Solution for aerosol inhalation of CK2 inhibitor CX4945 as well as preparation method and application of solution

Abstract

The invention discloses a solution for aerosol inhalation of a CK2 inhibitor CX4945 as well as a preparation method and application of the solution. The solution for aerosol inhalation, provided by the invention, is prepared from 10 to 20mg of CX4945, 10 to 20mg of an osmotic pressure regulator, a pH regulator and water for injection according to a single dose of 2mL. The CK2 inhibitor and the aerosol inhalation solution thereof provided by the invention can significantly inhibit neutrophil from generating NETs and the pro-inflammatory effect of the NETs, and can inhibit respiratory tract inflammations including chronic obstructive pulmonary disease (COPD), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), asthma and cystic fiber. The aerosol inhalation solution directly acts on the lung by being inhaled from the mouth and the nose, so that the metabolic process of the liver and the kidney to the medicine is reduced, the liver and kidney injury to patients is remarkably reduced, and the aerosol inhalation solution is particularly convenient for children, the old and severe patients to take.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a solution for aerosol inhalation of a CK2 inhibitor CX4945, and a preparation method and application thereof. Background Art

[0002] CX4945 (CAS NO.: 1009820-21-6) is the world's first and currently the only orally bioavailable CK2 small molecule inhibitor to enter human clinical trials. It has few side effects and strong patient compliance. Its structural formula is as follows:

[0003]

[0004] The CK2 inhibitor CX-4945 (trade name silmitasertib) has been designated an orphan drug by the FDA for the treatment of cholangiocarcinoma. However, for some children, the elderly, and critically ill patients, drug metabolism by the liver and kidneys can cause liver and kidney damage.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a solution for aerosol inhalation of a CK2 inhibitor CX4945, a preparation method thereof and a new application thereof.

[0007] In a first aspect, the present invention provides a solution for aerosol inhalation of the CK2 inhibitor CX4945, wherein a single dose of 2 mL comprises: 10-20 mg of CX4945, 10-20 mg of an osmotic pressure regulator, a pH regulator, and water for injection;

[0008] The osmotic pressure regulator is selected from one or more of sodium chloride, potassium chloride and mannitol;

[0009] The pH regulator is selected from one or more of sodium hydroxide, hydrochloric acid, sodium dihydrogen phosphate, citric acid and sodium citrate;

[0010] The pH range of the solution for aerosol inhalation is controlled at 4.0 to 5.0;

[0011] The osmotic pressure molar concentration of the solution for aerosol inhalation is controlled at 270mosm / l to 310mosm / l;

[0012] The solution for aerosol inhalation does not contain surfactants and metal complexes.

[0013] Preferably, the osmotic pressure regulator is sodium chloride.

[0014] Preferably, the pH adjuster is sodium hydroxide.

[0015] Preferably, the pH range of the solution for aerosol inhalation is controlled at 4.0 to 4.6.

[0016] In a second aspect, the present invention provides a method for preparing a solution for aerosol inhalation of the CK2 inhibitor CX4945, comprising the following steps: dissolving CX4945 powder in 1 mL of 0.1 M sodium hydroxide solution, dissolving and dispersing, adjusting the pH value and osmolarity, and filling.

[0017] In a third aspect, the present invention provides the use of the CK2 inhibitor CX4945 in the preparation of a drug for inhibiting the production of NETs by neutrophils and the pro-inflammatory effect of the generated NETs.

[0018] In a fourth aspect, the present invention provides use of the CK2 inhibitor CX4945 in the preparation of a medicament for inhibiting respiratory inflammatory diseases characterized by neutrophil imbalance.

[0019] The respiratory inflammatory diseases include chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, asthma, cystic fibrosis, upper respiratory tract infection and lower respiratory tract infection.

[0020] In a fifth aspect, the present invention provides the use of the above-mentioned CK2 inhibitor CX4945 solution for aerosol inhalation in the preparation of a drug for inhibiting the production of NETs by neutrophils and the pro-inflammatory effect of the generated NETs.

[0021] In a sixth aspect, the present invention provides use of the nebulized inhalation solution of the CK2 inhibitor CX4945 in the preparation of a medicament for inhibiting respiratory inflammatory diseases characterized by neutrophil imbalance.

[0022] The respiratory inflammatory diseases include chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, asthma, cystic fibrosis, upper respiratory tract infection and lower respiratory tract infection.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The CK2 inhibitor and its aerosol inhalation solution provided by the present invention can significantly inhibit the production of NETs by neutrophils and the pro-inflammatory effect of NETs, ​​thereby inhibiting respiratory inflammation, including chronic obstructive pulmonary disease (COPD), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), asthma, and cystic fibrosis. Specifically, it is manifested as:

[0025] 1) In vitro cell experiments have shown that CX4945 can effectively reduce the CK2 phosphorylation activity of neutrophils, reduce the level of ROS generation, reduce NETs production, and significantly inhibit the pro-inflammatory ability of NETs on macrophages.

[0026] 2) Experiments on a chronic obstructive pulmonary disease (COPD) mouse model showed that by using CX4945 aerosol inhalation inhibition, neutrophil infiltration in the mouse lungs was significantly reduced, the level of NETs complexes in the alveolar lavage fluid was significantly reduced, and the overall survival rate was increased.

[0027] 3) Experiments on the LPS-induced acute lung injury model in mice showed that the use of CX4945 aerosol inhalation agent to inhibit the lung edema in mice was alleviated, alveolar and interstitial inflammation was significantly reduced, lung injury scores were significantly reduced, neutrophil counts in alveolar lavage fluid were reduced, and cytokine levels were significantly reduced.

[0028] 2. The atomized inhalation solution provided by the present invention acts directly on the lungs through inhalation through the mouth and nose, reducing the metabolism of the drug by the liver and kidneys, significantly reducing liver and kidney damage to patients, and is particularly convenient for children, the elderly, and seriously ill patients.

[0029] 3. The preparation process of the aerosol inhalation solution provided by the present invention is simple, the equipment cost is low, it is convenient for industrial production, and it is also beneficial to improve the stability of the product batches. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a comparison chart showing that CX4945 significantly reduced the phosphorylation activity of CK2 in neutrophils in in vitro cell experiments.

[0031] Figure 2 This is a comparison chart showing that CX4945 significantly reduced the ROS generation levels of neutrophils and mouse bone marrow neutrophils in in vitro cell experiments.

[0032] Figure 3 This is a comparison chart showing that CX4945 significantly reduced NETs production in neutrophils in in vitro cell experiments.

[0033] Figure 4 This is a comparison chart showing that CX4945 significantly reduced the pro-inflammatory ability of neutrophils to produce NETs in in vitro cell experiments.

[0034] Figure 5 This is a comparison chart showing that lung tissue edema and inflammatory cell infiltration in ALI mice were significantly reduced after treatment with CX4945 nebulized inhalation solution.

[0035] Figure 6 This is a comparison chart showing that the level of NETs complex (MPO-DNA complex) in the alveolar lavage fluid of ALI mice was significantly reduced after treatment with CX4945 nebulized inhalation solution.

[0036] Figure 7 This is a comparison chart showing a significant increase in the survival rate of lupus mice after acute lung injury after treatment with CX4945 nebulized inhalation solution. DETAILED DESCRIPTION

[0037] The present invention will be further described below with reference to specific examples, but the present invention is not limited to the following examples.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0039] Unless otherwise specified, the reagents, materials, instruments, etc. used in the following examples can be obtained from commercial sources.

[0040] CX4945 used in the following examples was purchased from MedChemExpress (MCE) under the trade name Silmitasertib (HY-50855).

[0041] Example 1: In vitro cell experiments on the inhibition of NET production by CK2 inhibitor CX4945 and the pro-inflammatory effect of NET production by neutrophils are as follows:

[0042] 1) EDTA-anticoagulated blood samples were collected from healthy volunteers. Peripheral neutrophils were purified by Ficoll-pague combined with dextran (Dextron) sedimentation. The cells were lysed on ice for 30 minutes using RIPA high-performance protein lysis buffer (Solarbio, supplemented with protease and phosphatase inhibitors). The supernatant was collected after centrifugation at 10,000 rpm for 15 minutes to obtain the neutrophil protein sample.

[0043] Protein quantification was performed using a BCA kit (Thermo). Before protein electrophoresis, 5× protein loading buffer (Solarbio) was added and boiled in a 100°C metal bath block for 15 minutes.

[0044] 2) Load a total of 15 μg of protein onto a 12% precast gel (ACE) and run electrophoresis at 150 V for 40 min. Transfer the membrane using a Bio-rad rapid transfer system and pre-activated PVDF membrane according to the manufacturer's instructions. Block the transferred PVDF membrane in 5% skim milk (BD) for 1 hour. Incubate with the primary antibody (PS / T-CK2 substrates, CST, 1:1000) at 4°C overnight. Wash three times with TBST buffer and incubate with the secondary antibody (CST) at room temperature for 1 hour. Wash three times with TBST buffer and develop with a developing substrate (Millipore).

[0045] 3) ROS generation assay: Neutrophils were collected according to the method in 1), resuspended in HBSS, and pretreated with or without CX4945 (5 μM) in a 37°C cell incubator for 30 minutes. The probe was labeled according to the instructions of DHR123 (Invitrogen). Fluorescence values ​​were recorded every 10 minutes under PMA or LPS conditions, and ROS generation curves were plotted.

[0046] 4) NETs generation experiment: Neutrophils were collected according to the method in 1) and cultured in serum-free RPMI1640 medium at a concentration of 1×10 6 Resuspend the cells at a density of 100 μg / ml; add poly-lysine pretreated cell slides to a 24-well cell culture plate (Corning); carefully drop 50 μl of cell suspension onto the center of each cell slide, place in a 37°C cell incubator and let stand for 15 minutes to allow the cells to settle; carefully aspirate and discard the supernatant, add 50 μl of CX4945 (5 μM) or serum-free RPMI1640 medium, and pretreat in a 37°C cell incubator for 30 minutes; carefully aspirate and discard the supernatant, add 50 μl of PMA (50 nM), LPS (1 00ng / ml) or A23187 (4μM), incubated in a 37℃ cell incubator for 4 hours to stimulate NETs production; 500μl of 4% tissue fixative (Solarbio) was added for fixation at room temperature for 20 minutes, washed three times with PBS buffer, blocked with 0.2% Gelatin (Solarbio) at room temperature for 1 hour, and then stained with primary antibody (anti-NE antibody, Millipore, 1:400) at room temperature for 90 minutes; washed three times with PBS buffer, and then stained with secondary antibody (Donkey anti-Rabbit IgG (H+L) Alexa Fluor TM 555, Invitrogen, 1:500) at room temperature for 60 minutes; after washing three times with PBS buffer, use Hoechst33342 (Invitrogen, 1:1000) to stain at room temperature in the dark for 5 minutes; after washing three times with PBS buffer, use Prolong (Invitrogen) to cover the slides, wait for the slides to dry, and then take pictures and count them under a fluorescence microscope.

[0047] 5) Animal bone marrow neutrophil collection method: Femurs from C57BL / 6N mice were gently rubbed with gauze to remove the muscle and connective tissue adhering to the surface of the bones. The joint protuberances at both ends were cut with scissors. The bone marrow tissue was gently blown out using a 22G syringe needle and aspirated back and forth to form a single-cell suspension. After filtration, neutrophils were isolated using the Mouse Neutrophil Isolation Magnetic Bead Kit (Miltenyi Biotec) for ROS production assays. The experimental procedures were the same as those in 3) above.

[0048] 6) Culture of human peripheral monocyte-derived macrophages (MoDM): EDTA-anticoagulated peripheral blood was centrifuged on Ficoll density gradient to separate the upper PBMC cells. The cells were resuspended in RPMI medium (supplemented with 10% fetal bovine serum (Gibco), 1% penicillin-streptomycin complex (Gibco), and 50 ng / ml human M-CSF (Biolegend)) and cultured at a rate of 2 × 10 6 The cells were inoculated into a 12-well cell culture plate (Corning) at a density of 100 μg / ml and cultured in a 37°C cell incubator for 5 days. The culture medium was replaced on the third day. On the fifth day, the cells were observed under a microscope. Stimulation tests were performed when the cells were 90% full.

[0049] 7) Detection of NETs pro-inflammatory ability: According to the experimental method of NETs generation in 4), 1×10 6 Cells were seeded into 48-well cell culture plates (Corning) and pretreated with CX4945 and stimulated for NETs production according to the method in 6). After 4 hours, the supernatant was carefully discarded, 100 μL of preheated MNase (Invitrogen, 30 U / ml) was added, and the cells were incubated in a 37°C cell incubator for 30 minutes. The supernatant was collected and centrifuged at 5000 rpm for 5 minutes. The collected supernatant was the NETs sample; after protein quantification of CX4945-pretreated and untreated NETs samples using a BCA kit (Thermo), human MoDM cells were stimulated at a protein concentration of 1 μg / ml. After incubation in a 37°C cell incubator for 4 hours, the cells were collected, lysed with TRIzol, and RNA was extracted (ZYMO Tissue Cell RNA Extraction Kit). The cells were reverse transcribed into cDNA (TAKARA Reverse Transcription Kit) and then subjected to qPCR (PowerTrack SYBRGreen KIT, Invitrogen). The relative expression levels of inflammatory cytokines were calculated according to the ΔΔCT method using GAPDH (human) expression as an internal reference.

[0050] Example 2: Preparation of CX4945 solution for aerosol inhalation

[0051] The preparation method of CX4945 solution for nebulized inhalation, calculated as a single dose of 2 ml, comprises the following steps:

[0052] 1) Before preparing the solution, flush the pipe with 0.02-0.05% disodium EDTA solution;

[0053] 2) dissolving sodium hydroxide in 80-90% of the total volume of water for injection at a temperature of 30-60° C. and stirring until completely dissolved to form a 0.1 M sodium hydroxide aqueous solution (pH = 9);

[0054] 3) Add CX4945 powder to the sodium hydroxide aqueous solution and stir until completely dissolved. Then add a pH adjuster to adjust the pH to 4.0-4.6 and stir evenly.

[0055] 4) adding an osmotic pressure regulator to the CX4945 solution to control the osmotic pressure molarity of the solution to 270 mosm / l to 310 mosm / l;

[0056] 5) Add water for injection to make up to the total volume, stir evenly, prepare the intermediate drug solution, and perform quality inspection;

[0057] 6) The qualified intermediate drug solution is filtered through 0.45 μm and 0.22 μm filters in sequence, and filled into 2 ml polypropylene plastic ampoules using a blow-fill-seal integrated filling equipment;

[0058] 7) Sterilize with high pressure steam at 121°C for 15 minutes;

[0059] 8) Use high-voltage discharge leak detector to conduct leak detection, light inspection and labeling;

[0060] 9) Use polyester / aluminum / polyethylene pharmaceutical composite film for secondary sealing packaging;

[0061] 10) The finished products are packaged and put into storage, and then fully inspected for quality to obtain qualified CX4945 solution for nebulized inhalation.

[0062] The CX4945 nebulized inhalation solution prepared in this example was atomized using a parilc plus compression nebulizer. The mass median aerodynamic diameter of the inhalation solution was 2 to 5 μm, the percentage of fine particles was 30% to 70%, and the atomization administration time was 10 to 20 minutes.

[0063] Example 3: Inhibition of respiratory tract inflammation

[0064] 1. Chronic obstructive pulmonary disease (COPD) mouse model:

[0065] 1) SPF-grade C57BL / 6N male 8-week-old mice were obtained from Saiye (Suzhou) Biotechnology Co., Ltd.

[0066] 2) LPS (30 μg / 6 μL) was instilled intranasally on days 1 and 29. From days 2 to 30 (except day 29), mice were exposed to passive smoking by slowly inhaling 10 lit cigarettes in a sealed fumigation chamber for 1 hour, five days per week for four consecutive weeks. Treatment with CX4945 aerosol inhalation began on week 4, with 200 μL of the drug administered to each mouse every eight hours for one week. Euthanasia was performed at the end of the experiment.

[0067] 3) Lung tissue and alveolar lavage fluid samples: Mice were anesthetized with isoflurane, and alveolar lavage fluid was obtained by endotracheal intubation; the lungs were perfused with cold PBS by right cardiac puncture. OCT compound and frozen at -80 ° C until the slice thickness is 10 microns for immunofluorescence staining. The other lung lobe was made into paraffin sections and HE staining was performed according to the routine process. The content of NETs complex (MPO-DNA complex) in alveolar lavage fluid was detected by homemade ELISA method. The specific method is to first use anti-mouse MPO antibody (Abcam) to coat high adsorption 96-well plate, refrigerator overnight, use 1% BSA overnight blocking, alveolar lavage fluid diluted 100 times and added to the well, incubate in 4 degrees refrigerator overnight, wash three times and incubate with secondary antibody at room temperature for 1 hour, add TMB to develop color at room temperature in the dark for 30 minutes, add STOP to terminate the color reaction, and read the OD value under the condition of setting the wavelength of 450nm on the enzyme reader.

[0068] 2. LPS-induced acute lung injury model in mice:

[0069] 1) SPF-grade C57BL / 6N male 8-week-old mice were obtained from Saiye (Suzhou) Biotechnology Co., Ltd.

[0070] 2) Anesthesia was achieved by intraperitoneal injection of 1% phenobarbital. After anesthesia, LPS (Sigma) was instilled intratracheally through a #20 needle at a dose of 5 mg / kg body weight (or 10 μg / 50 μl LPS). Treatment was performed with CX4945 nebulizer inhalation, with 200 μl of drug per mouse administered every 8 hours for a total of 6 times. Euthanasia was performed at 72 hours.

[0071] 3) Lung tissue and bronchoalveolar lavage fluid samples: Follow the procedure in step 3) above for sampling, tissue section staining, and determination of NETs complexes in bronchoalveolar lavage fluid.

[0072] 4) Lung Injury Assessment Criteria: HE sections were used to semi-quantitatively analyze pulmonary edema, alveolar and interstitial inflammation, alveolar and interstitial hemorrhage, atelectasis, and hyaline membrane formation using the Smith score, with scores ranging from 0 to 4. No injury was scored as 0, lesion extent <25% = 1 point, lesion extent 25%-50% = 2 points, lesion extent 50%-75% = 3 points, and lesion extent of the entire field of view = 4 points. The total lung injury score was the sum of the above items. Ten high-power fields of view were observed for each animal, and the average score was taken.

[0073] Summarize:

[0074] (1) In vitro cell experiments have shown that CX4945 can significantly reduce the phosphorylation activity of protein kinase CK2 in neutrophils ( Figure 1 ), reduce the level of ROS generation ( Figure 2 ), inhibiting NETs generation ( Figure 3 ) and reduce the pro-inflammatory activity of generated NETs ( Figure 4 ).

[0075] (2) In animal experiments, CX4945 aerosol treatment can significantly reduce pulmonary edema and tissue neutrophil infiltration in mice ( Figure 5 ), significantly reduced the content of NETs complex in alveolar fluid ( Figure 6 ), increased the survival rate of mice with lung injury and COPD ( Figure 7 ).

[0076] (3) In previous animal models of CX4945 for tumor treatment, the dose of CX4945 used in animals was usually 25 mg / kg or 75 mg / kg, and was well tolerated.

[0077] (4) The present invention prepares CX4945 into a solution dosage form for aerosol inhalation, which can be directly inhaled through the mouth and nose through aerosolization, and can replace oral administration and other dosage forms, thereby reducing the metabolism of the drug by the liver and kidneys, significantly reducing liver and kidney damage to patients, and having high safety and strong patient compliance.

[0078] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A solution for aerosol inhalation of a CK2 inhibitor CX4945, characterized in that: A single dose of 2 mL includes: CX4945 10-20 mg, osmotic pressure regulator 10-20 mg, pH regulator, and water for injection; The osmotic pressure regulator is selected from one or more of sodium chloride, potassium chloride and mannitol; The pH regulator is selected from one or more of sodium hydroxide, hydrochloric acid, sodium dihydrogen phosphate, citric acid and sodium citrate; The pH range of the solution for aerosol inhalation is controlled at 4.0 to 5.0; The osmotic pressure molar concentration of the solution for aerosol inhalation is controlled at 270mosm / l to 310mosm / l; The solution for aerosol inhalation does not contain surfactants and metal complexes.

2. The solution for aerosol inhalation according to claim 1, characterized in that The osmotic pressure regulator is sodium chloride; The pH regulator is sodium hydroxide; The pH range of the solution for aerosol inhalation is controlled at 4.0-4.

6.

3. A method for preparing a solution for aerosol inhalation of the CK2 inhibitor CX4945 according to claim 1 or 2, comprising the steps of dissolving CX4945 powder in 1 mL of 0.1 M sodium hydroxide solution, dissolving and dispersing the solution, adjusting the pH and osmolarity, and filling the solution.

4. The use of CK2 inhibitor CX4945 in the preparation of drugs that inhibit the production of NETs by neutrophils and the pro-inflammatory effects of NETs.

5. Use of the CK2 inhibitor CX4945 in the preparation of drugs for inhibiting respiratory inflammatory diseases characterized by neutrophil imbalance.

6. The use according to claim 5, characterized in that The respiratory inflammatory diseases include chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, asthma, cystic fibrosis, upper respiratory tract infection and lower respiratory tract infection.

7. Use of the nebulized inhalation solution of the CK2 inhibitor CX4945 according to claim 1 or 2 in the preparation of a medicament for inhibiting the production of NETs by neutrophils and the pro-inflammatory effect of the generated NETs.

8. Use of the nebulized inhalation solution of the CK2 inhibitor CX4945 according to claim 1 or 2 in the preparation of a medicament for inhibiting respiratory inflammatory diseases characterized by neutrophil imbalance.

9. The use according to claim 8, characterized in that The respiratory inflammatory diseases include chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, asthma, cystic fibrosis, upper respiratory tract infection and lower respiratory tract infection.

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