JP2516365B2 - Periodontal disease test agent - Google Patents

Description

TECHNICAL FIELD The present invention relates to a periodontal disease test agent, and more specifically, to specifically and simply and rapidly detect a certain periodontal disease-causing bacterium in a sample, The present invention relates to a test drug capable of diagnosing or predicting morbidity or progression of periodontal disease.

2. Description of the Related Art In recent years, bacteriological research on periodontal diseases has progressed, and many spirochete have been detected in lesions of periodontal disease, and it has been revealed that they show high correlation with various clinical indicators. Also,
It is also known that anaerobic Gram-negative bacilli are major causative agents of periodontal disease, and among them, in particular, black pigment production (Bacteroides gingivalis) (Black-pigmented Bacte
roides), and there are many reports on its pathogenicity.

Therefore, attempts have been made to detect the presence of these causative bacteria in the oral cavity, diagnose or predict the occurrence 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 a bacteriological method requires the use of a dark-field microscope, advanced technology of handling anaerobic bacteria, special equipment, etc., and the operation is complicated,
There is a drawback that cultivation and judgment of the results also require a long time and skill, and there are many difficulties in clinically practicing. In addition, from the immunological point of view, the presence of the causative bacteria can be detected by measuring the antibody titer in the blood, which is humoral immunity against these causative bacteria, or measuring the lymphocyte blastogenesis, which is cellular immunity. However, there is a problem that a complicated operation is required for preparation of a sample, and it is still difficult to put the sample into practical use.

In view of such circumstances, the inventors of the present invention have conducted earnest studies to enable clinically practical detection of periodontal disease-causing bacteria. As a result, the oral spirochete has a very specific aminopeptidase-like enzyme activity, and Bacteroides gingivalis also has a similar activity.By using a certain substrate, this enzyme activity can be specifically determined. In addition, they have found that they can be detected easily and simply, and that the symptoms of periodontal disease are accurately reflected.

Until now, oral subilhoeta and Bacteroides
It is known that gingivalis produces trypsin-like enzymes and fibrinolytic enzymes [Journal of
Clinical Microbiology (Journal of Clini
cal Microbiology), 97-102, January 1982; Microbios Letters, 25 , 157-16.
0,1984; Jounal of Periodontal Research, 21 , 95-100,1
986], 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 provides a test agent for diagnosing or predicting morbidity or progression of periodontal disease by measuring aminopebutidase-like enzyme activity in a sample, comprising: ) Formula: X-T-Pro-S [1] [In the formula, Pro is a proline residue, X is a hydrogen or amino group-protecting group, S is a color-forming group bonded to the C-terminal of the proline residue,
T has a C-terminal bound to the N-terminal of a proline residue 0 to
A compound represented by 4 amino acids or an amino acid or peptide residue consisting of a protective group derivative thereof] and (b) Formula: X-Z-Arg-Y [2] [wherein Arg is an arginine residue , X is a hydrogen or amino protecting group, Y is a chromophore bound to the C-terminus of an arginine residue, Z is a 1 to 4 amino acid or its protected derivative whose C-terminus is bound to the N-terminus of an arginine residue. The present invention provides a test drug for periodontal diseases, which comprises a compound represented by the formula [1] as a substrate for the enzyme.

Using the test agent of the present invention, samples such as saliva, dental plaque, gingival crevicular fluid, etc., preferably under neutral conditions (pH 6.
0-8.5), with a substrate of formula [1] and formula [2],
By measuring the strength or weakness of the hydrolytic activity by a color development reaction, it is possible to easily and quickly diagnose and predict the morbidity or progression of periodontal disease.

The compound of the formula [1] used as a substrate is known,
At least, it can be easily produced by a known peptide synthesis method, and the amino group-protecting group represented by the X group in the formula [1] may be any of the known amino group-protecting groups used for peptide synthesis. , Formyl group, acetyl group, succinyl group, t-butoxycarbonyl group, benzoyl group, carvonebenzoxy group, p-toluenesulfonyl group and the like.

The chromophoric group of the S group may be any of those used for measuring the enzymatic activity by color development (including those by measuring the absorption in the ultraviolet region, visible region, infrared region, and fluorescence), for example, β
-Naphthylamine, 4-methoxy-2-naphthylamine, p-nitroaniline, p-nitrophenol, 7-
Examples thereof include groups derived from amino-4-methoxycoumarin, 5-aminoisophthalic acid dimethyl ester, 7-amino-4-trifluoromethylcoumarin and the like. In particular,
Groups derived from β-naphthylamine, 4-methoxy-2-naphthylamine, p-nitroaniline, and p-nitrophenol, which exhibit a color development visually recognizable by an appropriate coloring reagent, are preferred.

The T group may be any amino acid or peptide consisting of 0 to 4 amino acids whose C-terminal is bonded to the N-terminal of a brolin residue or a protected derivative thereof, but the C-terminal amino acid residue in the T group is glycine. , Lysine, phenylalanine or their protected derivative residues are preferred. The protected derivative includes serine OH group and cysteine group
SH group, β- or γ of aspartic acid and glutamic acid
Those in which the —COOH group is protected with a protecting group such as a benzyl group are included.

As long as the configuration of each amino acid residue in the compound of formula [1] can be a substrate for an aminopeptidase-like enzyme,
It is not particularly limited.

The compound of the formula [2] used as a substrate is known or can be easily produced at least by a known peptide synthesis method, and the amino group-protecting group represented by the X group of the formula [2] is a peptide synthesis compound. Any of the known amino group-protecting groups used in the above 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-toluenesulfonyl group. .

The chromophoric group of the Y group may be any of those used for measuring the enzymatic activity by color development (including those by measuring absorption in the ultraviolet region, visible region, infrared region, and fluorescence), for example, β
-Naphthylamine, 4-methoxy-2-naphthylamine, p-nitroaniline, p-nitrophenol, 7-
Examples thereof include groups derived from amino-4-methoxycoumarin, 5-aminoisophthalic acid dimethyl ester, 7-amino-4-trifluoromethylcoumarin and the like. In particular,
Groups derived from β-naphthylamine, 4-methoxy-2-naphthylamine, p-nitroaniline, and p-nitrophenol, which exhibit a color development visually recognizable by an appropriate coloring reagent, are preferred.

The Z group may be any amino acid or peptide consisting of 1 to 4 amino acids whose C-terminal is bonded to the N-terminal of an arginine residue or a protected derivative thereof, but the C-terminal amino acid residue in the Z group is glycine. , Lysine, arginine, phenylalanine or their protected derivative residues are preferred. The protected derivative includes OH of serine.
Group, SH group of cysteine, and β- or γ-COOH group of aspartic acid or glutamic acid are protected by a protecting group such as benzyl group.

As long as the configuration of each amino acid residue in the compound of formula [2] can serve as a substrate for an aminopeptidase-like enzyme,
It is not particularly limited.

The test agent of the present invention may be in any form as long as the compound of the formula [1] and the formula [2] can be reacted as a substrate of a sample-derived aminopeprase-like enzyme. It may be an aqueous solution in which the compound of [1] and the compound of the formula [2] coexist, and preferably contains a buffering agent so as to have a pH of 6.0 to 8.5 at the time of measurement. The buffer used may be any of those usually used, and for example, Tris-hydrochloric acid buffer, phosphate buffer, borate buffer, veronal, buffer, HEPES buffer and the like can be used. The aqueous solution is a compound of formula [1], formula [2] and, if desired,
It can be produced by a known method in which a buffering agent is dissolved in distilled water, and if necessary, other additives such as preservatives and antibiotics can be appropriately added.

Final mixed concentration of compounds of formula [1] and formula [2] 10 nM
To 10 mM, the compounding ratio is in the range of 1:10 to 10: 1, and the buffer is preferably used in the final concentration of 1 mM to 1 M, and the aqueous solution is used at the concentrations of the formula [1] and the formula [1]. The compound of 2], optionally containing a buffering agent, can be put into a form which can be directly subjected to an inspection as it is, or a concentrated liquid which is appropriately diluted with distilled water to a desired concentration at the time of use. You can also

The test agent of the present invention, in the form of these aqueous solutions, further in the form of solid powder such as dry powdered and granulated by a known method, powder mixed with powder components, and granules thereof. A solid form or a liquid form impregnated with a carrier such as a filter paper, a paper disk, a sponge, or a polymer is also included.

Furthermore, the test agent of the present invention also includes a kit in which a reagent containing the compound of the formula [1] and the formula [2] is combined with another reagent such as a buffering agent and a coloring reagent.

The coloring reagent can be appropriately selected according to the S group and the Y group in the compounds of the formula [1] and the formula [2], and S and Y are β-naphthylamine and 4-methoxy-2-.
In the case of a group derived from naphthylamine, p-nitroaniline, p-nitrophenol, for example, fast garnet
GBC, Fast Blue BB, or salts such as their diazonium salts or salts with zinc chloride are dissolved in water, ethanol, acetate buffer, 2-methoxyethanol, or a mixed solution thereof at a concentration of 0.01 to 5% by weight. Solution, 0.5-5M sodium hydroxide, potassium hydroxide,
An aqueous solution such as acetic acid is used. These solutions or concentrated solutions thereof, and further solidified products thereof are represented by the formula [1] and the formula [2].
It can be used as a kit in combination with a reagent containing the compound.

To perform a test using the test agent of the present invention, first, a specimen is collected. The specimen may be collected by a known method. For example, gingival crevicular fluid or saliva can be collected with a filter paper, capillary, paper point, etc., and plaque can be collected with a cotton swab, caret, scaler, or the like.

Then, the test agent of the present invention containing the compound adjusted to a final mixed concentration of 10 nM to 10 mM of the compound of the formula [1] and the compound of the formula [2] and the sample are, for example, a test tube, a microtiter plate, a cell, a vial bottle. , In a plastic cabbage or the like, and preferably the hydrolytic reaction is carried out at pH 6.0 to 8.5. This reaction is usually performed at 25-45 ℃, and the reaction time depends on the sample and reaction temperature, but at 37 ℃
Reaction for about 15 minutes to 72 hours is preferable.

After completion of the reaction, a coloring reagent is added, and the presence / absence of color development is judged with the naked eye or with a spectrophotometer or a fluorometer, and the presence or absence of aminopeptidase-like enzyme activity in the sample
Determine strength and strength to diagnose or predict morbidity or progression of periodontal disease.

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

Experiment 1 Aminopeptidase-like enzyme activity of various oral anaerobic bacteria Treponema dentica (Treponema denticola) 4 strains, Bacteroides
5 strains of Gingivalis, Actinobatillus actinomycetemcomitas
ns) 4 strains, Actinomyces Isla Ellie (Actinomy
ces israelli 2 strains and Fusobacterium nucleatum 3 strains were measured for their hydrolytic activity against aminopeptidase-like enzyme substrates as follows.

For Trebonema bacteria, use TYGUS medium at 37 ℃.
For the rest of the day, other bacteria were anaerobically cultivated using Brain Heart Infusion Broth at 37 ° C for 48-72 hours, and the culture solution was diluted to give a bacterial suspension with an absorbance of 1.0 at 660 nm. It was adjusted.

A substrate compound of an aminopeptidase-like enzyme having various chromophores derived from β-naphthylamine was dissolved in 0.1 M Tris-HCl buffer (pH 7.0) at a concentration of 0.2 mM to prepare a substrate solution.

0.3 ml of the above-mentioned bacterial suspension was added to 1.5 ml of this substrate solution, and the mixture was reacted at 37 ° C. for 60 minutes. After the reaction was completed, 0.6 ml of a coloring reagent (prepared by dissolving diazonium salt garnet GBC at a concentration of 0.5 mg / ml in 1 M acetate buffer (pH 4.2) containing 10% Tween 20) was added, and 15 minutes later 525 nm The absorbance at was measured with a spectrophotometer.

The hydrolytic activity was expressed as follows based on the average value of β-naphthylamine release amount of each bacterium.

−: Release amount <5 nmol / ml ±: Release amount 5 to <10 nmol / ml +: Release amount 10 to <20 nmol / ml ++: Release amount 20 to <40 nmol / ml +++: Release amount 40 to <80 Nanomol / ml +++++: Release amount 80 nmol / ml or more The results are shown in Table 1. In Table 1, the abbreviations of the substrate compounds are as follows.

GR: glycyl-arginine-β-naphthylamide BzGR: N-benzoyl-glycyl-arginine-β-naphthylamide CxVGR: N-carbobenzoxy-valyl-glycyl-arginine-β-naphthylamide GP: glycyl-proline-β- Naphthylamide CxKP: N-carbobenzoxy-lysyl-proline-β-naphthylamide BzRGFP: N-benzoyl-arginyl-glycyl-phenylalanyl-proline-β-naphthylamide As shown in Table 1, among oral anaerobic bacteria, spirochete (Trepoma denticola) and Bacteroides gingivalis, which are periodontal disease-causing bacteria, show specific aminopeptidase-like enzyme activity, and arginine at the C-terminus. The combined use of the substrate having the amino acid and the substrate having the proline has 2-3 times the activity as compared with each of them alone.

Experiment 2 Correlation with clinical findings-1 From the clinical findings, 5 persons who were recognized as healthy, 6 patients with gingivitis and 6 patients with periodontitis, respectively, collected gingival crevicular fluid samples by paper points, Ringer's solution 1.
After dispersing in 5 ml, using a phase contrast microscope, the relative amount of spirochete to the total number of bacteria Was measured. Further, 0.3 ml of this Ringer's solution was used as a substrate solution prepared in the same manner as in Experiment 1, and the hydrolytic activity was measured in the same manner. As the substrate, glycyl-proline-β-naphthylamide (G
P), N-benzoyl-arginyl-glycyl-phenylalanyl-proline-β-naphthylamide (BzRGFP)
And N-carbobenzoxy-, a compound of formula [1]
Valyl-glycyl-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 Table 2, the hydrolytic activity is 52.
Absorbance at 5 nm (OD ₅₂₅ ) was measured and displayed according to the following standard.

_{-: OD 525 <0.1 +:} OD 525 0.1~ <0.2 ++: OD 525 0.2~ <0.4 +++: OD 525 0.4~ <0.8 ++++: OD 525 <0.8 As shown in Table 2, the hydrolytic activity was correlated with the amount of spirochete and the clinical findings, and it was observed that the combination of the substrates increased the hydrolytic activity in the patient sample as compared with the case of using each alone.

Experiment 3 Correlation with clinical findings-2 Mixed whole saliva centrifugation of healthy group (10 persons), adult periodontitis patient group (10 persons), localized juvenile periodontitis disease group (4 persons) Using the supernatant as the sample, as above, but at 37 ° C
The reaction was carried out for 4 hours and the hydrolytic activity was measured. As a substrate,
N-carbobenzooxy-valyl-glycyl-arginine-β-naphthylamide (CxVGR-βNA), N-benzoyl-arginyl-glycyl-phenylalanyl-proline-β-naphthylamide (Bz-RGFP-βNA), N-carbobenzo Xy-valyl-glycyl-arginyl-P-nitroanilide (CxVGR-pNA), N-benzoyl-arginyl-glycyl-phenylalanyl-proline-P-nitroanilide (Bz-RGFP-pNA) were used individually and in combination. .. The results are shown in Table 3.

As shown in Table 3, both the adult periodontitis patient group and the localized juvenile periodontitis patient group were about 2 compared with the healthy subject group.
It shows a value (activity) that is more than twice as high, and a statistically significant difference is recognized. In particular, the combined use of the substrate having arginine at the C-terminus and the substrate having proline according to the present invention shows a high value (activity) of about 1.5 to 2 times in the patient group, as compared with each of them alone. Therefore, by measuring this activity, it is possible to objectively diagnose and predict periodontal disease.

Example 1 Equal volumes of a 2 mM distilled aqueous solution of N-carbobayzoxy-valyl-glycyl-arginine-4-methoxy-2-naphthylamide and a 2 mM distilled aqueous solution of N-benzoyl-lysyl-proline-4-methoxy-2-naphthylamide were mixed. To obtain a substrate solution. 0.1M Tris-HCl buffer (PH7.
0) was prepared and used as a buffer.

The diazonium salt garnet GBC was dissolved at a concentration of 0.5 mg / ml in a 1 M acetate buffer (pH 4.0) containing 10% Tween 20 to obtain a coloring reagent.

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

This kit can be used for diagnosis or prediction of periodontal disease as follows.

A specimen is collected by inserting a paper point into the gingival sulcus of the subject for 30 seconds. 0.1 ml of the substrate solution and 0.9 ml of the buffer solution are mixed, the sample is added to this, and the mixture is allowed to react at 37 ° C overnight. After the reaction is completed, 0.3 ml of the coloring reagent is added, and the mixture is left at room temperature for 15 minutes, and then the color tone is visually observed. Compare with the control without addition of the sample to determine the intensity of the brown color. A strong brown color indicates the prevalence of periodontal disease.

Example 2 N-benzoyl-valyl-glycyl-arginyl-4
-Methoxy-2-naphthylamide 500 nmol and N
A substrate reagent was prepared by impregnating a paver disc (diameter 0.6 cm) with 250 nmol of benzoyl-prolyl-alanyl-glycyl-proline-4-methoxy-2-naphthylamide.

A 0.1 M phosphate buffer (pH 7.2) was prepared and used as a buffer.

These were combined with a coloring reagent prepared in the same manner as in Example 1 to prepare a periodontal disease test drug kit of the present invention.

This kit can be used for examination or prediction of periodontal disease as follows.

400 μl of buffer solution is placed in a vial, 100 μl of mixed saliva collected from a subject is added thereto, and a paper disc of a substrate reagent is further added, and the mixture is reacted at 37 ° C. for 4 hours. After completion of the reaction, a coloring reagent is added, and the determination is made by visual observation as in Example 1.

Example 3 N-carbobenzoxy-arginyl-arginyl-4
-Methoxy-2-naphthylamide 500 nmol and N
500 nmoles of -benzoyl-arginyl-glycyl-phenylalanyl-proline-β-naphthylamide were mixed and freeze-dried in an ampoule (internal diameter 5 mm, length 3 cm) to give a substrate reagent.

A 0.05 M Tris-hydrochloric acid buffer solution (pH 7.5) was prepared and used as a buffer solution.

The diazonium salt Fast Blue B was dissolved at a concentration of 1 mg / ml in a 1 M acetate buffer (pH 4.2) containing 10% Tween 20, and used as a coloring reagent. By combining these, the periodontal disease test drug kit of the present invention was prepared.

This kit can be used for examination or prediction of periodontal disease as follows.

A sample is collected by inserting a paper strip into the gingival sulcus of the subject for 30 seconds. 1 ml of the buffer is placed in an ampoule containing the substrate to dissolve the substrate. A sample is added to this, and it is made to react overnight at 37 ° C. After the completion of the reaction, 0.4 ml of the coloring reagent is added, and the same as in Example 1, the observation is made by the naked eye to make a judgment.

Example 4 Nt-butoxycarbonyl-valyl-leucyl-glycyl-arginine-β-naphthylamide and lysyl-proline-β-naphthylamide were each adjusted to 400 nmol / ml in 0.05M phosphate buffer (pH 7.2). A circular filter paper (diameter: 1 cm) was impregnated with 100 μl of an equal amount of the mixture prepared as described above, dried and inserted into the bottom of a cuvette (inside diameter of 1 cm) to obtain a substrate reagent.

These were combined with the coloring reagent prepared in the same manner as in Example 3 to prepare the periodontal disease test drug kit of the present invention.

This kit can be used for examination or prediction of periodontal disease as follows.

100 μl of mixed saliva collected from a subject is added to a cuvette and reacted at 37 ° C. for 4 hours. After the reaction is completed, color reagent
40 μl was added, and judgment was made by visual observation in the same manner as in Example 1.

EFFECTS OF THE INVENTION By using the test agent of the present invention, it is possible to objectively diagnose or predict the morbidity or progression of periodontal disease simply and quickly without the need for special equipment or advanced technology. , Treatment and prevention of periodontal disease can be appropriately performed.

Claims (3)

(57) [Claims] 1. A test agent for diagnosing or predicting morbidity or progression of periodontal disease by measuring aminopeptidase-like enzyme activity in a sample, comprising: (a) Formula: XT-Pro- S [1] [in the formula, Pro is a proline residue, X is a hydrogen or amino group-protecting group, S is a color-forming group bonded to the C-terminal of the proline residue,
T has a C-terminal bound to the N-terminal of a proline residue 0 to
A compound represented by 4 amino acids or a protected derivative thereof or an amino acid or peptide residue thereof, and (b) formula: X-Z-Arg-Y [2] [wherein Arg is an arginine residue, X is a hydrogen- or amino-protecting group, Y is a chromophore bound to the C-terminus of an arginine residue, and Z is 1 to 4 amino acids whose C-terminus is bound to the N-terminus of an arginine residue or a protected derivative thereof. A compound representing a residue of an amino acid or a peptide] is used as a substrate for the enzyme to detect a periodontal disease. 2. The C-terminal amino acid residue in the T group is glycine,
The periodontal disease test drug according to the above (1), which is a residue of lysine, phenylalanine, or a protected derivative thereof. 3. The C-terminal amino acid residue in the Z group is glycine,
The periodontal disease test drug according to the above (1) or (2), which is a residue of lysine, arginine, phenylalanine or a protected derivative thereof.