Application of SSTR2 antagonist in preparation of medicine for preventing and / or treating periodontitis

Abstract

The invention belongs to the field of medicines, and particularly discloses application of a somatostatin receptor 2 (SSTR2) antagonist in preparation of a medicine for preventing and / or treating periodontitis. In-vivo experiments prove that the SSTR2 antagonist can reduce the expression level of periodontitis markers IL-1beta, TNF-alpha and IL-17, effectively reduce the number of osteoclasts, inhibit alveolar bone resorption and relieve periodontal tissue pathological damage, and has an obvious curative effect on periodontitis. Therefore, the invention provides a brand-new drug action target (SSTR2) and a therapeutic drug for treatment of periodontitis, and opens up a brand-new therapeutic approach.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to novel pharmaceutical uses of somatostatin receptor 2 (SSTR2) antagonists, and particularly to the use of SSTR2 antagonists in the preparation of drugs for the prevention and / or treatment of periodontitis. Background Technology

[0002] Periodontitis is a chronic inflammatory disease caused by plaque biofilm, which leads to the destruction of periodontal supporting tissues (gingiva, periodontal ligament, alveolar bone) and is a leading cause of tooth loss in adults. Current standard treatments include mechanical debridement (scaling and scaling), local or systemic antibiotics (such as doxycycline), and surgery. However, these methods have limitations: mechanical treatments are less effective for deep lesions and depend on patient maintenance; antibiotics are prone to resistance and may disrupt the oral microecological balance; and existing methods cannot completely block the persistent inflammatory response and pathological bone resorption.

[0003] Current treatments for periodontitis fail to precisely modulate the key molecular pathways underlying periodontal tissue destruction, particularly in controlling excessive immune inflammatory responses and osteoclast activity, necessitating the development of novel target-based therapeutic strategies. Somatostatin receptor 2 (SSTR2) is one of the main receptors for somatostatin. Known SSTR2 antagonists (e.g., CYN-154806 and its structural analogs) are primarily used in the prior art to study somatostatin-related physiological and pathological processes, or clinically for the diagnosis and treatment of diseases associated with somatostatin receptor overactivity, such as certain types of acromegaly and neuroendocrine tumors. Currently, no published literature or patents indicate or suggest that SSTR2 antagonists can be used for the prevention or treatment of periodontitis. Summary of the Invention

[0004] The present invention aims to overcome the above-mentioned shortcomings of existing periodontitis treatments and provide a novel treatment strategy based on a specific molecular target (SSTR2).

[0005] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides the use of an SSTR2 antagonist in the preparation of a medicament for the prevention and / or treatment of periodontitis.

[0007] Furthermore, the SSTR2 antagonist refers to a compound that can specifically bind to SSTR2 and inhibit its downstream signaling pathway, including but not limited to known peptide or non-peptide SSTR2 antagonists (such as CYN-154806, PRL-2903, etc.), pharmaceutically acceptable salts, esters, prodrugs, hydrates, solvates or isomers of the aforementioned peptide or non-peptide SSTR2 antagonists, as well as other known or newly discovered compounds with SSTR2 antagonistic activity.

[0008] Furthermore, the drug uses an SSTR2 antagonist as the active ingredient alone, or in combination with an existing periodontitis drug (such as antibiotics, nonsteroidal anti-inflammatory drugs, or osteoprotective agents) as the active ingredient.

[0009] Furthermore, the SSTR2 antagonist in the drug has the following functions: inhibiting the expression of key periodontal inflammatory factors IL-1β, TNF-α, and IL-17, thereby alleviating gingival inflammation; reducing the number of osteoclasts in periodontal tissues; and reducing alveolar bone resorption.

[0010] Furthermore, the drug comprises a therapeutically effective amount of the active ingredient and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means that when the carrier substance is appropriately administered to animals or humans, it does not produce adverse, allergic, or other adverse reactions. Pharmaceutically acceptable carriers include, but are not limited to, one or more of diluents, fillers, surfactants, absorption enhancers, disintegrants, wetting agents, and dispersants.

[0011] Furthermore, the dosage form of the drug is suitable for systemic administration or local oral administration. Preferably, it is a dosage form suitable for local oral administration to deliver the drug directly to the periodontal lesion, increasing local drug concentration and reducing systemic side effects. Specific examples of dosage forms suitable for systemic administration or local oral administration are as follows:

[0012] Dosage forms suitable for systemic administration: such as oral tablets, injections (subcutaneous injection), etc.

[0013] Dosage forms suitable for topical oral administration include gels, oral patches, microspheres, mouthwashes, periodontal sustained-release fibers, and injections (oral injections).

[0014] In a second aspect, the present invention provides a pharmaceutical composition for the prevention and / or treatment of periodontitis, the pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable carrier; said active ingredient is an SSTR2 antagonist, or an SSTR2 antagonist and an existing periodontitis treatment drug.

[0015] This invention is the first to discover and confirm the therapeutic effect of SSTR2 antagonists on periodontitis, with the following outstanding advantages and beneficial effects:

[0016] 1. Novel target: It provides a completely new drug target (SSTR2) for the treatment of periodontitis, opening up a new treatment approach.

[0017] 2. Clear therapeutic effect: In vivo experiments have confirmed that SSTR2 antagonists can significantly inhibit the production of key periodontal inflammatory factors (such as IL-1β, TNF-α, IL-17), reduce gingival inflammation, and reduce alveolar bone resorption.

[0018] 3. Potential advantages: Compared with existing antibiotic treatments, targeting host regulatory pathways is less likely to induce bacterial resistance. Local administration can further improve efficacy and safety.

[0019] 4. Clear application prospects: Based on clear experimental evidence, this invention lays a solid foundation for the development of a new generation of periodontitis treatment drugs. Attached Figure Description

[0020] Figure 1 Example 2: Expression of SSTR mRNA in gingival tissue of mice with periodontitis. The left figure shows the mRNA expression levels of SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 genes in gingival tissue of mice 2 weeks after ligation; the right figure shows the mRNA expression level of SSTR2 gene in gingival tissue of mice at different time points after ligation.

[0021] Figure 2 Comparison of Micro-CT three-dimensional reconstruction images of the maxillary molar region of mice in the antagonist treatment group (Perio+SSTR2 antagonist) and the control group (Perio+PBS) in Example 3, as well as the ABC-CEJ distance measurement results.

[0022] Figure 3 HE staining images of the maxillary molar region in mice of the antagonist treatment group and control group in Example 3.

[0023] Figure 4 Example 3: TRAP staining images and quantitative analysis results of osteoclasts in the maxillary molar region of mice in the antagonist treatment group and the control group.

[0024] Figure 5 Example 3: Expression levels of inflammatory factors (IL-1β, TNF-α and IL-17) in the gingival tissue of mice in the antagonist treatment group and the control group. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0026] Example 1: Constructing a mouse model of periodontitis

[0027] Six-week-old male mice were weighed and anesthetized by intraperitoneal injection of 1% sodium pentobarbital at a dose of 100 mg / kg in a clean bench. The maxillary second molars of the mice were ligated with 5-0 silk sutures. The mice were fixed and their upper and lower jaws were opened with an mouth gag. The maxillary molars were clearly visible. The 5-0 silk sutures were gently pressed into the mesial and distal parts of the second molars using microsurgical instruments and then tied on the palatal side for fixation.

[0028] Example 2: Detection of SSTR expression in model mice

[0029] Two weeks after ligation, gingival tissue from the molar region of mice was harvested and washed three times with PBS. The tissue was transferred to a thick-walled tube containing 350 μl of RNA lysis buffer, and stainless steel disruption beads were added. The tissue was disrupted using a high-throughput tissue homogenizer at a rate of 30 Hz for 3 minutes. RNA extraction was performed using the TaKaRa MiniBEST Universal RNA Extraction Kit (9767) according to the manufacturer's instructions. The volume corresponding to 500 ng of RNA was calculated based on the RNA concentration of each sample.

[0030] Reverse transcription was performed according to the Takara (RR036A) reverse transcription kit instructions. Real-time quantitative PCR was performed using an ABI 7500. After the reaction, the melting curve of the product was observed to determine the specificity of the PCR primers by observing whether it showed a tall single peak. The Ct value of each gene was derived, which is the initial cycle number when the fluorescence intensity of the PCR product showed a linear increase. The relative mRNA expression level of each gene was assessed using the ΔΔCt method, i.e., the Ct value of the target gene minus the Ct value of the housekeeping gene GAPDH, as ΔCt, and calculated using Equation 2. -ΔΔCt The expression level of the target gene relative to the housekeeping gene GAPDH was obtained. When comparing the gene expression level of the experimental group with that of the control group, the formula for calculating the gene expression level of the experimental group relative to the control group is: 2 -ΔΔCt =2 -(ΔCt 实验组-ΔCt 对照组) .

[0031] The mRNA expression levels of SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 genes in mouse gingival tissue were detected two weeks after ligation using the method described above. The mRNA expression level of the SSTR2 gene in mouse gingival tissue was also detected at different time points (0, 3, 5, 7, and 14 days) after ligation. The results are as follows: Figure 1 As shown, SSTR2 is specifically highly expressed in mouse gingival tissue, and its expression level increases significantly with the increase of modeling time, indicating that periodontitis is accompanied by an increase in SSTR2 expression level, and SSTR2 may become a target for the treatment of periodontitis.

[0032] Example 3: Animal experiments confirm the therapeutic effect of topical application of SSTR2 antagonists.

[0033] Mice were randomly divided into a Periodo+PBS (silk ligation + local injection of PBS into the gums) group and a Periodo+SSTR2antagonist (silk ligation + local injection of SSTR2 antagonist) group. Both groups underwent silk ligation. Starting on the first day of ligation, the Periodo+SSTR2antagonist group received daily local injections of the SSTR2 antagonist CYN-154806 TFA (purchased from MCE, catalog number HY-P1202A) into the gums at a dose of 20 μg / side for 7 consecutive days. The Periodo+PBS group received an equal volume of PBS. Two weeks after ligation, mice were sacrificed for sampling and analysis. The methods and results are as follows:

[0034] (1) Micro-CT detection

[0035] Mouse maxillae were collected and fixed in 4% paraformaldehyde tissue fixative at room temperature for 24 hours. Mouse maxillae samples were scanned using an Inveon MM Micro-CT scanner. Before use, the instrument underwent tube voltage and rotation center calibration. Samples were then removed from the fixative and placed in the specimen chamber for scanning. Instrument parameters were set as follows: voltage 60 kV, current 220 μA, exposure time 1500 ms, effective pixel size 8.89 μm, 360° rotation, and exposure every 1°. Data were imported into Inveon Research Workshop software. The alveolar bone in the maxillary molar region was selected as the Region of Interest (ROI) for 3D reconstruction, and the distance ABC-CEJ on the palatal side of the molar region was measured. ABC-CEJ is the straight-line distance from the apex of the alveolar bone (ABC) to the cementum-enamel junction (CEJ). An increase in this distance directly reflects alveolar bone destruction and resorption and is one of the core objective indicators for assessing the severity of periodontitis and treatment effectiveness.

[0036] The results are as follows Figure 2 As shown, compared with the Periodo+PBS group, the alveolar bone resorption in the maxillary molar region of mice in the Periodo+SSTR2 antagonist group was significantly inhibited (ABC-CEJ distance decreased).

[0037] (2) Histopathological analysis (HE staining)

[0038] Mouse maxillae were collected and fixed in 4% paraformaldehyde tissue fixative at room temperature for 24 hours, followed by EDTA decalcification for 4 weeks. The sections were then embedded in paraffin and sectioned. After dewaxing and hydration, the paraffin sections were stained with hematoxylin and eosin.

[0039] The results are as follows Figure 3As shown, both groups of mice exhibited pathological changes of periodontitis in their periodontal tissues, including root displacement of the epithelial attachment, formation of deep periodontal pockets, extensive inflammatory cell infiltration in the epithelium and subepithelial connective tissue, collagen fiber degeneration and dissolution, and significant bone resorption. Compared with the Periodo+PBS group, Periodo+SSTR2 antagonist mice showed significantly reduced alveolar bone resorption and inflammatory cell infiltration.

[0040] (3) Osteoclast activity assay (TRAP staining)

[0041] TRAP staining was used to detect osteoclasts. Paraffin sections were dewaxed and hydrated, and then stained using a TRAP staining kit (Sigma). The number of TRAP-positive cells between the first and second molars of mice was measured.

[0042] The results are as follows Figure 4 As shown, the number of TRAP-positive osteoclasts between the first and second molars in the Periodo+SSTR2 antagonist group mice was significantly less than that in the Periodo+PBS group.

[0043] (4) Detection of inflammatory factor levels (ELISA)

[0044] Gingival tissue from the molar region of mice was collected and washed three times with PBS. The gingival tissue was transferred to a 2 ml thick-walled centrifuge tube, and 100 μl of RIPA protein lysis buffer containing protease inhibitors was added. The tissue was minced, and stainless steel disruption beads were added. The tissue was then disrupted using a high-throughput tissue homogenizer. The disrupted tissue lysis buffer was transferred to a new 1.5 ml EP tube and centrifuged at 12,000 RPM for 30 min at 4°C. The supernatant was collected into a new 1.5 ml EP tube. The total protein sample was placed on ice, and the total protein concentration was determined using the Thermo Fisher Pierce™ BCA protein quantification kit. The concentrations of the cytokines IL-1β, TNF-α, and IL-17 were determined using an ELISA kit (Abclonal).

[0045] The results are as follows Figure 5 As shown, the protein expression levels of pro-inflammatory cytokines IL-1β, TNF-α, and IL-17 in the gingival tissue of mice in the Perio+SSTR2 antagonist group were significantly lower than those in the Perio+PBS group.

[0046] The above results indicate that SSTR2 antagonists can reduce the expression levels of periodontal inflammatory markers (IL-1β, TNF-α, IL-17), effectively reduce the number of osteoclasts, inhibit alveolar bone resorption, and alleviate periodontal tissue pathological damage, thus having a significant therapeutic effect on periodontitis.

[0047] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. Any changes made by those skilled in the art after reading the specification of the present invention, as long as they are within the scope of the claims of the present invention, will be protected by patent law.

Claims

1. Use of SSTR2 antagonists in the preparation of a medicament for preventing and / or treating periodontitis.

2. Use according to claim 1, characterized in that, The SSTR2 antagonist is selected from peptide or non-peptide SSTR2 antagonists, pharmaceutically acceptable salts, esters, prodrugs, hydrates, solvates or isomers thereof, or other compounds having SSTR2 antagonistic activity.

3. Use according to claim 2, characterized in that, The SSTR2 antagonist is one or more of CYN-154806, PRL-2903.

4. Use according to claim 1, characterized in that, The medicament uses the SSTR2 antagonist alone as the active ingredient, or uses the SSTR2 antagonist and existing periodontitis drugs as the active ingredient.

5. Use according to claim 4, characterized in that, The medicament has the following functions: inhibiting the expression of key inflammatory factors IL-1β, TNF-α and IL-17 in periodontal tissues, reducing gingival inflammation; reducing the number of osteoclasts in periodontal tissues; and reducing alveolar bone resorption.

6. Use according to claim 4, characterized in that, The existing periodontitis drugs include antibiotics, non-steroidal anti-inflammatory drugs, and bone protectants.

7. Use according to claim 4, characterized in that, The medicament contains a therapeutically effective amount of active ingredients and a pharmaceutically acceptable carrier.

8. Use according to claim 1, characterized in that, The dosage form of the medicament is suitable for systemic administration or oral local administration.

9. Use according to claim 1, characterized in that, The dosage form suitable for oral local administration includes gels, oral patch films, microsphere preparations, mouthwashes, periodontal sustained-release fibers, and injections.

10. A pharmaceutical composition for preventing and / or treating periodontitis, characterized by, The medicament contains active ingredients and a pharmaceutically acceptable carrier; the active ingredients are SSTR2 antagonists, or SSTR2 antagonists and existing periodontitis treatment drugs.

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