Antiviral combination drug of ifn-alpha combined with bortezomib (bortezomib) and application

Abstract

This invention relates to the field of biomedicine, specifically to the combined application of interferon-alpha (IFN-α) and NF-κB signaling pathway inhibitors, particularly the application of interferon-alpha combined with bortezomib in the preparation of drugs for the prevention and / or treatment of viral infections. Host-produced lactate is a key microenvironmental factor driving the shift of IFN-α from "anti-inflammatory" to "pro-inflammatory." Lactic acid, in conjunction with IFN-α, excessively activates the NF-κB signaling pathway, thereby triggering a cytokine storm. Based on this novel mechanism, this invention creatively proposes the combined use of IFN-α and bortezomib. This combination effectively blocks lactate / IFN-α-induced NF-κB overactivation, retaining the potent antiviral activity of IFN-α while completely reversing its side effect of exacerbating inflammation in the later stages of viral infection, achieving a dual effect of "antiviral" and "inflammatory control."

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the combined use of interferon α and NF-κB signaling pathway inhibitors, and more particularly to the use of interferon α combined with bortezomib in the preparation of drugs for the prevention and / or treatment of viral infections. Background Technology

[0002] Interferon-alpha (IFN-α) is a key effector molecule in the body's innate immune response. By activating the JAK-STAT signaling pathway, it induces the expression of a series of antiviral genes, thereby achieving broad-spectrum inhibition against DNA and RNA viruses. Based on its potent antiviral activity, IFN-α has been used clinically to treat diseases such as chronic hepatitis B (HBV) and chronic hepatitis C (HCV).

[0003] However, the clinical application of IFN-α has significant limitations. First, its clinical indications are limited. In high-incidence, emerging, and sudden acute viral infections such as influenza, COVID-19, and dengue fever, its limited efficacy and high risk of adverse reactions have prevented it from being included in routine treatment recommendations. Second, existing studies have shown that while pretreatment with IFN-α before viral infection can effectively inhibit viral replication, after viral infection is established, IFN-α treatment may exacerbate the inflammatory response, induce a "cytokine storm," and lead to more severe histopathological damage. This dual negative effect of "antiviral failure + enhanced inflammation" greatly limits its application in the treatment of acute viral infections.

[0004] Therefore, there is an urgent need to develop a new strategy that can effectively avoid or reverse the side effect of IFN-α exacerbating inflammation in the later stages of viral infection while retaining its broad-spectrum antiviral activity, thereby expanding its clinical application scenarios and providing a safer and more effective treatment option for viral infections, especially acute and severe viral infections. Summary of the Invention

[0005] The present invention aims to solve the technical problem that the use of IFN-α after viral infection is established will aggravate the inflammatory response and cause side effects, and provides a new scheme for the combined application of IFN-α and NF-κB signaling pathway inhibitors.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solutions: An antiviral combination drug comprising interferon-alpha and an NF-κB signaling pathway inhibitor. Specifically, the NF-κB signaling pathway inhibitor is bortezomib.

[0007] Furthermore, the combined drug is used to prepare a drug for the prevention and / or treatment of viral infections; preferably, the viral infection is an acute viral infection; more preferably, the acute viral infection is an influenza virus or a novel coronavirus infection.

[0008] A pharmaceutical composition for the prevention and / or treatment of viral infections, the pharmaceutical composition comprising an interferon α and an NF-κB signaling pathway inhibitor, and a pharmaceutically acceptable carrier.

[0009] A method for inhibiting viral replication and reducing the expression of inflammatory factors in vitro includes the step of contacting virus-infected cells with an effective amount of interferon-α and an NF-κB signaling pathway inhibitor; preferably, the NF-κB signaling pathway inhibitor is bortezomib.

[0010] Use of interferon α and NF-κB signaling pathway inhibitors in the preparation of a kit for the prevention and / or treatment of viral infections; the kit includes a second container for interferon and an NF-κB signaling pathway inhibitor.

[0011] An antiviral combination drug or drug composition wherein the interferon α and the NF-κB signaling pathway inhibitor are configured to be administered simultaneously, separately, or sequentially.

[0012] Compared with the prior art, the present invention has the following beneficial effects: This invention reveals for the first time that lactate produced by the host after viral infection is a key microenvironmental factor driving the shift of IFN-α from "anti-inflammatory" to "pro-inflammatory." Lactic acid, in conjunction with IFN-α, overactivates the NF-κB signaling pathway, thereby triggering a cytokine storm. Based on this novel mechanism, this invention creatively proposes the combined use of IFN-α and NF-κB signaling pathway inhibitors (especially bortezomib). Experiments have demonstrated that this combined regimen can effectively block lactate / IFN-α-induced NF-κB overactivation, completely reversing the side effect of exacerbating inflammation in the later stages of viral infection while retaining the potent antiviral activity of IFN-α, achieving the dual benefits of "antiviral + inflammation control." This invention successfully overcomes a key bottleneck in the clinical application of IFN-α, providing a novel, safe, and effective application strategy for its broad-spectrum antiviral treatment of acute and severe viral infections. Attached Figure Description

[0013] Figure 1: Interferon-α treatment significantly exacerbates inflammation after viral infection is established. (A) ELISA assay of IL-1β levels in A549 cells after adding IFN-α (2 nM) at different time points (-2 h, 24 h, 48 h). Data are expressed as mean ± standard deviation (Mean ± SD). n=3, ns = no significant difference, ***p<0.001, ****p<0.0001. (B) qPCR analysis of inflammatory factors induced by viral (VSV, H1N1) infection in PM and MLF cells after adding IFN-α (2 nM) at different time points (-2 h, 4 h, 12 h). (C) Histopathological analysis (HE staining), inflammatory response analysis (IHC, LY6G staining), and quantitative analysis of LY6G positive cell count in C57 mice after adding IFN-α (200 ng / mouse) at different time points (-2 h, 24 h, 48 h) induced by viral (VSV) infection. Data are expressed as mean ± standard deviation (Mean ± SD), n=3, **p<0.01, ***p<0.001, ****p<0.0001. (D)C57 mice supplemented with IFN-α (200 ng / mouse) at different time points (-2 h, 24 h, 48 h) underwent histopathological analysis (HE staining), inflammatory response analysis (IHC, LY6G staining), and quantitative analysis of LY6G-positive cell counts in lung injury induced by H1N1 virus infection. Data are expressed as mean ± standard deviation (Mean ± SD), n=3, ns = no significant difference, **p<0.01, ***p<0.001, ****p<0.0001.

[0014] Figure 2 Figure 1: Results of inflammatory response caused by excessive lactic acid produced by viral infection. (A) Correlation analysis of LAC, LDH, CRP, WBC, CK-MB, and IL-6 in COVID-19 and influenza patients. Data are expressed as mean ± standard deviation (Mean ± SD), n=91, **p<0.01, ***p<0.001. (B) Schematic diagram of viral infection combined with LAC treatment of infected cells. PM cells were infected with the virus (VSV or H1N1) and treated with LAC simultaneously, and cells were collected for analysis. (C) Western blot analysis of IL-1β, a downstream inflammatory factor of the NF-κB signaling pathway, after viral infection combined with LAC treatment of infected cells.

[0015] Figure 3The results of viral infection-induced excess lactate enhancing IFN-α activation of the NF-κB signaling pathway and promoting inflammatory response are illustrated in the diagram. (Left image) Schematic diagram of cell treatment with IFN-α after LAC stimulation. PM cells were stimulated with LAC for 24 hours and then treated with IFN-α, and cells were collected for analysis. (Right image) Western blot analysis of p65, a key upstream factor in the NF-κB signaling pathway, after LAC stimulation and IFN-α treatment.

[0016] Figure 4 Figure 1: Inhibitory effect of different concentrations of Bortezomib on H1N1 and VSV viral replication. (A) Detection of H1N1 (left) or VSV (right) viral replication levels after treatment of A549 cells with different concentrations of Bortezomib. n=3, ns = no significant difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0017] Figure 5 : Results of IFN-α combined with Bortezomib inhibiting viral infection and avoiding inflammatory side effects. (A) ELISA assay of IL-1β levels in H1N1-infected A549 cells after treatment with Bortezomib (0.5 μM) combined with IFN-α. Data are expressed as mean ± standard deviation (Mean ± SD). n=3, ns = no significant difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (B) Detection of H1N1 virus replication level in H1N1-infected A549 cells after treatment with Bortezomib combined with IFN-α. n=3, *p<0.05, ***p<0.001, ****p<0.0001. (C) ELISA assay of IL-1β levels in VSV-infected A549 cells after treatment with Bortezomib (0.5 μM) in combination with IFN-α. Data are expressed as mean ± standard deviation (Mean ± SD). n=3, **p<0.01, ***p<0.001, ***p<0.001, ****p<0.0001. (D) Detection of H1N1 virus replication levels in VSV-infected A549 cells after treatment with Bortezomib in combination with IFN-α. n=3, **p<0.01, ***p<0.001, ***p<0.001, ****p<0.0001. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0019] In the specific embodiments of this invention, the materials, reagents, cell lines, virus strains, and animal models involved can all be obtained commercially or by methods known in the art, unless otherwise specified. Experimental methods without specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0020] Example 1: IFN-α treatment exacerbates inflammation after viral infection is established. This embodiment aims to verify the problems existing in the prior art, namely that using IFN-α for treatment after viral infection is established not only fails to effectively suppress inflammation, but also aggravates the inflammatory response and tissue damage.

[0021] 1. Experimental Materials and Methods Cell lines: human lung adenocarcinoma epithelial cells (A549 cells), primary mouse peritoneal macrophages (PM cells), and mouse lung fibroblasts (MLF cells). Cells were cultured in DMEM or RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and incubated at 37°C in a 5% CO2 incubator.

[0022] Viral strains: Vesicular stomatitis virus (VSV, Indiana strain) and influenza A virus (H1N1, PR8 strain), both purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd. The viruses were amplified in A549 or Vero cells, and their titers were determined by plaque formation assay.

[0023] Animal model: SPF-grade C57BL / 6J female mice, 6-8 weeks old, purchased from the Model Animal Research Center. All animal experiments were conducted in accordance with the guidelines for the management and use of laboratory animals.

[0024] Main reagents: Recombinant mouse / human interferon-α (IFN-α), purchased from PeproTech; mouse anti-LY6G antibody, used for immunohistochemistry (IHC); HE staining kit; ELISA kit (for detecting IL-1β, IL-6, and TNF-α); TRIzol reagent, used for RNA extraction; SYBR Green qPCR Master Mix.

[0025] Experimental Design: Cellular experiments: A549, PM, and MLF cells were seeded into 6-well plates. IFN-α (final concentration 2 nM) was added at different time points: 2 hours before viral infection (-2h), 24 hours after viral infection (24h), and 48 hours after viral infection (48h). The multiplicity of infection (MOI) was 1. Cell supernatant and cell pellet were collected at 48 hours (for A549 cells) or 24 hours (for PM / MLF cells) after viral infection.

[0026] Animal experiments: C57 mice were infected with VSV or H1N1 virus via intranasal instillation. Following infection, IFN-α (200 ng / mouse) was administered intraperitoneally. Dosing was administered at different time points: 2 hours before infection (-2h), 24 hours after infection (24h), and 48 hours after infection (48h). Mice were sacrificed on day 5 (VSV) or day 7 (H1N1) post-infection, and lung and liver tissues were collected. The experiment was approved by the Experimental Animal Welfare and Ethics Review Committee of Hubei University of Medicine, approval number: Animal Welfare No. 2026-038.

[0027] Detection method: Detection of inflammatory factors: The concentrations of IL-1β, IL-6, and TNF-α in cell supernatant were detected using an ELISA kit according to the manufacturer's instructions. mRNA expression levels of inflammatory factors in cells: Total RNA was extracted from cells, reverse transcribed into cDNA, and analyzed by qPCR. Primer sequences were designed using standard techniques in this field.

[0028] Histopathological analysis: Mouse lung and liver tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) to observe histopathological changes (such as inflammatory cell infiltration and tissue structure destruction). Simultaneously, LY6G (neutrophil marker) immunohistochemical staining was performed to quantitatively analyze the degree of inflammatory cell infiltration.

[0029] 2. Experimental Results (1) Results of cell experiments like Figure 1 The ELISA results for A showed that, compared with the untreated control group, the addition of IFN-α 2 hours (-2 h) before VSV or H1N1 virus infection significantly inhibited the release of the inflammatory cytokine IL-1β. This indicates that prophylactic use of IFN-α before infection has an anti-inflammatory effect. However, the addition of IFN-α 24 hours or 48 hours after viral infection establishment resulted in a significant increase in IL-1β levels, showing the opposite trend (p<0.001).

[0030] Figure 1The qPCR results of B further confirmed the above findings. In PM and MLF cells, the addition of IFN-α before infection (-2h) significantly reduced the mRNA levels of IL-6 and TNF-α induced by the virus (VSV / H1N1) infection. Conversely, the addition of IFN-α at 4 or 12 hours after infection not only did not decrease the mRNA levels of these pro-inflammatory factors, but was significantly increased compared to the virus-infected group alone (p<0.01 or p<0.001). These data clearly indicate that the effect of IFN-α is time-dependent: prophylactic use has an anti-inflammatory effect, while use after viral infection is established transforms into a pro-inflammatory effect.

[0031] (2) Results of animal experiments Next, this embodiment verifies the above findings through in vivo experiments. For example... Figure 1 As shown in C and 1D, in the VSV and H1N1 infection models in C57 mice, the viral infection itself already caused a certain degree of lung damage (observable alveolar structure destruction and inflammatory cell infiltration). Mice given IFN-α 2 hours before infection (-2h) showed significantly less histopathological damage and neutrophil infiltration in their lungs according to HE staining and LY6G immunohistochemical staining compared to the virus-infected group alone, confirming its prophylactic protective effect.

[0032] However, consistent with in vitro results, mice given IFN-α 24 or 48 hours after viral infection showed more severe damage to their lung and liver tissues (data showed liver damage was similar to that of the lungs). HE staining revealed alveolar wall thickening, infiltration of numerous inflammatory cells (including lymphocytes and neutrophils), and even focal hemorrhage. Quantitative analysis with LY6G staining also confirmed that the number of neutrophils in the post-infection treatment group was significantly higher than that in the virus-only infection group (p<0.01 or p<0.001).

[0033] The above results indicate that after viral infection is established, the use of clinically therapeutic doses of IFN-α is not only ineffective, but also exacerbates the inflammatory response and histopathological damage. This is the key reason why the clinical application of IFN-α is limited in the current technology.

[0034] Example 2: Excess lactic acid (LAC) produced by viral infection is a core factor driving inflammation. This study aims to explore the potential mechanisms leading to the "post-infection IFN-α pro-inflammatory effect" described in Example 1, particularly the metabolic changes in the host microenvironment induced by viral infection.

[0035] 1. Experimental Materials and Methods Clinical samples: Peripheral blood samples were collected from 91 patients diagnosed with influenza or novel coronavirus infection upon admission. Informed consent from patients and approval from the ethics committee were obtained for all sample collection.

[0036] Serological testing: The levels of lactate (LAC), lactate dehydrogenase (LDH), high-sensitivity C-reactive protein (CRP), and creatine kinase isoenzyme (CK-MB) in patient serum were measured using a fully automated biochemical analyzer. Serum IL-6 levels were measured using an ELISA kit. Correlation analysis (Pearson correlation coefficient) was performed on the above indicators.

[0037] Cellular experiments: PM cells from C57BL / 6J mice were isolated and cultured. Cells were divided into a control group, a simple virus infection group (VSV or H1N1, MOI=1), a simple lactate (LAC) treatment group, and a combined "virus infection + LAC" treatment group. The LAC concentration was 10 mM (simulating the pathological concentration in vivo after viral infection). Cells were collected 24 hours after treatment.

[0038] Detection method: The protein expression level of IL-1β, a key downstream inflammatory cytokine of the NF-κB signaling pathway, in cell lysates was detected by Western blotting. Specific antibodies used: rabbit anti-mouse IL-1β antibody and HRP-labeled secondary antibody. GAPDH was used as an internal control.

[0039] 2. Experimental Results (1) Correlation analysis of clinical samples To investigate the association between LAC (Lower Scale Activity) and systemic inflammation after viral infection, this study performed a correlation analysis on clinical data from 91 patients with influenza and COVID-19. The results are as follows: Figure 2 As shown in Figure A, the level of lactate (LAC) in patients was significantly positively correlated with the pro-inflammatory factor IL-6 (r = 0.68, p < 0.001), the inflammation-related marker high-sensitivity CRP (r = 0.55, p < 0.01), and the disease severity-related markers LDH (r = 0.72, p < 0.001) and CK-MB (r = 0.61, p < 0.01). This result indicates that the increase in lactate induced by viral infection is closely related to the intensity of inflammation in the body.

[0040] (2) Results of cell experiments To verify whether LAC directly drives inflammation at the cellular level, in vitro cell experiments were performed in this embodiment. Figure 2 As shown in B, this embodiment establishes a PM cell infection model. Figure 2Western blot results from C showed that VSV or H1N1 virus infection alone could induce moderate expression of IL-1β. Treatment with 10 mM LAC alone did not significantly induce IL-1β expression. However, when virus infection was combined with LAC treatment, the protein expression level of IL-1β was greatly enhanced, far exceeding the additive effect of the virus infection group and the LAC group alone. This result strongly demonstrates that the excess lactate produced during viral infection is a key cofactor in causing and exacerbating the inflammatory response.

[0041] Example 3: Lactic acid promotes inflammation by enhancing IFN-α activation of the NF-κB pathway. Having confirmed that lactate is a key driver of post-infectious inflammation, this embodiment further explores the interaction mechanism between lactate and IFN-α, particularly its impact on the NF-κB signaling pathway.

[0042] 1. Experimental Materials and Methods Cellular experiments: PM cells from C57BL / 6J mice were isolated and cultured. The experimental design was as follows: First, PM cells were stimulated with lactate (LAC, 10 mM) for 24 hours to simulate the lactate microenvironment after viral infection. Subsequently, the cells were treated with IFN-α (2 nM). Control groups were set up: no treatment group, IFN-α treatment group (no LAC pretreatment), and LAC treatment group (no subsequent IFN-α treatment).

[0043] Detection method: Cells from each experimental group were collected, and total protein was extracted. The phosphorylation level (p-p65) of p65 protein, a key activation marker of the NF-κB signaling pathway, was detected by Western blotting. Total p65 protein was also detected as a control. Higher p-p65 levels indicate stronger NF-κB pathway activation.

[0044] 2. Experimental Results Western Blot results are as follows Figure 3 As shown in the figure, compared with the control group, treatment with IFN-α alone for 24 hours ("no LAC" group) only weakly induced p65 phosphorylation, or even showed almost no change. This is consistent with the traditional understanding that IFN-α mainly activates the JAK-STAT pathway rather than the NF-κB pathway. Treatment with LAC alone for 24 hours also showed a certain degree of increase in p-p65 levels, indicating that high concentrations of lactate can moderately activate NF-κB.

[0045] The key finding is that pretreatment of cells with LAC for 24 hours, followed by IFN-α treatment, resulted in a highly significant and substantial increase in p-p65 protein levels, far exceeding the additive effect of LAC alone and IFN-α alone. This result reveals a novel molecular mechanism: while lactate is not the sole factor in post-infection inflammation, it is a key "switch" altering the responsiveness of cellular signaling pathways. In a lactate-rich microenvironment, the IFN-α signaling pathway is "reprogrammed," transforming from a mild immunomodulator into a potent activator of the NF-κB signaling pathway, thus explaining why post-infection IFN-α treatment induces a "cytokine storm."

[0046] Example 4: Screening for the optimal concentration of NF-κB signaling pathway inhibitors Based on the findings of Example 3, inhibiting the overactivation of the NF-κB signaling pathway is key to reversing the side effects of IFN-α. This example aims to screen an NF-κB signaling pathway inhibitor and determine its optimal concentration.

[0047] 1. Experimental Materials and Methods Cell lines and viruses: A549 cells; H1N1 and VSV viruses.

[0048] Drug: Bortezomib, dissolved in DMSO, diluted with culture medium to the required concentration before use.

[0049] Experimental Design: Infect A549 cells with H1N1 or VSV virus with MOI=1.

[0050] Twelve hours after infection, different concentrations of bortezomib were added: 0, 0.1, 0.25, 0.5, 1.0, and 2.0 μM.

[0051] Cells were collected 12 hours after drug treatment.

[0052] Detection method: Total RNA was extracted from cells, reverse transcribed, and then subjected to qPCR to detect the copy number of the viral genome (M gene of H1N1 or N gene of VSV). The sequences of the VSV N gene are shown in SEQ ID NO:1-2, and the sequences of the H1N1 M gene are shown in SEQ ID NO:3-4. The relative levels of viral RNA after treatment with different concentrations of bortezomib were calculated, using the untreated group (0 μM) as a control.

[0053] Further experiments involved treating A549 cells with different concentrations of bortezomib (0, 0.1, 0.25, 0.5, 1.0, 2.0 μM) for 24 hours. Afterward, the culture medium was discarded, and working solution containing CCK-8 reagent was added. The cells were incubated for 2 hours, and the absorbance (OD value) was measured at 450 nm using a microplate reader. The relative cell viability of each group was calculated with the cell viability of the untreated group as 100%.

[0054] 2. Experimental Results Experimental results are as follows Figure 4 As shown. For the H1N1 virus ( Figure 4 (Left) Bortezomib exhibits dose-dependent antiviral activity in the concentration range of 0 to 0.5 μM. 0.1 and 0.25 μM bortezomib partially inhibited viral replication, while 0.5 μM bortezomib significantly inhibited viral RNA synthesis, reducing viral load by approximately 90% (p<0.0001). However, when the concentration was further increased to 1.0 μM and 2.0 μM, its antiviral effect decreased, possibly due to the cytotoxicity of high concentrations (data not shown).

[0055] For VSV virus ( Figure 4 (Right) This embodiment observed a completely consistent trend. 0.5 μM bortezomib also exhibited the best antiviral effect, significantly inhibiting VSV replication (p<0.001).

[0056] Furthermore, the results showed that when bortezomib concentrations ranged from 0 to 0.5 μM, the relative cell viability in all experimental groups remained above 95%, and no significant cytotoxicity was observed compared to the untreated control group (p>0.05). This indicates that bortezomib concentrations (0.1-0.5 μM), which significantly inhibit H1N1 and VSV viral replication, did not significantly damage host cells A549. However, when the bortezomib concentration increased to 1.0 μM and 2.0 μM, the relative cell viability decreased to approximately 78% and 62%, respectively, showing significant dose-dependent cytotoxicity. This trend of decreased cell viability is consistent with... Figure 4 The phenomenon of weakened antiviral effect in the medium and high concentration groups (1.0 μM and 2.0 μM) is highly consistent.

[0057] Therefore, in this embodiment, bortezomib at a concentration of 0.5 μM was selected for subsequent combination drug experiments. This concentration effectively inhibits the NF-κB pathway and viral replication while avoiding the potential cytotoxicity of high concentrations. The above data demonstrate that the preferred concentration (0.5 μM) of bortezomib described in this invention can exert synergistic antiviral and anti-inflammatory effects while completely maintaining host cell viability.

[0058] Example 5: Interferon α combined with bortezomib achieves dual effects of "antiviral + anti-inflammatory". After viral infection is established, can the combined use of IFN-α and the selected optimal concentration of bortezomib achieve effective antiviral effects while avoiding the inflammatory side effects of IFN-α used alone?

[0059] 1. Experimental Materials and Methods Cell lines and viruses: A549 cells; H1N1 and VSV viruses.

[0060] Drugs: IFN-α (2 nM), bortezomib (0.5 μM).

[0061] Experimental Groups: The experiment consisted of 6 groups: 1) Blank control group (no infection, no medication); 2) Viral infection group (infected with virus, no medication); 3) IFN-α treatment group (IFN-α added 12 hours after infection); 4) Bortezomib treatment group (bortezomib added 12 hours after infection); 5) Combined treatment group - sequential administration (bortezomib added 12 hours after infection, followed by IFN-α 1 hour later); 6) Combined treatment group - simultaneous administration (bortezomib and IFN-α added simultaneously 12 hours after infection).

[0062] Processing procedure: A549 cells were seeded in 24-well plates and infected with H1N1 or VSV virus at MOI=1. After 12 hours, the appropriate drug was added according to the group assignment. After 12 hours of drug treatment, the cell supernatant and cell pellet were collected.

[0063] Detection method: Inflammatory marker detection: The level of IL-1β in cell supernatant was detected using an ELISA kit.

[0064] Antiviral efficacy detection: Total RNA was extracted from cells, and the copy number of the viral genome was detected by qRT-PCR, using the same method as in Example 4.

[0065] 2. Experimental Results Experimental results are as follows Figure 5 As shown.

[0066] (1) Effects on the inflammatory response like Figure 5 A (H1N1 infection) and Figure 5As shown in C (VSV infection): Virus-only infection induced moderate levels of IL-1β release. The IL-1β level in the IFN-α-only treatment group was significantly higher than that in the virus-only group (p<0.05 or p<0.01), a result that replicates the findings of Example 1, namely that IFN-α alone after infection exacerbates inflammation. The IL-1β level in the bortezomib-only treatment group was not significantly different from that in the virus-only group, indicating that bortezomib alone does not have significant pro-inflammatory or anti-inflammatory effects.

[0067] In both sequential and simultaneous combination therapy groups (IFN-α + bortezomib), IL-1β levels in the cell supernatant were significantly inhibited, not only significantly lower than in the IFN-α-only group (p<0.001 or p<0.0001), but even reduced to levels almost insignificantly different from the blank control group (ns). This indicates that the combined use of bortezomib can completely reverse the inflammatory side effects induced by IFN-α in the later stages of infection.

[0068] (2) Impact on antiviral efficacy like Figure 5 B (H1N1 infection) and Figure 5 As shown in D (VSV infection): Both the IFN-α-only treatment group and the bortezomib-only treatment group effectively inhibited viral replication, and the viral RNA level was significantly lower than that of the virus-only group (p<0.01 or p<0.001).

[0069] Encouragingly, the combination therapy group (IFN-α + bortezomib) exhibited a more potent antiviral effect. Its viral RNA level was significantly lower than that of the IFN-α monotherapy group and the bortezomib monotherapy group (p<0.01 or p<0.001), and was the lowest among all treatment groups. This indicates that the combination therapy not only did not weaken, but rather enhanced, the individual antiviral effects, resulting in a synergistic effect.

[0070] 3. Conclusion The comprehensive experimental results of this embodiment clearly demonstrate that, in response to the problem of IFN-α treatment exacerbating inflammation after viral infection is established, the technical solution of "IFN-α combined with bortezomib" proposed in this invention has achieved unexpected technical effects.

[0071] This joint program is able to: Highly effective virus inhibition: It synergistically exerts the classic antiviral effect of IFN-α and the novel antiviral effect of bortezomib to achieve strong inhibition of viral replication.

[0072] Complete control of inflammation: By blocking the NF-κB pathway synergistically activated by lactate / IFN-α with bortezomib, the pro-inflammatory side effects of IFN-α used alone were completely reversed.

[0073] This solution successfully turns a potential drawback into a potential advantage, transforming IFN-α from a potentially harmful double-edged sword into a safe and highly effective broad-spectrum antiviral and anti-inflammatory weapon, perfectly achieving the beneficial effects described in this invention.

[0074] The combined drug of the present invention can be applied to the prevention or treatment of various acute viral infections, especially respiratory viruses that are prone to causing severe illness and excessive inflammatory response, such as influenza virus, novel coronavirus and its variants.

[0075] Scenario 1: Treatment of hospitalized critically ill patients When a patient is admitted to the hospital with severe pneumonia (caused by viruses such as H1N1 or SARS-CoV-2), high levels of viral replication and a significant inflammatory response are already present in their body (manifested as high fever, elevated CRP and IL-6, and even elevated lactate and respiratory distress). For such patients, the IFN-α described in this invention is used in combination with bortezomib. The preferred administration method is as follows: bortezomib is first administered intravenously (the dosage can be adjusted according to clinical practice, e.g., 0.7-1.3 mg / m²) to rapidly block the NF-κB signaling pathway and control inflammation. Following bortezomib administration (e.g., 1-6 hours later), a therapeutically effective dose of IFN-α is then administered (e.g., subcutaneous injection or nebulized inhalation) to exert a potent antiviral effect. Alternatively, the two can be formulated into a stable pharmaceutical composition and administered simultaneously via the same route (e.g., intravenous infusion).

[0076] Scenario 2: Prevention after high-risk exposure For healthcare workers, immunocompromised individuals, or those contraindicated for vaccination, they can be considered for emergency prevention after confirmed exposure to highly pathogenic viruses such as influenza or COVID-19. In this case, IFN-α and bortezomib can be administered immediately in combination. This regimen aims to utilize the early antiviral effect of IFN-α while simultaneously suppressing potential early inflammatory responses with bortezomib, thereby preventing the virus from establishing effective infection and reducing the severity of subsequent illness. In this scenario, a single-dose or short-course combination dosing regimen can be used.

[0077] The interferon α and bortezomib described in this invention can be administered simultaneously, separately, or sequentially.

[0078] Simultaneous administration: The two active ingredients are mixed or administered via the same route as part of a fixed-dose combination of pharmaceutical ingredients.

[0079] Separate / sequential administration: Both can be administered to the subject at different time points via the same or different routes of administration (e.g., subcutaneous injection of IFN-α, intravenous injection of bortezomib).

[0080] Those skilled in the art can select appropriate formulations (such as injections, lyophilized powder injections, nebulized inhalation solutions, etc.) and administration regimens based on factors such as the specific virus type, disease severity, and patient condition; all of these fall within the scope of protection of this invention.

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antiviral combination drug, characterized in that, The combination drugs include interferon-α and NF-κB signaling pathway inhibitors.

2. The antiviral combination drug according to claim 1, characterized in that, The NF-κB signaling pathway inhibitor is bortezomib.

3. The antiviral combination drug according to claim 1 or 2, characterized in that, The combined drug is used to prepare a drug for the prevention and / or treatment of viral infections; preferably, the viral infection is an acute viral infection; more preferably, the acute viral infection is an influenza virus or a novel coronavirus infection.

4. A pharmaceutical composition for the prevention and / or treatment of viral infections, characterized in that, The pharmaceutical composition comprises an inhibitor of the interferon α and NF-κB signaling pathways as described in claim 1 or 2, and a pharmaceutically acceptable carrier.

5. A method for inhibiting viral replication and reducing the expression of inflammatory factors in vitro, characterized in that, The method includes the step of contacting virus-infected cells with an effective amount of interferon-α and an NF-κB signaling pathway inhibitor; preferably, the NF-κB signaling pathway inhibitor is bortezomib.

6. The use of interferon α and NF-κB signaling pathway inhibitors in the preparation of a kit, characterized in that, The kit is used for the prevention and / or treatment of viral infections; the kit includes interferon-α and an inhibitor of the NF-κB signaling pathway.

7. The antiviral combination drug according to claim 1 or 2, or the pharmaceutical composition according to claim 4, characterized in that, The interferon α and the NF-κB signaling pathway inhibitor are configured to be administered simultaneously, separately, or sequentially.

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