CN114796215B

CN114796215B - Application of tirofiban in preparing medicine for treating human respiratory syncytial virus infection

Info

Publication number: CN114796215B
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: 2023-07-07
Anticipated expiration: 2042-05-27
Also published as: CN114796215A

Abstract

The invention discloses 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 drug support for clinically treating human respiratory syncytial virus infection, enriches the types of drugs for clinically interfering human respiratory syncytial virus infection, and widens the antiviral variety of tirofiban. In addition, as tirofiban has good safety and effectiveness as a clinical medicine, the inventor has good safety medicine base for treating human respiratory syncytial virus infection. The invention lays a foundation for further developing the medicine with antithrombotic capability into the anti-infective medicine. The invention fills the blank of medicines for treating human respiratory syncytial virus, especially pediatric medicines, and has great social value.

Description

Application of tirofiban in preparing 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 medicines for treating human respiratory syncytial virus infection.

Background

The normal vascular endothelium has adhered platelet Glycoprotein (GP), the complete endothelial system separates GP from platelets in the circulatory system, and GP on the platelet membrane is important for maintaining the morphology and integrity of platelets and constitutes various receptors for platelets, allowing platelets to exert hemostasis and related functions. Platelet GPIIb/IIIa is the last common effector protein of the above-mentioned activation pathway in the course of platelet aggregation after activation of platelets by fibrinogen, fibronectin, von Willebrand factor, ADP, collagen, thrombin and the like, and finally causes platelet crosslinking and platelet aggregation.

Tirofiban (Tirofiban) is an arginine in the arginine-glycine-aspartic acid (RGD) sequence replaced with an amidino or phenylamidino group. Tirofiban overcomes the defect of poor stability and short half-life of RGD peptide, and is a highly specific GPIIb/IIIa receptor antagonist (Glycoprotein Inhibitor, GPI). By reversibly combining with GPIIb/IIIa receptor, tirofiban prevents fibrinogen from combining with GPIIb/IIIa, thereby blocking platelet crosslinking and platelet aggregation, and having strong antiplatelet effect.

Meanwhile, it is reported that tirofiban has better anti-inflammatory effect. For example, tirofiban can significantly inhibit elevated levels of C-reactive protein (CRP), reduce inflammatory responses, impair chemokine action and expression of adhesion molecules in patients with acute myocardial infarction (Ercan E, 2004). Besides CRP, tirofiban can also reduce serum propylene glycol, IL-6, soluble intercellular adhesion factor (sICAM-1) and soluble vascular cell adhesion factor (sVCAM-1) levels (Eki A, 2007) of patients with acute coronary syndrome, which suggests that tirofiban has certain regulation effects on excessive inflammatory reaction, abnormal oxidative stress and vascular endothelial dysfunction of patients with acute coronary syndrome. Studies have shown that tirofiban can significantly reduce the magnitude of the elevation of sCD40L in the high risk group of stable angina (Azar RR, 2005). sCD40L is released from hydrolysis of CD40L, from activated lymphocytes and activated platelets, and is a common marker for inflammatory responses and procoagulant mechanisms. More than 95% of sCD40L in the circulation comes from platelets, and the activation degree of platelets and CD40-CD40L systems can be indirectly reflected. The TNF receptor (TNFR) family is a group of factors that reflect the severity of inflammatory responses, a typical representation of which is CD40L.

At present, no report of tirofiban against human respiratory syncytial virus (Human Respiratory Syncytial Virus, HRSV) has been retrieved. HRSV is the most important pathogen causing children's viral pneumonia, and is one of the important pathogens for long-term lower respiratory tract infection, and severe pneumonia caused by the virus infection can cause hundreds of thousands of children or long-term deaths every year, so far no vaccine is marketed.

Among the current respiratory viral infections, severe coronavirus 2019 (Coronavirus disease 2019, covd-19) caused by SARS-CoV-2 infection has been identified as being capable of causing significant platelet aggregation by binding to host cell membrane receptor ACE2, thereby causing thrombosis and inflammatory response, increasing patient mortality, and a clear medical record has been reported to significantly reduce mortality within 28 days after administration of low molecular weight heparin to 42 severe covd-19 complicated sepsis-induced coagulants (Tang N, 2020), while China has reported to significantly improve cerebral small vessel thromboembolism (Fu B, 2021) after administration of tirofiban, clopidogrel and heparin to 2 patients with covd-19 complicated acute ischemic stroke. In addition, it was also found in single-site clinical phase 2b experiments conducted by the university of italian milan Sacco that the blood oxygen saturation of patients was significantly improved after the use of tirofiban, clopidogrel and heparin in 5 severe patients, 4 patients were no longer treated with a ventilator and recovered after 7 days of treatment, and the control group of 5 patients died only after the treatment with heparin (Viecca M, 2020). Tirofiban and clopidogrel exert an anti-platelet aggregation effect by specifically inhibiting GPIIb/IIIa allosteric and ADP binding with platelet membrane receptors respectively, and heparin is anticoagulated mainly by activating antithrombin III to enhance the inhibition effect on thrombin. Thus, a series of clinical evidence confirm that thrombosis is a critical factor in the pathogenesis of severe viral pneumonia, and that timely intervention of platelet aggregation and thrombosis in the target population would likely reverse its great threat to patient life.

In view of this, the present invention has been made.

Disclosure of Invention

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

The invention is realized in the following way:

use of tirofiban for the preparation of a medicament for the treatment of human respiratory syncytial virus infection.

The inventor finds that tirofiban has good treatment effect on Human Respiratory Syncytial Virus (HRSV) infection, and the experiment on lung pathological section of mice shows that the mice in the tirofiban treatment group have good general state, the back Mao Guanghua is active, the feed intake is normal, the symptoms of shortness of breath and dyspnea are avoided, the weight is increased from the 3 rd day, and the survival rate of the mice is extremely higher than that of the mice in the non-treatment group. The immunofluorescence staining results show that the virus infection degree of the tirofiban treatment group is lower than that of the non-treatment group, and the virus platelet number of the tirofiban treatment group is obviously lower than that of the non-treatment group. These results all indicate that tirofiban has good in vivo anti-human respiratory syncytial virus effects. The inventors have also found that the mechanism of tirofiban against infection may be by way of inhibiting inflammation.

The invention provides that from the pathophysiology of interaction between platelets and neutrophils after human respiratory syncytial infection, the anti-platelet treatment can inhibit inflammatory reaction, thereby achieving the function of antivirus and protecting the organism, providing favorable drug support for clinically treating human respiratory syncytial virus infection, enriching the types of drugs for clinical drug intervention on human respiratory syncytial virus infection, and widening the variety of tirofiban antivirus. In addition, as tirofiban has good safety and effectiveness as a clinical medicine, the inventor has good safety medicine base for treating human respiratory syncytial virus infection. The invention lays a foundation for further developing the medicine with antithrombotic capability into the anti-infective medicine.

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

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

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

Symptoms of pneumonia include, but are not limited to: mild viral pneumonia, moderate viral pneumonia, severe viral pneumonia, e.g. severe viral pneumonia symptoms: numerous inflammatory cell infiltrates are seen around the small vessels and bronchi of the lung, most of the alveolar structure is destroyed, numerous erythrocytes leak from the small vessels to the alveolar space and the pulmonary interstitium, and extensive thrombosis is seen in the pulmonary interstitium.

In an alternative embodiment, the pneumonia is viral pneumonia, bacterial pneumonia, fungal pneumonia, or mycoplasma 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 manufacture 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 the group consisting of: at least one of heparin, aspirin, clopidogrel, eptifibatide, xuesaitong and ginkgo leaf.

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

The invention has the following beneficial effects:

the inventor finds that tirofiban has good treatment effect on human respiratory syncytial virus infection, and the experiment on lung pathological section of mice shows that the mice in the tirofiban treatment group have good general state, the back Mao Guanghua, the spirit is active, the feed intake is normal, the symptoms of shortness of breath and dyspnea are avoided, the weight is increased from the 3 rd day, and the survival rate of the mice is extremely higher than that of the mice in the non-treatment group. The immunofluorescence staining results show that the virus infection degree of the tirofiban treatment group is lower than that of the non-treatment group, and the virus platelet number of the tirofiban treatment group is obviously lower than that of the non-treatment group. These results all indicate that tirofiban has good in vivo anti-human respiratory syncytial virus effects. The inventors have also found that the mechanism of tirofiban against infection may be by way of inhibiting inflammation.

The invention provides favorable drug support for clinically treating human respiratory syncytial virus infection, enriches the types of drugs for clinically interfering human respiratory syncytial virus infection, and widens the antiviral variety of tirofiban. In addition, as tirofiban has good safety and effectiveness as one of clinical medicines, the inventor has good safety medicine base for treating human respiratory syncytial virus infection. The invention lays a foundation for further developing the medicine with antithrombotic capability into the anti-infective medicine. The invention fills the blank of medicines for treating human respiratory syncytial virus, especially 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 that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

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

FIG. 2 is a H & E staining image of whole lung or local lung pathology sections of HRSV high strain infected groups, HRSV high strain infected groups and control mice after infection with tirofiban;

FIG. 3 is H & E stained pictures of local lung pathology in R96-5 infected (A & B) and blank (C) mice;

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

Detailed Description

Reference now will 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. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C.Blackwell, inc.), gene transfer vectors for mammalian cells (Gene Transfer Vectors for Mammalian Cells) (J.M.Miller and M.P.calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

In vivo antiviral experiments: the high-virulent strain R96-5 of HRSV stored in the basic medical college of Guizhou medical university pathogenic biology laboratory is used for 3 times, and the dose of each challenge is 1x 10 ¹⁰ TCID ₅₀ Starting from 1d of virus attack at intervals of 12 hours, intravenously injecting tirofiban from the tail of a mouse according to the ratio of 50 mug/kg after daily virus attack to resist virus infection, and continuously injecting for 3d; simultaneously, a virus control group is arranged, and high-strain R96-5 is used for attacking the virus mice 3 times, and the dosage of each attack is 1x 10 ¹⁰ TCID ₅₀ At intervals of 12h, normal saline is injected from the tail of the mouse intravenously after daily toxin tapping from 1d, and the injection is continuously carried out for 3d. The endpoint was observed to be 21d post-infection (0 d post-infection with the last challenge day).

During the observation period of 21d after infection, we found that tirofiban treated mice were in good general status, were on the back Mao Guanghua, were psychoactive, had normal feed intake, had no shortness of breath and dyspnea, had body weight returned from day 3 to the point of observation had completely recovered to normal (fig. 1 a), and had a survival rate of 90% for 1 mouse at day 10 after infection (total 10 mice); in the HRSV high strain infected group, mice were not normally well-conditioned during the 21d observation period, were wrinkled in the dorsi hair, listlessness, decreased in feed intake, and with prolonged observation time, respiratory distress and dyspnea were gradually developed and aggravated, body weight was continuously decreased after infection, and after infection, mice died from day 11, observed from day 2 to day 14 after infection, with survival rates of only 20%, significantly lower than that of tirofiban treated group (B, p <0.001 in fig. 1).

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

In fig. 1, tirofiban: tirofiban; r96-5: HRSV high strain; * **: p < 0.001; each group n=10; experiments were independently repeated 2 times.

Example 2

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

The results of H & E staining of murine lung pathological sections are shown with reference to fig. 2, which suggests that the partial lung tissue of tirofiban treatment group is manifested as mild and moderate viral pneumonia, inflammatory cell infiltration is seen around small pulmonary vessels and bronchi, but most of alveoli are structurally intact, and red blood cell leakage and thrombosis are not seen. However, HRSV infection is manifested as severe viral pneumonia, massive inflammatory cell infiltration is seen around small vessels and bronchi of the lung, most of alveolus structures are destroyed, massive red blood cells leak from small vessels to alveolar spaces and pulmonary interstitium, and extensive thrombosis is seen in pulmonary interstitium. Tirofiban in fig. 2: tirofiban; r96-5: HRSV high strain; mock: a control; the scale bar is 2000 μm or 100 μm.

In FIG. 3, the A & B can see the obvious pathological changes of the lung of the HRSV high strain R96-5 virus-challenged mice, most of alveoli are fused and destroyed, pulmonary interstitium is thickened and oedema, a large amount of inflammatory cells infiltrate, mainly lymphocytes, and a large amount of thrombus is formed in pulmonary blood vessels of all stages, and the red blood cell leakage condition is obvious, which indicates that the obvious damage to the endothelial cells of the pulmonary blood vessels, the leakage of red blood cell platelets and the like can be caused by the infection of the HRSV high strain.

The results of the embodiment show that tirofiban can avoid severe viral pneumonia, and after the tirofiban is used for treatment, the alveolus structure is complete, and erythrocyte leakage and thrombosis are not seen, so that the treatment effect is good.

Fig. 3 a: black arrows indicate that extensive thrombosis is seen in the pulmonary interstitium; b: black arrows indicate massive red blood cell leakage from the lung 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 level is higher or lower than the HRSV high strain R96-5 virus challenge group, the CD42B protein is megakaryocyte and platelet specific membrane protein, the expression level and the expression position of the HRSV high strain virus challenge group are respectively expressed as the platelet number and the platelet position, the B and G results in fig. 4 show that the tirofiban treatment group virus infection level is lower than the HRSV high strain R96-5 virus challenge group, the C and H results in fig. 4 show that the tirofiban treatment group virus platelet number is lower than the R96-5 virus challenge group, and the HRSV high strain virus challenge group mouse platelets are distributed in alveolar-like tissues, and in combination with the B results in fig. 3, the HRSV high strain challenge group mouse lung vascular endothelial cells are damaged, erythrocytes and platelets leak to the alveolar space, and the HRSV F protein and CD42B protein are simultaneously expressed together, so that the mechanism of tirofiban infection may be inhibited by inhibiting inflammatory development.

In fig. 4, tirofiban: tirofiban; r96-5: HRSV high strain; HRSV F: red fluorescence; CD42b: pink fluorescence; DAPI: blue fluorescence; the scale bar is 100 μm or 2,000 μm.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. 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 for the manufacture of a medicament for the treatment of human respiratory syncytial virus infection.

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

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

4. The use according to any one of claims 1 to 3, wherein the medicament is in the form of a powder, a pill, a tablet, a capsule, an oral liquid, an aerosol or an injection.

5. The use according to claim 4, wherein the medicament is administered to the respiratory tract.