Application of alpha-terpilenol in preparation of medicine for resisting respiratory syncytial virus infection

Abstract

The invention discloses application of alpha-terpilenol in preparation of a medicine for resisting respiratory syncytial virus infection, and relates to the technical field of respiratory syncytial virus prevention and treatment. The alpha-terpilenol can inhibit the virus titer of the RSV in vitro, and obviously inhibits the infection of the RSV on cells. The alpha-terpilenol can maintain the normal alveolar structure of a mouse in vivo, inhibit inflammatory response and inhibit the expression of virus F protein in the lung, so that the alpha-terpilenol has remarkable anti-respiratory syncytial virus activity in vivo and in vitro. According to the invention, a new treatment scheme is provided for respiratory syncytial virus infection, and the treatment cost of respiratory syncytial virus infection is reduced.

Description

Technical Field

[0001] This invention relates to the field of respiratory syncytial virus (RSV) prevention and treatment technology, and more specifically, to the use of α-terpineol in the preparation of drugs for treating RSV infection. Background Technology

[0002] Human respiratory syncytial virus (RSV) is a non-segmented, single-stranded, negative-sense RNA virus with a genome of 10 genes sequentially encoding 11 proteins. Globally, an estimated 33 million cases of RSV-related acute lower respiratory tract infection (ARLI) occur annually in children under 5 years of age, resulting in 3.6 million hospitalizations and 26,300 in-hospital deaths. Besides infants and young children, RSV infection is also considered a significant cause of severe respiratory illness in the elderly and patients with underlying medical conditions.

[0003] Currently, RSV prevention mainly relies on passive immunization of high-risk infants, but its high cost and strict indications limit its widespread application. Clinical treatment remains primarily supportive therapy, with ribavirin and monoclonal antibodies showing limited efficacy only in specific populations. Therefore, existing RSV treatments have limitations such as side effects, high costs, and variability in efficacy among immunocompromised individuals. The application of medicinal plants (such as houttuynia cordata, scutellaria baicalensis, honeysuckle, and gardenia) or their extracts has significant research value due to their mild efficacy and low toxicity.

[0004] α-Terpineol (α-TPN), widely found in tea tree oil and pine needle oil, is a volatile monoterpene compound with strong inhibitory effects on various bacteria and fungi. Studies have shown that it can exert its pharmacological potential by alleviating mechanical hyperalgesia and inflammatory responses. In terms of antiviral activity, tea tree oil has been found to have activity against influenza A virus and herpes simplex virus type 1 (Astani et al., 2010; Garozzo et al., 2009). Furthermore, the volatile oil from the bark of *Taxus macrocarpa* has shown strong anti-SARS-CoV-2 activity in cells (Mohamed et al., 2022).

[0005] The main drawback of existing technologies and research is that studies on the antiviral effects of volatile oils (such as kauri bark volatile oil and tea tree oil) as a whole are rather general, lacking research on antiviral effects targeting α-terpineol as a single component. Furthermore, in vivo antiviral studies using α-terpineol as a single component are extremely rare, limiting the development of monoterpenoid compounds as antiviral natural products. Moreover, RSV belongs to different virus families than SARS-CoV-2, influenza A virus, and herpesvirus 1, and there are no specific drugs for RSV; therefore, the development of anti-RSV drugs is particularly important.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide the use of α-terpineol in the preparation of medicaments for treating respiratory syncytial virus infection.

[0008] This invention is implemented as follows: In a first aspect, the present invention provides the use of α-terpineol in the preparation of medicaments for treating respiratory syncytial virus infection.

[0009] In a second aspect, the present invention provides the use of α-terpineol and an antiviral drug in the preparation of a combined antirespiratory syncytial virus (RSV) remedy, wherein the antiviral drug is selected from at least one of ribavirin, nisvirumab, palizumab, and clarovirumab.

[0010] The present invention has the following beneficial effects: This invention demonstrates in vitro and in vivo experiments that α-terpineol can inhibit RSV viral titers in vitro and significantly suppress RSV infection of cells. In vivo, it can maintain normal alveolar structure in mice, inhibit inflammatory responses, and suppress viral F protein expression in the lungs. Therefore, α-terpineol exhibits significant anti-respiratory syncytial virus (RSV) activity both in vitro and in vivo. This invention provides a new treatment option for RSV infection and reduces the treatment cost of RSV infection. Attached Figure Description

[0011] 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.

[0012] Figure 1 The results of the in vitro anti-RSV infection experiment with α-terpineol (AB) were obtained by co-incubating HEp-2 cells with α-TPN for 48 h and measuring TC by MTT assay.50 Cells were then infected with RSV (GZ08-18) at an MOI of 0.1 for 1 hour, washed with PBS, and treated with different concentrations of α-TPN. The supernatant was collected 48 hours post-infection (hpi) and analyzed using TCID50. 50 Viral titer was determined by the following method: (CD) A549 cells were co-incubated with α-TPN for 48 h, and the TC50 value was determined by the MTT assay. Subsequently, cells were infected with RSV at an MOI of 0.1 for 1 h, washed with PBS, and treated with different concentrations of α-TPN; the supernatant was collected after 48 hpi and analyzed by TCID. 50 Viral titer was determined by the method; *p<0.05, **p<0.01, **p<0.001, 10 μg / mL ribavirin was used as a positive control). Figure 2 The results of the in vivo anti-RSV infection experiment with α-terpineol are as follows: (A) Change in body weight (%) of mice in each group, with the endpoint recorded at 21 dpi; (B) Survival rate (%) of mice in each group, with the endpoint recorded at 21 dpi; (C) Viral load in lung homogenates of mice in the RSV + Saline group, RSV + 50 mg / kg Ribavirin group, and RSV + 50 / 25 / 12.5 mg / kg α-TPN group 3 days after challenge, measured by qRT-PCR, with the viral N gene as the amplification target; *p<0.05, **p<0.01, ***p<0.001). Figure 3 H&E staining images of mouse lung pathological sections after α-terpineol treatment for RSV challenge; Figure 4 Immunofluorescence staining images of mouse lung pathological sections after α-terpineol treatment for RSV challenge. Detailed Implementation

[0013] 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.

[0014] The term "sample" refers to a biological specimen obtained from or derived from an individual for a purpose. The source of the biological specimen can be a fresh, frozen, and / or preserved organ or tissue sample or solid tissue derived from a biopsy or primer; blood or any blood component. The term "sample" includes biological samples that have been manipulated in any way after acquisition, such as by reagent treatment, stabilization, enrichment for certain components (e.g., proteins or polynucleotides), or embedding in a semi-solid or solid matrix for sectioning purposes. In this invention, samples are particularly peripheral blood samples, serum samples, alveolar samples, and lung tissue samples.

[0015] As used in this article, the term "subject" can be understood to refer to anyone who has been infected with respiratory syncytial virus. Subjects can be patients in a clinical setting.

[0016] In a first aspect, the present invention provides the use of α-terpineol in the preparation of medicaments for treating respiratory syncytial virus infection.

[0017] The inventors discovered that α-terpineol can significantly inhibit RSV infection in cells, significantly reduce the viral titer in mouse lungs in vivo, maintain the integrity of alveolar structure in mouse lungs, reduce inflammatory cell infiltration, decrease inflammatory response, and reduce viral F protein expression in mouse lungs. Therefore, α-terpineol exhibits significant anti-respiratory syncytial virus (RSV) activity both in vivo and in vitro. This invention provides a new treatment option for RSV infection and reduces the treatment cost of RSV infection.

[0018] In a preferred embodiment of the present invention, the drug is administered by injection or orally.

[0019] In a preferred embodiment of the present invention, the drug also includes pharmaceutically acceptable excipients.

[0020] Excipients include, but are not limited to, conventional pharmaceutical excipients, carriers, or diluents.

[0021] Pharmaceutically acceptable excipients include, but are not limited to, fillers, lubricants, disintegrants, binders, and flow aids.

[0022] In a preferred embodiment of the present invention, the pharmaceutically acceptable excipients include, but are not limited to, one or more of the following: polyvinylpyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methylcellulose and its derivatives, ethylcellulose and its derivatives, hydroxypropylcellulose and its derivatives, hydroxypropyl methylcellulose, starch and its derivatives, polyethylene glycol and its derivatives, lactose, lactose-starch complex, lactose-cellulose complex, sucrose, mannitol, mannitol-starch complex, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, povidone, copovidone, calcium hydrogen phosphate, calcium stearate, sodium stearoyl fumarate, silicon dioxide, titanium dioxide, talc, indigo, low-substituted hydroxypropyl cellulose, croscarmellose sodium cellulose, croscarmellose, magnesium stearate, sodium stearate fumarate, talc, and stearic acid, or a combination thereof.

[0023] In a preferred embodiment of the present invention, the drug is applied in the following ways: Reduce the viral titer of respiratory syncytial virus in the samples of the subjects.

[0024] In a preferred embodiment of the present invention, the drug is applied in the following ways: Improve the survival rate of test subjects.

[0025] In a preferred embodiment of the present invention, the drug is applied in the following ways: The viral load of respiratory syncytial virus (RSV) in the subjects' samples was reduced. Viral load was measured, for example, by detecting the transcriptional level of the viral N gene using qRT-PCR with the N gene as the amplification target.

[0026] In a preferred embodiment of the present invention, the drug is applied in the following ways: To maintain the integrity of the alveolar structure in the subjects.

[0027] In a preferred embodiment of the present invention, the drug is applied in the following ways: It reduces the inflammatory response in the lungs of the subjects and decreases the infiltration of inflammatory cells.

[0028] In a preferred embodiment of the present invention, the drug is applied in the following ways: It reduced the expression level of viral F protein in the lungs of the subjects.

[0029] In a second aspect, the present invention provides the use of α-terpineol and an antiviral drug in the preparation of a combined antirespiratory syncytial virus (RSV) remedy, wherein the antiviral drug is selected from at least one of ribavirin, nisvirumab, palizumab, and clarovirumab.

[0030] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0031] Example 1 This example demonstrates a cell experiment to assess anti-RSV infection activity. The reagents used in this experiment are as follows: α-terpineol, manufactured by Aladdin Reagent (Shanghai) Co., Ltd., product code: T111437, CAS: 10482-56-1, purity: greater than 98%. Ribavirin, manufactured by Aladdin Reagent (Shanghai) Co., Ltd., product code: R101754, CAS: 36791-0405, purity: 98%.

[0032] The experimental steps are as follows: First, the 50% cytotoxic concentration (TC) of α-terpineol (i.e., α-TPN) was determined in HEp-2 cells and A549 cells. 50 ), R 2 All values ​​were greater than 0.9, and based on this, the anti-RSV infection activity of α-terpineol in HEp-2 cells and A549 cells was tested.

[0033] HEp-2 cells were co-incubated with α-TPN for 48 h, and total cytogen (TC) was measured using the MTT assay. 50 Cells were then infected with RSV (GZ08-18) at an MOI of 0.1 for 1 hour, washed with PBS, and treated with different concentrations of α-TPN. The supernatant was collected 48 hours post-infection (hpi) and analyzed using TCID50. 50 Viral titer was determined using a method that uses 10 μg / mL ribavirin as a positive control.

[0034] A549 cells were co-incubated with α-TPN for 48 h, and total cytogen (TC) was measured using the MTT assay. 50 Cells were then infected with RSV at an MOI of 0.1 for 1 hour, washed with PBS, and treated with different concentrations of α-TPN. The supernatant was collected after 48 hours and analyzed by TCID50. 50 Viral titer was determined using a method. The dashed lines in the left and right panels represent the 50% cell viability level and TCID, respectively. 50 Limit of detection. 10 μg / mL ribavirin was used as a positive control.

[0035] Results reference Figure 1 As shown, Figure 1 Figure A shows the cytotoxicity results of α-TPN on Hep-2 cells, and Figure B shows the viral titer results after treating cells with 0.5, 2.5, and 5 mg / mL α-TPN. The dashed lines in the left and right panels represent the 50% cell viability level and TCID, respectively. 50 Legal detection limits.

[0036] Figure 1Figure C shows the cytotoxicity results of α-TPN on A549 cells, and Figure D shows the viral titer results after treating cells with 0.3 and 1.5 mg / mL α-TPN. The dashed lines in the left and right panels represent the 50% cell viability level and TCID, respectively. 50 Legal detection limits.

[0037] The results showed that α-terpineol had an effect on TC in HEp-2 and A549 cells. 50 The values ​​were 5.025 mg / mL and 2.682 mg / mL, respectively. Figure 1 (Figures A and C), and the goodness of fit (R²) 2 All values ​​are above 0.9, indicating that TC 50 The value is close to the true value.

[0038] Based on TC 50 As a result, TCID was detected 48 hours after infection (hpi). 50 The anti-RSV infection activity of α-terpineol was evaluated. The results showed that α-terpineol significantly inhibited RSV infection in two cell types in a dose-dependent manner. Specifically, 5 mg / mL α-terpineol inhibited approximately 90% of RSV infection in HEp-2 cells, while 1.5 mg / mL α-terpineol also inhibited approximately 90% of RSV infection in A549 cells. This indicates that α-terpineol can significantly inhibit RSV infection in cells.

[0039] Example 2 This embodiment conducts animal experiments to demonstrate the anti-RSV infection activity: First, a lethal RSV infection animal model was established in SPF-grade 8-month-old BALB / c female mice using a highly virulent RSV mouse-adapted strain (GZ08-18). Subsequently, infected mice were administered different concentrations of α-terpineol via intraperitoneal injection. Ribavirin intraperitoneal injection was used as a positive control, along with a virus control group, a solvent control group, and a blank control group. The body weight and survival rate of mice in each group were recorded daily. On the 3rd day after challenge, 3 mice from each of the drug treatment group, ribavirin group, and virus control group were collected, and the viral load in the lungs was measured. Simultaneously, mouse lung pathological sections were prepared, and H&E staining was used to observe the pathological changes in the mouse lungs. Immunofluorescence experiments were also conducted to evaluate the expression of viral F protein in the mouse lungs.

[0040] The specific experimental steps are as follows: (1) In vivo anti-RSV infection experiment of α-terpineol Seventy SPF-grade 8-month-old BALB / c female mice were randomly divided into 7 groups of 10 each, and were treated as follows: RSV + Saline group: 1×10 10 TCID 50Mice were challenged three times with RSV highly toxic strain GZ08-18 via tracheal intubation, with each challenge spaced 10-12 hours apart. One to two hours after each challenge, mice were intraperitoneally injected with 100 μL of DMSO / saline. The RSV + 50 mg / kg Ribavirin group received 1×10... 10 TCID 50 Mice were challenged three times with RSV highly toxic strain GZ08-18 via tracheal intubation, with each challenge spaced 10-12 hours apart. One to two hours after each challenge, mice were intraperitoneally injected with 50 mg / kg ribavirin (100 μL, physiological saline). The following groups were treated with RSV + 50 mg / kg α-TPN, RSV + 25 mg / kg α-TPN, and RSV + 12.5 mg / kg α-TPN: 1×10⁻⁶ mmol / L ribavirin was administered. 10 TCID 50 The RSV highly toxic strain GZ08-18 was used to challenge mice with laryngotracheal intubation three times, with each intubation occurring 10-12 hours apart. One to two hours after each intubation, mice were intraperitoneally injected with 50, 25, and 12.5 mg / kg α-terpineol (100 μL in DMSO + saline solution). The Mock group was a sham infection group, in which mice were laryngotracheally intubated with cell culture medium three times, 120 μL each time, with each intubation occurring 10-12 hours apart. One to two hours after each intubation, mice were intraperitoneally injected with 100 μL DMSO / saline solution. The Sham group was a blank control group.

[0041] The body weight and survival rate of mice in each group were recorded daily, with the recording endpoint at 21 days post-infection (dpi). The viral load in lung homogenates of mice in the RSV + Saline group, RSV + 50 mg / kg Ribavirin group, and RSV + 50 / 25 / 12.5 mg / kg α-TPN group was measured 3 days after challenge using qRT-PCR, with the viral N gene as the amplification target.

[0042] The primer sequences involved in qRT-PCR are as follows: NQF: 5'-CTCAATTTCCTCACTTCTC-3'; NQR: 5'-CCTCTGTATTCTCCCATT-3'. The standard detection plasmids for copy number calculation were serially diluted 10-fold with sterile double-distilled water. -2 ~10 -8Dilute the plasmid as template, perform 3 replicates for each concentration, and conduct qRT-PCR amplification to establish a standard curve. The qRT-PCR reaction system consisted of: 10.0 μl of TB Green® Fast qPCR Mix (2×), 0.2 μl each of 10 μmol / L NQ-F and NQ-R (Table 1), 4.2 μl of sterile double-distilled water, and 5.0 μl of viral cDNA template. The qRT-PCR amplification conditions were: 95 ℃ pre-denaturation for 30 s; 95 ℃ denaturation for 5 s; 60 ℃ annealing and extension for 10 s, collecting fluorescence signals, for a total of 40 cycles. After amplification, the temperature was increased from 65 to 95 ℃ in 0.5 ℃ increments, with results read after 5 s at each temperature. For specific qRT-PCR references: Liu Chunyuan, Yang Xiaomeng, Luo Yusi, et al. Establishment of absolute quantitative detection method for human respiratory syncytial virus qRT-PCR [J]. Chinese Journal of Pathogenic Biology, 2020, 15(09):1033-1037. DOI:10.13350 / j.cjpb.200908.

[0043] Results reference Figure 2 As shown, Figure 2 Figure A shows that, except for the Sham and Mock groups, the body weight of mice in the other groups began to recover around day 6 after the virus challenge, and basically returned to the baseline level by day 21. Figure 2 B in the study showed that mice treated with 50, 25, and 12.5 mg / kg α-terpineol had survival rates of 50%, 40%, and 30%, respectively. The survival rates of the 50 mg / kg α-terpineol group and the ribavirin group were significantly higher than those of the virus control group. Figure 2 The results showed that on day 3 post-challenge, the viral load in the lungs of mice treated with 50 mg / kg and 25 mg / kg α-terpineol was significantly lower than that in the viral control group.

[0044] (2) Observe the pathological changes in lung tissue by hematoxylin-eosin (H&E) staining.

[0045] Left lungs from mice at specific time points following infection were selected and fixed with 4% paraformaldehyde to ensure protein denaturation and coagulation while maintaining structural integrity. The tissues were first dehydrated and cleared with ethanol, then embedded and prepared into paraffin sections for subsequent staining and observation. A gradient ethanol dehydration method was used to remove paraffin while preserving the sample's structure. Lung tissue was cut into 6.0 μm thick sections, followed by hematoxylin and eosin staining. Finally, gradient dehydration and clearing were performed, and the sections were mounted with neutral resin. Pathological changes in the lung tissue were observed under a microscope, and images were acquired.

[0046] Microscopic observation and image acquisition, scale bar = 50 μm. H&E staining results of mouse lung pathological sections on day 3 post-infection. Figure 3 As shown, Figure 3 The results showed that the alveolar structure of the lungs of mice treated with 50 mg / kg and 25 mg / kg α-terpineol remained largely intact, with less inflammatory cell infiltration and a low inflammatory response.

[0047] (3) Immunofluorescence laser confocal assay: Left lung samples were taken from mice at designated time points after infection. Following the method described in the reference (Bian Wei. Sample Preparation Methods for Laser Confocal Microscopy (II) – Tissue Section Samples [J]. Journal of Electron Microscopy, 2010, 29(4): 4.), the following procedures were performed: Dewaxing and Dehydration: The waxed sections were attached to a glass slide and dewaxed and dehydrated. Antigen Retrieval: The tissue sections were placed on a plastic slide holder and sodium citrate buffer was added for antigen retrieval. Blocking: Blocking was performed with 3% BSA for 30 min. Primary Antibody Incubation: A 1:50 diluted primary antibody hRSV F was added to the sections and incubated overnight at 4°C. Secondary Antibody Incubation: Incubation was performed with fluorescent secondary antibody goat anti-rabbit (1:300) for 40 min. Counterstaining of Cell Nuclei with DAPI Staining Solution: Cell nuclei were stained with DAPI staining solution. Incubation with Tissue Autofluorescence Quencher: Incubation with autofluorescence quencher and thorough rinsing were performed. Mounting: Mount the slides with an anti-fluorescence quencher and neutral resin. Microscopic observation and image acquisition: Observe and acquire images under a laser confocal microscope. The virus F protein-specific monoclonal antibody was purchased from Santa Cruz Biotechnology, USA, product code: sc-101362.

[0048] Immunofluorescence staining was performed on mouse lung pathological sections treated with α-terpineol. Green fluorescent protein represents RSV F protein, and blue fluorescent protein represents DAPI-stained cell nuclei. Images were observed and acquired using a confocal fluorescence microscope (scale bar = 50 μm).

[0049] Immunofluorescence results ( Figure 4 The results were largely consistent with those above, namely, the expression of viral F protein in the lungs of mice in the α-terpineol treatment group was lower than that in the virus group.

[0050] 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.

Claims

1. Application of α-terpineol in the preparation of drugs against respiratory syncytial virus infection.

2. The application according to claim 1, characterized in that, The drug can be administered by injection or orally.

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

4. The application according to claim 1, characterized in that, The drug includes the following methods of application: Reduce the viral titer of respiratory syncytial virus in the samples of the subjects.

5. The application according to claim 1, characterized in that, The drug includes the following methods of application: Improve the survival rate of test subjects.

6. The application according to claim 1, characterized in that, The drug includes the following methods of application: Reduce the viral load of respiratory syncytial virus in the samples of the subjects.

7. The application according to claim 1, characterized in that, The drug includes the following methods of application: To maintain the integrity of the alveolar structure in the subjects.

8. The application according to claim 1, characterized in that, The drug includes the following methods of application: It reduces the inflammatory response in the lungs of the subjects.

9. The application according to claim 1, characterized in that, The drug includes the following methods of application: It reduced the expression level of viral F protein in the lungs of the subjects.

10. The use of α-terpineol and antiviral drugs in the preparation of a combined antiviral drug for respiratory syncytial virus infection, characterized in that, The antiviral drug is selected from at least one of ribavirin, nisevimob, palizumab, and clarovirumab.

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