JPH0357084B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a root canal treatment agent used in dental treatment, a kit for the treatment, and a composite kit.

Currently, in the treatment of dental caries, the tooth enamel is removed, the pulp is extracted, and then the remaining odontoblast layer that adheres to the inner wall of the root canal (pulp canal) is removed using mechanical means such as a microengine. A surgical method that involves first removing the tumor and then performing subsequent treatment is often used.

When using this method, the removal of odontoblasts must rely on the practitioner's intuition to determine the degree of odontoblast removal, which often results in incomplete removal. (Lysosomal Enzyme), etc., after a long period of time, problems such as induction of abscess in the root of the tooth will occur.

In view of the above-mentioned problems in dental treatment in the prior art, the main purpose of the present invention is to biochemically and extremely effectively remove the odontoblast layer that remains adhered to the inner wall of the root canal even after pulp extraction without the need for mechanical means. It is an object of the present invention to provide a new root canal treatment agent or a kit for root canal treatment that can be removed.

The above-mentioned object of the present invention is to provide a root canal treatment agent containing various proteases, especially collagenase as a main component, and buffers, enzyme activators, etc. as auxiliary components, or the present invention as described below for conveniently providing the same agent during treatment. This can be effectively achieved by various combination kits. That is, the present inventors discovered for the first time that root canal wall-adhered odontoblasts can be very effectively digested and removed by the action of proteases, particularly collagenase, and have thus arrived at the present invention.

Hereinafter, the composition, usage/dose, effects, etc. of the treatment agent or kits of the present invention will be explained in more detail.

Enzymes for odontoblast digestion and release processing, etc. Enzymes that can be used in the present invention include collagenase that specifically acts on undenatured collagen substrates, and other enzymes such as trypsin, chymotrypsin,
Proteases such as pepsin and papain (hereinafter referred to as
These are broadly classified into ``general proteases.''

According to the findings of the present inventors, odontoblasts are fixed to immature dentin via so-called collagen fibers, and therefore, by acting on collagenase, odontoblasts can be very effectively and specifically immobilized. It can cause detachment from young dentin. On the other hand, proteases in general are thought to act not only on collagen fibers but also on cell walls, etc., and their mechanism of action is different from that of collagenase in this respect, but it is necessary to use a sufficiently highly active enzyme solution. If these are used alone, almost complete digestion and removal of odontoblasts is possible.

On the other hand, in order to utilize these differences in the mechanism of action of the two more effectively, the root canal after pulp extraction is pre-digested with general proteases as much as possible,
A method of complete removal treatment using collagenase may then be proposed. That is, when this method is used, the penetration of collagenase into collagen fibers is further accelerated by the pre-digestion treatment, and as a result, prompt and reliable treatment can be achieved.

Although commercially available enzymes are usually sufficient as these enzymes that can be used in the present invention, their types, properties, buffer solutions, etc. are summarized as follows.

1 Collagenase Clositridium histolyticum, M.tuberculosis,
Bacteroides melaninogenicus, Streptomyces
madurae, Trichophyton schoenleinnii,
Typical enzymes include collagenases produced by various microorganisms such as Aspergillus oryzae, collagenases present in various animal tissues such as mouse fibroblasts, etc., but the present invention is not limited thereto, and is specific to undenatured collagen. It is clear that a wide variety of other collagenases capable of acting may be used as appropriate. These collagenases usually require calcium ions or magnesium ions as their activators, and on the other hand, the optimum pH thereof varies depending on the production source, but is approximately within the range of PH 6.5 to 9. Therefore, the treatment agent of the present invention is usually put into practical use by dissolving collagenase lyophilized powder or tablets in a buffer containing an activator such as calcium chloride (Cacl ₂ ) or calcium carbonate (CaCO ₃ ). Ru.

2 Proteases in general Mainly endopeptidases such as pepsin, trypsin, chymotrypsin, cathepsin, fuicin, papain, bromelain, etc. In addition to these, aminopeptidases are used as long as the action conditions such as pH value are approximately the same and the action is not inhibited. , carboxypeptidase, and other so-called exopeptidases may be used in combination.

All of these enzymes can be used as commercially available products.

On the other hand, when used in the above-mentioned pre-digestion treatment, trypsin is extremely suitable in that its optimum PH value can be approximately matched to that of a commercially available collagenase agent.

3 Buffers Phosphate, borate, Tris/HCL buffer, etc.
Various well-known buffers may be selected and used as long as they are acceptable for dental treatment.

Incidentally, since the treatment agent of the present invention can be used for a relatively short period of time, side effects such as toxicity are practically not a problem as long as the treatment agent is washed immediately after the reaction is completed.

4 Activator Collagenase usually uses Ca ²⁺ as its activator.
As mentioned above, these require _{CaCl 2} ^, CaCO ₃ , Ca
(CH ₃ COO) ₂ , MgCl ₂ , MgCO ₃ , etc., in the form of various inorganic or organic salts may be added in a trace amount of about 10 ⁻³ to ^{10 −1} %.

Even when a phosphate-based buffer is used, for example, about 5×10 ^-3 % of CaCl ₂ can be stably dissolved in the buffer, so it can be used satisfactorily.

5 Inhibitor The activity of collagenase is rapidly inhibited by chelating agents such as EDTA, EGTA, and σ-phenanthroline, and SH group protecting agents such as dithiothreitol and cysteine.

Therefore, these drugs can be used as a collagenase reaction stop solution, a cleaning solution, and an oral mucosal protectant.

Dosage and administration 1 Activity units of various enzymes The activity units of various enzymes used in the present invention are obtained from Sigma Corporation (POBOX 14508, ST.LOUIS, MO., USA).
63178 USA) as defined below in accordance with the regulations.

a One unit of collagenase reacts with ninhydrin coloration when it acts on undenatured collagen for 5 hours at 37°C at pH 7.4 in the presence of calcium ions.
It is defined as the activity that liberates an amount of amino acids equivalent to 1.0 μmole of leucine.

b One unit of trypsin is defined as the activity to hydrolyze 1.0 μmole of α-N-benzoyl-L-arginine ethyl ester (BAEE) per minute at 25° C. and pH 7.6.

c One unit of other non-specific protease is PH
7.4, when acting on casein for 5 hours at 37℃,
It is defined as the activity of releasing an amount of amino acid equivalent to 1.0 μmole of leucine by ninhydrin coloring.

2 Dosage/Usage The treatment agent of the present invention is used after cutting and extracting the pulp from the crown of a carious tooth, thoroughly cleaning the root canal, then injecting and filling the root canal in an amount that satisfies the root canal, allowing it to react for a predetermined period of time, and after completion of the treatment, It is used in a treatment method that involves re-cleaning.

For this cleaning, it is possible to use a dental sodium hypochlorite solution, but when collagenase is used as the enzyme, the above-mentioned inhibitors such as EDTA can also be used.

In dental practice, it is desirable that the time required for the above-mentioned static reaction be as short as possible, and in practice it should be within one hour, more preferably within a range of several tens of seconds to several minutes. From this point of view, the activities of various enzymes in the processing agent should be set as follows.

a Collagenase: at least 100 Units/ml,
More preferably 100 to 10000 Units/ml.

b Trypsin: at least 500 Units/ml, more preferably 10 ³ to 8×10 ⁵ Units/ml.

c Other non-specific proteases: at least
500 Units/ml, more preferably 1500~
50000 Units/ml.

It should be noted that the above prescriptions for dosage can be said to be appropriate also in the case of the aforementioned two-step treatment method, but lower activity can also be used.

In addition, the capacity of one root canal is usually about 1 to 10 microns, and the number of root canals per tooth is about 1 to 4, so the amount of treatment agent required for one treatment depends on the number of teeth to be treated. It depends, but generally it will be about 20 to 100μ.

Therefore, it is practical to prepare about 0.5 to 1 ml of the treatment agent for each treatment, but since it can be stably stored for about 1 month at 0 to 4℃, it is also possible to prepare a larger amount in advance and store it under cooling. good.

Kits Since enzyme solutions generally have the possibility of deactivation over time, it is particularly advantageous to provide the treatment agent of the present invention in the form of a kit. Some typical examples of the kits of the present invention are summarized below.

1 Collagenase kit [1st ampoule: 0.067M, PH7.4 phosphate buffer solution...1ml 2nd ampoule: Collagenase (136Units/mg)...5
mg and CaCl ₂ ...0.05mg [1st ampoule: 0.01M, PH8.0 phosphate buffer solution...1ml 2nd ampoule: Collagenase (136Units/mg)...10
mg and CaCl ₂ ...0.02 mg [First ampoule: 0.002% _CaCl2 containing 0.01M, PH8.0 phosphate buffer...1 ml Second ampoule: Collagenase (1000 Units/mg)...0.1
mg [1st ampoule: 0.05M _CaCl2 containing 0.05M, PH7.0 borate buffer...1ml 2nd ampoule: Collagenase (180Units/mg)...0.83
mg In place of the first ampoule, a vial with a larger capacity may be used to dispense the above amount when used.

2 General protease kit [1st ampoule: 0.067M, PH7.4 phosphate buffer...1ml 2nd ampoule: Trypsin (8500Units/mg)...1mg and CaCl ₂ ...0.01mg [1st ampoule: 0.01M , PH8.0 phosphate buffer...1mg Second ampoule: Trypsin (8500Units/mg)...0.5mg and _CaCl2 ...0.05mg [First ampoule: 0.01M containing 0.002% _CaCl2 , PH8.0 phosphorus] Salt buffer solution: 1 mg Second ampoule: Trypsin (8500 Units/mg): 0.1 mg Other protease kits are also provided in the same manner as above.

3. Complex kit The above "2. Protease general kit" is used as the first kit for pretreatment, and the same "1. Collagenase kit" is used as the first kit for pretreatment.
A combination kit with the second kit for main processing.

Hereinafter, the present invention will be explained in more detail using experimental examples.

Experimental Example 1 The action of collagenase in vitro was measured using type and collagen purified from bovine as substrates. The final concentration is 150 Units/ml.
Mix collagenase (manufactured by Wako Pure Chemical Industries, Ltd., 180 Units/mg) dissolved in Tris/HCl buffer and _CaCl2 .
℃. The reaction was started by adding collagen substrate at a final concentration of 1.5 mg/ml. After a certain period of reaction, stop the reaction, remove the undegraded collagen matrix in the reaction solution, and measure the amount of degraded collagen.
It was determined from absorption at 280 nm. Doses shown in Figure 10-
The reaction curve shows that in the in vitro system, collagen is degraded within a short time of 15 seconds, and the optimal concentration of CaCl ₂ is 50 to 100 mM.

Experimental Example 2 The crown of an adult cow's tooth, from which unnecessary tissues such as gums adhering to the surrounding area have been completely removed, is cut transversely with a grinder to remove the exposed pulp, then cut vertically with a grinder and soaked in physiological saline. The teeth were thoroughly washed and used as bovine sample teeth.

This sample tooth was treated with collagenase (Sigma grade, Type V-S; 136 Units/mg) at pH 7.4.
0.067M - phosphate buffer (containing 0.005% _CaCl2 )
Collagenase activity obtained by dissolving in 6,
37℃ in a test tube with 800 Units/ml treatment solution.
A treated sample tooth was obtained by immersion treatment for 60 minutes.

Next, this treated sample tooth was thoroughly washed with physiological saline and then immersed in a 2% glutaraldehyde solution diluted with a 0.067M phosphate buffer at pH 7.4 to prepare a sample for an electron microscope.

Attached figures 1 and 2 are magnified at 1000x and
Figures 3 and 4 are scanning electron micrographs of the untreated sample tooth (control) at 10,000x magnification, while Figures 3 and 4 are electron micrographs of the collagenase-treated tooth sample at 1,000x and 10,000x magnification. be.

As is clear from comparing these, it can be confirmed that not only odontoblasts but also collagen fibers were removed very clearly in the collagenase-treated sample teeth.

Experimental Example 3 Trypsin (Sigma Gray) was used as the treatment solution.
Type, 8500Units/mg) PH7.4,
Trypsin activity obtained by dissolving in 0.067M phosphate buffer (containing 0.005% _CaCl2 ) 4.25 x ¹⁰⁵
Experimental example 2 above except that enzyme solution of Units/ml was used.
In the same manner as above, bovine tooth samples were processed to obtain samples for electron microscopy. Figures 5 and 6 are electron micrographs (1000x and 10000x magnification, respectively) of the treated sample tooth.

As is clear from the figure, in the trypsin-treated sample tooth, odontoblasts were almost completely removed, but some collagen fibers remained undigested.

Experimental Example 4 Bovine sample teeth were treated with chymotrypsin, pepsin, and papain solutions (each protease activity: 140,000 Units/ml) in the same manner as in Experimental Example 2, except that the pH value of the buffer solution was adjusted to the optimal pH value for each protease. A sample for electron microscopy was obtained.

The results were determined to be approximately equivalent to trypsin treatment.

Experimental example 5 Bovine sample tooth was treated with the same trypsin treatment solution as above.
℃ for 10 minutes, then thoroughly washed with physiological saline, and washed with the same collagenase treatment solution as above for 37 minutes.
℃ for 10 minutes. As a result of electron microscopic observation, it was confirmed that both odontoblasts and collagen fibers had been completely removed.

Experimental Example 6 Under the same conditions as in Experimental Example 2 above, a bovine sample tooth was immersed in a test tube, and the optical density (OD) at 280 nm and protein content of the treatment solution (modified Folin-Lowry method; bovine serum albumin standard) were determined. ) was measured over time. The results are shown in Figures 7 and 8.

As is clear from the figure, in the case of treatment under the conditions, the predetermined purpose is achieved after 10 to 15 minutes.

Experimental Example 7 1 Under the same conditions as Experimental Example 2 above, human sample teeth (male
26 years old; location, lower left No. 8; symptom C-2) was treated and a sample for electron microscopy was obtained. As a result of electron microscopic observation, it was confirmed that both odontoblasts and collagen fibers had been completely removed.

In addition, the sample tooth was not cut vertically, but was cut with a micro engine and the pulp removed as per normal treatment, and the treatment solution was injected into the root canal (pulp cavity).
The same results as above were obtained when treated.

2 Human sample teeth were treated according to Experimental Examples 2 to 4 above, and the treated teeth were observed under an electron microscope. As a result, it was confirmed that almost the same results as in the case of bovine teeth were obtained.

Experimental Example 8 Bovine tooth samples were immersed in various active collagenase treatment solutions, and the optical density at 280 nm (O.
The time course (ΔO.D) of D) was measured to obtain the dose-response curve shown in FIG. From the figure, the minimum activity of the treatment solution that is practically effective for tooth odontoblast removal treatment is approximately
Approximately 100 Units/ml is recognized.

The treatment solution contains 1 to 10 mg each of the aforementioned Sigma collagenase (136 Units/mg) at pH 7.4 and 0.05 M.
-1 ml of Tris-HCl buffer (containing 0.005% _CaCl2 )
It was prepared by dissolving it in

On the other hand, experiments similar to those described above were conducted for trypsin and other proteases, and the doses (activities) that were substantially useful in treatment were determined as described above.

Experimental Example 9 Using a group of 10 ICR mice, collagenase, trypsin, chymotrypsin, pepsin, and papain were each dissolved or suspended in physiological saline.
0.5 ml was administered orally, and the mice were observed for 14 days to see if they were alive or dead. Regarding collagenase, a similar experiment was conducted in the presence of _100mMCaCl2 .

Calculated according to Litchfield & Wilcoxon method
LD ₅₀ value is 5000mg/Kg in all cases
It was more than the body weight and non-toxic.

[Brief explanation of drawings]

Attached FIGS. 1 to 6 are electron micrographs in an experimental example of the present invention, and FIGS. 7 to 10 are illustrations of the same experiment.

Claims (1)

[Claims] 1. A root canal treatment agent containing protease as a main component. 2. The processing agent according to claim 1, wherein the protease is an enzyme selected from the group consisting of trypsin, chymotrypsin, pepsin, and papain. 3. The processing agent according to claim 1, wherein the protease is collagenase. 4. The processing agent according to claim 3, wherein collagenase is dissolved in a buffer containing an activating substance if necessary. 5 The collagenase activity is at least
The processing agent according to claim 4, characterized in that the amount is 100 Units/ml. 6 The collagenase activity is 100−
The processing agent according to claim 5, characterized in that the amount is 10000 Units/ml. 7. The processing agent according to claims 4 to 6, wherein the activating substance is calcium or magnesium ion. 8. A kit for root canal treatment consisting of a combination of a collagenase agent and a buffer solution for maintaining it at its optimum pH. 9 When the collagenase agent is dissolved in a predetermined amount of the buffer solution, the collagenase activity is at least
The kit according to claim 8, wherein the kit has a concentration of 100 Units/ml. 10 Its collagenase activity is 100−
The kit according to claim 9, which has a concentration of 10000 Units/ml. 11. The kit according to any one of claims 8 to 10, wherein the buffer is one of a phosphate buffer, a Tris buffer, and a borate buffer. 12. The kit according to any one of claims 8 to 11, further comprising a collagenase activator, if necessary. 13. The kit according to claim 12, wherein the activator is added to the collagenase agent and/or the buffer in advance. 14. The kit according to claim 13, wherein the activator is a calcium salt or a magnesium salt. 15. A composite kit for root canal treatment comprising a first kit for preliminary treatment containing a protease agent other than collagenase as a main ingredient and a second kit for main treatment having a collagenase agent as a main ingredient. 16. The composite kit according to claim 15, wherein the protease is any one of trypsin, chymotrypsin, pepsin, and papain. 17 The first kit combines the protease agent and a buffer for maintaining it at its optimum pH, and the second kit combines the collagenase agent and a buffer for maintaining it at its optimum pH. The composite kit according to claim 15 or 16. 18 When the protease agent is dissolved in a predetermined amount of the buffer, its protease activity is 500-8
18. The composite kit according to claim 17, wherein the kit is 105 x ¹⁰⁵ Units/ml. 19 When the collagenase agent is dissolved in a predetermined amount of the buffer solution, the collagenase activity is 100-
18. The composite kit according to claim 17, which has a concentration of 10000 Units/ml. 20. The composite kit according to claim 18, wherein the protease agent is a trypsin agent.