JPS6336800A - Agent for examining periodontal disease - Google Patents

Abstract

PURPOSE:In an agent for detecting a causal mold of periodontal disease by measuring aminopeptidase-like enzyme activity in a test specimen, to examine the test specimen simply and rapidly, by using a beta-naphthylamide derivative as a substrate. CONSTITUTION:In an examining agent for measuring aminopeptidase-like enzyme activity in a test specimen, a compound shown by formula I is used as a substrate for enzyme. A carbon end amino acid residue in group Z is residue of glycine, lysine, arginine, phenylalanine or protective derivative thereof. The test specimen is brought into contact with an agent containing the compound whose concentration is adjusted to 10nM-10mM to carry out hydrolysis reaction. Then, a coloring reagent is added to the solution, occurrence of coloring and intensity of coloring are judged and presence of the aminopeptidase-like enzyme activity in the test specimen is judged.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention provides a periodontal disease test agent, more specifically, a periodontal disease-causing agent for specifically, simply, and rapidly detecting certain types of periodontal disease-causing bacteria in a specimen. It relates to a test drug that can diagnose or predict the onset and progression of periodontal disease.

BACKGROUND TECHNOLOGY AND PROBLEMS 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. In addition, anaerobic Durham-negative bacilli have been found to be the causative bacteria of major periodontal diseases, and among these, BacLeroides gingivalis (BacLeroides gingivalis) is particularly important.
vitalis) has attracted attention, and numerous reports have been made regarding its pathogenicity.

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.

However, the detection of these bacteria by mycological methods requires the use of a dark-field microscope, advanced techniques for handling anaerobic bacteria, and special equipment, making the operations complicated and the culture and results difficult. 'P-11 cutting also has the drawback of requiring a long time and skill, and there are many difficulties in clinically putting it into practical use. 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.

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.

Until now, has it been known that spirochetes and Bacteroides gingivalis in the oral cavity produce tribulenoid enzymes and fibrin-degrading enzymes [Journal of Clinical Microbiology]?
rClinical Microbiology).

97-102. January 1982; Microbios Letters,
25, 157-160. '1984; Journal of Periodontal Lizard
ofPeriodontal Research),
21, 95-100.

[1986], it is difficult to use these enzymes as indicators in terms of correlation with clinical symptoms and specificity of detection.

Means for Solving the Problems 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, which has the formula: % formula % [1] [In the formula, 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 a group whose C-terminus is the N-terminus of the arginine residue. Refers to residues of amino acids or peptides consisting of 1 to 4 bonded amino acids or protected derivatives thereof] The present invention provides a periodontal disease test agent characterized in that the compound represented by the following is used as a substrate for the enzyme. be.

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), by measuring the reaction with the substrate of formula [1] and the strength of its ice-melting activity by color reaction, it is possible to easily and quickly diagnose and predict the onset and progression of periodontal disease. be able to.

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 Z group in formula [I] is suitable for peptide synthesis. Any known amino group protecting group may be used, such as formyl group, acetyl group, succinyl group, t-butoxycarbonyl group, benzoyl group, carbobenzoxy group, p-toluenesulfonyl group, etc. .

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-methquine coumarin, 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.

The C-terminus of the Z group is bonded to the N-terminus of the arginine residue.
Any amino acid or peptide consisting of 1 to 4 amino acids or protected derivatives thereof may be used, but Z
Preferably, the C-terminal amino acid residue in the group is glycine, lysine, arginine, phenylalanine or a protected derivative thereof. The protected derivative includes O of serine.
The N group, the ST (group of instein), and those in which the β- or γ-COOH group of aspartic acid or glutamic acid are protected with a protecting group, such as a Hensyl group, are included.

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.

The test agent of the present invention may be a compound of formula [+] or any form that can react as a substrate for an aminopeptidase-like enzyme derived from a specimen.
An aqueous solution of compound No. 11 may be used, and preferably p
A buffer is contained so that Fr 6 is 0 to 85. The buffer used may be any of the commonly used buffers, such as 1.lis-hydrochloric acid buffer, phosphate buffer, borate buffer, veronal buffer, 14 EP E S buffer, and the like. The aqueous solution can be prepared by a known method such as dissolving the compound of formula [1] and, if desired, a buffer in distilled water, and if necessary, further adding other substances such as preservatives and antibiotics. Additives can be added as appropriate.

Compounds of formula [+] have a final concentration of 10 nM = 10 m
M range, and the buffer has a final concentration of 1 mtVl-I M
The aqueous solution of ll'l 32 contains the compound of formula [I] at these concentrations, optionally containing a buffer, and is in a form that can be directly subjected to the test. Alternatively, it can be in the form of a concentrated solution, which is diluted with distilled water to the desired concentration when used.

The test agent of the present invention includes these aqueous solution forms of radish,
Furthermore, the powder in solid form such as dried powder or granulated by a known method, the powder mixed with powder IiE, the solid form such as the granulated product, or the liquid form It also includes impregnating a carrier such as a paper disk, a sponge, or a polymer material with a carrier such as a paper disk, a sponge, or a polymer.

Furthermore, the test agent of the present invention also includes a kit comprising a combination of a reagent containing the compound of formula [1] and other reagents such as a buffer and a coloring reagent.

The coloring reagent can be appropriately selected depending on Y2J in the compound of the formula [1], where Y is a group derived from β-naphthylamine, 4-methquine-2-naphthylamine, p-2)-roaniline, or p-nitrophenol. For example, first garnet GBC, first blue B r3, or salts thereof such as diazonium salts or salts with zinc chloride are dissolved in water, ethanol, acetate buffer, 2-methquine ethanol, or a mixed solvent thereof. A solution dissolved at a concentration of 0.01 to 5% by weight or an aqueous solution of 0.5 to 5M sodium hydroxide, potassium hydroxide, acetic acid, etc. may be used. These solutions or their solidified products can be used as a kit in combination with a reagent containing the compound of formula [1].

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 vapor point, and 1-phase scale can be collected with a cotton swab, curetto, or scale.

Then, the concentration of the compound of formula [+] was set to IOnM-10mM
The test agent of the present invention containing the compound adjusted to p+- A hydrolysis reaction is carried out at t6°0 to 85 to complete four reactions. This reaction is usually carried out at 25-45°C.
The reaction time varies depending on the sample and reaction temperature, but the reaction time is 15 minutes to 72 hours at 3υC ['? , a degree of reaction is preferred.

After the reaction is complete, a coloring reagent is added, and the presence or absence of color development, strength or weakness, is determined with the naked eye or with a spectrophotometer or fluorometer, thereby determining the presence or absence, strength or weakness of aminopeptidase-like enzyme activity in the sample, Diagnose or predict the incidence and progression of periodontal disease.

The present invention will be explained in more detail with reference to experiments and examples.

Experiment I 6 species of oral anaerobic bacteria with aminobebutygose enzyme activity Trebonema denticola, an oral anaerobic bacterium 4
<5 strains of Cutilloides gingivalis, Actinobacillus actinomycetemcomitans (Actinobacillus actinomyc
eLemcomitans), 4 strains of Capnocytophaga nispi (CapnocyLophaga s)
p, ) 4 strains and Fucibacterium nucleum (F
usobacterium nucleatum)
The ice-melting activity for the three aminopeptidase-like enzyme substrates was measured as follows.

Bacteria of the genus Trebonema were grown at 37° using TYGUS medium.
Other bacteria were grown anaerobically for 48-72 hours at 37°C in plain heart infusion broth, and the cultures were diluted to reach an absorbance of 1.0 at 660 nm for each. A fine suspension was prepared.

Substrate compounds for aminopeptidase-like enzymes having various color-forming groups derived from β-saphthylamine were dissolved in 01M Tris-HCl buffer (pH 7.0) at a concentration of 0.2 mM to prepare substrate solutions.

To this substrate solution 1.5 mQ, the above bacteria @ suspension 0
3 mQ was added and the reaction was carried out at 37°C for 60 minutes. After the reaction was completed, the coloring reagent (prepared by dissolving diazonium salt garnet GI3c at a concentration of 0.5 m9/mQ in 1M acetate buffer (pI-r4,2) containing 10% Tween 20) was added. 15 minutes later, 525n
The absorbance at m was measured using a spectrophotometer.

The ice-melting activity was expressed as follows based on the average value of the amount of β-naphthylamine released by each bacterial strain.

−: Free amount 5 nmoles/11a ±: Release amount 5 ~ <lO nanomoles/ffc+・Free amount lO
~<20 nanomol/W&++: free ff120~<40
nanomoles/ffc+++: free amount 40 nanomoles/m12
The above results are shown in Table 1. In Table 1, the abbreviations of speech compounds are as shown.

A: Alanine-β-naphthylamide G Greenin-β-naphthylamide R: Arginine-β-naphthylamide: Lysine-β-naphthylamide GR nigericyl: Arginine-β-naphthylamide RR, Arginyl-arginine-β-naphthylamide RG: Arginyl -Glycine-β-naphthylamide KR Lysyl-arginine-β-naphthylamide FR: Phenylalanyl-arginine-β-naphthylamide GK Nigericyl-lysine-β-naphthylamide GF, Green-Phenylalanine-β-naphthylamide PFR: Prolyl -phenylalanyl-arginine-β
- Naphthylamide BzGR: N-henzoyl-grin-arginyl-β
-Naphthylamide CxRR・N-carbobenzoquine-arginyl-arginine-β-naphthylamide CxPfl: N-carbobenzoquine-phenylalanyl-arginine-β-naphthylamide CxKfl: N
-Carbobenzoxy-lysyl-arginine-β-naphthylamide CxVGR: N-carbobenzoquine-valyl-grinol-arginine-β-naphthylamide l3zVGR: N
-benzoyl-valyl-glynol-arginine-β-naphdylamide BcVLGR: N-t-butoxoluponyl-valyluroin-glycyl-arginine-β-naphthylamide 5cGPLGR: N-succinyl-grin-prolyruroin-glycyl-arginine -β-Naphdylamide As shown in Table 1, among the oral anaerobes, the periodontal disease-causing bacteria Aro spirochete (Trebonema denticola) and Batatheroides gingivalis exhibit specific aminopeptidase-like enzyme activity. Among various substrate compounds, the compound represented by the formula [+] is specifically hydrolyzed.

Experiment 2 Correlation with clinical findings-1 Five subjects who were found to be healthy based on clinical findings had gingivitis■
71 + gingival exudate samples were collected using a paper point from 6 patients with 1 periodontitis and 6 patients with periodontitis 1. After dispersing in 1.5 g of Ringer's solution (2), using a phase contrast microscope, the total number of bacteria was determined. Spirochete... Volume of Ringer's solution 1m12
The total number of bacteria was measured. In addition, 0.3 ml of this Ringer's solution was used in an experiment.
Ice-melting activity was measured in the same manner using a substrate solution prepared in the same manner as in . As substrates, the compounds of formula [1], N-henzoyl-valyluglycyl-arginine-β-naphthylamide (BzVGR) and N-carpobenzoyl-valyluglycyl-arginine-β-
Naphthylamide (CXVGR) was used.

The results are shown in Table 2. In Table 2, the ice-melting activity was determined by visual observation of the color development and was expressed according to the standards of GG.

12 orange + dark orange ++: brown +++: a brown Table 2 As shown in Table 2, the ice melting activity is spirochete j? 1,
Correlates with clinical findings. Furthermore, no difference in contrast was observed between both substrates.

Experiment 3 Correlation with clinical findings-2 Healthy subjects group (10 people), adult periodontitis patient 1 (10 people),
Using centrifuged mixed whole saliva lI'1 from each group of localized juvenile periodontitis patients (4 people) as a specimen, the ice-melting activity was measured in the same manner as above, but incubated at 37°C for 4 hours. did.

N-carbobenzoxy-valyl-glycyl-arginine-p-nitroanilide (CXVGR-pN
A), N-carbobenzoxyvalirugrinluarginine-4-methos-2-naphthylamide (CxVG
R-4NA) and N-carbobenzoxy-valyl-glycyl-arginine-β-naphthylamide (CXVGR
-pNA), and in the case of CxVGrt-I)NA, I
Color was developed using N sodium hydroxide, CxVGR-4NA, and, in the case of human CKVGfl-pNA, Fast Blue BB. The results are shown in Table 3.

Table 3 (nanomole/7Q) *・p < 0.01 **・p < 0.05 As shown in Table 3, adult periodontitis I and localized Y1
Both cases of adolescent periodontal deficiency showed a higher value (activity) of approximately 21&2'2 compared to healthy subjects, and a statistically significant difference was observed. Therefore, by measuring this activity, diagnosis and prediction of 1"44 disease can be objectively performed.
j;: Something is coming.

Real blood example 1 ゛＼J-Karoo 1jl＼2m of quinobalirutarinruarginine-4-methquin-2-naphthylamide
A distilled aqueous solution of M was prepared and used as a substrate solution.

A 0.1M Tris-HCl buffer (p+-17,0) was prepared and used as the buffer.

IM acetate buffer containing 10% Tween 20 (p[
(4,0)i,:Noazonium salt garnet Gr3C was dissolved at 0 degrees at 0.5 mg/:mQ to prepare a coloring reagent.

These were combined to form the periodontal group test drug kit of the present invention.

This kit can be used to diagnose or predict periodontal disease in the following manner.

A sample is collected by inserting a paper point into the subject's gingival sulcus for 30 seconds. Mix the substrate solution 0, 1 mQ and the buffer solution 0, 9 mQ, add the sample to this, and
Incubate overnight at 7°C. After the reaction is complete, color reagent 0
, 3 mQ was added, and after leaving it at room temperature for 15 minutes, the color and color were observed with the naked eye. The strength of the brown color is determined by comparing it with a control without the addition of the sample. strong? The eight colors indicate the full extent of the fit.

Actual Foam Example 2 N-Penzoyl Rubary Lugurin Luarginine-4-
A substrate reagent was prepared by impregnating a paper disk (0.6 cm in diameter) with 500 nmoles of Metquin-2-naphthylamide.

A 0.1M phosphate buffer (pl-17, 2) was prepared and used as a buffer.

These and a coloring reagent prepared in the same manner as in Example 1 were combined in U- to form a periodontal disease test kit of the present invention.

This kit can be used for diagnosing or predicting periodontal disease in the following manner.

Put 400 μQ of buffer into a vial, add 100 μρ of mixed saliva collected from the subject, add a paper disk of substrate reagent, and allow 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.

Example 3 N-carbobenzoxy-arginyl-arginine-4-
500% of methoxy-2-naphthylamide was dried and used as a substrate reagent. 0.05M Tris-HCl buffer (pH 7.
, 5) was prepared and used as a buffer solution.

1M acetate buffer (pH 4) containing 10% Tween 20
．． 2) 1 mg/diazonium salt Fast Blue B
It was dissolved at a concentration of mQ 15 and used as a coloring reagent. These were combined to form the periodontal disease test drug kit of the present invention.

This kit can be used for diagnosing or predicting periodontal disease in the following manner.

A sample is collected by inserting a paper strip into the subject's gingival sulcus for 30 seconds. Add 1 mρ of buffer solution to the substrate-containing ampoule to dissolve the substrate. Add the sample to this and allow to react at 37°C overnight. After the reaction is complete, add 0.4 m of coloring reagent.
Q is added and the judgment is made by visual observation in the same manner as in Example 1.

Example 4 N-t-butoxycarbonyl-valyl-loinruglycyl-arginine-β naphdylamide at 0.05M
200 nmol/IIg in phosphate buffer (pH 7.2)
100μσ of this was prepared using circular filter paper (diameter I
C. III) Impregnated with dry U, cuvette (inner diameter IC
2) It was inserted into the bottom and used as a substrate reagent.

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.

This kit can be used for diagnosis or prediction of periodontal disease in the following manner.

Add 100 µA of mixed saliva collected from the subject to the cuvette and react at 37°C for 4 hours. After the reaction is complete, add 40 µQ of the coloring reagent, and make a judgment by visual observation as in Example 1.

Effects of the Invention By using the tarnishing agent of the present invention, the onset of periodontal disease can be easily and quickly detected without the need for special equipment or advanced technology. However, it is possible to objectively diagnose or predict the progression of periodontal disease, thereby allowing appropriate treatment and prevention of periodontal disease.

Claims (2)

[Claims] (1) A test agent for diagnosing or predicting the morbidity or progression of periodontal disease by measuring aminopeptidase-like enzyme activity in a specimen, which has the formula: X-Z-Arg-Y [wherein, Arg is an arginine residue; or an amino acid or peptide residue consisting of a protected derivative thereof.] A periodontal disease test agent characterized in that the compound represented by the following formula is used as a substrate for the enzyme. (2) The periodontal disease test agent according to item (1) above, wherein the C-terminal amino acid residue in the Z group is glycine, lysine, arginine, phenylalanine, or a protected derivative thereof.