Application of Ipriflavone in resisting respiratory syncytial virus infection

Abstract

The invention discloses application of Ipriflavone in resisting respiratory syncytial virus infection, and belongs to the technical field of biological medicine. Specifically, a small molecule compound library is screened to obtain a compound Ipriflavone with a relatively good inhibition effect on the RSV virus, the effective and safe dosage of the compound Ipriflavone is explored, the obtained medicine can remarkably inhibit replication of the RSV virus, and the feasibility of the medicine in resisting RSV virus infection is proved through in-vivo experiments. The invention provides a new strategy for preventing and treating respiratory syncytial virus infection, which is expected to overcome the defects of traditional symptomatic treatment and treatment support, and provides a new choice for clinical prevention and treatment of RSV virus.

Description

Technical Field

[0001] The 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 main cause of hospitalization for lower respiratory tract infection (LRTI) in children. Globally, about 3.2 million children under the age of 5 are hospitalized for RSV infection each year, and about 60,000 die, posing a major health threat. The number of hospitalizations and deaths due to RSV infection in developing countries is significantly higher than that in developed countries. It is reported that about 2,500 children are hospitalized for RSV infection every day in my country, and up to 7,400 children die from RSV infection each year. Almost all children under the age of 2 have been infected with RSV, and 50% of children under the age of 1 have been infected twice. Since RSV infection does not lead to long-term immunity, children may have repeated infections. In addition, the impact of RSV infection on children can continue into adulthood, and severe RSV infection in infancy and childhood will significantly increase the risk of asthma. Therefore, it is of great clinical and social significance to establish an accurate diagnosis, treatment, and prevention system to reduce the incidence, hospitalization, and mortality of RSV infection in children.

[0003] RSV is a linear single-stranded negative-sense enveloped RNA virus, which is derived from the phospholipid membrane of the host cell membrane and encapsidates the virus nucleocapsid. The diameter ranges from 60-200nm, and it belongs to the Paramyxoviridae, Pneumovirinae, and Pneumovirus genus. It was first isolated from chimpanzees with rhinitis in 1955 and first found in respiratory infections in children in 1957. The genome is about 15.2kb long, contains 10 genes, and encodes 11 proteins, which is more complex than other members of the Paramyxoviridae. The adhesion protein (G) and fusion protein (F) located on the surface of the virus membrane mediate the attachment and fusion of RSV to host cells.

[0004] At present, no effective anti-RSV specific therapeutic drugs have been approved or recommended, and clinical treatment is mainly symptomatic and supportive. In recent years, some progress has been made in RSV vaccines for the elderly, and Nirsevimab, a long-acting monoclonal antibody injection, has been approved for infants. However, monoclonal antibodies are not vaccines, cannot provide long-term protection, and are expensive. Therefore, it is urgent to screen new, safe and effective anti-RSV virus compounds and prove their antiviral effects at the in vitro and animal levels. Early drug research on RSV was mostly nucleoside analogs, but has not been successful. Non-nucleoside inhibitors are currently in the early stages of research and development. RSV nucleoprotein is essential for viral assembly and replication, is one of the most conserved genes, and is an important target for antiviral therapy. In the past 10 years, progress has been made in the structure of the N protein, but the complexity of its structure has led to very slow research progress on this target. For example, the development of ALN-RSV01 and RSV604 has been stopped 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 for RSV would be a good solution, but currently no such small molecule compounds have been approved for use, and they are still in the early stages of development and clinical trials. Therefore, the research goal of the present invention focuses on finding natural small molecule drugs that have an inhibitory effect on RSV and further explores the potential mechanism of its 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 the replication of RSV virus, 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 auxiliary materials include one or more of fillers, excipients, stabilizers, diluents, binders, disintegrants, lubricants, glidants, wetting agents, effervescent agents, colorants, sweeteners, flavoring agents, 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 auxiliary materials.

[0014] The third object of the present invention is to provide an anti-respiratory syncytial virus preparation, 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 preparation containing the first active ingredient or the preparation containing the second active ingredient is in the form of capsules, tablets, powders, injections or oral preparations.

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

[0020] Beneficial effects of the present invention:

[0021] There is no clear and effective small molecule drug for RSV virus currently used in clinical practice. The present invention has discovered a new small molecule inhibitor with anti-RSV virus function through a large number of screenings. 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 role of lpriflavone in resisting RSV virus infection has been discovered and verified. The anti-RSV drug provided by the present invention has low preparation cost, new use of old drugs, small side effects, and can be quickly put into clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The results of screening for 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 is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0026] The scheme involved in the present invention 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, which 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 cells through fusion mediated by its F protein, while other viruses may enter cells through different surface proteins and / or receptors, which 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, has only one serotype, 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 existence of unclear mechanism of action, unobvious efficacy and other problems, its clinical application is very limited. Therefore, the present invention is studied for RSV virus.

[0029] The research on natural products as drugs or lead compounds has always attracted wide attention, and natural products from plants have always been the main source of drug development. The present invention found through antiviral drug screening experiments that compound No. 135 Ipriflavone has a strong anti-RSV virus effect, and verified this effect 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 the current drug screening field research. And reverse genetics technology can be based on the acquisition of the entire sequence of the organism genome, by the target gene is subjected to necessary processing and modification, such as site-directed mutagenesis, gene insertion\deletion, gene replacement, etc., and then the modified genome containing the essential elements of the organism is constructed in the order of composition, so that it can assemble individuals with life activity and study 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, and the RSV recombinant virus will carry a fluorescent marker or release a fluorescent signal in the infected cell, which can both track the virus in real time, complete the observation of the process of virus-infected cells, and 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 virus 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 viral genome, and RSV infection can be quantified by the mCherry expression level. The expression of the mCherry gene was quantitatively analyzed by inverted fluorescence microscopy. The virus can be quantified using the software ImageJ. The DMSO group was used as the control group, and the other groups were compared with the DMSO group.

[0034] (3) Verification experiment

[0035] To further verify the antiviral effect of the candidate compounds, we used RSV-infected cells and mouse models to detect their effects on viral replication. We found that compound No. 135, Ipriflavone, could significantly inhibit RSV viral protein expression.

[0036] Ipriflavone was originally a plant growth factor discovered in soybeans. It was later developed into a new anti-osteoporosis drug. It is commonly used in the clinic to treat and prevent postmenopausal osteoporosis, and has obvious pharmacological effects. Since its mechanism is a physiological metabolic process in the body, it does not have the various side effects produced by estrogen and has good application prospects. At present, it has not been reported to have an antiviral effect on RSV, so studying the viral effect 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, which contains the above-mentioned 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, and the pH value may vary with the properties of the formulated substance, and the technician can adjust it as needed. The formulated pharmaceutical composition can be administered by 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 (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned polypeptide of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): fillers, excipients, stabilizers, diluents, adhesives, lubricants, surfactants or combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. 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 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 condition, which are all within the skills of a skilled physician.

[0040] The present invention also provides an anti-RSV 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 virus 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 for compounds with anti-RSV virus function in the related small molecule compound library. Specifically, HEP-2 cells were inoculated in a 96-well plate, cultured overnight, and then different compounds were added. RSV virus was added 4 hours later. The selected drug concentration was 10 μmol. After 48 hours, the samples 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, substances including No. 49, 53, 59, and 135 in the screening library are 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 135 has less cytotoxicity and higher safety, is easier to obtain from plants, and has a better inhibitory effect on RSV. Therefore, we finally chose compound 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, followed by infection 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: HEP-2 cells were inoculated in 48-well plates, cultured overnight, treated with Ipriflavone, added virus 4 hours later, lysed cells 48 hours later, RNA was extracted, cDNA was reversed, and changes in viral RNA levels were detected by RT-PCR. In order to verify whether Ipriflavone has antiviral effects in other cell lines, we found that adding Ipriflavone to RAW264.7 cells infected with RSV can significantly inhibit RSV replication ( Figure 2 B). Ipriflavone (C18H16O3, CAS: 35212-22-7) has the 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 week 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 was injected with Ipriflavone (400mg / kg / d) intraperitoneally 2 hours later, and the other group was injected with PBS. The injections were continued for 4 days. On the fifth day, the mice were dissected and lung tissues were taken. 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 in the body increased ( Figure 3 B). In the pathological changes of lung tissue, the lung tissue morphology of the normal control group was normal, while the alveoli of the lung tissue of the virus infection group were uneven in size, the structure was severely damaged, the interval was thickened, and there were a large number of inflammatory cells infiltrating. The damage of lung tissue structure in the Ipriflavone-treated group was alleviated compared with the virus infection group, and the infiltration of inflammatory cells in the lung space was also significantly improved compared with the virus infection group ( Figure 4 ). These experiments fully show that Ipriflavone has a significant inhibitory effect on RSV virus replication.

[0051] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the 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 drug is used for preventing or treating respiratory syncytial virus infection.

3. A pharmaceutical composition for resisting respiratory syncytial virus, characterized in that: The pharmaceutical composition contains Ipriflavone.

4. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition also contains pharmaceutically acceptable excipients; the excipients include one or more of fillers, excipients, stabilizers, diluents, binders, disintegrants, lubricants, glidants, wetting agents, effervescent agents, colorants, sweeteners, flavoring agents, preservatives, dispersants, film formers, plasticizers, pore formers, opacifiers, retardants, and solvents.

5. A product for use against respiratory syncytial virus, characterized in that: It includes 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 virus substance other than Ipriflavone.

6. The anti-respiratory syncytial virus product according to claim 5, characterized in that: The second active ingredient includes one or more of cyclocytidine hydrochloride, DL-carnitine hydrochloride, kaempferol, myricetin, oridonin, quercetin dihydrate, tanshinone I, physcion methyl ether, palmatine hydrochloride, and chrysophanol.

7. The anti-respiratory syncytial virus product according to claim 5, characterized in that: The preparation containing the first active ingredient and the preparation containing the second active ingredient are administered simultaneously.

8. The anti-respiratory syncytial virus product according to claim 5, characterized in that: The preparation containing the first active ingredient and the preparation containing the second active ingredient are administered sequentially.

9. The anti-respiratory syncytial virus product according to claim 5, 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, injection or oral preparation.

10. The anti-respiratory syncytial virus product according to claim 5, 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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