WO2016064245A1 - Nouveau composé peptidique, son procédé de production et son utilisation - Google Patents

Nouveau composé peptidique, son procédé de production et son utilisation Download PDF

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Publication number
WO2016064245A1
WO2016064245A1 PCT/KR2015/011281 KR2015011281W WO2016064245A1 WO 2016064245 A1 WO2016064245 A1 WO 2016064245A1 KR 2015011281 W KR2015011281 W KR 2015011281W WO 2016064245 A1 WO2016064245 A1 WO 2016064245A1
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group
substituted
unsubstituted
asperphenin
pharmaceutically acceptable
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PCT/KR2015/011281
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English (en)
Korean (ko)
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신종헌
오기봉
오동찬
이상국
료리연
유민정
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서울대학교 산학협력단
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Priority claimed from KR1020150095365A external-priority patent/KR101723649B1/ko
Application filed by 서울대학교 산학협력단 filed Critical 서울대학교 산학협력단
Priority to EP15851785.4A priority Critical patent/EP3222629A4/fr
Priority to US15/521,222 priority patent/US10618934B2/en
Priority to CN201580070797.5A priority patent/CN107108693B/zh
Publication of WO2016064245A1 publication Critical patent/WO2016064245A1/fr
Priority to US16/805,423 priority patent/US10759831B2/en

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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
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
    • C07C49/76—Ketones containing a keto group bound to a six-membered aromatic ring
    • C07C49/782—Ketones containing a keto group bound to a six-membered aromatic ring polycyclic
    • C07C49/784—Ketones containing a keto group bound to a six-membered aromatic ring polycyclic with all keto groups bound to a non-condensed ring
    • C07C49/786—Benzophenone
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • 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
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus

Definitions

  • Novel peptide compounds methods for their production, and uses thereof.
  • Biologically active substances derived from microorganisms have generally been sources of antibiotics, antifungal and anticancer drugs, and have been developed as new drugs for treating various diseases including these or have been the basis for new drug development.
  • Antibiotics derived from microorganisms are for example amphotericin, erythromycin, streptomycin, tetratracycline, and vancomycin.
  • daptomycin isolated from actinomycetes Streptomyces, was approved by the US Food and Drug Administration (FDA) as the next-generation antibiotic.
  • anticancer agents derived from bacteria are, for example, doxorubicin, bleomycin, mithramycin, neocarzinostatin, pentostatin, and epothilone. There is this. As such, the study of bacterially derived physiologically active substances is very important in the development of antibacterial, antifungal and anticancer agents.
  • Novel peptide compounds, isomers, derivatives thereof, or pharmaceutically acceptable salts thereof are provided.
  • a peptide compound, an isomer, a derivative thereof, or a pharmaceutically acceptable salt comprising a novel lipopeptide and benzophenone.
  • a strain of genus Aspergillus F452 producing the peptide compound is provided.
  • a method of producing the peptide compound is provided.
  • compositions for preventing or treating cancer comprising the peptide compound, isomers, derivatives, or pharmaceutically acceptable salts thereof.
  • a method of preventing or treating cancer using the peptide compound, isomer, derivative thereof, or pharmaceutically acceptable salt thereof is provided.
  • novel peptide compounds including lipopeptides and benzophenones, have anticancer activity and can be used to prevent or treat various types of cancer.
  • inexpensive bulk culture media can be used to provide high yields of the peptide compounds.
  • 1A and 1B are structural formulas of asperphenin A and B, respectively.
  • Figure 2 is a photograph of the culture medium of the Aspergillus sp. F452 strain.
  • Figure 3a is a graph showing the percentage (%) of RKO cells in each cell cycle according to the concentration of asperphenin B
  • Figures 3b and 3c are cells according to the incubation time of 2.5 ⁇ M and 5 ⁇ M of asperphenin B, respectively It is a graph showing the percentage (%) of RKO cells in the cycle.
  • Figure 4 is a graph showing the percentage (%) of living cells, early cell death cells, terminal apoptosis cells or necrosis, or cell necrosis according to the concentration of asperphenin B.
  • 5A and 5B are immunoblotting images of cell cycle related proteins according to the concentration of asperphenin B and the incubation time (hours) of 5 ⁇ M of asperphenin B, respectively.
  • 6A and 6B are immunoblotting images of apoptosis-related proteins according to the concentration of asperphenin B and the incubation time (hours) of 5 ⁇ M of asperphenin B, respectively.
  • 7A to 7C are graphs showing cell survival rate (%) by a combination of asperphenin B and another anticancer agent, or another anticancer agent alone.
  • FIG 8 is a graph showing the tumor volume (mm 3 ) of mice according to days passed after administration of asperphenin B after administration of 4 mg / kg or 8 mg / kg of asperphenin B.
  • One aspect provides peptide compounds, isomers, derivatives, or pharmaceutically acceptable salts thereof, including lipopeptide and benzophenone.
  • peptide compound refers to a compound comprising a peptide.
  • Peptides are compounds in which two or more amino acids are linked by peptide bonds between carboxyl and amino groups. Dipeptide, tripeptide, tetrapeptide, etc., depending on the number of constituent amino acids, those having less than about 10 peptide bonds, oligopeptides, having multiple peptide bonds It is called a polypeptide.
  • isomer refers to a compound having the same molecular formula but not the same way of connection or spatial arrangement of the constituent atoms in the bonsai. Isomers include, for example, structural isomers, and stereoisomers.
  • derivative refers to a compound obtained by substituting a part of the structure of the compound with another atom or group of atoms.
  • pharmaceutically acceptable salts refers to inorganic and organic acid addition salts of compounds.
  • lipopeptide refers to a substance in which lipid is bound to a peptide. Lipopeptides may be lipids linked by amide bonds to peptides. Amide bonds, also called peptide bonds, refer to covalent bonds in which an amino group in one molecule and a carboxyl group in one molecule bond.
  • the lipid may be a substituted or unsubstituted C 12 to C C20 alkyl group, a substituted or unsubstituted C 2 to C C20 alkenyl group, or a substituted or unsubstituted C 2 to C C20 alkanyl group.
  • the alkyl group may be an alkyl group of C 2 to C 20 , C 5 to C 20 , C 10 to C 20 , or C 10 to C 15 .
  • the alkyl group may be an alkyl group of C 12 .
  • the alkenyl group may be an alkenyl group which is C 2 to C 20 , C 5 to C 20 , C 10 to C 20 , or C 10 to C 15 .
  • the alkanyl group may be an alkanyl group of C 2 to C 20 , C 5 to C 20 , C 10 to C 20 , or C 10 to C 15 .
  • “Substituted” in the “substituted” refers to the introduction of a substituted group, and “substituent” refers to the introduced atomic group when a derivative is formed by substituting a hydrogen atom in an organic compound with another atom group.
  • Substituents can include, for example, heterocyclic group of a hydroxy group, a halogen atom, C 1 to C C20 alkyl group, C 2 to C 20 alkenyl groups, C 2 to C 20 alkynyl group, C 1 to C 20 of the, C 6 to C 20 aryl group, C 6 to C 20 arylalkyl group, C 6 to C 20 heteroaryl group, or C 6 to C 20 heteroarylalkyl group, nitro group, cyano group, amino group, amidino group, hydrazine, hydra Zone, carboxyl group or salt thereof, sulfonic acid group or salt thereof, phosphoric acid or salt thereof.
  • a halogen atom C 1 to C C20 alkyl group, C 2 to C 20 alkenyl groups, C 2 to C 20 alkynyl group, C 1 to C 20 of the, C 6 to C 20 aryl group, C 6 to C 20 arylalkyl group, C 6 to C 20
  • the peptide may be one containing two, three, or four or more amino acids.
  • the peptide can be for example a tripeptide comprising three amino acids.
  • the peptide may comprise, for example, a peptide at the N-terminal-asparagine (Asp) -glutamine (Gln) -leucine-leu-C terminus.
  • the peptide may comprise one or more ⁇ (beta) -amino acids.
  • Amino acids consist of amino groups, carboxyl groups, and side chains specific for amino acids. Twenty kinds of standard biological amino acids have an amino group bonded to the ⁇ carbon of the carboxyl group, while ⁇ -amino acids have an amino group bonded to the ⁇ carbon of the carboxyl group.
  • ⁇ -amino acid is ⁇ 3 -amino acid when the side chain is bonded to the carbon next to the amine, and ⁇ 2 -amino acid when the side chain is bonded to the carbon next to the carboxyl group.
  • the ⁇ -amino acid may be ⁇ -leucine.
  • the ⁇ -amino acid may be ⁇ 3 -leucine.
  • the benzophenone is an organic compound having the general formula (C 6 H 5 ) 2 CO as diphenylmethanone.
  • Benzophenones can be, for example, compounds substituted with one, two or three or more hydroxy groups.
  • benzophenone may be a compound in which any one or more of carbon 5, carbon 9 and carbon 13 are substituted with a hydroxy group.
  • the lipopeptides and benzophenones can be linked by ketone bonds.
  • benzophenone can be linked to the C terminus of the lipopeptides.
  • the peptide compound may be a compound represented by Formula 1:
  • R 1 is a substituted or unsubstituted C 1 to C 20 alkyl group, a substituted or unsubstituted C 1 to C 20 alkenyl group, or a substituted or unsubstituted C 1 to C 20 alkanyl group and each R 1 is optionally substituted with a hydroxy group or unsubstituted,
  • R 5 may be a substituted or unsubstituted benzophenone.
  • alkyl group alkenyl group, alkynyl group, substitution, and benzophenone are as described above.
  • the peptide compound may be a compound represented by Formula 2:
  • the compound represented by Formula 2 may be a compound represented by Formula 3 or Formula 4, an isomer thereof, a derivative thereof, or a pharmaceutically acceptable salt thereof:
  • One aspect provides the Aspergillus genus F452 strain (Accession No .: KCTC12688BP) that produces a peptide compound comprising lipopeptides and benzophenones.
  • lipopeptides are as described above.
  • Such strains include variants thereof.
  • the variant may be, for example, a variant resulting from a natural mutation or an artificial mutation.
  • Artificial mutations can be caused by physical mutagens, such as ultraviolet light, or chemical mutagens, such as base compounds.
  • the strain comprises spores, cells, or culture thereof of the strain.
  • the strain may be isolated or derived from marine sediment.
  • One aspect includes the steps of culturing the Aspergillus genus F452 strain (Accession Number: KCTC12688BP); And it provides a method for producing a peptide compound comprising a lipopeptide and benzophenone comprising the step of separating the peptide compound comprising a lipopeptide and benzophenone from the culture.
  • the method includes culturing Aspergillus genus F452 strain (Accession Number: KCTC12688BP).
  • Aspergillus strain F452 is as described above.
  • the culturing step may be culturing the strain in liquid medium or solid medium.
  • the medium may comprise an artificial ocean.
  • the medium may include, for example, glucose, rice, syrup, dextrin, starch, molasses, animal oil, or vegetable oil as a carbon source.
  • the nitrogen source may include, for example, yeast extract, peptone, bran, soybean meal, wheat, malt, cottonseed meal, fishmeal, corn steep liquor, gravy, ammonium sulfate, sodium nitrate or urea.
  • the medium may include salts, potassium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid or other inorganic salts that promote the formation of ions.
  • the medium may include, for example, artificial seawater, yeast extract, peptone, and rice.
  • the culturing may be culturing while shaking or standing under aerobic conditions.
  • the culture temperature may be for example about 20 ° C to about 37 ° C, about 25 ° C to about 30 ° C, or 27 ° C.
  • Incubation time can be for example 1 day to 2 months, 1 week to 2 months, 2 weeks to 2 months, 1 month to 2 months, or 6 weeks.
  • the method includes the step of separating the peptide compound comprising the lipopeptide and benzophenone from the culture.
  • lipopeptides are as described above.
  • Separating the peptide compound from the culture may include concentration, centrifugation, filtration, or chromatography of the culture.
  • the culture solution may be extracted by ethyl acetate, water, or a combination thereof.
  • the concentrate obtained can be chromatographed to give eight fractions depending on the polarity.
  • the chromatography can be reverse phase flash chromatography, for example with water, acetonitrile, or a combination thereof as the mobile phase.
  • the fractions can be fractionated by reverse phase flash chromatography to give eight fractions.
  • the fraction eluted with water / acetonitrile in a volume ratio of 50:50 in the obtained fractions can be separated by performing high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the HPLC can be carried out using a reversed phase preparative HPLC column with water / methanol in a volume ratio of 70:30 as the mobile phase.
  • Isolated peptide compounds may be obtained with at least about 80%, about 90%, or about 99% purity.
  • One aspect provides a pharmaceutical composition for preventing or treating cancer, comprising a peptide compound comprising lipopeptides and benzophenone, isomers, derivatives, or pharmaceutically acceptable salts thereof.
  • lipopeptides benzophenones, peptide compounds, isomers, derivatives, and pharmaceutically acceptable salts are as described above.
  • Such cancers include, for example, intrahepatic bile duct cancer, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, myxosarcoma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, basal cell carcinoma , Ovarian epithelial cancer, ovarian germ cell tumor, male breast cancer, brain tumor, pituitary adenoma, multiple myeloma, gallbladder cancer, cholangiocarcinoma, colon cancer, retinoblastoma, choroidal melanoma, batterous swollen cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, Non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, childhood brain tumor, childhood lymphoma, childhood leukemia, small intestine cancer, meningioma,
  • prevention refers to any action that inhibits or delays the onset of a disease by administration of a composition.
  • treatment refers to any action by which administration of a composition improves or advantageously alters the condition of a disease.
  • the pharmaceutical composition may further include a known active ingredient having anticancer activity.
  • Known active ingredients having anticancer activity may be anticancer agents.
  • the anticancer agent may be irinotecan, 5-fluorouracil, gemcitabine, etoposide, paclitaxel, or a combination thereof.
  • the pharmaceutical composition may be a single composition or individual compositions.
  • the pharmaceutical composition may further comprise a carrier, excipient or diluent.
  • Carriers, excipients and diluents are, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, undetermined Vaginal cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.
  • compositions may each be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, or the like, external preparations, suppositories, or sterile injectable solutions according to conventional methods.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used.
  • the solid preparation for oral administration may be a tablet, pill, powder, granule, or capsule.
  • the solid preparation may further comprise an excipient. Excipients can be, for example, starch, calcium carbonate, sucrose, lactose, or gelatin.
  • the solid preparation may further comprise a lubricant such as magnesium stearate, or talc.
  • the liquid formulation for oral may be a suspension, a solution, an emulsion, or a syrup.
  • the liquid formulation may comprise water, or liquid paraffin.
  • the liquid formulation may include excipients such as wetting agents, sweetening agents, fragrances, or preservatives.
  • the preparation for parenteral administration may be sterile aqueous solution, non-aqueous solvent, suspension, emulsion, lyophilized, or suppository.
  • Non-aqueous solvents or suspending agents may contain vegetable oils or esters.
  • the vegetable oil may be, for example, propylene glycol, polyethylene glycol, or olive oil.
  • the ester may for example be ethyl oleate.
  • the base of the suppository may be witepsol, macrogol, tween 61, cacao butter, laurin butter, or glycerogelatin.
  • One aspect is a method of preventing cancer comprising administering to a subject a pharmaceutical composition for the prevention or treatment of cancer comprising a peptide compound comprising lipopeptide and benzophenone, an isomer, derivative thereof, or a pharmaceutically acceptable salt thereof. Or a method of treatment.
  • lipopeptides benzophenones, peptide compounds, isomers, derivatives, pharmaceutically acceptable salts, cancer, prevention, treatment, and pharmaceutical compositions are as described above.
  • the subject may be a mammal, including rats, mice, dogs, cattle, horses, monkeys, and humans.
  • the preferred dosage of the peptide compound depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art.
  • the peptide compound may be administered, for example, in an amount of about 0.0001 mg / kg to about 100 mg / kg, or about 0.001 mg / kg to about 100 mg / kg, once or several times daily.
  • the peptide compound in the pharmaceutical composition may be included in about 0.0001% to about 10% by weight, or about 0.001% to about 1% by weight based on the total weight of the total composition.
  • compositions of the peptide compounds may be in the form of their pharmaceutically acceptable salts.
  • the peptide compound may be used alone or in combination with other pharmaceutically active compounds.
  • the pharmaceutical composition can be administered to mammals including rats, mice, dogs, cattle, horses, monkeys, and humans by various routes.
  • the method of administration may be, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.
  • the method may further comprise administering an anticancer agent to the subject.
  • Peptide compounds, isomers, derivatives, or pharmaceutically acceptable salts and anticancer agents may be administered simultaneously, separately, or sequentially.
  • an anticancer agent may be administered to the subject after the peptide compound, isomer, derivative, or pharmaceutically acceptable salt thereof is administered to the subject.
  • F452 strains were isolated from marine sediments in the tropics. The whole genome of the F452 strain was isolated, 18S ribosomal DNA was cloned by polymerase chain reaction (PCR), and the nucleic acid sequence of 8S ribosomal DNA was analyzed (SEQ ID NO: 1).
  • F452 strain was a novel strain that was systematically similar to Aspergillus versicolor . This strain was named F452 strain of the genus Aspergillus, and the strain was deposited with the depository on October 13, 2014 (Accession No .: KCTC12688BP).
  • Aspergillus F452 strain was transferred to sterile YPG solid medium (5 g yeast extract, 5 g peptone, 10 g glucose, 16 g agar, 24.8 g Instant Ocean (Aquarium systems) per sterile distilled water). Inoculation and primary incubation for several days at 27 °C.
  • F452 strains cultured in primary cultured solid medium were sterilized in 100 ml of YPG liquid medium (5 g yeast extract per 1 L of distilled water, 5 g peptone, 10 g glucose, 24.8 g Instant Ocean (Aquarium systems) ) Was incubated for 2 days at 27 ° C. with shaking at 150 rpm.
  • YPG liquid medium 5 g yeast extract per 1 L of distilled water, 5 g peptone, 10 g glucose, 24.8 g Instant Ocean (Aquarium systems)
  • Example 1-2 a solid medium obtained by tertiary culturing of the F452 strain was obtained, and then immersed in 1 L of ethyl acetate (Daejung Chemical Co., Ltd.) per 100 g of the solid medium for one day, and the process was repeated three times in total.
  • the obtained ethyl acetate was filtered through a filter paper (Advantec), and the filtrate was decompressed to remove ethyl acetate as a solvent. This process was repeated to give 25 g of crude extract.
  • 200 ml of methanol (Daejunghwa Co., Ltd.) was added, and the methanol layer was depressurized to obtain 11.4 g of methanol extract.
  • the obtained methanol extract was divided into 8 fractions using reverse phase chromatography (MERCK, C18, 700 g, reversed-phase). Five fractions of the eluate were used with a 5% reduction in water from acetonitrile (Burdick & Jackson) in a volume ratio of 60:40. The final fraction was 100% methanol (Daejung Chemical Co., Ltd.), acetone. Fractions were obtained by (Dae Jung Hwa Gold Co., Ltd.) and ethyl acetate (Dae Jung Hwa Gold Co., Ltd.).
  • Fraction 3 fractionated with water / acetonitrile in a volume ratio of 50:50 was analyzed by liquid chromatography-mass spectrometry and hydrogen nuclear magnetic resonance spectroscopy.
  • the composition of the fractions was determined using LC / MS connected to Agilent Technologies' 1200 series LC and 6130 series mass spectrometer. Mass spectra were measured using a LTQ-Orbitrap ESI-MS mass spectrometer from Thermo-Finnigan, which was expressed in the form of mass / charge (m / z). Liquid chromatography-mass spectrometry and hydrogen nuclear magnetic resonance spectroscopic analysis confirmed that the culture medium of the strain contained new secondary metabolites. The new secondary metabolite was named Asperphenin.
  • Fraction 3 was subjected to C 18 reversed-phase semi-preparative HPLC column, particle diameter 5 ⁇ m, 250 x 10 mm (length x inner diameter), elution rate 2 ml / min, refractive index (RI) Separated by detector (Shodex)
  • the mobile phase used for the separation was water / methanol at a volume ratio of 70:30, and reverse phase semi-preparative HPLC was performed for about 1 hour 30 minutes, whereby 50.0 mg of asperphenin A And 46.0 mg of asperphenin B were obtained.
  • Asperphenin A and Asperphenin B are pale yellow, and soluble in methanol and acetone, which are stable at room temperature and are medium organic solvents.
  • the structures of asperphenin A and asperphenin B were determined by nuclear magnetic resonance spectrum, infrared and ultraviolet spectroscopy, optical luminosity, and high resolution mass spectrometry data.
  • Nuclear magnetic resonance spectra ( 1 H NMR, 13 C NMR) were used at 500 MHz NMR by Bruker, and DMSO- d 6 was used as the solvent.
  • Mass spectra were measured in the form of mass / charge (m / z) using a LTQ-Orbitrap ESI-MS mass spectrometer manufactured by Thermo-Finnigan.
  • Infrared spectra were used by Jasco's FT-IR-4200 spectrometer.
  • Ultraviolet spectrum was a U-3010 UV / VIS spectrometer manufactured by Hitachi.
  • the fluorescence was measured using a P-1020 polarimeter from Jasco.
  • Lung cancer cell line A549 (Korean Cell line Bank), colon cancer cell line HCT116 (ATCC), gastric cancer cell line SNU638 (Korean Cell Line Bank), liver cancer cell line SK-HEP-1 (Korean Cell line Bank), and breast cancer cell line MDA-MB-231
  • SRB Sulforhodamine B
  • 190 ⁇ l of a cell suspension at a concentration of 3.5 ⁇ 10 4 cells / ml was inoculated into each well of a 96 well microplate.
  • 0.8 ⁇ M, 4 ⁇ M, 20 ⁇ M, or 100 ⁇ M of asperphenin was added to the cell culture and incubated at 37 ° C. and 5% CO 2 for 72 hours.
  • 50 ⁇ l of 50% (v / v) trichloroacetic acid solution (Sigma Aldrich) per well was added and incubated at 4 ° C. for 30 minutes to fix cells. Fixed cells were washed five times with water and dried in air.
  • asperphenin A showed a strong cell growth inhibitory effect against cell lines of lung cancer, colon cancer, gastric cancer, liver cancer, and breast cancer.
  • Asperphenin B showed a strong cell growth inhibitory effect against colorectal cancer, gastric cancer, liver cancer, and breast cancer except A549 cell line.
  • both asperphenin A and asperphenin B showed similar or better anticancer activity to etoposide, a positive control against HCT116, a colorectal cancer cell line.
  • asperphenin B is not only expressed in colon cancer cell line HCT116 but also in other It was also confirmed that the colon cancer cell line had a cell growth inhibitory effect.
  • asperphenin B strongly inhibited cell growth of four colorectal cancer cell lines except SW480 cell line, and showed the strongest cell growth inhibitory effect against RKO cell line.
  • RKO cells (ATCC) were diluted to 1 ⁇ 10 5 cells / ml in medium containing 10% (v / v) FBS and seeded in 60 mm culture dishes. Inoculated cells were incubated at 37 ° C. and 5% CO 2 for about 24 hours. The cultured cells were washed once with PBS (phosphate-buffered saline) and exchanged with fresh medium. Asperphenin B at a final concentration of 0.625 ⁇ M, 1.25 ⁇ M, 2.5 ⁇ M, 5 ⁇ M or 10 ⁇ M was added to the cultured cells and incubated at 37 ° C. and 5% CO 2 for a period of time.
  • PBS phosphate-buffered saline
  • the cells attached to the culture dish and the cells not attached to the medium were collected to obtain cells.
  • the obtained cells were washed once with PBS, 1 ml of cold 70% (v / v) ethanol was added and incubated at 4 ° C. for at least 12 hours to fix the cells.
  • 70% (v / v) ethanol was removed and the fixed cells were washed once with PBS.
  • 500 ⁇ l of 50 ⁇ g / ml RNase A (Sigma Aldrich) was added to the cells and incubated for about 30 minutes at room temperature.
  • Propidium iodide (PI) at a final concentration of 50 ⁇ g / ml was added to the cells and incubated at room temperature for about 30 minutes with the reaction shielded.
  • G 2 / M phase cells When treated with 2.5 ⁇ M or 5 ⁇ M of asperphenin B for about 24 hours, G 2 / M phase cells increased compared to the control. When treated with 2.5 ⁇ M or 5 ⁇ M of asperphenin B for about 48 hours, the cells of G 2 / M phase decreased compared to the group treated with about 24 hours, and changed from 0 hours to 24 hours of asperphenin B treatment. The percentage of cells in sub-G 1 phase that did not have increased. Asperphenin B caused cell cycle arrest of G 2 / M phase until 24 hours after treatment, and induced cell death after about 48 hours.
  • 0.625 ⁇ M, 1.25 ⁇ M, 2.5 ⁇ M, 5 ⁇ M, or 10 ⁇ M of asperphenin B was added to RKO cells, and the cells were obtained after incubation for 48 hours.
  • 300 ⁇ l of 1 ⁇ binding buffer was added to the obtained cells and mixed well.
  • 5 ⁇ l of Annexin V and 5 ⁇ l of PI were added to 100 ⁇ l of the mixture, and the mixture was allowed to stand at room temperature for 15 minutes in a shielded state.
  • 400 ⁇ l of 1 ⁇ Binding Buffer was added to the reaction and cell death was analyzed using a FAX Caliber flow cytometer (BD FACSCalibur, BD Biosciences).
  • Example 2-3 As in the method described in Example 2-3, 0.625 ⁇ M, 1.25 ⁇ M, 2.5 ⁇ M, 5 ⁇ M, or 10 ⁇ M of asperphenin B was added to RKO cells, and the cells were obtained after incubation for 48 hours. Meanwhile, 5 ⁇ M of asperphenin B was added to RKO cells, and the cells were obtained after culturing for 0 to 48 hours. Protein was obtained from the obtained cells.
  • anti-ATM antibody Cell Signaling Technology
  • anti-p-Chk (Thr68) antibody Cell Signaling Technology
  • anti-Chk antibody Cell Signaling Technology
  • anti-p -H2AX antibody Cell Signaling Technology
  • anti-p53 antibody Santa Cruz
  • anti-Bax antibody Santa Cruz
  • anti-BID antibody Cell Signaling Technology
  • anti-Caspase-8 antibody Cell Signaling Technology
  • Immunoblotting with -Caspase-3 antibody Cell Signaling Technology
  • anti-Caspase-9 antibody Cell Signaling Technology
  • anti-cleaved PARP antibody BD Biosciences
  • anti- ⁇ -actin antibody Santa Cruz
  • ROS reactive oxygen species
  • Example 2-3 2.5 ⁇ M, 5 ⁇ M, or 10 ⁇ M of asperphenin B was added to RKO cells. The cells were obtained after culturing the cells for 24 hours. In addition, 5 ⁇ M and 10 ⁇ M asperphenin B were added to the control group, and 5 mM antioxidant N-acetylcysteine (NAC) (Sigma Aldrich) was added to the cell group, and asperphenin B and Cell groups without all NAC added were prepared.
  • NAC N-acetylcysteine
  • DCFH-DA 2 ', 7'-dichlorofluorescin diacetate
  • Sigma Aldrich 2 ', 7'-dichlorofluorescin diacetate
  • the unattached cells and attached cells in the cell medium were collected, washed twice with cold PBS, and then suspended again with 1 ml of PBS.
  • 2 ', 7' were analyzed using a BD FACSCalibur (BD Biosciences).
  • the intensity of Dichlorofluorescin (DCF) was measured.
  • the intensity of DCF was used to calculate the proportion of cells that produced reactive oxygen species, and the results are shown in Table 7.
  • RKO cells diluted with medium containing 10% (v / v) FBS were seeded in 96 well microplates at 7 ⁇ 10 3 cells per well and incubated for 24 hours at 37 ° C. and 5% CO 2 conditions.
  • a medium containing 10% (v / v) FBS irinotecan (Sigma Aldrich), 5-fluorouracil (Sigma Aldrich) or gemcitabine (Sigma Aldrich) is mixed with asperphenin B in a 1: 1 ratio, The mixture was added to the cultured cells. The cells were then incubated for 48 hours at 37 ° C. and 5% CO 2 conditions. Cell viability was measured using the SRB assay. The measured cell viability is shown in FIGS. 7A-7C.
  • FIG. 7A-7C The measured cell viability is shown in FIGS. 7A-7C.
  • FIG. 7A shows the cell viability (%) by irinotecan alone (•) of 1.25 ⁇ M to 10 ⁇ M or a combination of irinotecan and 2.5 ⁇ M of asperphenin B ( ⁇ )
  • FIG. 7B shows gemcitabine of 1 nM to 100 nM Cell survival rate (%) alone or in combination with gemcitabine and 8 ⁇ M of asperphenin B ( ⁇ )
  • FIG. 7C shows 5-fluorouracil alone ( ⁇ ) of 0.1 ⁇ M to 10 ⁇ M or Cell survival rate (%) by combination ( ⁇ ) of 5-fluorouracil and 8 ⁇ M of asperphenin B is shown.
  • (Dx) 1 Concentration of Asperphenin B, which is expected to be effective when Asperphenin B is administered alone.
  • Anticancer drugs Anticancer concentration ( ⁇ M ) Asperphenin B's density( ⁇ M ) Concomitant administration effect Combination Effect Rating symbol Irinotecan 1.25 2.5 0.792 ++ 2.5 2.5 0.751 ++ 5 2.5 0.691 +++ 10 2.5 0.650 +++ 5-fluorouracil 0.08 8 0.863 + 0.4 8 0.852 + 2 8 0.786 ++ 10 8 0.765 ++ Gemcitabine 0.32 8 0.907 ⁇ 1.6 8 0.882 + 8 8 0.801 ++ 40 8 0.714 ++
  • asperphenin B The anticancer effect of asperphenin B was confirmed in a tumor xenograft mouse model transplanted with a human colon cancer cell line.
  • RKO cells were injected subcutaneously into the right flank of nude mice (central laboratory animals, mice with a hairless whiskers and thymus without a thymus) at a concentration of 3.5 ⁇ 10 6 cells / 150 ⁇ l.
  • nude mice central laboratory animals, mice with a hairless whiskers and thymus without a thymus
  • Tumor volume was calculated by Equation 2 below, and tumor growth inhibition rate was calculated by Equation 3 from the calculated tumor volume.
  • Tumor volume (mm 3 ) (horizontal) ⁇ (vertical) ⁇ (height) ⁇ ⁇ / 6
  • % Tumor Growth Inhibition [1- (final mean tumor volume of asperphenin B treated group) / (final mean tumor volume of control)] ⁇ 100
  • the tumor volume (mm 3 ) of the mice according to the days elapsed after the administration of asperphenin B after the administration of asperphenin B is shown in FIG. 8 ( ⁇ : control, ⁇ : administration of asperphenin B at 4 mg / kg, ⁇ : 8 mg / kg of asperphenin B administration, *: p ⁇ 0.05, **: p ⁇ 0.01, ***: p ⁇ 0.005), and the tumor growth inhibition rate calculated therefrom is shown in Table 9.

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Abstract

Nouveau composé peptidique, son procédé de production, et son utilisation. Ledit composé a une activité anticancéreuse. Ainsi, ce composé peut être utilisé pour la prévention ou le traitement du cancer.
PCT/KR2015/011281 2014-10-23 2015-10-23 Nouveau composé peptidique, son procédé de production et son utilisation WO2016064245A1 (fr)

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EP15851785.4A EP3222629A4 (fr) 2014-10-23 2015-10-23 Nouveau composé peptidique, son procédé de production et son utilisation
US15/521,222 US10618934B2 (en) 2014-10-23 2015-10-23 Peptide compound, production method therefor, and use thereof
CN201580070797.5A CN107108693B (zh) 2014-10-23 2015-10-23 新的肽化合物、其制备方法及其用途
US16/805,423 US10759831B2 (en) 2014-10-23 2020-02-28 Peptide compound, production method therefor, and use thereof

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008144747A2 (fr) * 2007-05-21 2008-11-27 The Regents Of The University Of California Lipoprotéines, lipopeptides et analogues, et leurs procédés de fabrication et d'utilisation
US7671011B2 (en) * 2003-06-19 2010-03-02 Yeda Research & Development Co. Ltd. Antimicrobial and anticancer lipopeptides
US8394954B2 (en) * 2007-03-30 2013-03-12 Council Of Scientific & Industrial Research Benzophenone hybrids as anticancer agents and processes for the preparation thereof

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
US7671011B2 (en) * 2003-06-19 2010-03-02 Yeda Research & Development Co. Ltd. Antimicrobial and anticancer lipopeptides
US8394954B2 (en) * 2007-03-30 2013-03-12 Council Of Scientific & Industrial Research Benzophenone hybrids as anticancer agents and processes for the preparation thereof
WO2008144747A2 (fr) * 2007-05-21 2008-11-27 The Regents Of The University Of California Lipoprotéines, lipopeptides et analogues, et leurs procédés de fabrication et d'utilisation

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Title
DING ET AL.: "Genome-based characterization of two prenylation steps in the assembly of the stephacidin and notoamide anticancer agents in a marine-derived Aspergillus sp.", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 132, no. 36, 2010, pages 12733 - 12740, XP055275638 *
MATHAN ET AL.: "Anticancer and antimicrobial activity of Aspergillus protuberus SP1 isolated from marine sediments of South Indian coast", CHINESE JOURNAL OF NATURAL MEDICINES, vol. 9, no. 4, 2011, pages 286 - 292, XP055275633 *
PENG ET AL.: "Psychrophilins E-H and versicotide C, cyclic peptides from the marine-derived fungus Aspergillus versicolor ZLN-60", JOURNAL OF NATURAL PRODUCTS, vol. 77, no. 10, pages 2218 - 2223, XP055275639 *
See also references of EP3222629A4 *

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