Pharmaceutical application of rusotinib and / or S-rusotinib

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to new application of ruxotinib and / or S-ruxotinib in preparation of a medicine for treating nasosinusitis. According to the application of the ruxolitinib and / or the S-ruxolitinib in the chronic sinusitis, the application of the ruxolitinib and / or the S-ruxolitinib in the chronic sinusitis is provided for the first time, and the S-ruxolitinib is proved to have a remarkable curative effect in a mouse model with the chronic sinusitis through sufficient experimental data support. The invention verifies that S-Ruxolitinib can relieve nasal mucosa inflammation of mice with chronic sinusitis and protect the nasal mucosa function, verifies that S-Ruxolitinib has a very good treatment effect on chronic sinusitis, provides a new clinical choice for treatment of chronic sinusitis, particularly provides a new immunoregulation treatment strategy, and has a good application prospect. The blank in the field is filled, and the method has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a new use of Ruxolitinib and / or S-Ruxolitinib for preparing a drug for treating sinusitis. Background Art

[0002] Chronic rhinosinusitis (CRS) is a common upper respiratory tract disease. Patients usually present with symptoms such as nasal congestion, runny nose, headache, and loss of smell, which seriously affect people's quality of life. The etiology of chronic sinusitis is complex and is usually closely related to multiple factors such as environmental factors, infection, allergy, and abnormal immune function. Studies have shown that in the pathogenesis of chronic sinusitis, type II inflammatory response, epithelial barrier dysfunction, and mucociliary system dysfunction are key pathological factors, which interact with each other and jointly promote the persistence and aggravation of sinusitis symptoms. Type II inflammatory response is the main type of inflammatory response in chronic sinusitis, which is mainly mediated by Th2 cells and their secreted cytokines (such as IL-4, IL-5, and IL-13). The epithelial barrier plays an important role in normal sinus and respiratory function and is responsible for maintaining the immune defense of the respiratory tract. The occurrence of chronic sinusitis is closely related to the destruction of epithelial barrier function. The mucociliary system is an important defense mechanism of the nasal cavity and sinuses. It removes external pathogens, pollutants and allergens through the movement of cilia and normal secretion of mucus. In chronic sinusitis, dysfunction of the mucociliary system is an important reason for the persistence of the disease.

[0003] At present, the treatment methods for chronic sinusitis mainly include antibiotic treatment, nasal steroid drugs, saline irrigation and surgical operation. However, the above clinical treatment methods all have certain limitations, including: (1) unstable treatment effect: for some patients, the existing treatment methods are not ideal, or the symptoms recur during long-term treatment, which can easily lead to a decline in the patient's quality of life; (2) side effects: although steroid drugs can relieve inflammation of the nasal cavity and sinuses, long-term use can cause a series of side effects, such as immunosuppression and abnormal glucose metabolism; (3) insufficient individualization of treatment plans: existing treatment methods fail to fully consider individual differences among patients, resulting in differences in treatment effects among different patients; (4) lack of effective immunomodulatory treatment: the occurrence of chronic sinusitis is closely related to immune response disorders, and the JAK / STAT signaling pathway plays an important role in it. Therefore, the field expects to develop more suitable therapeutic drugs and treatment methods. Summary of the invention

[0004] To this end, the technical problem to be solved by the present invention is to provide a new use of Ruxolitinib and / or S-Ruxolitinib for preparing a drug for treating sinusitis. The Ruxolitinib and / or S-Ruxolitinib can inhibit the activity of JAK1 / JAK2 / Tyk2 kinases, thereby inhibiting the abnormal activation of the JAK / STAT signaling pathway, thereby regulating the immune response and inflammatory response, and can be used to treat chronic sinusitis.

[0005] In order to solve the above technical problems, the present invention provides a use of ruxolitinib and / or S-ruxolitinib for preparing a drug for treating sinusitis.

[0006] Specifically, the sinusitis includes chronic sinusitis.

[0007] Specifically, the ruxolitinib and / or S-ruxolitinib has at least one of the following uses:

[0008] (1) Inhibit the activity of JAK1, JAK2 and / or Tyk2 kinases;

[0009] (2) Inhibit abnormal activation of the JAK / STAT signaling pathway;

[0010] (3) Regulate immune and inflammatory responses;

[0011] (4) Relieve nasal mucosal inflammation in mice with chronic sinusitis;

[0012] (5) Improve the barrier function of the nasal mucosa;

[0013] (6) Improve the mucociliary function of the nasal mucosa.

[0014] Specifically, the ruxolitinib and / or S-ruxolitinib is the only active ingredient.

[0015] The present invention also discloses a pharmaceutical preparation for treating sinusitis, wherein the active ingredient of the pharmaceutical preparation comprises the ruxolitinib and / or S-ruxolitinib.

[0016] Specifically, the pharmaceutical preparation for treating sinusitis includes oral preparations, external preparations, injection preparations, respiratory tract administration preparations or intranasal administration preparations.

[0017] For example, the oral preparation may include conventional dosage forms in the art, such as tablets, pills, granules, powders, mixtures, etc.;

[0018] For example, the injection preparation may include conventional dosage forms in the art, such as injections, injection solutions, etc.;

[0019] For example, the respiratory tract administration preparation may include conventional dosage forms in the art, such as atomized preparations, spray preparations, etc.;

[0020] For example, the topical preparation may include conventional dosage forms in the art, such as tinctures, topical creams, etc.;

[0021] For example, the intranasal administration preparation includes conventional dosage forms in the art, such as nasal spray preparations or nasal drops;

[0022] In the present application, the preferred administration method of the pharmaceutical preparation is intranasal administration, and the preferred pharmaceutical dosage form is nasal spray preparation or nasal drops.

[0023] Specifically, the pharmaceutical preparation for treating sinusitis also includes clinically acceptable excipients.

[0024] The present invention also discloses a method for preparing the pharmaceutical preparation for treating sinusitis, comprising the steps of taking the ruxolitinib and / or S-ruxolitinib as active ingredients and adding conventional excipients to select a dosage form.

[0025] The present invention also discloses use of ruxolitinib and / or S-ruxolitinib and a composition thereof for preparing JAK1, JAK2 and / or Tyk 2 kinase inhibitors.

[0026] The present invention also discloses use of ruxolitinib and / or S-ruxolitinib and a composition thereof for preparing a JAK / STAT signaling pathway inhibitor.

[0027] The present invention proposes for the first time the use of ruxolitinib and / or S-Ruxolitinib in chronic sinusitis, and with sufficient experimental data support, proves the significant therapeutic effect of ruxolitinib and / or S-Ruxolitinib in a mouse model of chronic sinusitis. The present invention verifies the role and effect of ruxolitinib and / or S-Ruxolitinib in reducing nasal mucosal inflammation and protecting nasal mucosal function in mice with chronic sinusitis, verifies that it has a good therapeutic effect on chronic sinusitis, provides a new clinical option for the treatment of chronic sinusitis, and especially provides a new immunomodulatory treatment strategy, filling the gap in this field and having a good application prospect.

[0028] The new use of the drug of the present invention further verifies that the S-Ruxolitinib, as the S-isomer of Ruxolitinib, can inhibit the activity of JAK1 / JAK2 kinases and the abnormal activation of JAK / STAT signaling pathways, thereby regulating immune and inflammatory responses, and effectively fills the gap of S-Ruxolitinib in the treatment of chronic sinusitis.

[0029] The S-Ruxolitinib of the present invention, as the S-isomer of Ruxolitinib, has a different stereostructure from Ruxolitinib, and thus has different effects on its biological activity. As a JAK inhibitor, the S isomer of Ruxolitinib is the key configuration of some of its pharmacological activities, especially when the drug target is a protein or enzyme, different isomers may have significant differences in binding affinity, binding site and drug efficacy with the target. The S isomer structure of the drug S-Ruxolitinib of the present invention may show different selectivity and affinity in the binding with a specific receptor. In particular, the S isomer may show different characteristics in the metabolic pathway and absorption rate in the body due to the difference in molecular size, polarity or spatial configuration. The S isomer of some drugs has a longer half-life in the body than the R isomer, or has a lower clearance rate in liver metabolism. Therefore, the S isomer of the S-Ruxolitinib drug is safer.

[0030] In the pathogenesis of CRSwNP, the Tyk 2 gene may play a key role in the pathogenesis of CRSwNP. Interleukin IL-13 participates in regulating nasal mucosal inflammatory cell infiltration and epithelial dysfunction by activating Jak 1, Jak 2 and Tyk 2 kinases and signal transducers and activators of transcription (STATs). The results of the present invention found that in mouse transcriptome sequencing, S-Ruxolitinib can significantly inhibit the activation of JAK1, JAK2 and Tyk 2. Compared with Ruxolitinib, which exhibits activation performance on JAK1 and JAK2, S-Ruxolitinib has greater advantages in the treatment of CRSwNP.

[0031] The present invention uses ruxolitinib and / or S-Ruxolitinib as the only active ingredient to intervene in the JAK / STAT signaling pathway through nasal drops, and can intervene in the JAK / STAT signaling pathway based on the effect of local nasal drops to regulate the immune response and effectively reduce the inflammatory response. And because the nasal drops have a smaller impact on the immune system, local medication is safer and more suitable for the long-term management of patients with chronic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0033] Figure 1 This is a schematic diagram of the modeling of the CRSwNP mouse model in Example 1;

[0034] Figure 2The results of inflammatory cell infiltration in the nasal mucosa of mice with chronic sinusitis in Example 2;

[0035] Figure 3 The results of the levels of inflammatory factors related to nasal lavage fluid in Example 2;

[0036] Figure 4 The differential gene results of different groups of mice in Example 3;

[0037] Figure 5 The results are the screening results of the top 20 pathways in the GO enrichment analysis of differentially expressed genes in Example 3;

[0038] Figure 6 The results of screening the top 20 pathways in the KEGG enrichment analysis of differentially expressed genes in Example 3;

[0039] Figure 7 The cluster analysis results for the JAK / STAT pathway in Example 3;

[0040] Figure 8 The GSEA enrichment analysis results for the JAK / STAT pathway in Example 3;

[0041] Fig. 9 The results of immunofluorescence staining of barrier proteins in paraffin sections of mouse nasal mucosa in Example 4;

[0042] Fig.10 The results of immunofluorescence staining of β-Tubulin IV and MUC5AC on paraffin sections of mouse nasal mucosa in Example 4 are shown. DETAILED DESCRIPTION

[0043] Example 1

[0044] In this example, a CRSwNP mouse model was constructed. The specific process diagram is shown in the attached figure. Figure 1 shown.

[0045] Mouse modeling: CRSwNP mouse model, select 6-8 week old female C57BL / 6 mice, weighing 22-25g.

[0046] The mice in the modeling group and the S-Ruxonitilib treatment group were nasally dripped with a mixture of Aspergillus oryzaeprotease (AP) and ovalbumin (OVA). 2 units of AP and 75 μg of OVA were mixed in sterile PBS and diluted to a total volume of 20 μl for nasal drip, 3 times a week for 12 weeks (7 mice in each group). The mice in the control group were given 20 μl PBS nasal drops at the same time (7 mice in each group), and the mice in the S-Ruxonitilib treatment group were given S-Ruxonitilib 5 mg / Kg nasal drops for the last 8 weeks of the entire modeling period, 3 times a week for 8 weeks (7 mice in each group).

[0047] Example 2

[0048] This example is based on the mouse model in Example 1 to verify that S-Ruxolitinib can effectively relieve inflammation of the nasal mucosa of mice with chronic sinusitis and significantly reduce the performance of the level of related inflammatory factors. After the mice were anesthetized, they were killed by decapitation, and then the skin of the head and mandible was carefully removed and the mandible was removed. The skull was removed and fixed in 4% paraformaldehyde (PFA) for 12 hours. After fixation, the samples were decalcified in 5% nitric acid for 3 days, then dehydrated, paraffin-embedded, and cut into 4 μm thick coronal sections. The sections were stained with hematoxylin-eosin (H&E) to evaluate the infiltration of polypoid lesions and eosinophils. CD45 immunohistochemical staining was used to evaluate the infiltration of inflammatory cells, and myeloperoxidase (MPO) staining was used to detect the infiltration of neutrophils in nasal tissue. All histological operations, including H&E staining and immunohistochemistry, were performed by two independent pathologists, who kept the experimental group blind during the evaluation process to reduce potential bias and ensure the objectivity of the results.

[0049] like Figure 2 The results showed that the thickness of the nasal epithelium of chronic sinusitis mice treated with S-Ruxolitinib became thinner, and the infiltration of eosinophils, neutrophils and CD45-positive cells in the nasal mucosa was significantly reduced (n=4).

[0050] Furthermore, a No. 24 catheter was placed in the nasopharynx, and 200 μL of PBS was instilled into the nasal cavity. Subsequently, the nasal lavage fluid was collected from the nostrils. The concentrations of various cytokines and chemokines in the lavage fluid were quantitatively evaluated using a multiple cytokine detection kit (R&D Systems), which can simultaneously detect multiple inflammatory markers, including IFN-γ, TNF-α, IL-1β, IL-4, IL-5, IL-6, IL-13, IL-17A, eotaxin / CCL11, G-CSF, GM-CSF, and RANTES / CCL5. Sample analysis was performed using the Bio-Plex 200 system (Bio-Rad), which can perform high-throughput, multiple cytokine and chemokine detection, thereby comprehensively evaluating the concentration of inflammatory factors in the nasal lavage fluid.

[0051] like Figure 3 The results showed that the concentrations of IFN-γ, IL-4, IL-6, IL-13, CCL5, and GM-CSF in the nasal lavage fluid of chronic sinusitis mice treated with S-Ruxolitinib were significantly lower than those of the chronic sinusitis group mice (n=7).

[0052] Example 3

[0053] This example is based on the mouse model in Example 1 to verify the performance of S-Ruxolitinib in inhibiting the JAK / STAT pathway at the transcriptome level and alleviating nasal mucosal inflammation in mice with chronic sinusitis.

[0054] like Figure 4 (A) and (B) are the differentially expressed genes between the chronic sinusitis group and the control group, and the differentially expressed genes between the S-Ruxonitilib treatment group and the chronic sinusitis group, respectively. The results showed that after S-Ruxolitinib nasal drops treatment, the genes in the nasal mucosal transcriptome changed significantly.

[0055] In the present embodiment, mouse nasal mucosal tissue was collected, and total RNA was extracted using MJzol Animal RNA Isolation Kit (Shanghai, China), and the manufacturer's instructions were followed. RNA was purified by RNAClean XP Kit (Beckman Coulter, California, USA) and RNase-free DNase Kit (QIAGEN, Hilden, Germany). RNA integrity was assessed by Agilent 2100 Bioanalyzer / Agilent 4200 TapeStation (Agilent Technologies, California, USA), and RNA concentration and purity were measured by Qubit 2.0 Fluorometer (Thermo Fisher, Massachusetts, USA) and NanoDrop ND-2000 Spectrophotometer (Thermo Fisher, Massachusetts, USA). When constructing the library, mRNA was first isolated, fragmented and synthesized into cDNA (first and second chains), followed by end repair, 3'A tailing, adapter connection and enrichment. Library concentrations were measured by Qubit 2.0 Fluorometer (Thermo Fisher Scientific, MA, USA), and library fragment distribution was analyzed by Agilent 4200 TapeStation (Agilent Technologies, CA, USA). Gene expression levels were normalized by FPKM (fragments per kilobase of exon model per million aligned reads) to facilitate comparison across genes and samples. Differential gene expression was analyzed using edgeR, and P values ​​were adjusted for multiple hypothesis testing by controlling the false discovery rate (FDR). Fold changes were calculated based on FPKM. Differentially expressed genes (DEGs) were screened based on stringent thresholds of Q < 0.05 and log2 fold change (Log2FC) ≥ 1. The resulting gene lists (including Entrez gene IDs) were subsequently subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis using R software (version 4.0.3) and related bioinformatics packages. GSEA enrichment analysis was performed using GSEA software (version 4.3.3).

[0056] This example uses the above-mentioned method to screen differentially expressed genes and perform enrichment analysis on the differentially expressed genes. Figure 5 The results shown screened the top 20 pathways related to biological processes in GO analysis. Most of the pathways were related to inflammation, proving that S-Ruxolitinib treatment can reduce the inflammatory response of the nasal mucosa.

[0057] like Figure 6 The results shown screened the top 20 pathways in the KEGG analysis, and the results showed that the chronic sinusitis group could activate the JAK / STAT pathway, so the JAK / STAT pathway plays an important role in chronic sinusitis.

[0058] Furthermore, this example performed a cluster analysis on the JAK / STAT pathway, and the results were as follows: Figure 7 As shown. It can be seen that the chronic sinusitis group was found to activate the JAK / STAT pathway-related genes, while the S-Ruxolitinib treatment group could inhibit the activation of the JAK / STAT-related pathway.

[0059] Furthermore, this example performed GSEA enrichment analysis on the JAK / STAT pathway, and the results were as follows: Figure 8 As shown in Figure 3, the chronic sinusitis group activated 5 JAK / STAT-related pathways, while the S-Ruxolitinib treatment group inhibited 8 JAK / STAT-related pathways.

[0060] In summary, through the transcriptome results, this example discovered and verified the activation of the JAK / STAT pathway in chronic sinusitis mice and caused an inflammatory response, while the S-Ruxolitinib treatment group could inhibit the activation of the JAK / STAT pathway and thus reduce the inflammatory effect (n=3).

[0061] Example 4

[0062] This example verifies the role and effect of S-Ruxonitilib in improving the nasal mucosal barrier function of mice with chronic sinusitis, and verifies the improvement of the mucociliary function of the nasal mucosa of mice with chronic sinusitis by S-Ruxonitilib.

[0063] Mouse nasal tissue sections were fixed with a 1:1 mixture of acetone and methanol for 10 min and subsequently blocked with 5% skim milk powder to prevent nonspecific binding. Next, the sections were incubated with primary antibodies overnight at 4°C, including occludin (1:200), ZO-1 (1:200), β-Tubulin-IV (1:1000), and MUC5AC (1:100). The next day, the sections were incubated with secondary antibodies labeled with FITC (1:500) and rhodamine (1:500) for 1 h at room temperature. Nuclear contrast staining with DAPI was performed to visualize cell nuclei. Finally, the stained specimens were carefully observed under a confocal microscope (model IX81; Olympus, Tokyo, Japan) to observe the distribution and expression of the target protein and to ensure that high-resolution fluorescence images were obtained for accurate analysis. Subsequent image analysis was performed by 2 independent pathologists, and image fluorescence intensity quantification was analyzed by Image J software.

[0064] In this example, immunofluorescence staining was performed on paraffin sections of mouse nasal tissue. The results are shown in the attached figure. Fig. 9 As shown in the figure, the expression of tight junction proteins ZO-1 and Occludin related to the nasal mucosal barrier was detected. The results showed that the nasal mucosal barrier of mice in the chronic sinusitis group was damaged, while S-Ruxonitilib nasal drops had a good protective effect on the nasal mucosal barrier after treatment (n=4).

[0065] As attached Fig.10 The results shown in the figure were obtained by immunofluorescence staining of β-Tubulin IV and MUC5AC to study the effects on ciliary function and mucin secretion. The results showed that the epithelial mucociliary system of mice in the chronic sinusitis group was damaged, the cilia were reduced, and the secretion of mucin MUC5AC was increased. After nasal treatment with S-Ruxonitilib, the impaired epithelial mucociliary function was significantly improved (n=4).

[0066] Example 6

[0067] This example verifies the therapeutic effect of ruxolitinib on sinusitis, and the experiment is conducted in the manner of the above examples.

[0068] The results show that ruxolitinib has the effect of inhibiting the activity of JAK1 and JAK2 kinases, and can also regulate immune and inflammatory responses, effectively reduce inflammatory responses, and can relieve nasal mucosal inflammation in mice with chronic sinusitis, improve the nasal mucosal barrier function and nasal mucociliary function, has the efficacy of treating sinusitis, and can be used for the clinical treatment of sinusitis, especially chronic sinusitis.

[0069] Example 7

[0070] The drug for treating sinusitis, especially chronic sinusitis, described in this embodiment contains S-ruxolitinib as an active ingredient, and ruxolitinib and / or S-ruxolitinib can also be selected as the only active ingredient.

[0071] The drug for treating sinusitis, especially chronic sinusitis, of the present invention can be prepared into conventional dosage forms known in the art, including oral preparations, external preparations, injections, respiratory tract administration dosage forms, intranasal mucosal administration dosage forms, etc. Specific dosage forms include drops, tablets, capsules, granules, aqueous solutions, enteric-coated preparations, injections, roll-on formulations, caplets, chewable tablets, jelly, syrup, liquid solution, suspension, powder, solid crystal, orally disintegrating tablet or paste.

[0072] The drug for treating sinusitis, especially chronic sinusitis, of the present invention is preferably administered to the nasal mucosa, and suitable dosage forms are preferably nasal sprays or nasal drops.

[0073] In the therapeutic use of the pharmaceutical preparation, the daily dosage of ruxolitinib and / or S-ruxolitinib can be conventional dosages. These dosages can vary according to the needs of the patient, the severity of the disease being treated, and the drug used. Generally, treatment is started with a smaller dose than the optimal dose of the drug, and then the dose is increased in small amounts to achieve the best effect. For convenience, the total daily dose can be further divided into divided doses within a day if necessary.

[0074] The medicine of the present invention may also contain other Chinese and Western medicines or health products that have therapeutic or auxiliary therapeutic effects on sinusitis, especially chronic sinusitis, or the medicine of the present invention may be used in combination with these Chinese and Western medicines or health products.

[0075] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Use of ruxolitinib and / or S-ruxolitinib for preparing a medicament for treating sinusitis.

2. The use according to claim 1, characterized in that The sinusitis includes chronic sinusitis.

3. The use according to claim 1 or 2, characterized in that: The Ruxolitinib and / or S-Ruxolitinib has at least one of the following uses: (1) Inhibit the activity of JAK1, JAK2 and / or Tyk 2 kinases; (2) Inhibit abnormal activation of the JAK / STAT signaling pathway; (3) Regulate immune and inflammatory responses; (4) Relieve nasal mucosal inflammation in mice with chronic sinusitis; (5) Improve the barrier function of the nasal mucosa; (6) Improve the mucociliary function of the nasal mucosa.

4. The use according to any one of claims 1 to 3, characterized in that: The ruxolitinib and / or S-ruxolitinib is used as the sole active ingredient.

5. A pharmaceutical preparation for treating sinusitis, characterized in that: The active ingredient of the pharmaceutical preparation includes the ruxolitinib and / or S-ruxolitinib.

6. The pharmaceutical preparation for treating sinusitis according to claim 5, characterized in that: The pharmaceutical preparations include oral preparations, external preparations, injection preparations, respiratory tract administration preparations or intranasal administration preparations; Preferably, the pharmaceutical preparation comprises an intranasal preparation; Preferably, the pharmaceutical preparation comprises a nasal spray preparation or nasal drops.

7. The pharmaceutical preparation for treating sinusitis according to claim 5 or 6, characterized in that: The pharmaceutical preparation also includes clinically acceptable excipients.

8. A method for preparing the pharmaceutical preparation for treating sinusitis according to any one of claims 5 to 7, characterized in that: The method comprises the steps of taking the ruxolitinib and / or S-ruxolitinib as active ingredients and adding conventional auxiliary materials to process the selected dosage form.

9. Use of ruxolitinib and / or S-ruxolitinib and a composition thereof for preparing a JAK1, JAK2 and / or Tyk 2 kinase inhibitor.

10. Use of ruxolitinib and / or S-ruxolitinib and a composition thereof for preparing a JAK / STAT signaling pathway inhibitor.

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