Application of HIF-2alpha inhibitor in preparation of medicine for treating hypoxia-related stomatitis bone loss diseases

Abstract

The invention provides application of an HIF-2alpha inhibitor in preparation of a medicine for treating hypoxia-related stomatitis bone loss diseases. Experiments provided by the invention show that HIF-2alpha activates the expression of a key gene Camk4 for promoting osteoclast differentiation under the hypoxia condition, and the osteoclast differentiation promoting effect of the Camk4 is exerted; and the HIF-2 alpha inhibitor can be used for remarkably inhibiting osteoclast differentiation. Therefore, the HIF-2 alpha inhibitor can be used for treating the hypoxia-related stomatitis bone loss disease, and is expected to remarkably improve the treatment effect of the stomatitis bone loss disease when being used as an oral cavity-related treatment or prevention medicine.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the preparation of drugs for hypoxia-related oral inflammatory bone loss disease, particularly the application of HIF-2α inhibitors in the preparation of drugs for hypoxia-related oral inflammatory bone loss disease. Background Technology

[0002] Hypoxia-inducible factor (HIF) is considered a key regulatory protein in cellular responses to hypoxia. HIF-2α is expressed in specific organs such as the kidney, lung, and heart. Compared to HIF-1α, it has some overlap in its target genes, but plays diametrically opposed roles in tumorigenesis. Overexpression of HIF-2α is often closely associated with the progression of various malignant tumors, particularly in renal cell carcinoma (RCC), where approximately 90% of clear cell RCC tumors show VHL gene inactivation, leading to HIF-2α accumulation.

[0003] HIF-2α inhibitors primarily work by inhibiting the transcription and expression of HIF-2α, thereby blocking its interaction with downstream target genes and reducing tumor cell proliferation and angiogenesis. HIF-2α inhibitors have been developed as a class of drugs, and their publicly reported uses are mainly concentrated in the field of anti-tumor therapy, such as for the treatment of renal cell carcinoma.

[0004] Periapical periodontitis is an inflammatory response in the periapical tissues caused by microbial infection within the root canal. Its core pathological feature is alveolar bone resorption due to abnormal osteoclast activation. Conventional clinical treatment primarily involves root canal therapy, aiming to remove the source of infection and control inflammation, but it does not intervene in the regulatory pathways of bone destruction. Conventional treatments also fail to consider hypoxia, an important host risk factor.

[0005] Epidemiological studies have shown that individuals exposed to specific environments (such as high altitudes) may face a higher risk of periapical periodontitis; meanwhile, smoking (which can lead to chronic tissue hypoxia) has also been shown to be significantly associated with the occurrence of periapical periodontitis. However, the molecular mechanisms underlying these associations remain unclear, and targeted treatment strategies based on these mechanisms are lacking.

[0006] Therefore, for patients with concurrent systemic hypoxia, existing therapies have limited effectiveness and cannot effectively block the amplifying effect of hypoxia on bone destruction, leading to poor treatment outcomes or recurring symptoms. There is an urgent need in this field to identify the key driving factors and signaling pathways associated with hypoxia and the worsening of periapical periodontitis, thereby providing targeted therapies. Summary of the Invention

[0007] In view of the shortcomings of the existing technology, the purpose of this invention is to provide the application of HIF-2α inhibitors in the preparation of drugs for hypoxia-related oral inflammatory bone loss diseases.

[0008] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides the use of an HIF-2α inhibitor in the preparation of a medicament for the treatment or prevention of hypoxia-related inflammatory bone loss in the oral cavity.

[0009] In this invention, experiments have verified that the "hypoxia-HIF-2α-CAMK4 transcriptional regulatory axis" is the core molecular mechanism that exacerbates hypoxia-related oral inflammatory bone loss disease, providing a new scientific basis for the understanding and treatment of the disease. Prior to this, although studies have shown that the HIF pathway is involved in the regulation of bone metabolism, the specific functions and modes of action of HIF-1α and HIF-2α in osteoclasts are still controversial and unknown, especially in the context of hypoxia-related oral inflammatory bone loss disease, where the specific role of HIF subtypes is completely unknown.

[0010] Meanwhile, this invention confirms that HIF-2α inhibitors, which are already used in the field of oncology, can also be used to treat hypoxia-related oral inflammatory bone loss disease, uncovering their potential value in the treatment of oral diseases and opening up a completely new indication with huge clinical demand for this type of known anticancer drug.

[0011] In this invention, the drug for hypoxia-associated inflammatory bone loss in the oral cavity can be used to prevent or treat the related disease. Hypoxia-associated inflammatory bone loss in the oral cavity refers to a class of inflammatory oral diseases in which the pathological process (especially the absorption and destruction of hard tissues such as alveolar bone) is significantly aggravated under systemic or local hypoxic conditions. Hypoxic conditions refer to the physiological or pathological state in which the body is exposed to an environment with a concentration of oxygen below the normal physiological oxygen concentration (21% O2), such as exposure to high-altitude environments, sleep apnea syndrome, chronic obstructive pulmonary disease (COPD), and chronic or intermittent hypoxemia caused by heavy smoking.

[0012] As a preferred embodiment of the present invention, the HIF-2α inhibitor comprises any one or a combination of at least two of small molecule compounds, peptides, monoclonal antibodies or nucleic acids.

[0013] Preferably, the HIF-2α inhibitor includes any one or a combination of at least two of PT2399, HIF-2α-I2, PT2385, TC-S 7009 or Belzutifan (PT2977).

[0014] In this invention, the HIF-2α inhibitor is not limited to specific small molecule inhibitors, such as PT2399, HIF-2α-I2, PT2385, TC-S 7009, or Belzutifan (PT2977), but also includes all substances capable of inhibiting HIF-2α function or expression, including other small molecule compounds, peptides, monoclonal antibodies, and gene therapy methods, such as small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), and antisense oligonucleotides (ASO).

[0015] Preferably, the hypoxia-related oral inflammatory bone loss disease includes any one of hypoxia-related periapical periodontitis, hypoxia-related periodontitis, or hypoxia-related peri-implantitis.

[0016] The indications for this invention are hypoxia-related oral inflammatory bone loss diseases, not limited to periapical periodontitis, but can be extended to other hypoxia-related oral and maxillofacial inflammatory bone loss diseases with similar pathological mechanisms, such as periodontitis and peri-implantitis.

[0017] In a second aspect, the present invention provides a pharmaceutical composition comprising an HIF-2α inhibitor and a pharmaceutically acceptable carrier.

[0018] The pharmaceutical composition is a drug for treating or preventing hypoxia-related inflammatory bone loss in the oral cavity.

[0019] The pharmaceutical composition described in this invention is intended for a large number of patients with periapical periodontitis, periodontitis, and peri-implantitis who have combined hypoxia risk factors (such as heavy smoking, sleep apnea, high altitude exposure, chronic obstructive pulmonary disease, etc.). It provides the first precise targeted treatment strategy based on pathological mechanisms, which solves the clinical pain point that existing treatments are not effective for such patients.

[0020] As a preferred embodiment of the present invention, the pharmaceutical composition is a drug for oral administration.

[0021] Preferably, the dosage form of the pharmaceutical composition is any one of hydrogel, intracanal medication paste, biodegradable membrane or microspheres.

[0022] Preferably, the pharmaceutical composition is used alone or in combination with other drugs.

[0023] In this invention, HIF-2α inhibitors can also be used in combination with existing conventional treatment drugs, such as calcium hydroxide and antibiotics used in root canals; or anti-inflammatory drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs), to prepare compound preparations or propose combination drug regimens to achieve synergistic effects.

[0024] Thirdly, the present invention also provides Camk4 Application of (calmodulin-dependent protein kinase IV) gene initiation or transcription inhibitors in the preparation of drugs for hypoxia-related oral inflammatory bone loss disease.

[0025] Fourthly, the present invention also provides Camk4 Application of mRNA expression inhibitors in the preparation of drugs for hypoxia-related oral inflammatory bone loss disease.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention, through in vitro and in vivo experiments, demonstrates that the hypoxia-HIF-2α-CAMK4 transcriptional regulatory axis is the core molecular mechanism exacerbating hypoxia-related inflammatory bone loss in the oral cavity, providing a novel scientific basis for understanding and treating the disease. In other words, this invention is the first to confirm that HIF-2α is the core target mediating hypoxia-induced inflammatory bone loss in the oral cavity and verifies the effectiveness of HIF-2α inhibitors in treating this disease. Furthermore, in vivo experimental data show that inhibiting HIF-2α can reverse, rather than merely delay, hypoxia-induced bone destruction. Therefore, HIF-2α inhibitors can be used to treat hypoxia-related inflammatory bone loss in the oral cavity, and their use as adjunctive therapy in root canal treatment has significant potential to improve efficacy and reduce recurrence rates. Attached Figure Description

[0027] Figure 1 The images show the Micro-CT reconstruction and quantitative analysis of the periapical region in mice in the normoxic and hypoxic groups. Image I is the Micro-CT two-dimensional reconstruction of the periapical region in the normoxic group, Image II is the Micro-CT two-dimensional reconstruction of the periapical region in the hypoxic group, Image III is the low-density statistical area map of the periapical region, and Image IV is the low-density volume statistical map of the periapical region.

[0028] Figure 2 Figure 1 shows the staining and quantitative analysis of TRAP-positive multinucleated osteoclasts in the root apical lesion area of ​​mice in the normoxic and hypoxic groups; Figure 2 shows the cell staining results in the normoxic group, Figure 3 shows the cell staining results in the hypoxic group, and Figure 4 shows the statistical diagram of the number of osteoclasts in the root apical lesion area.

[0029] Figure 3 Injecting inhibitors Hif2a Micro-CT three-dimensional reconstruction and quantitative analysis images of periapical periodontitis mice in normoxic and hypoxic groups with gene-expressing viruses or negative viruses; where, Image I is the Micro-CT two-dimensional reconstruction image of the periapical region in the normoxic negative virus group, Image II is the Micro-CT two-dimensional reconstruction image of the periapical region in the hypoxic negative virus group, and Image III is the Micro-CT two-dimensional reconstruction image of the periapical region in the normoxic negative virus group. Hif2a Micro-CT two-dimensional reconstruction of the periapical region of the gene knockdown group, image IV shows hypoxia. Hif2a Micro-CT two-dimensional reconstruction of the periapical region of the gene knockdown group; V-plot is a statistical map of low-density volume in the periapical region.

[0030] Figure 4 Figure I shows the qRT-PCR results of osteoclasts cultured in vitro after hypoxia and / or LPS treatment, representing osteoclast differentiation markers. Nfatc1 The qRT-PCR results, Figure II is shown. Ctsk The qRT-PCR results, Figure III is shown. Trap The qRT-PCR results.

[0031] Figure 5 The images show the osteoclast formation results after in vitro cultured osteoclasts were treated with hypoxia and / or LPS; Figure I represents the normoxic group, Figure II the hypoxic group, Figure III the normoxic group treated with LPS, and Figure IV the hypoxic group treated with LPS.

[0032] Figure 6 The images show the Western Blot results of osteoclasts cultured in vitro after treatment with hypoxia and / or the HIF-2α inhibitor PT2399, representing osteoclast differentiation indices. Figure I is a Western Blot gel image, and Figure II is a statistical graph of NFATC1 and CTSK.

[0033] Figure 7 For HIF-2α and Camk4 Combine with peak diagrams.

[0034] Figure 8 This refers to the process of treating cultured osteoclasts with hypoxia and / or the HIF-2α inhibitor PT2399, and the downstream HIF-2α... Camk4 The image shows the qRT-PCR results. Detailed Implementation

[0035] The technical solution of the present invention will be further illustrated below through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be determined by the claims.

[0036] In the following embodiments, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the art; unless otherwise specified, all experimental methods and techniques used were conventional methods and techniques in the art.

[0037] Example 1 This embodiment demonstrates, using a mouse periapical periodontitis model, that hypoxia exacerbates bone destruction in periapical periodontitis and the core role of HIF-2α.

[0038] (1) Model building In this embodiment, an 8-week-old male C57BL / 6N mouse was used to establish a periapical periodontitis model by exposing the pulp of the maxillary first molar. The model mice were divided into a normoxic group (21% O2) and a chronic hypoxic group (10% O2, placed in an animal hypoxic chamber).

[0039] (2) Phenotypic verification Four weeks later, the results were obtained through Micro-CT reconstruction and quantitative analysis. Figure 1 The images show that Figures I and II are Micro-CT images of the periapical region in the normoxic and hypoxic groups, respectively. Figure III shows the low-density statistical area in the periapical region, and Figure IV compares the volumes in the normoxic and hypoxic groups. It can be seen that the volume of the low-density bone defect area in the periapical region is significantly larger in the hypoxic periapical group compared with that in the normoxic periapical group.

[0040] Meanwhile, histological TRAP staining showed ( Figure 2 Figure I and Figure II show the staining results of the normoxic and hypoxic groups, respectively. The arrows indicate TRAP-positive multinucleated osteoclasts. The number of TRAP-positive multinucleated osteoclasts in the periapical region of the hypoxic group (as shown in Figure III) was significantly increased.

[0041] This result confirms that hypoxia is indeed an important factor that exacerbates pathological bone loss in periapical periodontitis.

[0042] (3) Target validation To further verify the effect of HIF-2α, this embodiment also included local injection of HIF-2α into the periapical region of mice before modeling. Hif2a Adeno-associated virus vector (AAV9-sh-Hif2a) containing gene-specific shRNA locally knocks down HIF-2α expression.

[0043] result( Figure 3 The diagram shows that Figures I and II represent the normoxic and hypoxic negative virus groups, respectively; Figures III and IV represent the normoxic and hypoxic gene knockdown groups, respectively; and Figure V shows the statistical results of periapical low-density volume for each group. Under hypoxic conditions, compared with the group injected with the negative control virus, local knockdown of HIF-2α could completely reverse the aggravated bone destruction caused by hypoxia, and the bone defect volume was restored to the level of the normoxic group.

[0044] This result directly proves that HIF-2α is the core factor driving this pathological process, and that inhibiting HIF-2α is an effective treatment strategy.

[0045] Example 2 This embodiment demonstrates through a cell model that HIF-2α inhibitors can directly inhibit hypoxia-induced osteoclast differentiation.

[0046] (1) Cell model In this embodiment, mouse bone marrow-derived macrophages (BMMs) were isolated and differentiated into osteoclasts under the induction of M-CSF and RANKL.

[0047] The experimental groups include: ① Normoa group; ② Hypoxia group (3% O2); ③ Under normoxic conditions, lipopolysaccharide (LPS) was used (to simulate inflammation), and the group was designated as the normoxic + LPS group; ④ The group treated with lipopolysaccharide under hypoxic conditions was designated as the hypoxia + LPS group.

[0048] (2) Results of osteoclast differentiation 1. Marker genes related to osteoclast differentiation include Nfatc1, Ctsk, Trap The expression levels of relevant genes detected by qRT-PCR results can reflect the degree of osteoclast differentiation.

[0049] Figure 4 The results showed the expression levels of markers promoting osteoclast differentiation, with Figures I, II, and III representing... Nfatc1, Ctsk, Trap The relative expression levels of osteoclast differentiation markers were observed. The results showed that the expression levels of osteoclast differentiation markers were significantly increased in the hypoxia group and the hypoxia + LPS group. 2. Microscopic results of osteoclast formation, such as Figure 5 As shown, Figures I, II, III, and IV correspond to the normoxic group, hypoxic group, normoxic + LPS group, and hypoxic + LPS group, respectively. The microscopic results corresponded to the qRT-PCR results, and the results also showed that hypoxia and LPS together significantly promoted the expression of osteoclast differentiation markers and the formation of multinucleated osteoclasts.

[0050] (3) Drug validation In this embodiment, osteoclast differentiation was detected after adding the HIF-2α specific small molecule inhibitor PT2399 (2 μM) to the hypoxia + LPS group.

[0051] like Figure 6 As shown, Figure I is a Western blotting image of osteoclast differentiation-related proteins NFATC1 and CTSK, while Figures II and III are statistical graphs of the relative expression levels of NFATC1 and CTSK. The excessive osteoclast differentiation induced by both hypoxia and inflammation was significantly inhibited after the addition of the HIF-2α inhibitor PT2399.

[0052] The results of this embodiment demonstrate that small molecule HIF-2α inhibitors can effectively inhibit osteoclast differentiation caused by hypoxia at the cellular level.

[0053] Example 3 This embodiment is used to verify the mechanism of action of small molecule HIF-2α inhibitors.

[0054] (1) Target gene screening In this embodiment, osteoclasts induced under hypoxic and normoxic conditions were subjected to CUT&Tag sequencing to analyze the binding sites of HIF-2α across the entire genome.

[0055] The results are as follows Figure 7 As shown, under hypoxic conditions, HIF-2α plays a crucial role in promoting osteoclast differentiation. Camk4 The promoter region showed a specific and significantly enhanced binding peak.

[0056] (2) Mechanism verification Detected by qRT-PCR experiment Camk4 The relative expression level.

[0057] like Figure 8 The results showed that in in vitro cell models, the HIF-2α inhibitor PT2399 significantly downregulated hypoxia-induced ... Camk4 mRNA expression.

[0058] This result indicates that HIF-2α activates by directly binding to the promoter. Camk4 Gene transcription plays a role in promoting osteoclast differentiation.

[0059] In a specific embodiment of the present invention, the HIF-2α specific small molecule inhibitor used is PT2399; other compounds that are also HIF-2α specific small molecule inhibitors, such as HIF-2α-I2, PT2385, TC-S 7009 or PT2977, also have the same effect of inhibiting hypoxia-induced osteoclast differentiation.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of HIF-2α inhibitors in the preparation of drugs for the prevention or treatment of hypoxia-related inflammatory bone loss in the oral cavity.

2. The application according to claim 1, characterized in that, The HIF-2α inhibitors include any one or a combination of at least two of the following: small molecule compounds, peptides, monoclonal antibodies, or nucleic acids.

3. The application according to claim 1 or 2, characterized in that, The HIF-2α inhibitor is a small molecule compound.

4. The application according to claim 3, characterized in that, The small molecule compound includes any one or a combination of at least two of PT2399, HIF-2α-I2, PT2385, TC-S 7009 or PT2977.

5. The application according to claim 1, characterized in that, The hypoxia-associated oral inflammatory bone loss disease includes any one of hypoxia-associated periapical periodontitis, hypoxia-associated periodontitis, or hypoxia-associated peri-implantitis.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an HIF-2α inhibitor and a pharmaceutically acceptable carrier, and is a medicine for treating or preventing hypoxia-related inflammatory bone loss disease of the oral cavity.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition is a drug for oral administration; The dosage form of the drug composition is any one of hydrogel, root canal sealing paste, biodegradable membrane or microspheres.

8. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition may be used alone or in combination with other drugs.

9. Camk4 Application of gene initiation or transcription inhibitors in the preparation of drugs for hypoxia-related oral inflammatory bone loss disease.

10. Camk4 Application of mRNA expression inhibitors in the preparation of drugs for hypoxia-related oral inflammatory bone loss disease.

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