Technical field
[0001]
The present invention relates to an acellular root canal filling material and an acellular tooth tissue regeneration promotion kit that does not use stem cells or stem cell components and promotes regeneration of dental pulp, dentin and apical periodontal tissue.
Background technology
[0002]
In a super-aging society, it is important for healthy longevity to have healthy teeth and be able to chew properly. However, more than 20% of middle-aged and elderly people have a disease (infected root canal) in which a tooth whose nerve has already been removed (pulpectomy) is reinfected several decades later and pus accumulates under the root. Furthermore, about 25% of them are not cured completely even if they receive treatment. Such chronic foci of infection have a great impact on the whole body of elderly people with weakened immunity. Elderly people who are taking osteoporotic drugs often cannot have their teeth extracted. In addition, cases in which implants can be applied even after tooth extraction are decreasing among middle-aged and elderly people.
[0003]
On the other hand, tooth loss leads to deterioration of occlusion, pronunciation, taste, touch, and aesthetics, as well as deterioration of QOL. Recently, there is concern that oral frailty due to deterioration of dental and oral function may lead to sarcopenia, such as low muscle strength and low physical function, and deterioration of living functions due to malnutrition, etc., and eventually middle-aged and elderly people may fall into a state requiring nursing care. there is
[0004]
Therefore, in order to maintain teeth and oral cavity functions, it is possible to perform autologous transplantation of dental pulp stem cells isolated from one's own unwanted tooth, or allotransplantation of dental pulp stem cells isolated from another person's unwanted tooth, followed by pulp extraction. Dental pulp regeneration treatment methods have been developed to restore teeth to their original state so as not to lead to infected root canals and tooth extraction (Patent Documents 1 to 3). In addition to dental pulp stem cells, autologous or allogeneic transplantation of other tissue stem cells derived from bone marrow or adipose tissue can also regenerate the dental pulp. is inferior (Patent Document 4). Clinical studies have already confirmed the safety of dental pulp regeneration treatment using autologous dental pulp stem cell transplantation, suggesting its effectiveness (Non-Patent Document 1).
[0005]
In dental pulp regeneration treatment, in addition to the stem cell therapy (stem cell transplantation method) mentioned above, there is cell homing (cell migration method). For young human teeth with immature roots, the mainstream method is to fill the root canal with blood clots without using dental pulp stem cells (Non-Patent Document 2). Alternatively, there is also a method of injecting PRP (Platelet-Rich Plasma) instead of clot (Non-Patent Document 3). However, it is said that such a technique rarely regenerates pulp-specific tissue, and mainly regenerates fibrous and bone-like tissue rich in blood vessels (Non-Patent Document 4). In addition, in animal experiments, stromal cell-derived factor 1α (SDF1α), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), stem cell factor (SCF), and granulocyte A cell homing method using a cell growth factor such as colony stimulating factor (G-CSF) or a cytokine as a migration factor has also been developed (Non-Patent Document 5). However, it has been reported that such a technique does not lead to a sufficient amount of pulp regeneration, and that most of the pulp is fairly dense fibrous connective tissue with blood vessels, and in some cases the entire root canal is calcified. (Non-Patent Documents 6 and 7). Based on these findings, it is believed that stem cells are essential for pulp tissue regeneration, particularly in root-completed teeth (Non-Patent Document 8).
[0006]
However, autologous stem cell transplantation requires the use of one's own unused teeth such as wisdom teeth in order to use dental pulp stem cells. In addition, in middle-aged and elderly people, stem cell traits change, the total number of dental pulp stem cells that can be sorted is reduced, and it takes time to amplify them. In allogeneic transplantation, the cost required for safety confirmation after cell manufacturing and processing is reduced compared to autologous transplantation due to the increase in the number of cell products per lot. There are still unresolved issues, such as legal issues such as rights and remuneration for humans who have supplied teeth through the commercialization of stem cells, and issues related to the safety of immune reactions in humans.
[0007]
Therefore, it is desirable to develop a technology that promotes regeneration of tooth tissue after pulpectomy or infected root canal treatment, that is, dental pulp, dentin, and apical periodontal tissue, without using stem cells or stem cell-derived components. On the other hand, it is believed that further investigation of stem cell sources that promote host cell migration will enable the selection of appropriate signaling factors to be used for pulp regeneration. In other words, future development of a dental pulp regeneration method using appropriate signal factors that promote the migration of host stem cells with angiogenic and neuronal differentiation potentials and suppress the migration of cells with osteogenic and cementum-forming potentials is desired. (Non-Patent Document 9).
[0008]
In particular, pulp regeneration in middle-aged and elderly individuals is delayed compared to young individuals. In recent years, animal experiments using dogs have confirmed that dental pulp regeneration is promoted by transplantation of anti-CCL11 neutralizing antibody/CCR3 antagonist or ALK5 inhibitor together with dental pulp stem cells in dental pulp regeneration treatment by dental pulp stem cell transplantation in middle-aged and elderly patients. rice field. In addition, pretreatment of the root canals of middle-aged and older teeth with trypsin prior to transplantation of dental pulp stem cells promoted regeneration of the dental pulp (Patent Document 5). Anti-CCL11 neutralizing antibodies or CCR3 antagonists block signaling by inhibiting the binding of CCL11 to CCR3. Growth differentiation factor 11 (GDF11) also binds to the transforming growth factor-β (TGF-β) superfamily receptors ACVR1B (also known as ALK4), TGFBR1 (also known as ALK5) and ACVR1C (also known as ALK7), allowing ALK4 and Signals through ALK5, but ALK5 inhibitors block GDF11 signaling. During pulp regeneration, secreted factors from dental pulp stem cells accumulated in dentin are considered to be released from dentin, promoting pulp regeneration (Non-Patent Document 10). In vitro, the addition of CCR3 antagonists to the culture supernatant containing factors secreted by aging dental pulp stem cells significantly increased the neurite outgrowth-promoting and migration-promoting effects of the culture supernatant. In addition, addition of ALK5 inhibitor significantly increased the angiogenic activity and neurite outgrowth-promoting activity of the culture supernatant. These results suggest that CCR3 antagonists or ALK5 inhibitors promote dental pulp regeneration in middle-aged and elderly dogs by promoting blood vessel induction, neurite outgrowth, and migration. However, in young dogs, even if dental pulp stem cells were transplanted together with a CCR3 antagonist or ALK5 inhibitor, no dental pulp regeneration effect was observed (Patent Document 5).
[0009]
Trypsin is used as a pharmaceutical for the purpose of normalizing the wound surface and facilitating the action of antibiotics by decomposing necrotic tissue, blood clots, and denatured proteins (Non-Patent Document 11). In vitro, seeding of dental pulp stem cells on trypsin-treated dentin surfaces increased cell adherence and promoted odontoblast differentiation. In middle-aged dogs, trypsin pretreatment promotes dental pulp regeneration by inactivating inhibitory factors accumulated in middle-aged and elderly dentin, activating differentiation-promoting factors by cleaving precursors, and cell adhesion to dentin. This was suggested to be due to the increase in sex and the promotion of differentiation of dental pulp stem cells into odontoblasts. However, in young dogs, trypsin pretreatment was not found to promote dental pulp regeneration at all (Patent Document 5). Further, when only trypsin pretreatment was performed without transplantation of dental pulp stem cells, or when only CCR3 antagonist or ALK5 inhibitor was used without transplantation of dental pulp stem cells, almost no dental pulp regeneration was observed (Patent Document 5).
prior art documents
patent literature
[0010]
Patent Document 1: Japanese Patent No. 5621105
Patent Document 2: Japanese Patent No. 6031658
Patent Document 3: Japanese Patent No. 5748194
Patent Document 4: Japanese Patent No. 5939559
Patent Document 5: International Publication No. 2017/170996 Pamphlet
Non-patent literature
[0011]
Non-Patent Document 1: Nakashima M., Iohara K., Murakami M., Nakamura H., Sato Y., Ariji Y., Matsushita K.: Pulp regeneration by transplantation of dental pulp stem cells in pulpitis: A pilot clinical study. Stem Cell Res Therapy. 8(1):61, 2017.
Non-Patent Document 2: Galler KM.: Clinical procedures for revitalization: current knowledge and considerations. Int Endod J. 2016 Oct;49(10):926-36.
Non-Patent Document 3: Kontakiotis EG, Filippatos CG, Tzanetakis GN, Agrafioti A.: Regenerative endodontic therapy: a data analysis of clinical protocols. J Endod. 41(2):146-54. 2015.
Non-Patent Document 4: Del Fabbro M, Lolato A, Bucchi C, Taschieri S, Weinstein RL.: Autologous platelet concentrates for pulp and dentin regeneration: a literature review of animal studies. J Endod. 42(2):250-7, 2016.
Non-Patent Document 5 : Yang J., Yuan G., Chen Z.: Pulp Regeneration: Current Approaches and Future Challenges. Front Physiol.: 7:58, 2016.
Non-Patent Document 6: He L, Kim SG, Gong Q, Zhong J, Wang S, Zhou X, Ye L, Ling J, Mao JJ.: Regenerative endodontics for adult patients. J Endod. 43(9S):S57-S64 , 2017.
Non-Patent Document 7: Iohara K, Murakami M, Takeuchi N, Osako Y, Ito M, Ishizaka R, Utunomiya S, Nakamura H, Matsushita K, Nakashima M.: A novel combinatorial therapy with pulp stem cells and granulocyte colony-stimulating factor for total pulp regeneration. Stem Cells Transl. Med. 2(7): 521-533, 2013.
Non-Patent Document 8: Cao Y, Song M, Kim E, Shon W, Chugal N, Bogen G, Lin L, Kim RH, Park NH, Kang MK.: Pulp-dentin Regeneration: Current State and Future Prospects. J Dent Res 94(11):1544-51, 2015.
Non-Patent Document 9 : Yang J, Yuan G, Chen Z.: Pulp Regeneration: Current Approaches and Future Challenges. Front Physiol. 7:58, 2016. eCollection 2016.
Non-Patent Document 10: Kawamura R, Hayash i Y, Murakami H, Nakashima M.: EDTA soluble chemical components and the conditioned medium from mobilized dental pulp stem cells contain an inductive microenvironment, promoting cell proliferation, migration and odontoblastic differentiation. Stem Cell Res. Ther. 7(1):77 , 2016.
Non-Patent Document 11: Journal of Dermatology and Sexology Vol.64, Yoshikuni Noguchi et al., pp.497-506, 1954
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0012]
The present invention has been made in view of such problems. An object of the present invention is to provide an acellular root canal filling material capable of regenerating healthy tooth tissue. Another object of the present invention is to provide an acellular tooth tissue regeneration promoting kit using the acellular root canal filling material.
Means to solve problems
[0013]
As a result of extensive research, the present inventors discovered a tetrahydroisoquinoline compound that promotes regeneration of tooth tissue.
[0014]
That is, according to one embodiment of the present invention, the following formula (1):
[Chemical 1]

(In the formula,
R 1, R 2, R 3, and R 4 each independently represent -H, -halogen, substituted or unsubstituted C 1-6 alkyl, -OH, -O-C 1-6 alkyl, -SH, -S-C 1-6 alkyl, -COOH, -CO-C1-6 alkyl, -CO-O-C1-6 alkyl, -CO-NH-C1-6 alkyl, -NO2, -NH2, -NH-C1 -6alkyl, -N(C1-6alkyl)2, or -NH-CO-C1-6alkyl,
R 5 is substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted C 3-10 cycloalkyl, substituted or unsubstituted C 6-14 aryl, -C 1-6 alkylene-substituted or unsubstituted represents C3-10 cycloalkyl, or -C1-6 alkylene-substituted or unsubstituted C6-14 aryl,
R 6 represents -H, substituted or unsubstituted -C 1-6 alkyl, or -Y'-A',
　X represents C 1-6 alkylene,
Y and Y 'each independently represent a single bond or C 1-6 alkylene,
A and A' each independently represent a substituted or unsubstituted C6-14 aryl or a substituted or unsubstituted 3- to 15-membered heterocyclic group,
　n indicates 0 or 1. )
A non-cellular root canal filling material comprising a tetrahydroisoquinoline compound represented by or a pharmaceutically acceptable salt thereof or a solvate thereof.
[0015]
The acellular root canal filling material is (+)-4-[[2-[6-fluoro-3-(4-fluorobenzyl)-3,4-dihydroisoquinolin-2(1H)-yl]ethylamino ]methyl]-N-isopropylaniline 1 fumarate or (+)-N-[3-(methanesulfonylamino)benzyl]-2-[6-fluoro-3-(4-fluorobenzyl)-3,4 -dihydroisoquinolin-2(1H)-yl]ethanamine monocitrate.
[0016]
The acellular root canal filling material may further contain an extracellular matrix.
[0017]
The acellular root canal filling material may further contain an anti-CCL11 neutralizing antibody and/or an ALK5 inhibitor.
[0018]
The acellular root canal filling material may further contain at least one chemotactic factor selected from the group consisting of G-CSF, bFGF and SDF-1.
[0019]
The acellular root canal filling material may be used for regeneration of tooth tissue in young individuals or middle-aged and elderly individuals, but is preferably used in young individuals.
[0020]
In addition, according to one embodiment, the present invention provides a dental tissue regeneration promoting kit comprising a pretreatment agent containing a serine protease and the acellular root canal filling material described above.
[0021]
The tooth tissue regeneration promoting kit is preferably used for regeneration of tooth tissue of middle-aged and elderly individuals.
[0022]
The serine protease is preferably a chymotrypsin-like serine protease, more preferably trypsin.
Effect of the invention
[0023]
The acellular root canal filling material and dental tissue regeneration promoting kit according to the present invention enable effective regeneration of dental tissue without the need for transplantation of autologous or allogeneic dental pulp stem cells or their stem cell-derived components. useful and useful.
Brief description of the drawing
[0024]
[Fig. 1] Fig. 1 is a diagram showing the results of comparison between a kit for promoting acellular pulp regeneration using compound B and a kit for promoting acellular pulp regeneration using SB 328437. [Fig. (A) is an HE staining image of a tooth tissue section treated with a kit for promoting acellular pulp regeneration using compound B, and (B) is a tooth tissue treated with a kit for promoting acellular pulp regeneration using compound B. HE-stained image (high resolution) of the section, (C) is a HE-stained image of a tooth tissue section treated with a kit for promoting acellular pulp regeneration using SB 328437, and (D) is a HE-stained image using SB 328437. FIG. 10 is an HE-stained image (high resolution) of a tooth tissue section treated with a kit for promoting acellular dental pulp regeneration. FIG. (E) is a graph showing the results of quantitative analysis of the amount of dental pulp regeneration by an acellular dental pulp regeneration promoting kit using compound B and an acellular dental pulp regeneration promoting kit using SB 328437.
[Fig. 2] Fig. 2 is a diagram showing the results of comparison between a kit for promoting acellular pulp regeneration using compound B and a kit for promoting acellular pulp regeneration using SB 328437. [Fig. (A) is a lectin-stained image of a tooth tissue section treated with a kit for promoting acellular pulp regeneration using compound B, and (B) is a tooth tissue treated with a kit for promoting acellular pulp regeneration using SB 328437. It is a lectin staining image of the section, (C) is a PGP9.5 immunostaining image of a tooth tissue section treated with a kit for promoting acellular pulp regeneration using compound B, and (D) is a non-staining image using SB 328437. Fig. 4 is a PGP9.5 immunostained image of a tooth tissue section treated with a kit for promoting cellular dental pulp regeneration.
[Fig. 3] Fig. 3 is a diagram showing the results of comparison between an acellular dental pulp regeneration promoting kit using compound B and an acellular dental pulp regeneration promoting kit not using compound B. [Fig. (A) is an HE staining image of a tooth tissue section treated with a kit for promoting acellular pulp regeneration using compound B, and (B) is a tooth tissue treated with a kit for promoting acellular pulp regeneration using compound B. HE-stained image of the section (high resolution), (C) is a HE-stained image of a tooth tissue section treated with a compound B-free acellular pulp regeneration promotion kit, and (D) is a compound B-free. FIG. 10 is an HE-stained image (high resolution) of a tooth tissue section treated with the acellular dental pulp regeneration promotion kit used. FIG. (E) is a graph showing the results of quantitative analysis of the amount of dental pulp regeneration by an acellular dental pulp regeneration promoting kit using Compound B and an acellular dental pulp regeneration promoting kit not using Compound B. FIG.
[Fig. 4] Fig. 4 is a diagram showing the results of comparison between an acellular dental pulp regeneration promoting kit using Compound B and an acellular dental pulp regeneration promoting kit not using Compound B. [Fig. (A) is a lectin-stained image of a tooth tissue section treated with a compound B-using acellular pulp regeneration promoting kit, and (B) is a tooth treated with a compound B-free acellular pulp regeneration promoting kit. Lectin-stained images of tissue sections, (C) is PGP9.5 immunostained images of tooth tissue sections treated with a kit for promoting acellular pulp regeneration using compound B, and (D) is compound B-free. Fig. 10 is a PGP9.5 immunostaining image of a tooth tissue section treated with a kit for promoting acellular dental pulp regeneration.
[Fig. 5] Fig. 5 is a diagram showing the results of comparing the effects of acellular root canal filling materials using Compound B between the cases where trypsin pretreatment was performed and the case where trypsin pretreatment was not performed. (A) is an HE-stained image of a tooth tissue section pretreated with trypsin, and (B) is a HE-stained image (high resolution) of a tooth tissue section pretreated with trypsin. (C) is an HE-stained image of a tooth tissue section without trypsin pretreatment, and (D) is an HE-stained image (high resolution) of a tooth tissue section without trypsin pretreatment. be. (E) is a graph showing the results of quantitative analysis of the amount of dental pulp regeneration by the acellular root canal filling material using compound B with and without trypsin pretreatment.
[Fig. 6] Fig. 6 is a diagram showing the results of comparing the effects of acellular root canal filling materials using Compound B between cases in which trypsin pretreatment was performed and in which trypsin pretreatment was not performed. (A) is a lectin-stained image of a tooth tissue section treated with a kit for promoting acellular pulp regeneration when pretreated with trypsin; A lectin-stained image of a tooth tissue section treated with a dental pulp regeneration promotion kit, (C) is a PGP9.5 immunostained image of a tooth tissue section pretreated with trypsin, and (D) is before trypsin. It is a PGP9.5 immunostaining image of a tooth tissue section without treatment.
7] Fig. 7 is a diagram showing the results of comparison between an acellular dental pulp regeneration promoting kit using Compound C and an acellular dental pulp regeneration promoting kit not using Compound C. [Fig. (A) is an HE staining image of a tooth tissue section treated with a kit for promoting acellular pulp regeneration using compound C, and (B) is a tooth tissue treated with a kit for promoting acellular pulp regeneration using compound C. HE-stained image (high resolution) of the section, (C) is a HE-stained image of a tooth tissue section treated with a compound C-free acellular pulp regeneration promotion kit, and (D) is a compound C-free. FIG. 10 is an HE-stained image (high resolution) of a tooth tissue section treated with the acellular dental pulp regeneration promotion kit used. FIG. (E) is a graph showing the results of quantitative analysis of tooth tissue regeneration by an acellular dental pulp regeneration promoting kit using compound C and an acellular dental pulp regeneration promoting kit not using compound C, based on the area of ​​dentin. . (F) is a graph showing the results of quantitative analysis of tooth tissue regeneration by an acellular dental pulp regeneration promotion kit using compound C and an acellular dental pulp regeneration promotion kit not using compound C based on the density of odontoblasts. be.
MODE FOR CARRYING OUT THE INVENTION
[0025]
Although the present invention will be described in detail below, the present invention is not limited to the embodiments described herein.
[0026]
According to the first embodiment of the present invention, formula (1):
[Chemical 2]

(In the formula,
R 1, R 2, R 3, and R 4 each independently represent -H, -halogen, substituted or unsubstituted C 1-6 alkyl, -OH, -O-C 1-6 alkyl, -SH, -S-C 1-6 alkyl, -COOH, -CO-C1-6 alkyl, -CO-O-C1-6 alkyl, -CO-NH-C1-6 alkyl, -NO2, -NH2, -NH-C1 -6alkyl, -N(C1-6alkyl)2, or -NH-CO-C1-6alkyl,
R 5 is substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted C 3-10 cycloalkyl, substituted or unsubstituted C 6-14 aryl, -C 1-6 alkylene-substituted or unsubstituted represents C3-10 cycloalkyl, or -C1-6 alkylene-substituted or unsubstituted C6-14 aryl,
R 6 represents -H, substituted or unsubstituted -C 1-6 alkyl, or -Y'-A',
　X represents C 1-6 alkylene,
Y and Y 'each independently represent a single bond or C 1-6 alkylene,
A and A' each independently represent a substituted or unsubstituted C6-14 aryl or a substituted or unsubstituted 3- to 15-membered heterocyclic group,
　n is 0 or or 1. )
A noncellular root canal filling material comprising a tetrahydroisoquinoline compound represented by or a pharmaceutically acceptable salt thereof or a solvate thereof (hereinafter referred to as "compound group A" in this specification) is.
[0027]
"Non-cellular" means not containing cells or cell-derived components (eg, extracellular secretory proteins, exosomes, etc.). Also, the term "root canal" refers to a canal in which the dental pulp is accommodated in the root portion.
[0028]
The acellular root canal filling material of the present embodiment can contain a single compound or a mixture of two or more selected from compound group A as an active ingredient. The compounds of compound group A are CCR3 antagonists, bind to CCR3, have the effect of inhibiting the binding of CCL11 to CCR3, and can suppress CCL11 signal transduction.
[0029]
The compound of compound group A used in this 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, formula (2) below); 4-[[2-[6-fluoro-3-(4-fluorobenzyl)-3,4 -dihydroisoquinolin-2(1H)-yl]ethylamino]methyl]-N-isopropylaniline (Example 138 of WO2008/123582, formula (3) below); 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 (of WO2008/123582 Example 180, formula (5) below) or a pharmaceutically acceptable salt thereof.
[0030]
[Chemical 3]

　　　　
[0031]
Compounds of compound group A used in this embodiment are particularly preferably (+)-4-[[2-[6-fluoro-3-(4-fluorobenzyl)-3,4-dihydroisoquinoline-2 (1H)-yl]ethylamino]methyl]-N-isopropylaniline 1-fumarate (disclosed in JP-A-2009-173571, the fumarate of the above formula (3). In the present specification, it is also referred to as “compound B” ) or (+)-N-[3-(methanesulfonylamino)benzyl]-2-[6-fluoro-3-(4-fluorobenzyl)-3,4-dihydroisoquinolin-2(1H)-yl ] Ethanamine 1 citrate (disclosed in JP-A-2009-191048, the citrate of the above formula (2), also referred to herein as “compound C”).
[0032]
The compounds of compound group A can be produced by appropriately combining the chemical synthesis method described in WO2008/123582 and chemical synthesis methods based thereon with various conventionally known methods.
[0033]
The acellular root canal filling material of this embodiment may be composed of only the above active ingredients, but optional ingredients include extracellular matrix, anti-CCL11 neutralizing antibody and/or ALK5 inhibitor, and/or G - It may further comprise at least one chemotactic factor selected from the group consisting of CSF, bFGF and SDF-1.
[0034]
The extracellular matrix that can be used in the acellular root canal filling material of the present embodiment is not particularly limited, but examples include collagen, artificial proteoglycan, gelatin, hydrogel, fibrin, phosphophorin, heparan sulfate, and heparin. , laminin, fibronectin, alginic acid, hyaluronic acid, chitin, chitosan, PLA, PLGA, PEG, PGA, PDLLA, PCL, hydroxyapatite, β-TCP, calcium carbonate, and the like. Also, the extracellular matrix may be used by coating it on a metal substrate such as gold or titanium.
[0035]
The anti-CCL11 neutralizing antibody that can be used in the acellular root canal filling material of this embodiment may be any known anti-CCL11 antibody. An anti-CCL11 neutralizing antibody binds to CCL11, has the effect of inhibiting the binding of CCL11 to CCR3, and can suppress CCL11 signal transduction. Anti-CCL11 neutralizing antibodies are commercially available, and such commercially available products can be used in this embodiment.
[0036]
The ALK5 inhibitor that can be used in the acellular root canal filling material of this embodiment may be any known compound that inhibits GDF11 signaling. Various ALK5 inhibitors are commercially available, and such commercial products can be used in this embodiment. The ALK5 inhibitor in this embodiment is not particularly limited, and for example, the following compounds can be used.
[0037]
[Chemical 4]

[Chemical 5]

[Chemical 6]

[0038]
The chemotactic factors that can be used in the acellular root canal filling material of the present embodiment are not particularly limited, but examples include G-CSF, SDF-1, bFGF, TGF-β, NGF, PDGF, BDNF, and GDNF. , EGF, VEGF, SCF, MMP3, Slit, GM-CSF, LIF, HGF, etc., and can be used singly or in combination of two or more selected from these. Migratory factors can promote the migratory ability of stem cells around tooth tissue. A chemotactic factor that can be used in this embodiment is preferably selected from the group consisting of G-CSF, bFGF and SDF-1. All of the above chemotactic factors are commercially available, and such commercial products can be used in this embodiment.
[0039]
In the acellular root canal filling material of the present embodiment, the content of each of the above components may range, for example, from 50 ng/ml to 200 μg/ml, preferably from 3 μg/ml to 100 μg/ml. In addition, the mixing ratio of the active ingredient (compound of compound group A) and other optional ingredients is not particularly limited, and may be, for example, 10% by weight: 90% by weight to 90% by weight: 10% by weight. is possible. In addition, the acellular root canal filling material of the present embodiment may be prepared by combining the above-described components with known pharmaceutically acceptable diluents, carriers, excipients, and the like, if necessary.
[0040]
Although the acellular root canal filling material of the present embodiment can be applied to both young individuals and middle-aged and elderly individuals, it is preferably used for young individuals. Here, the young individual is not particularly limited, but for example, in the case of a human, it is 1 to 29 years old, and in the case of a rat, it is 1 to 29 weeks old. If it is a dog, it must be between 1 week and 1 year old.
[0041]
The acellular root canal filling material of this embodiment can be used by injecting it into the root canal in the same manner as conventionally known dental root canal filling materials.
[0042]
According to the second embodiment of the present invention, there is provided a tooth tissue regeneration promoting kit comprising a pretreatment agent containing a serine protease and the acellular root canal filling material.
[0043]
"Tooth tissue" in the present embodiment means tissue including at least one of dental pulp, dentin, and apical periodontal tissue.
[0044]
The "pretreatment agent" in this embodiment is used before inserting the acellular root canal filling material into the root canal. As a result, factors that inhibit tissue regeneration in tooth tissue and periodontal tissue can be decomposed, and/or latent forms of chemotactic factors and differentiation-promoting factors can be converted into active forms.
[0045]
The pretreatment agent used in the kit of this embodiment contains serine protease. Serine proteases are proteases (proteolytic enzymes) with serine residues that carry out nucleophilic attacks as catalytic residues. Serine proteases are classified into subtilisin-like serine proteases and chymotrypsin-like serine proteases based on similarities in amino acid sequences and three-dimensional structures. The former includes subtilisin BPN', thermitase, proteinase K, lantibiotic peptidase, kexin, cucumisin, etc. 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 used singly or in combination of two or more selected from the above, preferably a chymotrypsin-like serine protease, more preferably trypsin.
[0046]
The concentration of serine protease in the pretreatment agent used in the kit of the present embodiment can decompose factors that inhibit tissue regeneration in tooth tissue and periodontal tissue, and/or can reduce latent types of migration factors and differentiation-promoting factors. It is not particularly limited as long as it can be converted to the active form, and may be, for example, 10 μg/ml (0.001%) to 50 mg/ml (5%), preferably 500 μg/ml (0.05%). ~5 mg/ml (0.5%).
[0047]
The pretreatment agent used in this embodiment may further contain nanobubbles. Here, "nanobubble" refers to a bubble having a diameter in the order of nanometers or a lipid vesicle containing a gas or gas precursor in its lumen and having a diameter in the order of nanometers. The diameter of nanobubbles used in the pretreatment agent in the kit of this embodiment is, for example, 10 to 500 nm, preferably 70 to 300 nm. The diameter of nanobubbles can be measured, for example, with a nanoparticle distribution analyzer (SALD-7100, Shimadzu Corporation). The lipid composition, charge state, density, weight, etc. of nanobubbles can be determined as appropriate. Lipids used for the preparation of nanobubbles are not particularly limited, and may be, for example, phospholipids, glyceroglycolipids, glycosphingolipids, etc. These lipids have primary amino groups, secondary amino groups, and the like. , a cationic lipid into which a tertiary amino group or a quaternary ammonium group has been introduced. The concentration of nanobubbles in the pretreatment agent is not particularly limited, but can be, for example, 2×10 7 /cm 3 to 2×10 9 /cm 3 . The nanobubble concentration can be quantitatively analyzed, for example, by electron spin resonance (ESR).
[0048]
Pretreatment may be performed by injecting a pretreatment agent into the root canal. The pretreatment time can be appropriately determined according to the type and concentration of the serine protease used, and may be, for example, 3 to 30 minutes, preferably 5 to 20 minutes, more preferably 10 minutes.
[0049]
The kit of this embodiment may further include additional buffers, reagents, instructions, etc. in addition to the pretreatment agent and acellular root canal filling material.
[0050]
Although the kit of the present embodiment can be applied to both young and middle-aged individuals, it is preferably used for middle-aged and elderly individuals. Here, the middle-aged individuals are not particularly limited, but are, for example, 30 to 49 years of age in the case of humans, and 30 to 39 weeks of age in the case of rats. If it is a dog, it can be between 2 and 4 years old. Aged individuals are not particularly limited, but may be, for example, 50 years of age or older for humans, 40 weeks of age or older for rats, and 5 years of age for dogs. It can be more than Therefore, the kit of the present embodiment is preferably used for humans aged 30 years or older, rats aged 30 weeks or older, and dogs aged 2 years or older.
Example
[0051]
The present invention will be further described with reference to examples below. In addition, these do not limit this invention at all.
[0052]
[Example 1]
(Regeneration of pulp after pulpectomy in young dogs)
After general anesthesia, a young (12-month-old) dog was subjected to pulpectomy on the left and right anterior teeth of the upper and lower jaws. It was washed alternately with hydrogen water, washed with physiological saline, dried, and temporarily sealed with resin. 3 to 12 days after pulpectomy, the temporary seal is removed, Washing was performed again with alternating washings and physiological saline. Thereafter, the inside of the root canal was filled with 3% EDTA (Smear Clean, Nippon Dental Pharmaceutical Co., Ltd.), allowed to act for 2 minutes, washed with physiological saline, and dried. After that, a trypsin preparation (Francetin T powder (Mochida Pharmaceutical) (crystalline trypsin 2,500 USP per 10 mg)) 5 mg/ml (0.5%, prepared with nanobubble water (Foamest 8 (Nac)); Misako Nakashima, ``Study of the effect of drug-containing nanobubble water for sterilization of the root canal in intractable apical periodontitis,'' Nippon Dentistry Conservative Journal, Vol. 63, No. 1, p. Pretreatment was performed by applying for 1 minute followed by washing with saline. Next, 20 μl of extracellular matrix collagen (Koken atelocollagen implant, Koken) was added with 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- as a chemotactic factor. Acellular root canal filling materials containing CSF (Neutrogin, Chugai Pharmaceutical Co., Ltd.) were prepared and filled into root canals. After that, a hemostatic gelatin sponge (Sponzel, Astellas Pharma Inc.) was placed on it, and the cavity was completely sealed with glass ionomer cement and photopolymerization resin. Twenty-eight days after transplantation, the teeth were extracted, 5 μm longitudinal paraffin sections were prepared according to common methods, and the morphology was observed after H-E staining. The amount of pulp regeneration was evaluated by measuring the ratio of the area of ​​the regenerated pulp to the area of ​​the pulp cavity for 4 sections per sample, and calculating the average value of the 4 samples. Angiogenesis was determined by staining 28-day specimens with Fluorescein labeled Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL I, BSL I) and Fluorescein labeled Galanthus Nivalis (Snowdrop) Lectin (GNL) (Vector Laboratories) (20 μg/ml) for 15 minutes. and conducted a comparative study. Neurite outgrowth was compared by immunostaining 28-day specimens with anti-PGP9.5 antibody (UltraClone, 1:10,000).
[0053]
The results are shown in Figures 1 and 2. Acellular root canal filling materials containing SB328437 or compound B as a CCR3 antagonist induced pulp regeneration of loose connective tissue with abundant blood vessels, while no inflammatory cell infiltration or internal resorption was observed. (FIGS. 1A-D). Fig. 1E shows the ratio of the regenerated pulp to the pulp cavity. Acellular root canal filling materials containing SB328437 or compound B as CCR3 antagonists did not show a statistically significant difference in the amount of pulp regeneration (Fig. 1E). Furthermore, angiogenesis (Fig. 2A, B) and neurite outgrowth (Fig. 2C, D) were confirmed when using either SB328437 or acellular root canal filling material containing compound B as a CCR3 antagonist. It was clarified that odontoblast-like cells adhered to the lateral wall and dentin-like hard tissue was formed.
[0054]
[Example 2]
(Comparison of pulp regeneration after pulpectomy in young dogs with or without compound B)
After general anesthesia, a young (11-month-old) dog undergoes pulpectomy on the left and right anterior teeth of the upper and lower jaws. The root canal was washed alternately with hydrogen water, washed with physiological saline, completely dried with a paper point, and after bleeding was stopped, the root canal was completely temporarily sealed with cement and resin. Eight days after the pulpectomy, the temporary seal was removed, and the animals were again washed alternately and with physiological saline. Thereafter, the inside of the root canal was filled with 3% EDTA (Smear Clean, Nippon Dental Pharmaceutical Co., Ltd.), allowed to act for 2 minutes, washed with physiological saline, and dried. After that, a trypsin preparation (Francetin T powder (Mochida Pharmaceutical) (crystalline trypsin 2,500 USP per 10 mg)) 5 mg/ml (0.5%, prepared with nanobubble water (Foamest 8 (Nac)) was placed in the root canal, Example 1 Pretreatment was carried out by applying a solution of the same solution in ) for 10 minutes, followed by washing with saline. Next, to 20 μl of extracellular matrix collagen (Koken atelocollagen implant, Koken), 1.25 μg of compound B as a regeneration-promoting compound and 150 ng of G-CSF (Neutrogin, Chugai Pharmaceutical) as a chemotactic factor were added to acellular roots. A canal filling material was prepared and filled into the root canal. On the other hand, a root canal was filled with an acellular root canal filling material having the same composition except that compound B was not contained, and the same procedure was used as a control. After that, a hemostatic gelatin sponge (Sponzel, Astellas Pharma Inc.) was placed on it, and the cavity was completely sealed with glass ionomer cement and photopolymerization resin. Twenty-eight days after transplantation, the teeth were extracted, and 5 μm paraffin sections of longitudinal sections were prepared in the same manner as in Example 1, and after H-E staining, the morphology was observed. In the same manner as in Example 1, angiogenesis was confirmed by BS-1 lectin staining, and neurite outgrowth was confirmed by PGP9.5 immunostaining.
[0055]
The results are shown in Figures 3 and 4. When treated with the acellular root canal filling material containing compound B, sufficient pulp-like tissue regeneration was observed (Fig. 3A, B), but with the acellular root canal filling material not containing compound B Only a small amount of regeneration was seen when treated (Fig. 3C,D). A statistically significant difference was observed in the amount of pulp regeneration between the two (Fig. 3E). On the other hand, angiogenesis (Fig. 4A, B) and neurite outgrowth (Fig. 4C, D) were similarly seen in both cases. These results indicated that compound B was an effective ingredient for regeneration of pulp tissue.
[0056]
[Example 3]
(Comparison of pulp regeneration after pulpectomy in young dogs with or without trypsin pretreatment)
After general anesthesia, a young (11-month-old) dog underwent pulpectomy on the left and right anterior teeth of the lower jaw. The root canal was washed alternately with water, washed with physiological saline, completely dried with a paper point to stop bleeding, and then completely temporarily sealed with cement and resin. After the pulpectomy, the temporary seal was removed, and the animals were washed alternately and again with physiological saline. Thereafter, the inside of the root canal was filled with 3% EDTA (Smear Clean, Nippon Dental Pharmaceutical Co., Ltd.), allowed to act for 2 minutes, washed with physiological saline, and dried. After that, a trypsin preparation (Francetin T powder (Mochida Pharmaceutical) (crystalline trypsin 2,500 USP per 10 mg)) 5 mg/ml (0.5%, prepared with nanobubble water (Foamest 8 (Nac)) was placed in the left root canal. Pretreatment was carried out by applying the same solution as in Example 1) for 10 minutes, followed by washing with saline. On the other hand, the right root canal was not pretreated (control). Next, to 20 μl of extracellular matrix collagen (Koken atelocollagen implant, Koken), 1.25 μg of compound B as a regeneration-promoting compound and 150 ng of G-CSF (Neutrogin, Chugai Pharmaceutical) as a chemotactic factor were added to acellular roots. Canal fillers were prepared and filled into left and right root canals. After that, a hemostatic gelatin sponge (Sponzel, Astellas Pharma Inc.) was placed on it, and the cavity was completely sealed with glass ionomer cement and photopolymerization resin. Twenty-eight days after transplantation, the teeth were extracted, and 5 μm paraffin sections of longitudinal sections were prepared in the same manner as in Example 1, and after H-E staining, the morphology was observed. In the same manner as in Example 1, angiogenesis was confirmed by BS-1 lectin staining, and neurite outgrowth was confirmed by PGP9.5 immunostaining.
[0057]
The results are shown in Figures 5 and 6. Regeneration of dental pulp tissue was observed regardless of the presence or absence of trypsin pretreatment (Figs. 5A to 5D), and the amount of regeneration tended to increase slightly when trypsin pretreatment was performed, but the difference was not statistically significant. not seen (Fig. 5E). In addition, when trypsin pretreatment was performed, more odontoblast-like cells adhered to the dentin sidewall, and the amount of formation of dentin-like hard tissue tended to be slightly higher (Fig. 5A, B). . Furthermore, angiogenesis (Fig. 6A, B) and neurite outgrowth (Fig. 6C, D) were similarly seen in both cases. These results suggest that the injection of acellular root canal filling material without trypsin pretreatment results in regeneration of the dental pulp with neovascularization and nerve outgrowth, and similar to the case of trypsin pretreatment, the dentin lateral wall. It was clarified that odontoblast-like cells adhered to promote odontoblast differentiation and dentin-like hard tissue formation.
[0058]
[Example 4]
(Comparison of pulp regeneration after pulpectomy in young dogs with or without compound C)
After general anesthesia, a young (11-month-old) dog undergoes pulpectomy on the left and right anterior teeth of the upper and lower jaws. The root canal was washed alternately with hydrogen water, washed with physiological saline, completely dried with a paper point, and after bleeding was stopped, the root canal was completely temporarily sealed with cement and resin. After the pulpectomy, the temporary seal was removed, and the animals were washed alternately and again with physiological saline. Thereafter, the inside of the root canal was filled with 3% EDTA (Smear Clean, Nippon Dental Pharmaceutical Co., Ltd.), allowed to act for 2 minutes, washed with physiological saline, and dried. After that, a trypsin preparation (Francetin T powder (Mochida Pharmaceutical) (crystalline trypsin 2,500 USP per 10 mg)) 0.5 mg/ml (0.05%, prepared with nanobubble water (Foamest 8 (Nac)) was placed in the root canal, Example 1 Pretreatment was carried out by applying a solution of the same solution in ) for 10 minutes, followed by washing with saline. Next, to 20 μl of extracellular matrix collagen (Koken atelocollagen implant, Koken), 1.2 μg of compound C as a regeneration-promoting compound and 150 ng of G-CSF (Neutrogin, Chugai Pharmaceutical) as a chemotactic factor were added to acellular roots. A canal filling material was prepared and filled into the root canal. On the other hand, a root canal was filled with an acellular root canal filling material having the same composition except that it did not contain compound C, in the same procedure, as a control. After that, a hemostatic gelatin sponge (Sponzel, Astellas Pharma Inc.) was placed on it, and the cavity was completely sealed with glass ionomer cement and photopolymerization resin. Twenty-eight days after transplantation, the teeth were extracted, and 5 μm paraffin sections of longitudinal sections were prepared in the same manner as in Example 1, and after H-E staining, the morphology was observed. The area of ​​dentin was evaluated by measuring the ratio of the area of ​​dentin to the area of ​​teeth for one section out of 3 samples, and calculating the average value of the 3 samples. The density of dentin cells was calculated by counting the number of dentin cells contained in the range of 1 mm from the root canal wall for one section out of three samples.
[0059]
The results are shown in Figure 7. When treated with the acellular root canal filling material containing compound C, sufficient pulp-like tissue regeneration was observed (Fig. 7A, B), but with the acellular root canal filling material not containing compound C Only a small amount of regeneration was seen when treated (Fig. 7C,D). In addition, when treated with an acellular root canal filling material containing compound C, there was a tendency for the area of ​​dentin to increase compared to when treated with an acellular root canal filling material that did not contain compound C. (Fig. 7E) and increased density of dentin cells (Fig. 7F). These results indicated that Compound C was an effective ingredient for regeneration of pulp tissue.
The scope of the claims
[Claim 1]
Formula (1) below:
[Chemical 1]

(In the formula,
R 1, R 2, R 3, and R 4 each independently represent -H, -halogen, substituted or unsubstituted C 1-6 alkyl, -OH, -O-C 1-6 alkyl, -SH, -S-C 1-6 alkyl, -COOH, -CO-C1-6 alkyl, -CO-O-C1-6 alkyl, -CO-NH-C1-6 alkyl, -NO2, -NH2, -NH-C1 -6alkyl, -N(C1-6alkyl)2, or -NH-CO-C1-6alkyl,
R 5 is substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted C 3-10 cycloalkyl, substituted or unsubstituted C 6-14 aryl, -C 1-6 alkylene-substituted or unsubstituted C 3-10 cycloalkyl, or -C 1-6 alkylene- represents a substituted or unsubstituted C 6-14 aryl,
R 6 represents -H, substituted or unsubstituted -C 1-6 alkyl, or -Y'-A',
　X represents C 1-6 alkylene,
Y and Y 'each independently represent a single bond or C 1-6 alkylene,
A and A' each independently represent a substituted or unsubstituted C6-14 aryl or a substituted or unsubstituted 3- to 15-membered heterocyclic group,
　n indicates 0 or 1. )
A non-cellular root canal filling material comprising a tetrahydroisoquinoline compound represented by or a pharmaceutically acceptable salt thereof or a solvate thereof.
[Claim 2]
(+)-4-[[2-[6-fluoro-3-(4-fluorobenzyl)-3,4-dihydroisoquinolin-2(1H)-yl]ethylamino]methyl]-N-isopropylaniline 1 Fumarate or (+)-N-[3-(methanesulfonylamino)benzyl]-2-[6-fluoro-3-(4-fluorobenzyl)-3,4-dihydroisoquinolin-2(1H)-yl ] The acellular root canal filling material according to claim 1, comprising ethanamine.1 citrate.
[Claim 3]
The acellular root canal filling material according to claim 1 or 2, further comprising an extracellular matrix.
[Claim 4]
The acellular root canal filling material according to any one of claims 1 to 3, further comprising an anti-CCL11 neutralizing antibody and/or an ALK5 inhibitor.
[Claim 5]
The acellular root canal filling material according to any one of claims 1 to 4, further comprising at least one chemotactic factor selected from the group consisting of G-CSF, bFGF and SDF-1.
[Claim 6]
The acellular root canal filling material according to any one of claims 1 to 5, which is used for regeneration of tooth tissue of young individuals.
[Claim 7]
a pretreatment agent containing serine protease,
　Formula (1) below:
[Chemical 2]

(In the formula,
R 1, R 2, R 3, and R 4 each independently represent -H, -halogen, substituted or unsubstituted C 1-6 alkyl, -OH, -O-C 1-6 alkyl, -SH, -S-C 1-6 alkyl, -COOH, -CO-C1-6 alkyl, -CO-O-C1-6 alkyl, -CO-NH-C1-6 alkyl, -NO2, -NH2, -NH-C1 -6alkyl, -N(C1-6alkyl)2, or -NH-CO-C1-6alkyl,
R 5 is substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted C 3-10 cycloalkyl, substituted or unsubstituted C 6-14 aryl, -C 1-6 alkylene-substituted or unsubstituted represents C3-10 cycloalkyl, or -C1-6 alkylene-substituted or unsubstituted C6-14 aryl,
R 6 represents -H, substituted or unsubstituted -C 1-6 alkyl, or -Y'-A',
　X represents C 1-6 alkylene,
Y and Y 'each independently represent a single bond or C 1-6 alkylene,
A and A' each independently represent a substituted or unsubstituted C6-14 aryl or a substituted or unsubstituted 3- to 15-membered heterocyclic group,
　n indicates 0 or 1. )
A non-cellular root canal filling material comprising a tetrahydroisoquinoline compound represented by or a pharmaceutically acceptable salt thereof or a solvate thereof
A kit for promoting tooth tissue regeneration, comprising:
[Claim 8]
(+)-4-[[2-[6-fluoro-3-(4-fluorobenzyl)-3,4-dihydroisoquinolin-2(1H)-yl]ethylamino]methyl]-N-isopropylaniline 1 Fumarate or (+)-N-[3-(methanesulfonylamino)benzyl]-2-[6-fluoro-3-(4-fluorobenzyl)-3,4-dihydroisoquinolin-2(1H)-yl 8. The kit for promoting tooth tissue regeneration according to claim 7, comprising ethanamine monocitrate.
[Claim 9]
The kit for promoting tooth tissue regeneration according to claim 7 or 8, wherein the acellular root canal filling material further contains an extracellular matrix.
[Claim 10]
The kit for promoting tooth tissue regeneration according to any one of claims 7 to 9, wherein the acellular root canal filling material further contains an anti-CCL11 neutralizing antibody and/or an ALK5 inhibitor.
[Claim 11]
The dental tissue according to any one of claims 7 to 10, wherein said acellular root canal filling material further comprises at least one chemotactic factor selected from the group consisting of G-CSF, bFGF and SDF-1. regeneration promotion kit.
[Claim 12]
The kit for promoting tooth tissue regeneration according to any one of claims 7 to 11, wherein the serine protease is a chymotrypsin-like serine protease.
[Claim 13]
The kit for promoting tooth tissue regeneration according to any one of claims 7 to 12, wherein the chymotrypsin-like serine protease is trypsin.
[Claim 14]
The kit for promoting tooth tissue regeneration according to any one of claims 7 to 13, which is used for regeneration of tooth tissue of middle-aged and elderly individuals.