WO2010122946A1 - 歯周病マーカー - Google Patents

歯周病マーカー Download PDF

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WO2010122946A1
WO2010122946A1 PCT/JP2010/056778 JP2010056778W WO2010122946A1 WO 2010122946 A1 WO2010122946 A1 WO 2010122946A1 JP 2010056778 W JP2010056778 W JP 2010056778W WO 2010122946 A1 WO2010122946 A1 WO 2010122946A1
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periodontal disease
autoinducer
bacteria
furanone
agent
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French (fr)
Japanese (ja)
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初美 惣野
英剛 藤中
純二 中村
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Kao Corp
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Kao Corp
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Priority to US13/258,700 priority Critical patent/US8293478B2/en
Priority to EP10767003.6A priority patent/EP2423683B1/en
Publication of WO2010122946A1 publication Critical patent/WO2010122946A1/ja
Anticipated expiration legal-status Critical
Priority to US13/529,717 priority patent/US8568986B2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds containing boron, silicon or a metal, e.g. chelates or vitamin B12
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/18—Dental and oral disorders
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
    • Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Definitions

  • the present invention relates to a periodontal disease marker.
  • Periodontal tissue refers to tissue surrounding the teeth and supporting the teeth, and is composed of gingiva, periodontal ligament, cementum, and alveolar bone.
  • gingiva tissue surrounding the teeth and supporting the teeth
  • gingiva tissue surrounding the teeth and supporting the teeth
  • alveolar bone a tissue where the inflammation is confined to the gingiva
  • gingivitis the one where the inflammation is confined to the gingiva
  • periodontitis the case where the inflamed part extends beyond the gum and the periodontal ligament and alveolar bone are damaged or destroyed.
  • Periodontal disease unlike caries, is almost painless and often progresses without being noticed. However, if the periodontal disease is left untreated, the symptom progresses, and there is a high risk that the tooth will eventually fall out.
  • Periodontal disease measures include preventive actions such as correct tooth brushing, plaque control by regular checkups, and improvement of dietary habits.
  • preventive actions such as correct tooth brushing, plaque control by regular checkups, and improvement of dietary habits.
  • Diagnosis of periodontal disease is generally performed by measuring periodontal pockets, attachment level, X-ray image diagnosis, and the like.
  • these diagnostic methods place a heavy burden on the subject.
  • these periodontal disease diagnosis methods have a complicated operation procedure and have problems such as individual differences in judgment criteria because they are based on experience and skills of dentists.
  • gingival crevicular fluid is collected using a brush-like collection device, and biomarkers such as lactoferrin, ⁇ 1-antitrypsin, and hemoglobin contained in the gingival crevicular fluid are detected.
  • biomarkers such as lactoferrin, ⁇ 1-antitrypsin, and hemoglobin contained in the gingival crevicular fluid are detected.
  • the method of diagnosing periodontal disease is known (for example, refer patent documents 1 and 2).
  • the gingival crevicular fluid contains components other than the gingival crevicular fluid, and accuracy may be lost when saliva is mixed.
  • a method of diagnosing periodontal disease by detecting occult blood (hemoglobin) in saliva or mouthwash discharge liquid is also known (see Non-Patent Document 1, for example).
  • occult blood in saliva may contain other substances than those derived from periodontal disease.
  • Saliva volume varies greatly between individuals and diurnal variation, and since there is no hemoglobin quantitativeness, it is impossible to diagnose the degree of progression of periodontal disease.
  • This invention makes it a subject to provide the marker for periodontal disease onset determination which can determine the presence or absence of onset of periodontal disease simply and correctly.
  • Another object of the present invention is to provide a marker for determining the degree of progression of periodontal disease that can easily and accurately determine the degree of progression of periodontal disease.
  • this invention makes it a subject to provide the periodontal disease onset determination method which can determine the presence or absence of onset of a periodontal disease simply and correctly.
  • this invention makes it a subject to provide the periodontal disease progress determination method which can determine the extent of a periodontal disease progress simply and correctly.
  • the present invention provides a screening method for a periodontal disease ameliorating agent or preventive agent that is effective for simple and accurate screening of a periodontal disease ameliorating agent or prophylactic agent effective in preventing and improving periodontal disease. Let it be an issue.
  • Bacteria have acquired a mechanism for sensitively sensing changes in their environment. As one of such mechanisms, it is clear that it senses its own density in the environment through a specific information transmission substance and skillfully controls its various biological activities according to the density. ing. Such an information transmission mechanism between bacteria is called a quorum sensing system.
  • Bacteria with a quorum sensing system synthesize and release a signaling molecule called an autoinducer and respond to the signaling molecule to control gene expression as a function of bacterial density. So far, it has been reported that autoinducer-2 is used for communication between different bacterial species (see, for example, Bassler et al., Bacteriol. 179, pp. 4043-4045, 1997).
  • Japanese Patent Application Laid-Open No. 2008-214296 discusses the biofilm formation inhibitory effect when an antagonist of an autoinducer-2 receptor is added using a Porphyromonas gingivalis strain, which is one of periodontal pathogens.
  • Porphyromonas gingivalis strain which is one of periodontal pathogens.
  • there are many other bacterial species other than Porphyromonas gingivalis Moreover, there exists an effect
  • the present inventors have conducted intensive studies. As a result, the influence of autoinducer-2 on periodontal disease in vivo is clarified, and autoinducer-2 used for communication between different bacterial species released when resident bacteria present in the oral cavity grow. It was found that there is a correlation between the amount of cancer and periodontal disease. Specifically, it has been found that the presence or absence of periodontal disease and the progression of periodontal disease are related to the amount of autoinducer-2 in the oral cavity. Furthermore, the present inventors have found that a substance having an autoinducer-2 activity inhibitory effect is effective for periodontal disease improvement / prevention. The present invention has been completed based on these findings.
  • the present invention relates to a periodontal disease onset determination marker comprising autoinducer-2.
  • the present invention also relates to a marker for determining the degree of progression of periodontal disease comprising autoinducer-2.
  • the present invention also provides a sample collected from the oral cavity of a subject, quantifies autoinducer-2 contained in the collected sample, and periodontal disease develops when the quantified amount of autoinducer-2 is large.
  • the present invention relates to a method for determining periodontal disease onset.
  • the present invention collects a specimen from the oral cavity of a subject, quantifies autoinducer-2 contained in the collected specimen, and periodontal disease progresses as the amount of quantified autoinducer-2 increases.
  • the present invention relates to a periodontal disease progress determination method.
  • the present invention provides a test agent having an autoinducer-2 activity inhibitory effect or a preventive agent by mixing a test agent with a bacterium having a quorum sensing system via autoinducer-2 and a test agent.
  • the present invention relates to a screening method for a periodontal disease improving agent or a preventive agent to be selected.
  • the present invention also relates to the use of autoinducer-2 as a marker for determining periodontal disease onset.
  • the present invention relates to the use of autoinducer-2 as a marker for determining the progress of periodontal disease.
  • the presence or absence of the onset of periodontal disease can be determined easily and accurately.
  • the degree of progression of periodontal disease can be easily and accurately determined.
  • the method for determining the onset of periodontal disease of the present invention the presence or absence of the onset of periodontal disease can be determined easily and accurately.
  • the periodontal disease progression degree determination method of the present invention the degree of progression of periodontal disease can be easily and accurately determined.
  • the method for screening for a periodontal disease improving agent or preventive agent of the present invention can conveniently and accurately screen for a periodontal disease improving agent or prophylactic agent effective in preventing and improving periodontal disease.
  • FIGS. 1 (a) to 1 (c) show, in Example 2, a control (FIG. 1 (a)), 4-bromo-5-5 as a test agent on the mandibular first molar (circled portion) of Syrian hamster.
  • (4-methoxyphenyl) -2 (5H) -furanone (FIG. 1 (b)) and 3,4-dibromo-5-hydroxy-2 (5H) -furanone (FIG. 1 (c)) were administered, respectively. It is a drawing-substituting photograph taken under a stereomicroscope after extracting the mandible after 2 weeks.
  • the periodontal disease onset determination marker (also simply referred to as periodontal disease determination marker) and the periodontal disease progression determination marker of the present invention are obtained from autoinducer-2 collected from the oral cavity of a subject such as a human. Become. Autoinducer-2 (hereinafter also referred to as “AI-2”) in the present invention is not particularly limited as long as it is produced by bacteria and has AI-2 activity.
  • AI-2 activity refers to an activity in which AI-2 affects bacteria having a quorum sensing system, that is, an activity that promotes the function of bacteria brought about by quorum sensing via AI-2.
  • Bacteria are luminescent, swarming, biofilm formation, proteolytic enzyme production, antibiotic synthesis, gene acceptability development, plasmid conjugation transfer, pathogenic factor production and spore formation by quorum sensing via AI-2 Etc. are known to perform.
  • AI-2 activity in other words, bioluminescence, swarming, biofilm formation, production of proteolytic enzymes, antibiotic production by bacteria that recognize AI-2, ie, bacteria having an AI-2 receptor, It can be referred to as activities such as synthesis, development of gene acceptability, plasmid conjugation transmission, pathogenic factor production and sporulation.
  • pathogenic factor examples include enterotoxin, adenylate cyclase toxin, adhesin, alkaline protease, hemolytic toxin, anthrax toxin, APX toxin, ⁇ toxin, ⁇ toxin, ⁇ toxin, C2 toxin, C3 toxin, botulinum toxin, bundled line Hair structure subunit, C5A peptidase, cardiotoxin, chemotaxis, cholera toxin, ciliary toxin, clostridial cytotoxin, clostridial neurotoxin, collagen adhesion gene, cytolysin, vomiting toxin, endotoxin, epidermal exfoliating toxin, external Toxin, extracellular elastase, fibrinogen, fibronectin binding protein, fibrillar hemagglutinin, fimbria, gelatinase, hemagglutinin, leukotoxin,
  • AI-2 in the present invention include 4,5-dihydroxy-2,3-pentanedione (DPD) represented by the following formula.
  • DPD When DPD binds to the bacterial AI-2 receptor, it incorporates boron and is converted to furanosyl borate diester.
  • AI-2 in the present invention is the furanosyl borate diester.
  • specific examples of the furanosyl borate diester are shown below. However, the present invention is not limited to these.
  • the bacterium having an AI-2 receptor e.g., Porphyromonas (Porphyromonas) bacteria, Haemophilus (Haemophilus) bacteria, Bacillus (Bacillus) bacteria, Neisseria (Neisseria) bacteria, Streptococcus (Streptococcus) bacteria, Eikenera (Eikenella) bacteria belonging to the genus Lactobacillus (Lactobacillus) bacteria belonging to the genus Actinobacillus (Actinobacillus) bacteria belonging to the genus Actinomyces (Actinomyces) bacteria belonging to the genus Bacteroides (Bacteroides) bacteria belonging to the genus, Cap Roh site file moth (Capnocytophaga) bacteria belonging to the genus, Fusobacterium (Fusobacterium) bacteria, Peputokokkasu (Peptococcus) bacteria, Prevotella (Prevotella) bacteria, Seremonasu (
  • poly Philo Monas gingivalis Porphyromonas gingivalis
  • Streptococcus pyogenes Streptococcus pyogenes
  • Streptococcus mutans Streptococcus mutans
  • Eikenera-Korodensu Eikenella corrodens
  • Lactobacillus salivarius Lactobacillus salivarius
  • Streptococcus Sanginisu Streptococcus sanguinis
  • Streptococcus anginosus Streptococcus anginosus
  • Streptococcus oralis Streptococcus oralis
  • Streptococcus Gorudoni Streptococcus gordonii
  • Streptococcus mitis Streptococcus mitis
  • Actinobacillus actinomycetemcomitans Actinobacillus actinomycetemcomitans
  • AI-2 is collected from the oral cavity of a subject such as a human and used.
  • examples of the subject include mammals such as humans, monkeys, chimpanzees, dogs, cats, cows, pigs, rats, and mice.
  • the AI-2 is not particularly limited as long as it is collected from the oral cavity, and AI-2 contained in specimens such as saliva, plaque, tongue coating, and gingival crevicular fluid collected from the oral cavity can be used.
  • the saliva can be obtained as it is discharged as it is or whether it is impregnated with water, regardless of whether it is unstimulated or stimulated.
  • Plaque can be collected by cleaning the tooth surface with a dental scaler, swab, brush, or inserting a paper point. Tongue moss is obtained by brushing with a brush, cotton swab or gauze. The gingival crevicular fluid can be obtained by inserting a paper point or the like into the gingival sulcus.
  • the method for collecting AI-2 is not limited to this.
  • the present inventors have found that when the amount of AI-2 present in the oral cavity of the subject is large, the subject has developed periodontal disease. Furthermore, it has been found that periodontal disease progresses as the amount of AI-2 increases. Therefore, by quantifying the amount of AI-2 collected from the oral cavity, it is possible to determine the presence or absence of periodontal disease and the degree of progression (severity) of the periodontal disease in the subject. As an example, assuming that the AI-2 activity in the case of DPD 10 ⁇ M is 100, normal or mild periodontal disease when the AI-2 activity is 0 or more and less than 3, and moderate periodontal disease when the AI-2 activity is 3 or more and less than 10, 10 The above can be determined to be severe periodontal disease. The therapeutic effect can be determined by comparing the amount of AI-2 before the start of periodontal disease treatment and the amount of AI-2 after treatment.
  • AI-2 can be quantified by a bioassay using a reporter bacterium that emits light by recognizing AI-2, preferably a bacterium having AI-2 receptor and luciferase activity, and measuring the luminescence intensity. it can.
  • Vibrio Harvey BB170 strain is used as an AI-2 reporter bacterium, and calibration using 4-hydroxy-5-methyl-3 (2H) -furanone (HMF) standard and / or DPD standard as a standard sample for AI-2
  • HMF 4-hydroxy-5-methyl-3 (2H) -furanone
  • DPD DPD standard
  • a line can be prepared and AI-2 can be quantified from the luminescence intensity when a test sample is added (see, for example, Chen X. et al., Nature, vol. 415, p. 545-549, 2002) .
  • AI-2 contained in a test sample can be reacted with phenylenediamine and N-methyl-N- (trimethylsilyl) trifluoro-acetamide, and then AI-2 can be quantified by GC-MS (for example, Thiel V.
  • test sample for quantifying AI-2 it is preferable to remove impurities by ultrafiltration of a sample collected from the oral cavity with a filter having a cutoff molecular weight of 3000 or less after crushing / extraction treatment.
  • a container having low AI-2 adsorptivity such as a glass container, for preparing and storing a test sample.
  • a test agent having an autoinducer-2 activity inhibitory effect by mixing a test agent with a bacterium having a quorum sensing system via autoinducer-2 Is selected as a periodontal disease improving agent or preventive agent.
  • Vibrio Harvey BB170 strain was prepared to be diluted 1000 to 10,000 times (preferably 4000 to 5000 times) in a medium and preincubated with a test compound solution at room temperature for 1 to 30 minutes (preferably 10 minutes). Thereafter, AI-2 is added and aerobic shaking culture is performed at 30 ⁇ 5 ° C., and the luminescence intensity after 2 to 6 hours (preferably after 4 hours) is measured with a chemiluminescence meter.
  • the final concentration of AI-2 is 0.001 to 100 ⁇ M (preferably 10 ⁇ M), and each test compound solution is 0.0001 to 0.1%, or 1 to 100 ⁇ M. Each concentration is not limited to this.
  • the control compound is added with AI-2 alone, and the luminescence intensity when mixed with the test compound solution is measured as a relative value with respect to the luminescence intensity of the control, whereby the AI-2 activity inhibitory effect of the test compound can be measured.
  • Bacteria having a quorum sensing system via autoinducer-2 are not particularly limited, and reporter bacteria that recognize autoinducer-2 and emit light are preferable, and oral bacteria and periodontal pathogens can also be used.
  • bacteria having a quorum sensing system via autoinducer-2 collected from the oral cavity of a subject can also be used as a specimen.
  • the “autoinducer-2 activity inhibitory effect” in the present invention refers to an inhibitory effect on AI-2 activity. Specifically, bioluminescence, swarming, biofilm formation, production of proteolytic enzymes, synthesis of antibiotics, development of gene acceptability, plasmid conjugation transfer, pathogenesis by bacteria having AI-2 receptor It refers to the inhibitory effect on AI-2 activity such as factor production and sporulation.
  • the test agent having an AI-2 activity inhibitory effect selected by the screening method of the present invention can be used as an AI-2 activity inhibitor, a periodontal disease ameliorating agent or a preventive agent. Further, the AI-2 activity inhibitor can be suitably used as a material for preparing a periodontal disease ameliorating agent or preventive agent.
  • Example 1 Determination of Periodontal Disease Onset and Periodontal Disease Progression
  • AI-2 in plaque Vibrio Harvey BB170 strain (distributed from ATCC, AB (Autoinducer Bioassay) medium (10 mM potassium phosphate buffer [pH 7] 0.0], 0.3 M NaCl, 0.05 M MgSO 4 , 0.2% casamino acid, 2% glycerol, 1 mM L-arginine, 1 ⁇ g / mL thiamine, 0.01 ⁇ g / mL riboflavin) at 30 ° C. overnight. Culture) was used as AI-2 reporter bacteria and diluted 5000 times in AB medium to prepare a reporter bacteria solution.
  • AB Autoinducer Bioassay
  • a total amount of 0.001 to 100 mg of subgingival plaque adhering to the tooth surface of one tooth was collected with a dental scaler.
  • the collected plaque was dispersed in 1 mL of PBS, incubated at 90 ° C. for 10 minutes, and then crushed with 0.3 g of zirconia / silica beads (particle size: 0.1 mm, manufactured by Tommy Seiko Co., Ltd.). After centrifuging at 14,000 rpm for 5 minutes, the supernatant was passed through a 0.22 ⁇ m filter (Ultra Free MC, manufactured by Millipore) and further a filter with a molecular weight of 3000 cut (Microcon YM-3, manufactured by Millipore) to prepare a sample. .
  • the sample prepared in this manner was mixed with the reporter bacterial solution prepared in the same manner as described above at a reporter bacterial solution: standard volume ratio of 9: 1, cultured at 30 ° C. under aerobic shaking, and cultured for 4 hours.
  • the later emission intensity was measured with a chemiluminescence meter.
  • AI-2 contained in dental plaque was quantified from the measured luminescence intensity and the calibration curve.
  • Table 1 shows the relationship between the PD measured according to the above method and the AI-2 amount quantified in (1) above.
  • GI gingival inflammation index
  • Table 2 shows the relationship between the GI measured according to the above method and the amount of AI-2 quantified in (1) above.
  • Table 3 shows the relationship between BOP measured according to the above method and the amount of AI-2 quantified in (1) above.
  • AI-2 present in the oral cavity can be used as a marker for determining the onset of periodontal disease and a marker for determining the progression of periodontal disease.
  • Example 2 Screening for Periodontal Disease Improvement Agent or Preventive Agent Vibrio Harvey BB170 strain (cultured overnight at 30 ° C. in AB medium) was used as an AI-2 reporter bacterium, and diluted 4500 times in AB medium to produce a reporter bacterium. A liquid was prepared. After mixing the reporter bacterial solution and each test agent and preincubating at room temperature for 10 minutes, DPD was added, aerobic shaking culture was performed at 30 ° C., and the luminescence intensity after 4 hours was measured with a chemiluminescence meter (LB940 type, belt-type). Manufactured by Ludo Japan). From the measured emission intensity, AI-2 was quantified in the same manner as in Example 1.
  • compound 1 4-bromo-5- (4-methoxyphenyl) -2 (5H) -furanone (manufactured by Sigma)
  • compound 2 3,4-dibromo-5-hydroxy-2 (5H) -Furanone (manufactured by Sigma)
  • compound 3 4-bromo-5-methoxy-5- (4-methoxyphenyl) -2 (5H) -furanone (manufactured by Sigma)
  • compound 4 4- ⁇ [3- Bromo 2- (4-methoxyphenyl) -5-oxo-2,5-dihydro-2-furanyl] oxy ⁇ benzoic acid (manufactured by Sigma) was used.
  • AI-2 inhibitory compounds compound 5: 4-hydroxy-2,5-dimethyl-3 (2H) -furanone (manufactured by Sigma), compound 6: 2-methoxy- 2,4-diphenyl-3 (2H) -furanone (Sigma), compound 7: 2-pentyl-2-cyclopenten-1-one (Sigma)) (Bassseler et al., Bacteriol. 179, p. 4043-4045, 1997; Yoshida A. et al., Appl. Environ. Microbiol., 71 (5), p. 2372-2380, 2005; and Wen Z. T. et al., J. Bacteriol. 189 (9), p.2682-2691, 2004).
  • the AI-2 amount was a relative value to the control DPD amount (AI-2 amount) when the control agent was added with only DPD without adding the test agent.
  • the results are shown in Table 4.
  • the mandible of Syrian hamster (7 weeks old, 3 males each) was removed under anesthesia and photographed under a stereomicroscope.
  • the evaluation site was the lingual side of the extracted mandible, and the obtained photograph was subjected to image analysis, and the vertical distance from the first molar mesial cusp to the alveolar crest was measured.
  • FIGS. 1 (a) to 1 (c) The evaluation site was the lingual side of the extracted mandible, and the obtained photograph was subjected to image analysis. The obtained photograph was used for image analysis, and the vertical distance from the first molar mesial cusp to the alveolar crest was measured.
  • the alveolar bone resorption depth (value after addition for 2 weeks ⁇ value before addition) was measured from the vertical distance from the first molar mesial cusp to the alveolar crest before and after addition of the test agent. The results are shown in Table 5.

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PCT/JP2010/056778 2009-04-24 2010-04-15 歯周病マーカー Ceased WO2010122946A1 (ja)

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