CN114796215A

CN114796215A - Application of tirofiban in preparation of medicine for treating human respiratory syncytial virus infection

Info

Publication number: CN114796215A
Application number: CN202210594616.0A
Authority: CN
Inventors: 张科; 罗语思; 李伟; 刘刚; 张晗溪
Original assignee: Guizhou Medical University
Current assignee: Guizhou Medical University
Priority date: 2022-05-27
Filing date: 2022-05-27
Publication date: 2022-07-29
Anticipated expiration: 2042-05-27
Also published as: CN114796215B

Abstract

The invention discloses an application of tirofiban in preparing a medicament for treating human respiratory syncytial virus infection, and relates to the technical field of antiviral infection. The invention provides favorable medicine support for clinically treating the human respiratory syncytial virus infection, enriches the medicine types of clinical medicine intervention human respiratory syncytial virus infection and widens the antiviral variety of tirofiban. In addition, as tirofiban has good safety and effectiveness as a clinical medicament, the inventor has a good safety medicament basis for treating the human respiratory syncytial virus infection. The invention lays a foundation for further developing the antithrombotic drug into the anti-infective drug. The invention fills the blank of medicines for treating human respiratory syncytial virus, in particular to pediatric medicines, and has great social value.

Description

Application of tirofiban in preparation of medicine for treating human respiratory syncytial virus infection

Technical Field

The invention relates to the technical field of antiviral infection, in particular to application of tirofiban in preparing a medicament for treating human respiratory syncytial virus infection.

Background

Platelet Glycoprotein (GP) adheres to blood vessels under endothelium, the GP is separated from blood platelets in a circulatory system by an integral endothelial system, and the GP on a platelet membrane is important for maintaining the shape and integrity of the blood platelets and forms various receptors of the blood platelets to stop bleeding and perform related functions. In the process of causing platelet aggregation after activation of platelets by fibrinogen, fibronectin, von Willebrand factor, ADP, collagen, thrombin and the like, platelets GPIIb/IIIa are the final common effector proteins of the above activation pathways, and finally cause platelet cross-linking and platelet aggregation.

Tirofiban (Tirofiban) replaces arginine in the arginine-glycine-aspartic acid (RGD) sequence with an amidino or phenylamidine group. Tirofiban overcomes the defects of poor stability and short half-life of RGD peptide, and is a highly specific GPI (glycotoxin Inhibitor). Tirofiban prevents fibrinogen from being combined with GPIIb/IIIa through reversible combination with GPIIb/IIIa receptors, thereby blocking platelet cross-linking and platelet aggregation and having strong antiplatelet effect.

Meanwhile, it is reported that tirofiban has a good anti-inflammatory effect. For example, tirofiban significantly inhibits elevated levels of C-reactive protein (CRP) in patients with acute myocardial infarction, reduces inflammatory responses, impairs chemokine effects, and decreases adhesion molecule expression (Ercan E, 2004). Besides CRP, tirofiban can reduce the levels of serum propylene glycol, IL-6, soluble intercellular adhesion factor (sICAM-1) and soluble vascular cell adhesion factor (sVCAM-1) of patients with acute coronary syndrome (Eki A, 2007), and has a certain regulation effect on excessive inflammatory response, abnormal oxidative stress response and vascular endothelial dysfunction of patients with acute coronary syndrome. Research has shown that tirofiban can significantly reduce the rise of sCD40L in the high risk population with stable angina (Azar RR, 2005). sCD40L is released hydrolytically from CD40L, from activated lymphocytes and activated platelets, and is a common marker of inflammatory responses and procoagulant mechanisms. More than 78% of circulating sCD40L95 comes from platelets, and indirectly reflects the activation degree of platelets and the CD40-CD40L system. The TNF receptor (TNFR) family is a group of factors that reflect the severity of the inflammatory response, and is typically represented as CD 40L.

Currently, no report of tirofiban against Human Respiratory Syncytial Virus (HRSV) is retrieved. HRSV is the most important pathogen causing children viral pneumonia, and is also one of important pathogens for lower respiratory tract infection of long people, severe pneumonia caused by the viral infection can cause hundreds of thousands of children or long people to die every year, and no vaccine is available on the market so far.

In the current respiratory viral infection, severe Coronavirus 2019(coronavir disease 2019, CoV-19) caused by SARS-CoV-2 infection is confirmed to be capable of causing significant platelet aggregation by binding with host cell membrane receptor ACE2, thereby causing thrombosis and inflammatory reaction, increasing the mortality of patients, and there are definite cases that the mortality in 28 days can be significantly reduced after low molecular weight heparin is used for 42 patients with severe COVID-19 and sepsis-induced coagulopathy (TangN, 2020), and simultaneously, the small vessel thromboembolism in brain can be significantly improved after Tirofiban, clopidogrel and heparin are used for 2 patients with COVID-19 and acute ischemic stroke in China (Fu B, 2021). Furthermore, it was also found in the single-center clinical phase 2b trial conducted at the university hospital Sacco, milan, italy, that 5 patients with severe COVID-19 were treated with tirofiban, clopidogrel and heparin to significantly improve their blood oxygen saturation, 4 patients were no longer treated with a ventilator and recovered one after the other after 7 days of treatment, while 5 patients in the control group died only 3 after heparin treatment (vicca M, 2020). Tirofiban and clopidogrel respectively play a role in resisting platelet aggregation by specifically inhibiting GP IIb/IIIa allosteric and binding ADP with a platelet membrane receptor, and heparin is anticoagulated mainly by activating antithrombin III and enhancing the inhibition effect on thrombin. Thus, a series of clinical evidence confirms that thrombosis is a critical factor in the pathogenesis of severe viral pneumonia, and timely intervention in platelet aggregation and thrombosis for the target population would likely reverse its great threat to patient life.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide application of tirofiban in preparing a medicament for treating human respiratory syncytial virus infection.

The invention is realized by the following steps:

an application of tirofiban in preparing the medicines for treating the infection of human respiratory syncytial virus is disclosed.

The inventor finds that tirofiban has a good treatment effect on Human Respiratory Syncytial Virus (HRSV) infection, and experiments on pathological sections of the lungs of mice show that the mice in a tirofiban treatment group have good general state, smooth back hair, active spirit, normal feed intake, no tachypnea and dyspnea symptoms, the weight rises from day 3, and the survival rate of the mice is remarkably higher than that of the mice in a non-treatment group. The immunofluorescence staining result shows that the virus infection degree of the tirofiban treatment group is lower than that of the non-treatment group, and the number of virus platelets of the tirofiban treatment group is obviously lower than that of the non-treatment group. These results all indicate that tirofiban has a good in vivo effect against human respiratory syncytial virus. The inventors have also found that the mechanism of tirofiban against infection may proceed by way of inhibiting inflammation.

The invention provides an effective human respiratory syncytial virus vaccine which is still under development and has different immune attributes, starts from the pathophysiology that platelets and neutrophils interact after human respiratory syncytial infection, inhibits inflammatory reaction by antiplatelet treatment, achieves the effect of protecting organisms by resisting viruses, provides favorable medicine support for clinically treating the human respiratory syncytial virus infection, enriches the medicine types of clinical medicine intervention human respiratory syncytial virus infection, and widens the types of tirofiban viruses. In addition, as tirofiban has good safety and effectiveness as a clinical medicament, the inventor has a good safety medicament basis for treating the human respiratory syncytial virus infection. The invention lays a foundation for further developing the antithrombotic drug into the anti-infective drug.

In a preferred embodiment of the use of the invention, the subject infected with human respiratory syncytial virus is an infant, a child, an adult, an immunosuppressed patient or a patient receiving an organ transplant. Especially the elderly (over 65 years old).

Use of tirofiban in the manufacture of a medicament for the treatment of bronchiolitis caused by human respiratory syncytial virus.

Application of tirofiban in preparing a medicament for treating pneumonia caused by human respiratory syncytial virus.

Signs of pneumonia include, but are not limited to: mild viral pneumonia, moderate viral pneumonia, severe viral pneumonia, such as severe viral pneumonia signs: a large amount of inflammatory cell infiltration is seen around small vessels and bronchi of the lung, most alveolar structures are destroyed, and a large amount of red blood cells leak from the small vessels to alveolar cavities and pulmonary interstitium, so that extensive thrombosis is seen in the pulmonary interstitium.

In an alternative embodiment, the pneumonia is viral pneumonia, bacterial pneumonia, fungal pneumonia or mycoplasmal pneumonia.

The pneumonia is acute viral pneumonia, acute bacterial pneumonia, acute fungal pneumonia or acute mycoplasma pneumonia.

For example, acute viral pneumonia includes acute exudative pneumonia caused by one or more of influenza a virus, influenza b virus, avian influenza virus, SARS virus, MERS virus, zika virus, ebola virus, and SARS-CoV-2 virus.

Acute fungal pneumonia includes acute exudative pneumonia caused by one or more of cryptococcus, aspergillus, mucor and candida.

Acute bacterial pneumonia includes acute exudative pneumonia caused by one or more pathogens of Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Haemophilus influenzae, Legionella, and Pseudomonas aeruginosa.

Use of tirofiban in the manufacture of a medicament for the treatment of otitis media caused by human respiratory syncytial virus.

Use of tirofiban and a combination drug selected from antithrombotic drugs for the preparation of a medicament for the treatment of human respiratory syncytial virus infection.

In a preferred embodiment of the use of the invention, the antithrombotic agent is selected from: at least one of heparin, aspirin, clopidogrel, eptifibatide, Xuesaitong and ginkgo biloba leaves.

In the preferred embodiment of the present invention, the dosage form of the drug is powder, pill, tablet, capsule, oral liquid, aerosol or injection.

The invention has the following beneficial effects:

the inventor finds that tirofiban has a good treatment effect on human respiratory syncytial virus infection, and a lung pathological section experiment of a mouse shows that the mice in a tirofiban treatment group have good general state, smooth back hair, active spirit, normal feed intake, no tachypnea and dyspnea symptoms, the weight rises from day 3, and the survival rate of the mice is remarkably higher than that of the non-treatment group. The immunofluorescence staining result shows that the virus infection degree of the tirofiban treatment group is lower than that of the non-treatment group, and the number of virus platelets of the tirofiban treatment group is obviously lower than that of the non-treatment group. These results all indicate that tirofiban has a good in vivo effect against human respiratory syncytial virus. The inventors have also found that the mechanism of tirofiban against infection may proceed by way of inhibiting inflammation.

The invention provides favorable medicine support for clinically treating the human respiratory syncytial virus infection, enriches the medicine types of clinical medicine intervention human respiratory syncytial virus infection and widens the antiviral variety of tirofiban. In addition, the tirofiban has good safety and effectiveness as one of clinical medicines, so the inventor has good safety medicine basis for treating the human respiratory syncytial virus infection. The invention lays a foundation for further developing the antithrombotic drug into the anti-infective drug. The invention fills the blank of medicines for treating human respiratory syncytial virus, in particular to pediatric medicines, and has great social value.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a graph showing the statistical results of the body weight change (A) and the survival rate (B) of a tirofiban treatment group and an HRSV high-strain infection group after HRSV high-virus strain infection;

FIG. 2 is H & E staining pictures of whole or local lung pathological sections of HRSV high virus strain-infected Tirofiban treatment group, HRSV high virus strain-infected group and control mice;

FIG. 3 is a H & E stained image of a lung pathology section of a mouse in R96-5 infected group (A & B) and blank control group (C);

FIG. 4 is immunofluorescence pictures of whole lung (A & F) or local lung (B-E & G-J) of HRSV high strain infected group and HRSV high strain infected group after HRSV high strain infection.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are well explained in the literature, e.g. "molecular cloning: a Laboratory Manual, second edition (Sambrook et al, 1989); oligonucleotide Synthesis (oligo Synthesis) (eds. m.j. goal, 1984); animal Cell Culture (Animal Cell Culture), ed.r.i. freshney, 1987; methods in Enzymology (Methods in Enzymology), Handbook of Experimental Immunology (Handbook of Experimental Immunology) (ed. D.M.Weir and C.C.Black well), Gene Transfer Vectors for Mammalian Cells (ed. J.M.Miller and M.P.Calos) (ed. J.M.and M.P.Calos) (ed. 1987), Methods in Current Generation (Current Protocols in Molecular Biology) (ed. F.M.Ausubel.et al, 1987), PCR, Polymerase Chain Reaction (ed. PCR: The Polymerase Chain Reaction) (ed. Mullis et al, 1994), and Methods in Current Immunology (ed. J.1991).

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

In vivo antiviral experiments: HRSV high strain R96-5 challenge mice preserved in basic medical institute of Guizhou medical university laboratory in pathogenic biology 3 times, wherein the dose of each challenge is 1x 10 ¹⁰ TCID ₅₀ At an interval of 12h, starting from the 1 st day of tapping poison, and tapping from the tail vein of the mouse at a rate of 50 mu g/kg after every day of attacking poisonInjecting tirofiban for antiviral infection, and continuously injecting for 3 d; simultaneously setting virus control group, and using high-toxicity strain R96-5 to challenge mice for 3 times, wherein the dose of each challenge is 1x 10 ¹⁰ TCID ₅₀ And at an interval of 12h, starting from the 1 st d of tapping poison, continuously injecting normal saline from the tail vein of the mouse after tapping poison every day for 3 d. The observation endpoint was 21d post-infection (last challenge day was 0d post-infection).

In an observation period of 21d after infection, the mice in the tirofiban treatment group are generally good, smooth in back hair, active in spirit, normal in feed intake, free of tachypnea and dyspnea, the weight of the mice rises from day 3, and completely returns to normal (A in figure 1) until the observation end point, and 1 mouse died at day 10 after infection, and the survival rate of the mice is 90% (10 mice in total); however, in the HRSV high-toxicity strain infected group, the state of mice is generally poor, the back hair is shrunk, the spirit is low, the feed intake is reduced, the mice are rapidly breathed and the dyspnea gradually worsens along with the observation time extension, the weight continuously decreases after infection, the weight rises again from the 11 th day, the mice are dead during the 2 nd to the 14 th days after infection, the survival rate is only 20 percent, and the survival rate is obviously lower than that of the tirofiban treatment group (B in figure 1, p is less than 0.001).

The results of this example show that tirofiban has a good anti-HRSV infection effect.

In fig. 1, Tirofiban: tirofiban; r96-5: a high strain of HRSV; ***: p < 0.001; each group n is 10; the experiment was independently repeated 2 times.

Example 2

In the in vivo antiviral experiment of example 1, mouse lungs were collected at different time points for pathological sectioning and H & E staining.

The pathological section of the mouse lung and the H & E staining result are shown in figure 2, and the result indicates that local lung tissues of a tirofiban treatment group have light and medium viral pneumonia signs, inflammatory cell infiltration is visible around small pulmonary blood vessels and bronchi, but most of alveolar structures are complete, and erythrocyte leakage and thrombosis are not seen. However, the HRSV infection is manifested by severe viral pneumonia, a large amount of inflammatory cells infiltrate around small pulmonary blood vessels and bronchi, most of alveolar structures are destroyed, a large amount of red blood cells leak from the small blood vessels to alveolar spaces and pulmonary interstitium, and extensive thrombosis is observed in the pulmonary interstitium. Tirofiban in fig. 2: tirofiban; r96-5: a high strain of HRSV; mock: comparison; the scale bar is 2000 μm or 100 μm.

In the figure 3, A and B show that the HRSV high-toxicity strain R96-5 attacks the lung of a mouse to obviously cause the lung lesion, most of the lung alveoli are fused and destroyed, the lung interstitium is thickened and edematous, a large amount of inflammatory cells are infiltrated, mainly lymphocytes are used, in addition, a large amount of thrombus is formed in lung blood vessels at all levels, the erythrocyte leakage condition is obvious, and the HRSV high-toxicity strain infection can cause obvious damage to the lung blood vessel endothelial cells, and the erythrocyte platelets and the like are leaked to the alveolar space.

The results of the embodiment show that tirofiban can avoid severe viral pneumonia, and after the tirofiban is used for treatment, the alveolar structure is complete, and no erythrocyte leakage and thrombosis occur, so that the tirofiban has a good treatment effect.

In FIG. 3, A: the black arrows indicate extensive thrombus formation seen in the lung interstitium; b: black arrows indicate leakage of large numbers of red blood cells from the pulmonary interstitium and alveoli; the scale bar is 100 μm.

Example 3

In the in vivo antiviral experiment of example 1, mouse lungs were collected at different time points for immunofluorescent staining experiments.

The HRSV F protein expression quantity indicates the virus infection degree, while the CD42b protein is the specific membrane protein of megakaryocyte and platelet, the expression amount and the expression position respectively represent the number of platelets and the position of the platelets, the results of B in figure 4 and G in figure 4 show that the infection degree of the tirofiban treatment group virus is lower than that of the HRSV high strain R96-5 challenge group, the results C in FIG. 4 and H in FIG. 4 indicate that the number of virus platelets in the tirofiban treatment group is lower than that in the R96-5 challenge group, and the platelets of the HRSV high-toxicity strain challenge group mice are distributed in alveolar-like tissues, and the results B in figure 3 show that the pulmonary vascular endothelial cells of the HRSV high-toxicity strain challenge group mice are damaged, and the red blood cells and the platelets leak to alveolar cavities, meanwhile, the co-expression of HRSV F protein and CD42b protein can be seen, which suggests that the anti-infection mechanism of tirofiban can be developed by inhibiting inflammation.

In fig. 4, Tirofiban: tirofiban; r96-5: a high strain of HRSV; HRSV F: red fluorescence; CD42 b: pink fluorescence; DAPI: blue fluorescence; the scale bar is 100 μm or 2,000 μm.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of tirofiban in the manufacture of a medicament for the treatment of human respiratory syncytial virus infection.

2. The use of claim 1, wherein the subject infected with human respiratory syncytial virus is an infant, a child or an adult.

3. Use of tirofiban in the manufacture of a medicament for the treatment of bronchiolitis caused by human respiratory syncytial virus.

4. Application of tirofiban in preparing a medicament for treating pneumonia caused by human respiratory syncytial virus.

5. The use of claim 4, wherein the pneumonia is viral pneumonia, bacterial pneumonia, fungal pneumonia, or mycoplasmal pneumonia.

6. Use of tirofiban in the manufacture of a medicament for the treatment of otitis media caused by human respiratory syncytial virus.

7. Use of tirofiban and a combination drug for the preparation of a medicament for the treatment of human respiratory syncytial virus infection, characterized in that the combination drug is selected from antithrombotic drugs.

8. Use according to claim 7, wherein the antithrombotic agent is selected from: at least one of heparin, aspirin, clopidogrel, eptifibatide, Xuesaitong and ginkgo biloba leaves.

9. The use according to any one of claims 1 to 8, wherein the medicament is in the form of a powder, a pill, a tablet, a capsule, an oral liquid, an aerosol or an injection.

10. The use according to claim 9, wherein the medicament is administered to the respiratory tract.