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WO2016146026A1 - Method of increasing d-serine concentration and improving cognitive disorders - Google Patents

Method of increasing d-serine concentration and improving cognitive disorders Download PDF

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WO2016146026A1
WO2016146026A1 PCT/CN2016/076128 CN2016076128W WO2016146026A1 WO 2016146026 A1 WO2016146026 A1 WO 2016146026A1 CN 2016076128 W CN2016076128 W CN 2016076128W WO 2016146026 A1 WO2016146026 A1 WO 2016146026A1
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gallic acid
mice
serine
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brain
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Chia-Hung Hsieh
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CHINA MEDICAL UNIVERSITY
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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
    • 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
    • A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7024—Esters of saccharides
    • 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
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • 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
    • A61P25/24—Antidepressants
    • 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

Definitions

  • the disclosure relates to a method of increasing D-serine concentration and improving cognitive disorders.
  • D-serine is an endogenous co-agonist for the glycine modulatory binding site on the NR1 subunit of N-methyl-D-aspartate receptor (NMDAR) and potentiates glutamatergic neurotransmission in the central nervous system (CNS) (Mothet, J.P., et al. D-serine is an endogenous ligand for the glycine site of the N-methyl-D-aspartate receptor. Proceedings of the National Academy of Sciences of the United States of America 97, 4926-4931 (2000) ) .
  • D-serine High levels of D-serine are found in the brain via the serine racemase catalyzed conversion of L-serine into D-serine (Wolosker, H., Dumin, E., Balan, L. &Foltyn, V.N. D-amino acids in the brain: D-serine in neurotransmission and neurodegeneration. The FEBS journal 275, 3514-3526 (2008) ) . D-serine is degraded by D-amino acid oxidase (DAO) to maintain its balance (Pollegioni, L., Piubelli, L., Sacchi, S., Pilone, M.S. &Molla, G.
  • DAO D-amino acid oxidase
  • D-amino acid oxidases from yeast to humans. Cellular and molecular life sciences: CMLS 64, 1373-1394 (2007) ) .
  • D-serine regulates NMDAR-dependent long-term potentiation and/or long-term depression, which are basic processes of learning and memory, in the hypothalamic and hippocampal excitatory synapses (Panatier, A., et al. Glia-derived D-serine controls NMDA receptor activity and synaptic memory. Cell 125, 775-784 (2006) ; Rosenberg, D., et al.
  • the treatment of exogenous D-serine or the related compound D-cycloserine also rescues both synaptic plasticity and spatial memory in aged rodents (Baxter, M.G., et al. D-cycloserine, a novel cognitive enhancer, improves spatial memory in aged rats. Neurobiology of aging 15, 207-213 (1994) ; Potier, B., et al. Contribution of the d-Serine-Dependent Pathway to the Cellular Mechanisms Underlying Cognitive Aging. Frontiers in aging neuroscience 2, 1 (2010) ; Billard, J.M. &Rouaud, E. Deficit of NMDA receptor activation in CA1 hippocampal area of aged rats is rescued by D-cycloserine. The European journal of neuroscience 25, 2260-2268 (2007) ) .
  • D-amino acid oxidase is a peroxisomal enzyme containing flavin adenine dinucleotide (FAD) as cofactor that catalyzes the oxidative deamination of D-amino acids such as D-arginine, D-aspartate and D-serine (Pollegioni, L., Piubelli, L., Sacchi, S., Pilone, M.S. &Molla, G. Physiological functions of D-amino acid oxidases: from yeast to humans. Cellular and molecular life sciences: CMLS 64, 1373-1394 (2007) ) .
  • FAD flavin adenine dinucleotide
  • DAO D-arginine and D-aspartate
  • D-arginine and D-aspartate are not only affect pathways that regulate arterial pressure but also control melatonin and prolactin secretion
  • melatonin and prolactin secretion Nishimura, M., Takahashi, H., Nanbu, A., Sakamoto, M. &Yoshimura, M.
  • American journal of hypertension 10, 389-396 (1997) ; Pinilla, L., Gonzalez, D., Tena-Sempere, M., Aguilar, R.
  • D-amino acids in the brain D-serine in neurotransmission and neurodegeneration.
  • Regulation of NMDAR co-agonists through the pharmacological manipulation of DAO have been investigated as putative novel therapeutics to treat schizophrenia (Sacchi, S., Rosini, E., Pollegioni, L. &Molla, G. D-amino acid oxidase inhibitors as a novel class of drugs for schizophrenia therapy. Current pharmaceutical design 19, 2499-2511 (2013) ) . Since it has been demonstrated that psychiatric and cognitive disorders have a hypofunction of NMDAR (Paoletti, P., Bellone, C. &Zhou, Q.
  • NMDA receptor subunit diversity impact on receptor properties, synaptic plasticity and disease. Nature reviews. Neuroscience 14, 383-400 (2013) ; Lakhan, S.E., Caro, M. &Hadzimichalis, N. NMDA Receptor Activity in Neuropsychiatric Disorders. Frontiers in psychiatry 4, 52 (2013) ) , DAO inhibitors might be useful as novel therapeutics to treat these disorders.
  • Gallic acid (GA, 3, 4, 5-trihydroxybenzoic acid, chemical structure shown below) is one of the most important plant polyphenolic compounds, which can be abundantly found in gallnuts, sumac, witch hazel, tea leaves, oak bark, mango, and other plants or fruits (Ow, Y.Y. &Stupans, I. Gallic acid and gallic acid derivatives: effects on drug metabolizing enzymes. Current drug metabolism 4, 241-248 (2003) ) . It is also considered a putative active compound in tannin.
  • a method of increasing D-serine concentration in a brain of a subject includes administering an effective amount of gallic acid or a derivative of gallic acid, which is an inhibitor of D-amino acid oxidase, to increase D-serine concentration in the brain of the subject, so that the activation of the N-methyl-D-aspartate receptor in the brain can be increased.
  • a method of improving a cognitive disorder result from aging or a neuropsychiatric disorder includes administering an effective amount of gallic acid or a derivative of gallic acid to inhibit D-amino acid oxidase and increase an activation of a N-methyl-D-aspartate receptor and a cognitive function, so that the cognitive disorder result from aging or the neuropsychiatric disorder can be improved.
  • Fig. 1 shows gallic acid (GA) at different concentrations as an inhibitor of DAO, and a full dose response curve;
  • Figs. 2A and 2B respectively show the relative mRNA and protein levels of ectopic expression of human DAO in HEK293 cells (hDAO) at 48 h after lentiviral infection, and their control vector-infected cells (C. vector) are used as a reference;
  • Fig. 3 shows the D-Alanine concentration in human DAO overexpressed cells and control vector-infected cells with or without GA;
  • Fig. 4 shows the result of the DAO activity assay
  • Figs. 5A and 5B respectively show effects of orally feeding gallic acid on the daily diet and drinking water consumption of C57BL/6 mice;
  • Figs. 6A and 6B respectively show effects of orally feeding gallic acid on the body weight and organ weights of C57BL/6 mice;
  • Figs. 7A and 7B respectively shows the time courses of D-serine levels in plasma and brain after 0-100 mg/kg gallic acid administration
  • Fig. 8 shows relative DAO activity in the brain after 0-100 mg/kg gallic acid administration
  • Fig. 9 is a diagram showing the accumulation 18 F-FSAG in hippocampus tissues of C57BL/6 mice treated with vehicle and MK801, respectively.
  • Fig. 10A shows the results of the 18 F-FSAG accumulation amount in hippocampus tissues of 8-week-old (young) SAMP8 mice and 7-month-old (old) SAMP8 mice fed with various amounts of GA;
  • Fig. 10B shows the results of 18 F-FSAG accumulation amount in hippocampus tissues of wild type (WT) and DISC1 knockout (DISC1-/-) mice fed with various amounts of GA;
  • Fig. 10C shows the 18 F-FSAG accumulation in hippocampus of old SAMP8 mice and DISC1-/-mice with or without GA treatment derived from PET imaging studies;
  • Figs. 11A-11D respectively shows the test results of velocity, time in target quadrant, alternation rate and escape latency during Morris water maze performance
  • Fig. 12A shows the result of pass rate after the first three-day training period in wild type (WT) mice and DISC1 knockout (DISC1-/-) mice with or without GA treatment during the delayed non-match to place task;
  • Fig. 12B shows the result of correct rate in the delay times in VVT mice and DISC1-/-mice with or without GA treatment during the delayed non-match to place task.
  • derivatives of gallic acid include esters of gallic acid, tannic acid and catechin, wherein the esters of gallic acid includes methyl gallate, ethyl gallate, proply gallate and octyl gallate.
  • Gallic acid (GA) and esters of gallic acid have been used in food, cosmetics, and in pharmaceuticals as an antioxidant with nontoxic to mammals at pharmacological doses.
  • GA is an inhibitor of D-amino acid oxidase (DAO)
  • Fig. 1 shows GA at different concentrations as an inhibitor of DAO, and a full dose response curve. In Fig. 1, results are expressed as OD453 in function of the concentration of the inhibitor in the assay. GA inhibited the activity of human DAO on D-serine in a dose-dependent manner and the value for the 50%inhibitory concentration (IC 50 ) of GA was 46.22 ⁇ M.
  • Figs. 2A and 2B respectively show the relative mRNA and protein levels of ectopic expression of human DAO in HEK293 cells (hDAO) at 48 h after lentiviral infection, and their control vector-infected cells (C. vector) are used as a reference.
  • hDAO HEK293 cells
  • C. vector control vector-infected cells
  • Fig. 3 shows the D-Alanine concentration in human DAO overexpressed cells and control vector-infected cells with or without GA. Error bars denote the standard deviation within triplicate experiments. *P is ⁇ 0.0001 compared to control HEK293. # P is ⁇ 0.05 compared to vehicle.
  • ectopic expression of human DAO in HEK293 cells exhibited a decrease in intracellular D-alanine levels compared with control vector-infected cells.
  • the treatment of human DAO overexpressed cells with GA caused a significant increase in the intracellular D-alanine levels.
  • Fig. 4 shows the result of the DAO activity assay. Error bars denote the standard deviation within triplicate experiments. *P is ⁇ 0.0001 compared to control HEK293. In Fig. 4, it can be seen that GA also decreased cellular DAO activity in a dose-dependent manner. These results indicate that GA is an inhibitor of DAO.
  • C57BL/6 mice were respectively fed with regular drinking water and gallic acid solutions containing different dosages of GA for 12 weeks.
  • Control mice were supplied with regular drinking water and the treatment groups were fed with gallic acid solutions (0.1%, 0.5%and 1.0%gallic acid (w/v) in regular drinking water) for 12 weeks.
  • Figs. 5A and 5B respectively show effects of orally feeding gallic acid on the daily diet and drinking water consumption of C57BL/6 mice. Diet or drinking water consumption (g/d) per mouse is plotted as a function of time (weeks) for each group. From Figs. 5A and 5B, it was observed that GA feeding did not show any significant change in diet and fluid intake between control and GA-fed mice during the entire treatment regimen.
  • Figs. 6A and 6B respectively show effects of orally feeding gallic acid on the body weight and organ weights of C57BL/6 mice. Each group contains 12 -15 male and female mice. From Figs. 6A and 6B, it was also observed that no significant difference between control and GA-fed mice in body weight and organ weight after GA feeding. This result suggests GA feeding did not show any apparent toxicity.
  • GA increases D-serine level and inhibits DAO activity in vivo
  • Figs. 7A and 7B respectively shows the time courses of D-serine levels in plasma and brain after 0-100 mg/kg gallic acid administration. *P is ⁇ 0.05 compared to vehicle. Number of animals was 6 ⁇ 8.
  • GA increased D-serine levels in plasma and brain tissues in a dose dependent manner.
  • D-serine levels gradually increased, with peak expression at 4 h, after GA administration, then decreased from these peak levels, although expression remained significantly higher than in controls for up to 16 h.
  • Fig. 8 shows relative DAO activity in the brain after 0-100 mg/kg gallic acid administration. *P is ⁇ 0.05 compared to vehicle. Number of animals was 6 ⁇ 8. In Fig. 8, it was observed that a dose dependent decrease in the DAO activities versus the vehicle in brain.
  • GA is an inhibitor of DAO and increases D-serine levels in vitro and in vivo.
  • the effect of GA on the activation of NMDAR was assessed and tested whether GA is able to improve psychiatric and cognitive disorders-mediated hypofunction of NMDAR.
  • Senescence accelerated mice mice (Pallas, M., et al. From aging to Alzheimer′s disease: unveiling ′′the switch′′ with the senescence-accelerated mouse model (SAMP8) . Journal of Alzheimer′s disease : JAD 15, 615-624 (2008) ) and Disrupted-In-Schizophrenia-1 (DISC1) knockout mice (Jaaro-Peled, H. Gene models of schizophrenia: DISC1 mouse models. Progress in brain research 179, 75-86 (2009) ) are commonly used as models for aging and schizophrenia, respectively.
  • 18 F-FSAG 18 F-labelled alkylthiophenyl guanidine
  • PCP phencyclidine
  • 18 F-FSAG is a specific radioligand for PCP sites of the NMDA receptor. Therefore, after uptake of 18 F-FSAG, 18 F-FSAG can bind to the NMDA receptor. Then, the accumulation of 18 F-FSAG in the hippocampus tissues from C57BL/6 mice can be measured by PET or y-counter.
  • MK801 a non-competitive antagonist of the NMDA receptor.
  • MK801 phytocilpine
  • the treatment of MK801 can be used to show the radioligand specificity for the visualisation of activation of NMDA receptor.
  • the radioactivity levels in the hippocampus tissues were measured by ex vivo gamma counting.
  • Fig. 9 is a diagram showing the accumulation 18 F-FSAG in hippocampus tissues of C57BL/6 mice treated with vehicle (DMSO) and MK801, respectively. *P was ⁇ 0.01 compared to vehicle. Number of animals was 6 ⁇ 8. In Fig. 9, it can be observed that pretreatment of mice with MK801 blocked the binding of 18 F-FSAG. This result indicates the radioligand specificity for the activation of NMDAR.
  • Fig. 10A shows the results of the 18 F-FSAG accumulation amount in hippocampus tissues of 8-week-old (young) SAMP8 mice and 7-month-old (old) SAMP8 mice fed with various amounts of GA.
  • *P was ⁇ 0.01 compared to young mice.
  • # P was ⁇ 0.05 compared to vehicle.
  • Number of animals was 6 ⁇ 8.
  • 7-month-old (old) SAMP8 mice (old + vehicle) showed significantly decreased 18 F-FSAG accumulation in hippocampus compared to 8-week-old (young) SAMP8 mice.
  • old SAMP8 mice received intravenous injection of GA had a significant increase in the binding of 18 F-FSAG compared to mice with vehicle treatment.
  • Fig. 10B shows the results of 18 F-FSAG accumulation amount in hippocampus tissues of wild type (WT) and DISC1 knockout (DISC1-/-) mice fed with various amounts of GA.
  • *P was ⁇ 0.01 compared to wild type mice.
  • # P was ⁇ 0.05 compared to vehicle.
  • Number of animals was 6 ⁇ 8.
  • DISC1 knockout (DISC1-/-) mice showed significantly decreased 18 F-FSAG accumulation in hippocampus compared to wild type (VVT) mice.
  • DISC1-/- mice received intravenous injection ofGA had a significant increase in the binding of 18 F-FSAG compared to mice with vehicle treatment.
  • Fig. 10C shows the 18 F-FSAG accumulation in hippocampus of old SAMP8 mice and DISC1-/- mice with or without GA treatment derived from PET imaging studies. *Pwas ⁇ 0.01 compared to vehicle. Number of animals was 6 ⁇ 8. In Fig. 10C, it was observed that seven-month-old SAMP8 mice and DISC1 knockout mice received GA treatment exhibited an appreciably higher accumulation of radioactive substances in the hippocampus compared with control mice.
  • GA improves age and psychiatry-learning and memory deficits
  • the Morris Water Maze (MWM) task involves placing a mouse in a pool of water where it must use visual cues to remember the location of a hidden platform just below the water′s surface. Probe trials (transfer tests) are also used to assess the mouse′s ability to retrieve information learned in previous hidden platform tests.
  • the MWM test measures spatial learning and memory. This is one of the most popular tasks in behavioral neuroscience and is sensitive to both the amnestic and memory-enhancing effects of drugs, as well as gene manipulation dependent on intact hippocampal function.
  • Figs. 11A-11D respectively shows the test results of velocity, time in target quadrant, alternation rate and escape latency during Morris water maze performance.
  • *P was ⁇ 0.001 compared to young mice.
  • # P was ⁇ 0.05 compared to vehicle.
  • the used mice includes 8-week-old (young) SAMP8 mice and seven-month-old (old) SAMP8 mice with or without GA treatment. Number of animals was 6 ⁇ 8. Behavioral tests were performed 4 weeks after drug administration, and the drugs administration lasted until all tests were finished.
  • Fig. 11A seven-month-old SAMP8 mice swam more slowly than 8-week-old SAMP8 mice. GA treatment did not cause any significant difference in velocity.
  • Fig. 11B seven-month-old SAMP8 mice (old) took more time to find the platform compared with 8-week-old SAMP8 mice (young) , since the spent time of the seven-month-old SAMP8 mice in the target quadrant is less than the spent time of the 8-week-old SAMP8 mice. However, after receiving GA treatment for 4 weeks, the spent time of seven-month-old SAMP8 mice in the target quadrant is increased.
  • Fig. 12A shows the result of pass rate after the first three-day training period in wild type (WT) mice and DISC1 knockout (DISC1-/-) mice with or without GA treatment during the delayed non-match to place task.
  • *P was ⁇ 0.001 compared to VVT mice.
  • # P was ⁇ 0.05 compared to vehicle.
  • wild-type mice had a higher passing percentage after 3 days of training compared to DISC1 knockout mice during the training phase.
  • DISC1 knockout mice received GA treatment for four weeks showed a significant increase in passing percentage during training phase, compared to mice with vehicle feeding.
  • Fig. 12B shows the result of correct rate in the delay times in wild type (WT) mice and DISC1 knockout (DISC1-/-) mice with or without GA treatment during the delayed non-match to place task.
  • *P was ⁇ 0.001 compared to WT mice.
  • # P was ⁇ 0.05 compared to vehicle.
  • DISC1 knockout mice also had a lower correct rate compared to wild-type mice in the testing phase.
  • hDAO human DAO
  • cDNA was amplified in a reaction with Platinum Taq DNA polymerase (Invitrogen) using the hDAO primers as described previously (Chang, S.L., et al. The C-terminal region of G72 increases D-amino acid oxidase activity. Intemational journal of molecular sciences 15, 29-43 (2014) ) , which harboring 5’ Nhel and 3’and EcoRI sites. The fragments were subcloned into pAS2. EYFP. puro (National RNAi core facility, Academia Sinica, Taiwan) at the Nhel and EcoRI sites, respectively, and then the cDNA sequences were confirmed.
  • Lentiviral particles were generated by transiently cotransfecting 293T cells with the plasmids coding for (hDAO) in addition to plasmids encoding gag/pol and VSV-G envelope genes. Transfection was carried out with jetPEI reagent (Polyplus-Transfection) . Subconfluent cells were infected with lentivirus in the presence of 8 ⁇ g/ml polybrene (Sigma-Aldrich) . At 24 hours post-infection, medium were removed and replaced with fresh growth medium containing puromycin (0.5 ug/ml) select for infected ceils after 48 hours post-infection.
  • HEK293 cells stable transduced with control lentiviral vector and lentiviral vector encoding hDAO were termed control HEK293 and hDAO-overexpressing HEK293 cells, respectively.
  • the control HEK293 and hDAO-overexpressing HEK293 cells (1 ⁇ 10 6 cells) were seeded in 6-well plates and cultured in 2 mL of medium. On the next day, the medium was replaced with 1 mL of fresh medium, and then 100 ⁇ L of 0-60 ⁇ M gallic acid was added. After 30 min incubation, 100 ⁇ L of 20 mM D-alanine solution was added, and the cells were cultured for 24 h before extraction of amino acids in the cells.
  • the amino acids in the cells were extracted by methanol and the concentrations of D-and L-alanine were determined by high-performance liquid chromatography (HPLC) system in the cells as described previously (Katane, M., et al. Identification of novel D-amino acid oxidase inhibitors by in silico screening and their functional characterization in vitro. Journal of medicinal chemistry 56, 1894-1907 (2013) ) .
  • mice For toxicity study, eight-week-old male and female C57BL/6 mice were purchased from the Animal Facility of the National Science Counsel (NSC) and were randomly distributed into positive control and treatment groups. Positive control mice were supplied with regular drinking water and the treatment groups were fed with gallic acid (Sigma) solutions (0.1%, 0.5%and 1%gallic acid (w/v) in regular drinking water) for 12 weeks.
  • NSC National Science Counsel
  • mice were received intravenous injection of 0-100 mg/kg gallic acid for 16 ⁇ 32 hours.
  • 8-week-old and seven-month-old male SAMP8 mice eight-week-old male C57BL/6 mice and DISC1 knockout mice were received intravenous injection of 0-100 mg/kg gallic acid for 4 hours.
  • mice were treated intraperitoneal injection with either vehicle or gallic acid (0-100 mg/kg/day) for 4 weeks.
  • a guide cannula guide (outer diameter: 0.65 mm) was implanted in hippocampus (anterior-posterior, -3.0; lateral, +3.0; ventral, -1.8 mm from bregma) and secured to the skull with an anchor screw and acrylic dental cement.
  • a microdialysis probe (CMA10, Carnegie Medicin, Sweden; membrane length: 1 mm) was inserted and connected to a microinfusion pump set to a speed of 1 ⁇ l/min and then perfused with Ringer’s solution (147 mM NaCl, 4 mM KCI, 2.3 mM CaCl 2 ) . Probe positioning was histologically verified at the end of the experiments.
  • 18 F-labelled S-fluoroalkyl diarylguanidine-10 was performed by 18 F-fluorination of the protected precursor S-fluoroalkyl guanidine followed by acidic hydrolysis, as previously described (Robins, E.G., et al. Synthesis and in vitro evaluation of (18) F-labelled S-fluoroalkyl diarylguanidines: Novel high-affinity NMDA receptor antagonists for imaging with PET. Bioorganic &medicinal chemistry letters 20, 1749-1751 (2010) ) . The radiochemical purity of 18 F-FSAG was >95%.
  • mice were scanned on a small-animal positron emission tomography (PET) scanner (microPET; Concorde Microsystems) under isoflurane anesthesia. Static images (30 min) were obtained with a zoom factor of 2 in a 256 ⁇ 256 matrix. Calculations were corrected for radiation decay of 18 F and the amount of injected dose, and the consistent color scale was applied to all PET images.
  • PET positron emission tomography
  • Each animal was subjected to 4 trials per day for 6 consecutive days. After 6 days of training, the platform was removed from the pool and each animal was then placed in the pool at the same position and was allowed to swim for 1 minute. The swim velocity, latency in finding the platform, and time in the target quadrant were analyzed using the Any-maze. After 1 minute, the animal was removed from the maze, dried with a to wel, and returned to its cage beside an electric radiator.
  • mice were food-restricted to 85-90 %of their original weight. The body weight was maintained through the test period. Small pieces of food pellets were placed at two ends of the T-maze as a reward. After 2 days of habituation to the T-maze (10 min/day) , 2 days of forced alternation in the T-maze with the blocked opposite arm were conducted. In the training phase, a mouse was placed in the T-maze, forced turn to one arm, and the food reward at the terminal was consumed. After a 5-sdelay, the mouse was placed in the T-maze again.
  • the mouse was required to turn to the opposite side.
  • the testing phase was started. If not, the training phase was prolonged until the mouse fulfilled the required criteria. The number of days required to learn the criteria and the accuracy were recorded.
  • the delayed time was set to be 5, 15, 30, 45, and 60 s. The accuracy in each delay condition was calculated.
  • GA is a novel DAO inhibitor in vitro and in vivo.
  • GA treatment in senescence accelerated (SAMP8) mice and Disrupted-In-Schizophrenia-1 (DISC1) knockout mice demonstrated an improvement of age or schizophrenia-related abnormal glutamatergic neurotransmission and brain dysfunction such as D-serine decline, NMDA hypofunction and deterioration of memory and learning function, suggesting therapeutic potential of GA in psychiatric or cognitive disorders.
  • the derivatives of gallic acid such as tannic acid, catechin and esters of gallic acid, are able to further metabolize to gallic acid in living subjects. (Schantz M., Erk T. & Richling E.
  • gallic acid and gallic acid derivatives effects on drug metabolizing enzymes. Current drug metabolism 4, 241-248 (2003) ) . Therefore, the derivatives of gallic acid also have therapeutic potential in psychiatric or cognitive disorders by increasing D-serine concentration in the brain of the subject.
  • the pharmaceuticals or pharmaceutical compositions may contain pharmaceutically acceptable adjuvants which is compatible with the other ingredients of the formulation and is compatible with the living body.
  • pharmaceutically acceptable adjuvant′′ or ′′pharmaceutically acceptable carrier′′ means a pharmaceutically acceptable material, composition or vehicle, such as liquid or solid filler, diluent, excipients, a solvent or encapsulating material. It can be used in carrying or transporting the subject composition from the organ or part of the body to another organ or part of the body.
  • the term ′′acceptable′′ means a carrier which may contain other compatible components of composition.
  • the carrier may be solid, semi-solid, liquid, cream or capsule form.
  • the pharmaceutical composition including the GA or its derivatives as the main active ingredient is used to increase D-serine concentration in the brain of the subject can be administered enterally, mucosally and/or parenterally.
  • the enteral route of administration includes oral and rectal administration.
  • the mucosally route of administration includes buccal, sublingual, and intranasal administration.
  • the parenteral route of administration includes intravenous, intramuscular, intraarterial, intraperitoneal, intracerebroventricular, intraparenchymal, intrathecal, intracranial, subcutaneous, transdermal, intradermal, intrapulmonary, inhalation, topical, and instillation into a body of the subject.
  • the administration may be administered pharmaceutical alone or in combination with other conventional pharmaceutically acceptable adjuvants.
  • the pharmaceutical composition of the present disclosure is administered to a subject via the oral route (e.g., through food) .
  • GA or its derivatives also can be used as the pharmaceutical, the pharmaceutical composition or the food supplement for improving a cognitive disorder result from aging or a neuropsychiatric disorder by improving an impairment of the cognitive function.
  • the neuropsychiatric disorder can be schizophrenia, depression, attention deficit disorder, mild cognitive impairment, dementia, attention deficit hyperactivity disorder (ADHD) or bipolar disorder.

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Abstract

The present disclosure concerns the use of gallic acid or a derivative of gallic acid. The gallic acid or the derivative of gallic acid can be an inhibitor of D-amino acid oxidase. Therefore, N-methyl-D-aspartate receptor (NMDAR) can be activated to improve a cognitive result from aging or a neuropsychiatric disorder.

Description

METHOD OF INCREASING D-SERINE CONCENTRATION AND IMPROVING COGNITIVE DISORDERS
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the priority benefit of U.S. provisional application serial no. 62/132,502， filed March 13， 2015， the full disclosure of which is incorporated herein by reference.
BACKGROUND Technical Field
The disclosure relates to a method of increasing D-serine concentration and improving cognitive disorders.
Description of Related Art
D-serine is an endogenous co-agonist for the glycine modulatory binding site on the NR1 subunit of N-methyl-D-aspartate receptor (NMDAR) and potentiates glutamatergic neurotransmission in the central nervous system (CNS) (Mothet， J.P.， et al. D-serine is an endogenous ligand for the glycine site of the N-methyl-D-aspartate receptor. Proceedings of the National Academy of Sciences of the United States of America 97， 4926-4931 (2000) ) .
High levels of D-serine are found in the brain via the serine racemase catalyzed conversion of L-serine into D-serine (Wolosker， H.， Dumin， E.， Balan， L. &Foltyn， V.N. D-amino acids in the brain： D-serine in neurotransmission and neurodegeneration. The FEBS journal 275， 3514-3526 (2008) ) . D-serine is  degraded by D-amino acid oxidase (DAO) to maintain its balance (Pollegioni， L.， Piubelli， L.， Sacchi， S.， Pilone， M.S. &Molla， G. Physiological functions of D-amino acid oxidases： from yeast to humans. Cellular and molecular life sciences： CMLS 64， 1373-1394 (2007) ) . Several lines of evidence suggest that D-serine regulates NMDAR-dependent long-term potentiation and/or long-term depression， which are basic processes of learning and memory， in the hypothalamic and hippocampal excitatory synapses (Panatier， A.， et al. Glia-derived D-serine controls NMDA receptor activity and synaptic memory. Cell 125， 775-784 (2006) ； Rosenberg， D.， et al. Neuronal D-serine and glycine release via the Asc-1 transporter regulates NMDA receptor-dependent synaptic activity. The Journal of neuroscience ： the official journal of the Society for Neuroscience 33， 3533-3544 (2013) ； Henneberger， C.， Papouin， T.， Oliet， S.H. &Rusakov， D.A. Long-term potentiation depends on release of D-serine from astrocytes. Nature 463， 232-236 (2010) ) . These mechanisms suggest a physiological significance of D-serine in the regulation of the glutamatergic neurotransmission and further mediates brain functions. Despite its normal physiological functions， perturbation of D-serine levels in CNS has now been implicated in the pathophysiology of various neuropsychiatric disorders， including schizophrenia， Alzheimer’s disease and amyotrophic lateral sclerosis (ALS) (Nunes， E.A.， et al. D-serine and schizophrenia： an update. Expert review of neurotherapeutics 12， 801-812 (2012) ； Hashimoto， K.， et al. Possible role of D-serine in the pathophysiology of Alzheimer′s disease. Progress in neuro-psychopharmacology &biological psychiatry 28， 385-388 (2004) ； Sasabe， J.， et al. D-serine is a key determinant of glutamate toxicity in amyotrophic lateral sclerosis. The EMBOjournal 26， 4149-4159 (2007) ) .
A decrease in D-serine levels in CNS and the resultant dysfunction of NMDAR-mediated neurotransmission has now been postulated in the onset of various mental disorders including schizophrenia and aging brain (Nishikawa， T. [Metabolism and functions of brain D-serine in mammals： relevance to neuropsychiatric disorders] . Seikagaku. The Journal of Japanese Biochemical Society 80， 267-276 (2008) ) . Patients with schizophrenia have the decreased serum levels of D-serine (Hashimoto， K.， et al. Decreased serum levels of D-serine in patients with schizophrenia： evidence in support of the N-methyl-D-aspartate receptor hypofunction hypothesis of schizophrenia. Archives of general psychiatry 60， 572-576 (2003) ) ， providing an evidence in support of the NMDAR hypofunction hypothesis of schizophrenia. A number of clinical studies have demonstrated that D-serine has a significant symptom improvements in schizophrenia patients (Kantrowitz， J.T.， et al. High dose D-serine in the treatment of schizophrenia. Schizophrenia research 121， 125-130 (2010) ； Ermilov， M.， et al. A pilot double-blind comparison of d-serine and high-dose olanzapine in treatment-resistant patients with schizophrenia. Schizophrenia research 150， 604-605 (2013) ) ， indicating therapeutic potential of D-serine in schizophrenia. Besides， several studies indicate that aged rats have the lower availability of D-serine in the hippocampus and age-mediated impaired long-term potentiation (LTP) and NMDAR-mediated synaptic potentials are rescued by exogenous D-serine (Mothet， J.P.， et al. A critical role for the glial-derived neuromodulator D-serine in the age-related deficits of cellular mechanisms of learning and memory. Aging cell 5， 267-274 (2006) ； Turpin， F.R.， et al. Reduced serine racemase expression contributes to age-related deficits in hippocampal cognitive function. Neurobiology of aging 32， 1495-1504 (2011) ) .  The treatment of exogenous D-serine or the related compound D-cycloserine also rescues both synaptic plasticity and spatial memory in aged rodents (Baxter， M.G.， et al. D-cycloserine， a novel cognitive enhancer， improves spatial memory in aged rats. Neurobiology of aging 15， 207-213 (1994) ； Potier， B.， et al. Contribution of the d-Serine-Dependent Pathway to the Cellular Mechanisms Underlying Cognitive Aging. Frontiers in aging neuroscience 2， 1 (2010) ； Billard， J.M. &Rouaud， E. Deficit of NMDA receptor activation in CA1 hippocampal area of aged rats is rescued by D-cycloserine. The European journal of neuroscience 25， 2260-2268 (2007) ) .
D-amino acid oxidase (DAO) is a peroxisomal enzyme containing flavin adenine dinucleotide (FAD) as cofactor that catalyzes the oxidative deamination of D-amino acids such as D-arginine， D-aspartate and D-serine (Pollegioni， L.， Piubelli， L.， Sacchi， S.， Pilone， M.S. &Molla， G. Physiological functions of D-amino acid oxidases： from yeast to humans. Cellular and molecular life sciences： CMLS 64， 1373-1394 (2007) ) . High levels of DAO expression and enzyme activity are found in the mammalian liver， kidney， and brain although the expression pattern can change between species (Kawazoe， T.， Park， H.K.， lwana， S.， Tsuge， H. &Fukui， K. Human D-amino acid oxidase： an update and review. Chemical record 7， 305-315 (2007) ) . Although the physiological role of DAO in the brain is less clear， DAO mRNA and protein are localized to neurons in the prefrontal cortex， hippocampus and substantia nigra (Verrall， L.， et al. d-Amino acid oxidase and serine racemase in human brain： normal distribution and altered expression in schizophrenia. The European journal of neuroscience 26， 1657-1669 (2007) ) . DAO’s substrates， i.e. D-arginine and D-aspartate， are not only affect pathways that regulate arterial pressure but also control  melatonin and prolactin secretion (Nishimura， M.， Takahashi， H.， Nanbu， A.， Sakamoto， M. &Yoshimura， M. Cardiovascular regulation by L-arginine in the brain of rats： role of the brain renin-angiotensin system and nitric oxide. American journal of hypertension 10， 389-396 (1997) ； Pinilla， L.， Gonzalez， D.， Tena-Sempere， M.， Aguilar， R. &Aguilar， E. Effects of N-methyl-D-aspartate and kainic acid on prolactin secretion in prepubertal female rats. European journal of endocrinology /European Federation of Endocrine Societies 135， 464-468 (1996) ； lshio， S.， et al. D-aspartate modulates melatonin synthesis in rat pinealocytes. Neuroscience letters 249， 143-146 (1998) ) . Moreover， D-serine has been shown to play a role in learning， memory and cognitive function via functioning as co-agonists of the NMDAR (Wolosker， H.， Dumin， E.， Balan， L. & Foltyn， V.N. D-amino acids in the brain： D-serine in neurotransmission and neurodegeneration. The FEBS journal 275， 3514-3526 (2008) ) . Regulation of NMDAR co-agonists through the pharmacological manipulation of DAO have been investigated as putative novel therapeutics to treat schizophrenia (Sacchi， S.， Rosini， E.， Pollegioni， L. &Molla， G. D-amino acid oxidase inhibitors as a novel class of drugs for schizophrenia therapy. Current pharmaceutical design 19， 2499-2511 (2013) ) . Since it has been demonstrated that psychiatric and cognitive disorders have a hypofunction of NMDAR (Paoletti， P.， Bellone， C. &Zhou， Q. NMDA receptor subunit diversity： impact on receptor properties， synaptic plasticity and disease. Nature reviews. Neuroscience 14， 383-400 (2013) ； Lakhan， S.E.， Caro， M. &Hadzimichalis， N. NMDA Receptor Activity in Neuropsychiatric Disorders. Frontiers in psychiatry 4， 52 (2013) ) ， DAO inhibitors might be useful as novel therapeutics to treat these disorders.
Gallic acid (GA， 3， 4， 5-trihydroxybenzoic acid， chemical structure shown below) is one of the most important plant polyphenolic compounds， which can be abundantly found in gallnuts， sumac， witch hazel， tea leaves， oak bark， mango， and other plants or fruits (Ow， Y.Y. &Stupans， I. Gallic acid and gallic acid derivatives： effects on drug metabolizing enzymes. Current drug metabolism 4， 241-248 (2003) ) . It is also considered a putative active compound in tannin.
Figure PCTCN2016076128-appb-000001
It has been reported that oral feeding gallic acid to TRAMP (Transgenic Adenocarcinoma of the Mouse Prostate) mice has the chemopreventive effects on tumor growth and progression， suggesting GA possess antitumor effect. Moreover， GA-containing plant extracts have showed the antidiabetic， antiangiogenic and antimelanogenic effects， reduced heart infarction incidence as well as oxidative liver and kidney damage， and neuroprotective effects on chemical-induced oxidative stress or ischemia/reperfusion injury (Kim， Y.J. Antimelanogenic and antioxidant properties of gallic acid. Biological &pharmaceutical bulletin 30， 1052-1055 (2007) ； Jadon， A.， Bhadauria， M. &Shukla， S. Protective effect of Terminalia belerica Roxb. and gallic acid against carbon tetrachloride induced damage in albino rats. Journal of ethnopharmacology 109， 214-218 (2007) ； Rasool， M.K.， et al. Hepatoprotective and antioxidant effects of gallic acid in paracetamol-induced liver damage in  mice. The Journal of pharmacy and pharmacology 62， 638-643 (2010) ； Bayramoglu， G.， et al. Preventive role of gallic acid on hepatic ischemia and reperfusion injury in rats. Cytotechnology (2014) ； Sun， J.， et al. Neuroprotective effects of gallic acid against hypoxia/reoxygenation-induced mitochondrial dysfunctions in vitro and cerebral ischemia/reperfusion injury in vivo. Brain research 1589， 126-139 (2014) ) .
SUMMARY
According to one aspect of the present disclosure， a method of increasing D-serine concentration in a brain of a subject is provided. The method includes administering an effective amount of gallic acid or a derivative of gallic acid， which is an inhibitor of D-amino acid oxidase， to increase D-serine concentration in the brain of the subject， so that the activation of the N-methyl-D-aspartate receptor in the brain can be increased.
In another aspect， a method of improving a cognitive disorder result from aging or a neuropsychiatric disorder is provided. The method includes administering an effective amount of gallic acid or a derivative of gallic acid to inhibit D-amino acid oxidase and increase an activation of a N-methyl-D-aspartate receptor and a cognitive function， so that the cognitive disorder result from aging or the neuropsychiatric disorder can be improved.
BRIEF DESCRIPTION OF THE DRAWINGS
The disclosure can be more fully understood by reading the following detailed description of the embodiment， with reference made to the accompanying drawings as follows：
Fig. 1 shows gallic acid (GA) at different concentrations as an inhibitor of DAO， and a full dose response curve；
Figs. 2A and 2B respectively show the relative mRNA and protein levels of ectopic expression of human DAO in HEK293 cells (hDAO) at 48 h after lentiviral infection， and their control vector-infected cells (C. vector) are used as a reference；
Fig. 3 shows the D-Alanine concentration in human DAO overexpressed cells and control vector-infected cells with or without GA；
Fig. 4 shows the result of the DAO activity assay；
Figs. 5A and 5B respectively show effects of orally feeding gallic acid on the daily diet and drinking water consumption of C57BL/6 mice；
Figs. 6A and 6B respectively show effects of orally feeding gallic acid on the body weight and organ weights of C57BL/6 mice；
Figs. 7A and 7B respectively shows the time courses of D-serine levels in plasma and brain after 0-100 mg/kg gallic acid administration；
Fig. 8 shows relative DAO activity in the brain after 0-100 mg/kg gallic acid administration；
Fig. 9 is a diagram showing the accumulation 18F-FSAG in hippocampus tissues of C57BL/6 mice treated with vehicle and MK801， respectively.
Fig. 10A shows the results of the 18F-FSAG accumulation amount in hippocampus tissues of 8-week-old (young) SAMP8 mice and 7-month-old (old) SAMP8 mice fed with various amounts of GA；
Fig. 10B shows the results of 18F-FSAG accumulation amount in hippocampus tissues of wild type (WT) and DISC1 knockout (DISC1-/-) mice fed with various amounts of GA；
Fig. 10C shows the 18F-FSAG accumulation in hippocampus of old SAMP8 mice and DISC1-/-mice with or without GA treatment derived from PET imaging studies；
Figs. 11A-11D respectively shows the test results of velocity， time in target quadrant， alternation rate and escape latency during Morris water maze performance； and
Fig. 12A shows the result of pass rate after the first three-day training period in wild type (WT) mice and DISC1 knockout (DISC1-/-) mice with or without GA treatment during the delayed non-match to place task； and
Fig. 12B shows the result of correct rate in the delay times in VVT mice and DISC1-/-mice with or without GA treatment during the delayed non-match to place task.
DETAILED DESCRIPTION
According to embodiments of the present disclosure， derivatives of gallic acid include esters of gallic acid， tannic acid and catechin， wherein the esters of gallic acid includes methyl gallate， ethyl gallate， proply gallate and octyl gallate. Gallic acid (GA) and esters of gallic acid have been used in food， cosmetics， and in pharmaceuticals as an antioxidant with nontoxic to mammals at pharmacological doses. (Choubey S.， et al. Medicinal importance of gallic acid and its ester derivatives： a patent review. Pharmaceutical patent analyst 4： 305-315 (2015) ； EFSA Journal 2015； 13 (10) ： 4248； Mol Pharmacol. 1995 May； 47 (5) ： 1021-7. ； Biomed. Chromatogr. 2010； 24： 472-478) . Tannic acid and the ester of epigallocatechin and GA in catechin， epigallocatechin-3-gallate， have been found the most important plant polyphenolic compounds and are  abundant in tea. (J. Agric. Food Chem. 2009， 57， 8041-8048) . It has been showed that these compounds can be metabolized into GA in vivo. The structural formula of the derivatives of gallic acid are listed as following.
Figure PCTCN2016076128-appb-000002
Reference will now be made in detail to the present embodiments of the disclosure， examples of which are illustrated in the accompanying drawings.  Wherever possible， the same reference numbers are used in the drawings and the description to refer to the same or like parts.
GA is an inhibitor of D-amino acid oxidase (DAO)
Human recombinant DAO enzyme was used to investigate in vitro DAO inhibitory activity for GA. Fig. 1 shows GA at different concentrations as an inhibitor of DAO， and a full dose response curve. In Fig. 1， results are expressed as OD453 in function of the concentration of the inhibitor in the assay. GA inhibited the activity of human DAO on D-serine in a dose-dependent manner and the value for the 50％inhibitory concentration (IC50) of GA was 46.22 μM.
To further verify GA on inhibition of cellular DAO， human DAO was stably overexpressed in HEK293 cells using lentiviral expression vectors. The human DAO overexpressed cells and their control vector-infected cells were cultured for 24 h in the presence of exogenous D-alanine. Figs. 2A and 2B respectively show the relative mRNA and protein levels of ectopic expression of human DAO in HEK293 cells (hDAO) at 48 h after lentiviral infection， and their control vector-infected cells (C. vector) are used as a reference. In Figs. 2A and 2B， it can be clearly seen that in the DAO overexpressed HEK293 cells， the mRNA and protein levels is far more than the levels in the controlled vector-infected cells.
Next， human DAO overexpressed cells and control vector-infected cells were exposed to various concentrations of GA and exogenous D-alanine (18 mM) for 24 h. The concentrations of D-and L-alanine in the cells were then extracted from the cells， and then determined by high-performance liquid  chromatography (HPLC) . For intracellular L-alanine levels (data not shown) ， there was no significant difference between human DAO overexpressed cells and control cells. Addition of GA in media did not change intracellular L-alanine levels.
For intracellular D-alanine levels， the results are shown in Fig. 3. Fig. 3 shows the D-Alanine concentration in human DAO overexpressed cells and control vector-infected cells with or without GA. Error bars denote the standard deviation within triplicate experiments. ＊P is ＜ 0.0001 compared to control HEK293. #P is ＜ 0.05 compared to vehicle. In Fig. 3， ectopic expression of human DAO in HEK293 cells exhibited a decrease in intracellular D-alanine levels compared with control vector-infected cells. However， the treatment of human DAO overexpressed cells with GA caused a significant increase in the intracellular D-alanine levels.
Moreover， in vitro DAO activity assay of cell lysates was also performed. The cell lysates was derived from human DAO overexpressed cells exposed to various concentrations of GA for 24 h. Fig. 4 shows the result of the DAO activity assay. Error bars denote the standard deviation within triplicate experiments. ＊P is ＜ 0.0001 compared to control HEK293. In Fig. 4， it can be seen that GA also decreased cellular DAO activity in a dose-dependent manner. These results indicate that GA is an inhibitor of DAO.
GA feeding did not show any apparent toxicity
To explore in vivo toxicity of GA， C57BL/6 mice were respectively fed with regular drinking water and gallic acid solutions containing different dosages of GA for 12 weeks. Control mice were supplied with regular drinking water  and the treatment groups were fed with gallic acid solutions (0.1％， 0.5％and 1.0％gallic acid (w/v) in regular drinking water) for 12 weeks.
Figs. 5A and 5B respectively show effects of orally feeding gallic acid on the daily diet and drinking water consumption of C57BL/6 mice. Diet or drinking water consumption (g/d) per mouse is plotted as a function of time (weeks) for each group. From Figs. 5A and 5B， it was observed that GA feeding did not show any significant change in diet and fluid intake between control and GA-fed mice during the entire treatment regimen.
Moreover， the effect of oral feeding of gallic acid on the body weight and organ weights of C57BL/6 mice were also measured. Figs. 6A and 6B respectively show effects of orally feeding gallic acid on the body weight and organ weights of C57BL/6 mice. Each group contains 12 -15 male and female mice. From Figs. 6A and 6B， it was also observed that no significant difference between control and GA-fed mice in body weight and organ weight after GA feeding. This result suggests GA feeding did not show any apparent toxicity.
GA increases D-serine level and inhibits DAO activity in vivo
The in vivo effect of GA on D-serine levels was investigated. D-serine levels in plasma and brain tissues were measured at several time points after intravenous injection of GA. Figs. 7A and 7B respectively shows the time courses of D-serine levels in plasma and brain after 0-100 mg/kg gallic acid administration. ＊P is ＜ 0.05 compared to vehicle. Number of animals was 6～8. In Figs. 7A and 7B， GA increased D-serine levels in plasma and brain tissues in a dose dependent manner. Moreover， D-serine levels gradually  increased， with peak expression at 4 h， after GA administration， then decreased from these peak levels， although expression remained significantly higher than in controls for up to 16 h.
To further confirm in vivo effect of GA on DAO activity， ex vivo measurement of DAO activity was also measured. Fig. 8 shows relative DAO activity in the brain after 0-100 mg/kg gallic acid administration. ＊P is ＜ 0.05 compared to vehicle. Number of animals was 6～8. In Fig. 8， it was observed that a dose dependent decrease in the DAO activities versus the vehicle in brain. These results suggest that GA inhibits in vivo DAO activities and further increases D-serine levels in plasma and brain.
GA increases the activation of NMDAR in SAMP8 and DISC1 knockout mice
In the foregoing， GA is an inhibitor of DAO and increases D-serine levels in vitro and in vivo. Next， the effect of GA on the activation of NMDAR was assessed and tested whether GA is able to improve psychiatric and cognitive disorders-mediated hypofunction of NMDAR.
Senescence accelerated (SAMP8) mice (Pallas， M.， et al. From aging to Alzheimer′s disease： unveiling ″the switch″ with the senescence-accelerated mouse model (SAMP8) . Journal of Alzheimer′s disease ： JAD 15， 615-624 (2008) ) and Disrupted-In-Schizophrenia-1 (DISC1) knockout mice (Jaaro-Peled， H. Gene models of schizophrenia： DISC1 mouse models. Progress in brain research 179， 75-86 (2009) ) are commonly used as models for aging and schizophrenia， respectively. To observe the activation of glutamate receptor in vivo， a positron emission tomography (PET) radiotracer， 18F-labelled  alkylthiophenyl guanidine (18F-FSAG) ， was synthesized for imaging the phencyclidine (PCP) site of the NMDA ion channel (Robins， E.G.， et al. Synthesis and in vitro evaluation of (18) F-labelled S-fluoroalkyl diarylguanidines： Novel high-affinity NMDA receptor antagonists for imaging with PET. Bioorganic &medicinal chemistry letters 20， 1749-1751 (2010) ) . 18F-FSAG is a specific radioligand for PCP sites of the NMDA receptor. Therefore， after uptake of 18F-FSAG， 18F-FSAG can bind to the NMDA receptor. Then， the accumulation of 18F-FSAG in the hippocampus tissues from C57BL/6 mice can be measured by PET or y-counter.
First， the specificity of 18F-FSAG to the NMDA receptor was tested by MK801 (dizocilpine) ， a non-competitive antagonist of the NMDA receptor. Thus， the treatment of MK801 can be used to show the radioligand specificity for the visualisation of activation of NMDA receptor. The radioactivity levels in the hippocampus tissues were measured by ex vivo gamma counting.
Fig. 9 is a diagram showing the accumulation 18F-FSAG in hippocampus tissues of C57BL/6 mice treated with vehicle (DMSO) and MK801， respectively. ＊P was ＜ 0.01 compared to vehicle. Number of animals was 6～8. In Fig. 9， it can be observed that pretreatment of mice with MK801 blocked the binding of 18F-FSAG. This result indicates the radioligand specificity for the activation of NMDAR.
Fig. 10A shows the results of the 18F-FSAG accumulation amount in hippocampus tissues of 8-week-old (young) SAMP8 mice and 7-month-old (old) SAMP8 mice fed with various amounts of GA. ＊P was ＜ 0.01 compared to young mice. #P was ＜ 0.05 compared to vehicle. Number of animals was 6～8. In Fig. 10A， 7-month-old (old) SAMP8 mice (old + vehicle) showed  significantly decreased 18F-FSAG accumulation in hippocampus compared to 8-week-old (young) SAMP8 mice. However， old SAMP8 mice received intravenous injection of GA had a significant increase in the binding of 18F-FSAG compared to mice with vehicle treatment.
Fig. 10B shows the results of 18F-FSAG accumulation amount in hippocampus tissues of wild type (WT) and DISC1 knockout (DISC1-/-) mice fed with various amounts of GA. ＊P was ＜ 0.01 compared to wild type mice. #P was ＜ 0.05 compared to vehicle. Number of animals was 6～8. In Fig. 10B， DISC1 knockout (DISC1-/-) mice showed significantly decreased 18F-FSAG accumulation in hippocampus compared to wild type (VVT) mice. However， DISC1-/- mice received intravenous injection ofGA had a significant increase in the binding of 18F-FSAG compared to mice with vehicle treatment.
PET imaging is also used to evaluate the 18F-FSAG accumulation in hippocampus of old SAMP8 mice and DISC1-/- mice with or without GA treatment. Fig. 10C shows the 18F-FSAG accumulation in hippocampus of old SAMP8 mice and DISC1-/- mice with or without GA treatment derived from PET imaging studies. ＊Pwas ＜ 0.01 compared to vehicle. Number of animals was 6～8. In Fig. 10C， it was observed that seven-month-old SAMP8 mice and DISC1 knockout mice received GA treatment exhibited an appreciably higher accumulation of radioactive substances in the hippocampus compared with control mice.
Taken together， these findings indicate GA is able to improve psychiatric and cognitive disorders-mediated hypofunction of NMDAR.
GA improves age and psychiatry-learning and memory deficits
Finally， whether GA has the ability to improve age and psychiatry-related learning and memory deficits in SAMP8 and DISC1 knockout mice was tested via Morris water maze test or the delayed non-match to place task.
The Morris Water Maze (MWM) task involves placing a mouse in a pool of water where it must use visual cues to remember the location of a hidden platform just below the water′s surface. Probe trials (transfer tests) are also used to assess the mouse′s ability to retrieve information learned in previous hidden platform tests. The MWM test measures spatial learning and memory. This is one of the most popular tasks in behavioral neuroscience and is sensitive to both the amnestic and memory-enhancing effects of drugs， as well as gene manipulation dependent on intact hippocampal function.
Figs. 11A-11D respectively shows the test results of velocity， time in target quadrant， alternation rate and escape latency during Morris water maze performance. ＊P was ＜ 0.001 compared to young mice. #P was ＜ 0.05 compared to vehicle. The used mice includes 8-week-old (young) SAMP8 mice and seven-month-old (old) SAMP8 mice with or without GA treatment. Number of animals was 6～8. Behavioral tests were performed 4 weeks after drug administration， and the drugs administration lasted until all tests were finished.
In Fig. 11A， seven-month-old SAMP8 mice swam more slowly than 8-week-old SAMP8 mice. GA treatment did not cause any significant difference in velocity.
In Fig. 11B， seven-month-old SAMP8 mice (old) took more time to find the platform compared with 8-week-old SAMP8 mice (young) ， since the spent time of the seven-month-old SAMP8 mice in the target quadrant is less than the  spent time of the 8-week-old SAMP8 mice. However， after receiving GA treatment for 4 weeks， the spent time of seven-month-old SAMP8 mice in the target quadrant is increased.
In Fig. 11C the alternation rate of the seven-month-old SAMP8 mice was less than the 8-week-old SAMP8 mice. However， after receiving GA treatment for 4 weeks， the alternation rate of the seven-month-old SAMP8 mice is increased.
In Fig. 11D the escape latency of the seven-month-old SAMP8 mice was more than the 8-week-old SAMP8 mice. However， after receiving GA treatment for 4 weeks， the escape latency of the seven-month-old SAMP8 mice is decreased.
On the other hand， the effect of GA treatment on working memory of DISC1 knockout mice was examined using the delayed non-match recognition task by using T-maze. Behavioral tests were performed 4 weeks after drug administration， and the drugs administration lasted until all tests were finished. Number of animals was 6～8.
Fig. 12A shows the result of pass rate after the first three-day training period in wild type (WT) mice and DISC1 knockout (DISC1-/-) mice with or without GA treatment during the delayed non-match to place task. ＊P was ＜0.001 compared to VVT mice. #P was ＜ 0.05 compared to vehicle. In Fig. 12A， it was observed that wild-type mice had a higher passing percentage after 3 days of training compared to DISC1 knockout mice during the training phase. However， DISC1 knockout mice received GA treatment for four weeks showed a significant increase in passing percentage during training phase， compared to mice with vehicle feeding.
Fig. 12B shows the result of correct rate in the delay times in wild type (WT) mice and DISC1 knockout (DISC1-/-) mice with or without GA treatment during the delayed non-match to place task. ＊P was ＜ 0.001 compared to WT mice. #Pwas ＜ 0.05 compared to vehicle. In Fig. 12B， DISC1 knockout mice also had a lower correct rate compared to wild-type mice in the testing phase. However， DISC1 knockout mice received GA treatment for four weeks showed a significant increase in correct rate during the testing phase， compared to mice with vehicle feeding.
Taken together， these results suggest that GA has the ability to improve age and psychiatry-related learning and memory deficits.
Material and Methods
In vitro DAO Activity Assay
Human recombinant DAO enzyme (400 nM) derived from our previous study (Chang， S.L.， et al. The C-terminal region of G72 increases D-amino acid oxidase activity. International journal of molecular sciences 15， 29-43 (2014) ) was added into 3％ (w/v) o-phenylenediamine， 1 U horseradish peroxidase， 40 mM d-alanine (final volume， 200 μL) . The reaction at 25 ℃ generated hydrogen peroxide that was oxidized by the peroxidase and further converted， in the presence of o-phenylenediamine， to 2， 3-diaminophenazine， which was quantified by measuring OD450.
Plasmids
Full-length human DAO (hDAO) cDNA was amplified in a reaction with Platinum Taq DNA polymerase (Invitrogen) using the hDAO primers as  described previously (Chang， S.L.， et al. The C-terminal region of G72 increases D-amino acid oxidase activity. Intemational journal of molecular sciences 15， 29-43 (2014) ) ， which harboring 5’ Nhel and 3’and EcoRI sites. The fragments were subcloned into pAS2. EYFP. puro (National RNAi core facility， Academia Sinica， Taiwan) at the Nhel and EcoRI sites， respectively， and then the cDNA sequences were confirmed.
Lentivirus production and transduction
Lentiviral particles were generated by transiently cotransfecting 293T cells with the plasmids coding for (hDAO) in addition to plasmids encoding gag/pol and VSV-G envelope genes. Transfection was carried out with jetPEI reagent (Polyplus-Transfection) . Subconfluent cells were infected with lentivirus in the presence of 8 μg/ml polybrene (Sigma-Aldrich) . At 24 hours post-infection， medium were removed and replaced with fresh growth medium containing puromycin (0.5 ug/ml) select for infected ceils after 48 hours post-infection. HEK293 cells stable transduced with control lentiviral vector and lentiviral vector encoding hDAO were termed control HEK293 and hDAO-overexpressing HEK293 cells， respectively.
Cellular L-alanine and D-alanine assay
The control HEK293 and hDAO-overexpressing HEK293 cells (1 × 106 cells) were seeded in 6-well plates and cultured in 2 mL of medium. On the next day， the medium was replaced with 1 mL of fresh medium， and then 100 μL of 0-60 μM gallic acid was added. After 30 min incubation， 100 μL of 20 mM D-alanine solution was added， and the cells were cultured for 24 h before  extraction of amino acids in the cells. The amino acids in the cells were extracted by methanol and the concentrations of D-and L-alanine were determined by high-performance liquid chromatography (HPLC) system in the cells as described previously (Katane， M.， et al. Identification of novel D-amino acid oxidase inhibitors by in silico screening and their functional characterization in vitro. Journal of medicinal chemistry 56， 1894-1907 (2013) ) .
Cellular DAO Activity assay
Cell-based enzyme activity was evaluated as described (Brandish， P.E.， et al. A cell-based ultra-high-throughput screening assay for identifying inhibitors of D-amino acid oxidase. Journal of biomolecular screening 11， 481-487 (2006) ) . In brief， enzyme activity was evaluated in hDAO-overexpressing HEK293 cells. Enzyme activity was determined by monitoring hydrogen peroxide production， a by-product of the DAO reaction， using Amplex Red reagent (Invitrogen) together with horseradish peroxidase to produce the red fluorescent product resorufin. Fluorescence as detected at an excitation of 544 nm and an emission of 590 nm.
Animal treatments
For toxicity study， eight-week-old male and female C57BL/6 mice were purchased from the Animal Facility of the National Science Counsel (NSC) and were randomly distributed into positive control and treatment groups. Positive control mice were supplied with regular drinking water and the treatment groups were fed with gallic acid (Sigma) solutions (0.1％， 0.5％and 1％gallic acid (w/v) in regular drinking water) for 12 weeks.
For kinetic analysis of plasma D-serine， brain D-serine and DAO activity after GA treatments， C57BL/6 mice were received intravenous injection of 0-100 mg/kg gallic acid for 16 ～32 hours. In the observation of NMDAR activation in vivo， 8-week-old and seven-month-old male SAMP8 mice， eight-week-old male C57BL/6 mice and DISC1 knockout mice were received intravenous injection of 0-100 mg/kg gallic acid for 4 hours. For the behavior studies， mice were treated intraperitoneal injection with either vehicle or gallic acid (0-100 mg/kg/day) for 4 weeks.
Microdialysis
A guide cannula guide (outer diameter： 0.65 mm) was implanted in hippocampus (anterior-posterior， -3.0； lateral， +3.0； ventral， -1.8 mm from bregma) and secured to the skull with an anchor screw and acrylic dental cement. On the next day， a microdialysis probe (CMA10， Carnegie Medicin， Stockholm， Sweden； membrane length： 1 mm) was inserted and connected to a microinfusion pump set to a speed of 1 μl/min and then perfused with Ringer’s solution (147 mM NaCl， 4 mM KCI， 2.3 mM CaCl2) . Probe positioning was histologically verified at the end of the experiments.
Radiochemistry
Synthesis of 18F-labelled S-fluoroalkyl diarylguanidine-10 (18F-FSAG) was performed by 18F-fluorination of the protected precursor S-fluoroalkyl guanidine followed by acidic hydrolysis， as previously described (Robins， E.G.， et al. Synthesis and in vitro evaluation of (18) F-labelled S-fluoroalkyl diarylguanidines： Novel high-affinity NMDA receptor antagonists for imaging  with PET. Bioorganic &medicinal chemistry letters 20， 1749-1751 (2010) ) . The radiochemical purity of 18F-FSAG was ＞95％.
Biodistribution of 18F-FSAG
Animals received 29.6 MBq/kg of 18F-FSAG in 100 μL of PBS via lateral tail vein injection， and then were euthanized by CO2/O2 asphyxiation at 30 min after injection. After sacrifice， selected tissues of interest were then removed and weighed， and the radioactivity was measured using a y-counter. The percentage injected dose per gram (％ID/g) was then calculated.
MicroPET imaging
Each subject was injected with 9.25 MBq of 18F-FSAG. At 30 min after injection， mice were scanned on a small-animal positron emission tomography (PET) scanner (microPET； Concorde Microsystems) under isoflurane anesthesia. Static images (30 min) were obtained with a zoom factor of 2 in a 256 × 256 matrix. Calculations were corrected for radiation decay of 18F and the amount of injected dose， and the consistent color scale was applied to all PET images.
Morris water-maze task
The procedure of Morris water maze (MWM) was described previously (Morris， R. Developments of a water-maze procedure for studying spatial learning in the rat. Journal of neuroscience methods 11， 47-60 (1984) ) 39. Briefly， a plastic platform (diameter： 10 cm； height： 30 cm) was placed at the center of one quadrant in a pool with a diameter of 100 cm and height of 40 cm.  Before the experiment， the pool was filled with sufficient water so that the platform was approximately 1-2 cm beneath the water surface， and the water temperature was fixed at 22±1℃. During the experiment， all objects in the room were fixed in place to provide additional cues to enable the animals to locate the platform.
Each animal was subjected to 4 trials per day for 6 consecutive days. After 6 days of training， the platform was removed from the pool and each animal was then placed in the pool at the same position and was allowed to swim for 1 minute. The swim velocity， latency in finding the platform， and time in the target quadrant were analyzed using the Any-maze. After 1 minute， the animal was removed from the maze， dried with a to wel， and returned to its cage beside an electric radiator.
Delayed non-match to place task
The working memory of mice was evaluated using a delayed non-match to place task， which is considered to be an mPFC-related task. Prior to the experiment， mice were food-restricted to 85-90 ％of their original weight. The body weight was maintained through the test period. Small pieces of food pellets were placed at two ends of the T-maze as a reward. After 2 days of habituation to the T-maze (10 min/day) ， 2 days of forced alternation in the T-maze with the blocked opposite arm were conducted. In the training phase， a mouse was placed in the T-maze， forced turn to one arm， and the food reward at the terminal was consumed. After a 5-sdelay， the mouse was placed in the T-maze again. To receive the food reward， the mouse was required to turn to the opposite side. When a mouse exhibited an accuracy rate 80％for three  consecutive days， the testing phase was started. If not， the training phase was prolonged until the mouse fulfilled the required criteria. The number of days required to learn the criteria and the accuracy were recorded. In the testing phase， the delayed time was set to be 5， 15， 30， 45， and 60 s. The accuracy in each delay condition was calculated.
Statistical analysis
One-way analysis of variance with post hoc Scheffe analyses was carried out using the SPSS package (version 18.0) . The differences between control and experimental groups were determined by the two-sided， unpaired Student t test. P ＜ 0.05 was considered significant.
In this disclosure， it is showed that GA is a novel DAO inhibitor in vitro and in vivo. GA treatment in senescence accelerated (SAMP8) mice and Disrupted-In-Schizophrenia-1 (DISC1) knockout mice demonstrated an improvement of age or schizophrenia-related abnormal glutamatergic neurotransmission and brain dysfunction such as D-serine decline， NMDA hypofunction and deterioration of memory and learning function， suggesting therapeutic potential of GA in psychiatric or cognitive disorders. The derivatives of gallic acid， such as tannic acid， catechin and esters of gallic acid， are able to further metabolize to gallic acid in living subjects. (Schantz M.， Erk T. & Richling E. Metabolism of green tea catechins by the human small intestine. Biotechnology Journal 5， 1050-9 (2010) ； Ow， Y.Y. &Stupans， I. Gallic acid and gallic acid derivatives： effects on drug metabolizing enzymes. Current drug metabolism 4， 241-248 (2003) ) . Therefore， the derivatives of gallic acid  also have therapeutic potential in psychiatric or cognitive disorders by increasing D-serine concentration in the brain of the subject.
According to the above test results， GA or its derivatives can be used as a pharmaceutical， a pharmaceutical composition or a food supplement for increasing D-serine concentration in the brain of the subject. The pharmaceuticals or pharmaceutical compositions， according to a conventional pharmaceutically process prepared， may contain pharmaceutically acceptable adjuvants which is compatible with the other ingredients of the formulation and is compatible with the living body. The term ″pharmaceutically acceptable adjuvant″ or ″pharmaceutically acceptable carrier″ means a pharmaceutically acceptable material， composition or vehicle， such as liquid or solid filler， diluent， excipients， a solvent or encapsulating material. It can be used in carrying or transporting the subject composition from the organ or part of the body to another organ or part of the body. The term ″acceptable″ means a carrier which may contain other compatible components of composition. The carrier may be solid， semi-solid， liquid， cream or capsule form.
According to one embodiment of the present disclosure， the pharmaceutical composition including the GA or its derivatives as the main active ingredient is used to increase D-serine concentration in the brain of the subject can be administered enterally， mucosally and/or parenterally. The enteral route of administration includes oral and rectal administration. The mucosally route of administration includes buccal， sublingual， and intranasal administration. The parenteral route of administration includes intravenous， intramuscular， intraarterial， intraperitoneal， intracerebroventricular， intraparenchymal， intrathecal， intracranial， subcutaneous， transdermal，  intradermal， intrapulmonary， inhalation， topical， and instillation into a body of the subject. The administration may be administered pharmaceutical alone or in combination with other conventional pharmaceutically acceptable adjuvants. In the preferred embodiment， the pharmaceutical composition of the present disclosure is administered to a subject via the oral route (e.g.， through food) .
Furthermore， GA or its derivatives also can be used as the pharmaceutical， the pharmaceutical composition or the food supplement for improving a cognitive disorder result from aging or a neuropsychiatric disorder by improving an impairment of the cognitive function. The neuropsychiatric disorder can be schizophrenia， depression， attention deficit disorder， mild cognitive impairment， dementia， attention deficit hyperactivity disorder (ADHD) or bipolar disorder.
It will be apparent to those skilled in the art that various modifications and variations can be made to the structure of the present disclosure without departing from the scope or spirit of the disclosure. In view of the foregoing， it is intended that the present disclosure cover modifications and variations of this disclosure provided they fall within the scope of the following claims.

Claims (9)

  1. A method of increasing D-serine concentration in a brain of a subject， comprising：
    administering an effective amount of gallic acid or a derivative of gallic acid， which is an inhibitor of D-amino acid oxidase， to increase D-serine concentration in the brain of the subject， so that an activation of an N-methyl-D-aspartate receptor in the brain can be increased.
  2. The method of claim 1， wherein the derivative of gallic acid is tannic acid or catechin.
  3. The method of claim 1， wherein the derivative of gallic acid is an ester of gallic acid.
  4. The method of claim 3， wherein the ester of gallic acid is methyl gallate， ethyl gallate， proply gallate or octyl gallate.
  5. The method of claim 1， wherein the effective amount of the gallic acid or the derivative of gallic acid is between 1.6 to 81 mg/kg/day in a single dose or multiple doses.
  6. The method of claim 1， wherein the effective amount of the gallic acid or the derivative of gallic acid is administered to the subject in combination with a carrier or an adjuvant.
  7. A method of improving a cognitive disorder result from aging or a neuropsychiatric disorder， comprising：
    administering an effective amount of gallic acid or a derivative of gallic acid to inhibit D-amino acid oxidase and increase an activation of a N-methyl-D-aspartate receptor and a cognitive function， so that the cognitive disorder result from aging or the neuropsychiatric disorder can be improved.
  8. The method of claim 7， wherein the neuropsychiatric disorder is schizophrenia， depression， attention deficit disorder， mild cognitive impairment， dementia， attention deficit hyperactivity disorder (ADHD) or bipolar disorder.
  9. The method of claim 7， wherein the gallic acid or the derivative of gallic acid is used as a pharmaceutical or a food supplement for improving an impairment of the cognitive function.
PCT/CN2016/076128 2015-03-13 2016-03-11 Method of increasing d-serine concentration and improving cognitive disorders Ceased WO2016146026A1 (en)

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CN109444433A (en) * 2018-12-18 2019-03-08 深圳先进技术研究院 D-Ser is preparing application and Diagnosis of Depression kit in Diagnosis of Depression kit as marker
JP2019523286A (en) * 2016-08-04 2019-08-22 シニュークス インターナショナル（タイワン）コーポレイション Composition comprising benzoate compound and tannic acid for treatment of central nervous system disorders
JP2021525751A (en) * 2018-05-29 2021-09-27 シニュークス インターナショナル（タイワン）コーポレイション Strong inhibitor of D-amino acid oxidase (DAAO) and its use
CN114588180A (en) * 2018-12-18 2022-06-07 金相希 Composition for preventing or treating attention deficit hyperactivity disorder

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CN1891216A (en) * 2005-07-04 2007-01-10 中山大学 Use of catechin and its derivates for preparing medicine for preventing and treating neurodegenerative disease
CN101926790A (en) * 2009-06-26 2010-12-29 上海强圣医药科技有限公司 (-)-epigallocatechin gallate composition and application
CN102743367A (en) * 2012-05-31 2012-10-24 兰州理工大学 Application of liquidambar formosana hance fruit tannin monomer

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1891216A (en) * 2005-07-04 2007-01-10 中山大学 Use of catechin and its derivates for preparing medicine for preventing and treating neurodegenerative disease
CN101926790A (en) * 2009-06-26 2010-12-29 上海强圣医药科技有限公司 (-)-epigallocatechin gallate composition and application
CN102743367A (en) * 2012-05-31 2012-10-24 兰州理工大学 Application of liquidambar formosana hance fruit tannin monomer

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019523286A (en) * 2016-08-04 2019-08-22 シニュークス インターナショナル（タイワン）コーポレイション Composition comprising benzoate compound and tannic acid for treatment of central nervous system disorders
JP7204213B2 (en) 2016-08-04 2023-01-16 シニュークス インターナショナル（タイワン）コーポレイション Compositions containing benzoate compounds and tannic acid for treating central nervous system disorders
JP2021525751A (en) * 2018-05-29 2021-09-27 シニュークス インターナショナル（タイワン）コーポレイション Strong inhibitor of D-amino acid oxidase (DAAO) and its use
CN109444433A (en) * 2018-12-18 2019-03-08 深圳先进技术研究院 D-Ser is preparing application and Diagnosis of Depression kit in Diagnosis of Depression kit as marker
CN114588180A (en) * 2018-12-18 2022-06-07 金相希 Composition for preventing or treating attention deficit hyperactivity disorder

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