WO2008110034A1 - Acide gras 3-hydroxy et ses dérivés pour améliorer l'apprentissage et/ou la mémoire chez des individus - Google Patents

Acide gras 3-hydroxy et ses dérivés pour améliorer l'apprentissage et/ou la mémoire chez des individus Download PDF

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WO2008110034A1
WO2008110034A1 PCT/CN2007/000815 CN2007000815W WO2008110034A1 WO 2008110034 A1 WO2008110034 A1 WO 2008110034A1 CN 2007000815 W CN2007000815 W CN 2007000815W WO 2008110034 A1 WO2008110034 A1 WO 2008110034A1
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group
hydroxybutyrate
alkyl
cells
memory
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PCT/CN2007/000815
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English (en)
Chinese (zh)
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Guoqiang Chen
Xianghui Zou
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Shantou University
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Priority to PCT/CN2007/000815 priority Critical patent/WO2008110034A1/fr
Priority to CNA2007800264258A priority patent/CN101489543A/zh
Publication of WO2008110034A1 publication Critical patent/WO2008110034A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • 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.
  • 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).
  • 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).
  • human memory can be divided into three stages.
  • the first stage is sensory memory.
  • 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
  • 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.
  • NMDA N-methyl-D-aspartate, N-methyl aspartate
  • the NMDA 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
  • AD Alzheimer's disease
  • 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.
  • 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).
  • PHA polyhydroxyalkanoates
  • 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 ).
  • 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);
  • 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 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.
  • 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,
  • R 2 is selected from the group consisting of H, C 9 alkyl or cycloalkyl, alkoxy and aryl; and a pharmaceutically acceptable carrier.
  • the compounds of the invention are amphiphilic molecules, they are advantageous for crossing the blood-brain barrier.
  • 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.
  • the substituents in the compounds of the invention! ⁇ and R 2 generally contain no more than nine carbon atoms.
  • 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.
  • 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 .
  • 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.
  • 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 .
  • brain degenerative diseases such as Alzheimer's disease
  • 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 .
  • 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 ⁇ for 5 min, 60 ⁇ 240, 2.
  • 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.
  • Figure 5 is a fluorescence micrograph showing the effect of ethyl 3-hydroxybutyrate on apoptosis and death of rabbit cerebral cortical glial cells: 37 ⁇ , 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 ⁇ 1x10 s /ml cell suspension; cell adherent culture After 4 hours, add 9 00 ⁇ l 2 % FBS DMEM medium for 20 hours), and take a photo with a NIKON TE2000-E fluorescence microscope.
  • Figure 6 is a flow cytometry test showing the effect of ethyl 3-hydroxybutyrate on the glial cell cycle of rabbits.
  • Figure 7 shows the changes in learning and memory behavior in mice in the MWM experiment.
  • the positioning navigation test measures the ability of mice to learn water maze;
  • 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.
  • 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.
  • 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
  • the invention provides a pharmaceutical composition for improving the learning and/or memory ability of a subject, comprising:
  • 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)
  • 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.
  • the compound of the invention was fed to mice for one month, passed Morris Water Maze
  • mice Musris water maze
  • mice Musris 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.
  • 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.
  • 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).
  • 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.
  • 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-
  • subject 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.
  • pharmaceutically acceptable salt 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,
  • 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.
  • 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, ⁇ , 1995, and Design of Prodrugs ⁇ . Bundgaard, Described in Elsdvier, New York 1985).
  • biohydrolyzable amide Unless otherwise owned, 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.
  • 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).
  • biohydrolyzable amides include, but are not limited to, lower alkyl amides, alpha-amino acid amides, alkoxy amides, and alkylaminoalkyl carbonyl amides.
  • 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.
  • 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.
  • racemic refers to an optically inactive mixture of equal amounts of left-handed and right-handed bodies.
  • 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.
  • alkyl refers to both branched and straight-chain saturated aliphatic hydrocarbon groups bearing a given number of carbon atoms.
  • an alkyl group is defined as a straight or branched group having 1, 2, 3, 4, 5, 6, 7, 8 or 9 carbons.
  • C alkyl specifically includes Base, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, iso-butyl, pentyl, hexyl, heptyl, octyl, decyl and the like.
  • cycloalkyl refers to a monocyclic saturated aliphatic hydrocarbon group having a given number of carbon atoms.
  • C 3 -C 9 cycloalkyl includes cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl and the like.
  • alkoxy 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.
  • aryl 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.
  • a carbocyclic aryl group Is a phenyl group.
  • non-toxic metal ion 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.
  • an effective amount 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.
  • topical administration for example, to the skin or lungs and/or airways
  • 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.
  • compositions of the present invention can be obtained by conventional methods using conventional carriers well known in the art.
  • 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.
  • 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.
  • 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.
  • the injectable sterile preparation may also be an injectable sterile solution or suspension in a parenterally acceptable non-toxic diluent or solvent, for example
  • 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.
  • 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 ⁇ 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.
  • 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.
  • 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.
  • 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 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 ⁇ , 5% C0 2 culture; after the cells were grown con
  • 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).
  • PBS phosphate buffer pH 7.2
  • 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 ( ⁇ 0 ⁇ 05) (Fig.
  • MTT (5.0 g/l) 10 ⁇ l 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
  • 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.
  • 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 ⁇ lxlO 5 cells/ml was added; 37 ⁇ , 5% C0 2 cells were cultured for 4 hours, then 900 ⁇ l 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 ⁇ Binding Buffer (binding buffer), ⁇ Annexin-V-FITC, 5 ⁇ 1 ⁇ (propidium iodide), shake gently, react at room temperature in the dark 15 minute.
  • 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 ⁇ 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.
  • 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.
  • 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) ( ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ 1.24 ⁇ 10 6 , 20 mg/kg.d, 30 mg/kg.d and 40 mg/kg.d.
  • the number of bone marrow nucleated cells per femur was 8 ⁇ 5 ⁇ 0 ⁇ 89, 8.95 ⁇ 1 ⁇ 22, 8 ⁇ 7 ⁇ 1 ⁇ 06 ⁇ 10 6 , and the negative control mice
  • the number of nucleated cells in the bone marrow of each femur was only 5 ⁇ 975 ⁇ 1.
  • 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).
  • 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 ⁇ , 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 ⁇ 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).
  • GFAP glial fibrillary acidic protein
  • Immunocytochemical detection results 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
  • 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).
  • 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 ⁇ DAPI (2-(4-amidinophenyl)- were added to the plate according to the instructions of the DAPI reaction kit. 6-indolecarbamidine dihydrochloride ) Dyeing solution.
  • 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 ( ⁇ , 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).
  • 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.
  • 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.
  • 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.
  • Motoshio A Sakata T Alpha-amylase inhibitor increases plasma 3- hydroxybutyric acid in food-restricted rats.

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Abstract

L'invention concerne une composition pharmaceutique destinée à améliorer l'apprentissage et/ou la mémoire chez des individus, qui comprend un acide gras 3-hydroxy et ses dérivés comme composants actifs, et l'utilisation des composés dans la fabrication de médicaments pour améliorer l'apprentissage et/ou la mémoire chez des individus.
PCT/CN2007/000815 2007-03-14 2007-03-14 Acide gras 3-hydroxy et ses dérivés pour améliorer l'apprentissage et/ou la mémoire chez des individus WO2008110034A1 (fr)

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PCT/CN2007/000815 WO2008110034A1 (fr) 2007-03-14 2007-03-14 Acide gras 3-hydroxy et ses dérivés pour améliorer l'apprentissage et/ou la mémoire chez des individus
CNA2007800264258A CN101489543A (zh) 2007-03-14 2007-03-14 提高受试者的学习和/或记忆能力的3-羟基脂肪酸及其衍生物

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US20210015730A1 (en) * 2013-03-12 2021-01-21 Tdeltas Limited Compound for use in protecting skin
WO2016012657A1 (fr) 2014-07-21 2016-01-28 Oulun Yliopisto Formes oligomériques du 3-hydroxybutyrate
GB2567273A (en) * 2017-06-27 2019-04-10 Tdeltas Ltd Compounds for new use
GB2567273B (en) * 2017-06-27 2020-10-07 Tdeltas Ltd Compounds for use in reducing liver fat
US11648228B2 (en) 2017-09-27 2023-05-16 Tdeltas Limited Method of treatment

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