Application of Ipriflavone in treating respiratory syncytial virus infection

Abstract

The present invention discloses the use of ipriflavone in preventing respiratory syncytial virus infection, belonging to the field of biomedicine technology. Specifically, by screening a library of small molecule compounds, a compound ipriflavone with a good inhibitory effect on RSV virus was obtained, and its effective and safe dosage was explored. The obtained drug can significantly inhibit the replication of RSV virus, and in vivo experiments have confirmed the feasibility of the drug in preventing RSV virus infection. The present invention provides a new strategy for preventing and treating respiratory syncytial virus infection, which is expected to overcome the shortcomings of traditional symptomatic and supportive treatments and provide new options for the prevention and treatment of RSV virus in clinical practice.

Description

Technical Field

[0001] The present invention relates to application of ipriflavone in resisting respiratory syncytial virus infection, and belongs to the technical field of biomedicine. Background Art

[0002] Respiratory syncytial virus (RSV) is the leading cause of hospitalization for lower respiratory tract infection (LRTI) in children. Globally, RSV causes approximately 3.2 million hospitalizations and 60,000 deaths annually in children under the age of five, posing a significant health threat. The number of hospitalizations and deaths from RSV infection is significantly higher in developing countries than in developed countries. In my country, approximately 2,500 children are hospitalized daily for RSV infection, and up to 7,400 die annually. Almost all children under the age of two have been infected with RSV, and 50% of children under the age of one have been infected twice. Because RSV infection does not confer long-term immunity, children may experience recurrent infections. Furthermore, the impact of RSV infection on children can persist into adulthood, and severe RSV infection in infancy and childhood significantly increases the risk of asthma. Therefore, establishing a precise diagnostic, treatment, and prevention system to reduce the morbidity, hospitalization, and mortality rates of RSV infection in children is of great clinical and societal significance.

[0003] RSV is a linear, single-stranded, negative-sense, enveloped RNA virus. Its nucleocapsid is encapsidated by a phospholipid membrane derived from the host cell membrane. Its diameter ranges from 60 to 200 nm. It belongs to the family Paramyxoviridae, subfamily Pneumovirinae, and genus Pneumovirus. It was first isolated from a chimpanzee with rhinitis in 1955 and first described in pediatric respiratory infections in 1957. Its genome is approximately 15.2 kb long, containing 10 genes encoding 11 proteins, making it more complex than other members of the Paramyxoviridae family. Adhesion protein (G) and fusion protein (F) located on the viral membrane mediate RSV attachment and fusion with host cells.

[0004] Currently, no effective specific anti-RSV therapeutic has been approved or recommended, and clinical treatment primarily focuses on symptomatic and supportive care. In recent years, some progress has been made in developing RSV vaccines for the elderly, and the long-acting monoclonal antibody injection, nirsevimab, has been approved for infants. However, monoclonal antibodies are not vaccines, do not provide long-term protection, and are expensive. Therefore, the screening of new, safe, and effective anti-RSV compounds and the demonstration of their antiviral activity in vitro and in animals is urgent. Early drug research targeting RSV focused on nucleoside analogs, but this has not been successful. Non-nucleoside inhibitors are currently in the early stages of development. The RSV nucleoprotein is essential for viral assembly and replication, is one of the most conserved genes, and is an important target for antiviral therapy. While the structure of the N protein has been explored over the past decade, its structural complexity has slowed research progress. For example, the development of ALN-RSV01 and RSV604 was discontinued after entering clinical trials. Since 2010, the research on small molecule drugs for the treatment of RSV infection has accelerated, mainly compounds targeting the fusion process, inhibiting RSV fusion protein-mediated fusion with the host cell membrane and preventing viral entry, such as JNJ-53718678.

[0005] The development of small-molecule antiviral drugs targeting RSV would be a promising approach, but currently no such small-molecule compounds have been approved for use, and all remain in the early stages of development and clinical trials. Therefore, the present invention aims to identify natural small-molecule drugs that inhibit RSV and further explore the underlying mechanisms of their inhibition. Summary of the Invention

[0006] To solve the above problems, the present invention establishes a small molecule compound library to screen for RSV virus, and obtains a small molecule compound - Ipriflavone, which has a significant inhibitory effect on RSV virus replication, thereby achieving the function of resisting RSV virus infection.

[0007] The first object of the present invention is to provide the use of Ipriflavone in the preparation of anti-respiratory syncytial virus drugs.

[0008] Furthermore, the anti-respiratory syncytial virus drug is used to prevent or treat respiratory syncytial virus infection.

[0009] Furthermore, in the anti-respiratory syncytial virus drug, the concentration of Ipriflavone is 5-40 μM. Preferably, the concentration of Ipriflavone is 10-20 μM.

[0010] The second object of the present invention is to provide a pharmaceutical composition for resisting respiratory syncytial virus, wherein the pharmaceutical composition contains Ipriflavone.

[0011] Furthermore, the pharmaceutical composition also contains pharmaceutically acceptable excipients.

[0012] Furthermore, the excipients include one or more of fillers, excipients, stabilizers, diluents, binders, disintegrants, lubricants, glidants, wetting agents, effervescent agents, colorants, sweeteners, flavorings, preservatives, dispersants, film formers, plasticizers, pore formers, opacifiers, retardants, and solvents.

[0013] Furthermore, the composition consists of 0.1-100% of Ipriflavone and 99.9-0% of excipients.

[0014] The third object of the present invention is to provide an anti-respiratory syncytial virus product, comprising a container containing the following independently packaged preparations: a preparation containing a first active ingredient; and a preparation containing a second active ingredient; wherein the first active ingredient is ipriflavone, and the second active ingredient is an anti-RSV viral substance other than ipriflavone.

[0015] Furthermore, the second active ingredient includes cyclocytidine hydrochloride, DL-carnitine hydrochloride, kaempferol, myricetin, oridonin, quercetin dihydrate, tanshinone I, physcion methyl ether, palmatine hydrochloride, chrysophanol, and the like.

[0016] Furthermore, the preparation containing the first active ingredient and the preparation containing the second active ingredient are administered simultaneously.

[0017] Furthermore, the preparation containing the first active ingredient and the preparation containing the second active ingredient are administered sequentially.

[0018] Furthermore, the dosage form of the preparation containing the first active ingredient or the preparation containing the second active ingredient is a capsule, tablet, powder, injection or oral preparation.

[0019] In the present invention, the above definitions are only examples and are actually applicable to applications, pharmaceutical compositions and pharmaceutical products.

[0020] Beneficial effects of the present invention:

[0021] Currently, there are no clear and effective small molecule drugs for RSV virus used in clinical practice. The present invention has discovered a new small molecule inhibitor with anti-RSV virus function through extensive screening. The compound lpriflavone can significantly inhibit RSV virus replication, and its role in efficiently inhibiting RSV virus infection has been verified by in vivo experiments. The effect of lpriflavone on RSV virus infection has been discovered and verified. The anti-RSV drug provided by the present invention has low preparation cost, new uses for old drugs, few side effects, and can be quickly put into clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The results are from the screening of compounds that are anti-RSV replication.

[0023] Figure 2 These are the results of in vitro validation experiments on the effects of Ipriflavone on viruses.

[0024] Figure 3-4 These are the results of in vivo validation experiments on the effects of Ipriflavone on viruses. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0026] The scheme that the present invention relates to is as follows:

[0027] As we all know, different respiratory viruses differ in structure and replication mechanism. For example, RSV is an RNA virus with different RNA segments and replication strategies from other respiratory viruses. This requires the development of specific drugs for each virus to effectively inhibit its replication and infection. Moreover, each virus invades host cells in different ways. For example, RSV enters the cell through fusion mediated by its F protein, while other viruses may enter through different surface proteins and / or receptors. This requires drugs to be specific to target these different infection mechanisms. In addition, effective antiviral drugs usually target specific links in the viral life cycle. Since the life cycles and key proteins of different viruses are different, different drugs are needed to target these links. Therefore, due to differences in biological characteristics, infection mechanisms, immune responses, etc. between viruses, as well as challenges in drug development, the treatment of different respiratory viruses cannot rely on the same drug, and specific treatments for different viruses are still under continuous research and development.

[0028] Respiratory syncytial virus (RSV) belongs to the Paramyxoviridae family and has only one serotype. It is mainly transmitted through droplets and contact and can cause lower respiratory tract diseases in infants and young children. The current treatment for RSV is mainly supportive treatment, including oxygen inhalation and water supplementation, and there is no specific antiviral treatment drug. For specific high-risk infants, monoclonal antibodies such as Nirsevimab or Palivizumab can be used for passive immunization prevention. The broad-spectrum antiviral drug ribavirin is the only small molecule compound that can be applied to RSV, but due to the problems of unclear mechanism of action and unclear efficacy, its clinical application is very limited. Therefore, the present invention is studied for RSV virus.

[0029] The study of natural products as drugs or lead compounds has always attracted widespread attention, and plant-derived natural products have always been the main source of drug development. Through antiviral drug screening experiments, the present invention discovered that compound No. 135, Ipriflavone, has a potent anti-RSV effect, and this effect was verified through in vitro and in vivo experiments. Specifically:

[0030] (1) High-throughput screening of drugs against respiratory syncytial virus

[0031] High-throughput screening technology is an important topic in current drug screening research. Reverse genetics technology can be based on the complete sequence of the organism genome, by carrying out necessary processing and modification to the target gene, such as site-directed mutagenesis, gene insertion / deletion, gene replacement, etc., then constructing a modified genome containing the essential elements of the organism in the order of composition, allowing it to assemble an individual with vital activity and studying the structure and function of the organism genome. The present invention utilizes reverse genetics technology to insert the mCherry red fluorescent protein gene sequence into the genome of the RSV virus to form a recombinant virus. The RSV recombinant virus then carries a fluorescent marker or releases a fluorescent signal in the infected cell, which can both track the virus in real time and complete the observation of the process of the virus-infected cell. It can also provide help for the research of antiviral drug screening. We can observe red fluorescence under an inverted fluorescence microscope after 24h of recombinant RSV infected cells, and judge the situation of viral replication according to the strength of the red fluorescence.

[0032] (2) Screening of anti-RSV drugs using mCherry fluorescence

[0033] The mCherry gene was inserted into the RSV genome, and RSV infection can be quantified by mCherry expression levels. mCherry gene expression was quantitatively analyzed using an inverted fluorescence microscope. Viral counts were quantified using ImageJ software. The DMSO group served as the control, and all other groups were compared with the DMSO group.

[0034] (3) Verification experiment

[0035] To further validate the antiviral effects of candidate compounds, we used RSV-infected cells and mouse models to test their effects on viral replication. We found that compound 135, Ipriflavone, significantly inhibited RSV viral protein expression.

[0036] Ipriflavone, originally a plant growth factor discovered in soybeans, has been developed into a novel anti-osteoporosis drug, clinically used to treat and prevent postmenopausal osteoporosis. Its pharmacological activity is evident. Because its mechanism of action is physiological and metabolic, it is free of the side effects associated with estrogen, offering promising application prospects. Currently, there are no reports of antiviral activity against RSV, so studying the viral effects of this inhibitor may provide new insights into antiviral targets.

[0037] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition containing the aforementioned ipriflavone, and may also contain other active ingredients, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8. The pH value may vary depending on the properties of the formulated substance and can be adjusted as needed by a skilled practitioner. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intraperitoneal, intravenous, or topical administration.

[0038] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the polypeptide of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): fillers, excipients, stabilizers, diluents, binders, lubricants, surfactants, or combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents.

[0039] When using a pharmaceutical composition, a safe and effective amount of the drug is administered to an individual. The specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skills of a skilled physician.

[0040] The present invention also provides an anti-RSV viral product, comprising a container containing the following independently packaged preparations: a preparation containing a first active ingredient; and a preparation containing a second active ingredient; wherein the first active ingredient is ipriflavone, and the second active ingredient is an anti-RSV viral drug other than ipriflavone.

[0041] Preferably, the second active ingredient includes cyclocytidine hydrochloride, DL-carnitine hydrochloride, kaempferol, myricetin, oridonin, quercetin dihydrate, tanshinone I, physcion methyl ether, palmatine hydrochloride, chrysophanol, and the like.

[0042] Example 1 Screening of compounds with anti-RSV virus infection effects

[0043] RSV with mCherry red fluorescent protein was selected to screen compounds with anti-RSV virus function in the related small molecule compound library. Specifically, HEP-2 cells were inoculated in 96-well plates, cultured overnight, and different compounds were added. RSV virus was added 4 hours later. The selected drug concentration was 10 μmol. After 48 hours, the cells were observed under a microscope and photographed. The virus was quantified using the software ImageJ. The system was used to preliminarily detect whether the compounds in the inhibitor library had antiviral effects. Among them, the substances including No. 49, 53, 59, and 135 in the screening library were all flavonoids (including kaempferol, luteolin, myricetin, Ipriflavone, etc.). The results are as follows Figure 1 shown.

[0044] According to the screening results, we found some compounds 1, 32, 49, 59, 67, 77, 90, 121, 123, 132 and 135 that have obvious inhibitory effects on RSV virus. Among them, compound No. 135 has lower cytotoxicity and higher safety, is easier to obtain from plants, and has a better inhibitory effect on RSV. Therefore, we finally chose compound No. 135 for research.

[0045] Table 1 Information on some compounds that effectively inhibit RSV virus

[0046]

[0047]

[0048] Example 2 Verification of the antiviral effect of Ipriflavone

[0049] Ipriflavone was first added to HEP-2 cells, and then infected with RSV. RT-PCR\TCID 50 \WB detection found that Ipriflavone reduced viral load in a dose-dependent manner ( Figure 2A). The specific steps are as follows: HEP-2 cells were seeded into 48-well plates, cultured overnight, treated with ipriflavone, and virus was added 4 hours later. After 48 hours, the cells were lysed, RNA was extracted, cDNA was reversed, and changes in viral RNA levels were detected by RT-PCR. To verify whether ipriflavone has antiviral effects in other cell lines, we found that the addition of ipriflavone to RSV-infected RAW267.5 cells significantly inhibited RSV replication ( Figure 2 B). Ipriflavone (C18H16O3, CAS: 35212-22-7) has the following chemical structure: Figure 2 C. The effect of Ipriflavone on cytotoxicity was detected by CCK-8 kit. Finally, we selected 20uM drug concentration to study the antiviral effect of Ipriflavone ( Figure 2 D). Figure 2 The fluorescence image of E also confirmed that Ipriflavone had a significant inhibitory effect on RSV replication at 20uM.

[0050] We also conducted a validation study in Balb / c mice. First, we selected 6-8 weeks old female Balb / c mice weighing 18-20 g for infection. Specifically, we inoculated each mouse with 10 8 pfu of RSV 100μL. The mice were then divided into two groups. One group received an intraperitoneal injection of ipriflavone (400mg / kg / d) 2 hours later, while the other group received PBS. The injections were continued for 4 consecutive days. On the fifth day, the mice were dissected and lung tissue was obtained. The results showed that ipriflavone could significantly reduce the viral load in mice ( Figure 3 A), the levels of cytokines such as Ifn-β, IL-6, Tnf-α, Isg15, Ifit1, and Clg5 increased in the body ( Figure 3 B) Pathological changes in lung tissue: The normal control group showed normal lung tissue morphology, while the viral infection group showed uneven alveolar size, severe structural damage, thickened septa, and a large number of inflammatory cell infiltration. The ipriflavone-treated group showed less structural damage to lung tissue than the viral infection group, and significantly improved inflammatory cell infiltration in the lung spaces compared to the viral infection group. Figure 4 These experiments fully demonstrated that Ipriflavone has a significant inhibitory effect on RSV viral replication.

[0051] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Application of Ipriflavone in the preparation of anti-respiratory syncytial virus drugs.

2. The use according to claim 1, characterized in that The anti-respiratory syncytial virus treatment is the prevention or treatment of respiratory syncytial virus infection.

3. The use according to claim 1, characterized in that The anti-respiratory syncytial virus drug also contains pharmaceutically acceptable excipients; the excipients include one or more of fillers, excipients, stabilizers, binders, disintegrants, lubricants, glidants, wetting agents, effervescent agents, colorants, sweeteners, fragrances, preservatives, dispersants, film formers, plasticizers, pore-forming agents, opacifiers, and retardants.

4. The use according to claim 1, characterized in that The anti-respiratory syncytial virus drug includes an independently packaged container containing the following preparations: a preparation containing a first active ingredient; and a preparation containing a second active ingredient; wherein the first active ingredient is ipriflavone, and the second active ingredient is an anti-respiratory syncytial virus substance other than ipriflavone.

5. The use according to claim 4, characterized in that The preparation containing the first active ingredient and the preparation containing the second active ingredient are administered simultaneously.

6. The use according to claim 4, characterized in that The preparation containing the first active ingredient and the preparation containing the second active ingredient are administered sequentially.

7. The use according to claim 4, characterized in that The dosage form of the preparation containing the first active ingredient or the preparation containing the second active ingredient is capsule, tablet, powder or injection.

8. The use according to claim 4, characterized in that When the preparation containing the first active ingredient is a liquid preparation, the concentration of ipriflavone is 5-40 μM.

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