroxybutyrate treatment group (b), DL-3-hydroxybutyrate treatment group (c) and 3-hydroxybutyrate treatment group (d).

 Figure 13 shows the effects of sodium D-3-hydroxybutyrate, sodium DL-3-hydroxybutyrate and methyl 3-hydroxybutanoate on the apoptosis of murine glial cells by flow cytometry.

 Figure 14 is a fluorescence microscope for the in situ observation of the effects of sodium D-3-hydroxybutyrate, sodium DL-3-hydroxybutyrate and methyl 3-hydroxybutyrate on apoptosis of murine striatum. (a), (b), (c), and (d) were the negative control group, sodium D-3-hydroxybutyrate, sodium DL-3-hydroxybutyrate, and methyl 3-hydroxybutanoate, respectively.

Figure 15 shows the effect of calcium imaging on the intracellular calcium concentration of murine glial cells by sodium D-3-hydroxybutyrate, sodium DL-3-hydroxybutyrate and methyl 3-hydroxybutyrate. (a) The results of laser confocal microscopy were from left to right as a negative control group, sodium D-3-hydroxybutyrate, sodium DL-3-hydroxybutyrate and methyl 3-hydroxybutanoate. (b) Statistical results of intracellular calcium ion concentration in each control group. Figure 16 shows the effect of calcium imaging on the intracellular calcium concentration of glial cells at different concentrations of methyl 3-hydroxybutyrate.

Figure 17 shows the effect of different concentrations of D-3-hydroxybutyrate and DL-3-hydroxybutyrate on the intracellular calcium concentration of glial cells under different conditions by calcium imaging. ( _a ): Use NPM containing Ca ²⁺ and M ^ ; (b): Use NPM which does not contain Ca ²⁺ and Mg^.

 Figure 18 shows the calcium imaging method for the detection of intracellular calcium changes induced by the calcium ion analog nitredipine (nitrendipine) against sodium D- 3-hydroxybutyrate, sodium DL-3-hydroxybutyrate and methyl 3-hydroxybutyrate. The impact. Detailed ways

 In one aspect, the invention provides a pharmaceutical composition for improving the learning and/or memory ability of a subject, comprising:

 a compound of the formula (I) as an active ingredient,

0

(I)

 Or a pharmaceutically acceptable salt, solvate, hydrate, enantiomer, or prodrug thereof, wherein

a group consisting of an alkyl group of H, CC ₉ or a cycloalkyl group, an aryl group and a non-toxic metal ion;

R _{2 is} selected from the group consisting of H, C _r C ₉ alkyl or cycloalkyl, C alkoxy and aryl; and a pharmaceutically acceptable carrier.

 Preferred specific compounds of formula (I) include: methyl 3-hydroxybutyrate, ethyl 3-hydroxybutyrate, 3-hydroxybutyric acid (including salts thereof, such as sodium, calcium, potassium), 3- Methyl hydroxycaproate, ethyl 3-hydroxyhexanoate, 3-hydroxyhexanoic acid (including salts thereof, such as sodium salts, calcium salts, potassium salts).

The present inventors have found that the compound of the formula (I) (hereinafter referred to as "the compound of the present invention") is easily absorbed and acted by the body, and the drug effect is remarkable without side effects. The compound of formula (I) has been shown to significantly promote the proliferation of nerve cells in cytological experiments, and to increase the number of S phase and G2 M phase cells in the cell division cycle. Experiment in the water labyrinth experiment (Morris Water Maze) Among them, the compound of formula (I) significantly shortened the incubation period in the learning process, significantly enhanced the learning ability and spatial exploration ability of the mouse, and showed that the memory ability of the mouse was significantly enhanced in the memory retention step. The mice gained weight faster when taking this new type of drug, but their liver crude fat content and serum cholesterol content did not change significantly. The increase in body weight was reflected in the increase of bone density in mice.

In the process of nerve cell culture, the MTT assay showed that these small molecules can significantly increase the proliferation of nerve cells at low concentrations (Fig. 4a, 4b), and they can delay or inhibit the apoptosis and death of nerve cells (Fig. 4a, 4b). Figure 5a, 5b); Flow cytometry analysis shows that these small molecules promote the proliferation of nerve cells, which enhances the ability of nerve cells to divide, specifically in the S phase and G ₂ /M phase cells of the cell cycle. Significantly increased (Figures 6a, 6b).

 The compound of the invention was fed to mice for one month, passed Morris Water Maze

(Morris water maze) to detect learning and memory in mice (Morris et al, 1984). The results of the positioning navigation experiment showed that the EL of each group gradually shortened with the increase of the number of trials. The EL of each experimental group was shorter than the control group, and there was significant difference.

(P<0.05); In the space exploration experiment, the number of cross-platforms in each concentration of methyl 3-hydroxybutyrate group was more than that in the control group within 60 seconds, which was statistically significant (P<0.05). The memory retention test was performed 48 h after the end of the space exploration experiment. The results showed that the EL of the mice in each concentration of methyl ³ -hydroxybutyrate was significantly different from the control group (P<0.05), of which 30 mg/kg/ The mice in the 3-methylbutyrate methyl group showed the most significant performance (Fig. 7a, 7b).

 The mice fed the compound of the present invention showed a significant enhancement of learning ability and spatial localization ability in the Morris Water Maze behavioral experiment. The memory retention was longer, and it was easy to quickly find and climb on the water through enhanced memory and spatial localization ability. Under the platform

(Fig. 7c, 7d).

 In the course of feeding the compound of the present invention, the body weight of the mice was increased faster than that of the control group, and the mice were healthy and active compared with the control group, and the increase in body weight was reflected in the increase of bone marrow cells and bone density (Fig. 10).

Through the determination of serum cholesterol content, the cholesterol content in the serum of the mice in the administration group was similar to that in the control group, while in the determination of the crude fat content in the liver, the content of crude fat in the liver of the administration group was slightly lower than that in the control group. Rats, but not significantly different (Figures 8 and 9). Therefore, administration of the compound of the present invention does not increase the cholesterol content in the blood, nor does it increase the content of crude fat in the liver, and avoids the risk of cardiovascular disease and fatty liver. Another aspect of the invention provides a method for increasing the learning and/or memory ability of a subject, comprising administering to the subject an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, Solvates, hydrates, enantiomers, or prodrugs.

 A further aspect of the invention provides a compound of formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, enantiomer, or prodrug thereof, for use in the preparation of a subject for learning and/or Use in memory drugs.

The term "3-hydroxy fatty acid and its derivatives" as used herein has the structural formula of formula (I), including ³ -hydroxy fatty acids and esters, salts and the like. The term "3-hydroxy fatty acid and its derivatives" as used herein may be used interchangeably with the terms "compound of formula (I)" or "compound of the invention" unless otherwise stated or apparently inconsistent with the context.

 The compounds of the present invention can be obtained by various methods such as hydrolysis and alcoholysis of various polyhydroxy fatty acid esters (Chen and Wu, 2005), purified by distillation, and passed through FOR and GC-

MS analysis confirmed that the purity was extremely high, and there were no by-products such as double bonds that impair cell growth.

 The term "subject" as used herein refers to any mammal, for example, a mouse, a rat, a rabbit, a dog, a cow, and a primate such as a monkey and a human. "Subject," may refer to a diseased mammal, such as a mammal having memory loss or Alzheimer's disease, particularly a human; it may also refer to a healthy mammal that is not afflicted. It will be appreciated that for some purposes, such as improving learning outcomes or improving work performance, healthy subjects may also need to improve their learning and/or memory skills.

 The term "pharmaceutically acceptable salt" as used herein, unless otherwise stated, includes non-toxic acid and base addition salts of the compounds. Acceptable non-toxic acid addition salts include those derived from organic and inorganic acids or bases known in the art. The acid includes, for example, hydrochloric acid, phosphoric acid, sulfuric acid, acetic acid, lactic acid, succinic acid, citric acid, malic acid, maleic acid and the like. Compounds which are acidic in nature are capable of forming salts with various pharmaceutically acceptable bases. Bases which can be used in the preparation of pharmaceutically acceptable base addition salts are those which form non-toxic base addition salts, i.e., the salts contain pharmacologically acceptable cations such as, but not limited to, alkali metals Or an alkaline earth metal salt, especially a calcium, magnesium, sodium or potassium salt. Suitable organic bases include, but are not limited to,

N,N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, lysine, and procaine.

 The term "hydrate" as used herein, unless otherwise indicated, means that the compound of the invention is further combined with a quantity of water by non-covalent intermolecular forces.

Unless otherwise stated, the term "prodrug" as used herein means: Derivatives of the compounds of the invention are provided by hydrolysis, oxidation or other reaction under physical conditions (in vitro or in vivo). Examples of prodrugs include, but are not limited to, derivatives of the compounds of the invention, which include biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters or biohydrolyzable urethanes. section. Other examples of prodrugs include: Derivatives of a compound of formula (I) of the invention comprising a -NO, -N0 ₂ , -ONO or -ON0 ₂ moiety. Prodrugs can typically be prepared using well-known methods, for example, in Burger's Medicinal Chemistry and Drug Discovery, 172-178, 949-982 (edited by Manfred E. Wolff, 笫五版, 1995, and Design of Prodrugs Ά. Bundgaard, Described in Elsdvier, New York 1985).

 Unless otherwise obsessed, the terms "biohydrolyzable amide", "biohydrolyzable ester", "biohydrolyzable carbamate" as used herein mean amide, ester or carbamate of a compound, respectively. : 1) does not interfere with the biological activity of the compound, but may impart beneficial in vivo properties to the compound, such as uptake, time of action or action; or 2) be biologically inactive, but may be converted in vivo to a biologically active compound. Examples of biohydrolyzable esters include, but are not limited to, lower mercapto esters, lower acyloxyalkyl esters (e.g., acetoxymethyl, acetoxyethyl, aminocarbonyloxymethyl, pentanoyloxy) Alkyl and pentanoyloxyethyl), choline esters, and amidoalkyl esters (eg, acetamidomethyl ester). Examples of biohydrolyzable amides include, but are not limited to, lower alkyl amides, alpha-amino acid amides, alkoxy amides, and alkylaminoalkyl carbonyl amides. Examples of biohydrolyzable carbamates include, but are not limited to, lower alkylamines, substituted ethylenediamines, amino acids, hydroxyalkylamines, and polyetheramines.

"Enantiomers" of the compounds of the invention include racemic, enantiomerically enriched or enantiomerically pure compounds. The term "enantiomerically pure" as used herein, unless otherwise indicated, is meant to include one enantiomer of a compound and is substantially free of other enantiomers of the compound. Typical enantiomerically pure compounds include greater than about 95% by weight of one enantiomer of the compound, such as a D-form or an L-form enantiomer. The term "enantiomerically enriched" as used herein, unless otherwise indicated, is meant to include a pair of such compounds greater than about 50% by weight, preferably greater than about 70%, more preferably greater than about 80% by weight. Isomer. The term "racemic" or "racemate" as used herein, unless otherwise stated, refers to an optically inactive mixture of equal amounts of left-handed and right-handed bodies. For example, the racemate of 3-hydroxybutyric acid can be represented as DL- ³ -hydroxybutyric acid. The compound of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, enantiomer, or prodrug thereof (active ingredient) may be used in its own form, but will generally be in a pharmaceutical composition. In the form of a composition, the active ingredient is combined with a pharmaceutically acceptable carrier. The pharmaceutical composition will preferably comprise from 0.05 to 99 wt%, more preferably from 0.05 to 80 wt%, more preferably from 0.10 to 70 wt%, and even more preferably from 0.10 to 50 wt%, depending on the mode of administration. The active ingredients, all percentages by weight, are based on the weight of the total composition.

 The term "alkyl" as used herein, refers to both branched and straight-chain saturated aliphatic hydrocarbon groups bearing a given number of carbon atoms. For example, an alkyl group is defined as a straight or branched group having 1, 2, 3, 4, 5, 6, 7, 8 or 9 carbons. For example, "C alkyl" specifically includes Base, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, iso-butyl, pentyl, hexyl, heptyl, octyl, decyl and the like.

The term "cycloalkyl" as used herein refers to a monocyclic saturated aliphatic hydrocarbon group having a given number of carbon atoms. For example, "C ₃ -C ₉ cycloalkyl" includes cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl and the like.

 The term "alkoxy" as used herein denotes a fluorenyl or cycloalkyl group having the indicated number of carbon atoms attached through an oxygen bridge. "Alkoxy," thus includes the definitions of alkyl and cycloalkyl as described above.

 The term "aryl," as used herein, refers to a carbocyclic aryl group containing from 5 to 10 ring atoms. Representative examples include, but are not limited to, phenyl, tolyl, fluorenyl, fluorenyl, fluorenyl, pyridyl, and a naphthyl group, and a benzofused carbocyclic moiety comprising a 5,6,7,8-tetrahydronaphthalene. The carbocyclic aryl group can be unsubstituted or substituted. In one embodiment, a carbocyclic aryl group Is a phenyl group.

The term "non-toxic metal ion" as used herein refers to a metal ion that is not significantly toxic to a subject, such as, but not limited to, Na ⁺ , KU Ca ² \ Mg ² Zn ²⁺ , Fe ²⁺ , Fe ^{3+ ,} and the like.

 The term "effective amount" as used herein, refers to an amount of active compound that is sufficient to cause a biological or medical response sought by a veterinarian or clinician in an animal or human. The "effective amount" of the compound of the present invention can be determined by those skilled in the art depending on the route of administration, the weight of the subject, age, condition, and the like.

 The invention also provides a process for the preparation of a pharmaceutical composition of the invention comprising a compound of formula (I) as defined above, or a pharmaceutically acceptable salt, solvate, hydrate, enantiomer thereof, or The prodrug is mixed with a pharmaceutically acceptable carrier.

The pharmaceutical composition may be formulated, for example, as a cream, a solution, a suspension, an aerosol, and a dry powder. In the form of topical administration (for example, to the skin or lungs and/or airways); or for example, systemically by oral administration in the form of tablets, capsules, syrups, powders or granules; or in solution or The form of the suspension is administered parenterally; or it can be administered subcutaneously.

 The compositions of the present invention can be obtained by conventional methods using conventional carriers well known in the art. Thus, compositions for oral use may contain, for example, one or more coloring agents, sweetening agents, flavoring agents, and/or preservatives.

 Suitable pharmaceutically acceptable carriers for tablets include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate; granulating and disintegrating agents such as corn starch or alginic acid; binders such as starch; Lubricants such as magnesium stearate, stearic acid or talc; preservatives such as ethyl or propyl p-hydroxybenzoate; and antioxidants such as ascorbic acid. The tablets may be uncoated or coated to modify their disintegration and subsequent absorption of the active ingredient in the gastrointestinal tract, or to improve their stability and/or appearance, in either case using conventional coating agents and existing A method well known in the art.

 Compositions for oral use can be in the form of hard gelatin capsules or soft gelatin capsules. In hard gelatin capsules, the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin; in soft gelatin capsules, the active ingredient is combined with water or oil such as peanut oil, liquid paraffin, or olive The oil is mixed.

 Aqueous suspensions usually contain the active ingredient in the form of a fine powder and one or more suspensions, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone , tragacanth and gum arabic; a dispersing or wetting agent such as lecithin or a condensation product of an alkylene oxide with a fatty acid (for example polyoxyethylene stearate). Said aqueous suspensions may also contain one or more preservatives (such as ethyl or propyl p-hydroxybenzoate), antioxidants (such as ascorbic acid), colorants, flavoring agents, and/or sweetness. Agent (such as sucrose, saccharin or aspartame).

 An oily suspension can be prepared by suspending the active ingredient in a vegetable oil (such as peanut oil, olive oil, sesame oil or coconut oil) or a mineral oil (such as liquid paraffin). The oily suspensions may also contain a thickening agent such as beeswax, paraffin wax or cetyl alcohol. Sweetening agents such as those mentioned above, as well as flavoring agents, may be added to provide a palatable oral preparation. These compositions may be preservative treated by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for use in the preparation of aqueous suspensions by the addition of water usually comprise the active ingredient together a dispersing or wetting agent, a suspension, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are employed as described above for these materials. still alright Additional excipients such as sweetening, flavoring, and coloring agents are present.

 The pharmaceutical compositions of the invention may also be in the form of an oil-in-water emulsion. The oil phase may be a vegetable oil such as olive oil or peanut oil, or a mineral oil such as liquid paraffin, or a mixture of any of these. Suitable emulsifiers may be, for example, naturally occurring gums such as acacia or tragacanth, naturally occurring phospholipids such as soya lecithin, lecithin, esters derived from fatty acids and hexitol anhydrides or partial esters (eg sorbitan mono-oil) The acid ester) and the condensation product of the partial ester with ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavoring, and preservatives.

 Syrups and elixirs may be prepared with sweetening agents such as glycerol, propylene glycol, sorbitol, aspartame or sucrose, and may also contain a demulcent, preservative, flavoring and/or coloring agent.

 The pharmaceutical composition may also be in the form of an injectable sterile aqueous or oily suspension, and may contain, according to known methods, one or more suitable dispersing or wetting agents and suspensions already mentioned above. To prepare. The injectable sterile preparation may also be an injectable sterile solution or suspension in a parenterally acceptable non-toxic diluent or solvent, for example

A solution in 1,3-butanediol.

 Topical preparations, such as creams, ointments, gels, and aqueous or oily solutions or suspensions, may be usually formulated in the active ingredient using conventional excipients or diluents which are conventionally employed in the art using conventional methods known in the art. Preparation was carried out.

 The composition to be administered by the insufflation method may be in the form of a finely divided powder comprising particles having an average diameter of, for example, 30 μηη or less, the powder itself containing only the active ingredient or having one or more physiologically acceptable substances. The carrier is lactose. Then, the powder for the insufflation method can be conveniently held in a capsule containing, for example, 1 to 50 mg of the active ingredient, which is used by a turbo-inhaler device.

 The composition for administration by inhalation may be in the form of a conventional pressurized aerosol, the aerosol being arranged to contain the active ingredient in an aerosol comprising finely divided solid or liquid droplets. Forms are assigned. Conventional aerosol propellants such as volatile fluorinated hydrocarbons or hydrocarbons can be used and the aerosol device can be conveniently arranged to dispense a metered amount of the active ingredient.

It will be appreciated that the dosage administered will vary depending upon the compound employed, the mode of administration, the treatment desired, and the condition. Typical daily doses for mammals to be treated range from 0.05 mg to 75 mg active ingredient per kg body weight. For humans, the preferred daily dose is, for example L-10mg/kg. If desired, the daily dose can be administered in divided doses. The precise amount and route of administration of the active ingredient administered will depend on the weight, age, sex, and particular condition being treated, of the subject being treated, according to principles well known in the art.

In order to explain the present invention in more detail, embodiments of the invention will be given below in conjunction with the accompanying drawings. The examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention. Example 1: Preparation of ethyl 3-hydroxybutyrate 5 g of PHB and 50 ml of chloroform were placed in a round bottom flask. Install the water bath condenser tube and put it into the oil bath. Slowly heat and stir to dissolve. Another 100 ml of ethanol was placed in a 150 ml Erlenmeyer flask, and 2 ml of concentrated sulfuric acid was added and stirred at room temperature. The above ethanol-concentrated sulfuric acid solution was added to a round bottom flask of PHB chloroform solution, and mixed. The temperature is controlled at around 80 。. The reaction time is 48 hours. After the reaction was completed, the impurities were removed by suction filtration. The filtrate was transferred to a 250 ml separatory funnel, 25 ml of distilled water was added, and the layers were allowed to stand. The lower organic phase was collected, neutralized once with 25 ml of NaHC0 ₃ , once with 25 ml of distilled water, separated by a separating funnel, and organic phase. Dry over anhydrous sodium sulfate and filter off sodium sulfate. The organic phase was evaporated on a rotary evaporator and the chloroform was dried, distilled under reduced pressure (110 ± 10, 2900 Pa) to give ² .8 g product by Fourier transform infrared spectroscopy (FTIR) and praseodymium gas chromatography - mass spectrometry in conjunction (GC-MS) analysis determined The product was high purity ethyl 3-hydroxybutyrate (Figure 1, Figure 2). Example 2: Preparation of methyl 3-hydroxybutyrate 5 g of PHB and 50 ml of chloroform were placed in a round bottom flask. Install the water bath condenser tube and put it into the oil bath. Slowly heat and stir to dissolve. Another 100 ml of methanol was placed in a 150 ml Erlenmeyer flask, and 2 ml of concentrated sulfuric acid was added thereto at room temperature with stirring. The above methanol-concentrated sulfuric acid solution was added to a round bottom flask of PHB chloroform solution, and mixed. The temperature is controlled at around 80 。. The reaction time is 48 hours. After the reaction was completed, the impurities were removed by suction filtration. The filtrate was transferred to a 250 ml separatory funnel, 25 ml of distilled water was added, and the layers were allowed to stand. The lower organic phase was collected, neutralized once with 25 ml of NaHC0 ₃ , once with 25 ml of distilled water, separated with a separating funnel, and used for organic phase. Dry over anhydrous sodium sulfate and filter off sodium sulfate. The organic phase was evaporated to dryness with a rotary evaporator and dried, and distilled under reduced pressure (80 ± 10 ° C, 4800 Pa) to give 3.0 g of product, which was confirmed by FTIR analysis. The product was obtained as methyl 3-hydroxybutyrate (Fig. 3). Example 3: Preparation of rabbit cerebral cortex neuron shield cells After taking white rabbit brain tissue with sterile scissors, the fiber components such as meninges and blood vessels were carefully removed, and then cut into Hanks liquid (containing 100 U/mL penicillin + 100). Wash 1-2 times in mg/L streptomycin), place it in 30-50 volumes of Hanks solution, and make a cell suspension by repeated pipetting (the brain tissue is soft); inject the suspension into the centrifuge tube, After standing at room temperature for 5-10 minutes, the cells or cell clumps naturally sink, fat and other debris float on the surface of the suspension, and the supernatant is aspirated, repeated 2-3 times to obtain more cellular components; added to the last sediment Appropriate amount of DMEM medium (Dulbecco's Minimum Eagle's Medium) containing 20% FBS (fetal calf serum), filtered through a 300 mesh screen, count the cells and adjust the cell concentration, inoculate the flask, 37 Ό, 5% C0 ₂ culture; after the cells were grown confluent with ^0.2 passaged 5% trypsin digestion process. Example 4: MTT (thiazole blue) method for detecting the effect of ethyl 3-hydroxybutyrate on the proliferation of rabbit glial shield cells. Cells with good growth conditions (the cells obtained in Example 3, after 3 subcultures) were digested with trypsin. The solution was treated for 2 minutes, the digestive juice was aspirated, and fresh culture solution was added to prepare a single cell suspension. The number of cells was measured on a hemocytometer. The cells were seeded in 96-well plates (5×10 ³ cells/well), and 37 V, 5% C0 ₂ cells were cultured for 24 hours. The culture solution was removed and washed twice with PBS (phosphate buffer pH 7.2). Add filter-sterilized DMEM medium (containing 10% FBS) containing a series of concentration gradients (0, 0.00025, 0.0005, 0.001, 0.005, 0.01, 0.05 g/1) of 3-hydroxybutyrate, each Concentrations were made in 6 parallel experiments. After 24 hours and 48 hours of incubation, cell viability was measured by MTT. MTT (5.0 _δ /1 ) 10 μl was added to each well of a 96-well plate and incubated at 37 °C for 4 hours. Discard the supernatant, add DMSO (dimethyl sulfoxide) 100μ1, shake for a few minutes, and read the absorbance at 570 nm on a fully automated microplate reader within 30 minutes.

0.00025-0.01 g/1 ethyl 3-hydroxybutyrate treated with rabbit gelatin for 24 hours, the MTT absorbance values were 0.11 ± 0.01, 0.09 ± 0 · 01, 0.12 ± 0 · 01, 0.14 ± 0.02, respectively. 0.12±0.02, compared with the control group (0.06±0.01), there was a significant difference (Ρ<0·05) (Fig. 4a); 0.00025-0.01 g/1 of 3-hydroxybutyrate ethyl ester treated rabbit diglial cells 48 small After the time, the MTT absorbance values were 0.16±0·01, 0·19±0.01, and 0.2 0.01, respectively, compared with the control group (0·1 ² ±0·01), which was significantly different (Ρ<0·0 ⁵ ). (Fig. 4b); illustrates that low concentrations of ethyl 3-hydroxybutyrate can significantly promote the proliferation of rabbit glial cells. Example 5: MTT assay for the effect of methyl 3-hydroxybutyrate on the proliferation of rabbit glial cells Cells with good growth conditions (cells obtained in Example 3, after 3 subcultures) were treated with trypsin digest for 2 minutes. Aspirate the digestive juice and add a fresh medium to make a single cell suspension. The number of cells was measured on a hemocytometer. Inoculate in 96-well plates (5×10 ³ cells/well), and 37, 5% C0 ₂ cells were cultured for 24 hours. The culture solution was removed and washed twice with PBS. Add filter-sterilized DMEM medium (containing 10% FBS) containing a series of concentration gradients (0, 0.00025, 0.0005, 0.001, 0.005, 0.01, 0.05 g/1) of 3-hydroxybutyrate, each Concentrations were made in 6 parallel experiments. After 24 hours and 48 hours of incubation, cell viability was measured by MTT. MTT (5.0 g/l) 10 μl was added to each well of a 96-well plate and incubated at 37 ° C for 4 hours. Discard the supernatant, add DMSO lOO l, shake for a few minutes, and read the absorbance at 570 nm on a fully automated microplate reader within 30 minutes. MTT absorbance of all concentrations in rabbits treated with 3-hydroxybutyrate at each concentration for 24 hours (0.1 ³

±0.03, 0·15±0·02, 0.12±0.01, 0,12±0·01, 0.11±0.01, 0.10±0.02) were significantly different from the control group (0.06±0.01) (Ρ<0· 0 ⁵ ) (Fig.; Similarly, MTT absorbance values of all concentrations in the concentration of methyl 3-hydroxybutyrate for ⁴⁸ hours after treatment of rabbits (0.15 ± 0.01, 0.17 ± 0.01, 0 · 18 ± 0.01) , 0.16±0.01, 0.15±0.01, 0.15±0.01) compared with the control group (0·12±0·01), there are significant differences

(Ρ<0.05) (Fig. 4b). The results showed that all concentrations of methyl ³ -hydroxybutyrate in the experiment can significantly promote the proliferation of rabbit glial cells. Example 6: Effect of DL-3-hydroxybutyric acid on proliferation of rabbit glial cells by MTT assay Cells with good growth condition (cells obtained in Example 3, after 3 subcultures) were treated with trypsin digest for 2 minutes. Aspirate the digestive juice and add a fresh medium to make a single cell suspension. The number of cells was measured on a hemocytometer. The cells were seeded in 96-well plates (5×10 ³ cells/well), and cultured at 37 ° C for 5% C0 ₂ cells for 24 hours. The culture solution was removed and washed twice with PBS. Add filter-sterilized DMEM medium (containing 10% FBS), which contains a series of thick Degree gradients (0, 0.00025, 0.0005, 0.001, 0.005, 0.01, 0.05 g/1) of racemic DL-3-hydroxybutyrate were performed in 6 parallel experiments for each concentration. After 24 hours and 48 hours of incubation, cell viability was measured by MTT. MTT (5.0 g/l) 10 μΐ was added to each well of a 96-well plate and incubated at 37 ° C for 4 hours. Discard the supernatant, add DMSO 100 μΐ, shake for a few minutes, and read the absorbance at 570 nm on a fully automated microplate reader within 30 minutes. After 0.001, 0.005 g/1 of DL-3-hydroxybutyric acid treated rabbit gelatin cells for 24 hours, the MTT absorbance was 0.09±0.01 0.12 ± 0.03, which was significantly different from the control group (0.06±0.01). (P<0.05) (Fig. 4a); MTT absorbance (0·20±0·03) of the 0.005 g/1 concentration group was treated with DL-3-hydroxybutyric acid for 48 hours. The control group (0.12±0.01) had a significant difference (Ρ<0.05) (Fig. 4b). The results showed that low concentrations of DL- ³ -hydroxybutyric acid promoted the proliferation of nerve cells, but not as effective as methyl 3-hydroxybutyrate and ethyl 3-hydroxybutyrate. Example 7: In situ observation of the effect of ethyl 3-hydroxybutyrate on apoptosis of rabbit guinea shield cells by fluorescence microscopy Preparation of cell samples: cells with good growth conditions (cells obtained in Example 3, after 3 subcultures) After treatment with trypsin digest for 1 minute, the digestive juice was aspirated, and fresh culture solution was added to prepare a single cell suspension. The number of cells was measured on a hemocytometer. A 0.1;3-0.1 ⁷ mm thick coverslip was placed in a 12-well plate, and a cell suspension of ΙΟΟμΙ lxlO ⁵ cells/ml was added; 37 Ό, 5% C0 ₂ cells were cultured for 4 hours, then 900 μl was added. The 2% FBS DMEM medium was further cultured for 20 hours. Medium was removed, PBS washed ^twice. 1 ml of DMEM medium containing 0.004 g/13 ³ -hydroxybutyrate ethyl ester was added and cultured for ⁴⁸ hours. Discard the medium and wash the cells twice with 1 ml of water-cooled PBS (lightly adhering to and pouring out the liquid). Annexin-V-FITC Apoptosis Detection Kit Staining: Add 500 μΐ Binding Buffer (binding buffer), ΙΟμΙ Annexin-V-FITC, 5μ1 ΡΙ (propidium iodide), shake gently, react at room temperature in the dark 15 minute. Remove the coverslip and place it on a slide (NIKON inverted microscope TE2000-E). Fluorescence microscopy showed that 3-hydroxybutyrate significantly delayed and inhibited neuronal apoptosis and death. The control cells were essentially in the dead phase, and more cells were stained with PI for red fluorescence, and a small number of cells were Annexin staining is green fluorescent (Fig. 5a). The cells treated with 0.004 g/1 3-hydroxybutyrate were basically in the apoptotic phase, and most of the cells were stained with aimexin for green fluorescence, and very few cells. It was stained with PI and showed red fluorescence (Fig. 5b). Example 8 Flow cytometry detection of ethyl 3-hydroxyindolebutyrate on rabbit glial cell cycle

Preparation and staining of flow cytometry cell samples: Cells with good growth conditions (cells obtained in Example 3, after 3 subcultures) were treated with trypsin for 2 minutes, the digested solution was aspirated, and fresh culture medium was added. Single cell suspension. The number of cells was measured on a hemocytometer. Inoculated in a glass cell culture flask ( ⁵ ×10 ⁴ cells/ml), and 37, 5% C0 ₂ cells were cultured for 24 hours. The culture solution was removed and washed twice with PBS (phosphate buffer pH 7.2). Filter-sterilized DMEM medium (containing 10% FBS) containing 0.005 g/l of ethyl 3-hydroxybutyrate was added. The cells were trypsinized, 1 ml of the culture solution was blown, and centrifuged at 1000 rpm for 10 minutes. Discard the supernatant, wash twice with PBS, and mix well with 0.5 ml PBS. The cells were aspirated in a 5 ml syringe and pipetted into 5 ml of 70% pre-chilled ethanol and fixed overnight at 4 °C. The fixed cells were collected by centrifugation at 1000 rpm for 10 minutes, and washed twice with PBS. Resuspend the cells in 0.5 ml PBS and gently spread (to prevent cell breakage). Add 1.5 μΐ of RNase A to a final concentration of 60 g/ml and digest for 37 minutes at 37 °C. 0.25 ml of PI solution was added to a final concentration of 20 g/ml, and stained in the water bath for 30 minutes in the dark. On the machine (BECKMAN Coulter Epics XL flow cytometry) detection.

Analysis of the cell population collected by flow cytometry by software showed that ethyl 3-hydroxybutyrate increased the proportion of cells in the S phase and the G ₂ /M phase. The ratio of the number of cells in the S phase and the number of cells in the G ₂ /M phase to the total number of cell populations can reflect the degree of cell division. After glial cells were treated with 0.005 g/1 ethyl 3-hydroxybutyrate, the number of cells in S phase and the number of cells in G ₂ /M phase increased by 9.1% and 9.6%, respectively, indicating that ethyl 3-hydroxybutyrate It greatly promotes the division of glial cells into the S phase and G ₂ /M phase, which increases the degree of cell division and promotes cell proliferation (Fig. 6a, 6b). Example 9: Morris Water Maze (MWM) experimental mice feeding conditions and their grouping

KM mice: body weight 20 ± 2 g; female and male half; clean grade. Maintenance: clean mouse experimental feeding room; temperature: 25±2 °C; humidity: 40-60%; light: 12 hours Alternating light and dark; Drinking water: Ultrapure water, free to drink; Feed: Rats use full-price nutritional pellets; Feeding method: Cage.

 The experimental mice were grouped as follows:

 Negative control group: 11 mice, fed naturally for 30 consecutive days.

 Positive control group: 9 mice were treated with L-acetyl-carnitine aqueous solution (30 mg/g/d) for 30 consecutive days.

 Sample experimental group: 30 mice (10 in each concentration group, three in total) were administered with a solution of methyl 3-hydroxybutyrate (20, 30, 40 mg/Kg/d) for 30 consecutive days. Example 10: Morris water maze observed changes in learning behavior in mice Position navigation was used to measure the ability of mice to learn about water maze. The experiment lasted 5 days and was trained 4 times a day (4 training mice each entered the water from four different quadrants into the water; if the mouse did not find the platform within 60 seconds, it should be led to the platform, then the incubation period is recorded as 60 Seconds, each training interval is 60 seconds), randomly select a water inlet point during training, put the mouse into the pool wall, observe and record: The route and latency of the mouse to find and climb the platform (Escape Latency, EL) . The results showed that the EL of each group in Example 9 was gradually shortened as the number of tests increased, and the EL of the positive control group and the sample test group were shorter than the negative control group. One-way analysis of variance was performed for each group. The EL values of the 30 mg/kg/day L-acetylcarnitine group and the low concentration (20, 30 mg/kg.d) 3-hydroxybutyrate group were observed at 5 days. It was 12.63±4.58 seconds, 11.24±3.14 seconds, and 8.45±2.09 seconds, which was significantly different from the negative control group (19.55±4·94 seconds) (Ρ<0.05), and the route to climb the platform was also better than the negative control. The group is simple. This shows that the learning ability of the mouse has been significantly improved. The EL value of the 40 mg/kg.d 3-hydroxybutyrate methyl ester group was 12.21±3.05 seconds, which was not significantly different from the negative control group (P>0.05) (Fig. 7a).

The Spatial Probe Test is used to measure the ability of a mouse to learn the platform's spatial position after learning to find a platform. After the fifth day of the navigation test, the water platform was removed, and then a water inlet point was placed to place the mouse into the water in the pool wall, and the measurement data was divided into three times: the number of times to pass through the original platform position within 60 seconds; the recorded mouse was searched within 60 seconds. The platform roadmap results showed that the cross-platform times of each concentration (20, 30, 40 mg/kg.d) of 3-hydroxybutyrate methyl group in the 60 seconds were 6.07±2.22> 8.13±2.83, 5.85±2.19, respectively. More than the negative control group (4.20±1.16), of which 30 mg/kg.d 3-hydroxybutyrate methyl ester group Obvious, and all have statistical differences (Ρ<0·0 ⁵ ). The cross-platform frequency in the L-acetylcarnitine group was 5.52±1.94 within 60 seconds, slightly higher than the negative control group, but the difference was not significant (Ρ>0.05) (Fig. 7b).

All mice subjected to the Morris water maze test were subjected to a Retention Test 48 hours after the space exploration test. Performed in three separate intervals, 5 minutes apart. The mouse enters the water from a fixed position facing away from the platform, and the time from the inflow to the climb of the platform is EL in a time limit of 60 seconds. After the mouse climbed onto the platform, it was quickly removed into the cage until the next test. The results showed that the EL values of the L-acetylcarnitine group and the mice in each concentration (20, 30, 40 mg/kg.d) 3-hydroxybutyrate methyl group were 20.05±4.25 seconds, 19.01±4.41 seconds, 15.51±6.17, respectively. Seconds and 19.45±7.52 seconds, compared with the negative control group ( 28.08±10.84 seconds), there was significant difference (Ρ<0.05), among which 30 mg/kg.d 3-hydroxybutyrate methyl group EL and negative control group Compared with the very significant difference (Ρ<0·01) (Fig. 7c). The 30 mg/kg.d methyl 3-hydroxybutyrate group and the negative control group continued their memory retention test after 45 days of natural feeding. The EL value of this group of mice was 8.78±3.21 sec, still more than the negative control group ( 18.98). ±7.52 s) is much lower, and the memory retention effect is significantly better than that of the negative control group (the difference is extremely significant 箸Ρ<0·01) (Fig. 7d). Example 11: Quantitative determination of crude liver fat in mice The washed Soxhlet extractor flask was dried at 103-105 for 2 hours, cooled in a desiccator, and weighed (this weight is denoted as B). Weigh a certain amount of sample (take the liver tissue of each group of mice in Example 9, dry at 103-105 ° C to constant weight) (the weight is recorded as C), grind, wrap it with filter paper, put Dip in the tube. Add 1/2 volume of petroleum ether to the small flask, connect the parts of the Soxhlet extractor, and heat and distill for 14-20 hours in a water bath at ⁸⁰ °C. After the extraction is completed, when the petroleum ether completely flows into the small flask, take out the filter paper bag, and then reflux once, wash the leaching tube and continue heating. After the petroleum ether liquid surface in the leaching tube approaches the upper end of the siphon tube and does not flow into the small flask, pour out the leaching tube. PetroChina ether. If petroleum ether is left in the small flask, continue heating and evaporate until the solvent has evaporated. The small flask was dried at 103-105 ° C for 0.5 hours, taken out and placed in a desiccator to cool to room temperature, and weighed (the weight is denoted as A"). Calculation method: Crude fat content % = (AB) / Cxl00% (A: co-weight of small flask and crude fat; B: weight of small flask; C: weight of sample). The results showed that the crude fat content of the negative control group was 9.71±1.46%, L-acetylcarnitine group and each concentration ( 20, 30, 40 mg/kg.d) 3-hydroxybutyrate methyl ester group The crude fat content of the mice was 10.24±3.35 %, 9.85±2.76%, 10.86±0.79%, 8.00±1.28%, and there was no significant difference in the crude fat content of the mice in each group (Fig. 8). Example 12: Quantitative determination of serum cholesterol Solution reagent: 10% ferric chloride solution: 10 g FeCl ₃ .6H ₂ 0 dissolved in phosphoric acid, adjusted to 100 ml, stored in a brown bottle; phosphorus iron reagent: 10 1.5 ml of ferric chloride solution in a 100 ml brown volumetric flask, add concentrated sulfuric acid to the mark; cholesterol standard stock solution: cholesterol 80 mg, dissolved in absolute ethanol, to a volume of 100 ml; cholesterol standard solution: the stock solution Dilute 10 times with absolute ethanol, this standard contains 0.08 mg of cholesterol per ml.

Experimental procedure: Take 75 L of each group of mouse serum in Example 9 and place it in a centrifuge tube. Shake well with 0.3 ml of absolute ethanol, then add 1.5 ml of absolute ethanol and shake well. After 10 min, centrifuge at 3000 rpm for 5 min. Take the supernatant for use (ethanol extract). Another centrifuge tube number, blank tube: 0.75 ml absolute ethanol; standard tube: 0.75 ml cholesterol standard solution; sample tube: 0.75 ml ethanol extract. Each tube was added with 0.75 ml of phosphorus pyrite reagent, and after 10 minutes, OD _{56() was} measured by spectrophotometer. The results showed that the serum of the negative control group had a serum cholesterol content of 0.154±0.03%, and the serum of the L-acetylcarnitine group and each concentration (20, 30, 40 mg/kg.d) of the methyl 3-hydroxybutyrate group. The cholesterol levels were 0.154±0.05%, 0·125±0·03%, 0·158±0.02%, 0.102±0.01%, among which 40 mg/kg.d 3-hydroxybutyrate methyl group was serum cholesterol. The content was significantly lower than that of the negative control group, but there was no significant difference between the other experimental groups and the control group (Fig. 9). The level of serum cholesterol is significantly related to the prevalence of coronary heart disease. If the serum cholesterol level is increased, it will undoubtedly increase the probability of coronary heart disease in mammals. The methyl ³ -hydroxybutyrate and L-acetylcarnitine used in our experiments did not increase the serum cholesterol level of the mice and therefore did not cause the risk of coronary heart disease in animals. Example 13: Bone marrow cell count The mice in each group of Example 9 were subjected to cervical dislocation and sacrifice. Each mouse was taken with 2 femurs, and each femur was washed out with bone marrow cells with 10 ml of 3 % acetic acid solution on a hemocytometer. Count the number of cells in 4 large squares, multiply the number of cells obtained by 2.5x100 000, which is 1 femur. The number of nucleated cells in the bone marrow. The experimental results showed that the number of bone marrow nucleated cells per femur in the L-acetylcarnitine group was 8.125±1.24χ10 ⁶ , 20 mg/kg.d, 30 mg/kg.d and 40 mg/kg.d. ^{In the 3} -hydroxybutyric acid methyl ester group, the number of bone marrow nucleated cells per femur was 8·5±0·89, 8.95±1·22, 8·7±1·06χ10 ⁶ , and the negative control mice The number of nucleated cells in the bone marrow of each femur was only ⁵ · ⁹⁷⁵ ± 1. ³⁹ <10 ^δ , and the bone marrow nucleated cells of each femur of mice in the L-acetylcarnitine group and each concentration of 3-hydroxybutyrate methyl group The number was significantly different from the negative control group (Ρ<0·01) (Fig. 10). Example 14: Preparation of rat brain glial cells Bal/c mice were recruited for 1 day, anesthetized with ether and decapitated under sterile conditions. Whole rat brains were taken and stored under sterile conditions in sterile D-Hanks (containing 50 U/mL penicillin + 50 mg/L streptomycin). Carefully remove the fibrous components such as meninges and blood vessels with a sterile scissors under a dissecting microscope, transfer the brain tissue to a sterile plastic culture sub-zone, and add 6 ml of pre-warmed 20% fetal bovine serum (FBS) pre-warmed at 37 °C. DMEM medium. The brain tissue was chopped into small pieces of about 15 mm ³ with a sterile scalpel and filtered through a 0.05 mm pore size filter. The filtered cells were centrifuged at 1200 rpm for 10 minutes. Discard the supernatant and resuspend in DMEM containing 20% FBS. The above centrifugation step was repeated, and the finally collected cells were suspended in DMEM medium containing 20% FBS, the cells were counted and the cell concentration was adjusted, and inoculated into a culture flask, and cultured at 37 ° C, ⁵ % CO ₂ . After the cells were confluent, they were subcultured with 0.25% trypsin. Example 15: Immunocytochemistry The cells in which the mouse glial cells were well-growed (the cells obtained in Example 14 were subcultured for 3 times) were treated with trypsin for 2 minutes, and the digested solution was aspirated and added. The fresh medium is made into a single cell suspension. The number of cells was measured on a hemocytometer. The cells were inoculated in a 96-well plate (5×10 ³ cells/well), 37 Ό, and 5% C0 ₂ cells were cultured for 24 hours, and the culture medium was changed, and the culture was continued for 48 hours. After gettering culture solution (phosphate buffer ρΗ 7 · ²⁾ was washed with 0.1 M PBS 2 times culture plate, a solution of 4% paraformaldehyde fixed cells for 30 minutes. The fixative was removed and the plate was washed 2 times with 0.1 M PBS solution. Identification of glial cells was performed by detecting whether the cells contained glial fibrillary acidic protein (GFAP). Primary antibody using rabbit anti-GFAP anti- The body was diluted 1:100 in PBS containing 0.3% Triton X-100 and 5% sheep serum. Using goat anti-rabbit secondary antibodies Cy3- IgG antibody, diluted 1: 30 ratio in a solution of PBS containing 0. ^3% Triton X-100 in. Primary antibody is at 4. The reaction was carried out overnight under C conditions, and the reaction was washed with 0.1 M PBS; the secondary antibody was reacted at room temperature for 2 hours, and then washed with 0.1 M PBS. The nuclei were counterstained using DAPI, stained for 30 minutes at room temperature; washed three times with PBS. Immunocytochemical detection results (magnification: 10 x 20) were observed under a fluorescent inverted microscope (NIKON Elipse TE2000). Randomly selected fluorescence micrographs showed that the primary antibody binds to GFAP, and the secondary antibody with Cy3 fluorescent luminescent group binds to the primary antibody, and red fluorescence (Cy3 fluoresces) can be observed under fluorescence inverted microscope. Dyeed by DAPI, emitting green fluorescence (Figure 11). Immunocytochemistry results confirmed that the cells isolated in Example 14 were mouse brain glial cells. Example 16: Flow cytometry detection of sodium D-3-hydroxybutyrate, sodium DL-3-hydroxybutyrate and

Effect of methyl 3-hydroxybutyrate on inhibition of apoptosis of mouse glial cells Cells with good growth condition (cells obtained in Example 14 after 3 subcultures) were treated with trypsin for 2 minutes to absorb digestive juice. Add fresh medium to make a single cell suspension. The number of cells was measured on a hemocytometer and seeded in 6-well plates (1 x 10 ^s cells/well). A negative control group, and a treatment group of sodium D-3-hydroxybutyrate, sodium DL-3-hydroxybutyrate and methyl 3-hydroxybutanoate (concentration of 10 mM) were separately set. Use DMEM + 20% FBS medium, 37. (, 5% C0 ₂ cells were cultured for 24 hours, the culture medium was aspirated, washed twice with PBS (phosphate buffer pH 7.2), and added to serum-free DMEM medium for 24 hours, respectively, to D- ³ -hydroxybutyrate. Sodium, DL- ³ -hydroxybutyrate and methyl 3-hydroxybutyrate were added to the group containing 10 mM sodium D-3-hydroxybutyrate, 10 mM sodium DL-3-hydroxybutyrate and 10 mM 3-hydroxyl Methyl butyrate was further cultured for 48 hours.

Cells were stained with Flowncytometry using the Annexin-V-FITC Apoptosis Detection Kit: Trypsinized cells, 1 ml of culture medium, and centrifuged at 1000 rpm for 10 minutes. Discard the supernatant, wash twice with PBS, and mix well with 0.5 ml PBS. The cells were aspirated in a 5 ml syringe and pipetted into 5 ml of 70% pre-chilled ethanol, 4 fixed overnight. The fixed cells were collected by centrifugation at 1000 rpm for 10 minutes, and washed twice with PBS. Resuspend the cells in 0.5 ml PBS and gently spread (to prevent cell breakage). Add 1.5μ1 RNase A to a final concentration of 60 g/ml, digest at 37 ° C for 30 minutes; add 0.25 ml PI solution to a final concentration of 20 g / inl, water bath In the dark, staining for 30 minutes; on the machine (BECKMAN Coulter Epics XL flow cytometry) detection.

The results of flow cytometry showed that the percentage of apoptosis in the negative control group was 94.3%, and the cells in the treatment group of D-3-hydroxybutyrate, DL-3-hydroxybutyrate and methyl 3-hydroxybutanoate were withered. The proportion of death was 84.3 %, 86.1% and 87.9% (Fig. 12a, 12b, 12c, 12d), sodium D-3-hydroxybutyrate, sodium DL-3-hydroxybutyrate and methyl 3-hydroxybutanoate treatment group The proportion of apoptosis was lower than that of the negative control group (Fig. 13). Example 17: In situ observation of the effects of sodium D-3-hydroxybutyrate, sodium DL-3-hydroxybutyrate and methyl 3-hydroxybutanoate on the apoptosis of murine striatum by fluorescence microscopy Cell culture conditions and control group The setting is the same as in the embodiment 16. The cultured 6-well plate was washed 3 times with PBS solution, and 100 DAPI Binding Buffer (binding buffer) and 5 μΐ DAPI (2-(4-amidinophenyl)- were added to the plate according to the instructions of the DAPI reaction kit. 6-indolecarbamidine dihydrochloride ) Dyeing solution. After the staining, the staining solution was removed, and the PBS solution was washed 3 times, and the cell culture plate was observed using a fluorescent inverted microscope (NIKON Elipse fluorescence inverted microscope TE2000). Randomly selected fluorescence micrographs showed that the nucleus of apoptotic cells was stained with DAPI and emitted blue fluorescence, sodium D-3-hydroxybutyrate, sodium DL-3-hydroxybutyrate and methyl 3-hydroxybutyrate. The number of apoptotic cells was reduced in the treatment group compared with the negative control group, and the number of apoptotic cells in the 3-hydroxybutyrate treatment group was significantly lower than that in the control group (Fig. 14). Example 18: Calcium imaging assay for the effects of sodium D-3-hydroxybutyrate, sodium DL-3-hydroxybutyrate and methyl 3-hydroxybutanoate on intracellular calcium concentration in murine glial cells The cells (the cells obtained in Example 14 were subcultured for 3 times) were subjected to trypsin digestion for 1 minute, the digested solution was aspirated, fresh culture medium was added to prepare a single cell suspension; and the number of cells was measured by a hemocytometer; 0.1: 3-0.17 mm thick poly-L-lysine coated coverslips were placed in 6-well plates, and 1x10 ^s /ml cell suspension and DMEM+20% FBS were added to the plates. The culture medium was incubated with 5% C0 ₂ cells for 24 hours. The control group setting was the same as in Example 16. After removing the culture solution, add 2 mM calcium ion-specific dye fura-4/AM and stain at 37 °C. Minute; normal physiological solution (ΝΡΜ, 45 mM NaCl, 5 mM KC1, 1.8 mM CaCl ₂ , 0.8 mM MgCl ₂ , 10 mM glucose, and 10 mM Hepes pH 7.4); preparation of sodium D-3-hydroxybutyrate with NPM , DL-3-hydroxybutyrate and methyl 3-hydroxybutyrate solution; the three solutions diffuse through the cells at a rate of 1 ml/min, and control the solution concentration by controlling the solution flow rate (10 mM D-3-hydroxyl Sodium butyrate, 10 mM sodium DL-3-hydroxybutyrate and 10 mM methyl 3-hydroxybutyrate). Calcium imaging results were recorded using a laser confocal microscope (Zises LSM 510).

 Calcium imaging test showed that D-3-hydroxybutyrate, DL-3-hydroxybutyrate and 3-hydroxybutyrate could cause a sudden increase in intracellular calcium concentration, which was compared with the negative control group. There was a statistically significant difference (P<0.05), the increase was 60 units in the sodium hydroxybutyrate treatment group, 120 units in the sodium hydroxybutyrate treatment group, and 160 units in the 3-hydroxybutyrate treatment group ( Figure 15). In addition, compared with sodium DL-3-hydroxybutyrate and sodium D-3-hydroxybutyrate, methyl 3-hydroxybutyrate has a concentration of only one thousandth of the former two, and thus has a more significant effect. (Fig. 15b). In addition, the increase in intracellular calcium concentration has a corresponding relationship with the concentration of methyl 3-hydroxybutyrate. 10 mM 3-hydroxybutyrate can cause an increase in intracellular calcium concentration of 240 units, while 5 mM 3-hydroxybutyrate Methyl esters only cause an increase in intracellular calcium concentration of approximately 100 units (Figure 16). It can be concluded that the effect of methyl 3-hydroxybutyrate on intracellular calcium ion concentration is concentration dependent.

To investigate the mechanism of intracellular calcium concentration increase caused by sodium D-3-hydroxybutyrate, sodium DL-3-hydroxybutyrate and methyl 3-hydroxybutyrate, to investigate whether the intracellular increased calcium ion is derived from extracellular The NPM solution, or the release of the source and intracellular calcium pool, was performed using NPM containing Ca ²⁺ and Mg ²⁺ and NPM not consuming these two cations, respectively. The results showed that 5 mM, 10 mM sodium D-3-hydroxybutyrate and 5 mM, 10 mM DL-3-hydroxybutyrate could cause intracellular calcium when using NPM containing Ca ²⁺ and Mg ²⁺ . Sudden increase in ion concentration: 5 mM D-3-hydroxybutyrate (abbreviated as (D)-3-HBNa or D-3-HBNa) compared to the negative control, maximum increase in calcium ion concentration at 69.7 seconds Is 111 units, 5 mM DL-3-hydroxybutyrate (abbreviated as (DL)-3-HBNa or DL-3-HBNa). At 59.2 seconds, the maximum increase in calcium ion concentration is 142.5 units, 10 mM D- 3-hydroxybutyrate sodium butyrate, when 57.4 seconds, the maximum increase calcium concentration of 96 units, 10 mM D-3- hydroxybutyrate sodium butyrate, when 61.5 ^seconds, the maximum increase calcium concentration of 1 unit ^53.4 (Fig. 17a). It can be concluded that the effect of sodium DL-3-hydroxybutyrate is stronger than that of sodium D-3-hydroxybutyrate, and there is a statistical difference (P<0.05), but there is no significant difference in the effect of different concentrations of the same substance. (P<0.05). 5mM, 10 mM sodium D-3-hydroxybutyrate and 5 mM, 10 mM DL-3-hydroxybutyrate can also cause intracellular calcium concentration when using NPM without Ca ²⁺ and Mg ²⁺ sudden rise: compared to negative control, 5 mM D-3- hydroxybutyrate sodium butyrate, when 53.3 seconds, the maximum increase calcium concentration of 6 units ^3.4, 5 mM DL-3- hydroxybutyrate sodium butyrate, 36 sec, the maximum increase calcium concentration of ^6.5 units ^3, 10 mM D-3- hydroxybutyrate sodium butyrate, when 73.8 seconds, the maximum increase calcium concentration of 91 units, 10 mM DL-3- hydroxybutyrate For sodium, at 7 ⁵ · ⁹ seconds, the maximum increase in calcium ion concentration was 54.1 units (Fig. 17b). It therefore follows, when using the NPM containing no Ca ²⁺ and M _g ^2+, the two substances causes intracellular calcium concentration effect has declined.

 Combined with the above analysis, it can be concluded that sodium D-3-hydroxybutyrate, sodium DL-3-hydroxybutyrate and methyl 3-hydroxybutyrate can not only induce intracellular calcium ion elevation by promoting extracellular calcium influx. High, can also increase intracellular calcium ion concentration by causing the release of intracellular calcium stores.

For further study of sodium D-3-hydroxybutyrate, sodium DL-3-hydroxybutyrate and methyl 3-hydroxybutanoate (abbreviated as M(D)-3-HB or M-3-HB) caused intracellular calcium The mechanism of the increase in ion concentration is the use of the calcium ion analog Nitredipine to competitively suppress calcium channel. The results showed that the increase of intracellular calcium concentration caused by sodium D-3-hydroxybutyrate, sodium DL-3-hydroxybutyrate and methyl 3-hydroxybutyrate was significantly reduced after adding 10 μΜ Nitredipine. The maximum increase in calcium ion concentration caused by mM 3-hydroxybutyrate decreased from 250 units to 120 units, and the maximum increase in calcium ion concentration caused by 3 mM D-3-hydroxybutyrate decreased from ⁷⁰ units to 42 units. units, ³ mM DL- 3 - hydroxybutyrate sodium calcium concentration caused by the increase in maximum decrease of 90 units ⁴⁵² units (FIG. 1, 18b, 18c).. The results show that Nitredipine competitively inhibits the L-type voltage-gated calcium channel, causing a decrease in the effects of the three substances. Therefore, it can be concluded that the three substances further cause an increase in intracellular calcium ion concentration by affecting the L-type voltage-gated calcium channel.

 The references referred to herein, including patent documents, academic papers, publications, etc., are hereby incorporated by reference in their entirety.

It will be appreciated that various changes and modifications can be made in the form and details without departing from the spirit and scope of the invention, which are considered to fall within the protection of the present invention. range. references:

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Claims

' ·, '","

A pharmaceutical composition for improving the learning and/or memory ability of a subject, comprising:

 a compound of the formula (I) as an active ingredient,

0

0H— C —CH ₂ — C— 0R】

(I)

 Or a pharmaceutically acceptable salt, solvate, hydrate, enantiomer, or prodrug thereof, wherein

a group selected from the group consisting of H, C _r C ₉ alkyl, C ₃ -C ₉ cycloalkyl, aryl and non-toxic metal ions;

R _{2 is} selected from the group consisting of H, CC ₉ alkyl, C ₃ -C ₉ cycloalkyl, C _r C ₉ alkoxy, and aryl;

 A pharmaceutically acceptable carrier.

2. The pharmaceutical composition according to claim 1, wherein the group consists of an alkyl group of H, CC ₇ and a non-toxic metal ion.

3. The pharmaceutical composition according to claim 2, wherein the group consists of an alkyl group of H, CC ₅ and a non-toxic metal ion.

The pharmaceutical composition according to claim 3, which is selected from the group consisting of H, (C ₃ alkyl group and non-toxic metal ion).

5. The pharmaceutical composition according to claim 4, wherein the non-toxic metal ions are Na+, K+ and Ca2 ⁺ .

The pharmaceutical composition according to any one of claims 1 to 5, wherein R _{2 is} selected from the group consisting of H, c ₇ alkyl, c _r c ₇ alkoxy and aryl.

The pharmaceutical composition according to claim 6, wherein R _{2 is} selected from the group consisting of pit groups of H, ac ₅ alkoxy groups.

The pharmaceutical composition according to claim 7, wherein R _{2 is} selected from the group consisting of an alkyl group of H, CC ₃ and an alkoxy group of cc ₃ .

The pharmaceutical composition according to claim 1, wherein the group consists of a group consisting of an alkyl group of H, C ₂ , Na ⁺ and K + , and R _{2 is} free from a C ₂ alkyl group.

 10. The pharmaceutical composition according to claim 1, wherein the compound of formula (I) is selected from the group consisting of:

 Ethyl 3-hydroxybutyrate;

 Ethyl 3-hydroxybutyrate;

 3-hydroxybutyric acid;

 Sodium D-3-hydroxybutyrate;

 Sodium DL-3-hydroxybutyrate.

 The pharmaceutical composition according to any one of claims 1 to 10, wherein the pharmaceutical composition is for treating a memory-related disease in a subject.

 12. A method for increasing a subject's learning and/or memory ability, comprising administering to the subject an effective amount of a compound of formula (I),

OH— CH— CH ₂ — C— OR

R ₂

(I)

 Or a pharmaceutically acceptable salt, solvate, hydrate, enantiomer, or prodrug thereof, wherein

a group consisting of H, (a wide alkyl group, a C ₃ -C ₉ cycloalkyl group, an aryl group, and a non-toxic metal ion);

R ₂ selected from the group consisting of H, (: wide ^ alkyl, cycloalkyl ₉ CC, CC alkoxy group and aryl group ₉ constituted.

13. The method according to claim 12, wherein the group consisting of an alkyl group of H, C ₇ and a non-toxic metal ion is selected.

 14. The method according to claim 13, wherein R is selected from the group consisting of alkyl groups of H, C Cs and non-toxic metal ions.

15. A method according to claim 14, wherein selected from the group consisting of H, (^ - alkyl group and a non-toxic metal ions ₍₃ constituted.

16. The method according to claim 15, wherein said non-toxic metal ion is Na+, K ⁺ And Ca ²⁺ .

The method according to any one of claims 12 to 16, wherein R _{2 is} selected from the group consisting of H, CVC ₇ alkyl, cc ₇ alkoxy and aryl.

18. The method according to claim 17, wherein R ₂ is selected from the group consisting of H, (: wide ^ alkyl, group c _s a group consisting of embankment.

19. The method according to claim 18, wherein R ₂ is selected from the group consisting of H, alkyl group, alkoxy group having ₃ to the CC configuration.

Alkyl 20. The method of claim 12, wherein selected from the group consisting of H, (^ a, Na ⁺ and K ⁺ from the group constituted, and R ₂ is selected from the group consisting of C ₂ alkyl constituted.

 21. A method according to claim 12 wherein the compound of formula (I) is selected from the group consisting of:

 Methyl 3-hydroxybutyrate;

 Ethyl 3-hydroxybutyrate;

 3-hydroxybutyric acid;

 Sodium D-3-hydroxybutyrate;

 Sodium DL-3-hydroxybutyrate.

 The method according to any one of claims 12 to 21, wherein the method is for treating a memory-related disease in a subject.

 23. Compounds of formula (I)

OH— CH— C3⁄4— C— OR,

 R,

(I)

 Or a pharmaceutically acceptable salt, solvate, hydrate, enantiomer, or prodrug thereof, for use in the manufacture of a medicament for improving the learning and/or memory ability of a subject, wherein

a group consisting of H, (a broad group of ₉ alkyl, C ₃ -C ₉ cycloalkyl, aryl and non-toxic metal ions;

R _{2 is} selected from the group consisting of H, an alkyl group, a C ₃ -C ₉ cyclodecyl group, a C ₉ alkoxy group, and an aryl group; A pharmaceutically acceptable carrier.

24. Use according to claim 23, wherein selected from the group consisting of H, C _r C alkyl group and non-toxic metal ion composed of _7.

 25. The use according to claim 24, wherein a group consisting of H, (: ^ alkyl and non-toxic metal ions is selected.

26. The use according to claim 25, wherein selected from the group consisting of H, (alkyl and non-toxic metal ion of the group consisting of C _3.

27. The use according to claim 26, wherein the non-toxic metal ions are Na+, K ⁺ and Ca2 ⁺ .

The use according to any one of claims 23 to 27, wherein R _{2 is} selected from the group consisting of H, C ₇ alkyl, alkoxy and aryl.

29. The use according to claim 28, wherein R ₂ is selected from the group consisting of H, (^ - (^ alkyl, C ₅ alkoxy group constituted.

30. Use according to claim 29, wherein selected from the group consisting of H, (^ - alkyl _<3 cc alkoxy group composed of _3.

The use according to claim 23, wherein a group consisting of an alkyl group of H, CC ₂ , Na+ and K+ is selected, and R _{2 is} selected from the group consisting of alkyl groups of CC ₂ .

 32. The use according to claim 23, wherein the compound of formula (I) is selected from the group consisting of:

 Methyl 3-hydroxybutyrate;

 Ethyl 3-hydroxybutyrate;

 3-hydroxybutyric acid;

 Sodium D-3-hydroxybutyrate;

 Sodium DL-3-hydroxybutyrate.

 The use according to any one of claims 23 to 32, wherein the medicament is for treating a memory-related disease in a subject.