Inhalation type ZK-22 preparation administrated through airway and preparation method and application of inhalation type ZK-22 preparation

Abstract

The invention relates to an inhalation type ZK-22 preparation for airway administration as well as a preparation method and application of the inhalation type ZK-22 preparation. The inhalation type ZK-22 preparation comprises a ZK-22 phospholipid complex, in the ZK-22 phospholipid complex, the mass ratio of ZK-22 to phospholipid molecules is 1: 3, the ZK-22 phospholipid complex is dispersed in a water phase to obtain a suspension, and particles are obtained after spray drying. The invention also relates to the use of said inhalation-type ZK-22 formulation in the treatment or prevention of respiratory viral infections.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and specifically relates to an inhalation-type ZK-22 preparation for administration through the airway, a preparation method thereof and uses thereof. Background Art

[0002] Currently, there are no specific drugs for acute respiratory viral infections except influenza virus and coronavirus, nor are there broad-spectrum prevention and treatment drugs. There is an urgent need to find new, safe, and broad-spectrum anti-respiratory viral drugs.

[0003] Piperazine substituted-2-methylbenzonitrile compound ZK-22, whose chemical name is 2-((4-(4-chlorophenyl)piperazin-1-yl)methyl)-4-((2-(dimethylamino)ethyl)(methyl)amino)benzonitrile, has a structure as shown in formula (1):

[0004]

[0005] A number of prior studies, including the applicant's work, have found that ZK-22 has good function and activity against various viruses such as hepatitis C virus and coronavirus (J Med Chem. 2020; 63(11): 5972-5989; Benzylpiperazine compounds and their preparation methods and applications in antiviral treatment, Chinese Patent No.: ZL201910905738.5, U.S. Patent No.: US11939307; Application of a piperazine-substituted 2-methylbenzonitrile compound ZK-22 in the preparation of anti-coronavirus drugs, Chinese Patent No.: ZL202111092666.0).

[0006] The respiratory tract is the body's first line of defense against respiratory pathogens. Although there are currently several oral small-molecule viral drugs, there are no anti-respiratory viral drugs on the market that are administered through the airway, and there are also relatively few inhaled anti-respiratory viral drugs in the clinical trial stage (mainly including large molecule drugs or immune regulation-related molecules, such as hACE2 nasal spray, etc.). Considering that respiratory viruses mainly replicate in the respiratory tract in the early stages, inhalation therapy can deliver drugs directly into the respiratory tract and lungs. On the one hand, inhaled preparations can achieve effective concentrations in the respiratory tract with a smaller dose, instantly achieve high airway exposure, and take effect more quickly; on the other hand, local administration has a smaller systemic exposure, which can avoid systemic side effects of drugs and reduce drug interactions.

[0007] Therefore, in order to optimize and improve the lung bioavailability of ZK-22, enhance the therapeutic effect and overall drugability of the drug, it is very necessary to develop a safe and effective inhaled broad-spectrum anti-respiratory viral compound formulation of ZK-22.

[0008] Based on this, the present invention is proposed. Summary of the invention

[0009] The present invention firstly relates to a ZK-22 phospholipid complex, which can be used to prepare an inhaled ZK-22 preparation for administration through the airway.

[0010] In the ZK-22 phospholipid complex, the mass ratio of ZK-22 to phospholipid molecules is 1:3;

[0011] The phospholipid molecules include, but are not limited to, soybean lecithin, egg yolk lecithin, dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC); preferably soybean lecithin, DPPC, and DSPC.

[0012] The present invention also relates to a method for preparing the ZK-22 phospholipid complex, which comprises the following steps:

[0013] (1) dissolving ZK-22 and phospholipid in an organic solvent at a mass ratio of 1:3 and performing a heat reflux reaction to obtain a ZK-22 phospholipid complex; preferably, the heat reflux reaction conditions are: heat reflux at 50° C. for 3 h; preferably, the organic solvent is chloroform;

[0014] (2) The ZK-22 phospholipid complex is subjected to reduced pressure distillation combined with vacuum drying to completely remove the organic solvent to obtain a ZK-22 phospholipid complex dry powder.

[0015] The present invention also relates to a suspension of ZK-22 phospholipid complex, which is prepared according to the following method:

[0016] The ZK-22 phospholipid complex dry powder is re-dissolved with deionized water or physiological saline, and then treated by a particle size control method such as ultrasonic dispersion, high-pressure homogenization, microfluidics, etc. to obtain the ZK-22 phospholipid complex suspension.

[0017] Preferably, the ultrasonic dispersion parameters are: 150W ultrasonic dispersion 5min

[0018] In the suspension of the ZK-22 phospholipid complex, the average particle size of the complex particles is 100-400 nm, preferably 150-250 nm, the dispersion index (PDI) is 0.1-0.4, and the potential is 10-25 mV.

[0019] The present invention also relates to a method for preparing a suspension of a ZK-22 phospholipid complex, comprising the following steps:

[0020] (1) dissolving ZK-22 and phospholipid in an organic solvent at a mass ratio of 1:3 and performing a heat reflux reaction to obtain a ZK-22 phospholipid complex; preferably, the heat reflux reaction conditions are: heat reflux at 50° C. for 3 h; preferably, the organic solvent is chloroform;

[0021] (2) performing vacuum distillation and vacuum drying of the ZK-22 phospholipid complex to completely remove the organic solvent, thereby obtaining a ZK-22 phospholipid complex dry powder;

[0022] (3) The ZK-22 phospholipid complex dry powder is re-dissolved in deionized water or physiological saline, and then treated by ultrasonic dispersion, high-pressure homogenization, microfluidics or other particle size control methods to obtain a suspension of the ZK-22 phospholipid complex; preferably, the ultrasonic dispersion parameters are: 150W ultrasonic dispersion for 5 min.

[0023] The present invention also relates to a ZK-22-containing spray-dried particle, which is prepared by the following method:

[0024] (1) stirring and mixing the ZK-22 phospholipid complex suspension or the ZK-22 suspension and the carrier emulsion uniformly;

[0025] (2) Spray drying to prepare ZK-22 spray-dried microparticles.

[0026] The preparation method of the carrier emulsion is:

[0027] The phospholipids are dispersed in a heated deionized water solution, and the oil phase perfluorooctyl bromide (PFOB) is added dropwise and stirred at high speed to form colostrum, and then the carrier emulsion is formed by high pressure homogenization;

[0028] Preferably,

[0029] Add lecithin at 60-80°C, disperse under the shear force of 8000 rpm, drop perfluorooctyl bromide (PFOB) and continue shearing for 5 minutes to obtain colostrum;

[0030] The colostrum was homogenized under high pressure at 18,000 psi to obtain a carrier emulsion;

[0031] More preferably, the phospholipid is distearoylphosphatidylcholine (DSPC);

[0032] The preparation method of the ZK-22 suspension is:

[0033] ZK-22 was micronized by high pressure homogenization and dispersed in a physiological saline solution containing 0.04% (w / w) Tween 80.

[0034] Preferably, 20.0 mg of ZK-22 solid powder is dispersed in 5 ml of physiological saline containing 0.04% (w / w) Tween 80 and ultrasonicated for 30 min; and 950 bar high pressure homogenization is cycled 20 times to obtain a ZK-22 suspension.

[0035] Preferably, the spray drying conditions are: inlet temperature 110° C., outlet temperature 67° C., fan efficiency 100%, pump rate 8%, and spray air flow rate 1750 L / h.

[0036] The present invention also relates to the following applications of the ZK-22 phospholipid complex, the suspension of the ZK-22 phospholipid complex, and the ZK-22 spray-dried microparticles:

[0037] (1) Preparation of dry powder inhalation dosage form;

[0038] (2) preparing aerosol inhalation drugs;

[0039] The medicine is used to prevent or treat respiratory viral infection; preferably, the respiratory virus is influenza virus, new coronavirus, or rhinovirus.

[0040] The present invention also relates to an inhalation-type ZK-22 preparation for administration through the airway, wherein the inhalation-type ZK-22 preparation comprises a therapeutically effective amount of the ZK-22 phospholipid complex according to claim 1, a suspension of the ZK-22 phospholipid complex according to claim 3, or the ZK-22 spray-dried microparticles according to any one of claims 5 to 7; and necessary pharmaceutical excipients.

[0041] The present invention also relates to the use of the ZK-22 phospholipid complex, a suspension of the ZK-22 phospholipid complex or ZK-22 spray-dried particles in the treatment of respiratory viral infections. Preferably, the respiratory virus is influenza virus, new coronavirus, or rhinovirus.

[0042] The beneficial effects of the present invention are:

[0043] (1) Designed and prepared an inhalable ZK-22 phospholipid complex, which significantly improved the local bioavailability of ZK-22 in the lungs and enhanced the antiviral effect of ZK-22 in mice.

[0044] (2) It was demonstrated that the inhaled ZK-22 phospholipid complex has broad-spectrum anti-respiratory virus capabilities and provides a drug reserve for the prevention and treatment of broad-spectrum respiratory viruses in the future.

[0045] (3) It was demonstrated that ZK-22 also has a significant inhibitory effect on the replication of the new coronavirus variants that appeared after 2022, influenza virus (Orthomyxoviridae) and rhinovirus (Enterovirus genus, Picornaviridae) that infect and spread through the respiratory tract, suggesting that ZK-22 has a broad-spectrum anti-respiratory virus ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 , particle size distribution of ZK-22 phospholipid complex suspension.

[0047] Figure 2 , particle size distribution of ZK-22 micron suspension.

[0048] Figure 3 , tissue distribution of ZK-22 phospholipid complex suspension and ZK-22 micron suspension after pulmonary administration (ZK-22-pc in the figure is ZK-22 phospholipid complex suspension, and ZK-22 suspension is ZK-22 micron suspension), 3A, lung tissue after lavage; 3B, lung epithelial lining fluid (ELF); 3C, immune cells in lung epithelial lining fluid; 3D, drug distribution in plasma at different time points (n=6, mean±SD).

[0049] Figure 4 , ZK-22 micron suspension and ZK-22 phospholipid complex suspension on the level of viral RNA in the lung tissue of mice infected with influenza virus (ZK-22-pc in the figure is ZK-22 phospholipid complex suspension, and ZK-22 suspension is ZK-22 micron suspension).

[0050] Figure 5 , The results of the effect of ZK-22 on viral RNA and protein in influenza virus-infected cells, 5A, viral RNA; 5B, viral protein M2.

[0051] Figure 6 , the results of ZK-22's effect on viral RNA in cells infected with three new coronavirus variants, 6A, variant BA.5; 6B, variant XBB.1.16; 6C, variant EG.5.

[0052] Figure 7 , ZK-22's effect on viral RNA in human rhinovirus-infected cells. DETAILED DESCRIPTION

[0053] Example 1. Preparation of ZK-22 phospholipid complex suspension

[0054] 1. Preparation of ZK-22 phospholipid complex:

[0055] (1) 20 mg of ZK-22 and soybean lecithin for injection or DPPC were dissolved in 5 mL of chloroform solution at a mass ratio of 1:3 and heated under reflux at 50 °C for 3 h to generate ZK-22 phospholipid complex;

[0056] (2) The obtained ZK-22 phospholipid complex was distilled under reduced pressure and then further dried under vacuum for 24 hours to obtain a ZK-22 phospholipid complex dry powder;

[0057] Determination of the phospholipid complexing rate in the ZK-22 phospholipid complex: After cyclohexane extraction, the ratio of the ZK-22 content in the total ZK-22 input was determined by high performance liquid chromatography, and the calculated complexing rate was 85.6±2.49%.

[0058] Extraction process: Add 10 mL of cyclohexane to the round-bottom flask containing the dry product (ZK-22 phospholipid complex powder) and shake appropriately to allow the ZK-22 phospholipid complex to be fully dissolved, then filter with a 0.45 μm filter membrane, and the filtrate is spin-dried and vacuum-dried to obtain the ZK-22 phospholipid complex obtained by the reaction.

[0059] 2. Preparation of ZK-22 phospholipid complex suspension:

[0060] The phospholipid complex dry powder prepared in the above step 1 was re-dissolved in deionized water and then subjected to 150W ultrasonic dispersion for 5 minutes to obtain a ZK-22 phospholipid complex suspension.

[0061] The average particle size of the suspension is about 214.0±16.54nm ( Figure 1 ), the dispersion degree (PDI) is 0.265±0.09, and the potential is 15.3mV.

[0062] In addition, after the ZK-22 phospholipid complex suspension was stored at room temperature for one month, the particle size and phospholipid complexation rate did not show significant changes, indicating that the suspension has good storage stability.

[0063] Example 2. Preparation of ZK-22 micron suspension

[0064] 1. Preparation process of ZK-22 micron suspension:

[0065] (1) Accurately weigh 20.0 mg of ZK-22 solid powder, disperse it in 5 ml of saline containing 0.04% Tween-80, and sonicate for 30 min;

[0066] (2) 950 bar high pressure homogenization cycle 20 times to obtain ZK-22 micron suspension.

[0067] The average particle size of the suspension is 1.647 μm ( Figure 2 ). In addition, the particle size of the suspension did not change significantly after being stored at room temperature for 1 month, indicating that it has good storage stability.

[0068] Example 3. Preparation of spray-dried microparticles

[0069] 1. Add 3.5 mg of calcium chloride to 3 mL of deionized water at 70°C, disperse 50 mg of DSPC therein under the shear force of 8000 rpm, then slowly add 1.25 g of perfluorooctyl bromide (PFOB) and continue shearing for 5 minutes to obtain colostrum; the colostrum is immediately homogenized at 18000 psi for 5 times to obtain a carrier emulsion.

[0070] 2. Using the method in Example 1, prepare a phospholipid complex suspension of ZK-22 and DSPC (mass ratio 1:3).

[0071] 3. Mix 10 mL of 2 mg / mL ZK-22 phospholipid complex suspension with the carrier emulsion (containing 30 mg DSPC) in step 1 above, and obtain spray-dried particles loaded with ZK-22 phospholipid complex by spray drying; the spray drying conditions are: inlet temperature 110°C, outlet temperature 67°C, fan efficiency 100%, pump rate 8%, and spray air flow rate 1750 L / h.

[0072] 4. Weigh 20 mg of ZK-22 solid powder, disperse it with 5 ml of deionized water containing 1.2% DSPC and ultrasonicate for 30 min; cycle 950 bar high pressure homogenization 20 times to obtain a ZK-22 micron suspension, and dilute it with deionized water to obtain 10 mL of 2 mg / mL ZK-22 micron suspension.

[0073] 5. Mix 10 mL of 2 mg / mL ZK-22 micron suspension with the carrier emulsion (containing 30 mg DSPC) in step 1 above, and obtain spray-dried particles loaded with ZK-22 by spray drying; the spray drying conditions are: inlet temperature 110°C, outlet temperature 67°C, fan efficiency 100%, pump rate 8%, and spray air flow rate 1750 L / h.

[0074] Experimental Example 4: Tissue distribution characteristics of ZK-22 phospholipid complex suspension

[0075] The efficacy of pulmonary anti-inflammatory drugs is usually not correlated with plasma drug concentration, but is directly related to the drug concentration in lung tissue, so it is necessary to clarify the distribution of drugs after pulmonary administration. This example compares the tissue distribution of ZK-22 phospholipid complex (prepared in Example 1) and its reference preparation ZK-22 micron suspension (prepared in Example 2) in lung and plasma after pulmonary administration.

[0076] 1. Lung tissue distribution experiment:

[0077] (1) Male BALB / c mice were randomly divided into two groups and administered with ZK-22 phospholipid complex suspension and ZK-22 micron suspension via tracheal administration, respectively, with the dosage of both groups being equivalent to 2.5 mg / kg (based on the amount of ZK-22);

[0078] (2) Blood was collected at 1, 3, 6, and 12 hours after administration, and the animals were sacrificed and intubated for alveolar lavage, followed by removal of lung tissue (n=6). The alveolar lavage fluid was centrifuged at low speed to separate the lower layer of cells, and the drug content in plasma, alveolar lavage fluid supernatant, lower layer of cells, and lung tissue was determined.

[0079] After rats were given ZK-22 micron suspension and ZK-22 phospholipid complex suspension via trachea at a dose of 2.5 mg / kg, the drug content in plasma, alveolar lavage fluid, lavage fluid cells and lavage lung tissue was Figure 3 . Result analysis:

[0080] (1) After administration of the two dosage forms, there was no significant difference in the distribution of the drug in plasma.

[0081] (2) After administration of the two dosage forms, there were significant differences in the distribution of the drug in the alveolar lavage fluid, lavage fluid cells, and lung tissue after lavage. Specifically:

[0082] Compared with ZK-22 micron suspension, ZK-22 phospholipid complex suspension in alveolar lavage fluid, lavage fluid cells and Drug levels were higher in lung tissue after lavage .

[0083] More specifically,

[0084] (1) After intratracheal instillation of ZK-22 micron suspension, the drug was rapidly cleared from the pulmonary epithelial lining fluid (ELF), while intratracheal administration of ZK-22 phospholipid complexes showed a slowed absorption and clearance in ELF, indicating that ZK-22 has a sustained release effect in the respiratory tract after being made into phospholipid complexes.

[0085] (2) The results of lavage fluid cells showed that compared with the administration of ZK-22 micron suspension, ZK-22 phospholipid complexes made the drug more easily phagocytosed by macrophages. This may be related to the prolonged exposure time of the phospholipid complex group in ELF and the phagocytic characteristics of macrophages. The increase in its content in lung tissue may be mainly due to the phospholipid complex increasing the affinity of the drug to the lungs.

[0086] The results of lung tissue distribution showed that compared with ZK-22 suspension, ZK-22 phospholipid complex suspension group drug The residence time in the lungs is significantly prolonged .

[0087] Experimental Example 5: Efficacy test of ZK-22 phospholipid complex suspension

[0088] This example compares the effects of ZK-22 phospholipid complex suspension (prepared in Example 1) and its reference preparation ZK-22 micron suspension (prepared in Example 2) on the viral RNA content in the lung tissue of mice infected with influenza virus subtype A (PR8 strain, H1N1).

[0089] The specific experimental operations are as follows:

[0090] 1. Animal grouping

[0091] The experimental animals were 2-week-old female KM mice without specific pathogens (SPF), which were randomly divided into 8 groups (n=7-9). All animals were raised in a biosafety level 2 laboratory (ABSL-2). Except for normal control mice, the remaining mice were anesthetized with isoflurane and given 10 μL influenza virus (PR8, H1N1) (3LD 50 ) infection, and the experimental animals were divided into groups and given medication as follows:

[0092] (1) Virus control group: 40 μL normal saline was administered intranasally 3 times, 24 h before, 2 h after, and 24 h after virus infection;

[0093] (2) ZK-22 micron suspension group: 40 μL of ZK-22 micron suspension equivalent to 2.5 mg / kg was administered intranasally 3 times, 24 h before, 2 h after, and 24 h after virus infection;

[0094] (3) Low-dose group of ZK-22 phospholipid complex suspension (-24h, -2h, +24h): 40 μL of drug equivalent to 2.5 mg / kg ZK-22 phospholipid complex was administered intranasally 24h before virus infection, 2h before infection, and 24h after infection, for a total of 3 times;

[0095] (4) ZK-22 phospholipid complex suspension (-24h, -2h, +24h) high-dose group: 40 μL of drug equivalent to 5 mg / kg ZK-22 phospholipid complex was administered intranasally 24h before virus infection, 2h before infection, and 24h after infection, for a total of 3 times;

[0096] (5) Low-dose group of ZK-22 phospholipid complex suspension (-24h, 0h, +24h): 40 μL of drug equivalent to 2.5 mg / kg ZK-22 phospholipid complex was administered intranasally 24h before virus infection, immediately after infection, and 24h after infection, for a total of 3 times;

[0097] (6) ZK-22 phospholipid complex suspension (-24h, 0h, +24h) high-dose group: 40 μL of drug equivalent to 5 mg / kg ZK-22 phospholipid complex was administered intranasally 24h before virus infection, immediately after infection, and 24h after infection, for a total of 3 times;

[0098] (7) Oseltamivir phosphate (Tamiflu) control group: 27.5 mg / kg Tamiflu was given 2 hours after virus infection, once a day;

[0099] (8) Normal control group: normal feeding.

[0100] All mice were euthanized 96 hours after virus infection, and lung tissues were obtained and stored at -80°C.

[0101] 2. Quantification of viral RNA in each group of samples

[0102] (1) Lung tissue was placed in a tissue grinding tube, and RNA was extracted using an animal tissue RNA extraction kit to detect RNA concentration. The influenza virus RNA content was detected using the HiScript II One Step qRT-PCR SYBR Green Kit on an ABI 7500 Fast high-throughput real-time fluorescence quantitative PCR (qPCR) instrument, and each RNA sample was measured twice.

[0103] (2) Using influenza virus NP protein particles as standard, perform gradient dilutions to achieve a final concentration of 8.4 × 10 9 , 8.4×10 8 , 8.4×10 7 , 8.4×10 6 , 8.4×10 5 , 8.4×10 4 and 8.4×10 3 copies / uL.

[0104] Primers used for one-step RNA content detection:

[0105] Influenza virus NP protein: 5'-TGCTTCAAAACAGCCAAGTG-3' (upstream) (SEQ ID NO. 1);

[0106] 5′-GATGCCCTCTGTTGATTGGT-3′ (downstream) (SEQ ID NO. 2);

[0107] The reaction system is as follows:

[0108]

[0109]

[0110] The reaction conditions are as follows:

[0111]

[0112] (3) A standard curve is made based on the gene copy number and Ct value of the plasmid standard expressing influenza NP protein, and the copy number can be further calculated from the Ct value of viral RNA in each lung tissue obtained in the experiment.

[0113] 3. Result analysis:

[0114] (1) Compared with the virus control group, the viral RNA content in the lung tissue of mice in the oseltamivir group was significantly reduced (P<0.001). This result is consistent with the literature reports and previous experimental results of our research group.

[0115] (2) In the group given ZK-22 micron suspension (ZK-22) at a dose of 2.5 mg / kg, the viral RNA content in the lung tissue of mice was significantly lower than that in the virus control group (P<0.05);

[0116] (3) The content of viral RNA in the lung tissue of mice in the two dose groups of ZK-22 phospholipid complex suspension (ZK-22-pc) was also significantly lower than that in the virus control group (P<0.01 or P<0.001); compared with the ZK-22 micron suspension, ZK-22 Phospholipid Complex The suspension reduced the viral RNA content in lung tissue to a lesser extent ( Figure 4 ) .

[0117] Experimental Example 6: In vitro efficacy test of ZK-22 against influenza virus

[0118] The example examined the effect of ZK-22 on viral RNA and protein levels in cells infected with influenza virus subtype A (PR8 strain, H1N1).

[0119] The specific experimental operations are as follows:

[0120] 1. Cell infection and drug administration

[0121] All experiments were performed in a biosafety level 2 laboratory (BSL-2). MDCK canine kidney cells were seeded in 12-well plates and inoculated with influenza virus (100 TCID 50 ) infection and give corresponding drugs at the same time. After 2 hours, the virus solution was discarded and replaced with maintenance solution containing drugs for continued culture.

[0122] The cell groups and drug administration are as follows:

[0123] (1) Blank control group: not infected with the virus;

[0124] (2) Virus control group: infected with virus, and replaced with normal cell maintenance medium 2 h later;

[0125] (3) Positive control group: infected with virus, and replaced with cell maintenance medium containing 10 nM baloxavir 2 h later;

[0126] (4) High dose of ZK-22: ZK-22 10 μM was administered simultaneously with virus infection, and the cell maintenance medium containing 10 μM ZK-22 was replaced after 2 h;

[0127] (5) Medium dose of ZK-22: 5 μM ZK-22 was administered simultaneously with virus infection, and the cell maintenance medium containing 5 μM ZK-22 was replaced after 2 h;

[0128] (6) Low dose of ZK-22: ZK-22 2.5 μM was administered simultaneously with virus infection, and the cell maintenance medium containing 2.5 μM ZK-22 was replaced after 2 h.

[0129] 24 h after infection, the supernatant was discarded, TRIzol or protein lysis buffer was added to the cells, and the cells were stored at -80°C.

[0130] 2. Quantification of viral RNA in each group of samples

[0131] (1) 24 hours after virus infection, RNA was extracted using the RNeasy Mini kit RNA extraction kit (purchased from QIAGEN), and then the influenza virus RNA content was detected using the HiScript II One Step qRT-PCR SYBR Green Kit on an ABI7500Fast high-throughput real-time fluorescence quantitative PCR (qPCR) instrument. Two replicate wells were set for each sample during the test, and the experiment was repeated three times.

[0132] The primers used for influenza virus M2 protein and canine GAPDH are shown below, and the reaction system and conditions are the same as above.

[0133]

[0134] (2) qPCR relative quantitative calculation method: The target gene (gene of interest, GOI) was analyzed for relative quantitative analysis, and the housekeeping gene GAPDH was used as the internal control (IC) to normalize the Ct value. -ΔΔCt The differential expression of target genes was calculated.

[0135] sample represents the experimental group; control represents the control group; Ct-GOI S Represents the Ct value of the target gene in the experimental group sample; Ct-GOI C Represents the Ct value of the target gene in the control group sample; Ct-IC S Represents the Ct value of the internal reference of the experimental group sample; Ct-IC C Represents the Ct value of the internal reference of the control group sample; Fold difference represents the fold difference of the target gene expression between the experimental group and the control group. The calculation method is as follows:

[0136] ΔCt sample =Ct-GOI S -Ct-IC S

[0137] ΔCt control =Ct-GOI C -Ct-IC C

[0138] ΔΔCt=ΔCt sample -ΔCt control

[0139] Fold difference = 2 -ΔΔCt

[0140] 3. Quantification of viral proteins in each group of samples

[0141] Total cell proteins were extracted and then subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then the proteins were transferred to a PVDF membrane.

[0142] After electroporation, immerse the PVDF membrane in 5% skim milk powder blocking solution and oscillate slowly at room temperature for 1 hour. Cut the membrane according to the size of the target protein and the marker position, put in the corresponding primary antibody (internal reference β-actin: 1:1000 dilution (CST Company), influenza virus M2: 1:400 dilution (Santa Company)), and oscillate overnight at 4°C. Rinse with TBST 3 times, 10 minutes each time. Add 1:5000 secondary antibody and incubate at room temperature for 1 hour. Rinse with TBST 3 times, 10 minutes each time. Finally, use exposure solution to expose in gel imager.

[0143] 4. Results Analysis

[0144] (1) Compared with the virus control group, the positive drug baloxavir significantly inhibited the levels of influenza virus RNA and M2 protein, which is consistent with the literature reports and previous experimental results of our research group ( Figure 5 );

[0145] (2) Compared with the virus control group, ZK-22 can significantly inhibit the expression of influenza virus RNA and M2 protein in a dose-dependent manner. flat ( Figure 5 ).

[0146] Experimental Example 7: In vitro efficacy test of ZK-22 against novel coronavirus variants

[0147] 1. Cell infection and drug administration

[0148] All experiments were performed in a biosafety level 4 laboratory (BSL-3).

[0149] Vero E6 cells were inoculated in a 96-well plate; on the next day, the culture medium was discarded, and culture medium containing different concentrations of ZK-22 (2.5, 5 and 10 μM) was added, and DMSO was added to the control group; the cells were cultured at 37°C for 1 hour, the supernatant was discarded, and then the virus solution containing the new coronavirus variant (BA.5, XBB.1.16 or EG.5) (infection dose was MO=0.05) and culture medium containing ZK-22 or DMSO were added at the same time; after culturing at 37°C for 24 hours, the supernatant was discarded, TRIzol was added to the cells, and the cells were stored at -80°C.

[0150] 2. Quantification of viral RNA in each group of samples

[0151] (1) Total RNA was extracted from cells using an RNA extraction kit and the RNA concentration was detected. The content of viral RNA and GAPDH was detected using the TransScriptProbe One-Step qRT-PCR SuperMix kit on an ABI7500Fast high-throughput qPCR instrument. Each RNA sample was measured twice.

[0152] (2) Primers used in one-step detection of RNA content:

[0153] Upstream of SARS-CoV-2: 5'-GGGGAACTTCTCCTGCTAGAAT-3' (SEQ ID NO.7);

[0154] Probe: 5'-6-FAM-TTGCTGCTGCTTGACAGATT-TAMRA-3'(SEQ ID NO.8);

[0155] Downstream: 5'-CAGACATTTTGCTCTCAAGCTG-3' (SEQ ID NO. 9).

[0156] Monkey GAPDH upstream: 5'-CTGTTGCTGTAGCCAAATTCGT--3' (SEQ ID NO.10);

[0157] Downstream: 5'-ACCCACTCCTCCACCTTTGAC-3' (SEQ ID NO. 11).

[0158] The reaction system is as follows:

[0159]

[0160] The reaction conditions are as follows:

[0161]

[0162] (3) The qPCR relative quantification method was used to calculate the changes in the SARS-CoV-2 RNA content in each group, using the same method as above.

[0163] 3. Results Analysis

[0164] Compared with the virus control group, The viral RNA content of the three new coronavirus variants in the ZK-22 treatment group increased with the drug The concentration of the substance gradually decreases ( Figure 6 ) .

[0165] Experimental Example 8: In vitro efficacy test of ZK-22 against rhinovirus

[0166] 1. Cell infection and drug administration

[0167] All experiments were performed in a biosafety level 2 laboratory (BSL-2).

[0168] HeLa cells were inoculated in 48-well plates. On the second day, the culture medium was discarded, and then a virus solution containing human rhinovirus 14, Strain 1059 and a culture medium containing ZK-22 or DMSO were added simultaneously. After culturing at 37°C for 24 hours, the supernatant was discarded, TRIzol was added to the cells, and the cells were stored at -80°C.

[0169] 2. Quantification of viral RNA in each group of samples

[0170] (1) RNA was extracted using an animal tissue RNA extraction kit and the RNA concentration was detected. The viral RNA and GAPDH content were detected using the HiScript II OneStep qRT-PCR SYBR Green Kit on an ABI 7500 Fast high-throughput real-time fluorescence quantitative PCR (qPCR) instrument. Each RNA sample was measured twice.

[0171] (2) Primers used in one-step detection of RNA content:

[0172] Human rhinovirus: 5'-TTGGGTCGTGCAGCTTGTGTG-3' (upstream) (SEQ ID NO. 12)

[0173] 5'-CTAAGTTGGACAAGGCTGGACAGG-3' (downstream) (SEQ ID NO. 13)

[0174] Human GAPDH: 5'-GAAGGTGAAGGTCGGAGTC-3' (upstream) (SEQ ID NO. 14)

[0175] 5'-GAAGATGGTGATGGGATTTC-3' (downstream) (SEQ ID NO. 15)

[0176] The reaction system and reaction conditions are the same as above.

[0177] (3) The qPCR relative quantitative method was used to calculate the changes in the rhinovirus RNA content in each group, using the same method as above.

[0178] 3. Results Analysis

[0179] Compared with the virus control group, The rhinovirus RNA content in the ZK-22-treated group gradually decreased with the increase of drug concentration. Gradually decrease ( Figure 7 ) .

[0180] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention and are not used to limit the protection scope of the present invention.

Claims

1. A ZK-22 phospholipid complex, which can be used to prepare an inhaled ZK-22 preparation for administration through the airway, characterized in that: In the ZK-22 phospholipid complex, the mass ratio of ZK-22 to phospholipid molecules is 1:3; The phospholipid molecules include, but are not limited to, soybean lecithin, egg yolk lecithin, dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC); preferably soybean lecithin, DPPC, and DSPC.

2. The method for preparing the ZK-22 phospholipid complex according to claim 1, characterized in that: The method comprises the following steps: (1) dissolving ZK-22 and phospholipid in an organic solvent at a mass ratio of 1:3 and performing a heat reflux reaction to obtain a ZK-22 phospholipid complex; Preferably, the heat reflux reaction conditions are: heat reflux at 50°C for 3h; preferably, the organic solvent is chloroform; (2) The ZK-22 phospholipid complex is subjected to reduced pressure distillation combined with vacuum drying to completely remove the organic solvent to obtain a ZK-22 phospholipid complex dry powder.

3. A suspension of ZK-22 phospholipid complex, wherein the suspension is prepared according to the following method: The ZK-22 phospholipid complex dry powder of claim 1 is re-dissolved with deionized water or physiological saline, and then treated by ultrasonic dispersion, high-pressure homogenization, microfluidics or other particle size control methods to obtain a suspension of the ZK-22 phospholipid complex; Preferably, the ultrasonic dispersion parameters are: 150W ultrasonic dispersion for 5 min. In the suspension of the ZK-22 phospholipid complex, the average particle size of the complex particles is 100-400 nm (preferably 150-250 nm), the dispersion index (PDI) is 0.1-0.4, and the potential is 10-25 mV.

4. The method for preparing the suspension of ZK-22 phospholipid complex according to claim 3 comprises the following steps: (1) dissolving ZK-22 and phospholipid in an organic solvent at a mass ratio of 1:3 and performing a heat reflux reaction to obtain a ZK-22 phospholipid complex; preferably, the heat reflux reaction conditions are: heat reflux at 50° C. for 3 h; preferably, the organic solvent is chloroform; (2) performing vacuum distillation and vacuum drying of the ZK-22 phospholipid complex to completely remove the organic solvent, thereby obtaining a ZK-22 phospholipid complex dry powder; (3) The ZK-22 phospholipid complex dry powder is re-dissolved in deionized water or physiological saline, and then treated by ultrasonic dispersion, high-pressure homogenization, microfluidics or other particle size control methods to obtain a suspension of the ZK-22 phospholipid complex; preferably, the ultrasonic dispersion parameters are: 150W ultrasonic dispersion for 5 min.

5. A spray-dried particle containing ZK-22, wherein the spray-dried particle containing ZK-22 is prepared by the following method: (1) stirring and mixing the ZK-22 phospholipid complex suspension or the ZK-22 suspension and the carrier emulsion as described in claim 3; (2) Spray drying to prepare ZK-22 spray-dried microparticles; The preparation method of the carrier emulsion is: The phospholipids are dispersed in a heated deionized water solution, and the oil phase perfluorooctyl bromide (PFOB) is added dropwise and stirred at high speed to form colostrum, and then the carrier emulsion is formed by high pressure homogenization; The preparation method of the ZK-22 suspension is as follows: ZK-22 is micronized by high-pressure homogenization and then dispersed in a physiological saline solution containing 0.04% (w / w) Tween 80; preferably, 20.0 mg of ZK-22 solid powder is dispersed in 5 ml of physiological saline containing 0.04% (w / w) Tween 80 and ultrasonicated for 30 minutes; 950 bar high-pressure homogenization is repeated 20 times to obtain the ZK-22 suspension.

6. The spray-dried microparticles containing ZK-22 according to claim 5, characterized in that The spray drying conditions are: inlet temperature 110° C., outlet temperature 67° C., fan efficiency 100%, pump rate 8%, and spray air flow rate 1750 L / h.

7. The spray-dried microparticles containing ZK-22 according to claim 5 or 6, characterized in that Lecithin is added at 60-80°C, dispersed under the shear force of 8000 rpm, and perfluorooctyl bromide (PFOB) is added dropwise and shearing is continued for 5 minutes to obtain colostrum; the colostrum is homogenized under high pressure at 18000 psi to obtain a carrier emulsion; preferably, the lecithin is distearoylphosphatidylcholine (DSPC).

8. The following uses of the ZK-22 phospholipid complex of claim 1, the suspension of the ZK-22 phospholipid complex of claim 3, or the ZK-22 spray-dried microparticles of any one of claims 5 to 7: (1) Preparation of dry powder inhalation dosage form; (2) preparing aerosol inhalation drugs; The medicine is used to prevent or treat respiratory viral infection; preferably, the respiratory virus is influenza virus, new coronavirus, or rhinovirus.

9. An inhalation-type ZK-22 preparation for administration through the airway, characterized in that: The inhalable ZK-22 preparation comprises a therapeutically effective amount of the ZK-22 phospholipid complex of claim 1, a suspension of the ZK-22 phospholipid complex of claim 3, or the ZK-22 spray-dried microparticles of any one of claims 5 to 7, and necessary pharmaceutical excipients.

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