Application of cinacalcet hydrochloride in preparation of medicine for treating allergic rhinitis

Abstract

The invention relates to cinacalcet hydrochloride, in particular to application of cinacalcet hydrochloride in preparation of drugs for treating allergic rhinitis. The invention discovers that cinacalcet hydrochloride has the effect of treating allergic rhinitis for the first time. Specifically, firstly, drugs capable of effectively inhibiting the NO release amount of RAW264.7 cells (using LPS to stimulate the RAW264.7 cells to establish an in-vitro inflammation model) are screened from 1430 small molecule compounds approved by FDA, and it is found for the first time that cinacalcet hydrochloride can inhibit NO release in the in-vitro inflammation model in a dose-dependent mode. On the other hand, in-vivo treatment experiments also find that cinacalcet hydrochloride can effectively inhibit release of allergic media in vivo without affecting the weight of mice, and has good safety.

Description

Technical Field

[0001] The invention relates to cinacalcet hydrochloride, and in particular to application of cinacalcet hydrochloride in preparing a drug for treating allergic rhinitis Background Art

[0002] Allergic rhinitis (AR), also known as allergic rhinitis, is caused by external allergens stimulating the body's nasal mucosal innate immune cells to produce specific IgE antibodies, and IgE antibodies combine with basophils and mast cells to make the body enter a sensitized state. When the same allergen is felt again, the effector cells release inflammatory factors, which act on the small blood vessels, smooth muscles and glands of the respiratory tract, causing the body's nasal mucosa to swell and nasal concha edema, which belongs to type I allergic reaction. Patients often show symptoms of nasal itching, runny nose, and sneezing. Some patients will also experience symptoms such as itchy eyes, itchy ears, itchy throat, hyposmia, and asthma. Some patients may be very weak, leading to irritability, insomnia and fatigue, and reducing patients' work efficiency and learning ability. The risk factors for allergic rhinitis patients mainly include genetic factors, air pollution factors, work environment pollution, smoking, frequent respiratory tract infections, large-scale use of antibiotics, drugs, or a history of pollen allergies. Moreover, the recurrence of allergic rhinitis is often the result of multiple factors, so its pathological phenomenon is relatively complex and individual-specific.

[0003] Modern medicine mainly treats allergic rhinitis through drug therapy, surgical treatment and immunotherapy. Drug therapy mainly uses antihistamines, glucocorticoids, mast cell stabilizers, decongestants and other drugs; these drugs have different degrees of efficacy, but usually can only control symptoms and cannot cure patients, and are often accompanied by side effects such as dry mouth, pharyngitis, headache, dizziness, drowsiness, palpitations, neuropsychiatric events, etc. Surgical treatment can only solve the symptoms of abnormal nasal hyperplasia and polyps in patients with severe rhinitis, but does not have the effect of completely curing allergic rhinitis; although immunotherapy is currently the only specific treatment method, it also faces problems such as low safety and poor patient compliance.

[0004] Cinacalcet is the first drug in a new class of compounds called calcimimetics, which can activate calcium receptors in the parathyroid glands, thereby reducing the secretion of parathyroid hormone (PTH) and is used to treat secondary hyperparathyroidism in patients with chronic kidney disease (CKD) undergoing maintenance dialysis. There are no literature reports on the use of cinacalcet hydrochloride in the treatment of allergic rhinitis. Summary of the invention

[0005] Purpose of the Invention

[0006] The present invention provides a new use of Cinacalcet hydrochloride, namely, provides the effect of Cinacalcet hydrochloride in treating allergic rhinitis.

[0007] Technical Solution

[0008] The use of Cinacalcet hydrochloride in the preparation of a drug for treating allergic rhinitis, wherein the structural formula of Cinacalcet hydrochloride is as follows:

[0009]

[0010] In the technical solution of the present invention, the aerosolized dosage of Cinacalcet Hydrochloride is 0.1% (w / v) and 0.5% (w / v), the spraying volume is 2 mL / 10 min, and the time is 10 min / time.

[0011] In the technical scheme of the present invention, adult Balb / c mice are used for systemic sensitization and nasal sensitization. After the mice are systemically sensitized, cinacalcet hydrochloride is administered by atomization during the nasal sensitization stage, the behavior of the mice is observed, and their allergic symptoms are scored to evaluate the therapeutic effect of the drug on allergic rhinitis. Studies have found that cinacalcet hydrochloride can inhibit the release of inflammatory factors in allergic rhinitis and alleviate the nasal allergy symptoms of AR modeling mice.

[0012] Beneficial effects of the present invention:

[0013] New drug development has disadvantages such as long cycle, high cost and low success rate, while new uses of old drugs can significantly shorten the drug development cycle and reduce development risks and costs. Compounds that have been approved by the FDA for marketing are safe, effective and quality-controlled. Developing new functions for old drugs can greatly shorten the drug development cycle, cost and reduce risks. We screened FDA-approved compounds by high-throughput screening, and determined the anti-inflammatory effect of the drugs by measuring the inhibitory effect of each drug on the NO release content in the inflammatory model established by RAW264.7 cells.

[0014] The present invention first discovered that Cinacalcet hydrochloride has the effect of inhibiting and treating allergic rhinitis. Specifically, the drug that effectively inhibits the NO release of RAW264.7 cells (using LPS to stimulate RAW264.7 cells to establish an in vitro inflammatory model) was first screened from 1430 small molecule compounds approved by the FDA, and it was first discovered that Cinacalcet hydrochloride can dose-dependently inhibit the release of NO in the in vitro inflammatory model. On the other hand, it was also found through in vivo treatment experiments that Cinacalcet hydrochloride can effectively inhibit the release of allergic mediators in vivo without affecting the weight of mice, and has good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 .Screening of drugs for the treatment of allergic rhinitis: A. Drug screening flow chart; B. Ranking of inhibition rates of different drugs on the NO content produced by RAW264.7 cells.

[0016] Figure 2 .In vitro efficacy and safety verification of Cinacalcet HCl: A. CCK-8 method was used to determine the effects of different concentrations of Cinacalcet HCl on RAW264.7 cell viability; B. Cinacalcet HCl inhibited the production of NO in a dose-dependent manner. ## P<0.01vsCTL and ** P<0.01vsLPS, n=3; C. Mouse primary hepatocytes (PHs); D. Cell viability of A549 cells after drug addition; ns: No significance vs0group.

[0017] Figure 3 .Cinacalcet HCl improves the behavioral phenotype of mice with allergic rhinitis: A. Flowchart of mouse modeling; B. Number of sneezing in mice during atomization; C. Number of nose scratching; D. Number of sneezing in mice on the last day of modeling; E. Number of nose scratching; F. Score. ## P<0.01vsCTL, * P<0.05and ** P<0.01vsAR,n=8.

[0018] Figure 4 .In vivo efficacy verification of Cinacalcet HCl: A. Effect of Cinacalcet HCl on the expression of IL-4, a Th1 cell-related inflammatory factor in the nasal mucosa of mice; B. Effect of Cinacalcet HCl on the expression of IL-33, a Th1 cell-related inflammatory factor in the nasal mucosa of mice; C. Effect of Cinacalcet HCl on the expression of Th2 cell-related inflammatory factors in the nasal mucosa of mice; D. Effect of Cinacalcet HCl on the balance of Th1 / Th2 cells; E. Inflammatory factor NO in serum; F. IL-6; G. OVA-IgE content detection; H. OVA-IgE content detection in nasal lavage fluid. ns: No significance vsCTL. # P<0.05and ## P<0.01vsCTL, * P<0.05and ** P<0.01vsAR,n=5.

[0019] Figure 5 .Pathological examination of mouse nasal mucosa tissue: A. H&E staining of mouse nasal mucosa tissue; B. AB-PAS staining of mouse nasal mucosa tissue; C. Statistics of nasal septum thickness of mouse nasal mucosa tissue; ## P<0.01vsCTL, *P<0.05vsAR,n=5.

[0020] Figure 6 . Detection of various indicators of mouse aerosol modeling: A. Body weight of mice during aerosol modeling; B. Food intake; C. Water intake statistics; D. H&E staining of tissue sections; E. Serum AST of mice; F. ALT level;

[0021] ns: No significance vs CTL. DETAILED DESCRIPTION

[0022] Abbreviations: Cinacalcet HCl; Budesonide (Budesonide is clinically used for patients with glucocorticoid-dependent or -independent bronchial asthma and asthmatic chronic bronchitis.)

[0023] The present invention is further described below in conjunction with examples, but the protection scope of the present invention is not limited thereto: Example 1 Drug screening to determine the efficacy of Cinacalcet HCl in improving allergic rhinitis

[0024] 1. Cell Culture

[0025] Mouse macrophage RAW264.7 was purchased from the American Type Culture Collection. RAW264.7 cells were cultured in a cell culture incubator at 37°C, 5% CO2, constant temperature and humidity. Primary mouse hepatocytes were isolated and inoculated into 96-well plates, and drug treatment was added 12 hours later.

[0026] 2. Drug screening experiments

[0027] NO kit was purchased from Bio-Technology Co., Ltd. RAW264.7 cells were seeded in 24-well plates at a density of approximately 2×10 5 Cells / well, cultured in an incubator for 10 hours. Use serum-free DMEM to replace the medium for 2 hours to synchronize the cells. Use DMEM medium containing 100ng / mL LPS to culture for 12 hours, replace the medium with serum-free DMEM medium containing 100ng / mL LPS and 5μM drug (small molecule compound library, Selleck, USA, catalog number: L1300) or serum-free medium containing 0.1% DMSO (control group), and continue to incubate in a 37°C incubator for 12 hours. After the drug treatment, collect the supernatant of each well in a 1.5mL centrifuge tube and centrifuge at 1000r and room temperature for 5 minutes. Add 50μL of the supernatant of each sample to a 96-well plate (3 replicates), and then add 50μL of Griess Reagent I and Griess Reagent II respectively, and use an enzyme reader to detect the absorbance at 540nm.

[0028] 3. Animal husbandry and treatment

[0029] 40 6-week-old Balb / c male mice were purchased from Jicui Yaokang. After one week of adaptation, they were randomly divided into 5 groups, namely the control group, modeling group, positive control group (Budesonide), low-dose administration group and high-dose administration group. Except for the control group, the rest of the mice were intraperitoneally injected with ovalbumin and aluminum hydroxide suspension (200μg ovalbumin + 2mg aluminum hydroxide / 200μL normal saline / mouse), and the control group was intraperitoneally injected with the same volume of normal saline. The injection was once every 3 days, for a total of 5 injections, which was the basic sensitization stage. After the basic sensitization stage, ovalbumin solution (20μg / 20μL / mouse) was used for local nasal drops for sensitization every day, and normal saline was used for nasal drops in the control group. During this period, nebulization was performed at the same time, and the nebulization doses were: 5% (w / v) Budesonide, 0.1% (w / v) Cinacalcet HCl and 0.5% (w / v) Cinacalcet HCl; the spray volume was 2mL / 10min, and the nebulization was 10min / time. During the local sensitization process, behavioral observations were performed, and the number of sneezing and scratching of the mice within 10min was recorded immediately after nasal drops, and the behavioral data were scored. The scoring criteria are: 1 point for 1 to 3 sneezes, 2 points for 4 to 10 sneezes, and 3 points for more than 10 sneezes; 1 point for 1 to 5 scratching of the nose, 2 points for 6 to 15 scratching of the nose, and 3 points for more than 15 scratching of the nose; 1 point for runny nose at the nostrils, 2 points for runny nose to the mouth, and 3 points for runny nose all over the face. A behavioral score of 5 points or more proves that the model is successful, and the drug is administered for a total of 14 days. On the last day of modeling, behavioral scores were performed again. 24 hours later, the mice were euthanized by spinal decapitation, and blood, nasal lavage fluid, nasal mucosa and other tissues were collected.

[0030] 4. Total RNA Extraction from Tissues

[0031] After the modeling, the mice were killed by dislocating the neck, and their nasal mucosa was separated and placed in a 1.5mL centrifuge tube and stored in a -80℃ refrigerator. When extracting tissue, 2 magnetic beads and 1mL Trizol were added to the centrifuge tubes respectively, and the tissue grinder was used for grinding and crushing. The grinding procedure was to grind for 1min, stop for 1min, and a total of three cycles. After grinding, 200μL of chloroform was added to each centrifuge tube, vortex 10s, and the sample was fully mixed. Centrifuge at 13000rpm, 4℃, 10min. 400μL of the supernatant was aspirated and transferred to a new RNase-free centrifuge tube, and then an equal volume of isopropanol was added, vortex 10s to mix it thoroughly, and let it stand at -20℃ for 20min. Centrifuge at 13000rpm, 4℃, 10min, and a white flocculent precipitate appeared at the bottom of the tube. The supernatant was discarded, and the supernatant was absorbed as much as possible with an inverted absorbent paper, and care was taken to prevent the white precipitate from slipping. Add 1mL of 75% ethanol DEPC water solution, vortex to suspend the white precipitate, centrifuge at 13000rpm, 4℃, 10min, discard the supernatant as much as possible, turn it upside down, use absorbent paper to absorb the supernatant as much as possible, be careful to prevent the white precipitate from sliding, and place it in a fume hood at room temperature for about 10min to dry. After the ethanol evaporates completely, the RNA white precipitate will become transparent. According to the size of the RNA white clumps in the previous step, add 25-100μL DEPC water, vortex to mix, put it in a 65℃ metal bath for 7min, observe whether the RNA is completely dissolved, vortex for 10s, centrifuge instantly, and store at -80℃. Use agarose gel method to detect whether the extracted RNA is qualified.

[0032] 5. Reverse Transcription PCR

[0033] The extracted total RNA concentration was detected by Nanodrop, and the reverse transcription kit instructions of Nanjing Novozyme Biotechnology Co., Ltd. were followed. The program settings are shown in the following table:

[0034] Table 1 PCR reverse transcription program

[0035]

[0036]

[0037] After the program is completed, add 90 μL RNase-free water and perform RT-qPCR detection immediately or store at -20°C for a long time. 6. Real-time fluorescence quantitative PCR (RT-qPCR)

[0038] Using the PrimerBank website ( https: / / pga.mgh.harvard.edu / primerbank / ) designed primers and commissioned Beijing Qingke Xinye Biotechnology Co., Ltd. to synthesize them. The primer sequences are shown in Table 3. The PCR reaction products were subjected to electrophoresis and melting curve analysis to verify the specificity of the primers. The expression abundance of cDNA was determined from the Ct value, and GAPDH was used as an internal reference. -△△Ct The relative gene expression was calculated.

[0039] Use 2×ChamQ Universal SYBR qPCR Master Mix Kit (purchased from Nanjing Novozyme Biotechnology Co., Ltd.) to prepare the reaction system according to the instructions, as shown in the following table:

[0040] Table 2 Fluorescence quantitative PCR system formula

[0041]

[0042] Table 3 Primer sequences

[0043]

[0044] After mixing, centrifuge briefly and place in RT-qPCR amplification tube for reaction. The program settings are as follows:

[0045] Table 4 Fluorescence quantitative PCR program

[0046]

[0047]

[0048] 7. Alanine aminotransferase and aspartate aminotransferase assay

[0049] Mouse serum collection and pretreatment: Use 1.5mL centrifuge tubes to collect mouse blood and place it at room temperature for more than 30 minutes. Serum precipitation can be observed. Centrifuge the centrifuge tube at 3000rpm, 4℃, and centrifuge for 20 minutes. Pipette the serum supernatant into a new tube, and store the aliquoted serum at -80℃. Follow the instructions in the alanine aminotransferase (alanine aminotransferase / ALT / GPT) and aspartate aminotransferase (aspartate aminotransferase / AST / GOT) detection kits of Nanjing Jiancheng Company to prepare the corresponding reagents and consumables. Pipette 20μL of ALT / AST matrix solution preheated to 37℃ into a 96-well plate, add 5μL of the serum sample to be tested, and mix it repeatedly with a pipette tip to avoid bubbles. Gently shake the plate and place it in a 37℃ air bath for 30min. Add 20μL of phenylhydrazine colorimetric solution to each well one by one, and mix it repeatedly with a pipette tip again to avoid bubbles. Gently shake the plate and place it in a 37℃ air bath for 20min. Add 200 μL of 0.4 M sodium hydroxide solution to each well to terminate the reaction and develop color. Gently shake the well plate, let it stand at room temperature for 15 minutes, and use a multifunctional microplate reader to measure OD505 / OD510nm to calculate the ALT / AST content in the serum.

[0050] 8. Hematoxylin–Eosin (H&E) staining analysis

[0051] Remove the tissue fixed with 4% paraformaldehyde, embed it with paraffin, and then slice it with a slicer, with a slice thickness of 5μm. Bake the slices until the paraffin in the slices melts, about 30 minutes. Transfer the slices to xylene for dewaxing for 5 minutes, and then use filter paper to absorb excess liquid. Transfer the slices to xylene and continue dewaxing for 10 minutes until the slices are transparent, and then use absorbent paper to absorb excess liquid. Transfer the slices to ethanol for 5 minutes, absorb excess liquid, and repeat 2 times, changing fresh ethanol each time. Soak the slices in 95% ethanol for 3 minutes, wash with running water for 5 minutes, and use filter paper to absorb excess water. Add hematoxylin staining solution to the slice sample, cover it completely, stain it at room temperature for 5 minutes, and wash it slightly. Use 1% HCl solution to differentiate for 8 seconds until the color of the slice changes from blue to red, and then rinse with tap water for about 30 minutes to make the slice return to blue. Add 0.5% eosin to the slice sample for staining for 1 minute, then wash the slice twice with 95% and 100% ethanol, 5 minutes each time; soak the slice in xylene for 5 minutes to decolorize. Take out the slice, absorb the excess liquid and seal it with neutral resin. Under microscopic examination, the cell nucleus appears blue, and other tissue components appear red with varying shades. Observe the H&E-stained slices under a microscope and take pictures.

[0052] 9. AB-PAS staining analysis

[0053] The tissue fixed with 4% paraformaldehyde was removed, embedded in paraffin, and then sliced ​​using a microtome with a thickness of 5 μm. The paraffin sections were routinely dewaxed to water, first stained with Alcian blue for 8-10 minutes, and the excess dye was washed off with running water, so that the acidic mucopolysaccharide in the cytoplasm was blue, while the middle-aged mucin was not stained. Then the periodic acid stain was dripped for 20 minutes, and the distilled water was rinsed 3 times, 1 minute each time, to oxidize the hydroxyl groups on the two adjacent carbon atoms in the carbohydrate to aldehyde groups. Then the Schiff reagent was dripped for 40 minutes, and the distilled water was rinsed 3 times, 1 minute each time. The Schiff reagent reacted with the aldehyde group to generate a purple-red substance. Then the Mayer hematoxylin stain was dripped for 2-3 minutes and rinsed with running water. Finally, the sections were transparentized by three cylinders of anhydrous ethanol and xylene, and then neutral gum sealing agent was added to seal the sections. Alcian blue and PAS staining can distinguish acidic glycoproteins from neutral glycoproteins. Acidic mucus is blue, glycogen and neutral mucus are red, and mixed mucus is blue-purple. The AB-PAS-stained sections were observed under a microscope and photographed.

[0054] result:

[0055] like Figure 1 As shown in Figure 2, the NO method was used to screen out drugs with high anti-inflammatory effects from 1430 small molecule compounds approved by the FDA. Figure 2 As shown in the results, it was first discovered that Cinacalcet HCl can dose-dependently inhibit the release of NO in the RAW264.7 cell inflammation model. The NO release rate in the high-dose group dropped from 18.89 μM to 15.82 μM. However, this drug concentration did not kill mouse primary hepatocytes and human non-small cell lung cancer cells A549. This shows that Cinacalcet HCl has good safety. Figure 3 As shown in the figure, we found through animal experiments that Cinacalcet HCl can significantly inhibit the behavioral phenotype of mice with allergic rhinitis. The behavioral indicators on the last day of modeling showed that the high-dose group sneezed and scratched their nose less frequently than the positive control group. The scores of the two groups were the same and both were below 5 points, indicating that the allergic symptoms of mice were significantly improved after treatment. Figure 4As shown in the results, Cinacalcet HCl can effectively inhibit the production of inflammatory mediators in the nasal mucosa and serum of mice with allergic rhinitis in vivo, and the high-dose group was superior to the positive control group in some indicators. For example, at the mRNA level, the Th1 cell-related inflammatory factor TNF-β in the high-dose group was higher than that in the positive control group, while the Th2 cell-related inflammatory factor IL-4 was lower than that in the positive control group. Therefore, the high-dose group significantly increased Th1 / Th2, that is, it improved the Th1 / Th2 cell imbalance caused by allergic rhinitis. In addition, the expression levels of inflammatory mediators NO and IL-6 in the serum of the high-dose group and the positive control group were similar, and the positive control group had a better effect on the inhibition of OVA-IgE in serum and nasal lavage fluid. As Figure 5 As shown in the results, Cinacalcet HCl can improve the swelling of the nasal mucosa and the number of goblet cells in mice with allergic rhinitis. The nasal mucosa structure of mice in the control group was intact, the epithelial cells were evenly arranged, and there was no obvious swelling and vasodilation in the basal layer. In the sensitized group, the nasal mucosal epithelial cells were desquamated, the ciliary layer was missing, the basal layer was thickened, the small blood vessels under the mucosa were dilated, and eosinophil infiltration was visible in the propria. The nasal mucosal structure of the Cinacalcet HCl group and the positive control group improved to varying degrees. The statistics of the nasal septum thickness showed that the nasal septum thickness decreased more significantly in the high-dose group than in the positive control group. At the same time, the goblet cells in the nasal mucosa of mice in the sensitized group proliferated, and the positive control group was better than the high-dose group in inhibiting goblet cell hyperplasia. As shown in the figure Figure 6 As shown in the results, Cinacalcet HCl also did not affect the body weight of mice, and there was no statistical difference in the amount of food and water consumed among the groups. Compared with the control group, the morphological and structural characteristics of H&E sections of heart, liver, spleen, lung, and kidney tissues in the Cinacalcet HCl-treated group did not change, and there was no difference in serum AST and ALT levels, indicating that Cinacalcet HCl has good in vivo safety.

Claims

1. Use of Cinacalcet hydrochloride in the preparation of a drug for treating allergic rhinitis, wherein the structural formula of Cinacalcet hydrochloride is as follows: Cinacalcet hydrochloride.

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