An inhaled formulation of ZK-22 for administration via the airway, its method of preparation and use

Abstract

The present application relates to an inhalation type ZK-22 preparation for airway administration, a preparation method and use thereof. The inhalation type ZK-22 preparation comprises a ZK-22 phospholipid complex, wherein the mass ratio of ZK-22 to phospholipid molecules in the ZK-22 phospholipid complex is 1:3, and the ZK-22 phospholipid complex is dispersed in an aqueous phase to obtain a suspension, and the suspension is spray dried to obtain microparticles. The present application also relates to use of the inhalation type ZK-22 preparation in treating or preventing respiratory viral infection.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, specifically relating to an inhaled ZK-22 formulation administered via the airway, its preparation method, and its uses. Background Technology

[0002] Currently, there are no specific drugs for acute respiratory viral infections other than influenza virus and coronavirus, nor are there any broad-spectrum preventive and therapeutic drugs. There is an urgent need to find new, safe, and broad-spectrum antiviral drugs for respiratory infections.

[0003] The piperazine-substituted 2-methylbenzyl nitrile compound ZK-22, with the chemical name 2-((4-(4-chlorophenyl)piperazin-1-yl)methyl)-4-((2-(dimethylamino)ethyl)(methyl)amino)benzyl nitrile, has the structure shown in formula (1):

[0004]

[0005] Multiple prior studies, including the applicant's work, have found that ZK-22 has good antiviral activity against hepatitis C virus and coronaviruses (J Med Chem. 2020; 63(11):5972-5989; Benzylpiperazine compounds and their preparation methods and applications in antiviral therapy, Chinese Patent No.: ZL201910905738.5, US Patent No.: US11939307; Application of a piperazine-substituted 2-methylbenzyl nitrile compound ZK-22 in the preparation of anticoronavirus drugs, Chinese Patent No.: ZL202111092666.0).

[0006] The respiratory tract is the body's first line of defense against respiratory pathogens. Although several oral small-molecule antiviral drugs are available, there are currently no antiviral drugs for airway administration on the market, and there are also few inhaled antiviral drugs in clinical trials (mainly including large-molecule drugs or immunomodulatory molecules, such as hACE2 nasal spray). 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. Inhaled formulations can achieve effective concentrations in the respiratory tract with smaller doses, achieve high airway exposure instantly, and have a faster onset of action. On the other hand, local administration results in less systemic exposure, avoiding systemic drug side effects and reducing drug interactions.

[0007] Therefore, in order to optimize and improve the pulmonary bioavailability of ZK-22, enhance the therapeutic effect and overall drug quality, it is essential to develop a safe and effective inhaled broad-spectrum antiviral inhalable ZK-22 compound formulation.

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

[0009] This invention first relates to a ZK-22 phospholipid complex, which can be used to prepare an inhaled ZK-22 formulation for inhalation via 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 distearate phosphatidylcholine (DSPC); preferably soybean lecithin, DPPC, and DSPC.

[0012] This invention also relates to a method for preparing the ZK-22 phospholipid complex, the method comprising the following steps:

[0013] (1) ZK-22 and phospholipids are dissolved in an organic solvent at a mass ratio of 1:3 and subjected to a hot reflux reaction to obtain ZK-22 phospholipid complex; preferably, the hot reflux reaction conditions are: hot reflux at 50°C for 3 hours; preferably, the organic solvent is chloroform;

[0014] (2) The organic solvent was completely removed by vacuum distillation combined with vacuum drying of ZK-22 phospholipid complex to obtain ZK-22 phospholipid complex dry powder.

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

[0016] The ZK-22 phospholipid complex dry powder was reconstituted with deionized water or physiological saline, and then processed by particle size control methods such as ultrasonic dispersion, high pressure homogenization, and microfluidics to obtain a suspension of the ZK-22 phospholipid complex.

[0017] Preferably, the parameters for ultrasonic dispersion are: 150W ultrasonic dispersion for 5 minutes.

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

[0020] (1) ZK-22 and phospholipids are dissolved in an organic solvent at a mass ratio of 1:3 and subjected to a hot reflux reaction to obtain ZK-22 phospholipid complex; preferably, the hot reflux reaction conditions are: hot reflux at 50°C for 3 hours; preferably, the organic solvent is chloroform;

[0021] (2) The organic solvent was completely removed by vacuum distillation and vacuum drying of ZK-22 phospholipid complex to obtain ZK-22 phospholipid complex dry powder;

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

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

[0024] (1) Stir and mix the ZK-22 phospholipid complex suspension or ZK-22 suspension and carrier emulsion evenly;

[0025] (2) Preparation of ZK-22 spray-dried microparticles by spray drying.

[0026] The method for preparing the carrier emulsion is as follows:

[0027] Phospholipids were dispersed in a heated deionized aqueous solution, and oil-phase perfluorobromooctane (PFOB) was added dropwise while stirring at high speed to form a primary emulsion. Then, a carrier emulsion was formed by high-pressure homogenization.

[0028] Preferred,

[0029] Phospholipids were added at 60-80℃ and dispersed under a shear force of 8000 rpm. Perfluorooctane (PFOB) was added dropwise and sheared continuously for 5 minutes to obtain the colostrum.

[0030] The colostrum was homogenized under high pressure at 18000 psi to obtain the carrier emulsion;

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

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

[0033] ZK-22 is obtained by micronizing it using a high-pressure homogenization method and then dispersing it 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 sonicated for 30 min; ZK-22 suspension is obtained by high-pressure homogenization and cycling at 950 bar for 20 cycles.

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

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

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

[0038] (2) Preparation of nebulized inhalation drugs;

[0039] The drug is used to prevent or treat respiratory viral infections; preferably, the respiratory virus is influenza virus, SARS-CoV-2, or rhinovirus.

[0040] The present invention also relates to an inhaled ZK-22 formulation administered via the airway, the inhaled ZK-22 formulation comprising, in a therapeutically effective amount, 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 5-7; and necessary pharmaceutical excipients.

[0041] The present invention also relates to the application of the ZK-22 phospholipid complex, the suspension of the ZK-22 phospholipid complex, or the ZK-22 spray-dried microparticles in the treatment of respiratory viral infections, preferably, the respiratory viruses being influenza virus, SARS-CoV-2, or rhinovirus.

[0042] The beneficial effects of this invention are as follows:

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

[0044] (2) This study demonstrates that the inhaled ZK-22 phospholipid complex has broad-spectrum antiviral activity 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 SARS-CoV-2 variants that emerged after 2022, as well as influenza viruses (Orthomyxoviridae) and rhinoviruses (Picovirusidae Enteroviruses), which are transmitted through the respiratory tract, suggesting that ZK-22 has a broad-spectrum antiviral ability against respiratory viruses. Attached Figure Description

[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 3A. Tissue distribution of ZK-22 phospholipid complex suspension and ZK-22 micron suspension after administration to the lungs (ZK-22-pc in the figure represents ZK-22 phospholipid complex suspension, and ZK-22 suspension represents ZK-22 micron suspension). 3B. Lung tissue after lavage. 3C. Lung epithelial lining fluid (ELF). 3D. Immune cells in lung epithelial lining fluid. 3D. Drug distribution in plasma at different time points (n=6, mean ± standard deviation).

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

[0050] Figure 5 Figure 1 shows the effects of ZK-22 on viral RNA and proteins in influenza virus-infected cells. 5A: Viral RNA; 5B: Viral protein M2.

[0051] Figure 6 Figure 6A shows the effect of ZK-22 on viral RNA in cells infected by three SARS-CoV-2 variants. Variant BA.5; Variant XBB.1.16; Variant EG.5.

[0052] Figure 7 Figure showing the effect of ZK-22 on viral RNA in human rhinovirus-infected cells. Detailed Implementation

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

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

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

[0056] (2) The obtained ZK-22 phospholipid complex was further vacuum dried for 24 hours after vacuum distillation to obtain ZK-22 phospholipid complex dry powder;

[0057] Determination of phospholipid complexation rate in ZK-22 phospholipid complex: After extraction with cyclohexane, the content of ZK-22 in the complex was determined by high performance liquid chromatography to obtain the complexation rate, which was calculated to be 85.6±2.49%.

[0058] Extraction process: Add 10 mL of cyclohexane to the round-bottom flask containing the dried product (ZK-22 phospholipid complex dry powder) and shake appropriately to ensure that the ZK-22 phospholipid complex is fully dissolved. Then filter it through a 0.45 μm filter membrane. The filtrate is evaporated and vacuum dried to obtain the ZK-22 phospholipid complex obtained from the reaction.

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

[0060] The phospholipid complex powder prepared in step 1 above was reconstituted with deionized water and then ultrasonically dispersed at 150W for 5 minutes to obtain ZK-22 phospholipid complex suspension.

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

[0062] Furthermore, after one month of storage at room temperature, the particle size and phospholipid complex ratio of the ZK-22 phospholipid complex suspension 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 physiological saline containing 0.04% Tween-80 and sonicate for 30 min;

[0066] (2) ZK-22 micron suspension was obtained by high-pressure homogenization and cycling at 950 bar for 20 cycles.

[0067] The average particle size of the suspension was 1.647 μm. Figure 2 Furthermore, no significant change in particle size was observed in the suspension after one month of storage at room temperature, indicating 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 in it under shear force of 8000 rpm, then slowly add 1.25 g of perfluorooctane (PFOB) and continue shearing for 5 min to obtain the pre-emulsion; immediately homogenize the pre-emulsion under high pressure at 18000 psi 5 times to obtain the carrier emulsion.

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

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

[0072] 4. Weigh 20mg of ZK-22 solid powder, disperse it in 5ml of deionized water containing 1.2% DSPC and sonicate for 30min; homogenize and cycle at 950bar for 20 times to obtain ZK-22 micron suspension, and dilute with deionized water to obtain 10mL of 2mg / 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) from step 1 above, and obtain ZK-22-loaded spray-dried microparticles by spray drying. The spray drying conditions are: inlet temperature 110℃, outlet temperature 67℃, fan efficiency 100%, pump speed 8%, and spray airflow 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 generally not correlated with plasma drug concentration, but is directly related to drug concentration in lung tissue. Therefore, it is necessary to clarify the distribution of drugs after pulmonary administration. This example compares the tissue distribution in the lungs and plasma after pulmonary administration of ZK-22 phospholipid complex (prepared in Example 1) and its reference preparation ZK-22 micron suspension (prepared in Example 2).

[0076] 1. Lung tissue distribution experiment:

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

[0078] (2) Blood samples were collected at 1, 3, 6 and 12 hours after drug administration. After euthanasia, tracheal intubation was performed for bronchoalveolar lavage, and lung tissue was then removed (n=6). The bronchoalveolar lavage fluid was centrifuged at low speed to separate the lower layer cells, and the drug content in plasma, bronchoalveolar lavage fluid supernatant and lower layer cells, and lung tissue was measured.

[0079] Rats were administered ZK-22 micron suspension and ZK-22 phospholipid complex suspension via tracheal intubation at a dose of 2.5 mg / kg. Drug concentrations in plasma, bronchoalveolar lavage fluid, lavage fluid cells, and post-lavage lung tissue were as follows: Figure 3 Results analysis:

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

[0081] (2) Significant differences were observed in drug distribution in the bronchoalveolar lavage fluid, lavage fluid cells, and post-lavage lung tissue after administration of the two dosage forms. Specifically:

[0082] Compared to ZK-22 micron suspension, ZK-22 phospholipid complex suspension in bronchoalveolar lavage fluid, lavage fluid cells and The drug content in lung tissue is higher 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 the intratracheal administration of ZK-22 phospholipid complex showed slowed absorption and clearance in ELF, indicating that ZK-22 has a sustained-release effect in the respiratory tract after being made into a phospholipid complex.

[0085] (2) Results from lavage fluid analysis showed that the ZK-22 phospholipid complex facilitated drug phagocytosis by macrophages compared to administration of ZK-22 micron suspension. This may be related to the prolonged exposure time of the phospholipid complex group within the ELF and the phagocytic properties of macrophages. The increased content of the phospholipid complex in lung tissue may primarily be due to the increased affinity of the phospholipid complex for the lungs.

[0086] The results of lung tissue distribution indicate that, compared with ZK-22 suspension, ZK-22 phospholipid complex suspension group drug in The retention 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 A virus subtype (PR8 strain, H1N1).

[0089] The specific experimental procedure is as follows:

[0090] 1. Animal grouping

[0091] The experimental animals were 2-week-old female specific pathogen-free (SPF) KM mice, randomly divided into 8 groups (n = 7-9). All animals were housed in a biosafety level 2 laboratory (ABSL-2). Except for normal control mice, the other mice were anesthetized with isoflurane and then intranasally administered 10 μL of influenza virus (PR8, H1N1) (3LD). 50 The following is a summary of the grouping and administration of drugs to the experimental animals following the infection.

[0092] (1) Virus control group: 40 μL of physiological saline was administered nasally 24 h before, 2 h after and 24 h after viral infection, for a total of 3 times;

[0093] (2) ZK-22 micron suspension group: 40 μL of the drug equivalent to 2.5 mg / kg ZK-22 micron suspension was administered nasally 24 h before, 2 h after and 24 h after viral infection, for a total of 3 administrations;

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

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

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

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

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

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

[0100] All mice were euthanized 96 hours after viral infection, and lung tissue was collected and stored at -80°C.

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

[0102] (1) Lung tissue was placed in a tissue grinding tube and extracted using an animal tissue RNA extraction kit. The RNA concentration was then detected. 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. Each RNA sample was measured twice.

[0103] (2) Using influenza virus NP protein particles as a standard, serial dilutions were performed to achieve a final concentration of 8.4 × 10⁻⁶. 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 in 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 shown below:

[0108]

[0109]

[0110] The reaction conditions are as follows:

[0111]

[0112] (3) A standard curve was plotted based on the gene copy number and Ct value of the plasmid standard expressing influenza NP protein. The copy number could then be calculated from the Ct value of viral RNA in each lung tissue obtained from the experiment.

[0113] 3. Results 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), which is consistent with the literature reports and previous experimental results of our group.

[0115] (2) In the group treated with 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 viral control group (P<0.05);

[0116] (3) The viral RNA content in the lung tissue of mice in both dosage groups of ZK-22 phospholipid complex suspension (ZK-22-pc) was significantly lower than that in the viral control group (P<0.01 or P<0.001); compared with ZK-22 micron suspension, ZK-22 Phospholipid Complex The suspension had a lower degree of reduction in viral RNA levels in lung tissue. Figure 4 ) .

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

[0118] The example investigated the effect of ZK-22 on the intracellular viral RNA and protein content of influenza A subtype (PR8 strain, H1N1) infection.

[0119] The specific experimental procedure is as follows:

[0120] 1. Cell infection and drug administration

[0121] All experiments were performed in a biosafety level 2 (BSL-2) laboratory. Canine kidney cells (MDCK) were seeded in 12-well plates and cultured overnight before being inoculated with influenza virus (100 TCID50). 50 If infected, administer the appropriate medication. After 2 hours, discard the viral solution and replace it with a maintenance solution containing the medication for continued culture.

[0122] The cell grouping and drug administration details 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 hours later;

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

[0126] (4) High dose of ZK-22: 10 μM ZK-22 was administered at the same time as the virus infection, and the cell maintenance medium containing 10 μM ZK-22 was replaced after 2 hours;

[0127] (5) Medium dose of ZK-22: 5 μM ZK-22 was administered at the same time as the virus infection, and the cell maintenance medium containing 5 μM ZK-22 was replaced after 2 hours;

[0128] (6) Low dose of ZK-22: 2.5 μM ZK-22 was administered at the same time as the virus infection, and the cell maintenance medium containing 2.5 μM ZK-22 was replaced after 2 hours.

[0129] 24 hours 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. Quantitative analysis of viral RNA in each group of samples

[0131] (1) RNA was extracted 24 hours after viral infection using the Rneasy Mini kit RNA extraction kit (purchased from QIAGEN). The influenza virus RNA content was then detected using the HiScript II One Step qRT-PCR SYBR Green Kit on an ABI 7500 Fast high-throughput real-time quantitative PCR (qPCR) instrument. Two replicates were set for each sample, and the experiment was repeated in three batches.

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

[0133]

[0134] (2) qPCR relative quantification method: Relative quantification analysis of the target gene (GOI) was performed. The Ct value was normalized using the housekeeping gene GAPDH as an internal control (IC). A 2-1 qPCR method was used. -ΔΔCt The method calculates the difference in expression of the target gene.

[0135] sample represents the experimental group; control represents the control group; Ct-GOI S Ct value representing the target gene in the experimental group samples; Ct-GOI C Ct value representing the target gene in the control group sample; Ct-IC S The Ct value representing the internal reference value of the experimental group samples; Ct-IC C The Ct value represents the internal reference value in the control group sample; the fold difference represents the fold change in 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. Quantitative analysis of viral proteins in each group of samples

[0141] Total protein was extracted from the cells and then subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), after which the protein was transferred to a PVDF membrane.

[0142] After electroporation, the PVDF membrane was immersed in 5% skim milk blocking buffer and gently shaken at room temperature for 1 hour. The membrane was then cut to the appropriate size according to the target protein and marker position, and incubated with the corresponding primary antibody (internal control β-actin: 1:1000 dilution (CST Biotech), influenza virus M2: 1:400 dilution (Santa Clinic)) at 4°C with shaking overnight. The membrane was washed three times with TBST for 10 minutes each time. A 1:5000 dilution of secondary antibody was added and incubated at room temperature for 1 hour. The membrane was then washed three times with TBST for 10 minutes each time. Finally, the membrane was exposed using an exposure buffer in a gel imaging system.

[0143] 4. Results Analysis

[0144] (1) Compared with the virus control group, the positive control drug baloxavir significantly inhibited the levels of influenza virus RNA and M2 protein, consistent with 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 influenza virus RNA and M2 protein in a dose-dependent manner. flat ( Figure 5 ).

[0146] Experiment Example 7: In vitro efficacy test of ZK-22 against SARS-CoV-2 variant

[0147] 1. Cell infection and drug administration

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

[0149] Vero E6 cells were seeded in 96-well plates. 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. DMSO was added to the control group. The cells were incubated at 37°C for 1 h, and the supernatant was discarded. Then, virus solution containing SARS-CoV-2 variants (BA.5, XBB.1.16, or EG.5) (infectious dose MO = 0.05) and culture medium containing ZK-22 or DMSO were added simultaneously. After incubation at 37°C for 24 h, the supernatant was discarded, TRIzol was added to the cells, and the cells were stored at -80°C.

[0150] 2. Quantitative analysis 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 viral RNA and GAPDH content was detected using the TransScriptProbe One-Step qRT-PCR SuperMix kit on an ABI 7500 Fast high-throughput qPCR instrument. Each RNA sample was measured twice.

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

[0153] The upstream of the novel coronavirus: 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] Upstream of monkey-derived GAPDH: 5'-CTGTTGCTGTAGCCAAATTCGT--3' (SEQ ID NO.10);

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

[0158] The reaction system is shown below:

[0159]

[0160] The reaction conditions are as follows:

[0161]

[0162] (3) The changes in the RNA content of the novel coronavirus in each group were calculated by qPCR relative quantification, and the method was the same as above.

[0163] 3. Results Analysis

[0164] Compared with the virus control group, The viral RNA levels of three COVID-19 variants in cells of the ZK-22-treated group all increased with the drug's effect. The concentration of the substance gradually decreases as the concentration increases. Figure 6 ) .

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

[0166] 1. Cell infection and drug administration

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

[0168] HeLa cells were seeded in 48-well plates. The next day, the culture medium was discarded, and then a virus solution containing human rhinovirus 14 (Strain1059) 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. Quantitative analysis 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 was detected using the HiScript II OneStep qRT-PCR SYBR Green Kit 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 the one-step RNA content detection method:

[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 changes in rhinovirus RNA content in each group were calculated by qPCR relative quantification, and the method was the same as above.

[0178] 3. Results Analysis

[0179] Compared with the virus control group, The rhinovirus RNA content in cells of the ZK-22-treated group gradually increased with increasing 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 intended to limit the scope of protection of the present invention.

Claims

1. A suspension of a ZK-22 phospholipid complex, wherein the suspension is prepared according to the following method: (1) ZK-22 and phospholipids were dissolved in an organic solvent at a mass ratio of 1:3 and subjected to a hot reflux reaction to obtain ZK-22 phospholipid complex; (2) The organic solvent was completely removed by vacuum distillation and vacuum drying of ZK-22 phospholipid complex to obtain ZK-22 phospholipid complex dry powder; (3) The ZK-22 phospholipid complex dry powder is reconstituted with deionized water or physiological saline and then treated with a particle size control method to obtain a suspension of the ZK-22 phospholipid complex. In the suspension of the ZK-22 phospholipid complex, the average particle size of the ZK-22 phospholipid complex particles is 100-400 nm, the dispersion is 0.1-0.4, and the potential is 10-25 mV.

2. The method for preparing the suspension of the ZK-22 phospholipid complex according to claim 1, characterized in that, Includes the following steps, (1) ZK-22 and phospholipids were dissolved in an organic solvent at a mass ratio of 1:3 and subjected to a hot reflux reaction to obtain ZK-22 phospholipid complex; the hot reflux reaction conditions were: hot reflux at 50°C for 3 hours; the organic solvent was chloroform. (2) The organic solvent was completely removed by vacuum distillation and vacuum drying of ZK-22 phospholipid complex to obtain ZK-22 phospholipid complex dry powder; (3) The ZK-22 phospholipid complex dry powder is reconstituted with deionized water or physiological saline and then processed by a particle size control method to obtain a suspension of the ZK-22 phospholipid complex; the particle size control method is ultrasonic dispersion, and the ultrasonic dispersion parameters are: 150W ultrasonic dispersion for 5 min.

3. A spray-dried microparticle containing ZK-22, wherein the ZK-22-containing spray-dried microparticle is prepared by the following method: (1) The ZK-22 phospholipid complex suspension and the carrier emulsion described in claim 1 are stirred and mixed evenly; (2) Spray drying to prepare spray-dried microparticles containing ZK-22; The method for preparing the carrier emulsion is as follows: Phospholipids were dispersed in a heated deionized aqueous solution, and oil-phase perfluorobromooctane was added dropwise while stirring at high speed to form a primary emulsion. Then, a carrier emulsion was formed by high-pressure homogenization.

4. The spray-dried microparticles containing ZK-22 according to claim 3, characterized in that, The spray drying conditions are as follows: inlet temperature 110℃, outlet temperature 67℃, fan efficiency 100%, pump speed 8%, and spray airflow rate 1750 L / h.

5. The spray-dried microparticles containing ZK-22 according to claim 3 or 4, characterized in that, The method for preparing the carrier emulsion is as follows: Phospholipids were added at 60-80°C and dispersed under a shear force of 8000 rpm. Perfluorooctane was added dropwise and sheared continuously for 5 minutes to obtain the primary emulsion. The primary emulsion was homogenized under high pressure at 18000 psi to obtain the carrier emulsion. The phospholipids were distearate phosphatidylcholine.

6. The following applications of the suspension of the ZK-22 phospholipid complex according to claim 1 or the spray-dried microparticles containing ZK-22 according to any one of claims 3-5: (1) Preparation of dry powder inhalation formulation; (2) Preparation of nebulized inhalation drugs.

7. An inhaled ZK-22 formulation administered via the airway, characterized in that, The inhaled ZK-22 formulation comprises a therapeutically effective amount of a suspension of the ZK-22 phospholipid complex of claim 1 or spray-dried microparticles containing ZK-22 as described in any one of claims 3-5; and necessary pharmaceutical excipients.

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