Application of cycloastragenol-flower type lactose nanoparticles in preparation of medicine for resisting pneumonia caused by new coronavirus infection

Abstract

The invention discloses application of cycloastragenol-flower type lactose nanoparticles in preparation of a medicine for resisting pneumonia caused by new coronavirus infection. A large number of experiments prove that the astragenol-flower type lactose particles can inhibit inflammatory response in vivo, reduce apoptosis of target organ cells and improve related symptoms of SARS-CoV-2 pneumonia mice, so that the astragenol-flower type lactose particles can be applied as a treatment medicine for SARS-CoV-2 pneumonia related diseases.

Description

Technical Field

[0001] The present invention relates to a new use of cyclocophorol-flower-shaped lactose nanoparticles (CAG-FL), and particularly to the use of cyclocophorol-flower-shaped lactose nanoparticles (CAG-FL) in the preparation of a drug for treating pneumonia caused by COVID-19 infection. Background Art

[0002] Since the large-scale outbreak of coronavirus disease 2019 (COVID-19) caused by the novel coronavirus infection, there has been no clear conclusion on the origin of the virus so far. Seeking comprehensive and effective treatment strategies is the top priority and an urgent clinical problem to be solved. The novel coronavirus SARS-CoV-2 is the 7th known coronavirus that can infect humans. The detailed pathogenic mechanism of SARS-CoV-2 has not been thoroughly understood yet, but organ damage caused by the acute inflammatory response induced by SARS-CoV-2 infection is the main factor leading to severe cases. At present, the discovery of direct antiviral compounds mainly focuses on the screening of existing compounds and the repurposing of old drugs. Although these compounds targeting the virus or host cells show certain inhibitory effects on SARS-CoV-2, their drug safety and exact efficacy still need to be confirmed by later clinical trials. At present, drug development still faces huge challenges. Clinical studies have found that traditional Chinese medicine has played an active role in the prognosis of patients, especially showing good treatment effects in the treatment of some severe or critical patients. Cyclocophorol is the aglycone of astragaloside IV, an active ingredient of astragalus, and belongs to the cyclolanostane-type tetracyclic triterpenoid compound ( Figure 1 ). Research shows that cyclocophorol has anti-aging, antiviral, anti-inflammatory, and immunomodulatory effects, but its water-insoluble property limits its clinical use. We have recently prepared medicinal flower-shaped carrier lactose microparticles with nanostructured pores ( Figure 2 Figure 3 ), and preliminarily explored its adsorption and desorption capabilities for cyclocophorol ( Figure 4 ). On this basis, a cyclocophorol-flower-shaped lactose preparation was prepared, which greatly improved the water solubility and bioavailability of the drug and reduced the drug toxicity. This novel nano-preparation has obtained an invention patent authorization (patent number

[0003] ZL202111026322.X). Astragalus is one of the active ingredients of traditional Chinese medicine preparations for treating pneumonia caused by COVID-19. However, there is no report on the treatment of pneumonia caused by COVID-19 with the active ingredient cyclocophorol of astragalus. The clinical and basic research on using nano-carriers to transport cyclocophorol against the novel coronavirus is even blank. The present invention mainly discovers that cyclocophorol-flower-shaped lactose particles have a significant improvement effect on the inflammatory response and target organ lesions in a mouse model of pneumonia caused by COVID-19 infection. Summary of the Invention ​

[0004] Objective of the Invention: The objective of the present invention is to provide the use of cycloastragenol-flower-shaped lactose nanoparticles in the preparation of a drug for treating pneumonia caused by COVID-19 infection.

[0005] Technical Solution: On the one hand, the present invention provides the use of cycloastragenol-flower-shaped lactose nanoparticles in the preparation of a drug for treating pneumonia caused by COVID-19 infection.

[0006] Furthermore, the drug effect is dose-dependent.

[0007] Furthermore, the dosage is 5 mg / kg, 10 mg / kg, 20 mg / kg.

[0008] Furthermore, the dosage is 10 mg / kg, 20 mg / kg.

[0009] Furthermore, the dosage is 20 mg / kg.

[0010] Furthermore, the drug reduces the contents of inflammatory factors IL-6, TNF-α and neutrophil extracellular traps (NETs).

[0011] Furthermore, the purity of the cycloastragenol raw material drug is greater than 98%.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The research of the present invention proves the use of cycloastragenol-flower-shaped lactose nanoparticles in the preparation of a drug for treating pneumonia caused by COVID-19 infection. The present invention proves through a large number of experiments that cycloastragenol-flower-shaped lactose particles can inhibit the in vivo inflammatory response, reduce the apoptosis of target organ cells, and improve the related symptoms of SARS-CoV-2 pneumonia mice. Therefore, it can be used as a therapeutic drug for SARS-CoV-2 pneumonia-related diseases. Description of the Drawings

[0014] Figure 1 is the structural formula of cycloastragenol (CAG);

[0015] Figure 2 is the structure I of the medicinal flower-shaped carrier lactose microparticle containing nanostructured pores;

[0016] Figure 3 is the structure II of the medicinal flower-shaped carrier lactose microparticle containing nanostructured pores;

[0017] Figure 4 is the adsorption and desorption capacity curve of flower-shaped lactose carrying cycloastragenol;

[0018] Figure 5The results and pathological scores of HE staining of the lungs of mice killed 24 hours after the establishment of the SARS-CoV-2 pneumonia model induced by Poly(I:C)+SP by cycloastragenol-floral lactose particles;

[0019] Figure 6 24 hours after modeling, bronchoalveolar lavage fluid was collected from mice in each group to detect the levels of inflammatory factors IL-1α, TNF-α and neutrophil extracellular traps (NETs);

[0020] Figure 7 In the in vitro cell model, compared with the normal control group, the cell survival rate of the model group (SARS-CoV-2 pseudovirus group) was significantly decreased. After administration of CAG-FL, the cell survival rate was significantly improved and the cell activity was increased.

[0021] Figure 8 In an in vitro cell model, compared with the normal cell group, the high, medium, and low doses of CAG-FL treatment groups could significantly reduce cell apoptosis induced by viral infection. Specific implementation plan

[0022] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be further described below.

[0023] The method for preparing cycloastragenol-floral lactose particles in the following experiments adopts the method of patent number ZL202111026322.X.

[0024] 1. Pharmacological experiments of cycloastragenol-floral lactose nanoparticles

[0025] Poly(I:C)+SP-induced humanized hACE2 mouse SARS-CoV-2 pneumonia model.

[0026] Pneumonia model caused by COVID-19 infection in mice was established by referring to the method of Professor Dong Zigang's team. Male Balb / C mice, 8 weeks old, were kept in an SPF animal room with a room temperature of 20°C ± 2°C, free access to water and food, 12h day and night alternation, and a relative humidity of 50% ± 10%. All experimental procedures involving animals were approved by the Ethics Review Committee of the Laboratory Animal Science Department of Fudan University.

[0027] The mice were injected with adenoviral vector encoding hACE2 (Ad5-hACE2, 3.32×10 11PFU / ml, Hanheng Biotechnology Co., Ltd.) established humanized hACE2 mice. The hACE2 mice were randomly divided into 6 groups: normal group, normal mice + high-dose cyclocarya pallida alcohol-flower-shaped lactose granules group, model group, and low-, medium-, and high-dose CAG-FL groups (5 mg / kg, 10 mg / kg, 20 mg / kg, calculated based on the effective components of CAG). The normal group and the model group were given an equal amount of normal saline, and the remaining groups were administered by gavage according to the dose, once a day, 0.1 ml each time. After continuous administration for 3 days, except for the normal control group and the normal mice + high-dose CAG-FL group, the mice in the other groups were anesthetized with sodium pentobarbital (50 mg / kg, IP), and then fresh mixed poly(I:C) 2.5 mg / kg and recombinant SARS-CoV-2 S protein 15 μg were injected into the trachea of the mice for modeling.

[0028] At 24 hours after modeling, bronchoalveolar lavage fluid was collected from the mice in each group. After centrifugation at 3000 revolutions per minute for 5 minutes, the contents of TNF-α, IL-1α, and neutrophil extracellular traps (NETs) in the bronchoalveolar lavage fluid were detected.

[0029] At 24 hours after modeling, the lung tissues of the mice in each group were fixed by paraffin embedding for pathological sectioning. HE staining was used to observe the pathological changes, and the pathological scores were 0-4 points according to the degree of inflammatory cell infiltration and alveolar tissue lesions: 0 points: no lesions and inflammatory cell infiltration; 1 point: mild degree of lesions or inflammatory cell infiltration; 2 points: moderate degree of lesions or inflammatory cell infiltration; 3 points: severe degree of lesions or inflammatory cell infiltration; 4 points: very severe degree of lesions or inflammatory cell infiltration.

[0030] Intervention effect of CAG-FL on the in vitro model of pneumonia caused by SARS-CoV-2 infection

[0031] Experimental grouping: Primary isolated humanized ACE2 mouse alveolar type II epithelial cells were inoculated into 96-well plates at a density of 5×10 5 / ml, 100 μl per well. It was divided into a normal control group, a virus infection group, a low-dose group (1 μg / ml), a medium-dose group (2 μg / ml), and a high-dose group (4 μg / ml), all calculated based on the effective components of cyclocarya pallida alcohol carried. There were 3 samples in each group, and 2 replicates were set for each sample.

[0032] Cells were treated with high-, medium-, and low-dose CAG-FL. 100 μl of normal saline was added to each well in the normal control group and the virus infection group. After 12 hours, TCID50 = 10 was added to the cells in each group 4.68SARS-CoV-2 pseudovirus (Heyuan Biotechnology Co., Ltd.), 100 μl / well, the virus solution was discarded after infecting the cells for 1 hour. The normal control group and the virus infection group added 100 μl / well of culture medium without bovine serum, and the intervention group added high, medium, and low doses of CAG-FL and continued to culture for 24 hours. The treated cells in each well were tested as follows:

[0033] ① Add 10 μl of CCK8 reagent to each well, react at 37°C for 1 hour, measure the OD value at 450nm, and calculate the cell activity of each group;

[0034] ② Flow cytometry was used to detect the apoptotic proportion of cells in each group (Annexin / V kit, Bio-Tech Biotechnology Co., Ltd.).

[0035] 2. Analysis of pharmacological experimental results of cycloastragenol-floral lactose nanoparticles

[0036] (1) Effects of cycloastragenol-floral lactose nanoparticles on the Poly(I:C)+SP-induced SARS-CoV-2 pneumonia model in humanized hACE2 mice

[0037] like Figure 5 As shown in the figure, HE staining results showed that CAG-FL could significantly improve the degree of lung tissue damage and inflammatory cell infiltration in model mice, and this improvement showed obvious dose-dependency. Compared with the normal control group, the pathological score of the model group (Poly (I: C) + SP group) was significantly increased; CAG-FL 5mg / kg, 10mg / kg, 20mg / kg groups could dose-dependently reduce the HE staining pathological score. There was no difference in the HE staining pathological score between the normal control group and the normal control + high-dose drug group.

[0038] like Figure 6 As shown in the results, compared with the normal control group, the contents of IL-1α, TNF-α and neutrophil extracellular traps (NETs) in the bronchoalveolar lavage fluid of the model group were significantly increased; cycloastragenol administration could reduce the expression level of inflammatory factors in a dose-dependent manner. CAG-FL administration could significantly reduce the expression level of inflammatory factors and neutrophil extracellular traps (NETs), while there was no difference in bronchoalveolar lavage fluid between the normal control group and the normal control + high-dose drug group.

[0039] like Figure 7 As shown, in the in vitro cell model, compared with the normal control group, the cell survival rate of the model group (SARS-CoV-2 pseudovirus group) was significantly decreased. The CAG-FL low-dose group (1μg / ml), medium-dose group (2μg / ml), and high-dose group (4μg / ml) were all calculated based on the effective ingredient of cycloastragenol. After administration, they were able to significantly improve cell survival rate and increase cell activity.

[0040] As shown Figure 8 in the in vitro cell model, compared with the normal cell group, the high, medium, and low-dose treatment groups of CAG-FL could significantly reduce the apoptosis induced after virus infection.

[0041] The results of in vitro cell experiments suggest that cycloastragenol-flower-shaped lactose nanoparticles can not only inhibit the immune storm of the body by regulating the immune system, but may also play an antiviral role by activating the intrinsic defense mechanism of target organ cells.

[0042] The cycloastragenol-flower-shaped lactose nanoparticles described in the present invention can be used for the treatment of SARS-CoV-2 pneumonia-related diseases and the preparation of corresponding drugs.

[0043] Note: The raw material of cycloastragenol was purchased from Nanjing Daosifu Biotechnology Co., Ltd., with a purity greater than 98%.

[0044] Results:

[0045] For the SARS-CoV-2 pneumonia mouse model induced by Poly(I:C)+SP, intragastric administration of cycloastragenol-flower-shaped lactose particles at 5 mg / kg, 10 mg / kg, and 20 mg / kg could significantly reduce the pathological score of HE staining, alleviate the tissue damage and inflammatory cell infiltration of target organs, and reduce the contents of inflammatory factors IL-1α, TNF-α, and neutrophil extracellular traps (NETs) in bronchoalveolar lavage fluid, and the effect was dose-dependent.

[0046] In vitro cell experiments showed that after administration of CAG-FL, the cell viability of the SARS-CoV-2 pseudovirus group could be significantly improved and the cell activity could be enhanced; compared with the normal cell group, the high, medium, and low-dose treatment groups of CAG-FL could significantly reduce the apoptosis induced after virus infection.

[0047] The above results suggest that cycloastragenol-flower-shaped lactose particles can inhibit the inflammatory response in vivo, reduce the apoptosis of target organ cells, and improve the related symptoms of SARS-CoV-2 pneumonia mice, so it can be used as a therapeutic drug for SARS-CoV-2 pneumonia-related diseases.

[0048] The above are only the preferred embodiments of the present invention and do not impose any limitation on the present invention. Any person skilled in the art, without departing from the technical solution of the present invention, makes any form of equivalent substitution or modification and other changes to the technical solution and technical content disclosed by the present invention, which are all within the content of the technical solution of the present invention and still fall within the protection scope of the present invention.

Claims

1. Use of cycloastragenol-floral lactose nanoparticles in the preparation of drugs for treating pneumonia caused by novel coronavirus infection.

2. The use according to claim 1, characterized in that The drug effect is dose-dependent.

3. The use according to claim 2, characterized in that: The dosage is 5 mg / kg, 10 mg / kg, and 20 mg / kg.

4. The use according to claim 3, characterized in that The dosage is 10 mg / kg and 20 mg / kg.

5. The use according to claim 4, characterized in that The dose was 20 mg / kg.

6. The use according to claim 1, characterized in that: The drug reduces the content of inflammatory factors IL-6, TNF-α and neutrophil extracellular traps (NETs).

7. The use according to claim 1, characterized in that The purity of the cycloastragenol raw material drug is greater than 98%.

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