Application of cinacalcet hydrochloride in the preparation of drugs for treating allergic rhinitis

Abstract

This invention relates to cinacalcet hydrochloride, specifically to its application in the preparation of drugs for treating allergic rhinitis. This invention is the first to discover that cinacalcet hydrochloride has a therapeutic effect on allergic rhinitis. Specifically, drugs that effectively inhibit NO release from RAW264.7 cells (an in vitro inflammation model was established using LPS stimulation of RAW264.7 cells) were screened from 1430 FDA-approved small molecule compounds, and cinacalcet hydrochloride was discovered for the first time to dose-dependently inhibit NO release in the in vitro inflammation model. Furthermore, in vivo therapeutic experiments also showed that cinacalcet hydrochloride effectively inhibits the release of allergic mediators in vivo without affecting the body weight of mice, demonstrating good safety.

Description

Technical Field

[0001] This invention relates to cinacalcet hydrochloride, and more specifically to the use of cinacalcet hydrochloride in the preparation of drugs for treating allergic rhinitis. Background Technology

[0002] Allergic rhinitis (AR), also known as hay fever, is caused by external allergens stimulating the body's nasal mucosa's innate immune cells, causing them to produce specific IgE antibodies. These IgE antibodies then bind to basophils and mast cells, sensitizing the body. When the same allergen is encountered again, effector cells release inflammatory factors. These factors act on small blood vessels, smooth muscle, and glands in the respiratory tract, leading to swelling of the nasal mucosa and turbinate edema, classifying it as a type I hypersensitivity reaction. Common symptoms include nasal itching, runny nose, and sneezing. Some patients may also experience itchy eyes, ears, throat, decreased sense of smell, and asthma. It can be very debilitating for some patients, leading to irritability, insomnia, and fatigue, reducing work efficiency and learning ability. Risk factors for allergic rhinitis mainly include genetic factors, air pollution, work environment pollution, smoking, frequent respiratory infections, extensive use of antibiotics, medication use, or a history of pollen allergies. Furthermore, the recurrence of allergic rhinitis is often the result of multiple factors working together, so its pathological phenomena are also quite complex and have individual specificity.

[0003] Modern medicine treats allergic rhinitis primarily through medication, surgery, and immunotherapy. Medication mainly uses antihistamines, corticosteroids, mast cell stabilizers, and decongestants; these drugs have varying degrees of efficacy, but usually only control symptoms and cannot cure the condition, often accompanied by side effects such as dry mouth, pharyngitis, headache, dizziness, drowsiness, palpitations, and neuropsychiatric events. Surgery can only address symptoms such as abnormal nasal growths and polyps in severe rhinitis patients, but it does not completely cure allergic rhinitis. While immunotherapy is currently the only specific treatment method, it also faces challenges such as low safety and poor patient compliance.

[0004] Cinacalcet is the first drug in a new class of compounds called calcimimetics. It activates 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. No literature reports on cinacalcet hydrochloride for the treatment of allergic rhinitis. Summary of the Invention

[0005] Purpose of the invention

[0006] This invention provides a new use for cinacalcet hydrochloride, namely, the role of cinacalcet hydrochloride in the treatment of allergic rhinitis.

[0007] Technical solution

[0008] The use of cinacalcet hydrochloride in the preparation of drugs for treating allergic rhinitis, wherein the structural formula of cinacalcet hydrochloride is shown below:

[0009]

[0010] In the technical solution of the present invention, the nebulized dosage of cinacalcet hydrochloride is 0.1% (w / v) and 0.5% (w / v), the spray volume is 2 mL / 10 min, and the time is 10 min / time.

[0011] In this invention, adult Balb / c mice are used for systemic and nasal sensitization. After systemic sensitization, cinacalcet hydrochloride is administered via nebulization during the nasal sensitization phase. Mouse behavior is observed, and allergy symptoms are scored to evaluate the therapeutic effect of the drug on allergic rhinitis. Studies have shown that cinacalcet hydrochloride can inhibit the release of inflammatory factors in allergic rhinitis and alleviate nasal allergy symptoms in AR model mice.

[0012] The beneficial effects of this invention are:

[0013] New drug development is characterized by long development cycles, high costs, and low success rates, while drug repurposing can significantly shorten the development cycle and reduce development risks and costs. Compounds already approved by the FDA are characterized by safety, efficacy, and controllable quality. Developing new functions for existing drugs can greatly shorten the drug development cycle, reduce costs, and lower risks. We used high-throughput screening to screen FDA-approved compounds and determined their anti-inflammatory effects by measuring the inhibitory effect of each drug on NO release in an inflammation model established in RAW264.7 cells.

[0014] This invention is the first to discover that cinacalcet hydrochloride has an inhibitory and therapeutic effect on allergic rhinitis. Specifically, drugs that effectively inhibit NO release in RAW264.7 cells (an in vitro inflammation model was established using LPS stimulation of RAW264.7 cells) were screened from 1430 FDA-approved small molecule compounds, and cinacalcet hydrochloride was found for the first time to inhibit NO release in an in vitro inflammation model in a dose-dependent manner. Furthermore, in vivo therapeutic experiments also showed that cinacalcet hydrochloride effectively inhibits the release of allergic mediators in vivo without affecting the body weight of mice, demonstrating good safety. Attached Figure Description

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

[0016] Figure 2 In vitro efficacy and safety validation of Cinacalcet HCl: A. CCK-8 assay to determine the effect of different concentrations of Cinacalcet HCl on RAW264.7 cell viability; B. Cinacalcet HCl dose-dependently inhibits NO production. ## P<0.01 vs CTL and ** P < 0.01 vs LPS, n = 3; C. Primary mouse hepatocytes (PHs); D. Cell viability of A549 cells after drug administration; ns: No significance vs 0 group.

[0017] Figure 3 Cinacalcet HCl improved the behavioral phenotype of allergic rhinitis in mice: A. Flowchart of mouse modeling; B. Number of sneezes during nebulization; C. Number of nose scratches; D. Sneezing on the last day of modeling; E. Number of nose scratches; F. Scoring. ## P<0.01 vsCTL, * P < 0.05 and ** P < 0.01 vs AR, n = 8.

[0018] Figure 4 In vivo efficacy validation of Cinacalcet HCl: A. Effect of Cinacalcet HCl on the expression of IL-4, a Th1 cell-related inflammatory cytokine, in mouse nasal mucosa; B. Effect of Cinacalcet HCl on the expression of IL-33, a Th1 cell-related inflammatory cytokine, in mouse nasal mucosa; C. Effect of Cinacalcet HCl on the expression of Th2 cell-related inflammatory cytokines in mouse nasal mucosa; D. Effect of Cinacalcet HCl on the Th1 / Th2 cell balance; E. Serum inflammatory cytokine NO; F. IL-6; G. Detection of OVA-IgE content; H. Detection of OVA-IgE content in nasal lavage fluid. ns: No significance vs. CTL. # P<0.05 and ## P<0.01 vsCTL, * P < 0.05 and ** P < 0.01 vs AR, 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. Statistical analysis of nasal septum thickness in mouse nasal mucosa tissue; ## P<0.01 vsCTL, *P < 0.05 vs AR, n = 5.

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

[0021] ns: No significance vsCTL. Detailed Implementation

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

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

[0024] 1. Cell Culture

[0025] RAW264.7 mouse macrophages were purchased from the American Culture Collection. RAW264.7 cells were cultured in a cell culture incubator at 37°C, 5% CO2, under constant temperature and humidity conditions. Primary mouse hepatocytes were isolated and seeded into 96-well plates, and treated with medication 12 hours later.

[0026] 2. Drug screening experiments

[0027] The NO kit was purchased from Beyotime Biotechnology Co., Ltd. RAW264.7 cells were seeded in 24-well plates at a density of approximately 2 × 10⁻⁶ cells / well. 5 Cells per well were incubated for 10 h. Cells were then treated with serum-free DMEM for 2 h to synchronize them. After incubation for 12 h in DMEM containing 100 ng / mL LPS, the medium was replaced with serum-free DMEM containing 100 ng / mL LPS and 5 μM drug (small molecule compound library, Selleck, catalog number: L1300) or serum-free medium containing 0.1% DMSO (control group). Incubation continued at 37°C for 12 h. After drug treatment, the supernatant from each well was collected into a 1.5 mL centrifuge tube and centrifuged at 1000 rpm for 5 min at room temperature. 50 μL of supernatant from each sample was added to each well of a 96-well plate (3 replicates), followed by 50 μL of Griess Reagent I and Griess Reagent II. The absorbance at 540 nm was measured using a microplate reader.

[0028] 3. Feeding and handling of laboratory animals

[0029] Forty 6-week-old male Balb / c mice were purchased from Jicui Pharmaceutical Co., Ltd. After one week of acclimatization, they were randomly divided into five groups: a control group, a model group, a positive control group (Budesonide), a low-dose treatment group, and a high-dose treatment group. Except for the control group, the other mice received intraperitoneal injections of ovalbumin and aluminum hydroxide suspension (200 μg ovalbumin + 2 mg aluminum hydroxide / 200 μL saline / mouse), while the control group received the same volume of saline intraperitoneally. Injections were given every 3 days for a total of 5 times, constituting the basal sensitization phase. After the basal sensitization phase, ovalbumin solution (20 μg / 20 μL / mouse) was administered daily via nasal drops for sensitization, while the control group received saline nasal drops. During this period, nebulized drug administration was performed simultaneously at the following doses: 5% (w / v) budesonide, 0.1% (w / v) Cinacalcet HCl, and 0.5% (w / v) Cinacalcet HCl; the spray volume was 2 mL / 10 min, and the nebulization was performed every 10 min. During local sensitization, behavioral observations were conducted. Immediately after nasal instillation, the number of sneezes and nose scratchings in mice within 10 min were recorded, and scores were assigned based on the behavioral data. The scoring criteria were as follows: 1-3 sneezes = 1 point, 4-10 sneezes = 2 points, more than 10 sneezes = 3 points; 1-5 nose scratchings = 1 point, 6-15 nose scratchings = 2 points, more than 15 nose scratchings = 3 points; nasal discharge = 1 point, discharge to the mouth = 2 points, and full-face discharge = 3 points. A behavioral score of 5 or higher was considered a successful model establishment. The drug administration lasted for 14 days. On the last day of modeling, behavioral scores were assessed again. 24 hours later, the mice were euthanized by spinal cord dissection, and blood, nasal irrigation fluid, and nasal mucosa were collected.

[0030] 4. Total RNA extraction from tissues

[0031] Mice were euthanized by cervical dislocation after modeling. Nasal mucosa was separated and placed in 1.5 mL centrifuge tubes, stored at -80°C. For tissue extraction, two magnetic beads and 1 mL of Trizol were added to each centrifuge tube. The tissue was then homogenized using a tissue homogenizer with a 1-min, 1-min pause cycle for three cycles. After homogenization, 200 μL of chloroform was added to each centrifuge tube, vortexed for 10 s to thoroughly mix the sample, and centrifuged at 13000 rpm, 4°C, for 10 min. 400 μL of the supernatant was transferred to a new RNase-free centrifuge tube, and an equal volume of isopropanol was added, vortexed for 10 s to thoroughly mix, and incubated at -20°C for 20 min. Centrifugation was then performed at 13000 rpm, 4°C, for 10 min. A white flocculent precipitate appeared at the bottom of the tube; the supernatant was discarded, and the supernatant was blotted away with inverted absorbent paper, taking care to prevent the white precipitate from slipping. Add 1 mL of 75% ethanol-DEPC aqueous solution, vortex to suspend the white precipitate, centrifuge at 13000 rpm, 4°C, for 10 min, discard as much supernatant as possible, invert the container, and absorb as much supernatant as possible with absorbent paper, taking care to prevent the white precipitate from slipping. Air dry at room temperature in a fume hood for about 10 min. After the ethanol has completely evaporated, the white RNA precipitate will become transparent. Add 25–100 μL of DEPC water according to the size of the white RNA clumps from the previous step, vortex to mix well, place in a 65°C metal bath for 7 min, observe whether the RNA has completely dissolved, vortex for 10 s, centrifuge briefly, and store at -80°C. Test the extracted RNA for quality using the agarose gel method.

[0032] 5. Reverse transcription PCR

[0033] The concentration of extracted total RNA was detected using Nanodrop, following the instructions for the reverse transcription kit from Nanjing Novizan Biotechnology Co., Ltd. The program settings are shown in the table below:

[0034] Table 1 PCR Reverse Transcription Procedure

[0035]

[0036]

[0037] After the procedure is complete, add 90 μL of RNase-free water. RT-qPCR detection can be performed immediately, or the sample can be stored long-term at -20°C. 6. Real-time quantitative PCR (RT-qPCR)

[0038] Use the PrimerBank website ( https: / / pga.mgh.harvard.edu / primerbank / Primers were designed and synthesized by Beijing Qingke Xinyue Biotechnology Co., Ltd. Primer sequences are shown in Table 3. The specificity of the primers was verified by electrophoresis and melting curve analysis of the PCR reaction products. The cDNA expression abundance was determined from the Ct value, using GAPDH as an internal control. -△△Ct The method calculates the relative gene expression level.

[0039] The reaction system was prepared according to the instructions using the 2×ChamQ Universal SYBR qPCR Master Mix kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.), as shown in the table below:

[0040] Table 2 Formulas for Real-Time PCR System

[0041]

[0042] Table 3 Primer sequences

[0043]

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

[0045] Table 4. Quantitative Real-Time PCR Procedure

[0046]

[0047]

[0048] 7. Determination of alanine aminotransferase and aspartate aminotransferase

[0049] Mouse serum collection and pretreatment: Collect mouse blood using 1.5 mL centrifuge tubes and allow to stand at room temperature for at least 30 minutes until serum separation is observed. Centrifuge the tubes at 3000 rpm and 4°C for 20 minutes, then transfer the serum supernatant to a new tube. Store the aliquoted serum at -80°C. Prepare the necessary reagents and consumables according to the instructions in the alanine aminotransferase (ALT / GPT) and aspartate aminotransferase (AST / GOT) detection kits from Nanjing Jiancheng Company. Pipette 20 μL of ALT / AST matrix solution preheated to 37°C into a 96-well plate, add 5 μL of the serum sample to be tested, and mix thoroughly by pipetting repeatedly to avoid air bubbles. Gently shake the plate and incubate at 37°C for 30 minutes. Add 20 μL of phenylhydrazine chromogenic solution to each well, and mix thoroughly by pipetting repeatedly to avoid air bubbles. Gently shake the plate and incubate at 37°C for 20 minutes. Add 200 μL of 0.4 M sodium hydroxide solution to each well to terminate the reaction and develop color. Gently shake the plate and let it stand at room temperature for 15 min. Then, use a multi-mode microplate reader to measure the OD505 / OD510nm ratio and calculate the serum ALT / AST content.

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

[0051] Tissue fixed with 4% paraformaldehyde was extracted, embedded in paraffin, and sectioned using a microtome to a thickness of 5 μm. The sections were baked until the paraffin melted, approximately 30 min. The sections were then transferred to xylene for dewaxing for 5 min, followed by blotting with filter paper to remove excess liquid. The sections were then transferred back to xylene for further dewaxing for 10 min until transparent, and blotted with absorbent paper to remove excess liquid. The sections were then treated with ethanol for 5 min, blotting to remove excess liquid, and this process was repeated twice, using fresh ethanol each time. The sections were then immersed in 95% ethanol for 3 min, rinsed with running water for 5 min, and blotted with filter paper to remove excess water. Hematoxylin staining solution was added to the section samples, completely covering them, and stained at room temperature for 5 min, followed by a slight rinse with water. Differentiation was performed using 1% HCl solution for 8 s until the section color changed from blue to red, followed by rinsing with tap water for approximately 30 min to allow the sections to regain their blue color. Add 0.5% eosin to the slide sample for 1 min, then wash the slide twice with 95% and 100% ethanol, 5 min each time; immerse the slide in xylene for 5 min to decolorize. Remove the slide, aspirate excess liquid, and mount with neutral resin. Microscopic examination reveals that cell nuclei appear blue, while other tissue components show varying shades of red. Observe and photograph the H&E-stained slides under a microscope.

[0052] 9. AB-PAS staining analysis

[0053] Tissue fixed in 4% paraformaldehyde was extracted, embedded in paraffin, and then sectioned using a microtome to a thickness of 5 μm. Paraffin sections were routinely dewaxed to water, stained with Alcian blue for 8-10 min, and rinsed with running water to remove excess stain, resulting in acidic mucopolysaccharides in the cytoplasm turning blue while middle-aged mucins remained unstained. Next, periodic acid was stained for 20 min, followed by rinsing three times with distilled water for 1 min each time, oxidizing the hydroxyl groups on adjacent carbon atoms of the sugars to aldehyde groups. Then, Schiff's reagent was stained for 40 min, followed by rinsing three times with distilled water for 1 min each time, as Schiff's reagent reacts with the aldehyde groups to produce a purple-red substance. Finally, Mayer's hematoxylin was stained for 2-3 min, followed by rinsing with running water. Finally, the sections were cleared with three baths of anhydrous ethanol and xylene, and then mounted with neutral resin mounting medium. The combined staining with Alcian blue and PAS can distinguish between acidic and neutral glycoproteins. Acidic mucus appears blue, glycogen and neutral mucus appear red, and mixed mucus appears bluish-purple. The AB-PAS-stained sections were observed and photographed under a microscope.

[0054] result:

[0055] like Figure 1 As shown, drugs with highly effective anti-inflammatory effects were screened from 1430 FDA-approved small molecule compounds using a NO determination method. Figure 2 As shown, this study is the first to demonstrate that Cinacalcet HCl can dose-dependently inhibit NO release in a RAW264.7 cell inflammation model, reducing the NO release rate from 18.89 μM to 15.82 μM in the high-dose group. Furthermore, this drug concentration did not kill mouse primary hepatocytes or human non-small cell lung cancer cells A549. This indicates that Cinacalcet HCl has good safety profile. Figure 3 As shown, our animal experiments revealed that Cinacalcet HCl significantly inhibited the behavioral phenotype of allergic rhinitis in mice. On the last day of modeling, behavioral indicators showed that the high-dose group sneezed and scratched their noses less frequently than the positive control group. The scores of both groups were similar and below 5, indicating that the allergy symptoms in the mice were significantly improved after treatment. Figure 4As shown, Cinacalcet HCl effectively inhibited the production of inflammatory mediators in the nasal mucosa and serum of mice with allergic rhinitis in vivo, with the high-dose group showing superior performance compared to the positive control group in some indicators. For example, at the mRNA level, the high-dose group had higher levels of Th1 cell-related inflammatory factor TNF-β than the positive control group, while the high-dose group had lower levels of Th2 cell-related inflammatory factor IL-4. Therefore, the high-dose group significantly increased the Th1 / Th2 ratio, thus improving the Th1 / Th2 cell imbalance caused by allergic rhinitis. Furthermore, 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, while the positive control group showed better inhibitory effects on OVA-IgE in serum and nasal lavage fluid. Figure 5 As shown, Cinacalcet HCl can improve nasal mucosal swelling and goblet cell count in mice with allergic rhinitis. In the control group, the nasal mucosa structure was intact, epithelial cells were evenly arranged, and there was no significant swelling or vasodilation in the basal layer. In the sensitized group, the nasal mucosa showed epithelial cell shedding, ciliary layer loss, basal layer thickening, submucosal small blood vessel dilation, and eosinophil infiltration in the lamina propria. Both the Cinacalcet HCl group and the positive control group showed varying degrees of improvement in nasal mucosal structure. Statistical analysis of nasal septum thickness showed a more significant decrease in nasal septum thickness in the high-dose group compared to the positive control group. Simultaneously, goblet cell proliferation was observed in the nasal mucosa of the sensitized mice, and the positive control group showed a better inhibitory effect on goblet cell proliferation than the high-dose group. Figure 6 As shown, Cinacalcet HCl did not affect the body weight of mice, and there were no statistically significant differences in food and water intake 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 groups were unchanged, and there were no differences in serum AST and ALT levels, indicating that Cinacalcet HCl has good in vivo safety.

Claims

1. The use of cinacalcet hydrochloride in the preparation of drugs for treating allergic rhinitis, wherein the structural formula of cinacalcet hydrochloride is shown below: Cinacalcet hydrochloride.

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