Application of 2,4-di-tert-butylphenol in the preparation of drugs against human respiratory syncytial virus

Abstract

This invention discloses the application of 2,4-di-tert-butylphenol in the preparation of anti-human respiratory syncytial virus (RSV) drugs, belonging to the field of biomedical technology. 2,4-Di-tert-butylphenol exhibits good anti-RSV effects both in vitro and in vivo. In vitro, it can inhibit viral infection of cells, and in vivo, it can alleviate lung damage caused by viral infection and reduce lung inflammation. This invention provides experimental evidence for the development of highly effective and safe anti-RSV drugs and also offers more drug options for their development.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the use of 2,4-di-tert-butylphenol in the preparation of drugs against human respiratory syncytial virus. Background Technology

[0002] Human respiratory syncytial virus (hRSV) is a non-segmental single-stranded negative-sense RNA virus belonging to the genus Pneumovirus of the family Orthopviridae [1]. hRSV infection can lead to acute lower respiratory tract infection (ALRTI) and even death in infants, immunocompromised individuals, and the elderly [2]. Almost all children have been infected with RSV at least once before the age of 2 [3]. The severity of hRSV infection in newborns is more severe than in older children, and outbreaks are more likely to occur [4]. Acute lower respiratory tract infection (i.e., pneumonia) caused by hRSV is one of the leading causes of neonatal death [5]. Currently, although there has been extensive and in-depth research on the structure and function of hRSV, the mechanism of infection, and the immune response of the host after infection, there is still no hRSV vaccine available in China. The only FDA-approved treatment drug, ribavirin, has some efficacy, but due to its potential for serious adverse reactions and teratogenic effects, it is no longer recommended for clinical use [6-7]. The only approved monoclonal antibody, palizil, is mainly used for passive immunization, but its application is limited due to its high cost and its restriction to high-risk infants [8-9].

[0003] There is an urgent need to explore a highly effective and safe treatment to address the social burden of hRSV and alleviate treatment costs.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide the application of 2,4-di-tert-butylphenol in the preparation of anti-human respiratory syncytial virus drugs, and to provide more drug options for the development of highly effective and safe anti-human respiratory syncytial virus drugs.

[0006] This invention is implemented as follows:

[0007] In a first aspect, the present invention provides the use of 2,4-di-tert-butylphenol and its pharmaceutically acceptable salts in the preparation of anti-human respiratory syncytial virus drugs.

[0008] 2,4-Di-tert-butylphenol, CAS No.: 96-76-4, molecular formula is C 14 H 22 O, English name: 2,4-Di-tert-butylphenol, also abbreviated as 2,4-DTBP.

[0009] The inventors discovered that 2,4-di-tert-butylphenol exhibits good antiviral activity against human respiratory syncytial virus (RSV) both in vitro and in vivo. In vitro, it can inhibit the virus's ability to infect cells, and in vivo, it can alleviate lung damage caused by viral infection and reduce lung inflammation. This invention provides experimental evidence for the development of highly effective and safe antiviral drugs against RSV, offering more drug options for this field.

[0010] In other embodiments, the above-mentioned compounds that can be used as drugs are pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" include pharmaceutically acceptable inorganic acid salts or organic acid salts; inorganic acid salts are preferably sulfates, sulfites, hydrochlorides, hydrobroms, nitrates, phosphates, and dihydrogen phosphates; organic acid salts are preferably acetates, maleates, fumarates, succinates, citrates, p-toluenesulfonates, tartrates, formates, propions, heptanoates, oxalates, benzoates, malonates, succinates, maleates, hydroxybutyrates, citrates, methanesulfonates, benzenesulfonates, lactates, or mandelates.

[0011] In a preferred embodiment of the present invention, the drug is used to prevent human respiratory syncytial virus infection and / or to alleviate or eliminate inflammation caused by human respiratory syncytial virus infection.

[0012] The aforementioned relief or elimination refers to the process of intervening in or altering a specific health condition, including curing or suppressing at least one of the inflammations caused by human respiratory syncytial virus infection.

[0013] In one alternative implementation, the above-mentioned relief refers to the transition of inflammatory symptoms from the active phase to the remission phase. For example, the maintenance of remission for lung inflammation refers to the state in which the remission phase continues without repeated occurrences of the active phase.

[0014] Prevention refers to the preventive therapeutic effect on mammals that are not yet sick but are susceptible to human respiratory syncytial virus (HRSV) and may be at risk of HSV infection.

[0015] In a preferred embodiment of the present invention, the inflammation is selected from at least one of bronchitis, lung inflammation, and pulmonary fibrosis.

[0016] In a preferred embodiment of the invention, the drug is a vaccine composition. For example, the vaccine composition may also include a pharmaceutically acceptable adjuvant.

[0017] In a preferred embodiment of the present invention, the drug has at least one of the following uses:

[0018] (1) Reduce the viral titer of cells after infection with human respiratory syncytial virus;

[0019] (2) Maintain the integrity of the alveolar structure;

[0020] (3) Inhibits viral F protein expression in lung tissue;

[0021] (4) Reduce or eliminate inflammatory cell infiltration in the lungs;

[0022] (5) Inhibits human respiratory syncytial virus replication.

[0023] In a preferred embodiment of the present invention, maintaining the integrity of the alveolar structure includes at least one of the following uses: preventing thickening of the alveolar septa, reducing or avoiding alveolar fusion, and mitigating lung damage.

[0024] In a preferred embodiment of the present invention, the drug also includes pharmaceutically acceptable excipients.

[0025] In a preferred embodiment of the present invention, the pharmaceutically acceptable excipient is selected from at least one of fillers, disintegrants, lubricants, flavoring agents, binders, suspending agents, solubilizers, and flavorings.

[0026] Pharmaceutically acceptable excipients include, but are not limited to, pharmaceutically acceptable carriers, excipients, or solvents. Pharmaceutically acceptable excipients include a variety of organic or inorganic carriers and / or excipients because they are commonly used for pharmaceutical purposes, particularly for solid drug formulations. Examples include: excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylene pyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, hydrolyzed starch, carboxymethylcellulose, calcium carboxymethylcellulose, hydroxypropyl starch, sodium glycol starch, sodium bicarbonate, calcium phosphate, and calcium citrate; lubricants such as magnesium stearate, talc, and sodium lauryl sulfate; and flavorings such as citric acid, menthol, and glycine. Orange powder; preservatives, such as sodium benzoate, sodium bisulfite, parabens (e.g., methylparaben, ethylparaben, propylparaben, butylparaben); stabilizers, such as citric acid, sodium citrate, acetic acid, and polycarboxylic acids from the titriplex series, such as diethylenetriaminepentaacetic acid (DTPA); suspending agents, such as methylcellulose, polyvinylpyrrolidone, aluminum stearate; dispersants; diluents, such as water, organic solvents; waxes, fats and oils, such as beeswax, cocoa butter; polyethylene glycol; white petrolatum, etc.

[0027] In a preferred embodiment of the present invention, the pharmaceutically acceptable excipient is a solubilizer, such as micelles prepared from polymeric materials.

[0028] In one optional embodiment, the solubilizer is selected from PEG-PLA micelles, PEG-PCL micelles, or PEG-PLGA micelles. In another optional embodiment, the particle size of the micelles can be adjusted as needed; for example, after loading with 2,4-di-tert-butylphenol, the particle size of the micelles is less than 100 nm. Small particle size is beneficial for mucus penetration and tissue distribution.

[0029] In an alternative embodiment, the PDI of the micelles is set to be less than 0.3 after loading with 2,4-di-tert-butylphenol. A low PDI value (<0.3) indicates that the micromicelles have a narrow particle size distribution and good uniformity.

[0030] In one alternative implementation, the lower the zeta potential of the micelles after loading with 2,4-di-tert-butylphenol, the better. A high absolute value of the zeta potential leads to enhanced repulsion between nanoparticles, which contributes to better stability in vivo.

[0031] In a preferred embodiment of the present invention, the dosage form of the drug is tablet, pill, powder, suspension, gel, emulsion, cream, granule, nanoparticle, capsule, suppository, injection or spray.

[0032] In an alternative embodiment, the aforementioned drug is a liquid pharmaceutical preparation (such as an injectable formulation), such as a solution, suspension, or gel, which typically contains a liquid carrier, such as water, and / or a pharmaceutically acceptable organic solvent. Furthermore, such liquid preparations may also contain pH adjusters, emulsifiers or dispersants, buffers, preservatives, wetting agents, gelling agents (e.g., methylcellulose), dyes, and / or flavoring agents, as defined above. The drugs may be isotonic, i.e., they may have the same osmotic pressure as blood. The isotonicity of the drug can be adjusted by using sodium chloride and other pharmaceutically acceptable reagents, such as glucose, maltose, boric acid, sodium tartrate, propylene glycol, and other inorganic or organic soluble substances. The viscosity of the liquid composition can be adjusted by a pharmaceutically acceptable thickener, such as methylcellulose. Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of the thickener depends on the reagent selected.

[0033] In a preferred embodiment of the present invention, the drug is formulated for oral or injectable administration.

[0034] In a preferred embodiment of the present invention, respiratory syncytial virus (RSV) is human respiratory syncytial virus (HRSV).

[0035] Secondly, the present invention also provides the use of 2,4-di-tert-butylphenol and its pharmaceutically acceptable salts in the preparation of a combination drug against human respiratory syncytial virus.

[0036] The present invention has the following beneficial effects:

[0037] The inventors discovered that 2,4-di-tert-butylphenol exhibits good antiviral activity against human respiratory syncytial virus (RSV) both in vitro and in vivo. In vitro, it can inhibit the virus's ability to infect cells, and in vivo, it can alleviate lung damage caused by viral infection and reduce lung inflammation. This invention provides experimental evidence for the development of highly effective and safe antiviral drugs against RSV and offers more drug options for this purpose. Attached Figure Description

[0038] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 Figure 1 shows the experimental results of 2,4-DTBP treatment inhibiting viral replication of the highly virulent strain GZ08-18 in HEp-2 and Vero E6 cells. (A: Different concentrations of 2,4-DTBP treatment reduced viral titer in HEp-2 cells; B: Different concentrations of 2,4-DTBP treatment reduced viral titer in Vero E6 cells; C: Different concentrations of 2,4-DTBP treatment reduced CPE in HEp-2 cells; D: Different concentrations of 2,4-DTBP treatment reduced CPE in Vero E6 cells; (25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL: different concentrations of drug treatment groups; GZ08-18: virus control group; Ribavirin: positive control group; *: compared with the virus control group; #: compared with the Ribavirin positive control group; ★: compared with 25 μg / mL 2,4-DTBP; ▲: compared with 12.5 μg / mL 2,4-DTBP) Comparison with 2,4-DTBP; ◆: Comparison with 6.25 μg / mL 2,4-DTBP; *: P<0.05; **: P<0.01; #: P<0.05; ##: P<0.01; ★: P<0.05; ★★: P<0.01; ▲: P<0.05; ▲▲: P<0.01; ◆: P<0.05.) DL: Limit of detection);

[0040] Figure 2Figure 1 shows the results of an in vivo experiment on 2,4-DTBP against the highly virulent strain GZ08-18 infection (A: Changes in body weight of mice in the 2,4-DTBP treatment group and the virus control group at different concentrations (n=6-8); B: Survival rate of mice in the 2,4-DTBP treatment group and the virus control group (n=6-8). (10.0 mg / kg, 5.0 mg / kg, 2.5 mg / kg: different concentrations of drug treatment groups; GZ08-18: virus control group; *: comparison between the 10.0 mg / kg and 5.0 mg / kg 2,4-DTBP treatment groups and the virus control group; #: comparison with the positive control ribavirin; *: P<0.05; ***: P<0.001; #: P<0.05; ###: P<0.001).

[0041] Figure 3 The statistical results of viral gene expression in lung tissue homogenates of mice infected with the highly virulent strain GZ08-18 on day 2 after infection with 2,4-DTBP are shown in the figure. (10.0 mg / kg, 5.0 mg / kg, 2.5 mg / kg: different concentrations of 2,4-DTBP treatment groups; PBS: virus control group; n=3 per group. *: comparison with virus control; *: P<0.05).

[0042] Figure 4 The image shows the hematologic and epithelial results (H&E) of lung tissue sections from mice infected with the highly virulent strain GZ08-18 in the 2,4-DTBP treatment group on day 2 (10.0 mg / kg, 5.0 mg / kg, 2.5 mg / kg: different concentrations of 2,4-DTBP treatment groups; PBS: virus control group; Mock: blank control group; A: scale bar = 1,000 μm; B: scale bar = 50 μm; C: scale bar = 10 μm; n = 3 per group, and the experiment was independently repeated twice).

[0043] Figure 5 Immunofluorescence results of lung tissue in mice infected with the highly virulent strain GZ08-18 in the 2,4-DTBP treatment group on day 2 (A: 10.0 mg / kg, 5.0 mg / kg, 2.5 mg / kg: different concentrations of 2,4-DTBP treatment groups; PBS: virus control group; Mock: blank control group; DAPI scale bar = 50 μm; hRSV F protein (red fluorescence) scale bar = 50 μm; Merge scale bar = 50 μm; B: immunofluorescence intensity of lung tissue; *: compared with the virus control group; #: compared with the 10.0 mg / kg 2,4-DTBP treatment group; ▲: compared with the 2.5 mg / kg 2,4-DTBP treatment group; ***: P<0.001; ****: P<0.0001; ####: P<0.0001; ▲▲▲▲: P<0.0001; n=3 per group);

[0044] Figure 6Statistical results of diameter, PDI, Zeta potential, loading efficiency (LE), and loading capacity (LC) for liposomes loaded with 2,4-DTBP (Liposom), PEG-PLA, PEG-PCL, and PCLG are shown in the figure for each group n=3.

[0045] Figure 7 The biodistribution of 3-hydroxyisoquinoline (HIQ) micelles in vivo is shown in the following graphs: (A shows the distribution and content of HIQ-loaded micelles in various animal tissues over time; B shows the content of HIQ-loaded micelles in bronchoalveolar lavage fluid and plasma over time, with n=3 for each group). Detailed Implementation

[0046] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0047] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0048] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0049] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0050] 2,4-DTBP (CAS: 96-76-4) was purchased from Sigma Aldrich (Shanghai) Trading Co., Ltd. (lot no. 137731, purity ≥ 99%).

[0051] The GZ08-18 dilution was based on the strain disclosed by Zhang K, Li C, Luo YS, Wen L, Yuan S, Wang D, Ho-Yin WongB, Zhao X, Chiu MC, Ye ZW, Sun Z, Zhao H, Zhang X, Hu M, Yang D, Shuai H, Wang Y, He J, Bose ME, Henrickson KJ, Huang JD, Zheng B, Chu H, Zhou J. Establishment of a lethalaged mouse model of human respiratory syncytial virus infection. Antiviral Res. 2019 Jan;161:125-133. doi:10.1016 / j.antiviral.2018.11.015.Epub2018Nov29.PMID:30503888.

[0052] BALB / c mice were purchased from Jiangsu Huachuang Xinno.

[0053] Example 1

[0054] This embodiment conducts an in vitro antiviral experiment:

[0055] First, a CCK8 cytotoxicity assay was used to construct a regression equation (R²). 2 =0.9772), calculate the effect of 2,4-DTBP on TC in CCK8 cells. 50The concentration was 0.6169 mg / mL. Based on the cytotoxicity results of 2,4-DTBP and ribavirin, GZ08-18 dilution with a multiplicity of infection (MOI) of 0.01 was incubated with pre-cultured HEp-2 (human laryngeal carcinoma cells) and VERO E6 (African green monkey kidney cells) cells at 37°C for 1 hour. The supernatant was discarded, and the cells were washed with PBS to remove unbound viruses. HEp-2 cells were then incubated with 2,4-DTBP (non-cytotoxic concentrations of 3.125, 6.25, 12.5, and 25 μg / mL) and 100 μg / mL ribavirin for 48 hours; Vero E6 cells were incubated with 2,4-DTBP (non-cytotoxic concentrations of 3.125, 6.25, 12.5, and 25 μg / mL) and 100 μg / mL ribavirin for 48 hours, respectively. Cells were then incubated with TCID-1 for 48 hours. 50 The inhibitory effect of the drug on the virus was measured. Its ability to inhibit viral infection of HEp-2 and VERO E6 cells was examined.

[0056] Results reference Figure 1 As shown, TCID 50 The results showed that all concentrations of 2,4-DTBP exhibited a significant decrease in viral titer in both HEp-2 and Vero E6 cells (e.g., Figure 1 The results (A and B in the table) indicate that 2,4-DTBP treatment significantly inhibited viral infection, and the inhibitory effect shown by HEp-2 cells was positively correlated with concentration, while 12.5 μg / mL 2,4-DTBP had the best inhibitory effect on Vero E6 cells.

[0057] The cytopathic effect (CPE) results after 2,4-DTBP treatment are shown in the figure below. Figure 1 As shown in C and D. Figure 1 In this context, "Mock" refers to a blank control, which means that only the transfection reagent is added without the pathogen, in order to eliminate the influence of the transfection reagent on the cells.

[0058] Microscopic images also showed a reduction in cytopathic effects after 2,4-DTBP treatment. These experimental results indicate that 2,4-DTBP treatment can significantly inhibit viral infection of HEp-2 and Vero E6 cells.

[0059] Example 2

[0060] This embodiment describes the screening and preparation of 2,4-DTBP-loaded micelles.

[0061] 2,4-DTBP-loaded micelles were prepared by solvent evaporation. PEG-PLA, PEG-PCL, and PLGA were dissolved in acetone and then mixed with 2,4-DTBP at a volume ratio of 1:5. After stirring for 30 minutes, the acetone was removed using a rotary evaporator (RE52CS, Shanghai Yarong Biochemical Instrument Co., Ltd., Shanghai, China). Free 2,4-DTBP was then removed through a 0.45 μm filter membrane to obtain 2,4-DTBP-loaded micelles. For biodistribution studies, hydroxyisoquinoline (HIQ) was used instead of 2,4-DTBP, and fluorescently labeled micelles were prepared using the same method.

[0062] The particle size, particle density (PDI), and zeta potential of 2,4-DTBP-loaded micelles were measured using a NanoBrook 90Plus PALS (Brookhaven, GA, United States). The concentration of 2,4-DTBP in the micelles was measured using a UV-2700 spectrophotometer (Shimadzu, Japan) at a detection wavelength of 277 nm. Drug loading efficiency (LE) and drug loading capacity (LC) were calculated using formulas (1) and (2):

[0063] LE(%)=W(drug loaded)*100 / W(total micelle), formula (1);

[0064] LC(%)=W(drug loaded)*100 / W(drug added), formula (2).

[0065] Wherein, W(drug loaded) represents the mass of drug encapsulated in micelles, W(total micelle) represents the mass of micelles containing encapsulated drug, and W(drug added) represents the mass of drug added during the preparation of micelles.

[0066] Compared to liposomes and PEG-PLGA micelles, both PEG-PLA and PEG-PCL exhibit smaller diameters and higher loading efficiency (LE) and loading capacity (LC) for 2,4-dinitroperoxybenzoic acid ester (2,4-DTBP). Figure 6 (A, D). The diameter of PEG-PLA and PEG-PCL micelles is less than 100 nm. The PDI of PEG-PLA is less than 0.3 ( Figure 6 B). The Zeta potential of PEG-PLA is close to -9 mV, which is the lowest among all groups (the Zeta parameter reflects the surface charge of the micelles, determining their stability and interaction with endogenous substances). Figure 6C) Small particle size is beneficial for mucus penetration and tissue distribution. A low PDI value (<0.3) indicates that the micelles have a narrow particle size distribution and good uniformity. A high absolute value of zeta potential leads to enhanced repulsion between nanoparticles, contributing to better stability in vivo.

[0067] Overall, the results show that PEG-PCL and PEG-PLA micelles loaded with 2,4-DTBP exhibit uniform particle size and good dispersibility. For LE and LC studies, PEG-PLA was chosen to prepare 2,4-DTBP-loaded micelles for further investigation.

[0068] Example 3

[0069] This embodiment conducts an in vivo antiviral experiment:

[0070] BALB / c mice were first challenged with the highly virulent strain GZ08-18, and then treated with 2,4-DTBP encapsulated in PEG-PLA micelles at doses of 2.5 mg / kg, 5.0 mg / kg, and 10.0 mg / kg, respectively, based on body weight. Ribavirin served as the drug control group (7.5 mg / kg). Mock served as the blank control group, specifically by intubation with an equal volume of physiological saline.

[0071] The changes in body weight and survival of mice within 21 days after infection were observed to reflect the protective effect of the drug on mice.

[0072] Results reference Figure 2 As shown, Figure 2 The results showed that the survival rates of the virus control group, ribavirin positive control group, and the 2.5 mg / kg, 5.0 mg / kg, and 10.0 mg / kg treatment groups were 0%, 100%, 28.6%, 33.3%, and 66.7%, respectively, in the 2,4-DTBP treatment group. Furthermore, the survival rates of the 5.0 mg / kg and 10.0 mg / kg treatment groups were significantly different from those of the virus control group (P<0.001, P<0.05). These results demonstrate that all concentrations of 2,4-DTBP have significant antiviral effects in vivo.

[0073] The changes in viral titer in mouse lung tissue homogenate were observed using qRT-PCR to reflect the inhibitory effect of drugs on the virus.

[0074] Results reference Figure 3 As shown, the viral titer results of lung tissue homogenates indicate that all concentrations of 2,4-DTBP can downregulate viral titers.

[0075] The pathological changes and differences in viral F protein expression in mice of different groups were observed by combining H&E staining and immunofluorescence experiments on lung tissue pathological sections with laser confocal imaging.

[0076] Figure 4 H&E staining results showed that, compared with the positive control group, the alveolar structure of the different concentrations of 2,4-DTBP treatment groups was basically maintained, the alveolar septa were not thickened, there was no obvious alveolar fusion, and the lung damage was reduced; while the alveolar structure of the virus control group was obviously fused, the alveolar septa were thickened, and there were a large number of inflammatory cell infiltrations and microthrombi.

[0077] Figure 5 The results of immunofluorescence of lung tissue showed that the expression of viral F protein in the lung tissue of mice treated with different concentrations of 2,4-DTBP decreased, and the difference was statistically significant compared with the viral control group (P<0.0001).

[0078] Example 4

[0079] This example studies the tissue distribution of 3-hydroxyisoquinoline (HIQ) micelles.

[0080] First, standard curves were plotted for plasma, bronchoalveolar lavage fluid, and various tissues (heart, liver, spleen, lung, and kidney). After randomization, mice were anesthetized with isoflurane inhalation and administered 22G... Intratracheal intubation was performed via IV catheter, and mice were administered 3.12 mg / kg of 3-hydroxyisoquinoline (HIQ) in micelle form. Mice were then exposed to HIQ micelles for 10 minutes, 0.5 hours, 1 hour, 2 hours, 4 hours, and 8 hours. After anesthesia, mice were euthanized, and abdominal aortic blood was collected and centrifuged at 3500 rpm for 10 minutes to obtain plasma. Bronchoalveolar lavage was performed, and tissue samples (heart, liver, spleen, lung, and kidney) were collected. The tissue surfaces were washed with PBS to remove residual blood and dried with filter paper. Simultaneously, the tissues were homogenized with PBS. Then, propionitrile was added to extract HIQ from the plasma, bronchoalveolar lavage fluid, and homogenized tissue samples in a specific ratio. Finally, the HIQ content in the supernatant was determined using a fluorescence spectrophotometer (Varian CaryEclipse, Palo Alto, USA) at excitation / emission wavelengths of 400 / 491 nm.

[0081] The biodistribution of PEG-PLA micelles after inhalation was investigated using HIQ-loaded PEG-PLA micelles (PEG-PLA micelles prepared in Example 2). Plasma, bronchoalveolar lavage fluid (BALF), post-lavage lung tissue, and other organs (heart, liver, spleen, and kidney) were collected at 10 min, 0.5 h, 1 h, 2 h, 4 h, and 8 h after inhalation of HIQ-loaded PEG-PLA micelles to determine HIQ concentration. Figure 7 ).

[0082] like Figure 7As shown in Figure A, the HIQ concentration in BALF decreased rapidly within 1 hour, then the decline slowed. Simultaneously, the highest HIQ concentrations in lung tissue and plasma were observed 0.5 hours after inhalation. High HIQ concentrations were also observed in the kidneys, heart, and liver during the initial 1 hour, then gradually decreased. Figure 7 B). The distribution behavior of HIQ in BALF indicates the drug's retention capacity in the respiratory tract and alveolar air, while the change in HIQ over time in lung tissue indicates the drug's distribution in the lung parenchyma. This suggests that PEG-PLA micelles can facilitate the inhalation of 2,4-DTBP, and that most of the 2,4-DTBP crosses the respiratory tract and lung tissue within 1 hour and is rapidly distributed to metabolic organs.

[0083] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0084] References:

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Claims

1. The use of 2,4-di-tert-butylphenol and its pharmaceutically acceptable salts in the preparation of drugs against human respiratory syncytial virus.

2. The application according to claim 1, characterized in that, The drug is used to prevent human respiratory syncytial virus infection.

3. The application according to claim 1, characterized in that, The drug has at least one of the following uses: (1) Reduce the viral titer of cells after infection with human respiratory syncytial virus; (2) Inhibits the replication of human respiratory syncytial virus.

4. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that, The pharmaceutically acceptable excipients are selected from at least one of fillers, disintegrants, lubricants, flavoring agents, binders, suspending agents, and solubilizers.

6. The application according to claim 4, characterized in that, The pharmaceutically acceptable excipient is a solubilizer.

7. The application according to claim 6, characterized in that, The solubilizer is selected from PEG-PLA micelles or PEG-PCL micelles.

8. The application according to any one of claims 1-7, characterized in that, The dosage form of the drug is tablet, pill, powder, suspension, gel, emulsion, cream, granule, capsule, suppository, injection or spray.

9. The application according to claim 1, characterized in that, The drug is formulated for oral or injectable administration.

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