JPS63277966A - Drug for inspecting periodontal disease - Google Patents
Drug for inspecting periodontal diseaseInfo
- Publication number
- JPS63277966A JPS63277966A JP11312287A JP11312287A JPS63277966A JP S63277966 A JPS63277966 A JP S63277966A JP 11312287 A JP11312287 A JP 11312287A JP 11312287 A JP11312287 A JP 11312287A JP S63277966 A JPS63277966 A JP S63277966A
- Authority
- JP
- Japan
- Prior art keywords
- soln
- group
- formula
- periodontal disease
- residue
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 208000028169 periodontal disease Diseases 0.000 title claims abstract description 34
- 239000003814 drug Substances 0.000 title abstract description 6
- 229940079593 drug Drugs 0.000 title abstract description 6
- 239000000758 substrate Substances 0.000 claims abstract description 32
- 230000000694 effects Effects 0.000 claims abstract description 21
- 102000004190 Enzymes Human genes 0.000 claims abstract description 18
- 108090000790 Enzymes Proteins 0.000 claims abstract description 18
- 238000012360 testing method Methods 0.000 claims description 27
- 150000001875 compounds Chemical class 0.000 claims description 24
- 239000003795 chemical substances by application Substances 0.000 claims description 16
- 150000001413 amino acids Chemical group 0.000 claims description 11
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 claims description 9
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 8
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 claims description 7
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 5
- 108090000765 processed proteins & peptides Chemical group 0.000 claims description 5
- 125000006239 protecting group Chemical group 0.000 claims description 5
- 239000004475 Arginine Substances 0.000 claims description 4
- 125000001433 C-terminal amino-acid group Chemical group 0.000 claims description 4
- 239000004471 Glycine Substances 0.000 claims description 4
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 4
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 4
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 4
- 239000004472 Lysine Substances 0.000 claims description 4
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052739 hydrogen Inorganic materials 0.000 claims description 4
- 239000001257 hydrogen Substances 0.000 claims description 4
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 4
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 4
- 125000003277 amino group Chemical group 0.000 claims description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 19
- 241000894006 Bacteria Species 0.000 abstract description 14
- 239000000872 buffer Substances 0.000 abstract description 14
- 238000006243 chemical reaction Methods 0.000 abstract description 13
- 238000004040 coloring Methods 0.000 abstract description 12
- 239000012954 diazonium Substances 0.000 abstract description 5
- 150000001989 diazonium salts Chemical class 0.000 abstract description 5
- 239000000203 mixture Substances 0.000 abstract description 4
- 239000002223 garnet Substances 0.000 abstract description 3
- 238000002156 mixing Methods 0.000 abstract description 2
- 230000015572 biosynthetic process Effects 0.000 abstract 1
- 235000019646 color tone Nutrition 0.000 abstract 1
- 238000007689 inspection Methods 0.000 abstract 1
- 238000000034 method Methods 0.000 description 10
- 235000001014 amino acid Nutrition 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 239000007864 aqueous solution Substances 0.000 description 7
- 238000002844 melting Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- JBIJLHTVPXGSAM-UHFFFAOYSA-N 2-naphthylamine Chemical compound C1=CC=CC2=CC(N)=CC=C21 JBIJLHTVPXGSAM-UHFFFAOYSA-N 0.000 description 6
- 241000605862 Porphyromonas gingivalis Species 0.000 description 6
- 241000589970 Spirochaetales Species 0.000 description 6
- -1 t-butoxycarbonyl group Chemical group 0.000 description 6
- SFKZPTYRENGBTJ-UHFFFAOYSA-N 4-methoxynaphthalen-2-amine Chemical compound C1=CC=C2C(OC)=CC(N)=CC2=C1 SFKZPTYRENGBTJ-UHFFFAOYSA-N 0.000 description 5
- TYMLOMAKGOJONV-UHFFFAOYSA-N 4-nitroaniline Chemical compound NC1=CC=C([N+]([O-])=O)C=C1 TYMLOMAKGOJONV-UHFFFAOYSA-N 0.000 description 5
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 5
- 210000003296 saliva Anatomy 0.000 description 5
- 239000008351 acetate buffer Substances 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 238000010647 peptide synthesis reaction Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 210000003731 gingival crevicular fluid Anatomy 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 3
- 230000000007 visual effect Effects 0.000 description 3
- FTOAOBMCPZCFFF-UHFFFAOYSA-N 5,5-diethylbarbituric acid Chemical compound CCC1(CC)C(=O)NC(=O)NC1=O FTOAOBMCPZCFFF-UHFFFAOYSA-N 0.000 description 2
- JBNOVHJXQSHGRL-UHFFFAOYSA-N 7-amino-4-(trifluoromethyl)coumarin Chemical compound FC(F)(F)C1=CC(=O)OC2=CC(N)=CC=C21 JBNOVHJXQSHGRL-UHFFFAOYSA-N 0.000 description 2
- AKASINKCIWHQEP-UHFFFAOYSA-N 7-amino-4-methoxychromen-2-one Chemical compound C1=C(N)C=CC2=C1OC(=O)C=C2OC AKASINKCIWHQEP-UHFFFAOYSA-N 0.000 description 2
- 208000010266 Aggressive Periodontitis Diseases 0.000 description 2
- 208000002064 Dental Plaque Diseases 0.000 description 2
- 241000605909 Fusobacterium Species 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 2
- 208000033608 aggressive 1 periodontitis Diseases 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 239000003708 ampul Substances 0.000 description 2
- 235000003704 aspartic acid Nutrition 0.000 description 2
- 230000000721 bacterilogical effect Effects 0.000 description 2
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 description 2
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 2
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 2
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 208000034391 chronic adult periodontitis Diseases 0.000 description 2
- 208000001277 chronic periodontitis Diseases 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- DEKPYXUDJRABNK-UHFFFAOYSA-N dimethyl 5-aminobenzene-1,3-dicarboxylate Chemical compound COC(=O)C1=CC(N)=CC(C(=O)OC)=C1 DEKPYXUDJRABNK-UHFFFAOYSA-N 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 210000000214 mouth Anatomy 0.000 description 2
- UUQKDNWDIYWCAE-NRFANRHFSA-N n-[2-[[(2s)-5-(diaminomethylideneamino)-1-(naphthalen-2-ylamino)-1-oxopentan-2-yl]amino]-2-oxoethyl]benzamide Chemical compound N([C@@H](CCCNC(=N)N)C(=O)NC=1C=C2C=CC=CC2=CC=1)C(=O)CNC(=O)C1=CC=CC=C1 UUQKDNWDIYWCAE-NRFANRHFSA-N 0.000 description 2
- FBYVDJCBBGMJHI-UHFFFAOYSA-N n-methoxynaphthalen-2-amine Chemical compound C1=CC=CC2=CC(NOC)=CC=C21 FBYVDJCBBGMJHI-UHFFFAOYSA-N 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 125000002730 succinyl group Chemical group C(CCC(=O)*)(=O)* 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 2
- RUFPHBVGCFYCNW-UHFFFAOYSA-N 1-naphthylamine Chemical compound C1=CC=C2C(N)=CC=CC2=C1 RUFPHBVGCFYCNW-UHFFFAOYSA-N 0.000 description 1
- XNWFRZJHXBZDAG-UHFFFAOYSA-N 2-METHOXYETHANOL Chemical compound COCCO XNWFRZJHXBZDAG-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 241000186046 Actinomyces Species 0.000 description 1
- 241000606749 Aggregatibacter actinomycetemcomitans Species 0.000 description 1
- 241000606125 Bacteroides Species 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241001058146 Erium Species 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- DICSXQHGGOAUNU-AWEZNQCLSA-N L-arginine 2-naphthylamide Chemical compound C1=CC=CC2=CC(NC(=O)[C@H](CCCN=C(N)N)N)=CC=C21 DICSXQHGGOAUNU-AWEZNQCLSA-N 0.000 description 1
- 241000237503 Pectinidae Species 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- 241000589892 Treponema denticola Species 0.000 description 1
- PUDCZUQFOPHIGU-UHFFFAOYSA-N [2-methyl-4-[(2-methylphenyl)diazenyl]phenyl]azanium;chloride Chemical compound Cl.C1=C(N)C(C)=CC(N=NC=2C(=CC=CC=2)C)=C1 PUDCZUQFOPHIGU-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 229960002319 barbital Drugs 0.000 description 1
- DDCSGCFMHGKUCB-BDYUSTAISA-N benzyl n-[(2s)-1-[[2-[[(2s)-5-(diaminomethylideneamino)-1-(naphthalen-2-ylamino)-1-oxopentan-2-yl]amino]-2-oxoethyl]amino]-3-methyl-1-oxobutan-2-yl]carbamate Chemical compound N([C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NC=1C=C2C=CC=CC2=CC=1)C(=O)OCC1=CC=CC=C1 DDCSGCFMHGKUCB-BDYUSTAISA-N 0.000 description 1
- WYVUDEJWEXQWJV-LSYYVWMOSA-N benzyl n-[(2s)-1-[[2-[[(2s)-5-(diaminomethylideneamino)-1-[(4-methoxynaphthalen-2-yl)amino]-1-oxopentan-2-yl]amino]-2-oxoethyl]amino]-3-methyl-1-oxobutan-2-yl]carbamate Chemical compound N([C@H](C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NC=1C=C(C2=CC=CC=C2C=1)OC)C(C)C)C(=O)OCC1=CC=CC=C1 WYVUDEJWEXQWJV-LSYYVWMOSA-N 0.000 description 1
- HKVJXEIFVWURCA-UIOOFZCWSA-N benzyl n-[(2s)-6-amino-1-[(2s)-2-(naphthalen-2-ylcarbamoyl)pyrrolidin-1-yl]-1-oxohexan-2-yl]carbamate Chemical compound N([C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)NC=1C=C2C=CC=CC2=CC=1)C(=O)OCC1=CC=CC=C1 HKVJXEIFVWURCA-UIOOFZCWSA-N 0.000 description 1
- 230000035584 blastogenesis Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
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- 239000006172 buffering agent Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 1
- 208000007565 gingivitis Diseases 0.000 description 1
- WKQUUNDBFODDFP-HNNXBMFYSA-N glycyl-L-proline 2-naphthylamide Chemical compound NCC(=O)N1CCC[C@H]1C(=O)NC1=CC=C(C=CC=C2)C2=C1 WKQUUNDBFODDFP-HNNXBMFYSA-N 0.000 description 1
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- 230000003902 lesion Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- JVXXKQIRGQDWOJ-UHFFFAOYSA-N naphthalene-2-carboxamide Chemical compound C1=CC=CC2=CC(C(=O)N)=CC=C21 JVXXKQIRGQDWOJ-UHFFFAOYSA-N 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 230000003239 periodontal effect Effects 0.000 description 1
- 201000001245 periodontitis Diseases 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- SYUHGPGVQRZVTB-OIOBTWANSA-N radon-219 atom Chemical compound [219Rn] SYUHGPGVQRZVTB-OIOBTWANSA-N 0.000 description 1
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- ZQCOOZZRVINOLC-NSVAZKTRSA-N tert-butyl n-[(2s)-1-[[(2s)-1-[[2-[[(2s)-5-(diaminomethylideneamino)-1-(naphthalen-2-ylamino)-1-oxopentan-2-yl]amino]-2-oxoethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]carbamate Chemical compound C1=CC=CC2=CC(NC(=O)[C@H](CCCN=C(N)N)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)OC(C)(C)C)C(C)C)CC(C)C)=CC=C21 ZQCOOZZRVINOLC-NSVAZKTRSA-N 0.000 description 1
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Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
本発明は歯周疾患検査薬、さらに詳しくは、検体中のあ
る種の歯周疾患原因菌を特異的に、かつ、簡便、迅速に
検出し、歯周疾患の1患や進行を診断あるいは予測する
ことのできる検査薬に間する。DETAILED DESCRIPTION OF THE INVENTION The present invention provides a periodontal disease test agent, more specifically, a periodontal disease-causing agent that specifically, simply, and quickly detects certain types of periodontal disease-causing bacteria in a sample, and which detects one type of periodontal disease. Use test drugs that can diagnose or predict the disease and its progression.
/ s 、11
近年、歯周疾患に間する細菌学的研究が進み、歯周疾患
病巣部に多くのスピロヘータが検出され、種々の臨床的
指標と高い相関性を示すことが判明している。また、嫌
気性のダラム陰性畳菌が主要な歯周疾患の原因菌である
ことも判明しており、その中でも特に、バクテロイデス
・ジンジバリス(B acteroides gin
gival is)などの黒色色素産生バクテロイデス
(B lack−pign+ented B acte
roides)が注目され、その病原性について多数の
報告がなされている。/s, 11 In recent years, bacteriological research on periodontal diseases has progressed, and it has been found that many spirochetes have been detected in periodontal disease lesions and show a high correlation with various clinical indicators. It has also been found that the anaerobic Durham-negative Bacterium is a major causative agent of periodontal diseases, and among these, Bacteroides gingivalis (Bacteroides gingivalis) is particularly important.
Black-pign+entered Bacteroides, such as
roides) has attracted attention, and numerous reports have been made regarding its pathogenicity.
そこで、口腔内におけるこれらの原因菌の存在を検知し
、歯周疾患の罹患や進行を診断あるいは予測し、歯周疾
患の治療、予防に、臨床的に応用する試みがなされてい
る。Therefore, attempts are being made to detect the presence of these causative bacteria in the oral cavity, diagnose or predict the onset and progression of periodontal disease, and apply it clinically to the treatment and prevention of periodontal disease.
しかしな佛ら、細菌学的方法によるこれらの原因菌の検
知には、暗視野顕V&鏡の便所、嫌気性菌の取扱という
高度な技術、特殊な設備等を必要とし、操作が煩雑で、
培養や結果の判断にも長い時間や熟練を要するという欠
点があり1、臨床的に実用化するには困難な点が多い。However, the detection of these causative bacteria by bacteriological methods requires a dark-field microscope and mirror toilet, advanced techniques for handling anaerobic bacteria, special equipment, etc., and is complicated to operate.
It has the disadvantage of requiring a long time and skill to culture and judge the results1, making it difficult to put it into clinical practice.
また、免疫学的な面から、これらの原因菌に対する液性
免疫である血中の抗体価を測定したり、細胞性免疫であ
るリンパ球幼若化反応を測定し、原因菌の存在を検知す
る試みもなされているが、検体試料の調整に煩雑な操作
を必要とする問題があり、やはり、実用化はなかなか困
難である。In addition, from an immunological perspective, we can detect the presence of causative bacteria by measuring antibody titers in the blood, which is humoral immunity, against these causative bacteria, and by measuring lymphocyte blastogenesis, which is cell-mediated immunity. Although attempts have been made to do so, there is a problem in that the preparation of specimen samples requires complicated operations, and it is still difficult to put them into practical use.
このような事情にかんがみ、本発明者らは、臨床的に実
用化できる歯周疾患原因菌の検知を可能とすべく、鋭意
研究を重ねた。その結果、口腔内スピロヘータが非常に
特異的なアミノペプチダーゼ様酵素活性を有し、また、
バクテロイデス・ジンジバリスも同様な活性を有し、あ
る種の基質を用いることにより、この酵素活性を特異的
に、かつ、簡便、迅速に検出でき、しかも、歯周疾患の
症状が正確に反映されることを見出した。In view of these circumstances, the present inventors have conducted extensive research in order to make it possible to detect periodontal disease-causing bacteria that can be put to practical use clinically. As a result, oral spirochetes possess highly specific aminopeptidase-like enzymatic activity;
Bacteroides gingivalis also has similar activity, and by using a certain type of substrate, this enzyme activity can be detected specifically, easily, and quickly, and moreover, it accurately reflects the symptoms of periodontal disease. I discovered that.
これまで、口腔内のスピロヘータやバクテロイデス・ジ
ンジバリスがトリプシン様酵素やフィブリン分解酵素を
産生ずることは知られているが[ジャーナル・オブ・ク
リニカル・マイクロバイオロジー(J ournal
of C11nical Microbiology)
+97〜102.1982年1月;マイクロパイオス・
レターズ(Microbios Letters)、λ
旦、 157〜160,1984年;ジャーナルφオ
ブ番ベリオドンタルΦリサーチ(Jounal of
PeriodontalResearch)、 gよ、
95〜100.1986年]、臨床症状との相関性や検
出の特異性の点でこれらの酵素を指標とすることは困難
である。It has been known that spirochetes and Bacteroides gingivalis in the oral cavity produce trypsin-like enzymes and fibrin-degrading enzymes [Journal of Clinical Microbiology].
of C11nical Microbiology)
+97~102. January 1982; Micro Pius・
Letters (Microbios Letters), λ
157-160, 1984; Journal of Beryodontal Φ Research (Journal of
Periodontal Research), g.
95-100, 1986], it is difficult to use these enzymes as indicators in terms of correlation with clinical symptoms and specificity of detection.
、パ
本発明は、検体中のアミノペプチダーゼ様酵素活性を測
定することにより、歯周疾患の臘患や進行を診断あるい
は予測するための検査薬であって、(a)式: X−T
−P r o−S Elコ[式中、P
roはプロリン残基、Xは水素またはアミノ基保護基、
Sはプロリン残基のC末端に結合する発色基、Tはその
C末端がプロリン残基のN末端と結合する0〜4個のア
ミノ酸またはそ・の保護誘導体からなるアミノ酸または
ペプチドの残基を意味する]
で示される化合物および
(b)式: X−Z−Ar g−Y
[2コ[式中、Argはアルギニン残基、Xは水素
またはアミノ基保護基、Yはアルギニン残基のC末端に
結合する顔色基、ZはそのC末端がアルギニン残基のN
末端と結合する1〜4個のアミノ酸またはその保Fi誘
導体からなるアミノ酸またはペプチドの残基を意味する
コ
で示される化合物を該酵素の基質としてなることを特徴
とする歯周疾患検査薬を提供するものである。The present invention is a test agent for diagnosing or predicting the onset and progression of periodontal disease by measuring aminopeptidase-like enzyme activity in a specimen, comprising: (a) formula:
-P r o-S Elco [wherein, P
ro is a proline residue, X is hydrogen or an amino group protecting group,
S is a chromogenic group that is bonded to the C-terminus of the proline residue, and T is an amino acid or peptide residue consisting of 0 to 4 amino acids or their protected derivatives whose C-terminus is bonded to the N-terminus of the proline residue. ] and the formula (b): X-Z-Ar g-Y
[2 Co[In the formula, Arg is an arginine residue, X is hydrogen or an amino group protecting group, Y is a complexion group bonded to the C-terminus of the arginine residue, and Z is the N of the arginine residue whose C-terminus is
Provided is a periodontal disease test agent, characterized in that the compound represented by the symbol , which means an amino acid or peptide residue consisting of 1 to 4 amino acids or their Fi-preserving derivatives bonded to the terminal, serves as a substrate for the enzyme. It is something to do.
本発明の検査薬を用いれば、唾液、歯垢、歯肉溝浸出液
などのような検体を、好ましくは、中性条件下(pH6
,0〜8.5)、式[1]および式[2コの基質と反応
させ、その氷解活性の強弱を発色反応により測定するこ
とにより、簡便かつ迅速に、歯周疾患の鳳患や進行を診
断、予測することができる。By using the test agent of the present invention, samples such as saliva, dental plaque, gingival crevicular fluid, etc. can be tested under neutral conditions (pH 6.
, 0 to 8.5), Formula [1] and Formula [2] are reacted with a substrate, and the strength of the ice-melting activity is measured by a color reaction, thereby easily and quickly detecting the onset and progression of periodontal disease. can be diagnosed and predicted.
基質として用いる式[1コの化合物は公知であるか、少
なくとも、公知のペプチド合成法によって容易に製造で
きるものであり、式[1コのX基で示されるアミノ基保
護基はペプチド合成に用いられる公知のアミノ基保護基
のいずれのものでもよく、例えば、ホルミル基、アセチ
ル基、スクシニル基、t−ブトキシカルボニル基、ベン
ゾイル基、カルボベンゾキシ基、p−)ルエンスルホニ
ル基などが挙げられる。The compound of formula [1] used as a substrate is known or at least can be easily produced by a known peptide synthesis method, and the amino group-protecting group represented by the X group of formula [1] is a compound that can be used in peptide synthesis. Any of the known amino group-protecting groups may be used, and examples thereof include a formyl group, an acetyl group, a succinyl group, a t-butoxycarbonyl group, a benzoyl group, a carbobenzoxy group, and a p-)luenesulfonyl group.
S基の発色基は、発色による酵素活性の測定(紫外部、
可ti部、赤外部の吸収、蛍光の測定によるものを包含
する)に用いられるものいずれでもよく、例えば、β−
ナフチルアミン、4−メトキシ−2−ナフチルアミン、
p−ニトロアニリン、p−二トロフェノール、7−アミ
ノ−4−メトキシクマリン、5−アミノイソフタル酸ジ
メチルエステル、7−アミノ−4−トリフルオロメチル
クマリンなどから由来する基が挙げられる。特に、適当
な発色試薬により、肉眼的に判定可能な発色を示すβ−
ナフチルアミン、4−メトキシ−2−ナフチルアミン、
p−ニトロアニリン、p−ニトロフェノール由来の基が
好ましい。The chromogenic group of the S group can be used to measure enzyme activity by color development (ultraviolet light,
For example, any method used in β-
naphthylamine, 4-methoxy-2-naphthylamine,
Examples include groups derived from p-nitroaniline, p-nitrophenol, 7-amino-4-methoxycoumarin, 5-aminoisophthalic acid dimethyl ester, 7-amino-4-trifluoromethylcoumarin, and the like. In particular, β-
naphthylamine, 4-methoxy-2-naphthylamine,
Groups derived from p-nitroaniline and p-nitrophenol are preferred.
T基はそのC末端がプロリン残基のN末端と結合する0
〜4個のアミノ酸またはその保護誘導体からなるアミノ
酸またはペプチドであればいずれでもよいが、T基中の
C末端アミノ酸残基がグリシン、リジン、フェニルアラ
ニンまたはこれらの保護誘導体残基であることが好まし
い。該保護誘導体には、セリンのOH基、システィンの
SH基、アスパラギン酸やグルタミン酸のβ−あるいは
γ−COOH基が保護基、例えば、ベンジル基などで保
護されたものが包含される。The T group has a C-terminus bonded to the N-terminus of the proline residue.
Any amino acid or peptide consisting of ~4 amino acids or protected derivatives thereof may be used, but it is preferable that the C-terminal amino acid residue in the T group is glycine, lysine, phenylalanine, or a protected derivative thereof. The protected derivatives include those in which the OH group of serine, the SH group of cysteine, and the β- or γ-COOH group of aspartic acid or glutamic acid are protected with a protective group such as a benzyl group.
式[1コの化合物における各アミノ酸残基の立体配置は
、アミノペプチダーゼ様酵素の基質となりうる限り、特
に限定するものではない。The configuration of each amino acid residue in the compound of formula [1] is not particularly limited as long as it can serve as a substrate for an aminopeptidase-like enzyme.
また、基質として用いる式[2コの化合物は公知である
か、少なくとも、公知のペプチド合成法によって容易に
製造できるものであり、式[2]のY基で示されるアミ
ノ基保護基はペプチド合成に用いられる公知のアミノ基
保護基のいずれのものでもよく、例えば、ホルミル基、
アセチル基、スクシニル基、t−ブトキシカルボニル基
、ベンゾイル基、カルボベンゾキシ基、p−)ルエンス
ルホニル基などが挙げられる。In addition, the compound of formula [2] used as a substrate is known or at least can be easily produced by a known peptide synthesis method, and the amino group-protecting group represented by the Y group in formula [2] is a compound that can be easily produced by a known peptide synthesis method. Any of the known amino group-protecting groups used in
Examples include an acetyl group, a succinyl group, a t-butoxycarbonyl group, a benzoyl group, a carbobenzoxy group, and a p-)luenesulfonyl group.
Y基の発色基は、発色による酵素活性の測定(紫外部、
可視部、赤外部の吸収、蛍光の測定によるものを包含す
る)に用いられるものいずれでもよく、例えば、β−ナ
フチルアミン、4−メトキシ−2−ナフチルアミン、p
−ニトロアニリン、p−ニトロフェノール、7−アミノ
−4−メトキシクマリン、5−アミノイソフタル酸ジメ
チルエステル、7−アミノ−4−トリフルオロメチルク
マリンなどから由来する基が挙げられる。特に、適当な
発色試薬により、肉眼的に判定可能な発色を示すβ−ナ
フチルアミン、4−メトキシ−2−ナフチルアミン、p
−ニトロアニリン、p−ニトロフェノール由来の基が好
ましい。The chromogenic group of the Y group can be used to measure enzyme activity by color (ultraviolet light,
(including measurements of absorption in the visible region, infrared region, and fluorescence), such as β-naphthylamine, 4-methoxy-2-naphthylamine, p
-Nitroaniline, p-nitrophenol, 7-amino-4-methoxycoumarin, 5-aminoisophthalic acid dimethyl ester, 7-amino-4-trifluoromethylcoumarin, and the like. In particular, β-naphthylamine, 4-methoxy-2-naphthylamine, p
-Nitroaniline, groups derived from p-nitrophenol are preferred.
Z基はそのC末端がアルギニン残基のN末端と結合する
1〜4個のアミノ酸またはその保護誘導体からなるアミ
ノ酸またはペプチドであればいずれでもよいが、Z基中
のC末端アミノ酸残基がグリシン、リジン、アルギニン
、フェニルアラニンまたはこれらの保護誘導体残基であ
ることが好ましい。該保護誘導体には、セリンのOH基
、システィンのSH基、アスパラギン酸やグルタミン酸
のβ−あるいはγ−COOH基が保護基、例えば、ベン
ジル基なとで保護されたものが包含される。The Z group may be any amino acid or peptide consisting of 1 to 4 amino acids or protected derivatives thereof whose C-terminus is bonded to the N-terminus of an arginine residue, but the C-terminal amino acid residue in the Z group may be glycine. , lysine, arginine, phenylalanine or a protected derivative thereof. The protected derivatives include those in which the OH group of serine, the SH group of cysteine, and the β- or γ-COOH group of aspartic acid or glutamic acid are protected with a protective group such as a benzyl group.
式[2コの化合物における各アミノ酸残基の立体配置は
、アミノペプチダーゼ様酵素の基質となりうる限り、特
に限定するものではない。The configuration of each amino acid residue in the compound of formula [2] is not particularly limited as long as it can serve as a substrate for an aminopeptidase-like enzyme.
本発明の検査薬は、式[l]および式[2]の化合物が
検体由来のアミノペプチダーゼ様酵素の基質として反応
できる形態のものであればいずれでもよく、もっとも基
本的には、式[11および式[2]の化合物が共存する
水溶液でよく、好ましくは、測定時、pH8,0〜8.
5となるように緩衝剤を含有させる。用いる緩衝剤は通
常用いられるものいずれでもよく、例えば、トリス塩酸
緩衝剤、リン酸緩衝剤、ホウ酸緩衝剤、ベロナール、緩
衝剤、HEPES緩衝剤などを用いることができる。該
水溶液は、式[1コ、式[2]の化合物および、所望に
より、緩衝剤を蒸留水に溶解するような公知の方法で製
造することができ、要すれば、さらに、防腐剤、抗生物
質などの他の添加物を適宜配合することができる。The test agent of the present invention may be in any form as long as the compounds of formula [1] and formula [2] can react as substrates for aminopeptidase-like enzymes derived from the specimen, and most basically, compounds of formula [11 An aqueous solution in which the compound of formula [2] and the compound of formula [2] coexist may be used, preferably at a pH of 8.0 to 8.0 at the time of measurement.
A buffering agent is included so that the amount of water is 5. The buffer used may be any commonly used buffer, such as Tris-HCl buffer, phosphate buffer, borate buffer, veronal buffer, HEPES buffer, and the like. The aqueous solution can be prepared by a known method such as dissolving the compound of formula [1], formula [2] and, if desired, a buffer in distilled water, and if necessary, preservatives and antibiotics. Other additives such as substances can be added as appropriate.
式[1]および式[2]の化合物の最終混合濃度10n
M〜10mM、配合比率1:10〜10:1の範囲で、
また、緩衝剤は最終濃度1mM〜IMの範囲で使用する
ことが好ましく、前記の水溶液は、これらの濃度の式[
1]および式[2コの化合物、所望により緩衝剤を含有
し、そのまま直接、検査に供することのできる形態にす
ることができ、あるいは、使用時、適宜蒸留水で所望の
濃度に希釈する濃厚液の形態とすることもできる。Final mixed concentration of compounds of formula [1] and formula [2] 10n
M to 10mM, in a blending ratio of 1:10 to 10:1,
Further, it is preferable to use the buffer at a final concentration in the range of 1mM to IM, and the aqueous solution described above has the following concentration formula [
Compounds of formulas 1 and 2, optionally containing a buffer, can be made into a form that can be directly subjected to testing, or can be diluted with distilled water to the desired concentration at the time of use. It can also be in liquid form.
本発明の検査薬には、これらの水溶液の形態のものを、
さらに、公知の方法により乾燥粉末化、粒化したものの
ごとき固形の形態のもの、粉末成分を混合した粉末、そ
の敷粒化物のごとき固形の形態のもの、あるいは、液状
の形態のものをろ紙、ペーパーディスク、スポンジ、高
分子物などの担体に含浸させるものも包含される。The test agent of the present invention includes these in the form of an aqueous solution,
Furthermore, solid forms such as dry powders and granules obtained by known methods, solid forms such as powders mixed with powder components, and granulated products thereof, or liquid forms may be filter paper, It also includes those impregnated into carriers such as paper disks, sponges, and polymers.
さらに、本発明の検査薬には、式[lコおよび式[2]
の化合物を含有する試薬と、緩衝剤、発色試薬などの他
の試薬を組合わせてなるキットも包含される。Furthermore, the test agent of the present invention includes formulas [1] and [2]
Also included are kits comprising a reagent containing the compound in combination with other reagents such as a buffer and a coloring reagent.
発色試薬は、式[1]および式[2コの化合物における
S基およびY基に応じて適宜選択でき、SおよびYがβ
−ナフチルアミン、4−メトキシ−2−ナフチルアミン
、p−ニトロアニリン、p−ニトロフェノール由来の基
の場合、例えば、ファーストガーネットGBCやファー
ストアル−BBあるいはこれらのジアゾニウム塩や塩化
亜鉛との塩のごとき塩などを、水、エタノール、酢酸緩
衝液、2−メトキシエタノールあるいはこれらの混合溶
液などに0.01〜5重量%の濃度で溶解した溶液や、
0.5〜5Mの水酸化ナトリウム、水酸化カリウム、酢
酸などの水溶液が用いられる。The coloring reagent can be appropriately selected depending on the S group and Y group in the compounds of formula [1] and formula [2, and S and Y are β
- In the case of groups derived from naphthylamine, 4-methoxy-2-naphthylamine, p-nitroaniline, p-nitrophenol, for example, salts such as Fast Garnet GBC, Fast Al-BB, or their diazonium salts or salts with zinc chloride. etc. in water, ethanol, acetate buffer, 2-methoxyethanol, or a mixed solution of these at a concentration of 0.01 to 5% by weight,
A 0.5-5M aqueous solution of sodium hydroxide, potassium hydroxide, acetic acid, etc. is used.
これらの溶液またはその濃厚液、さらには固形化物を式
[11および式[2コの化合物を含有する試薬と組合わ
せてキットとして用いることができる。These solutions, concentrated liquids, and solidified products thereof can be used as a kit in combination with reagents containing compounds of formulas [11 and 2].
本発明の検査薬を用いて検査を行なうには、まず、検体
を採取する。検体の採取は公知の方法で行なってもよく
、例えば、歯肉溝浸出液や唾液はろ紙、キャピラリー、
ペーパーポイントなどで採取でき、歯垢は綿棒、キュレ
ット、スケラーなどで採取できる。To conduct a test using the test agent of the present invention, first, a specimen is collected. Samples may be collected using known methods; for example, gingival crevicular fluid and saliva may be collected using filter paper, capillaries,
It can be collected with a paper point, etc., and dental plaque can be collected with a cotton swab, curette, scaler, etc.
ついで、式[11および式[2]の化合物の最終混合濃
度10nM−10mMに調整した該化合物を含有する本
発明の検査薬と検体を、例えば、試験管、マイクロタイ
タープレート、セル、バイアル瓶、プラスチック・キュ
ベツトなどの中で接触させ、好ましくは、pH6,0〜
8.5で氷解反応を行なわせる。この反応は、通常25
〜45℃でおこなわれ、反応時間は検体や反応温度によ
り異なるが、37℃で15分〜72時間程度の反応が好
ましい。Next, the test agent of the present invention and the specimen containing the compounds of formula [11 and formula [2] adjusted to a final mixed concentration of 10 nM to 10 mM, for example, in test tubes, microtiter plates, cells, vials, etc. Contact in a plastic cuvette etc., preferably at pH 6.0~
8.5 to perform the ice-thawing reaction. This reaction usually takes place at 25
Although the reaction time varies depending on the specimen and the reaction temperature, the reaction is preferably carried out at 37°C for about 15 minutes to 72 hours.
反応終了後、発色試薬を添加し、発色の有無・強弱を肉
眼あるいは分光光度計や蛍光光度計で判定し、これによ
り、検体中のアミノペプチダーゼ様酵素活性の有無・強
弱を判断し、歯周疾患の湿患や進行を診断あるいは予測
する。After the reaction is complete, a coloring reagent is added and the presence/absence and intensity of color development is determined with the naked eye or with a spectrophotometer or fluorometer.This allows the presence/absence/intensity of aminopeptidase-like enzyme activity in the sample to be determined. Diagnose or predict the onset and progression of diseases.
つぎに、実験および実施例を挙げて本発明をさらに詳し
く説明する。Next, the present invention will be explained in more detail with reference to experiments and examples.
実」1
各種口腔内嫌気性細菌のアミノペプチダーゼ様酵素活性
口腔内の嫌気性細菌であるトレボネーマ争デンテイコー
ラ(Treponema denticola)4株、
バクテロイデス・ジンジバリス5株、アクチノバチルス
・アクチノマイセテムコミタンス(Actinobat
illus actinon+ycetemcomit
ans)4株、アクチノバチルス・イスラエリー(
Actinomyces 1sraelli)2株およ
びフシバクテリウム・ヌクレータム(Fusobact
erium nucleatum)3株のアミノペプチ
ダーゼ様酵素基質に対する氷解活性をつぎのとおり測定
した。1. Aminopeptidase-like enzyme activity of various oral anaerobic bacteria. 4 strains of Treponema denticola, which is an oral anaerobic bacterium.
Bacteroides gingivalis 5 strains, Actinobacillus actinomycetemcomitans (Actinobat
illus actinon+ycetemcomit
ans), 2 strains of Actinomyces israelli, and 2 strains of Fusobacterium nucleum (Fusobacterium nucleum).
The ice-melting activity of three strains of E. erium nucleatum against aminopeptidase-like enzyme substrates was measured as follows.
トレボネーマ属の細菌はTYGUS培地を用い、37℃
で7日間、他の細菌はプレイン・ハート・インフュージ
ョン・ブロスな用い、37℃で48〜72時間嫌気的に
培養し、培養液を希釈して、各々、660nmにおける
吸光度が1−00の細菌懸濁液を調整した。。Bacteria of the genus Trebonema were grown at 37°C using TYGUS medium.
For 7 days, other bacteria were incubated anaerobically at 37°C for 48-72 hours using plain heart infusion broth, and the cultures were diluted to obtain bacteria with an absorbance of 1-00 at 660 nm. A suspension was prepared. .
β−ナフチルアミン由来の種々の発色基を有するアミノ
ペプチダーゼ様酵素の基質化合物をO01M)リス塩m
緩衝液(pH7,0)に0.2mMの濃度で溶解し、基
質溶液を調整した。Substrate compounds of aminopeptidase-like enzymes having various chromogenic groups derived from β-naphthylamine are
A substrate solution was prepared by dissolving it in a buffer solution (pH 7,0) at a concentration of 0.2 mM.
この基質溶解1.5mlに、前記の細菌懸濁液0、am
tを加え、37℃で60分間反応させた。To 1.5 ml of this substrate solution, add 0 am of the above bacterial suspension.
t was added, and the mixture was reacted at 37°C for 60 minutes.
反応終了後、発色試薬(10%ツイーン20を含有する
1M酢酸緩衝液(pH4,2)にジアゾニウム塩ガーネ
ットGBCを0.5mg/mlの濃度で溶解して調整)
0.amtを加え、15分後に525nmにおける吸光
度を分光光度計にて測定した。After the reaction, color reagent (adjusted by dissolving diazonium salt garnet GBC at a concentration of 0.5 mg/ml in 1M acetate buffer (pH 4,2) containing 10% Tween 20)
0. amt was added, and 15 minutes later, the absorbance at 525 nm was measured using a spectrophotometer.
氷解活性は、各細面のβ−ナフチルアミン遊離量の平均
値に基づき、つぎのとおり表示した。The ice-melting activity was expressed as follows based on the average value of the amount of β-naphthylamine released on each thin surface.
−:遊Ha<5ナノモル/ml
±:遊離量5′〜く10ナノモル/ml+:遊離量lO
〜〈20ナノモル/ml++:遊離量20〜<40ナノ
モル/ml+÷+:遊離ff140〜<80ナノモル/
ml++÷+:遊離tsoナノモル/m1以上結果を第
1表に示す。第1表中、基質化合物の略号はつぎのとお
りである。−: Free Ha<5 nanomol/ml ±: Free amount 5'~10 nanomol/ml+: Free amount 1O
~<20 nanomol/ml++: free amount 20-<40 nanomol/ml+÷+: free ff140-<80 nanomol/
ml++÷+: free tso nanomoles/ml or more The results are shown in Table 1. In Table 1, the abbreviations of the substrate compounds are as follows.
GRニゲリシル−アルギニン−β−ナフチルアミド
BzGR:N−ベンゾイル−グリシル−アルギニン−β
−ナフチルアミド
CxVGR: N−カルポベンゾキ2−バリルーグリシ
ル−アルギニン−β−ナフチルアミドGPニゲリシル−
プロリン−β−ナフチルアミド
CxKP:N−カルボベンゾキシ−リジル−プロリン−
β−ナフチルアミド
BzRGFP:N−ペンゾイルーアルギニルーグリシル
ーフェニルアラニルーブロリンーβ−ナフチルアミド
(以下余白)
第1表に示すごとく、口腔内嫌気性細菌のうち、歯周疾
患原因菌であるスピロヘータ(トレボネー、 マ・デ
ンティコーラ)およびバクテロイデス・ジンジバリスが
特異的なアミノペプチダーゼ様酵素活性を示し、C末端
にアルギニンをもつ基質とプロリンをもつ基質とを併用
することにより、各々単独のものにくらべ2〜3倍の活
性を有する。GR nigericyl-arginine-β-naphthylamide BzGR: N-benzoyl-glycyl-arginine-β
-Naphthylamide CxVGR: N-carpobenzoki2-valyluglycyl-arginine-β-naphthylamide GP nigericyl-
Proline-β-naphthylamide CxKP: N-carbobenzoxy-lysyl-proline-
β-naphthylamide BzRGFP: N-penzoyl-arginyl-glycyl-phenylalanyl-broline-β-naphthylamide (see the margin below) As shown in Table 1, among the oral anaerobic bacteria, it is a periodontal disease-causing bacterium. Spirochetes (Trebone, Ma denticola) and Bacteroides gingivalis exhibit specific aminopeptidase-like enzymatic activity, and the combined use of a substrate with arginine and a substrate with proline at the C-terminus results in a higher ~3 times more active.
見肱ユ
臨床所見との相関−1
臨床所見上、健常であると認められる者5名、歯肉炎患
者6名および歯周炎患者6名から、各々、ペーパーポイ
ントにより歯肉溝浸出液検体を採取し、リンガ−液1.
5mlに分散後、位相差顕微鏡を用い、全菌数に対する
スピロヘータの相対量を測定した。また、このリンガ−
液0.3mlを、実験lにおけると同様にして調整した
基質溶液を用い、同様にして氷解活性を測定した。基質
としては、式[1]の化合物であるグリシル−プロリン
−β−ナフチルアミド(GP) 、N−ペンゾイルーア
ルギニルーグリシルーフェニルアラニルーブロリンーβ
−ナフチルアミド(BZRGFP)および式[11の化
合物であるN−カルボベンゾキシ−バリル−グリシル−
アルギニン−β−ナフチルアミド(CxVGR) 、N
−ベンゾイル−グリシル−アルギニン−β−ナフチルア
ミド(BzGR)を各々単独、併用して用いた。Correlation with clinical findings - 1 Samples of gingival crevicular fluid were collected using paper points from 5 patients who were considered to be healthy based on clinical findings, 6 patients with gingivitis, and 6 patients with periodontitis. , Ringer's solution 1.
After dispersing in 5 ml, the relative amount of spirochetes to the total number of bacteria was measured using a phase contrast microscope. Also, this ringer
Ice-melting activity was measured in the same manner using 0.3 ml of the substrate solution prepared in the same manner as in Experiment 1. As a substrate, glycyl-proline-β-naphthylamide (GP), which is a compound of formula [1], N-penzoyl-arginyl-glycyl-phenylalanyl-broline-β
- naphthylamide (BZRGFP) and N-carbobenzoxy-valyl-glycyl- which is a compound of formula [11]
Arginine-β-naphthylamide (CxVGR), N
-Benzoyl-glycyl-arginine-β-naphthylamide (BzGR) was used alone or in combination.
結果を第2表に示す。なお、第2表中、水解活性は52
5nmにおける吸光度(ODΩj)を測定し、つぎの基
準により表示した。The results are shown in Table 2. In addition, in Table 2, the water-splitting activity is 52
The absorbance at 5 nm (ODΩj) was measured and expressed according to the following criteria.
−: OD、1..5−<0. 1
+:ODお、0.1〜<0.2
++:ODぬ夕0.2〜<0.4
+++ : OD、、0 、4〜<0.8++++:0
0よよr> 0 、 8(以下余白)
第2表に示すごとく、氷解活性はスピロヘータ量および
臨床所見と相関し、基質を組合わせることにより各々単
独で用いた場合より患者サンプル中の氷解活性の増大が
認められた。-: OD, 1. .. 5-<0. 1 +: OD, 0.1~<0.2 ++: OD, 0.2~<0.4 +++: OD,,0,4~<0.8++++:0
As shown in Table 2, ice-melting activity correlates with the amount of spirochetes and clinical findings, and by combining substrates, the ice-melting activity in patient samples is lower than when each is used alone. An increase was observed.
!1ユ
臨床所見との相関−2
健常者群(10名)、成人性歯周炎患者群(10名)、
限局性若年性歯周炎患者群(4名)の各群の混合全唾液
遠心上清を検体として用い、前記と同様に、ただし、3
7℃で4時間反応させて氷解活性を測定した。基質とし
て、N−カルボベンゾキシ−バリル−グリシル−アルギ
ニン−β−ナフチルアミド(CxVGR−pNA) 、
N−ペンゾイルーアルギニルーグリシルーフェニルアラ
ニルーブロリンーβ−ナフチルアミド(Bz−RGFP
−pNA) 、N−カルボベンゾキシ−バリル−グリシ
ル−アルギニン−P−ニトロアニリド(CxVGR−p
NA)、N−ペンゾイルーアルギニルーグリシルーフェ
ニルアラニルーブロリンーP−ニトロアニリド(Bz−
RGFP−pNA)を各々単独および併用して用いた。! 1 Correlation with clinical findings-2 Healthy subjects group (10 people), adult periodontitis patient group (10 people),
The mixed whole saliva centrifuged supernatant of each group of localized juvenile periodontitis patients (4 patients) was used as the sample, and the same procedure as above was performed except for 3
The reaction was carried out at 7°C for 4 hours and the ice-melting activity was measured. N-carbobenzoxy-valyl-glycyl-arginine-β-naphthylamide (CxVGR-pNA) as a substrate,
N-penzoyl-arginyl-glycyl-phenylalanyl-broline-β-naphthylamide (Bz-RGFP
-pNA), N-carbobenzoxy-valyl-glycyl-arginine-P-nitroanilide (CxVGR-p
NA), N-penzoyl-arginyl-glycyl-phenylalanyl-broline-P-nitroanilide (Bz-
RGFP-pNA) were used alone and in combination.
結果を第3表に示す。The results are shown in Table 3.
(以下余白)
第3表に示すごとく、成人性歯周炎患者群および限局性
若年性歯周炎患者群はいずれも健常者群と比べて約2倍
以上の高い値(活性)を示し、統計的にも有意差が認め
られる。特に、本発明のC末端にアルギニンをもつ基質
とプロリンをもつ基質とを併用することにより、患者群
において、各々単独のものに比べて約1.5〜2倍の高
い値(活性)を示す。したがって、この活性の測定によ
り、歯周疾患の診断、予測を客観的に行なうことができ
る。(Margin below) As shown in Table 3, both the adult periodontitis patient group and the localized juvenile periodontitis patient group showed values (activity) more than twice as high as the healthy group. A statistically significant difference is also observed. In particular, the combined use of the substrate having arginine and the substrate having proline at the C-terminus of the present invention shows a value (activity) about 1.5 to 2 times higher than that of each alone in the patient group. . Therefore, by measuring this activity, periodontal disease can be objectively diagnosed and predicted.
実施例I
N−カルボベンゾキシ−バリル−グリシル−アルギニン
−4−メトキシ−2−ナフチルアミドの2mM蒸留水溶
液およびN−ベンゾイル−リジル−プロリン−4−メト
キシ−2−ナフチルアミドの2mM蒸留水溶液を等量混
合し、基質溶液とした。0.1M)リス−塩酸緩衝液(
pH7,0)を調整し、緩衝液として用いた。Example I A 2mM distilled aqueous solution of N-carbobenzoxy-valyl-glycyl-arginine-4-methoxy-2-naphthylamide and a 2mM distilled aqueous solution of N-benzoyl-lysyl-proline-4-methoxy-2-naphthylamide, etc. The amounts were mixed and used as a substrate solution. 0.1M) Lis-HCl buffer (
pH 7.0) was adjusted and used as a buffer.
10%ツイーン20を含有する1M酢酸緩衝液(pH4
,0)にジアゾニウム塩ガーネットGBC1t0.5m
g/mlの濃度で溶解し、発色試薬とした。1M acetate buffer (pH 4) containing 10% Tween 20
,0) with diazonium salt garnet GBC1t0.5m
It was dissolved at a concentration of g/ml and used as a coloring reagent.
これらを組合わせて、本発明の歯周疾患検査薬キットと
した。These were combined to form the periodontal disease test drug kit of the present invention.
このキットは、つぎのようにして歯周疾患の診断あるい
は予測に用いることができる。This kit can be used for diagnosing or predicting periodontal disease in the following manner.
被検者の歯肉溝にペーパーポイントを30秒間挿入して
検体を採取する。基質溶液0.1mlおよび緩衝液0.
9mlを混合し、これに検体を加え、37℃で一昼夜反
応させる。反応終了後、発色試薬0.3mlを加え、室
温で15分間放置後、色調を肉眼観察する。検体を添加
しない対照と比較し、褐色の強弱を判定する。強い褐色
の呈色は歯周疾患の簾患を示す。A sample is collected by inserting a paper point into the subject's gingival sulcus for 30 seconds. 0.1 ml of substrate solution and 0.1 ml of buffer solution.
Mix 9 ml, add the sample to this, and react at 37°C overnight. After the reaction is complete, add 0.3 ml of coloring reagent, leave at room temperature for 15 minutes, and observe the color tone with the naked eye. The strength of the brown color is determined by comparing it with a control without the addition of the sample. A strong brown color indicates a periodontal disease.
実施例2
N−ベンゾイル−バリル−グリシル−アルギニン−4−
メトキシ−2−ナフチルアミド500ナノモルおよびN
−ベンゾイル−プロリル−アラニル−グリシル−プロリ
ン−4−メトキシ−2−ナフチルアミド250ナノモル
をペーパーディスク(径0.6cm)に含浸させて基質
試声を調整した。Example 2 N-benzoyl-valyl-glycyl-arginine-4-
500 nmoles of methoxy-2-naphthylamide and N
A substrate test was prepared by impregnating a paper disk (0.6 cm in diameter) with 250 nmoles of -benzoyl-prolyl-alanyl-glycyl-proline-4-methoxy-2-naphthylamide.
0.1Ml7ン*緩mn (pH7,2) をm整し、
Ji1′4jwIとして用いた。Adjust 0.1Ml7*mildly (pH 7,2),
It was used as Ji1'4jwI.
これらと、実施例1におけると同様に調整した発色試薬
を組合わせ、本発明の歯周疾患検査薬キットとした。These and a coloring reagent prepared in the same manner as in Example 1 were combined to form a periodontal disease test kit of the present invention.
このキットは、つぎのようにして歯周疾患の検査あるい
は予測に用いることができる。This kit can be used for testing or predicting periodontal disease in the following manner.
緩衝液400μlをバイアル瓶に入れ、これに被検者か
ら採取した混合唾液100μlを加え、さらに基質試薬
のペーパーディスクを加え、37℃で4時間反応させる
。反応終了後、発色試薬を加え、実施例1と同様に肉眼
観察して判定する。400 μl of the buffer solution is placed in a vial, 100 μl of mixed saliva collected from the subject is added thereto, a paper disc of substrate reagent is added, and the mixture is allowed to react at 37° C. for 4 hours. After the reaction is completed, a coloring reagent is added, and judgment is made by visual observation in the same manner as in Example 1.
実施例3
N−カルボベンゾキシ−アルギニル−アルギニン−4−
メトキシ−2−ナフチルアミド500ナノモルおよびN
−ペンゾイルーアルギニルーグリシルーフェニルアラニ
ルーブロリンーβナフチルアミド500ナノモルを混合
し、アンプル(内径5mm、長さ3cm)内で凍結乾燥
させて基質試薬とした。Example 3 N-carbobenzoxy-arginyl-arginine-4-
500 nmoles of methoxy-2-naphthylamide and N
-Penzoyl-arginyl-glycyl-phenylalanyl-broline-500 nanomoles of β-naphthylamide were mixed and freeze-dried in an ampoule (inner diameter 5 mm, length 3 cm) to obtain a substrate reagent.
0.05M)リス−塩酸緩衝液(pH7,5)を調整し
、緩衝液とした。A 0.05M) Lis-HCl buffer (pH 7.5) was prepared and used as a buffer solution.
10%ツイーン20を含有する1M酢酸緩衝液(pH4
,2)にジアゾニウム塩ファーストアル−Bを1mg/
mlの濃度で溶解し、発色試薬とした。これらを組合わ
せて、本発明の歯周疾患検査薬キットとした。1M acetate buffer (pH 4) containing 10% Tween 20
, 2) with 1 mg of diazonium salt Fastal-B/
It was dissolved at a concentration of 1.0 ml and used as a coloring reagent. These were combined to form the periodontal disease test drug kit of the present invention.
このキットは、つぎのようにして歯周疾患の検査あるい
は予測に用いることができる。This kit can be used for testing or predicting periodontal disease in the following manner.
被験者の歯肉溝にペーパーストリップを30秒間挿入し
て検体を採取する。緩衝液1mlを基質含有アンプルに
入れ基質を溶解させる。これに検体を加え、37℃で一
昼夜反応させる。反応終了後、発色試薬0.4mlを加
え、実施例1と同様に肉眼観察して判定する。A sample is collected by inserting a paper strip into the subject's gingival sulcus for 30 seconds. Add 1 ml of buffer to the ampoule containing the substrate to dissolve the substrate. Add the sample to this and allow to react at 37°C overnight. After the reaction is completed, 0.4 ml of coloring reagent is added, and judgment is made by visual observation in the same manner as in Example 1.
実施例4
N−t−ブトキシカルボニル−バリル−ロイシル−グリ
シル−アルギニン−β−ナフチルアミドおよびリジル−
プロリン−β−ナフチルアミドを0.05Mリン酸緩衝
液(pH7,2)でそれぞれ400ナノモル/mlとな
るように調整し、等量混合したもの100μlを円形ろ
紙(径1cm)に含浸乾燥させ、キュベツト(内径1c
m)底に挿入して基質試薬とした。Example 4 N-t-butoxycarbonyl-valyl-leucyl-glycyl-arginine-β-naphthylamide and lysyl-
Proline-β-naphthylamide was adjusted to 400 nanomol/ml with 0.05 M phosphate buffer (pH 7, 2), and 100 μl of the mixed equal amount was impregnated into a circular filter paper (diameter 1 cm) and dried. Cuvette (inner diameter 1c
m) It was inserted into the bottom and used as a substrate reagent.
これらと実施例3におけると同様に調整した発色試薬を
組合わせて、本発明の歯周疾患検査薬キットとした。These and a coloring reagent prepared in the same manner as in Example 3 were combined to form a periodontal disease test drug kit of the present invention.
このキットは、つぎのようにして歯周疾患の検査あるい
は予測に用いることができる。This kit can be used for testing or predicting periodontal disease in the following manner.
被験者から採取した混合唾液100μmをキュベツトに
加え、37℃で4時間反応させる。反応終了後、発色試
薬40μlを加え、実施例1と同様に肉眼観察して判定
する。100 μm of mixed saliva collected from the subject is added to the cuvette and allowed to react at 37° C. for 4 hours. After the reaction is completed, 40 μl of a coloring reagent is added, and judgment is made by visual observation in the same manner as in Example 1.
l帆立11
本発明の検査薬を用いれば、特殊な設備や高度な技術を
必要とせずに、簡便かつ迅速に歯周疾患の胤患や進行を
客観的に診断あるいは予測することができ、これにより
、歯周疾患の治療や予防を適切に行なうことができる。l Scallops 11 By using the test agent of the present invention, it is possible to objectively diagnose or predict the onset and progression of periodontal disease easily and quickly without the need for special equipment or advanced technology. Thereby, periodontal disease can be appropriately treated and prevented.
Claims (3)
ることにより、歯周疾患の罹患や進行を診断あるいは予
測するための検査薬であって、(a)式:X−T−Pr
o−S[1] [式中、Proはプロリン残基、Xは水素またはアミノ
基保護基、Sはプロリン残基のC末端に結合する発色基
、TはそのC末端がプロリン残基のN末端と結合する0
〜4個のアミノ酸またはその保護誘導体からなるアミノ
酸またはペプチドの残基を意味する] で示される化合物および (b)式:X−Z−Arg−Y[2] [式中、Argはアルギニン残基、Xは水素またはアミ
ノ基保護基、Yはアルギニン残基のC末端に結合する発
色基、ZはそのC末端がアルギニン残基のN末端と結合
する1〜4個のアミノ酸またはその保護誘導体からなる
アミノ酸またはペプチドの残基を意味する] で示される化合物を該酵素の基質としてなることを特徴
とする歯周疾患検査薬。(1) A test agent for diagnosing or predicting the onset and progression of periodontal disease by measuring aminopeptidase-like enzyme activity in a specimen, the test agent having the formula (a): X-T-Pr
o-S [1] [In the formula, Pro is a proline residue, X is hydrogen or an amino group protecting group, S is a chromogenic group bonded to the C-terminus of the proline residue, and T is the N of the proline residue whose C-terminus is 0 that connects to the end
Refers to an amino acid or peptide residue consisting of ~4 amino acids or protected derivatives thereof] and (b) a compound represented by the formula: X-Z-Arg-Y [2] [wherein, Arg is an arginine residue , X is hydrogen or an amino group-protecting group, Y is a chromogenic group bonded to the C-terminus of the arginine residue, and Z is from 1 to 4 amino acids or protected derivatives thereof whose C-terminus is bonded to the N-terminus of the arginine residue. A periodontal disease test agent characterized in that the compound represented by the following formula is used as a substrate for the enzyme.
、フェニルアラニンまたはこれらの保護誘導体残基であ
る前記第(1)項の歯周疾患検査薬。(2) The periodontal disease test agent according to item (1) above, wherein the C-terminal amino acid residue in the T group is glycine, lysine, phenylalanine, or a protected derivative thereof.
、アルギニン、フェニルアラニンまたはこれらの保護誘
導体残基である前記第(1)項または第(2)項の歯周
疾患検査薬。(3) The periodontal disease test agent according to item (1) or item (2) above, wherein the C-terminal amino acid residue in the Z group is glycine, lysine, arginine, phenylalanine, or a protected derivative thereof.
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP62113122A JP2516365B2 (en) | 1987-05-09 | 1987-05-09 | Periodontal disease test agent |
AT87306663T ATE85362T1 (en) | 1986-07-29 | 1987-07-28 | REAGENTS FOR TESTING PERIODENTAL DISEASES. |
EP87306663A EP0255341B1 (en) | 1986-07-29 | 1987-07-28 | Reagent for testing periodontal diseases |
ES87306663T ES2053547T3 (en) | 1986-07-29 | 1987-07-28 | REAGENT TO SUBMIT PERIODONTAL DISEASES TO TEST. |
DE8787306663T DE3783966T2 (en) | 1986-07-29 | 1987-07-28 | REAGENTS FOR THE EXAMINATION OF PERIODENTAL DISEASES. |
CA000543277A CA1310893C (en) | 1986-07-29 | 1987-07-29 | Reagent for detecting the presence and extent of periodontal diseases |
US07/459,185 US5137811A (en) | 1986-07-29 | 1989-12-29 | Method for diagnosing periodontal diseases with a substrate specific for aminopeptidase activity of periodontopathic bacteria |
GR920403143T GR3006962T3 (en) | 1986-07-29 | 1993-02-04 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP62113122A JP2516365B2 (en) | 1987-05-09 | 1987-05-09 | Periodontal disease test agent |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS63277966A true JPS63277966A (en) | 1988-11-15 |
JP2516365B2 JP2516365B2 (en) | 1996-07-24 |
Family
ID=14604079
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP62113122A Expired - Fee Related JP2516365B2 (en) | 1986-07-29 | 1987-05-09 | Periodontal disease test agent |
Country Status (1)
Country | Link |
---|---|
JP (1) | JP2516365B2 (en) |
Cited By (1)
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JP2021009040A (en) * | 2019-06-28 | 2021-01-28 | サンスター スイス エスエー | Periodontal disease progression risk measuring method and kit |
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JP4603557B2 (en) * | 2007-01-15 | 2010-12-22 | 有限会社 ミクロデント | Quantitative standardized culture method |
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JPS6171000A (en) * | 1984-08-27 | 1986-04-11 | ジヨンソン・アンド・ジヨンソン・デンタル・プロダクツ・カンパニー | Discovery of presence of tooth gum disease |
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---|---|---|---|---|
JPS523494A (en) * | 1975-06-23 | 1977-01-11 | Pentapharm Ag | Reagents and method of proteolytic enzyme determination |
JPS549273A (en) * | 1977-06-23 | 1979-01-24 | Ajinomoto Co Inc | 7-arginylamono-4-methylcoumarin |
JPS56121499A (en) * | 1979-12-03 | 1981-09-24 | Abbott Lab | Alpha hydroxy tripeptide receptor memia |
JPS6171000A (en) * | 1984-08-27 | 1986-04-11 | ジヨンソン・アンド・ジヨンソン・デンタル・プロダクツ・カンパニー | Discovery of presence of tooth gum disease |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2021009040A (en) * | 2019-06-28 | 2021-01-28 | サンスター スイス エスエー | Periodontal disease progression risk measuring method and kit |
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JP2516365B2 (en) | 1996-07-24 |
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