阅读说明：本技术 肝素钠用于新型冠状病毒SARS-CoV-2感染的新用途 (New use of heparin sodium for infection of novel coronavirus SARS-CoV-2 ) 是由 王升启 王学军 杨扬 杨鹏 吴玉明 于 2020-06-02 设计创作，主要内容包括：本发明涉及肝素钠在抑制新型冠状病毒SARS-CoV-2感染方面的应用,属于生物医药技术领域。本发明研究了肝素钠对新型冠状病毒SARS-CoV-2感染的抑制作用,结果显示肝素钠能够抑制SARS-CoV-2假病毒颗粒对宿主细胞的感染。具体是通过构建新型冠状病毒SARS-CoV-2假病毒系统和HEK293的人血管紧张素转换酶2(hACE2)稳转细胞系(HEK293/hACE2),研究了肝素钠对于新型冠状病毒SARS-CoV-2假病毒感染宿主细胞的影响,结果发现随肝素钠浓度的增加,假病毒颗粒对细胞的感染率逐渐降低,肝素钠的50％抑制浓度(IC50)在100μM左右。此研究证明了肝素钠作为新型冠状病毒SARS-CoV-2感染抑制剂和临床治疗药物及制剂的新用途。(The invention relates to application of heparin sodium in inhibiting novel coronavirus SARS-CoV-2 infection, belonging to the technical field of biological medicine. The invention researches the inhibition effect of heparin sodium on SARS-CoV-2 infection of novel coronavirus, and the result shows that the heparin sodium can inhibit the infection of SARS-CoV-2 pseudovirion on host cells. Specifically, by constructing a novel coronavirus SARS-CoV-2 pseudovirus system and a human angiotensin converting enzyme 2(hACE2) stable transfer cell line (HEK293/hACE2) of HEK293, the influence of heparin sodium on a novel coronavirus SARS-CoV-2 pseudovirus infected host cell is researched, and as a result, the infection rate of pseudovirus particles on the cell is gradually reduced along with the increase of the concentration of the heparin sodium, and the 50% inhibition concentration (IC50) of the heparin sodium is about 100 mu M. The research proves the new application of heparin sodium as the novel coronavirus SARS-CoV-2 infection inhibitor and clinical treatment medicine and preparation.)

1. Use of heparin sodium for inhibiting infection by novel coronavirus SARS-CoV-2.

2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is formulated into injection or lyophilized injection, or formulated into clinically acceptable dosage forms by adding pharmaceutically acceptable adjuvants.

Technical Field

The invention relates to the field of pharmacy and novel medicaments for treating coronavirus SARS-CoV-2 infection, in particular to application of heparin sodium serving as an inhibitor in resisting novel coronavirus SARS-CoV-2 infection.

Background

Pneumonia caused by the novel coronavirus SARS-CoV-2 is mainly manifested by fever, dry cough, hypodynamia, dyspnea and the like, and severe patients rapidly develop acute respiratory distress syndrome, septic shock, metabolic acidosis which is difficult to correct, bleeding dysfunction and the like. Until now, no specific medicine is available clinically, and the main purposes are symptomatic treatment and preventive intervention. Research shows that in severe new coronary pneumonia patients, the effective components are Reidesvir and chloroquine, but more clinical data support is needed. The coptidis and antipyretic capsule is proved to be effective for common patients with new coronary pneumonia. As a highly infectious virus, the novel coronavirus SARS-CoV-2 has the characteristics of certain mutability, many potential hosts, long incubation period and the like, and in addition, a plurality of characteristics are yet to be clarified. Therefore, there is an urgent need to find more safe and effective candidate drugs against the novel coronavirus as soon as possible.

Viral infection of a host cell is a highly complex process, typically involving various molecules on the cell surface. The first step is usually the binding of the virus to low affinity attachment sites on the cell membrane surface, followed by binding of the virus to its specific receptor, and thus invasion of the cell. Heparan Sulfate Proteoglycan (HSPG), a proteoglycan widely distributed on mammalian cell membranes, has been shown to provide an initial attachment site for many viruses such as SARS coronavirus, dengue virus, hepatitis b virus, etc., to promote aggregation of viral particles on the cell surface and subsequent binding of the virus to specific receptors. Angiotensin converting enzyme 2(ACE2) has been shown to be a specific receptor for the novel coronavirus SARS-CoV-2 infected host cell.

Heparin is a polymer formed by alternately connecting two polysaccharides, has an average molecular weight of about 15000D, has an anticoagulation effect both in vivo and in vitro, is mainly clinically used for anticoagulation of thromboembolic diseases, disseminated intravascular coagulation, cardiovascular surgery, cardiac catheter examination, hemodialysis and the like, and also has various effects of lipid reduction, inflammation resistance, allergy resistance and the like. The research shows that the exogenous heparin can reduce the binding capacity of SARS coronavirus and cell surface, and raise the resistance of cell to virus infection. The main principle is that heparin competes with HSPG for binding to viral particles, thereby preventing initial contact of viral particles with cells. However, no clear report has been made as to whether heparin can also inhibit the binding of the novel coronavirus SARS-CoV-2 to cells. Heparin sodium is the sodium salt form of heparin, which is more stable than free heparin.

Accordingly, the present invention is particularly set forth.

Disclosure of Invention

The invention aims to provide application of heparin sodium as a novel coronavirus SARS-CoV-2 infection inhibitor. The invention adopts heparin sodium with different concentrations to act on a human angiotensin converting enzyme 2(hACE2) stable cell line (HEK293/hACE2) of HEK293, then utilizes a novel coronavirus SARS-CoV-2 pseudovirus to infect cells, finds that the infection rate of virus particles is reduced along with the increase of the concentration of the heparin sodium, and IC₅₀About 100. mu.M. The research can provide a new way for inhibiting the infection of the novel coronavirus SARS-CoV-2.

The specific technical scheme of the invention is as follows:

one, constructing new type coronavirus SARS-CoV-2S protein pseudovirus system

A novel coronavirus SARS-CoV-2S protein pseudovirus system is constructed by using a lentivirus three-plasmid packaging system, wherein the pseudovirus system takes SARS-CoV-2 spike protein as surface membrane protein and firefly luciferase as a reporter gene.

Secondly, HEK293/hACE2 cell is infected with novel coronavirus SARS-CoV-2 pseudovirus after pre-incubation of heparin sodium

HEK293/hACE2 cells were incubated with different concentrations of heparin sodium for 0.5h at 37 ℃ while heparin sodium was incubated with the novel coronavirus SARS-CoV-2 pseudovirus for 0.5h at 37 ℃ after which the cells were infected with the novel coronavirus SARS-CoV-2 pseudovirus. The infection condition of the pseudovirus particles is judged by detecting the activity of luciferase in the cells.

Drawings

FIG. 1 shows the inhibitory effect of heparin sodium on SARS-CoV-2 pseudovirus infection of HEK293/hACE2 cells.

Detailed Description

1) Construction of novel coronavirus SARS-CoV-2 pseudovirus system

A novel coronavirus SARS-CoV-2 pseudovirus system is constructed by using a lentivirus three-plasmid packaging system. The pseudovirus system takes SARS-CoV-2 spike protein as surface membrane protein and firefly luciferase as reporter gene. Spike envelope plasmid, packaging plasmid psPAX2 and HIV plasmid containing luciferase reporter gene were co-transfected into HEK293T cells using GenJet Ver II transfection reagent, and the medium was replaced with fresh medium after 12 h. After 36, 48, 60h of transfection, the supernatant medium was collected and centrifuged at 800 Xg for 5min, then filtered through a 0.45 μm filter to remove cell debris and the resulting pseudovirus was stored at-80 ℃ in aliquots.

2) HEK293/hACE2 cell infection with novel coronavirus SARS-CoV-2 pseudovirus after pre-incubation of heparin sodium

HEK293/hACE2 cells grown in the logarithmic growth phase in DMEM medium containing 10% fetal bovine serum at 1X 10⁴Uniformly inoculating the cells/well into a 96-well plate, after overnight adherence, incubating the cells and heparin sodium with different concentrations for 0.5h at 37 ℃, incubating the heparin sodium and SARS-CoV-2 pseudovirus for 0.5h at 37 ℃, then infecting the cells with the novel coronavirus SARS-CoV-2 pseudovirus incubated with the heparin sodium, continuously culturing for 6h, and then replacing fresh DMEM culture solution containing 5% fetal calf serum to continuously culture for 60 h.

3) Luciferase Activity detection

Taking out the cell culture plate, balancing at room temperature for 10min, adding 100 μ L firefly luciferase detection reagent into each hole, incubating at room temperature for 10min, and performing chemiluminescence detection by using a chemiluminescence immunoassay analyzer.