JPS63277966A - Drug for inspecting periodontal disease - Google Patents

Abstract

PURPOSE:To obtain an inspection drug which specifically, easily and rapidly detects the causative bacteria for periodontal diseases by mixing a compd. expressed by specific formula and a compd. expressed by another specific formula and measuring the aminopeptitase-like enzyme activity in a specimen. CONSTITUTION:An aq. soln. of the N-carbobanzoy-valyl-glycyl-alginine-4- methoxy-naphthyl amide expressed by formula II and an aq. soln. of the N- benzoyl-lysyl-proline-4m-methoxy-2-naphthyl amide are mixed to prepare a substrate soln. The specimen sampled by inserting a paper point into the gingival trough of a person to be inspected is added to a soln. mixture composed of this substrate soln. and buffer soln. and the soln. is rested a whole day and night to effect reaction at about 37 deg.C. A coloring reagent prepd. by dissolving diazonium salt garnet is thereafter added to the soln. and after the soln. is rested for about 15min at a room temp., the color tones thereof are observed by naked eyes. The formation of a strong brown color is indicative of the apart affected by the periodontal diseases.

Description

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 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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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].

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.

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.

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.

Next, the present invention will be explained in more detail with reference to experiments and examples.

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.

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. .

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.

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.

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.

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

−: 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 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.

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.

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, 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 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.

The results are shown in Table 3.

(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.

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.

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.

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 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.

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.

Adjust 0.1Ml7*mildly (pH 7,2),
It was used as Ji1'4jwI.

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.

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

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.

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.

A 0.05M) Lis-HCl buffer (pH 7.5) was prepared and used as a buffer solution.

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.

This kit can be used for testing 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 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.

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.

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 testing or predicting periodontal disease in the following manner.

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 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)

[Claims] (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. (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. (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.