Application of NTSR1 inhibitor in treatment of chronic nasosinusitis

Abstract

The invention relates to application of an NTSR1 inhibitor SR48692 in treatment of chronic sinusitis (CRS), in particular to application of the NTSR1 inhibitor SR48692 in treatment of chronic sinusitis (CRSwNP) accompanied with nasal polyp. Chronic sinusitis is a long-term inflammatory disease and is divided into a nasal polyp type and a nasal polyp-free type. By inhibiting a Wnt3a / beta-catenin signal channel, the SR48692 reduces abnormal deposition of an extracellular matrix and inhibits epithelial-mesenchymal transition (EMT), so that mucous membrane tissue remodeling is improved. Compared with a traditional treatment method, the SR48692 can reduce the recurrence rate of CRSwNP, provides a new treatment target for CRSwNP patients difficult to treat, and achieves a more lasting treatment effect.

Description

Technical Field

[0001] The present application relates to a new use of neurotensin receptor 1 (NTSR1) inhibitors; specifically, to the use of NTSR1 inhibitors in the preparation of drugs for treating or preventing chronic sinusitis and nasal polyps. Background Art

[0002] Chronic rhinosinusitis (CRS) refers to chronic inflammation of the nasal cavity and sinus mucosa, with a course of more than 12 weeks. CRS is a heterogeneous disease. EPOS2012 divided CRS into two types according to the presence or absence of nasal polyps: chronic rhinosinusitis with nasal polyps (CRSwNP) and chronic rhinosinusitis without nasal polyps (CRSsNP). CRSwNP is the focus of clinical research because of its more severe symptoms and higher recurrence rate after surgery. Although the pathogenesis of CRSwNP has not yet been elucidated, it has been recognized that CRS is not a simple disease, but a heterogeneous disease with multiple manifestations under the combined action of multiple factors.

[0003] Neurotensin (NTS) is a brain-gut peptide widely present in the central nervous system (CNS) and peripheral nervous system (such as the gastrointestinal tract). It is unevenly distributed in the nerve cells, fibers and terminals of brain tissue, and can act on multiple organs endocrinely and paracrinely. This peptide was first isolated from the bovine hypothalamus by American physiologists Carraway and Leeman in 1973. Since this peptide was first discovered in the brain and is closely related to neurons, and can cause vasodilation and lower blood pressure after local administration to the epidermal blood vessels of pre-anesthetized rats, it is named neurotensin, also known as neurotensin. NTS is a neurotransmitter and neuromodulator of the central nervous system, and is also a local hormone of peripheral tissues (mainly the gastrointestinal tract). Recently, a literature found that researchers revealed for the first time through high-throughput sequencing that NTS is highly expressed in tissue samples of eosinophilic chronic sinusitis with nasal polyps. So, why is NTS expressed at a high abundance in eosinophilic chronic sinusitis, and does it play an important regulatory role in the pathogenesis of CRSwNP?

[0004] There are many studies on the pathogenesis of chronic sinusitis, but it is still not fully elucidated. In recent years, the research focus has been on tissue remodeling. Tissue remodeling will change the tissue structure of the target organ, causing irreversible damage, and bringing difficulties to the treatment of sinusitis. The key to CRS tissue remodeling lies in the abnormal deposition of submucosal matrix. The extracellular matrix (ECM) is mainly degraded by matrix metalloproteinases and synthesized by myofibroblasts. When degradation and synthesis are disordered, ECM can be deposited excessively under the mucosa, causing changes in tissue structure. Epithelial-mesenchymal transition (EMT) refers to a biological process in which polarized epithelial cells undergo a series of biochemical changes to acquire the phenotype of mesenchymal cells. This process is a benign process of damage repair and tissue remodeling, but excessiveness leads to pathological consequences. Studies have reported that nasal mucosal EMT plays an important role in mucosal remodeling in chronic sinusitis and is involved in the occurrence and development of chronic rhinosinusitis.

[0005] In order to further study the mechanism of NTSR1 signaling pathway regulating the occurrence of EMT in chronic sinusitis, this application first revealed that the Wnt pathway is in an activated state in chronic rhinosinusitis with nasal polyps, and found that there is a mutual regulation between NTS and Wnt: At the same time, it was found that the promoter region of neurotensin (NTS) contains the binding site of the transcription complex β-catenin / TCF (T cellfactor, TCF). The use of Wnt signaling pathway agonist Wnt3a or inhibitor can significantly increase or inhibit the secretion of tumor cell NTS, so it is believed that the Wnt / β-catenin signaling pathway plays an important role in regulating the occurrence of epithelial-mesenchymal transition and sinusitis. Summary of the invention

[0006] This application first discovered that the NTSR1 inhibitor SR48692 can be used to treat chronic sinusitis. Experiments have shown that SR48692 works by inhibiting the Wnt3a / β-catenin signaling pathway, thereby inhibiting the epithelial-mesenchymal transition process of nasal mucosal tissue and improving the occurrence and development of chronic sinusitis.

[0007] The first aspect of the present application is to provide a use of a neurotensin receptor 1 inhibitor in the preparation of a medicament or a pharmaceutical composition for preventing or treating chronic sinusitis.

[0008] Further, the neurotensin receptor 1 inhibitor is in the form of SR48692 (CAS No. 146362-70-1) or a tautomer, a pharmaceutically acceptable salt, a solvate, an ester, a polymorph, a tautomer, a racemate, an enantiomer or a diastereomer or a mixture thereof;

[0009] Specifically, the chemical name of SR48692 is

[0010] 2-[[[1-(7-Chloro-4-quinolinyl)-5-(2,6-dimethoxyphenyl)-1H-pyrazol-3-yl]carbonyl]amino]-tricyclo[3.3.1.13,7]decane-2-carboxylic acid, chemical formula see formula I:

[0011]

[0012] In some embodiments, the chronic sinusitis is chronic sinusitis with nasal polyps (CRSwNP).

[0013] Further, the SR48692 is administered to the patient orally, by injection or by inhalation;

[0014] Preferably, the SR48692 is administered by intraperitoneal injection.

[0015] Further, the administration dose of SR48692 is 1-100 mg / kg body weight, once or twice a week;

[0016] Preferably, the administration dose of SR48692 is 10 mg / kg body weight, once a week;

[0017] Furthermore, the subject of administration of SR48692 is a vertebrate, such as a mammal;

[0018] Further, the mammals include mice, rats, rabbits, cats, dogs, pigs, sheep, horses, non-human primates (e.g., macaques, chimpanzees);

[0019] Preferably, the mammal is a mouse.

[0020] The second aspect of the present application is to provide a pharmaceutical composition for preventing or treating chronic sinusitis.

[0021] Further, the pharmaceutical composition comprises the SR48692, and the SR48692 is used in combination with at least one anti-inflammatory drug;

[0022] Furthermore, the chronic sinusitis is chronic sinusitis with nasal polyps, and the anti-inflammatory drug is dexamethasone.

[0023] Furthermore, the pharmaceutical composition is administered by intraperitoneal injection.

[0024] In some embodiments, the pharmaceutical composition is in solid form, such as a lyophilized powder, liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation or powder suitable for reconstitution. In some embodiments, a delivery vehicle such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. can be used.

[0025] Compared with the prior art, this application has the following technical effects:

[0026] This application innovatively confirms the effect of NTSR1 inhibitor SR48692 in treating chronic rhinosinusitis (CRS), especially chronic rhinosinusitis with nasal polyps (CRSwNP). SR48692 effectively reduces the abnormal deposition of extracellular matrix (ECM) and significantly inhibits the epithelial-mesenchymal transition (EMT) process by targeting and inhibiting the Wnt3a / β-catenin signaling pathway. The inhibition of EMT plays a key role in reducing the abnormal deposition of submucosal matrix and improving tissue remodeling.

[0027] Given the high recurrence rate of CRSwNP after surgery, the SR48692 proposed in this application can reduce the recurrence rate of chronic sinusitis with nasal polyps through its unique mechanism of action, providing a potential target for the treatment of this disease. In particular, for CRSwNP patients who do not respond well to traditional treatments, SR48692 can achieve a more lasting therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It was shown that the expression of NTS and NTSR1 was elevated in the CRS mouse model.

[0029] (A) HE staining of pathological changes in mouse nasal mucosa; scale bar, 100 μm, n=4.

[0030] (B) mRNA expression of NTS and NTSR1 in mouse sinus mucosa tissue, n=4.

[0031] (C) The expression of NTSR1 in nasal mucosa was detected by immunohistochemical staining; scale bar, 100 μm, n=4.

[0032] (D) Protein expression of NTSR1 in mouse sinus mucosal tissues assessed by Western blot analysis, n = 4. Data are presented as mean ± SEM. * represents p < 0.05 compared with the control group; *** represents p < 0.001 compared with the control group.

[0033] Figure 2SR48692 improves nasal symptoms in the CRS mouse model. Nasal symptoms of CRS include (A) the number of nose scratching and (B) the number of sneezing. Data are presented as mean ± SEM. **p<<0.01, *p<<0.001vs control, * represents p<=0.05, ** represents p<0.01, #p<0.001vs CRS, repeated measures ANOVA followed by Bonferroni post hoc test was used to compare data at different times; n=8.

[0034] Figure 3 The effect of SR48692 on the NTS / NTSR1 signaling pathway and nasal mucosal pathology in the mouse CRS model. (A, B) Immunohistochemistry to detect NTSR1 expression and grayscale value statistics; scale bar is 100μm, n=4; (C) Western blot to detect NTSR1 protein expression and statistics; (D) RT-PCR to detect the mRNA levels of NTS and NTSR1, n=4; (E) HE staining of mouse nasal mucosal pathological changes; scale bar is 100μm, n=4.

[0035] Figure 4 The effect of SR48692 on the mRNA expression of eosinophilic inflammatory cytokines in the mouse CRS model. (AD) mRNA expression levels of Th2 cytokines (IL-4 and IL-5), IFN-γ, and IL-17 in the sinus mucosa of CRS mice, n = 4. Data are expressed as mean ± SEM. *p<<0.05, *p<<0.001 vs control group. #p<<0.05, ##p<<0.01, #p<<0.001 vs CRS.

[0036] Figure 5 The effect of SR48692 on EMT of nasal mucosal tissue in CRS mice. (A, B) Immunohistochemistry to detect the expression of EMT-related proteins (E-cadherin, vimentin and α-SMA) and grayscale value statistics; scale bar is 100μm, n=4; (CE) Western blot to detect the protein expression of E-cadherin and vimentin and statistics.

[0037] Figure 6 SR48692 inhibits the expression of Wnt3a / β-Catenin signaling pathway in the nasal mucosa of CRS mice. (AD) RT-PCR detected the mRNA levels of Wnt pathway molecules Wnt1, Wnt3a, Wnt5a and β-Catenin;

[0038] (E, F) Western blot detection of Wnt3a and β-catenin protein expression in nasal mucosa and statistics. DETAILED DESCRIPTION

[0039] The terms "treatment" or "treating" or "amelioration" are used interchangeably. These terms refer to an approach to obtaining beneficial or desired results, including but not limited to therapeutic benefit and / or prophylactic benefit. A therapeutic benefit refers to any treatment-related improvement in or effect on one or more diseases, conditions or symptoms under treatment.

[0040] The term "treat" includes: (1) preventing or delaying the onset of clinical symptoms of a state, disorder or condition that develops in a patient who may have or is susceptible to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; (2) inhibiting the state, disorder or condition (e.g., arresting, alleviating or delaying the development of the disease or the recurrence of at least one of its clinical or subclinical symptoms while treatment is maintained); (3) alleviating the condition (e.g., by causing regression or reducing the severity of at least one of the state, disorder or condition or its clinical or subclinical symptoms).

[0041] The term "effective amount" or "therapeutically effective amount" refers to an amount of a pharmaceutical agent sufficient to achieve a result, e.g., a beneficial or desired result. The therapeutically effective amount may vary depending on one or more of the following factors: the subject and disease condition being treated, the subject's weight and age, the severity of the disease condition, the mode of administration, etc.

[0042] Example 1 Establishment of a mouse chronic sinusitis (CRS) model

[0043] Animals: C57BL / 6 mice

[0044] Gender: Male

[0045] Age: 5 weeks

[0046] Methods for modeling mouse CRS model

[0047] 2U of AP and 75μg of OVA were mixed together and prepared with PBS to a final volume of 20μL. The mixture was dripped into the nose bilaterally (20μL on each side) 3 times a week for 12 weeks. After nasal instillation, the mice were placed head down to prevent the reagent from flowing into the lungs. An equal amount of PBS was dripped into the control group.

[0048] Experimental groups:

[0049] 1) Control group (n=6)

[0050] 2) CRS model group (n = 8)

[0051] 3) CRS model group + NTSR1 inhibitor group (SR48692 10 mg / kg) (n=6)

[0052] 4) CRS model group + dexamethasone group (1 mg / kg) (n=6)

[0053] Evaluation of sneezing / scratching symptoms in mice: At 4, 6, 8, 10, and 12 weeks, at 5 time points, after the nasal drop treatment, each mouse rested for 10 minutes, and the number of sneezing / scratching in the mouse within 10 minutes was observed and recorded.

[0054] To examine the role of NTSR1 signaling in the progression of CRS, mice were treated with the NTSR1 inhibitor SR48692 (10 mg / kg) once a week for a total of 8 times.

[0055] The dexamethasone group (Dex, 1 mg / kg) was used as a positive control group.

[0056] After the treatment, the mice were deeply anesthetized with 2% sodium pentobarbital and then killed by dislocating the neck. Immediately, the mouse nasal cavity was completely removed after cardiac perfusion with 20 mL 4% PFA, fixed with 4% PFA, and sliced. The method for obtaining the mouse nasal mucosa was to dislocate the neck after deep anesthesia with 2% sodium pentobarbital after the experiment. The mouse head was removed, the skin was removed, and the mouse nasal cavity and sinus mucosa were obtained and stored at -80°C.

[0057] The results showed that CRS mice had increased sneezing and nose rubbing, accompanied by severe runny nose and secretions. However, SR49692 treatment significantly reduced the frequency of snoring and nose rubbing after 8 weeks (Appendix Figure 2 ). The data showed that SR48692 could improve the occurrence of nasal symptoms in CRS mice.

[0058] Example 2 Tissue Hematoxylin-Eosin Staining (hereinafter referred to as H&E Staining)

[0059] The staining steps include:

[0060] (1) Preparation of 0.5% eosin solution: 0.5 g eosin was completely dissolved in 75 mL ddH2O, 25 mL 95% ethanol was added, and 1 drop of glacial acetic acid was added.

[0061] (2) After the paraffin sections were dewaxed and hydrated, hematoxylin was added to cover the samples and stained at room temperature for 5 min.

[0062] (3) Rinse with ddH2O and add 1% hydrochloric acid ethanol solution for differentiation for 5-10 seconds (second, sec). The slices will turn from blue to red.

[0063] (4) Rinse with ddH2O to return the color to blue for about 30 minutes.

[0064] (5) Stain with 0.5% eosin solution for 2 minutes.

[0065] (6) Wash twice with 95% and 100% ethanol, each time for 5 min.

[0066] (7) Use xylene to clear the sample twice, 5 min each time.

[0067] (8) Microscopic observation results: The cell nucleus is blue, and the cytoplasm and fibrous tissue are red of varying shades.

[0068] H&E staining was used to observe the pathological changes of the sinus mucosa of CRS mice. The epithelial layer of the sinus mucosa of CRS mice was thickened, the structure was loose, the goblet cells, epithelial cells and submucosal glands were hyperplastic, and there was a large amount of eosinophil infiltration in the submucosal tissue. The mucosal epithelium of normal mice was intact, without thickening, the goblet cells were slightly hyperplastic, and there were fewer inflammatory cells, indicating that the CRS mouse model was successfully established (see Appendix). Figure 1 A).

[0069] Example 3 Immunohistochemistry experiment

[0070] CRS is characterized by an uneven and complex pathogenesis, and various helper T cell responses are associated with the pathogenesis of CRS. In order to clarify the expression of NTSR1 pathway molecules in CRS and explore the effects of SR48692 on the pathological state of nasal mucosal tissue and the role of the NTSR1 signaling pathway, this application performed immunohistochemical analysis to evaluate whether SR48692 inhibits NTSR1.

[0071] Immunohistochemistry experimental steps include:

[0072] (1) Dewax and hydrate the paraffin sections, then soak the sections in 3% H2O2 for 10-15 min to avoid nonspecific background staining caused by catalase in the subsequent staining process;

[0073] (2) Wash with PBS three times, 5 minutes each time. Then, add antigen repair solution to cover the tissue sample sections and incubate at room temperature for 5-8 minutes;

[0074] (3) Wash with PBS three times, 5 min each time. Then, add 10% goat serum and block at room temperature for 0.5 h.

[0075] (4) After blocking, wash twice with PBS, 5 minutes each time. Then, add the primary antibody working solution and incubate at room temperature for 2 hours. After incubation, wash three times with PBST, 10 minutes each time. Then, add the secondary antibody working solution and continue incubation at room temperature for 1 hour. After incubation, wash three times with PBST, 10 minutes each time;

[0076] (5) Add freshly prepared DAB solution to the treated sections for color development for 1-2 min;

[0077] (6) Wash slowly with ddH2O, add hematoxylin to counterstain, then wash with water to return to blue, and dry;

[0078] (7) Dehydration and drying were performed using gradient ethanol and xylene, and sealing was performed using a neutral resin;

[0079] (8) Microscopic examination.

[0080] The results of immunohistochemistry (IHC) analysis of tissue sections showed that the expression of NTSR1 in the CRS group was significantly higher than that in the control group (Appendix Figure 1 C)

[0081] The expression level of E-cadherin in the CRS group was significantly lower than that in the control group, while the expression of vimentin and α-SMA was significantly higher than that in the control group. Compared with the CRS group, the expression of E-cadherin in the SR48692 and Dex treatment groups showed a significant decrease, while the expression of Vimentin and a-SMA was significantly increased. The mRNA expression of NTS and NTSR1 in the nasal mucosal tissue of mice in the CRS group was significantly upregulated. SR48692 treatment effectively inhibited the mRNA expression of NTS and NTSR1 in the nasal mucosal tissue of CRS mice. Histological analysis showed that the sinus mucosal epithelium of mice in the CRS group was disordered, some areas were necrotic or blurred, and the submucosal glands and granulocytes proliferated, accompanied by inflammatory cell infiltration. However, after treatment with SR48692 and Dex, the above results were improved (Appendix Figure 3 )

[0082] The present application shows that treatment with SR48692 and dexamethasone reduces the expression of NTSR1 in nasal mucosal tissue. The effect of SR48692 on the expression of inflammatory cytokines in nasal mucosal tissue of CRS mice is shown in the attached Figure 4 A,4B.

[0083] Example 4 Western blot detection:

[0084] In this example, the expression level of NTSR1 was detected by western blot analysis. Specifically:

[0085] The total protein of mouse nasal mucosa tissue was extracted using RIPA lysis buffer, and the protein concentration was determined using a BCA protein assay kit. Proteins of different molecular weights were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a PVDF membrane. After completing the above steps, the primary antibody diluent (1:1000) was added and incubated with PVDF at 4°C overnight; the next day, it was washed with TBST three times for 10 minutes each time, incubated with the secondary antibody at room temperature for 1 hour, washed with TBST three times for 10 minutes each time, and the prepared ECL luminescent solution was dropped on the PVDF membrane, which was then placed in a gel imaging system, and the image results were analyzed using Image J software.

[0086] Western blot analysis showed that the protein levels of Wnt3a and β-Catenin were increased in the CRS group, and these effects were significantly reversed after SR48692 treatment. These results indicate that NTSR1 inhibitors improve EMT in CRS mice by inhibiting the Wnt3a / β-catenin signaling pathway, and that inhibition of the NTS / NTSR1 pathway can improve pathological changes and CRS symptoms in mice.

[0087] This experiment showed that the expression level of NTSR1 in the sinus mucosa of the CRS group was increased compared with the normal group (Appendix Figure 5 In the CRS group, the protein level of E-cadherin decreased, while the protein expression of vimentin increased, and these effects were significantly reversed after SR48692 and Dex treatment (Appendix Figure 5 ). These results indicate that SR48692 treatment attenuates EMT in the nasal mucosa of CRS mice.

[0088] In summary, this application shows that NTS and NTSR1 are highly expressed in CRS mice, indicating that dysregulated NTSR1 signaling may have a potential role in the occurrence and development of CRS.

[0089] Example 5 RT-qPCR detection:

[0090] Trizol was used to extract RNA from mouse nasal mucosa tissue and reverse transcribed into cDNA. According to the operating instructions of the amplification kit, the obtained cDNA was amplified using a fluorescent quantitative PCR instrument, and the CT values ​​were compared and the 2 -ΔΔCt The values ​​were statistically analyzed.

[0091] The results of RT-qPCR detection of NTS and NTSR1 mRNA showed that the expression levels of NTS and NTSR1 mRNA in the sinus mucosa tissue of the CRS group were significantly higher than those in the normal group (Appendix Figure 1 B).

[0092] The mRNA expression levels of interleukin IL-17 and Th2 cytokines (IL-4 and IL-5) in the CRS group were significantly higher than those in the control group. Compared with the CRS group, the levels of IL-17, IL-4 and IL-5 were downregulated in the SR48692 group and the Dex group; there was no significant difference in the expression of IFN-γ between the groups. The above results show that treatment with the NTSR1 inhibitor SR48692 can reduce the levels of eosinophilic inflammatory cytokines, including IL-4 and IL-5. For example, IL-4 can induce Th2 and Tc 2 cell differentiation. IL-5 can promote the accumulation of eosinophils in nasal mucosal tissue. IL-17A, as a typical type 3 cytokine, is highly expressed in nasal polyps and is associated with the severity of symptoms and the adverse outcomes of corticosteroid treatment in CRS. The data from this experiment showed that SR48692 treatment can significantly reduce the occurrence of eosinophilic inflammatory response in CRS mice (see Appendix Figure 4 ).

[0093] RT-qPCR analysis was performed to determine the expression of Wnt signaling molecules. Compared with the normal group, the mRNA expression levels of Wnt3a and β-Catenin in the CRS group were significantly increased, while other Wnt ligands did not change significantly. Compared with the CRS group, the mRNA expression of Wnt3a and β-Catenin in the SR48692 and Dex treatment groups was significantly decreased (Supplementary Figure 5). Figure 6 AD).

Claims

1. Use of a neurotensin receptor 1 inhibitor in the preparation of a medicament or a pharmaceutical composition for preventing or treating chronic sinusitis, It is characterized in that The neurotensin receptor 1 inhibitor is in the form of SR48692 or its tautomer, pharmaceutically acceptable salt, solvate, ester, polymorph, tautomer, racemate, enantiomer or diastereomer or a mixture thereof; the chemical formula of SR48692 is shown in Formula I.

2. The use according to claim 1, It is characterized in that The chronic sinusitis is chronic sinusitis with nasal polyps.

3. The use according to claim 1, It is characterized in that The SR48692 is administered to the subject orally, by injection or by inhalation; preferably, the SR48692 is administered by intraperitoneal injection.

4. The use according to claim 3, It is characterized in that The SR48692 is administered at a dosage of 1-100 mg / kg body weight, once or twice a week.

5. The use according to claim 3, It is characterized in that The SR48692 was administered at a dose of 10 mg / kg body weight, once a week.

6. The use according to claim 5, It is characterized in that The subject of administration of SR48692 is a mammal, including mice, rats, rabbits, cats, dogs, pigs, sheep, horses or non-human primates.

7. The use according to claim 6, It is characterized in that The subjects of administration of SR48692 are mice.

8. A pharmaceutical composition for preventing or treating chronic sinusitis, It is characterized in that The drug or pharmaceutical composition comprises the SR48692 as claimed in claim 1, wherein the SR48692 is used in combination with at least one anti-inflammatory drug; preferably, the anti-inflammatory drug is dexamethasone.

9. The pharmaceutical composition according to claim 8, It is characterized in that The chronic sinusitis is chronic sinusitis with nasal polyps.

10. The pharmaceutical composition according to claim 9, It is characterized in that The pharmaceutical composition is administered by intraperitoneal injection.

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