Use of a bitter taste receptor agonist composition in the manufacture of a medicament for alleviating and / or treating apical periodontitis

Abstract

The application provides a use of a bitter taste receptor agonist composition in preparation of a medicine for relieving and / or treating periapical periodontitis, and belongs to the technical field of oral medicine, wherein the bitter taste receptor agonist composition comprises benzalkonium, chlorpheniramine and quinine. The application applies the synergistic effect of the three bitter taste receptor agonists to the treatment of periapical periodontitis, can significantly relieve the inflammatory absorption of the periapical periodontal bone, promote the repair and reconstruction of the absorbed periodontal bone, and provides a new thought for the clinical treatment of periapical periodontitis.

Description

Technical Field

[0001] This invention belongs to the field of oral medicine technology, specifically relating to the use of bitter taste receptor agonist compositions in the preparation of medicaments for relieving and / or treating periapical periodontitis. Background Technology

[0002] Periapical periodontitis is a common chronic inflammatory disease of the oral cavity, usually caused by pulp infection due to dental caries, dental trauma, or iatrogenic factors. Bacteria and their metabolic products spread to the periapical tissues through the apical foramen or lateral canals. Its main pathological features are inflammation of the periapical soft tissues and progressive alveolar bone resorption. In severe cases, it can lead to tooth tenderness, loosening, or even loss, significantly impacting the patient's masticatory function and quality of life. The occurrence and development of periapical periodontitis are closely related to the host's immune inflammatory response. Continuous bacterial stimulation can activate immune cells such as macrophages and neutrophils in the periapical tissues, inducing the release of various inflammatory cytokines and chemokines, thereby exacerbating the local inflammatory response and promoting osteoclast differentiation and activation, ultimately leading to the destruction of periapical bone tissue.

[0003] Currently, the main clinical treatment for periapical periodontitis is root canal therapy. This involves a combination of mechanical preparation and chemical irrigation to remove infected tissue and bacterial biofilm from the root canals, followed by thorough filling of the root canal system to block the continuous stimulation of the periapical tissues by pathogenic microorganisms, thereby promoting inflammation resolution and bone repair. However, due to the complex structure of the root canal system, including areas such as lateral canals, isthmuses, and dentinal tubules that are difficult to clean thoroughly, conventional root canal therapy alone often fails to completely remove residual microorganisms and inflammatory irritants. In some cases, persistent inflammation or even recurrence may occur after treatment, leading to a recurrence of chronic periapical lesions. To improve treatment efficacy, topical medications are often used in conjunction with root canal therapy in clinical practice to enhance the control of periapical inflammation and infection. Currently, commonly used adjunctive treatments mainly include antibiotics and anti-inflammatory drugs, whose mechanisms of action are primarily focused on inhibiting local bacterial growth or reducing the inflammatory response. However, these drugs have limited ability to directly intervene in the immune regulation imbalance and bone metabolism abnormalities involved in periapical periodontitis. Furthermore, long-term or repeated use of antibacterial drugs may lead to problems such as microbial resistance and local flora imbalance. Therefore, they still have certain limitations in inhibiting periapical bone resorption and promoting tissue repair.

[0004] Recent studies have shown that certain small molecule compounds can indirectly affect inflammation-related bone destruction by regulating the immune status of macrophages. Further research indicates that bitter taste receptors are not only expressed in taste cells of traditional taste organs such as the tongue, but are also widely distributed in various non-taste tissues and immune cells. Various bitter taste receptor agonists can regulate the inflammatory responses of immune cells such as macrophages and dendritic cells through the bitter taste receptor signaling pathway, affecting the production of inflammatory factors and the phenotypic transformation of immune cells, thereby playing an immunomodulatory role in various inflammatory diseases. However, current research largely focuses on the biological effects of single bitter taste receptor agonists or single signaling pathways, and systematic studies on the potential complementary or synergistic effects between different types of bitter taste receptor agonists are still lacking.

[0005] Although studies have revealed the use of bitter taste receptor agonists in the treatment of periodontitis, periodontitis and periapical periodontitis differ fundamentally in their etiology, pathological characteristics, and pathological microenvironment. Therefore, the applicability of this existing technology to periapical periodontitis is significantly limited: First, regarding etiology and infection routes, periodontitis is primarily caused by periodontal pathogens in the gingival plaque biofilm, with infection originating in the periodontal pocket and spreading along the periodontal ligament towards the root apex. Periapical periodontitis, on the other hand, originates from anaerobic bacterial infection within the pulp cavity, spreading retrogradely through the apical foramen or lateral canals to the periapical tissues, with the root canal system being the primary infection pathway. Second, regarding the pathological microenvironment, periodontitis is characterized by an imbalance of anaerobic bacteria within the periodontal pocket, with inflammatory responses mainly occurring in the gingiva and periodontal ligament region, and bone resorption often presenting as horizontal. In contrast, the periapical lesions of periapical periodontitis are surrounded by bone tissue and dense connective tissue, forming a relatively closed inflammatory microenvironment characterized by macrophage-mediated chronic inflammation, with bone resorption presenting as localized vertical resorption, often accompanied by a need for bone defect repair. Secondly, regarding the route of administration, periodontitis can be treated locally through the periodontal pocket, allowing the drug to directly contact the diseased tissue; however, medication for periapical periodontitis needs to penetrate through the root canal to reach the periapical tissue, placing specific requirements on the physicochemical properties of the drug and the administration method, making it difficult to directly apply with existing technologies. Furthermore, in terms of treatment goals, periodontitis focuses on controlling plaque and preventing periodontal attachment loss; while the core goal of periapical periodontitis is to clear residual infection within the root canal, suppress the inflammatory response, and promote bone regeneration and repair, requiring drugs with higher bone metabolism regulation capabilities. Therefore, current technologies do not provide drug combination schemes suitable for periapical periodontitis that can improve periapical bone metabolism through synergistic regulation of immune inflammatory responses and osteoclast activity, nor have they revealed the synergistic mechanism of different types of bitter taste receptor agonists under this specific pathological condition. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides the use of a bitter taste receptor agonist composition in the preparation of medicaments for relieving and / or treating periapical periodontitis, addressing issues such as persistent inflammatory response, abnormal macrophage activation, and difficulty in effectively controlling periapical bone resorption in periapical periodontitis lesions.

[0007] The present invention provides the use of bitter taste receptor agonist compositions in the preparation of medicaments for relieving and / or treating periapical periodontitis, said bitter taste receptor agonist compositions comprising dinatamine benzoate, chlorpheniramine and quinine, or pharmaceutically acceptable salts thereof.

[0008] Furthermore, the molar ratio of the benzodiazepine, chlorpheniramine, and quinine is 500:5:1.

[0009] At this preferred concentration, the three drugs exhibit good synergistic effects and biocompatibility. For ease of description, the bitter taste receptor agonist composition consisting of denatonium benzoate (Den or D), chlorpheniramine (Chl or C), and quinine (Qun or Q) is referred to as DCQ in this invention.

[0010] This invention further provides different administration methods for DCQ.

[0011] Firstly, DCQ can be prepared as a solution, wherein the DCQ is dissolved in a pharmaceutically acceptable solvent or buffer solution for topical administration or related in vitro experimental studies. Preferably, the solvent or buffer solution is selected from at least one of HBSS, PBS, HEPES buffer, physiological saline, cell culture medium, or water for injection.

[0012] Furthermore, the effective concentration range of each component in the bitter taste receptor agonist composition is as follows: benzyl dinatamine 0.5-5 mM; chlorpheniramine 5-50 μM; quinine 1-10 μM; Preferably, the final concentrations of benzodiazepine, chlorpheniramine, and quinine are 1.25 mM, 12.5 μM, and 2.5 μM, respectively.

[0013] Secondly, DCQ can be loaded onto a hydrogel carrier to form a DCQ-loaded hydrogel formulation for local in vivo administration. Preferably, the hydrogel carrier comprises tetrazine-modified alginate (Tz-VLVG) and norbornene-modified alginate (Nb-VLVG), which are cross-linked through a bioorthogonal click chemistry-mediated reaction to form a DCQ-loaded tetrazine-norbornene alginate hydrogel (abbreviated as DCQ@Tz-Nb ​​drug-loaded hydrogel). This hydrogel has good injectability and local retention capacity, which is beneficial for the local delivery of DCQ to the periapical lesion area. Further, the concentration of norbornene-modified alginate is 5-25 g / L, and the concentration of tetrazine-modified alginate is 5-25 g / L; preferably, the concentration of norbornene-modified alginate is 15 g / L, and the concentration of tetrazine-modified alginate is 15 g / L. Furthermore, the drug-loaded hydrogel formulation of the bitter taste receptor agonist composition is an injectable hydrogel for local delivery of DCQ to periapical tissues.

[0014] The preparation method of the aforementioned bitter taste receptor agonist composition includes the following steps: dissolving benzodiazepines, chlorpheniramine and quinine in a pharmaceutically acceptable solvent or buffer solution to obtain a solution formulation of the bitter taste receptor agonist composition; Alternatively, the preparation method of the bitter taste receptor agonist composition includes the following steps: dissolving benzodiazepine, chlorpheniramine, and quinine in a pharmaceutically acceptable buffer solution to obtain a bitter taste receptor agonist composition solution; mixing the bitter taste receptor agonist composition solution with a tetrazine-modified alginate solution; and then adding a norbornene-modified alginate solution to allow the tetrazine groups and norbornene groups to undergo a bioorthogonal click chemistry-mediated crosslinking reaction to obtain a drug-loaded hydrogel formulation of the bitter taste receptor agonist composition.

[0015] Furthermore, the volume ratio of the bitter taste receptor agonist composition solution, the tetrazine-modified alginate solution, and the norbornene-modified alginate solution is 4:3:3.

[0016] Studies have found that the DCQ described in this invention can effectively regulate the inflammatory microenvironment associated with periapical periodontitis, reduce pro-inflammatory activation of macrophages, regulate the M1 / M2 macrophage phenotype ratio, and inhibit periapical periodontitis-related inflammatory bone resorption. Therefore, this invention also provides the use of DCQ and its pharmaceutical compositions in the preparation of drugs for relieving and / or treating periapical periodontitis, and in the preparation of drugs for relieving periapical periodontitis-related inflammatory responses, inhibiting periapical bone resorption, or promoting the repair of periapical lesions.

[0017] The present invention has achieved the following beneficial effects: (1) The present invention obtains a bitter receptor agonist composition for the prevention and treatment of periapical periodontitis by rationally combining benzyl dinatamine, chlorpheniramine and quinine. (2) Compared with single bitter taste receptor agonists, the DCQ described in this invention has a better synergistic effect in regulating macrophage inflammatory response; (3) Under the preferred concentration conditions, namely 1.25 mmol / L of benzyldenafil, 12.5 μmol / L of chlorpheniramine, and 2.5 μmol / L of quinine, the DCQ can reduce the expression of pro-inflammatory factors in macrophages and improve the inflammatory phenotype of macrophages; (4) The DCQ described in this invention can improve the local inflammatory microenvironment of periapical lesions, thereby inhibiting periapical inflammatory bone resorption; (5) The DCQ described in this invention can be prepared as a solution or a DCQ@Tz-Nb ​​drug-loaded hydrogel, which is suitable for both topical and in vivo local administration. (6) The DCQ@Tz-Nb ​​drug-loaded hydrogel of the present invention has good injectability and local retention ability, which is conducive to achieving precise delivery and continuous effect in the periapical lesion area; (7) The bitter taste receptor agonist composition and its drug-loaded hydrogel preparation described in this invention have good application prospects in the relief and / or treatment of periapical periodontitis.

[0018] Obviously, based on the above description of the present invention, and according to the knowledge and conventional techniques of those skilled in the art, various modifications, substitutions, or alterations can be made to the present invention without departing from its basic technical concept. All technical solutions implemented based on the above description of the present invention fall within the protection scope of the present invention.

[0019] The following embodiments further illustrate specific implementations of the present invention, but should not be construed as limiting the scope of protection of the present invention to the following embodiments. All technical solutions implemented based on the above description of the present invention should fall within the protection scope of the present invention. Attached Figure Description

[0020] Figure 1 Experimental results on the synergistic effect of DCQ on macrophage calcium signaling; (A) Activation effects of benzodiazepine (D), chlorpheniramine (C), and quinine (Q) on different bitter taste receptor subtypes; (B) Activation spectra of D, C, and Q on bitter taste receptor subtypes; (C) Fluorescence maps of changes in calcium signaling in macrophages after treatment with D, C, and Q alone and after combined treatment with DCQ; (D) Representative dynamic curves of calcium ion response induced by different treatments in macrophages; (E) Dose-dependent analysis of calcium ion response induced by DCQ in macrophages; (F) Bliss synergistic analysis of calcium ion response induced by combined application of DCQ.

[0021] Figure 2Experimental results on the regulation of macrophage pro-inflammatory (M1) / anti-inflammatory (M2) phenotypes and their synergistic effects by DCQ; (A) DCQ inhibits LPS-induced expression of macrophage pro-inflammatory factors TNF, IL-1β and IL-6; (B) Bliss synergistic analysis of DCQ inhibition of pro-inflammatory factor expression; (C) DCQ promotes the expression of macrophage anti-inflammatory / repair phenotype markers Arg-1, Mrc1 and Ym1; (D) Bliss synergistic analysis of DCQ promotion of anti-inflammatory / repair phenotype marker expression.

[0022] Figure 3 To investigate the effect of intracanal local delivery of DCQ@Tz-Nb ​​hydrogel on inhibiting periapical periodontitis-related bone resorption in mice; (A) Schematic diagram of the establishment of a mouse model of periapical periodontitis and local drug administration process; (B) Observation of the gel-forming properties of DCQ@Tz-Nb ​​hydrogel; (C) Observation of the injectability and gel-forming state of DCQ@Tz-Nb ​​hydrogel; (D) Micro-CT analysis showing that DCQ@Tz-Nb ​​hydrogel is more effective than single bitter agent-loaded hydrogel in alleviating periapical periodontitis bone resorption. Detailed Implementation

[0023] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0024] Example 1: Preparation of a bitter taste receptor agonist composition (DCQ) solution Denatum benzylamine, chlorpheniramine, and quinine were weighed in a molar ratio of 500:5:1 to prepare an 80-fold stock solution of DCQ compound bittering agent. Taking the preparation of a 500 mL stock solution as an example, the concentrations of denatum benzylamine, chlorpheniramine, and quinine in the stock solution were 100 mM, 1 mM, and 0.2 mM, respectively. 22.3 g of denatum benzylamine (MW 446.58 g / mol, Sigma), 195 mg of chlorpheniramine (MW 390.86 g / mol, Sigma), and 39.7 mg of quinine (MW 396.91 g / mol, Sigma) were weighed out, and the three components were added to a certain volume of phosphate buffer solution or Hanks' balanced salt solution (HBSS). The solution was then ultrasonically dispersed at room temperature to ensure complete dissolution and uniform dispersion of each component. The volume was then adjusted to 500 mL to obtain a clear, transparent, and stable DCQ stock solution. When used, the final concentrations of benzodiazepine, chlorpheniramine, and quinine in the system are 1.25 mM, 12.5 μM, and 2.5 μM, respectively, to obtain the DCQ composite bittering agent.

[0025] Example 2: Preparation of DCQ@Tz-Nb ​​hydrogel Ultra-low viscosity sodium alginate VLVG (Provona UP VLVG, MW 30000-35000 g / mol, NovaMatrix) was dissolved in 0.1 M MES / 0.3 M NaCl buffer to prepare a 1% (w / v) solution, and the pH was adjusted to 6.5. Under stirring at 4°C, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, MW 191.70 g / mol, Sigma) and N-hydroxysuccinimide (NHS, MW 115.90 g / mol, Sigma) were added sequentially to activate the carboxyl groups. Then, 5-norbornene-2-methylamine (Nb, MW 123.19 g / mol, TCI America) or (4-(6-methyl-1,2,4,5-tetraazin-3-yl)phenyl)methylamine hydrochloride (Tz, MW 237.69 g / mol, KareBay) were added dropwise. (Biochem), reacted overnight at 4℃, and after centrifugation, filtration through a 0.22 μm filter membrane, dialysis purification, and lyophilization, Nb-VLVG and Tz-VLVG powders were obtained for later use.

[0026] Weigh out Tz-VLVG powder and Nb-VLVG powder, and dissolve them separately in HBSS to prepare Tz-VLVG solutions and Nb-VLVG solutions with a concentration of 50 g / L. Shake at high speed (2000 rpm, 30 minutes) at room temperature to ensure complete dissolution. Separately, take 80 times the amount of DCQ stock solution (Den 100 mM, Chl 1 mM, Qun 200 μM), dilute with HBSS to prepare DCQ working solution, so that the concentrations of Den, Chl, and Qun are 3.125 mM, 31.25 μM, and 6.25 μM, respectively.

[0027] Before administration, DCQ working solution, Tz-VLVG solution, and Nb-VLVG solution were mixed in a volume ratio of 4:3:3. Taking the preparation of 1 mL DCQ@Tz-Nb ​​hydrogel as an example, 400 μL of DCQ working solution was mixed with 300 μL of Tz-VLVG solution, followed by 300 μL of Nb-VLVG solution. This allowed the tetrazine groups to undergo a bioorthogonal click reaction with the norbornene groups, gradually forming the DCQ@Tz-Nb ​​hydrogel. In the resulting hydrogel system, the final concentrations of Den, Chl, and Qun were 1.25 mM, 12.5 μM, and 2.5 μM, respectively, and the final concentrations of Tz-VLVG and Nb-VLVG were both 15 g / L. For local injection, the system could be placed on ice, and the injection should be completed within 10 minutes after mixing.

[0028] The following is a comparison example.

[0029] Preparation of control sample for DCQ solution (Example 1) Phosphate buffer solution or HBSS without DCQ was used as a control solution, and the remaining treatment conditions were the same as those for DCQ solution.

[0030] Preparation of control samples for DCQ@Tz-Nb ​​hydrogel (Example 2) The control hydrogel was prepared according to the method of Example 2, except that the 80-fold DCQ stock solution was replaced with an equal volume of HBSS, Den stock solution, Chl stock solution or Qun stock solution, and the other treatment conditions were the same as those for DCQ@Tz-Nb ​​hydrogel, resulting in blank Tz-Nb ​​hydrogel, Den@Tz-Nb ​​hydrogel, Chl@Tz-Nb ​​hydrogel and Qun@Tz-Nb ​​hydrogel, respectively.

[0031] The following experimental examples demonstrate the beneficial effects of the present invention.

[0032] Experiment 1: DCQ synergistically induces macrophages to produce a stronger calcium ion response. 1. Experimental Methods (1) Culture and passage of HEK293 cells HEK293 cells were cultured under standard conditions. When the cells reached 80-90% confluence, they were passaged using trypsin digestion.

[0033] (2) Heterologous expression of bitter taste receptors and signal-coupled proteins HEK293 cells were seeded in 96-well plates with a black transparent bottom coated with polylysine, achieving a cell confluence of 60-80% at transfection. Mouse bitter taste receptor expression plasmids (…) were then applied to the cells. Tas2r102, 105, 108, 109, 115, 118, 119, 126, 130、131、135、137、140、144 Gα16-gust44 was co-transfected into cells at a predetermined ratio, and an empty vector control group was set up. Transfection was performed using Lipofectamine 2000 transfection reagent, and cells were cultured for subsequent functional assays after transfection.

[0034] (3) Detection of calcium ion response of bitter taste receptors using the FlexStation system After transfection, HEK293 cells were loaded with the Fluo-4 calcium ion fluorescent probe. Benzyldenafil, chlorpheniramine, or quinine were added to the detection wells, and intracellular calcium ion fluorescence changes were continuously monitored. Empty vector transfection group and solvent control group were used as negative controls to confirm the specific response of bitter taste receptors.

[0035] (4) Perfusion-type calcium ion imaging Mouse bone marrow-derived macrophages (BMDMs) were seeded into polylysine-pretreated perfusion imaging chambers. After cell adhesion, Fluo-4 fluorescent probes were loaded. The cells were sequentially stimulated with benzodiazepines, chlorpheniramine, quinine, a combination of these three drugs (DCQ), and ATP via a multi-channel perfusion system. Real-time monitoring of calcium ion fluorescence changes in the same cells under different stimulation conditions was recorded.

[0036] (5) Calcium ion fluorescent probe loading and high-throughput imaging detection Mouse BMDMs were seeded in 96-well cell culture plates with a black transparent bottom. Fluo-4 AM was used as a fluorescent probe for calcium ions, and the cells were incubated in the dark. After staining, real-time imaging was performed under an inverted fluorescence microscope, and intracellular calcium ion fluorescence signals were continuously acquired by adding different stimuli or corresponding solvents. The stimuli included benzyldiamine, chlorpheniramine, quinine, and other candidate bitter substances; ATP was used as a positive control, and HBSS and DMSO were used as blank controls.

[0037] (6) Analysis of the concentration-reaction relationship of bitter substances Following the calcium ion imaging method described above, mouse BMDMs were stimulated with different concentrations of benzodiazepines, chlorpheniramine, and quinine, and changes in calcium ion fluorescence peaks were recorded. Dose-response curves were then plotted to obtain the EC50 values ​​of benzodiazepines, chlorpheniramine, and quinine. 20 EC 50 and EC 80 parameter.

[0038] (7) Synergistic effect analysis of DCQ combined stimulation Based on dose-response results of individual bitter substances, the synergistic effect of benzodiazepines, chlorpheniramine, and quinine on calcium ion signaling under combined DCQ stimulation was evaluated using a Bliss independence model. This was achieved by comparing different concentrations (0, EC50, ...) of different bitter substances. 20 EC 50 and EC 80 The measured and theoretical expected effects of combined stimuli were compared to determine the synergistic, additive, or antagonistic effects of three bitter substances at different concentration combinations.

[0039] 2. Experimental Results like Figure 1 A, Den, Chl, and Qun can all activate specific bitter taste receptors in mice and induce an increase in calcium ions, among which Tas2r105 and Tas2r108 are receptors that are activated by all three. Figure 1 B. The receptor activation profiles of the three bitter substances partially overlap, suggesting that they may mediate downstream signaling through common and differential bitter receptors. For example... Figure 1As shown in Figure C, in mouse BMDMs, individual stimulation with Den, Chl, and Qun can induce calcium ion responses, while the combined stimulation of DCQ produces a stronger calcium ion response. Figure 1 As shown in Figure D, perfusion-based calcium ion imaging further confirmed that the same cell responded to Den, Chl, Qun, and DCQ, with DCQ inducing the most significant calcium ion peak. Figure 1 As shown in E, Den, Chl, and Qun all induce a concentration-dependent increase in calcium ions in BMDMs. Figure 1 As shown in F, Bliss synergistic analysis revealed that the DCQ triple combination exhibited positive ΔBliss values ​​across multiple concentration combinations, with EC... 20 The synergistic effect of the horizontal combination was the most obvious, indicating that DCQ can synergistically enhance calcium ion signaling in macrophages.

[0040] Experiment Example 2: Co-regulatory effect of DCQ on macrophage M1 / M2 phenotypic switching 1. Experimental Methods C57BL / 6 mouse BMDMs cells were seeded in 12-well plates and treated with lipopolysaccharide (LPS) at a final concentration of 100 ng / mL for 24 hours. After treatment, the culture medium was discarded and the cells were washed with PBS. Then, benzodiazepines, chlorpheniramine, quinine, or a combination of DCQ were added to the cells for treatment. After treatment, total RNA was extracted from each group of cells, and cDNA was obtained by reverse transcription. qPCR was used to detect M1-related genes. Tnf , Il1b and Il6 and M2-related genes Arg1 , Mrc1 and Ym1 mRNA expression levels. Based on the gene expression changes in the single-component and combined treatment groups, the synergistic effect of DCQ on the regulation of the above gene expression was evaluated using the ΔBliss independence model.

[0041] 2. Experimental Results like Figure 2 As shown in Figure A, compared with single-component treatments, the DCQ combination treatment significantly downregulated M1-related genes in LPS-induced mouse BMDMs. Tnf , Il1b and Il6 The expression level. The results of the ΔBliss synergistic effect analysis are as follows: Figure 2 As shown in B, DCQ suppresses Tnf and Il6 All expression levels showed positive ΔBliss values, and the results differed statistically from the calculated predicted effects (dashed line). This indicates that DCQ has a synergistic effect in inhibiting M1-type inflammatory genes.

[0042] like Figure 2 As shown in C, DCQ combination treatment can upregulate M2-related genes. Arg1 , Mrc1 and Ym1 The expression level was significantly higher than that of the individual component treatment groups. Further ΔBliss analysis results are as follows: Figure 2 As shown in D, DCQ promotes Arg1 , Mrc1 and Ym1 Both showed a clear positive synergistic effect in terms of expression.

[0043] The above results indicate that the DCQ combination can synergistically inhibit the M1 type inflammatory response in mouse BMDMs and promote their conversion to the M2 type phenotype.

[0044] Experimental Example 3: Synergistic therapeutic effect of DCQ@Tz-Nb ​​hydrogel on periapical periodontitis bone resorption 1. Experimental Methods (1) Animal grouping and establishment of periapical periodontitis model C57BL / 6 mice were used as experimental animals, with the left mandibular first molar selected as the experimental tooth site for each mouse. Mice were randomly divided into four groups: blank Tz-Nb ​​hydrogel group, Den@Tz-Nb ​​hydrogel group, Chl@Tz-Nb ​​hydrogel group, Qun@Tz-Nb ​​hydrogel group, and DCQ@Tz-Nb ​​hydrogel group. After anesthesia, the left mandibular first molar underwent pulpotomy to expose the root canal system to the oral environment, and this process was continued for 3 weeks to establish a periapical periodontitis model. After model establishment, the experimental teeth underwent root canal preparation, during which the root canals were irrigated with sodium hypochlorite solution to remove infected material and debris. Subsequently, blank Tz-Nb ​​hydrogel, Den@Tz-Nb ​​hydrogel, Chl@Tz-Nb ​​hydrogel, Qun@Tz-Nb ​​hydrogel, or DCQ@Tz-Nb ​​hydrogel were placed into the root canals according to the group, and the pulpotomy site was filled with resin. After administering the medication, continue feeding and observation for 3 weeks.

[0045] (2) Sample collection and Micro-CT detection Mice were sacrificed after the observation period, and mandibular bone specimens were collected. Micro-CT scans and three-dimensional reconstructions were performed on the mandibles, and three-dimensional quantitative analysis was conducted on the periapical bone resorption area of ​​the distal root of the left mandibular first molar. The volume of the bone resorption area and other indicators were calculated to evaluate the effects of different hydrogel treatments on periapical bone resorption.

[0046] 2. Experimental Results like Figure 3As shown in Figure A, this experiment used a mouse periapical periodontitis model to evaluate the therapeutic effects of different hydrogels. Periapical periodontitis was induced in the left mandibular first molar of mice after pulpotomy. Three weeks later, root canal preparation was performed, and blank Tz-Nb ​​hydrogel, Den@Tz-Nb ​​hydrogel, Chl@Tz-Nb ​​hydrogel, Qun@Tz-Nb ​​hydrogel, or DCQ@Tz-Nb ​​hydrogel were sealed in the root canal. After another three weeks of observation, samples were taken for analysis.

[0047] like Figure 3 As shown in Figure B, the Tz-Nb ​​hydrogel gradually gels after mixing, forming a stable semi-solid gel, indicating that this material has good in-situ gelation ability. Figure 3 As shown in Figure C, the DCQ@Tz-Nb ​​hydrogel can be smoothly ejected with a syringe and forms a stable gel morphology in vitro, suggesting that it has good injectability and potential for clinical local drug delivery.

[0048] like Figure 3 As shown in Figure D, Micro-CT sagittal and coronal images revealed a more pronounced low-density bone resorption area around the periapical region in the blank Tz-Nb ​​hydrogel group. The bone resorption in the Den@Tz-Nb, Chl@Tz-Nb, and Qun@Tz-Nb ​​single-bitter-agent hydrogel groups was improved compared to the blank group, but significant bone defects still remained. In contrast, the DCQ@Tz-Nb ​​hydrogel group exhibited the smallest periapical bone resorption area and relatively intact periapical bone structure, suggesting that the DCQ combined loaded hydrogel has a superior effect in alleviating periapical periodontitis and bone resorption compared to single-bitter-agent hydrogels.

[0049] In summary, the ΔBliss synergistic analysis results in in vitro calcium ion imaging (Experiment 1) and macrophage phenotype regulation (Experiment 2) are consistent with the results of periapical periodontitis bone resorption treatment (Experiment 3). This clearly demonstrates that the DCQ triple combination consisting of dinatamine benzoate, chlorpheniramine, and quinine exhibits significant synergistic effects in activating macrophage bitter receptor signaling, synergistically inhibiting M1-type inflammatory polarization, promoting M2-type tissue repair phenotype, and ultimately alleviating periapical periodontitis bone resorption. Its overall therapeutic effect is significantly better than the use of any single component alone.

Claims

1. The use of a bitter taste receptor agonist composition in the preparation of a medicament for relieving and / or treating periapical periodontitis, characterized in that: The bitter taste receptor agonist composition includes benzodiazepines, chlorpheniramine, and quinine, or pharmaceutically acceptable salts thereof.

2. The use according to claim 1, characterized in that: The molar ratio of benzyldiamine, chlorpheniramine and quinine is 500:5:

1.

3. The use according to claim 1 or 2, characterized in that: The bitter taste receptor agonist composition is a solution of benzodiazepines, chlorpheniramine, and quinine dissolved in a pharmaceutically acceptable solvent or buffer solution.

4. The use according to claim 3, characterized in that: The effective concentration range of each component in the bitter taste receptor agonist composition is as follows: benzyl dinatamine 0.5-5 mM; chlorpheniramine 5-50 μM; quinine 1-10 μM.

5. The use according to claim 4, characterized in that: The final concentrations of each component in the bitter taste receptor agonist composition are as follows: benzodiazepine 1.25 mM; chlorpheniramine 12.5 μM; quinine 2.5 μM.

6. The use according to claim 1, characterized in that: The bitter taste receptor agonist composition is a drug-loaded hydrogel formulation in which benzodiazepine, chlorpheniramine, and quinine are loaded onto a hydrogel carrier.

7. The use according to claim 6, characterized in that: The hydrogel carrier comprises norbornene-modified alginate and tetraazine-modified alginate.

8. The use according to claim 7, characterized in that: The concentration of norbornene-modified alginate is 5-25 g / L, and the concentration of tetraazine-modified alginate is 5-25 g / L. Preferably, the concentration of norbornene-modified alginate is 15 g / L, and the concentration of tetrazine-modified alginate is 15 g / L.

9. The use according to any one of claims 1 to 5, characterized in that: The method for preparing the bitter taste receptor agonist composition includes the following steps: dissolving benzodiazepines, chlorpheniramine and quinine in a pharmaceutically acceptable solvent or buffer solution to obtain a solution formulation of the bitter taste receptor agonist composition.

10. The use according to any one of claims 6 to 8, characterized in that: The preparation method of the bitter taste receptor agonist composition includes the following steps: dissolving benzodiazepine, chlorpheniramine, and quinine in a pharmaceutically acceptable buffer solution to obtain a bitter taste receptor agonist composition solution; mixing the bitter taste receptor agonist composition solution with a tetrazine-modified alginate solution; and then adding a norbornene-modified alginate solution to allow the tetrazine groups and norbornene groups to undergo a bioorthogonal click chemistry-mediated crosslinking reaction to obtain a drug-loaded hydrogel formulation of the bitter taste receptor agonist composition.

Images