Use of a pharmaceutical composition in the treatment of respiratory viral infections

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the application of pharmaceutical compositions in the treatment of respiratory viral infections. Experimental studies using this invention have demonstrated that the combined use of drugs targeting upstream metabolic damage (ZLN005) and downstream chemokine-mediated cell recruitment (BX471) and Oseltamivir can significantly reduce disease severity in a severe influenza model. This finding highlights the translational potential of host-guided therapy strategies, which are not only unaffected by viral mutations but also overcome the narrow therapeutic window problem inherent in traditional antiviral therapies. This study is the first to construct a synergistic intervention strategy combining "metabolic and immune dual-targeting" with antiviral small molecule drugs, proposing a treatment concept of "epithelial repair and immunopathogenesis inhibition," breaking the traditional antiviral treatment window limitations, and significantly improving the treatment efficacy of severe pneumonia, providing a new cross-mechanism combined treatment strategy for severe viral pneumonia.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of pharmaceutical compositions in the treatment of respiratory viral infections. Background Technology

[0002] Acute respiratory viral infections (such as influenza virus and SARS-CoV-2) continue to pose a significant challenge to global public health. Lethal immunopathological damage and cytokine storms are key contributing factors to patient deterioration and even death. After severe lung tissue damage, the regeneration and repair pathways of alveolar epithelial cells deviate, resulting in various abnormal epithelial progenitor cell intermediates (e.g., Krt8+ADI, PATS, DATP). The persistent presence and retention of these abnormal alveolar progenitor cells are core drivers of pathological tissue remodeling and ultimately pulmonary fibrosis. However, whether these dysfunctional alveolar progenitor cell intermediates participate in regulating the explosive, lethal, and acute inflammatory response in the progression of severe viral pneumonia remains unclear. Uncontrolled cytokine release is a crucial factor driving the transformation of respiratory viral infections into severe illness. Previous studies have largely focused on the imbalances in the regulation of immune cells, cytokine networks, and interferon signaling pathways, while the role of non-immune cells (especially epithelial cells) in this process remains unclear. Host metabolic disorders have been identified as an important risk factor for the development of severe viral infections, but the direct causal relationship between epithelial cell metabolic reprogramming, abnormal progenitor cell differentiation, and lethal immune overactivation remains unclear.

[0003] Antiviral small molecule drugs are currently one of the important intervention methods for dealing with acute respiratory viral infections. These drugs usually act on key enzymes or functional proteins in the viral life cycle, such as RNA polymerase, protease, or neuraminidase, thereby directly inhibiting the viral replication process in host cells. Taking influenza virus as an example, neuraminidase inhibitors (such as oseltamivir) and RNA polymerase inhibitors (such as favipiravir and baloxavir) have been widely used in clinical practice. Initiating treatment in the early stage of infection (usually within 48 hours of symptom onset) can significantly reduce the peak viral load, shorten the course of the disease, reduce the severity of symptoms, and reduce the risk of severe illness and transmission. For SARS-CoV-2, 3CL protease inhibitors (such as nematvir / ritonavir) and RdRp inhibitors (such as remdesivir and monopravir) have also shown effects in reducing the risk of severe illness and death in high-risk populations. However, although small molecule antiviral drugs have clear efficacy under ideal conditions, their clinical application still has several significant drawbacks and limitations. First, the treatment window is highly dependent on early drug administration; the efficacy decreases significantly after the window period is missed. The exponential replication rate of the virus in the early stages of infection far outpaces the initiation of the host's adaptive immunity. Most small-molecule drugs can only inhibit viral replication, but cannot clear integrated or released viral particles, nor reverse existing tissue damage. Therefore, if patients seek medical attention more than 48 hours after symptom onset (especially for influenza), the viral load has often reached a plateau or is declining, and medication at this point offers very limited clinical benefit. This characteristic significantly diminishes the public health value of these drugs in real-world clinical settings (especially for primary care or patients with delayed medical attention). Second, they lack positive regulation of the host immune response and cannot repair existing immunopathological damage. When viral infection exceeds the 48-hour window, tissue damage is often not only directly caused by the virus but also closely related to excessive or dysregulated host inflammatory responses (such as cytokine storms and immune cell infiltration). Existing small-molecule drugs only inhibit viral replication and have no direct repair capability for initiated inflammatory cascades and epithelial barrier damage. This means that even if the viral load decreases in late-stage treatment, clinical symptoms may continue to worsen due to immunopathological damage.

[0004] Therefore, a deeper understanding and development of dual intervention strategies targeting abnormal epithelial repair and acute inflammatory response, as well as treatment regimens targeting metabolic regulation and excessive inflammatory response, have potential clinical translational significance in improving severe cases. Summary of the Invention

[0005] This study is the first to construct a synergistic intervention strategy of "dual targeting of metabolism and immunity" with antiviral small molecule drugs, and proposes a treatment concept of "repairing the epithelium and inhibiting immunopathology". It breaks through the traditional window period limitation of antiviral treatment and significantly improves the treatment effect of severe pneumonia, providing a new strategy for cross-mechanism combined treatment of severe viral pneumonia.

[0006] To achieve the above-mentioned strategy of this invention, the technical solution adopted by this invention is: A first aspect of the present invention provides the use of a pharmaceutical composition in the preparation of a treatment for respiratory viral infections; In some embodiments of the present invention, the pharmaceutical composition includes any one of 1) to 3). 1) Oseltamivir and CCR1 antagonists; 2) Oseltamivir and fatty acid oxidation agonists; 3) Oseltamivir, CCR1 antagonists and fatty acid oxidation agonists.

[0007] In some embodiments of the present invention, the CCR1 antagonist includes BX471, AZD-4818, MLN3897, or nucleic acid molecules that negatively regulate CCR1.

[0008] In some embodiments of the present invention, the nucleic acid molecules that negatively regulate CCR1 include shRNA, siRNA, and sgRNA.

[0009] In some embodiments of the present invention, the fatty acid oxidation agonist includes ZLN005, GW501516, or a nucleic acid molecule that positively regulates fatty acid oxidation.

[0010] In some embodiments of the present invention, the nucleic acid molecules that positively regulate fatty acid oxidation include vectors that overexpress PGC-1α.

[0011] In some embodiments of the present invention, the respiratory virus includes influenza virus.

[0012] In some embodiments of the present invention, the influenza virus includes influenza A virus and influenza B virus.

[0013] In some embodiments of the present invention, the pharmaceutical composition comprises a pharmaceutically acceptable salt.

[0014] In some embodiments of the present invention, the pharmaceutically acceptable salt includes acid addition salts and base addition salts.

[0015] "Pharmaceutically acceptable acid addition salts" refer to salts that retain the biological effectiveness and properties of the free base, are not undesirable in biological or other respects, and are formed from an inorganic acid and an organic acid, such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., and such as, but not limited to, acetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, camphoric acid, camphor 10 sulfonic acid, decanoic acid, hexanoic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexanesulfonic acid, dodecyl sulfate, ethane 1,2 disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, fumaric acid, galactopyric acid, gentian acid, gluconic acid, glucuronic acid, glutamic acid, glutamate, 2-oxoglutamate, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, etc.

[0016] "Pharmaceutically acceptable base addition salts" refer to salts that retain the biological effectiveness and properties of the free acid and are not undesirable in biological or other respects. These salts are prepared by the addition of an inorganic or organic base to a free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, the following: primary, secondary, and tertiary amines; substituted amines (including naturally occurring substituted amines); cyclic amines; and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, thiazoline, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0017] In some embodiments of the present invention, the pharmaceutically acceptable salts of oseltamivir include oseltamivir phosphate and oseltamivir hydrochloride.

[0018] In some embodiments of the invention, the pharmaceutically acceptable salt of BX471 includes BX471 hydrochloride.

[0019] In some embodiments of the present invention, the pharmaceutically acceptable salt of ZLN005 includes ZLN005 hydrochloride.

[0020] In some embodiments of the present invention, the medicament includes pharmaceutically acceptable excipients.

[0021] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of diluents, binders, wetting agents, lubricants, disintegrants, solvents, cosolvents, solubilizers, preservatives, surfactants, coating materials, antioxidants, antibacterial agents, or buffers.

[0022] The pharmaceutically acceptable excipients mentioned above are generally recognized for use in this purpose and as inactive ingredients in the pharmaceutical preparation. Compilations of pharmaceutically acceptable excipients can be found in reference books such as the *Handbook of Pharmaceutical Excipients* (2nd edition, edited by A. Wade and PJ Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994) and the *Pharmacopoeia of the People's Republic of China - List of Pharmaceutical Excipients*.

[0023] In some embodiments of the present invention, the dosage form of the drug includes at least one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pellets, injections, oral preparations, suppositories, enemas, aerosols, patches, or drops.

[0024] In some embodiments of the present invention, the route of administration of the drug includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, nebulized administration, or transdermal administration.

[0025] In some embodiments of the present invention, the drug further includes one or more other active ingredients.

[0026] In some embodiments of the present invention, the drug is suitable for mammals.

[0027] A second aspect of the invention provides the use of BX471 and ZLN005 in the preparation of a medicament that enhances the efficacy of oseltamivir in treating respiratory viral infections.

[0028] The beneficial effects of this invention are: This invention's therapeutic experiments demonstrated that targeting upstream metabolic damage (ZLN005) in combination with downstream chemokine-mediated cell recruitment (BX471) and Oseltamivir significantly reduced disease severity in a severe influenza model. This finding highlights the translational potential of host-directed therapy strategies, which are not only unaffected by viral mutations but also overcome the narrow therapeutic window problem inherent in traditional antiviral therapies. ZLN005 is a fatty acid oxidation agonist. Currently, the regulation of fatty acid oxidation has been extensively explored clinically in chronic metabolic diseases and cardiovascular fields; however, its role in acute hyperinflammatory lung injury remains unclear. This study provides preclinical evidence for the re-utilization of ZLN005 in severe viral pneumonia. Meanwhile, CCR1 antagonists have completed clinical trials in various chronic inflammatory diseases (such as rheumatoid arthritis, endometriosis-related pelvic pain, and chronic obstructive pulmonary disease) (NCT01404585, NCT00185341, NCT00629239), demonstrating good safety and targeted binding properties. This invention further reveals that BX471 can effectively alleviate cytokine storms by inhibiting the recruitment of myeloid macrophages to lung tissue, thereby improving mouse survival and promoting lung tissue repair. This discovery provides a basis for re-evaluating the therapeutic potential of CCR1 antagonists in the context of acute viral infection and opens up new avenues for improving clinical outcomes in patients with severe viral pneumonia. Furthermore, this invention demonstrates that combining host-guided interventions targeting epithelial cell metabolic regulation or myeloid inflammatory macrophage recruitment with antiviral therapy can significantly prolong the therapeutic window, delay disease progression, and achieve optimal protective effects in severe pneumonia models. Attached Figure Description

[0029] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The results of combination therapy for severe PR8 influenza virus infection in a mouse model are shown, including: (A) Schematic diagram of the combined use of host immune modulation and antiviral therapy. (BC) Changes in body weight (B) and survival rate (C) of infected mice after treatment with PBS, Oseltamivir, Oseltamivir+BX471, Oseltamivir+ZLN005, and Oseltamivir+BX471+ZLN005 (n=10 per group). (D) Representative H&E staining images of lung tissue pathology, with the right image showing the quantitative results of lung injury scoring (n=6 per group), scale bar: 500 μm. Data represent the results of three independent experiments, expressed as ±SEM. Statistical analysis: multiple two-tailed t-test (D), two-way ANOVA (B), and log-rank test (C).

[0030] Figure 2 Immunofluorescence and immunohistochemical results of lung tissue from mice with severe PR8 influenza virus infection treated with combination drugs. Multicolor immunofluorescence (IF) and IHC staining results of AT2, AT1, and MDM markers in lung tissue sections from H1N1 virus-infected mice under different combination drug treatments show the changes after different combination treatments. IF scale bar: 500 μm; Inset scale bar: 20 μm; IHC scale bar: 200 μm.

[0031] Figure 3 Results of inflammatory factors, lung epithelial marker genes, and viral load assays in mice treated with combination drugs for severe PR8 influenza virus infection. (AB) Quantitative analysis of proteins and inflammatory factors in BALF (A) and serum (B) after treatment with different drug combinations in mice (n=4 per group). (C) RT-qPCR analysis of transcriptional levels of AT1 and AT2 marker genes in H1N1-infected mice treated with different drug combinations (n=4 per group). (D) Detection of viral vRNA levels in mouse lungs (n=4 per group). Data represent results from three independent experiments, expressed as ±SEM. Statistical analysis: multiple two-tailed t-test (AD). Detailed Implementation

[0032] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0033] Example 1: Combination therapy improves survival rate and alleviates pathological damage in severe influenza virus infections. 1. Experimental Materials Influenza virus strain: A / Puerto Rico / 8 / 1934 (H1N1) (hereinafter referred to as PR8 strain), ordinary C57 mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., serial number N000013.

[0034] 2. Experimental Methods Common 6-8 week old C57 mice were randomly divided into four groups: control group, virus infection-solvent group, virus infection-Oseltamivir treatment group, virus infection-Oseltamivir + BX471 treatment group, virus infection-Oseltamivir + ZLN005 treatment group, and virus infection-Oseltamivir + BX471 + ZLN005 treatment group (Oseltamivir, MCE-HY-13318; ZLN005, MCE-HY-17538; BX471, MCE-HY-12080A), with 10 mice in each group. The mice were then lightly anesthetized with isoflurane and infected with the PR8 strain nasally at a dose of 50 μL DMEM, with a viral titer of 150 PFU / mouse. The control group received nasal DMEM. Subsequently, 48 hours after viral infection, the mice were administered the drugs intraperitoneally at doses of Oseltamivir (20 mg / kg), BX471 (20 mg / kg), and ZLN005 (15 mg / kg), once daily for six consecutive days. Body weight and survival were monitored daily. Samples were collected at designated locations for analysis. The animals were euthanized humanely via cervical dislocation, and blood samples were collected from the orbital artery and orbital vein. The lungs were then removed from the mice. The left lung lobe was fixed with 4% paraformaldehyde and stained with hematoxylin and eosin (HE). The remaining lung tissue was aliquoted and immersed in PBS or TRIzol reagent for further analysis and detection.

[0035] 3. Experimental Results Two-way ANOVA analysis of weight changes in a mouse model of severe illness showed that, compared with Oseltamivir monotherapy, both combination regimens (Oseltamivir + BX471 and Oseltamivir + ZLN005) improved the pathological condition of mice. The three-drug combination regimen (Oseltamivir + BX471 + ZLN005) showed a synergistic effect and brought the most significant improvement in efficacy, including reduced weight loss (p < 0.001). Log-rank test analysis of survival changes in the mouse model of severe illness showed that, compared with Oseltamivir monotherapy, both combination regimens (Oseltamivir + BX471 and Oseltamivir + ZLN005) and the three-drug combination regimen (Oseltamivir + BX471 + ZLN005) improved the pathological condition of mice, showing a synergistic effect, increasing mouse survival rate, and alleviating the aggravation of lung epithelial damage and inflammatory infiltration. Figure 1 ).

[0036] Example 2: Combined medication can improve and reduce epithelial cell damage and inflammatory infiltration. 1. Experimental Methods Lung tissues from mice in each group in Example 1 were collected.

[0037] Mouse lung tissue was fixed overnight in 4% paraformaldehyde at room temperature after paraffin embedding. It was then washed four times with PBS buffer (30 minutes each time at 4°C), dehydrated sequentially with a series of ethanol solutions (30%, 50%, 70%, 95%, 100%), cleared with xylene for 1 hour, and then sectioned at 4 μm after paraffin embedding. For immunofluorescence staining, the dewaxed and rehydrated sections underwent antigen retrieval (30 minutes at 95°C). Sections were washed with TBST (TBS solution containing 0.1% Tween-20), blocked with TBST containing 3% donkey serum for 1 hour, and incubated with primary antibody overnight at 4°C. After TBST washing, sections were incubated with species-matched secondary antibody at room temperature for 1 hour, washed, counterstained with DAPI, and mounted. Image acquisition was performed using a Nikon D-Eclipse C1 confocal microscope. Immunohistochemical processing followed the same steps. After primary antibody incubation, endogenous peroxidase was quenched with 3% HO2 (25 minutes at room temperature). Sections were blocked with 3% donkey serum, incubated with HRP-labeled secondary antibody for 30 minutes, and developed using a DAB substrate kit. Whole-section images were acquired using an AperioVersa 8 system.

[0038] 2. Experimental Results Immunofluorescence staining of lung tissue revealed changes in AT1 and AT2 cells in the lungs of mice treated with different combination drugs. The results showed that Oseltamivir + BX471, Oseltamivir + ZLN005, and Oseltamivir + BX471 + ZLN005 improved the therapeutic effect of Oseltamivir and alleviated epithelial cell damage in AT1 (PDPN) and AT2 (SFTPC). Simultaneously, immunohistochemistry showed a significant reduction in iNOS+ inflammatory infiltration. Figure 2 ).

[0039] Example 3: Combined medication can improve the reduction of inflammatory factors in bronchoalveolar lavage fluid and serum, as well as lung tissue damage. 1. Experimental Methods Lung tissues from mice in each group in Example 1 were collected.

[0040] The levels of cytokines and chemokines in mouse serum and BALF were detected using commercially available ELISA kits. The specific procedures were as follows: 50 μL of serum or BALF sample was taken, and IL1β, TNFα, and IL6 were detected using a mouse-specific kit according to the manufacturer's instructions. Colorimetric detection was performed using a BioTek microplate reader, with optical density (OD) measured at 450 nm, and background correction performed using 630 nm as a reference reading.

[0041] Lung tissue was homogenized using a tissue homogenizer, and total RNA was extracted using the TRIzol kit (Invitrogen, catalog number 15596018CN) according to the manufacturer's instructions. 1 μg of total RNA was reverse transcribed into cDNA using M-MLV (Invitrogen, catalog number 28025021). Quantitative RT-PCR was performed using PowerUp SYBR Green reagent (Applied Biosystems, catalog number A25743) in a PCR instrument. Subsequently, qPCR experiments were conducted according to the SYBR Green Pro Taq HS Premix (catalog number: AG11733) instructions to detect the relative mRNA expression levels of the samples, and the levels were standardized using GAPDH as a control.

[0042] 2. Experimental Results The levels of protein and inflammatory factors in bronchoalveolar lavage fluid and serum of mice were detected. The results showed that the combined administration of Oseltamivir + BX471, Oseltamivir + ZLN005, and Oseltamivir + BX471 + ZLN005 improved the therapeutic effect of Oseltamivir alone in the Oseltamivir group. Figure 3 (AB). Furthermore, RT-qPCR detection of AT2 and AT1 marker gene expression further confirmed reduced lung tissue damage in the combined drug administration group (AB). Figure 3 (C), but there was no significant difference in viral load between the combined treatment group and the single treatment group (C). Figure 3 (D). In summary, these results indicate that host-directed interventions targeting epithelial cell metabolism or inhibiting the recruitment of myeloid inflammatory macrophages, combined with antiviral therapy strategies, can significantly prolong the treatment window, slow disease progression, and achieve better protective effects in severe viral pneumonia.

[0043] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Application of the pharmaceutical composition in the preparation of a treatment for respiratory viral infections; The pharmaceutical composition comprises any one of 1) to 3), 1) Oseltamivir and CCR1 antagonists; 2) Oseltamivir and fatty acid oxidation agonists; 3) Oseltamivir, CCR1 antagonists and fatty acid oxidation agonists.

2. The application according to claim 1, characterized in that: The CCR1 antagonists include BX471, AZD-4818, MLN3897, or nucleic acid molecules that negatively regulate CCR1; The fatty acid oxidation agonists include ZLN005, GW501516, or nucleic acid molecules that positively regulate fatty acid oxidation.

3. The application according to claim 1, characterized in that: The respiratory viruses include influenza viruses; Preferably, the influenza virus includes influenza A virus and influenza B virus.

4. The application according to claim 1, characterized in that: The pharmaceutical composition comprises a pharmaceutically acceptable salt; The drug includes pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that: The pharmaceutically acceptable excipients include at least one of the following: diluents, binders, wetting agents, lubricants, disintegrants, solvents, cosolvents, solubilizers, preservatives, surfactants, coating materials, antioxidants, antibacterial agents, or buffers.

6. The application according to claim 4, characterized in that: The dosage forms of the drug include those administered via the gastrointestinal tract or those administered outside the gastrointestinal tract.

7. The application according to claim 4, characterized in that: The drug can be administered orally, subcutaneously, intravenously, or intraperitoneally.

8. The application according to claim 4, characterized in that: The drug is suitable for mammals.

9. The application according to any one of claims 1 to 8, characterized in that: The drug includes other active ingredients for treating respiratory viral infections.

10. Application of CCR1 antagonists and fatty acid oxidation agonists in the preparation of drugs that enhance the efficacy of oseltamivir in treating respiratory viral infections.

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