NON-CELLULAR ROOT CANAL FILLER AND NON-CELLULAR DENTAL TISSUE REGENERATION PROMOTING KIT

Abstract

A non-cellular root canal filler comprises a tetrahydroisoquinoline compound or a pharmaceutically acceptable salt thereof, or a solvate of the compound or the salt, and a dental tissue regeneration promotion kit comprises a pretreatment agent comprising a serine protease, and the non-cellular root canal filler.

Claims

1. A non-cellular root canal filler comprising a tetrahydroisoquinoline compound represented by the following formula (1): ##STR00005## wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are each independently —H, -halogen, substituted or unsubstituted C.sub.1-6 alkyl, —OH, —O—C.sub.1-6 alkyl, —SH, —S—C.sub.1-6 alkyl, —COOH, —CO—C.sub.1-6 alkyl, —CO—O—C.sub.1-6 alkyl, —CO—NH—C.sub.1-6 alkyl, —NO.sub.2, —NH.sub.2, —NH—C.sub.1-6 alkyl, —N(C.sub.1-6 alkyl).sub.2, or —NH—CO—C.sub.1-6 alkyl, R.sup.5 is substituted or unsubstituted C.sub.1-6 alkyl, substituted or unsubstituted C.sub.3-10 cycloalkyl, substituted or unsubstituted C.sub.6-14 aryl, —C.sub.1-6 alkylene-substituted or unsubstituted C.sub.3-10 cycloalkyl, or —C.sub.1-6 alkylene-substituted or unsubstituted C.sub.6-14 aryl, R.sup.6 is —H, substituted or unsubstituted —C.sub.1-6 alkyl, or —Y′-A′, X is C.sub.1-6 alkylene, Y and Y′ are each independently a single bond or C.sub.1-6 alkylene, A and A′ are each independently substituted or unsubstituted C.sub.6-14 aryl or a substituted or unsubstituted 3- to 15-membered heterocyclic group, and n is 0 or 1; or a pharmaceutically acceptable salt thereof or a solvate thereof.

2. The non-cellular root canal filler according to claim 1, comprising (+)-4-[[2-[6-fluoro-3-(4-fluorobenzyl)-3,4-dihydroisoquinolin-2(1H)-yl]ethylamino]methyl]-N-isopropylaniline monofumarate or (+)-N-[3-(methanesulfonylamino)benzyl]-2-[6-fluoro-3-(4-fluorobenzyl)-3,4-dihydroisoquinolin-2(1H)-yl]ethanamine monocitrate.

3. The non-cellular root canal filler according to claim 1, further comprising extracellular matrix.

4. The non-cellular root canal filler according to claim 1, further comprising an anti-CCL11 neutralizing antibody and/or an ALK5 inhibitor.

5. The non-cellular root canal filler according to claim 1, further comprising at least one chemotactic factor selected from the group consisting of G-CSF, bFGF and SDF-1.

6. The non-cellular root canal filler according to claim 1 for use in the dental tissue regeneration in a young individual.

7. A dental tissue regeneration promotion kit comprising: a pretreatment agent comprising a serine protease; and a non-cellular root canal filler comprising a tetrahydroisoquinoline compound represented by the following formula (1): ##STR00006## wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are each independently —H, -halogen, substituted or unsubstituted C.sub.1-6 alkyl, —OH, —O—C.sub.1-6 alkyl, —SH, —S—C.sub.1-6 alkyl, —COOH, —CO—C.sub.1-6 alkyl, —CO—O—C.sub.1-6 alkyl, —CO—NH—C.sub.1-6 alkyl, —NO.sub.2, —NH.sub.2, —NH—C.sub.1-6 alkyl, —N(C.sub.1-6 alkyl).sub.2, or —NH—CO—C.sub.1-6 alkyl, R.sup.5 is substituted or unsubstituted C.sub.1-6 alkyl, substituted or unsubstituted C.sub.3-10 cycloalkyl, substituted or unsubstituted C.sub.6-14 aryl, —C.sub.1-6 alkylene-substituted or unsubstituted C.sub.3-10 cycloalkyl, or —C.sub.1-6 alkylene-substituted or unsubstituted C.sub.6-14 aryl, R.sup.6 is —H, substituted or unsubstituted —C.sub.1-6 alkyl, or —Y′-A′, X is C.sub.1-6 alkylene, Y and Y′ are each independently a single bond or C.sub.1-6 alkylene, A and A′ are each independently substituted or unsubstituted C.sub.6-14 aryl or a substituted or unsubstituted 3- to 15-membered heterocyclic group, and n is 0 or 1, or a pharmaceutically acceptable salt thereof, or a solvate of the compound or the salt.

8. The dental tissue regeneration promotion kit according to claim 7, comprising (+)-4-[[2-[6-fluoro-3-(4-fluorobenzyl)-3,4-dihydroisoquinolin-2(1H)-yl]ethylamino]methyl]-N-isopropylaniline monofumarate or (+)-N-[3-(methanesulfonylamino)benzyl]-2-[6-fluoro-3-(4-fluorobenzyl)-3,4-dihydroisoquinolin-2(1H)-yl]ethanamine monocitrate.

9. The dental tissue regeneration promotion kit according to claim 7, wherein the non-cellular root canal filler further comprises extracellular matrix.

10. The dental tissue regeneration promotion kit according to claim 7, wherein the non-cellular root canal filler further comprises an anti-CCL11 neutralizing antibody and/or an ALK5 inhibitor.

11. The dental tissue regeneration promotion kit according to claim 7, wherein the non-cellular root canal filler further comprises at least one chemotactic factor selected from the group consisting of G-CSF, bFGF and SDF-1.

12. The dental tissue regeneration promotion kit according to claim 7, wherein the serine protease is a chymotrypsin-like serine protease.

13. The dental tissue regeneration promotion kit according to claim 7, wherein the chymotrypsin-like serine protease is trypsin.

14. The dental tissue regeneration promotion kit according to claim 7 for use in the dental tissue regeneration in middle-aged and elderly individuals.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0030] FIG. 1 shows results of comparing a non-cellular dental pulp regeneration promotion kit using compound B with a non-cellular dental pulp regeneration promotion kit using SB 328437. FIG. 1A is an image of HE staining of a dental tissue section treated with the non-cellular dental pulp regeneration promotion kit using compound B. FIG. 1B is an image (high resolution) of HE staining of the dental tissue section treated with the non-cellular dental pulp regeneration promotion kit using compound B. FIG. 1C is an image of HE staining of a dental tissue section treated with the non-cellular dental pulp regeneration promotion kit using SB 328437. FIG. 1D is an image (high resolution) of HE staining of the dental tissue section treated with the non-cellular dental pulp regeneration promotion kit using SB 328437. FIG. 1E is a graph showing results of quantitatively analyzing the amounts of the dental pulp regenerated by the non-cellular dental pulp regeneration promotion kit using compound B and the non-cellular dental pulp regeneration promotion kit using SB 328437.

[0031] FIG. 2 shows results of comparing a non-cellular dental pulp regeneration promotion kit using compound B with a non-cellular dental pulp regeneration promotion kit using SB 328437. FIG. 2A is an image of lectin staining of a dental tissue section treated with the non-cellular dental pulp regeneration promotion kit using compound B. FIG. 2B is an image of lectin staining of a dental tissue section treated with the non-cellular dental pulp regeneration promotion kit using SB 328437. FIG. 2C is an image of PGP9.5 immunostaining of a dental tissue section treated with the non-cellular dental pulp regeneration promotion kit using compound B. FIG. 2D is an image of PGP9.5 immunostaining of a dental tissue section treated with the non-cellular dental pulp regeneration promotion kit using SB 328437.

[0032] FIG. 3 shows results of comparing a non-cellular dental pulp regeneration promotion kit using compound B with a non-cellular dental pulp regeneration promotion kit using no compound B. FIG. 3A is an image of HE staining of a dental tissue section treated with the non-cellular dental pulp regeneration promotion kit using compound B. FIG. 3B is an image (high resolution) of HE staining of the dental tissue section treated with the non-cellular dental pulp regeneration promotion kit using compound B. FIG. 3C is an image of HE staining of a dental tissue section treated with the non-cellular dental pulp regeneration promotion kit using no compound B. FIG. 3D is an image (high resolution) of HE staining of the dental tissue section treated with the non-cellular dental pulp regeneration promotion kit using no compound B. FIG. 3E is a graph showing results of quantitatively analyzing the amounts of the dental pulp regenerated by the non-cellular dental pulp regeneration promotion kit using compound B and the non-cellular dental pulp regeneration promotion kit using no compound B.

[0033] FIG. 4 shows results of comparing a non-cellular dental pulp regeneration promotion kit using compound B with a non-cellular dental pulp regeneration promotion kit using no compound B. FIG. 4A is an image of lectin staining of a dental tissue section treated with the non-cellular dental pulp regeneration promotion kit using compound B. FIG. 4B is an image of lectin staining of a dental tissue section treated with the non-cellular dental pulp regeneration promotion kit using no compound B. FIG. 4C is an image of PGP9.5 immunostaining of a dental tissue section treated with the non-cellular dental pulp regeneration promotion kit using compound B. FIG. 4D is an image of PGP9.5 immunostaining of a dental tissue section treated with the non-cellular dental pulp regeneration promotion kit using no compound B.

[0034] FIG. 5 shows results of comparing the effect of a non-cellular root canal filler using compound B between with and without trypsin pretreatment. FIG. 5A is an image of HE staining of a dental tissue section with trypsin pretreatment. FIG. 5B is an image (high resolution) of HE staining of the dental tissue section with trypsin pretreatment. FIG. 5C is an image of HE staining of a dental tissue section without trypsin pretreatment. FIG. 5D is an image (high resolution) of HE staining of the dental tissue section without trypsin pretreatment. FIG. 5E is a graph showing results of quantitatively analyzing the amounts of the dental pulp regenerated by the non-cellular root canal filler using compound B between with and without trypsin pretreatment.

[0035] FIG. 6 shows results of comparing the effect of a non-cellular root canal filler using compound B with and without trypsin pretreatment. FIG. 6A is an image of lectin staining of a dental tissue section treated with a non-cellular dental pulp regeneration promotion kit with trypsin pretreatment. FIG. 6B is an image of lectin staining of a dental tissue section treated with the non-cellular dental pulp regeneration promotion kit without trypsin pretreatment. FIG. 6C is an image of PGP9.5 immunostaining of a dental tissue section with trypsin pretreatment. FIG. 6D is an image of PGP9.5 immunostaining of a dental tissue section without trypsin pretreatment.

[0036] FIG. 7 shows results of comparing a non-cellular dental pulp regeneration promotion kit using compound C with a non-cellular dental pulp regeneration promotion kit using no compound C. FIG. 7A is an image of HE staining of a dental tissue section treated with the non-cellular dental pulp regeneration promotion kit using compound C. FIG. 7B is an image (high resolution) of HE staining of a dental tissue section treated with the non-cellular dental pulp regeneration promotion kit using compound C. FIG. 7C is an image of HE staining of a dental tissue section treated with the non-cellular dental pulp regeneration promotion kit using no compound C. FIG. 7D is an image (high resolution) of HE staining of a dental tissue section treated with the non-cellular dental pulp regeneration promotion kit using no compound C. FIG. 7E is a graph showing results of quantitatively analyzing dental tissue regeneration by the non-cellular dental pulp regeneration promotion kit using compound C and the non-cellular dental pulp regeneration promotion kit using no compound C, on the basis of the area of the dentin. FIG. 7F is a graph showing results of quantitatively analyzing dental tissue regeneration by the non-cellular dental pulp regeneration promotion kit using compound C and the non-cellular dental pulp regeneration promotion kit using no compound C, on the basis of the density of odontoblasts.

DESCRIPTION OF EMBODIMENTS

[0037] Hereinafter, the present invention will be described in detail. However, the present invention is not limited to the embodiments described in the present specification.

[0038] According to the first embodiment, the present invention provides a non-cellular root canal filler comprising a tetrahydroisoquinoline compound represented by the formula (1):

##STR00002##

wherein

[0039] R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are each independently —H, -halogen, substituted or unsubstituted C.sub.1-6 alkyl, —OH, —O—C.sub.1-6 alkyl, —SH, —S—C.sub.1-6 alkyl, —COOH, —CO—C.sub.1-6 alkyl, —CO—O—C.sub.1-6 alkyl, —CO—NH—C.sub.1-6 alkyl, —NO.sub.2, —NH.sub.2, —NH—C.sub.1-6 alkyl, —N(C.sub.1-6 alkyl).sub.2, or —NH—CO—C.sub.1-6 alkyl,

[0040] R.sup.5 is substituted or unsubstituted C.sub.1-6 alkyl, substituted or unsubstituted C.sub.3-10 cycloalkyl, substituted or unsubstituted C.sub.6-14 aryl, —C.sub.1-6 alkylene-substituted or unsubstituted C.sub.3-10 cycloalkyl, or —C.sub.1-6 alkylene-substituted or unsubstituted C.sub.6-14 aryl,

[0041] R.sup.6 is —H, substituted or unsubstituted —C.sub.1-6 alkyl, or —Y′-A′,

[0042] X is C.sub.1-6 alkylene,

[0043] Y and Y′ are each independently a single bond or C.sub.1-6 alkylene,

[0044] A and A′ are each independently substituted or unsubstituted C.sub.6-14 aryl or a substituted or unsubstituted 3- to 15-membered heterocyclic group, and

[0045] n is 0 or 1;

or a pharmaceutically acceptable salt thereof or a solvate thereof (hereinafter, referred to as “compound group A” in the present specification).

[0046] The term “non-cellular” means that cells or cell-derived components (e.g., extracellularly secreted proteins and the exosome) are not involved. The term “root canal” refers to a canal in which the dental pulp is housed in the root portion of the tooth.

[0047] The non-cellular root canal filler of the present embodiment can comprise one compound alone or a mixture of two or more compounds selected from compound group A as an active ingredient. The compound of compound group A is a CCR3 antagonist which has an effect of inhibiting the binding of CCL11 to CCR3 by binding to the CCR3, and can suppress the signal transduction of CCL11.

[0048] The compound of compound group A used in the present embodiment is preferably N-[3-(methanesulfonylamino)benzyl]-2-[6-fluoro-3-(4-fluorobenzyl)-3,4-dihydroisoquinolin-2(1H)-yl]ethanamine (Example 126 of WO2008/123582; the following formula (2)); 4-[[2-[6-fluoro-3-(4-fluorobenzyl)-3,4-dihydroisoquinolin-2(1H)-yl]ethylamino]methyl]-N-isopropylaniline (Example 138 of WO2008/123582; the following formula (3)); 4-[[2-[6-fluoro-3-(4-fluorobenzyl)-3,4-dihydroisoquinolin-2(1H)-yl]ethylamino]methyl]-N-(2-methoxyethyl)aniline (Example 150 of WO2008/123582; the following formula (4)); or N-(pyridin-4-yl)methyl-2-[6-fluoro-3-(4-fluorobenzyl)-3,4-dihydroisoquinolin-2(1H)-yl]ethanamine (Example 180 of WO2008/123582; the following formula (5)) or a pharmaceutically acceptable salt thereof.

##STR00003##

[0049] The compound of compound group A used in the present embodiment is particularly preferably (+)-4-[[2-[6-fluoro-3-(4-fluorobenzyl)-3,4-dihydroisoquinolin-2(1H)-yl]ethylamino]methyl]-N-isopropylaniline monofumarate (disclosed in JP 2009-173571 A, fumarate of the formula (3); also referred to as “compound B” in the present specification) or (+)-N-[3-(methanesulfonylamino)benzyl]-2-[6-fluoro-3-(4-fluorobenzyl)-3,4-dihydroisoquinolin-2(1H)-yl]ethanamine monocitrate (disclosed in JP 2009-191048 A, citrate of the formula (2); also referred to as “compound C” in the present specification).

[0050] The compound of compound group A can be produced by appropriately combining a chemical synthesis method described in WO2008/123582 and a chemical synthesis method equivalent thereto with various conventional methods known in the art.

[0051] The non-cellular root canal filler of the present embodiment may consist of only the active ingredient and may further comprise extracellular matrix, an anti-CCL11 neutralizing antibody and/or an ALK5 inhibitor, and/or at least one chemotactic factor selected from the group consisting of G-CSF, bFGF and SDF-1 as an optional component.

[0052] Examples of the extracellular matrix that can be used in the non-cellular root canal filler of the present embodiment include, but are not particularly limited to, collagen, artificial proteoglycan, gelatin, hydrogel, fibrin, phosphophoryn, heparan sulfate, heparin, laminin, fibronectin, alginic acid, hyaluronic acid, chitin, chitosan, PLA, PLGA, PEG, PGA, PDLLA, PCL, hydroxyapatite, β-TCP, and calcium carbonate. Alternatively, the extracellular matrix may be used in the form of coating on a metal substrate such as gold or titanium.

[0053] The anti-CCL11 neutralizing antibody that can be used in the non-cellular root canal filler of the present embodiment can be any antibody known in the art. The anti-CCL11 neutralizing antibody has an effect of inhibiting the binding of CCL11 to CCR3 by binding to the CCL11, and can suppress the signal transduction of CCL11. The anti-CCL11 neutralizing antibody is commercially available, and such a commercially available product can be used in the present embodiment.

[0054] The ALK5 inhibitor that can be used in the non-cellular root canal filler of the present embodiment can be any compound known in the art which inhibits GDF11 signal transduction. Various ALK5 inhibitors are commercially available, and such a commercially available product can be used in the present embodiment. Examples of the ALK5 inhibitor according to the present embodiment include, but are not limited to, any of the following compounds.

##STR00004##

[0055] Examples of the chemotactic factor that can be used in the non-cellular root canal filler of the present embodiment include, but are not particularly limited to, G-CSF, SDF-1, bFGF, TGF-β, NGF, PDGF, BDNF, GDNF, EGF, VEGF, SCF, MMP3, Slit, GM-CSF, LIF, and HGF. One chemotactic factor alone, or a combination of two or more chemotactic factors selected therefrom, can be used. The chemotactic factor can promote the chemotactic activity of stem cells surrounding dental tissues. The chemotactic factor that can be used in the present embodiment is preferably selected from the group consisting of G-CSF, bFGF and SDF-1. All of these chemotactic factors are commercially available, and such a commercially available product can be used in the present embodiment.

[0056] In the non-cellular root canal filler of the present embodiment, the content of each of the components can fall within the range of, for example, 50 ng/ml to 200 μg/ml, and is preferably 3 μg/ml to 100 μg/ml. The mixing ratio between the active ingredient (compound of compound group A) and additional optional components is not particularly limited and may be, for example, 10% by weight:90% by weight, to 90% by weight:10% by weight. The non-cellular root canal filler of the present embodiment may be prepared from the components appropriately combined with a pharmaceutically acceptable diluent, carrier, excipient, or the like known in the art, as necessary.

[0057] The non-cellular root canal filler of the present embodiment is applicable both to young individuals and to middle-aged and elderly individuals and is preferably used in a young individual. In this context, the young individual is not particularly limited, and is, for example, a human 1 year of age or older and 29 years of age or younger, a rat at 1 week or more and 29 weeks or less after birth, or a dog at 1 week or more and 1 year or less after birth.

[0058] The non-cellular root canal filler of the present embodiment can be used by injection into the root canal, as in conventional dental root canal fillers known in the art.

[0059] According to the second embodiment, the present invention provides a dental tissue regeneration promotion kit comprising a pretreatment agent comprising a serine protease, and the non-cellular root canal filler.

[0060] The term “dental tissue” in the present embodiment means a tissue that encompasses at least one of dental pulp, dentin, and periapical tissues.

[0061] The “pretreatment agent” according to the present embodiment is used before insertion of the non-cellular root canal filler into the root canal. This can decompose a factor inhibiting tissue regeneration in dental tissues and periodontal tissues, and/or convert a latent form of a chemotactic factor or a differentiation enhancing factor to an active form.

[0062] The pretreatment agent used in the kit of the present embodiment comprises a serine protease. The serine protease is a protease (proteolytic enzyme) having a serine residue that performs nucleophilic attack as a catalytic residue. The serine protease is classified into subtilisin-like serine protease and chymotrypsin-like serine protease from similarity in amino acid sequence or conformation. The former includes subtilisin BPN′, thermitase, proteinase K, lantibiotic peptidase, kexin, cucumisin, and the like, and the latter includes trypsin, chymotrypsin, thrombin, factor Xa, elastase, and the like. The serine protease that can be used in the present embodiment can be one enzyme alone, or a combination of two or more enzymes selected therefrom, and is preferably a chymotrypsin-like serine protease, more preferably trypsin.

[0063] The concentration of the serine protease in the pretreatment agent used in the kit of the present embodiment is not particularly limited as long as the serine protease at that concentration can decompose a factor inhibiting tissue regeneration in dental tissues and periodontal tissues, and/or convert a latent form of a chemotactic factor or a differentiation enhancing factor to an active form. The concentration can be, for example, 10 μg/ml (0.001%) to 50 mg/ml (5%), and it is preferably 500 μg/ml (0.05%) to 5 mg/ml (0.5%).

[0064] The pretreatment agent used in the present embodiment may further comprise nanobubbles. In this context, the “nanobubble” refers to an air bubble having a diameter in nanometers, or a lipid vesicle containing a gas or a gas precursor in its lumen and having a diameter in nanometers. The diameter of the nanobubble used in the pretreatment agent in the kit of the present embodiment is, for example, 10 to 500 nm, preferably 70 to 300 nm. The diameter of the nanobubble can be measured with, for example, a nanoparticle distribution measurement apparatus (SALD-7100, Shimadzu Corp.). The lipid composition, charged state, density, weight, etc. of the nanobubble can be appropriately determined. The lipid for use in the preparation of the nanobubbles is not particularly limited and can be, for example, phospholipid, glyceroglycolipid, and/or sphingoglycolipid or may be cationic lipid containing a primary amino group, a secondary amino group, a tertiary amino group or a quaternary ammonium group introduced in such lipid. The concentration of the nanobubbles in the pretreatment agent is not particularly limited and can be, for example, 2×10.sup.7 nanobubbles/cm.sup.3 to 2×10.sup.9 nanobubbles/cm.sup.3. The nanobubble concentration can be quantitatively analyzed by, for example, electron spin resonance (ESR).

[0065] The pretreatment can be performed by injecting the pretreatment agent into the root canal. The pretreatment time can be appropriately determined according to the type and concentration of the serine protease to be used. The pretreatment time can be, for example, 3 to 30 minutes, is preferably 5 to 20 minutes, and more preferably is 10 minutes.

[0066] The kit of the present embodiment may further comprise an additional buffer solution, reagent, instruction, and the like, in addition to the pretreatment agent and the non-cellular root canal filler.

[0067] The kit of the present embodiment is applicable both to young individuals and to middle-aged and elderly individuals, and is preferably used for middle-aged and elderly individuals. In this context, a middle-aged individual is not particularly limited and can be, for example, a human 30 years of age or older and 49 years of age or younger, a rat at 30 weeks or more and 39 weeks or less after birth, or a dog at 2 years or more and 4 years or less after birth. The elderly individual is not particularly limited and can be, for example, a human 50 years of age or older, a rat at 40 weeks or more after birth, or a dog at 5 years or more after birth. Thus, the kit of the present embodiment is preferably used in an individual which is a human 30 years of age or older, a rat at 30 weeks or more after birth, or a dog at 2 years or more after birth.

EXAMPLES

[0068] Hereinafter, the present invention will be further described with reference to Examples. However, the present invention is not by any means limited to these examples.

Example 1

Dental Pulp Regeneration after Pulpectomy in Young Dogs

[0069] After general anesthesia, pulpectomy was performed for maxillary and mandibular right and left anterior teeth in young (12-month-old) dogs. The openings were enlarged to the apex of the root with #55, then washed alternately with a 5% sodium hypochlorite solution and a 3% hydrogen peroxide solution, further washed with saline, dried, and then temporarily sealed with a resin completely. At 3 to 12 days after the pulpectomy, the temporary seals were removed, and the openings were washed alternately and washed with saline again. Then, the root canals were filled with 3% EDTA (Smear Clean, Nippon Shika Yakuhin Co., Ltd.), treated for 2 minutes, further washed with saline, and dried. Then, the root canals were pretreated by the application of trypsin preparation (5 mg of Francetin T powder (2,500 USP crystal trypsin per 10 mg, Mochida Pharmaceutical Co., Ltd.)/ml of 0.5% nanobubble water (prepared with Foamest 8 (NAC Corp.); see Koichiro Iohara and Misako Nakajima “Enhanced Delivery of Antibacterial Nanopolymers with Nanobubbles for the Complete Disinfection of the Root Canal System in a Canine Model of Intractable Periapical Disease”, The Japanese Journal Of Conservative Dentistry, Vol. 63, No. 1, p. 73-82) for 10 minutes, and then washed with saline. Subsequently, a CCR3 antagonist (1.25 μg of compound B or 0.83 μg of SB328437 (Tocris Bioscience)) as a regeneration promoting compound and 150 ng of G-CSF (Neutrogin, Chugai Pharmaceutical Co., Ltd.) as a chemotactic factor were added to 20 μl of extracellular matrix collagen (Koken Atelocollagen Implant, Koken Co., Ltd.) to prepare a non-cellular root canal filler, which was then filled into the root canals. Then, a gelatin sponge for hemostasis (Spongel, Astellas Pharma Inc.) was placed thereon, and the cavities were completely sealed with glass ionomer cement and a photopolymerizable resin. Then, 28 days after transplantation, the teeth were extracted, and 5 μm paraffin sections were prepared on longitudinal sections according to a standard method, stained with H-E, and then morphologically observed. The amount of the regenerated dental pulp was evaluated by measuring the ratio of the area of regenerated dental pulp to the area of a dental pulp cavity as to four sections per sample, and calculating the mean of four samples. Angiogenesis was evaluated by staining day 28 specimens with Fluorescein labeled Griffonia simplicifolia (Bandeiraea simplicifolia) Lectin I (GSL I, BSL I) and Fluorescein labeled Galanthus nivalis (Snowdrop) Lectin (GNL) (Vector Laboratories) (20 μg/ml) for 15 minutes, followed by comparative examination. Neurite extension was evaluated by immunostaining day-28 specimens with an anti-PGP9.5 antibody (UltraClone, 1:10,000), followed by comparative examination.

[0070] The results are shown in FIGS. 1 and 2. The non-cellular root canal fillers containing SB328437 or compound B as the CCR3 antagonist were found to cause dental pulp regeneration of loose connective tissues rich in blood vessels, whereas neither the infiltration nor internal absorption of inflammatory cells was observed (FIGS. 1A to 1D). The ratio of the regenerated dental pulp to a dental pulp cavity is shown in FIG. 1E. No statistically significant difference in the amount of the regenerated dental pulp was observed between the non-cellular root canal fillers containing SB328437 or compound B as the CCR3 antagonist (FIG. 1E). Angiogenesis (FIGS. 2A and 2B) and neurite extension (FIGS. 2C and 2D) were confirmed for both the non-cellular root canal fillers containing SB328437 or compound B as the CCR3 antagonist, demonstrating that odontoblast-like cells adhere to the side wall of the dentin to form dentin-like hard tissues.

Example 2

Comparison of Dental Pulp Regeneration after Pulpectomy in Young Dogs Between Presence and Absence of Compound B

[0071] After general anesthesia, pulpectomy was performed for maxillary and mandibular right and left anterior teeth in young (11-month-old) dogs. The openings were enlarged to the apex of the root with #55, then washed alternately with a 5% sodium hypochlorite solution and a 3% hydrogen peroxide solution, and further washed with saline. The root canals were thoroughly dried with a paper point and temporarily sealed with cement and a resin completely after hemostasis. Then, 8 days after the pulpectomy, the temporary seals were removed, and the openings were washed alternately and washed with saline again. Then, the root canals were filled with 3% EDTA (Smear Clean, Nippon Shika Yakuhin Co., Ltd.), treated for 2 minutes, further washed with saline, and dried. Then, the root canals were pretreated by the application of a trypsin preparation (5 mg of Francetin T powder (2,500 USP crystal trypsin per 10 mg), Mochida Pharmaceutical Co., Ltd.)/ml of 0.5% nanobubble water (prepared with Foamest 8 (NAC Corp.); as described in Example 1)) for 10 minutes, and then washed with saline. Subsequently, 1.25 μg of compound B as a regeneration promoting compound and 150 ng of G-CSF (Neutrogin, Chugai Pharmaceutical Co., Ltd.) as a chemotactic factor were added to 20 μl of extracellular matrix collagen (Koken Atelocollagen Implant, Koken Co., Ltd.) to prepare a non-cellular root canal filler, which was then filled into the root canals. On the other hand, a non-cellular root canal filler having the same composition as above except for the absence of compound B was filled into the root canals by the same procedures as above and used as a control. Then, a gelatin sponge for hemostasis (Spongel, Astellas Pharma Inc.) was placed thereon, and the cavities were completely sealed with glass ionomer cement and a photopolymerizable resin. Then, 28 days after the transplantation, the teeth were extracted, and 5 μm paraffin sections were prepared on longitudinal sections according to a usual method, stained with H-E, and then morphologically observed in the same way as in Example 1. Angiogenesis and neurite extension were confirmed by BS-1 lectin staining and PGP9.5 immunostaining, respectively, in the same way as in Example 1.

[0072] The results are shown in FIGS. 3 and 4. Treatment with the non-cellular root canal filler containing compound B was found to sufficiently regenerate dental pulp-like tissues (FIGS. 3A and 3B), whereas treatment with the non-cellular root canal filler containing no compound B was found to regenerate such tissues only in a very small amount (FIGS. 3C and 3D). Statistically significant difference in the amount of the regenerated dental pulp was observed therebetween (FIG. 3E). On the other hand, similar angiogenesis (FIGS. 4A and 4B) and neurite extension (FIGS. 4C and 4D) were observed in both the cases. These results indicated that compound B is an effective component for the regeneration of dental pulp tissues.

Example 3

Comparison of Dental Pulp Regeneration after Pulpectomy in Young Dogs Between Presence and Absence of Trypsin Pretreatment

[0073] After general anesthesia, pulpectomy was performed for maxillary and mandibular right and left anterior teeth in young (11-month-old) dogs. The openings were enlarged to the apex of the root with #50, then washed alternately with a 5% sodium hypochlorite solution and a 3% hydrogen peroxide solution, and further washed with saline. The root canals were thoroughly dried with a paper point and temporarily sealed with cement and a resin completely after hemostasis. After the pulpectomy, the temporary seals were removed, and the openings were washed alternately and washed with saline again. Then, the root canals were filled with 3% EDTA (Smear Clean, Nippon Shika Yakuhin Co., Ltd.), treated for 2 minutes, further washed with saline, and dried. Then, the left root canals were pretreated by the application of a trypsin preparation (5 mg of Francetin T powder (2,500 USP crystal trypsin per 10 mg), Mochida Pharmaceutical Co., Ltd.)/ml of 0.5% nanobubble water (prepared with Foamest 8 (NAC Corp.); as described in Example 1)) for 10 minutes, and then washed with saline. On the other hand, the right root canals were not subjected to the pretreatment (control). Subsequently, 1.25 μg of compound B as a regeneration promoting compound and 150 ng of G-CSF (Neutrogin, Chugai Pharmaceutical Co., Ltd.) as a chemotactic factor were added to 20 μl of extracellular matrix collagen (Koken Atelocollagen Implant, Koken Co., Ltd.) to prepare a non-cellular root canal filler, which was then filled into the right and left root canals. Then, a gelatin sponge for hemostasis (Spongel, Astellas Pharma Inc.) was placed thereon, and the cavities were completely sealed with glass ionomer cement and a photopolymerizable resin. Then, 28 days after the transplantation, the teeth were extracted, and 5 μm paraffin sections were prepared on longitudinal sections according to a usual method, stained with H-E, and were then morphologically observed in the same way as in Example 1. Angiogenesis and neurite extension were confirmed by BS-1 lectin staining and PGP9.5 immunostaining, respectively, in the same way as in Example 1.

[0074] The results are shown in FIGS. 5 and 6. The regeneration of dental pulp tissues was observed, regardless of whether trypsin pretreatment was performed or not (FIGS. 5A to 5D). The trypsin pretreatment performed tended to slightly increase the amount of regenerated dental pulp, albeit with no statistically significant difference (FIG. 5E). Also, the trypsin pretreatment performed tended to allow more odontoblast-like cells to adhere to the side wall of the dentin and form dentin-like hard tissues in a slightly higher amount (FIGS. 5A and 5B). Angiogenesis (FIGS. 6A and 6B) and neurite extension (FIGS. 6C and 6D) were similarly observed in both these cases. These results demonstrated that the injection of the non-cellular root canal filler without trypsin pretreatment regenerates the dental pulp with angiogenesis and neurite extension, and as in with trypsin pretreatment, allows odontoblast-like cells to adhere to the side wall of the dentin and promotes differentiation into odontoblasts and formation of dentin-like hard tissues.

Example 4

Comparison of Dental Pulp Regeneration after Pulpectomy in Young Dogs Between Presence and Absence of Compound C

[0075] After general anesthesia, pulpectomy was performed for maxillary and mandibular right and left anterior teeth in young (11-month-old) dogs. The openings were enlarged to the apex of the root with #60, then washed alternatingly with a 5% sodium hypochlorite solution and a 3% hydrogen peroxide solution, and further washed with saline. The root canals were thoroughly dried with a paper point and temporarily sealed with cement and a resin completely after hemostasis. After the pulpectomy, the temporary seals were removed, and the openings were washed alternatingly and washed with saline again. Then, the root canals were filled with 3% EDTA (Smear Clean, Nippon Shika Yakuhin Co., Ltd.), treated for 2 minutes, further washed with saline, and dried. Then, the root canals were pretreated by the application of a trypsin preparation (5 mg of Francetin T powder (2,500 USP crystal trypsin per 10 mg), Mochida Pharmaceutical Co., Ltd.)/ml of 0.5% nanobubble water (prepared with Foamest 8 (NAC Corp.); as described in Example 1)) for 10 minutes, and was then washed with saline. Subsequently, 1.2 μg of compound C as a regeneration promoting compound and 150 ng of G-CSF (Neutrogin, Chugai Pharmaceutical Co., Ltd.) as a chemotactic factor were added to 20 μl of extracellular matrix collagen (Koken Atelocollagen Implant, Koken Co., Ltd.) to prepare a non-cellular root canal filler, which was then filled into the root canals. On the other hand, a non-cellular root canal filler having the same composition as above except for the absence of compound C was filled into the root canals by the same procedures as above and was used as a control. Then, a gelatin sponge for hemostasis (Spongel, Astellas Pharma Inc.) was placed thereon, and the cavities were completely sealed with glass ionomer cement and a photopolymerizable resin. Then, 28 days after the transplantation, the teeth were extracted, and 5 μm paraffin sections were prepared on longitudinal sections according to a usual method, stained with H-E, and then morphologically observed in the same way as in Example 1. The area of the dentin was evaluated by measuring the ratio of the area of the dentin to the area of a tooth as to one section per three samples, and calculating the mean of the three samples. The density of dentin cells was calculated by measuring the number of dentin cells included in the range of 1 mm from the side wall of the root canal as to one section per three samples.

[0076] The results are shown in FIG. 7. Treatment with the non-cellular root canal filler containing compound C was found to sufficiently regenerate dental pulp-like tissues (FIGS. 7A and 7B), whereas treatment with the non-cellular root canal filler containing no compound C was found to regenerate such tissues only in very small amounts (FIGS. 7C and 7D). The treatment with the non-cellular root canal filler containing compound C tended to increase the area of the dentin (FIG. 7E) and increased the density of dentin cells (FIG. 7F), as compared with the treatment with the non-cellular root canal filler containing no compound C. These results indicated that compound C is an effective component for the regeneration of dental pulp tissues.