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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/465—Streptomyces

Definitions

• This invention relates to chemical substance for protection of nerve cell and method for producing thereof.
• this invention focuses on chemical substance for protection of nerve cell with new activated prevention function and method for producing thereof, by classifying and identifying Streptomyces sp. 60910 strain, which produces complestatin, an aromatic peptide chemical compound with protection activation of brain nerve cell, from which the prevention active material is separated and purified, and its physicochemical structure and biochemical activity is measured.
• apoplexy (palsy, paroxysm) is a disease, which causes regional neurologic disturbances induced by bleeding into ventricle tissue and subarachnoid intracavitary from cerebral artery of intracranial portion caused by sudden blocking of cerebral blood flow or trauma, and also which accompanies successive brain .dysfunction and its equivalent neurologic and mental symptoms. It was reported that about 75% of those apoplexy cases were caused by occlusion of cerebral artery, about 11% by cerebral hemorrhage, and 5% by bleeding in subarachnoid intracavitary [Boulton et al, Neuromethods, Vol.22 (1992)]. Every year many apoplexy patients were broken out and about one third of them consequently died. But development activities for ischemic apoplexy therapeutics were not sufficient.
• neurotrophic factor such as BFGF (basic fibroblast growth factor), NMDA (N-methyl-D-aspartate), non-NMDA receptor antagonist, lazaroid-series drug such as 21-aminosteroid, free radical scavanger, nitric oxide inhibitor, interleukin-1, 9 inhibitor, polyamine binding area receptor existing in NMDA receptor area, antisense oligonucleotide for glutamate receptor, body temperature depression, or ganglioside.
• neurotrophic factor such as BFGF (basic fibroblast growth factor), NMDA (N-methyl-D-aspartate), non-NMDA receptor antagonist
• lazaroid-series drug such as 21-aminosteroid
• free radical scavanger such as 21-aminosteroid
• nitric oxide inhibitor such as interleukin-1, 9 inhibitor
• polyamine binding area receptor existing in NMDA receptor area such as antisense oligonucleotide for glutamate receptor, body temperature depression,
• excitatory amino acid receptor such as NMDA and non-NMDA were known to be closely related to brain nerve cell damages caused by ischemic apoplexy, delayed paroxysm, hypoglycemia- or hypoxia-induced ischemia, and acute brain trauma.
• brain nerve cell kill from brain disease such as apoplexy is caused by glutamate, a nerve transmitter substance.
• glutamate secreted at optimum concentration executes the function as a nerve transmitter substance, while in cerebral ischemic state it excessively secretes and thus causes diseases such as apoplexy by inducing excitotoxicity, which kills nerve cell.
• excitotoxicity is caused by excessively secreted glutamate, which -activates gmtamate-receptor, NMDA, and ⁇
• AMPA/Kainate receptor through those receptors large amount of Ca flows into cells. That is, Ca 2+ ion flowed excessively into cell activates Ca 2+ - dependent enzymes such as phospholipase A 2 (PLA 2 ), nitric oxide synthetase
• NMDA-type receptor antagonist such as MK-801 (Buchan et al, Stroke 21:9, 1990), Ketamine, PCP, D-AP5, and CPP, AMPA/KA-type receptor antagonist such as CNQX, DNQX, NBQX, and LY215490, metabolism control-type receptor antagonist such as L-AP3.
• First method is for searching glutamate receptor antagonist as a blocking material to directly cut off excitotoxicity of nerve cell, and second one for searching antioxidant chemical compound to block oxidative toxicity induced within cell.
• this invention is about glutamate receptor antagonist, a chemical substance for protection of brain nerve cell, produced through primary cultivation of mouse cortical neuron.
• the glutamate receptor antagonists known up to now are mostly synthetic chemical compounds with strong toxicity, and thus there have been many problems in developing those as treatment drug for apoplexy.
• this invention found out glutamate receptor antagonist from Streptomyces sp. culture solution, by primary cultivating, injecting, and surveying low toxic or nontoxic chemical substance for protection of brain nerve cell and glutamate-receptor antagonist extracted from microorganism metabolic products or natural substance into mouse cortical neuron and chick telencephalic neuron.
• complestatin was identified as complestatin, by finding out its chemical structure and physicochemical characteristics, by establishing manufacturing methodology, and by identifying pharmacological activation. Also this invention showed that, through new modus operandi, this chemical substance has special features in suppressing brain nerve cell kill caused by excitotoxicity of nontoxic NMDA and AMPA/KA and brain nerve cell kill caused by hypoxia and anoxia.
• complestatin with special features of nontoxically and completely suppressing brain nerve cell kill caused by glutamate through new modus operandi [Kaneko, I., Kamoshida, K. and Takahashi, S. J.Antibiotics, 42:236-241, (1989)], is an aromatic peptide chemical compound reported as containing anticomplement activation, anti-HIV antibiotics, and new vasculogenesis inhibition activation.
• This invention succeeded in separating and purifying complestatin, a low toxic chemical substance for protection of brain nerve cell with new mechanism to be reported yet,, which acts as antagonist to glutamate-receptor including both NMDA- and AMPA/KA-receptor and suppresses brain nerve cell kill caused by hypoxia and anoxia, by effectively separating complestatin from Streptomyces sp. containing protection activation for brain nerve cell and by identifying activation mechanism.
• drugs including complestatin compound invented here as effective ingredient they could be injection or oral inoculation such as tablet, pill, granulum, soft • hard capsule, pulvis, granum, power, emulsion, syrup, pellet, and so on. Pharmaceutical preparation containing complestatin compounds as an effective ingredient can also be easily done in accordance with commercial laws.
• the objective of this invention is to provide Streptomyces sp. 60910 strain, which produces chemical substance for protection of brain nerve cell.
• Another objective of this invention is to provide chemical substance for protection of brain nerve cell produced by the above
• Streptomyces sp. 60910 strains and its method for producing thereof are known in the art.
• Streptomyces sp. 60910 strains and its method for producing thereof are known in the art.
• the objectives of this invention were accomplished by identifying and cultivating Streptomyces sp. 60910 strain with protection activation of brain nerve cell, then analyzing the structure and characteristics of chemical substance for protection of brain nerve cell produced by the strain after separating this substance from centrifuged equivalent liquid and bacterial cell, and then measuring the inhibitory effect of this active material against nerve cell kill caused by excitotoxin such as NMDA (N-methyl-D-aspartate), AMPA ( ⁇ -amino-3-hydroxy-5-methyl-4-isoxazole propionate), and Kainate and caused by oxygen- glucose, deprivation as ischemic brain disease model.
• excitotoxin such as NMDA (N-methyl-D-aspartate), AMPA ( ⁇ -amino-3-hydroxy-5-methyl-4-isoxazole propionate)
• Kainate caused by oxygen- glucose, deprivation as ischemic brain disease model.
• Figure 1 shows the graph generated by LDH measurement on the death rate of mouse brain nerve cell after 24 hours of treating 20 ⁇ M NMDA, 40 ⁇ M Kainate(KA), and 30 ⁇ M AMPA and chemical substance invented here for protection of brain nerve cell at the same time, after cultivating mouse cortical neuron.
• Figure 2a shows the graph generated by LDH measurement on the death rate of mouse brain nerve cell after 24 hours of treating 10-1000 ⁇ M NMDA and chemical substance invented here for protection of brain nerve cell at the same time, after cultivating mouse cortical neuron.
• Figure 2b shows the graph generated by LDH measurement on the death rate of mouse brain nerve cell after 24 hours of treating 10-100 ⁇ M Kainate(KA) and chemical substance invented here for protection of brain nerve cell at the same time, after cultivating mouse cortical neuron.
• Figure 2c shows the graph generated by LDH measurement on the death rate of mouse brain nerve cell after 24 hours of treating 10-100 ⁇ M AMPA and chemical substance invented here for protection of brain nerve cell at the same time, after cultivating mouse cortical neuron.
• Figure 3a is the graph showing measurements on cytotoxicity after 80 minutes of cultivation from adding chemical substance for protection of nerve cell invented here 110 ⁇ M, NMDA receptor antagonist MK-801 10 ⁇ M or AMPA/kainate receptor antagonist CNQX 20 ⁇ M into culture medium, when in vitro cultivation of nerve cell with oxygen-glucose deprivation.
• Figure 3b is the graph showing measurements on cytotoxicity after 20 hours of cultivation from adding chemical substance for protection of nerve cell invented here 0.1, 1, 5, 10 ⁇ M and apoptosis inhibitor Z-VAD, when cell kill was induced by hypoxia after endothelial cell was cultivated in vitro.
• Figure 4a shows measurements on Ca 2+ amount flowed into cell by fura-2 dye after inducing Ca 2+ flow into cell by treating 100 ⁇ M NMDA to mouse cortical neuron cultivated.
• Figure 4b shows measurements on Ca 2+ amount flowed into cell by fura-2 dye after inducing Ca 2+ flow into cell by treating 100 ⁇ M NMDA and chemical substance for protection of brain nerve cell invented here simultaneously to mouse cortical neuron cultivated.
• Figure 4c shows measurements on Ca 2+ amount flowed into cell by
• This invention is composed of several stages; stage for classification and identification of Streptomyces sp. strain with protection activity for brain nerve cell and thus identification of new Streptomyces sp. 60910 strain producing chemical substance for protection of brain nerve cell and analyses of cultivation condition and characteristics of the strain; stage for separation of active material from equivalent liquid and bacterial cell by centrifugation after cultivating Streptomyces sp.
• G.S.S culture medium (glucose lOg, soluble starch 20g, soybean meal 25g, meat extract lg, yeast extract 4g, NaCl 2g, K 2 HP0 4 0.25g, CaC0 3 2g, pH 7.2, D.W 1L); stage for identifying the active material as complestatin, an aromatic peptide ' chemical compound by surveying UV and IR spectrum absorption, by measuring molecular weight of this active material through FAB and ESI- mass measurement, and by deciding molecular structure from NMR spectrum; stage for excitotoxicity inhibition capability of chemical substance for protection of brain nerve cell by quantifying amount of lactate dehydrogenase (LDH) after treating NMDA of acute toxicity, non-NMDA excitotoxin AMPA with prolonged neurotoxicity and Kainate, and chemical substance for protection of brain nerve cell produced by Streptomyces sp.
• LDH lactate dehydrogenase
• stage 60910 strain simultaneously after cultivating chick telencephalic neuron separated from embryo of fertilized egg and mouse cortical neuron separated from mouse embryo; stage for measuring nerve cell protection activity of chemical substance for protection of brain nerve cell by conducting oxygen-glucose derivation experimentation as in vitro ischemia model and by conducting in vivo mouse retinal ischemia model; stage for surveying if nerve cell protection activation of this invented chemical compound is induced by antioxidation activation, from measurement of antioxidation activation after treating Fe 2+ 50 ⁇ M, H 2 0 2 150 ⁇ M, BSO 10 mM to mouse cortical tissue of primary cultured mouse; and stage for surveying effect of this chemical substance for protection of brain nerve cell on Ca 2+ accumulation into cell caused by excitotoxin such as NMDA, by using Fura-2, a Ca 2+ -binding dye.
• stage 60910 strain simultaneously after cultivating chick telencephalic neuron separated from embryo of fertilized egg and mouse cortical neuron separated from mouse embryo;
• EXAMPLE 1 Classification and identification of the strain producing chemical substance for protection of brain nerve cell
• the Streptomyces sp. strain showing protection activation for brain nerve cell was classified and identified, by surveying its cultural, morphologic, and physiologic characteristics. For cultural characteristics, color of aerial hyphae, color and viability of basal hypha, and water soluble pigment formation were surveyed every 7 days, while cultivating for 21 days on yeast extract-malt extract agar medium, oatmeal agar medium, inorganic salts- starch agar medium, glycerol-asparagine agar medium, peptone-yeast extract- iron agar medium, tyrosine agar medium, glucose-asparagine agar medium, Bennet's agar medium, and nutrient's agar medium.
• the color of basal hypha was light yellow and did not form water-soluble pigment.
• the spore was cylinder-like shape and its size was 0.7-0.8 x 0.8-0.9 ⁇ m, with the smooth surface.
• the spore chain was spiral and formed with 10-20 resting spores. It did not form melanin and showed negative reaction to milk peptonization and nitric acid reduction, but positive to milk agglutination and hydrolysis of starch and skim milk.
• the diaminopymeric acid of cell wall was L,L shape.
• EXAMPLE 2 Culture of the above Streptomyces sp. 60910 producing chemical substance for protection of brain nerve cell, and isolation and purification of chemical substance for protection of brain nerve cell
• the G.S.S culture medium of 100 ml was split into the 500 ml Erlenmeyer flask, in which Streptomyces sp. 60910 strain producing protection active material for brain nerve cell as above was inoculated and pre-cultivated for 2 days (28 ° C, 150 rp ).
• Second step Isolation and identification of chemical substance for protection of brain nerve cell -
• the protection active material for brain nerve cell was isolated from the above Streptomyces sp. 60910 strain and purified.
• the culture solution in 2 nd step, 3L was centrifuged into equivalent liquid and bacterial cell.
• the equivalent liquid was adjusted at pH 4 by using IN HC1, and then extracted by ethyl acetate and solvent layer was concentrated, then silica gel column chromatography was applied by using CHCl 3 -MeOH (10: 1-1:4) as developing solvent.
• the activated fraction was stored for one day at low temperature and then sediment generated was filtered and purified to get active material separated.
• the bacterial cell resulted from centrifugation of the above material was extracted by 70% acetone, put under decompressed concentration, adjusted to be at pH 4 by applying IN HC1, and then extracted by ethyl acetate through solvent extraction.
• the ethyl acetate was put under decompressed concentration, dissolved in MeOH, and stored at low temperature, and consequently white sediment was generated. This sediment was filtered and purified, and then same active material as compound separated from the above equivalent liquid was isolated.
• EXAMPLE 3 Physicochemical characteristics of complestatin, chemical substance for protection of brain nerve cell produced by Streptomyces sp. 60910
• the compound of the above example 2 showed the physicochemical characteristics as following. It was yellow powder with molecular weight of 1325 determined by FAB and ESI-mass measurement, and its molecular formula was determined as C 6 iH 5 N 7 Oi5 ⁇ 6 from Mass data and l H, 13 C NMR spectrum, and NMR spectrum for other structural interpretation. The measurement of its UV spectrum by using MeOH as solvent showed that maximum absorption rate was at 280 and 290 nm. Also, survey of its solubility showed that it dissolved well in DMSO, but did not dissolve in other solvent. Its Rf value on TLC was measured at 0.09, when using 40% of ACN and 60% of citrate buffer (0.05M, pH6.0) as developing solvent.
• EXAMPLE 4 Chemical structure of chemical substance for protection of brain nerve cell produced by Streptomyces sp. 60910
• protection of brain nerve cell one dimensional NMR including H, C, and DEPT, and two dimensional NMR spectrum including COSY and HMBC were measured. Based on those NMR data and physicochemical characteristics of practical application example 3, chemical structure of the above protection active material for brain nerve cell was interpreted and consequently protection active material for brain nerve cell was identified as complestatin, an aromatic peptide chemical compound. Its structural chart was as general formula shown below.
• EXAMPLE 5 Bio-activation examination of chemical substance for protection of brain nerve cell produced by Streptomyces sp. 60910
• First step Activation measurement of chemical substance for protection of brain nerve cell produced by Streptomyces sp. 60910 through LDH assay
• chick telencephalic neuron separated from embryo of fertilized egg and mouse cortical neuron separated from mouse fetus were cultivated.
• the chick telencephalic neuron, separated from embryo of fertilized egg was cultivated in 48 well microplate for 5 days at concentration of 5* 10 5 cell and then 300 ⁇ M Kainate inducing brain nerve cell kill and complestatin, a protection active material for brain nerve cell were treated simultaneously. Then 24 hours later, viable cells were counted per unit area, by surveying kill inhibition degree of nerve cell through optical microscope.
• mouse cortical neuron separated from mouse fetus of 15 day-pregnant, was cultivated for 12-14 days and then NMDA (N-methyl-D-aspartate), AMPA ( ⁇ -amino-3-hydroxy-5-methyl-4-isoxazole propionate), and Kainate, all inducing nerve cell kill, and complestatin, a protection active material for brain nerve cell were treated simultaneously. After 24 hours of treatment, viability of cell was surveyed by optical microscope and LDH assay was conducted to quantify the amount of lactate dehydrogenase (LDH), and their results were shown in Figures 1 and 2.
• LDH lactate dehydrogenase
• Second step Activation measurement of chemical substance for protection of brain nerve cell produced by Streptomyces sp. 60910 using In vitro and in vivo ischemia model
• the in vivo experiment was conducted using mouse retinal ischemia model.
• intraocular pressure of a mouse was kept high at 160-180 mmHg for 90 minutes, thus ischemia was induced and then this invented compound was treated, chemical substance for protection of brain nerve cell invented here inhibited cell kill, depending on concentration.
• mouse retinal ischemia was induced and nerve cell protection activation for inner nuclear layer and ganglion cell layer was measured, about 50-60% of nerve cells in control group not treated with chemical substance for protection of brain nerve cell invented here were killed, because of retinal ischemia. Meanwhile over 90% of nerve cells in experimental group treated with intravitreal injection of 0.5-1 nM of chemical substance for protection of brain nerve cell survived, depending on the concentration.
• Third step Antioxidation activation measurement of chemical substance for protection of brain nerve cell produced by Streptomyces sp. 60910 against oxidative toxicity
• 04- primary cultured mouse cortical cell was treated with Fe 50 ⁇ M, H 2 0 2 150 ⁇ M, BSO lOmM, respectively, and then antioxidation activation was measured. The result showed that the oxidative nerve cell death caused by 04-
• Glutamate-receptor Activation of Glutamate-receptor caused Ca 2+ accumulation within cell, and increased Ca 2+ in cell promoted the in-flow of other ions and water molecule, and finally nerve cell kill was induced due to oxidative toxicity.
• this invention was effective in providing chemical substance for protection of brain nerve cell containing nerve cell protection activation, which inhibited brain nerve cell kill induced by excitotoxin such as NMDA, AMPA, and Kainate, and in providing aromatic peptide chemical compounds containing new brain nerve cell protection activation, not existent before, through chemical modification or diverse alteration of this invented chemical compound.
• this invented chemical compound had different modus operandi from other chemical compounds already known, and could be of wide and effective use in prevention and treatment drugs for brain disease such as apoplexy, food, and reagent of brain nerve cell-related researches because of no toxicity in drugs. This invention, therefore, would be of great use in health improvement of the general public and pharmaceutical manufacturing industry.

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