CN117064893A

CN117064893A - Application of ametinib in preparation of anti-coronavirus drugs

Info

Publication number: CN117064893A
Application number: CN202311065167.1A
Authority: CN
Inventors: 李长志; 钱朝南; 陈金东; 周红娟; 黄婕
Original assignee: Guangzhou Chaoliliang Biological Technology Co ltd
Current assignee: Guangzhou Chaoliliang Biological Technology Co ltd
Priority date: 2023-08-22
Filing date: 2023-08-22
Publication date: 2023-11-17

Abstract

The invention relates to application of amotinib in preparing anti-coronavirus medicaments, and the invention verifies that the amotinib can obviously reduce the infection efficiency of SARS-COV-2 through in vivo and in vitro experiments, and inhibit virus invasion cells, which shows that the amotinib has obvious inhibition effect on coronaviruses of at least one of 2019-nCoV, HCoV-229E, HCoV-OC43, SARS-CoV and MERS-CoV, and is suitable for preparing medicaments for treating coronaviruses and complications, including simple infection, pneumonia, acute respiratory tract infection, severe acute respiratory tract infection, hypoxic respiratory failure, acute respiratory distress syndrome, sepsis and septic shock.

Description

Application of ametinib in preparation of anti-coronavirus drugs

Technical Field

The invention belongs to the technical field of pharmacy, and particularly relates to application of amotinib in preparation of anti-coronavirus medicaments.

Background

Coronaviruses belong to the order of the family Coronaviridae, genus Coronavirales, the genus Coronavirales, are a class of RNA viruses with a envelope and a linear single positive strand genome, and some coronaviruses can infect humans and cause diseases, such as pneumonia caused by Middle East Respiratory Syndrome (MERS), severe Acute Respiratory Syndrome (SARS) and novel coronaviruses (SARS-CoV-2), and symptoms thereof can range from common cold to severe pulmonary infection, seven historically known coronaviruses capable of infecting humans, four of HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 cause only mild or moderate cold symptoms. SARS-CoV, MERS-CoV and 2019-nCoV are similar in pathology, and can be combined with cell surface receptor by means of glycoprotein on virus surface to release RNA into cytoplasm so as to synthesize new virus particles, so that at present, no approved therapeutic method for coronavirus exists in the global scope, and the development of anti-coronavirus medicine is urgent.

The pneumonia caused by SARS-CoV-2 mainly takes fever, hypodynamia and dry cough as main manifestations, and few patients are accompanied with symptoms such as nasal obstruction, nasal discharge, diarrhea and the like. Based on the fact that enzymes and proteins in the current viral replication cycle are potential acting targets of anti-coronavirus drugs, research on anti-coronavirus drugs is focused on S protein, rdRp, 3CLpro, PLpro and the like. Spike (S) protein belongs to structural protein of virus, mature S protein is stable trimer, has receptor and membrane fusion activity, cleavage site of protease exists between S1 and S2, and cleavage activation of S protein is key for virus invasion into host cell. The receptor-binding domain (RBD) on the S protein binds to the cell surface receptor, so that the virus adheres to the cell surface, different coronaviruses have different receptors and different affinities, MERS-CoV takes human cell surface protein dipeptidyl peptidase 4 (dipeptidyl peptidase-4, DPP 4) as an access receptor, SARS-CoV-2 and SARS-CoV take angiotensin converting enzyme II (ACE 2) as receptors, and as the S protein of the SARS-CoV-2 has 20 times higher affinity with the ACE2 receptor than the SARS-CoV, the transmission speed of the virus in human is faster, and the SARS-CoV-2 has the characteristics of a certain mutation, more potential hosts, long latency and the like, and other characteristics are still needed, so that more safe and effective candidate drugs for resisting novel coronaviruses are urgently found as soon as possible.

Disclosure of Invention

Aiming at the prior art problems, the invention provides application of the amotinib in preparing anti-coronavirus medicaments.

In a first aspect, the invention provides the use of almitinib for the preparation of an anti-coronavirus medicament.

Further, the structural formula of the almitinib (almonetinib) is shown as (I);

further, the anti-coronavirus drug also comprises geometric isomers of ametinib or pharmaceutically acceptable salts thereof and/or solvates thereof and/or hydrates thereof.

Further, the medicine also comprises pharmaceutically acceptable auxiliary materials.

Further, the administration route of the anti-coronavirus drug includes, but is not limited to, oral administration, injection or respiratory tract inhalation.

Further, the anti-coronavirus drug is formulated into suspension, granule, capsule, powder, tablet, emulsion, solution, drop pill, injection, aerosol or drop.

Further, the coronavirus includes at least one of 2019-nCoV, HCoV-229E, HCoV-OC43, SARS-CoV and MERS-CoV.

Further, the anti-coronavirus drugs include, but are not limited to, simple infections that inhibit fever, cough and sore throat, pneumonia, acute respiratory infections, severe acute respiratory infections, hypoxic respiratory failure, acute respiratory distress syndrome, sepsis and septic shock.

Compared with the prior art, the invention has the beneficial effects that:

(1) Ametinib can effectively inhibit in-vitro SARS-COV-2 infection, when the administration concentration is 0.1-10 mu M, the infection rate of SARS-COV-2 in the 293T ACE2 over-expression cell strain and H1299 ACE2 over-expression cell strain can be obviously reduced, the growth of the H1299 ACE2 over-expression cell strain is non-toxic, the growth toxicity effect on the 293T ACE2 over-expression cell strain is small, wherein the inhibition of 293T cell from sensing the IC of SARS-COV-2 pseudovirus is low ₅₀ IC for inhibiting infection of H1299 cells with SARS-COV-2 pseudovirus at 2.052. Mu.M ₅₀ Is 2.195 mu M, has remarkable inhibitory activity on SARS-COV-2 coronavirus.

(2) The amotinib has remarkable effect of inhibiting SARS-COV-2 virus activity in vivo, has no toxicity on cell growth, and can be used for preparing anti-coronavirus medicines.

Drawings

FIG. 1 is a molecular structural formula of armetinib.

FIG. 2 is a graph showing the effect of amotinib on the growth of a 293T ACE2 overexpressing cell line.

Figure 3 is the effect of amotinib on the growth of H1299 ACE2 overexpressing cell lines.

FIG. 4 is a graph showing the infection efficiency of SARS-COV-2 pseudovirus by Amatinib into 293T ACE2 overexpressing cells.

FIG. 5 shows the inhibition of SARS-COV-2 pseudovirus by amotinib on entry into 293T ACE2 overexpressing cells.

FIG. 6 shows the infection efficiency of SARS-COV-2 pseudovirus by Amatinib into H1299 ACE2 overexpressing cells.

FIG. 7 shows the inhibition of SARS-COV-2 pseudovirus by Amatinib against H1299 ACE2 overexpressing cells.

FIG. 8 is a fluorescent image of mice in the control group infected with SARS-COV-2 pseudovirus.

FIG. 9 is a fluorescence image of the in vivo inhibition of SARS-COV-2 pseudovirus by Ameitinib.

FIG. 10 is a bar graph of in vivo inhibition of SARS-COV-2 pseudovirus by Ameitinib.

Detailed Description

The experimental methods of the present invention, in which specific conditions are not specified in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The terms "comprising" and "having" and any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to the elements or modules listed but may alternatively include additional steps not listed or inherent to such process, method, article, or device.

The present invention will be further described in detail with reference to the following embodiments, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the description is only illustrative and is not intended to limit the scope of the invention. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the present invention.

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.

Example 1 Effect of Amatinib on cell growth

First part, test method

The experimental object: a 293t ACE2 overexpressing cell line, a H1299 ACE2 overexpressing cell line.

Experimental drugs: almitinib (almonetinib), compound solvent DMSO.

The experimental method comprises the following steps:

the first pm plating: collecting log phase cells, adjusting cell suspension concentration, adding 90uL,293T ACE2 and H1299 ACE2 into each well, and spreading 1×10 respectively ⁴ And 6X 10 ³ Each cell per well.

Dosing the next morning: adding 10 μl of the drug with concentration gradient into each well, arranging 3 compound wells for each drug concentration, and placing in 5% CO ₂ Incubate at 37 ℃.

Plate collection after 48h of dosing: the reaction was stopped after incubation for 4h by first visual inspection under an inverted microscope, followed by addition of 20ul MTT solution (5 mg/ml, i.e., 0.5% MTT) per well, careful aspiration of the in-well broth, addition of 100. Mu.L of dimethyl sulfoxide per well, and shaking on a shaker for 10min at low speed to allow the crystals to dissolve well.

OD value detection: detecting the absorbance value of each hole at the 490nm wavelength of the enzyme-labeled instrument, and calculating the drug inhibition rate, wherein the calculation method is shown in the formula (1):

in the experiment, a culture medium without cells is used as a blank control group, a DMSO solution with the same dilution ratio as that of the compound is added into each hole to be used as a negative control group, and an experiment group containing the amotinib is also provided.

Second part, test results

The results obtained by the test method according to the first section above are shown in FIGS. 2 to 3, in which it is known from the results that the drug inhibits 293T cell growth IC ₅₀ 7.688. Mu.M, without toxicity to H1299 cells.

EXAMPLE 2 Amatinib pair blocking SARS-COV-2 pseudovirus entry into cell assay

First part, test method

Experimental materials: SARS-CoV-2_S (D614G) protein pseudolentivirus (expressing green fluorescent protein GFP and Luciferase within the virus) was purchased from Nanoboat Biotechnology (Guangzhou) Inc.

Polybrene (5 mg/mL, 200. Mu.L) was purchased from the cloud navicular Biotechnology (Guangzhou) Co., ltd.

Experimental drugs: almitinib (almonetinib);

the experimental method comprises the following steps:

1 day (day 0) prior to SARS-COV-2 pseudovirus transduction, cells were seeded into new sterile black opaque 96-well plates, and each well of 293T ACE2 and H1299 ACE2 overexpressing cells was seeded 1X 10 individually ⁴ And 6X 10 ³ Every 100 μl of complete medium (DMEM+10% FBS) per well, preferably about 50% of the cell density at the next day of infection, is placed at 37deg.C and 5% CO ₂ Is cultured overnight in a carbon dioxide incubator;

on the day of SARS-COV-2 pseudovirus transduction (day 1), firstly thawing frozen virus liquid on ice, gently blowing for several times, uniformly mixing the thawed virus particles, and then adding 10mL of virus liquid into 10mL of fresh complete culture containing 7.5 mug/mL Polybrene, so that the volume ratio of virus liquid to complete culture medium is 1:1, gently mixing, and then equally dividing into the following 10 groups (except the control group);

the experiment was carried out by setting a Control group (Control), a Pseudovirus liquid group (Pseudovirus), a test group [ Pseudovirus liquid+candidate drug (BTRX-335140 working concentration was set to 8. Mu.M, 4. Mu.M, 2. Mu.M, 1. Mu.M, 0.5. Mu.M, 0.25. Mu.M), preparing a slightly larger amount of 4 compound wells each, incubating on ice for 30min after preparing, sucking out the original medium, adding the complete medium to the cells to obtain the complete medium containing the Pseudovirus particles (finally, 60. Mu.L of fresh medium was added to each well of the Control group, 60. Mu.L of medium containing the virus particles was added to each Pseudovirus group and the test group, respectively), and finally gently shaking the culture plate to allow the virus liquid to cover each cell, and then placing at 37℃and 5% CO ₂ Is cultured overnight in a carbon dioxide incubator;

on day 2 of SARS-COV-2 pseudovirus transduction, the solution was changed 24h after virus transfection to 100 μl per well in complete medium containing 1% double antibody.

On day 4 of SARS-COV-2 pseudovirus transduction, namely after virus transfection for 72 hours, sucking the culture medium, adding 30 mu L of lysate into each hole, shaking for 10min, adding 20 mu L of luciferase reaction substrate in a dark place, detecting luciferase activity by using an enzyme-labeling instrument, and finally calculating infection efficiency and drug inhibition rate, wherein the calculation method of the infection rate is shown in a formula (2), and the calculation method of the drug inhibition rate is shown in a formula (3);

infection rate% = absorbance of experimental group/absorbance of pseudovirome x 100% (2)

Drug inhibition ratio% = 100% -infection ratio% (3)

Second part, test results

The test results obtained according to the test method of the first section described above are shown in tables 2 to 3 and fig. 4 to 7, and are specifically as follows:

TABLE 2

TABLE 3 Table 3

As is apparent from the results of tables 2 to 3 and FIGS. 4 to 7, the effect of blocking the entry of SARS-COV-2 pseudovirus into cells after the administration of Ametinib was remarkable, wherein the activity of inhibiting the infection of 293T cells and H1299 cells with SARS-COV-2 pseudovirus was increased with the increase of the administration concentration, and the inhibitory activity was exhibited at a trace dose of 0.25. Mu.M, wherein the IC of inhibiting the infection of 293T cells with SARS-COV-2 pseudovirus was exhibited ₅₀ IC for inhibiting infection of H1299 cells with SARS-COV-2 pseudovirus at 2.052. Mu.M ₅₀ The experimental result shows that the inhibition activity is 2.195 mu M, and the experimental result has extremely remarkable difference.

EXAMPLE 3 in vivo inhibition of SARS-COV-2 pseudovirus assay by Ametinib

First part, test method

The experimental object: c57BL/6J (B6J) mice, females, 6-8 weeks old, were kept in SPF rearing room for 5 days while isolation observations were made.

Experimental materials:

adeno-associated virus (AAV): AAV9[ ssAAV.CAG.human ACE2.WPRE.SV40Pa ], 5E+12GC in total, and titres of not less than 1E+13GC/mL, 100. Mu.L/tube were purchased from Paizhen organisms.

SARS-CoV-2 pseudovirus: FNV-SARS-CoV-2-S (BA.4) (Omicron) pseudovirus purchased from Beijing Boolong with titre > 10 ⁷ TU/mL, 200. Mu.L/tube.

Experimental drugs: kappa opioid receptor antagonist BTRX-335140.

The virus infection and administration mode is nasal cavity perfusion: the mice were anesthetized with inhaled isoflurane, fixed in the hands, held against the chin with the tip of the thumb, the thumb joints were bent upward in order to completely shut off the mouth-type breath of the mice, 50 μl of liquid was aspirated with a 100 μl pipette, placed on the nostrils of the mice, and the mice were aspirated into the nasal cavities by nasal breathing.

Viral infection and administration steps: AAV9 (titre 10) was used in experimental mice ¹¹ GC/mL) for 5 days, 30 mu L of drug with nasal cavity perfusion concentration of 7.5mg/kg is adopted, SARS-CoV-2 pseudovirus infection is carried out after 1h of administration, and AAV9 infection and SARS-CoV-2 pseudovirus infection are carried out in a control group without administration;

in vivo imaging monitoring: the nasal cavity was perfused with D-luciferin (potassium salt) at a concentration of 10mg/mL, 20. Mu.L, at day 5 post viral infection and administration, and isoflurane was inhaled for anaesthesia, and luciferase signals were detected 10 minutes later using an IVscope8500 small animal in vivo imaging system.

Second part, test results

The results obtained by the test method according to the first part are shown in figures 8-10, and compared with the fluorescence imaging of SARS-CoV-2 pseudovirus in mice in a control group and an administration group, the fluorescence signal of SARS-CoV-2 pseudovirus in the mice after the administration of the amotinib is found to be obviously reduced, which indicates that the amotinib can obviously inhibit the infection of SARS-COV-2 pseudovirus in the mice.

It should be noted that, in the present specification, specific features, structures, materials, or characteristics may be arbitrarily combined, and in order to simplify the description, all possible combinations of the features in the foregoing embodiments are not described, and those skilled in the art may combine and combine the features of the different embodiments and the different embodiments described in the present specification without contradiction.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. The application of the amotinib in preparing anti-coronavirus medicaments is characterized in that the structure of the amotinib is shown as (I);

2. use of ametinib for the preparation of an anti-coronavirus drug according to claim 1, characterized in that said anti-coronavirus drug further comprises geometric isomers of ametinib or pharmaceutically acceptable salts thereof and/or solvates thereof and/or hydrates thereof.

3. Use of almitinib according to claim 1 or 2 for the preparation of an anti-coronavirus medicament, wherein the medicament further comprises pharmaceutically acceptable excipients.

4. Use of almitinib according to claim 1 or 2 for the preparation of an anti-coronavirus medicament, wherein the route of administration of the medicament is oral, injectable or respiratory inhalation.

5. Use of amotinib for the preparation of an anti-coronavirus medicament according to claim 1 or 2, characterized in that the dosage form of the medicament comprises a suspension, a granule, a capsule, a powder, a tablet, an emulsion, a solution, a drop pill, an injection, an aerosol or a drop.

6. Use of ametinib for the preparation of an anti-coronavirus drug according to claim 1 or 2, characterized in that said coronavirus comprises at least one of 2019-nCoV, HCoV-229E, HCoV-OC43, SARS-CoV and MERS-CoV.

7. Use of ametinib for the preparation of an anti-coronavirus drug according to claim 1 or 2, characterized in that said drug is a drug for inhibiting fever, cough and sore throat in simple infections, pneumonia, acute respiratory infections, severe acute respiratory infections, hypoxic respiratory failure, acute respiratory distress syndrome, sepsis and septic shock.