CN113318115A

CN113318115A - Application of metacycline in preparation of medicine for preventing and treating bovine parainfluenza virus type 3 virus infection

Info

Publication number: CN113318115A
Application number: CN202110692012.5A
Authority: CN
Inventors: 程凯慧; 于志君; 楚会萌; 杨宏军
Original assignee: Institute Animal Science and Veterinary Medicine of Shandong AAS; Poultry Research Institute Shandong Academy of Agricultural Sciences
Current assignee: Institute Animal Science and Veterinary Medicine of Shandong AAS; Poultry Research Institute Shandong Academy of Agricultural Sciences
Priority date: 2021-06-22
Filing date: 2021-06-22
Publication date: 2021-08-31
Anticipated expiration: 2041-06-22
Also published as: CN113318115B

Abstract

The invention provides application of metacycline in preparing a medicament for preventing and treating bovine parainfluenza virus type 3 infection, belonging to the field of medical medicaments. The invention proves that the metacycline can effectively prevent the proliferation of BPIV3 for the first time, and has relatively low toxicity to cells, and experiments prove that the half cytotoxic concentration (CC50) of the metacycline to MDBK cells is more than 100 mu M, and the half effective concentration (EC50) to BPIV3 viruses is 9.08 mu M; the therapeutic index of the methacycline to the BPIV3 is more than 10, which shows that the methacycline has the prospect of being developed into a BPIV3 resistant medicament, opens up new medicament application for the methacycline, lays an experimental foundation for developing a high-efficiency and specific BPIV3 resistant medicament and provides a new visual field, thereby having good value of practical application.

Description

Application of metacycline in preparation of medicine for preventing and treating bovine parainfluenza virus type 3 virus infection

Technical Field

The invention belongs to the technical field of medicine, and particularly relates to application of metacycline in preparation of medicines for preventing and treating BPIV3 infection.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Bovine parainfluenza virus type 3 (BPIV 3) belongs to the family of paramyxoviridae and the genus pneumovirus, is the most important pathogen of Bovine respiratory syndrome (BRDC), is clinically characterized by fever, cough, asthma, anorexia, increased eye and nose secretions, few accompanied by diarrhea, and finally shows pneumonia, and can seriously cause abortion. Currently, BPIV3 infection is distributed worldwide, causing serious economic losses to the world cattle industry. In 1959, the virus is first isolated in the United states, and the report of BPIV3 in China is relatively late.

BPIV3 is one of the important pathogens responsible for upper respiratory tract infections in calves and adult cattle, and members of the BPIV3 genus also include Human parainfluenza virus type 3 (HPIV 3) and Sendai virus (SeV). Under natural conditions, the disease only infects cattle, most of which are bred in barn, sick cattle and cattle with virus are main infection sources, and susceptible cattle are infected through air-droplets via respiratory tracts due to contact with the cattle with toxin expelling, and intrauterine infection can also occur. Viruses have been found in bull semen and cow reproductive tract and may cause infertility. In addition, BPIV3 was isolated by researchers from dairy cow clinical mastitis. The disease is common in late autumn and winter, and the cattle infected with BPIV3 are often damaged by respiratory epithelium to cause the defense capability of respiratory mucosa to be reduced and cause immunosuppression, so that the sick cattle infected with BPIV3 are often susceptible to secondary infection with serious bacterial or mycoplasma diseases to cause serious pneumonia, and the death rate is greatly increased.

Metacycline is a semi-synthetic tetracycline, with broad-spectrum antibacterial activity. The antibacterial mechanism is the same as that of tetracycline, the antibacterial effect is stronger than that of tetracycline, and the antibacterial composition has high efficiency and long-acting property. Has stronger antibacterial activity to gram positive or negative bacteria; has strong antagonistic effect on rickettsia, chlamydia, mycoplasma, atypical mycobacterium and amebiasis, but has no report on the application of the composition in preventing or treating bovine parainfluenza virus type 3.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the application of metacycline in preventing and/or treating BPIV3 related diseases, and the metacycline is proved to be capable of effectively inhibiting the proliferation of BPIV3 for the first time and has low toxicity to cells, so the metacycline has the prospect of being developed into a medicament for preventing and/or treating BPIV3 infection. The application of the invention is disclosed for the first time and is different from the known clinical application of metacycline.

Specifically, the invention relates to the following technical scheme:

in a first aspect of the invention, there is provided the use of metacycline in the manufacture of a medicament for the prevention and/or treatment of infection by bovine parainfluenza virus type 3.

According to the present invention, the concept of "prevention and/or treatment" means any measure suitable for treating a BPIV 3-related disease, or to prophylactically treat such an manifested disease or manifested symptoms, or to avoid recurrence of such a disease, such as recurrence after the end of a treatment period or treatment of symptoms of an already-developed disease, or to prevent or inhibit or reduce the occurrence of such a disease or symptoms with prior intervention.

Meanwhile, in the medicine for preventing and/or treating bovine parainfluenza virus type 3 infection, the metacycline has no less than half Effective Concentration (EC)₅₀) The half Effective Concentration (EC) of methacycline to BPIV3₅₀) 9.08. mu.M; the therapeutic index of methacycline to BPIV3 is greater than 10.

In a second aspect of the invention, there is provided the use of metacycline in the preparation of a medicament against bovine parainfluenza virus type 3.

In a third aspect of the invention, the application of metacycline in the preparation of a medicament for inhibiting and/or killing bovine parainfluenza virus type 3 is provided.

According to the present invention, not only is the use of metacycline in the manufacture of a medicament for the prevention and/or treatment of bovine parainfluenza virus type 3 infection disclosed, but it is also disclosed that this effect may be enhanced when combined with at least one other pharmaceutically active ingredient. As an alternative or in addition to other pharmaceutically active ingredients, metacycline may also be used in combination with other non-pharmaceutically active ingredients.

In view of the above, according to a fourth aspect of the present invention, there is provided a pharmaceutical composition for preventing and/or treating bovine parainfluenza virus type 3 infection, which comprises metacycline and at least one other pharmaceutically active ingredient and/or at least one other non-pharmaceutically active ingredient.

Wherein the metacycline has an Effective Concentration (EC) of not less than half₅₀) The half Effective Concentration (EC) of methacycline to BPIV3₅₀) 9.08. mu.M; the therapeutic index of methacycline to BPIV3 is greater than 10.

In the sense of the invention, the pharmaceutical composition provided by the invention represents a substance, and the contained metacycline has obvious inhibition and/or killing effects on BPIV3, can realize direct inactivation on BPIV3, and can block the adsorption of BPIV3 on cells.

In a fifth aspect of the present invention, there is provided a method for preventing and/or treating a disease associated with infection with bovine parainfluenza virus type 3, said method comprising: administering to the subject a therapeutically effective dose of metacycline or the pharmaceutical composition described above.

The beneficial technical effects of the technical scheme are as follows:

the technical scheme firstly discovers that the compound metacycline can effectively prevent the proliferation of BPIV3, and has relatively low toxicity to cells, and experiments prove that the half cytotoxic concentration (CC50) of the metacycline to MDBK cells is more than 100 mu M, and the half effective concentration (EC50) to BPIV3 viruses is 9.08 mu M; the therapeutic index of the methacycline to the BPIV3 is more than 10, which shows that the methacycline has the prospect of being developed into a BPIV3 resistant medicament, opens up new medicament application for the methacycline, lays an experimental foundation for developing a high-efficiency and specific BPIV3 resistant medicament and provides a new visual field, thereby having good value of practical application.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

Fig. 1 is a graph of the effect of methacycline of the present invention in preventing BPIV3 damaged cells;

wherein: wherein A is a virus control group; b is MDBK normal cell group; c is infected cell drug test group (using 10 μ M metacycline);

FIG. 2 is a graph of the half-cytotoxic concentration of metacycline on MDBK cells (CC50) in example 2;

figure 3 is a graph of the half-effective concentration (EC50) of metacycline versus BPIV3 in example 3;

figure 4 is a graph of the effect of metacycline administration at various time points on BPIV3 inhibition in example 4;

figure 5 example 5 graph of the effect of metacycline on the blockade of BPIV3 replication.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. 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 application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions, or according to the conditions recommended by the sales companies; materials, reagents and the like used in examples were commercially available unless otherwise specified.

As described in the background, there has been no report in the prior art of the use of metacycline for the prevention and/or treatment of BPIV3 related diseases.

In view of the above, the invention establishes a BPIV3 drug screening system on the cell (MDBK cell) level, and determines the half toxicity concentration (CC) of methacycline on MDBK cells₅₀) Greater than 100. mu.M, half the Effective Concentration (EC) of BPIV3₅₀) At 9.08 μ M, the therapeutic index of methacycline to BPIV3 was greater than 10.

The invention also provides an in vitro antiviral inhibition test which is carried out by respectively adopting 3 different action modes of different time point administration experiments, addition of the virus after the medicine is added and pre-action of the virus according to the pathogenic mechanism of the virus.

In one exemplary embodiment of the invention, there is provided a use of metacycline in the manufacture of a medicament for the prevention and/or treatment of BPIV3 infection. And thus, metacycline is effective for the prevention and/or treatment of BPIV 3-related diseases.

The compound metacycline (CAS number: 914-00-1) of the invention has the following structural formula:

wherein, in the medicine for preventing and/or treating BPIV3 infection, the metacycline has no less than half Effective Concentration (EC)₅₀) The half Effective Concentration (EC) of methacycline to BPIV3₅₀) 9.08. mu.M;

In a further embodiment of the invention, there is provided the use of methacycline or a composition comprising methacycline, or a formulation thereof, in the manufacture of a medicament against BPIV 3.

In a further embodiment of the invention there is provided the use of metacycline or a composition comprising metacycline, or a formulation thereof, in the manufacture of a medicament for the inhibition and/or killing of BPIV 3.

According to the present invention, not only is the use of metacycline in the manufacture of a medicament for the prevention and/or treatment of BPIV3 infection disclosed, but it is also disclosed that this effect may be enhanced when administered in combination with at least one other pharmaceutically active ingredient. As an alternative or in addition to other pharmaceutically active ingredients, metacycline may also be used in combination with other non-pharmaceutically active ingredients.

In yet another embodiment of the present invention, there is provided a pharmaceutical composition for the prevention and/or treatment of BPIV3 infection, said pharmaceutical composition consisting of metacycline and at least one other pharmaceutically active ingredient and/or at least one other non-pharmaceutically active ingredient.

Wherein the metacycline has an Effective Concentration (EC) not less than half that of metacycline₅₀) The half Effective Concentration (EC) of methacycline to BPIV3₅₀) 9.08. mu.M; of course, when metacycline is used in combination with other drugs or active ingredients having inhibitory and/or killing properties or assisting in the inhibition and/or killing of BPIV3, etc. for the same applications as described above in the present invention, the drug concentration may theoretically be lower than the above effective concentration, without excluding special exceptions.

According to the embodiment of the invention, the inhibition or killing effect of the metacycline is improved along with the increase of the concentration within the safe range of the drug concentration. The medicine of the invention has no harm to susceptible cells of BPIV3 when the medicine inhibits or kills BPIV3 within a safe concentration range.

In the sense of the invention, the pharmaceutical composition provided by the invention represents a substance, and the metacycline contained in the pharmaceutical composition has obvious inhibition and/or killing effects on BPIV3, can realize direct killing effect on BPIV3, and can block the adsorption effect of BPIV3 on cells.

In yet another embodiment of the present invention, the other pharmaceutically active ingredient comprises a substance having the ability to inhibit and/or kill BPIV3 or to assist in inhibiting and/or killing BPIV 3.

In a further embodiment of the invention, the other non-pharmaceutically active ingredient comprises a pharmaceutically acceptable adjuvant and/or carrier.

The pharmaceutical compositions of the present invention containing such metacycline may be administered in unit dosage form. The administration dosage form can be liquid dosage form or solid dosage form. The liquid dosage forms can be true solutions, colloids, microparticles, emulsions, and suspensions. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, emulsion, granule, suppository, lyophilized powder for injection, clathrate, landfill, patch, liniment, etc.

The pharmaceutical combination or pharmaceutical preparation of the present invention may further comprise a conventional carrier, wherein the pharmaceutically acceptable carrier includes but is not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbates, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin and the like. The carrier may be present in the pharmaceutical composition in an amount of from 1% by weight to 98% by weight, typically about 80% by weight. For convenience, the local anesthetic, preservative, buffer, etc. may be dissolved directly in the vehicle.

Oral tablets and capsules may contain excipients such as binding agents, for example syrup, acacia, sorbitol, tragacanth, or polyvinylpyrrolidone, fillers such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, glycine, lubricants such as magnesium stearate, talc, polyethylene glycol, silica, disintegrants such as potato starch, or acceptable wetting agents such as sodium lauryl sulfate. The tablets may be coated by methods known in the art of pharmacy.

The oral liquid can be made into water and oil suspension, solution, emulsion, syrup, or dried product, and supplemented with water or other suitable medium before use. Such liquid preparations may contain conventional additives such as suspending agents, sorbitol, cellulose methyl ether, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gelatin, hydrogenated edible fats and oils, emulsifying agents such as lecithin, sorbitan monooleate, gum arabic; or a non-aqueous carrier (which may comprise an edible oil), such as almond oil, an oil such as glycerol, ethylene glycol, or ethanol; preservatives, e.g. methyl or propyl p-hydroxybenzoates, sorbic acid. Flavoring or coloring agents may be added if desired.

In another embodiment of the present invention, there is provided a method for preventing and/or treating a disease associated with infection of bovine parainfluenza virus type 3, the method comprising: administering to the subject a therapeutically effective dose of metacycline or the pharmaceutical composition described above.

The subject refers to an animal, preferably a mammal, most preferably a bovine, who has been the object of treatment, observation or experiment. By "therapeutically effective amount" is meant an amount of active compound or pharmaceutical agent, including a compound of the present invention, that elicits the biological or medical response in a tissue system, animal, or other subject sought by a researcher, veterinarian, medical doctor or other medical professional, which includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition, or disorder being treated. It will be appreciated that the optimum dosage and interval for administration of the active ingredients of the invention will be determined by the nature and external conditions, such as the form, route and site of administration and the particular mammal being treated, and that such optimum dosage may be determined by conventional techniques. It should also be recognized that the optimal course of treatment, i.e., the daily dosage of the compound over a nominal period of time, may be determined by methods known in the art.

The technical solution of the present invention is further described with reference to the following specific examples. It should be noted that the BPIV3 used in the examples of the present application was isolated from the research center for dairy cows of the academy of agricultural sciences of shandong province and was identified as belonging to the BPIV 3C type.

The specific separation and identification method is as follows: diluting fresh nasal swab with PBS, repeatedly freezing and thawing for 3 times, centrifuging at 5000 r/min for 5min, collecting supernatant, adding penicillin (200IU/mL) and streptomycin (100 μ g/mL), collecting 200 μ L supernatant, extracting BPIV3 virus genome RNA according to virus genome DNA/RNA extraction kit specification, reverse transcribing into cDNA, performing PCR amplification with BPIV3 specific primer, filtering and sterilizing the nasal swab liquid identified as positive by PCR with 0.22 μm filter membrane, collecting 1mL inoculated MDBK single layer cell, adsorbing at 37 deg.C for 1h, discarding, adding into DMEM culture medium containing 2% FBS, and culturing at 37 deg.C and 5% CO for 5min₂Culturing in a cell culture box, observing cytopathic condition every 12h, and continuously observing for 3 d. Freezing and thawing cells with pathological Changes (CPE) for 3 times repeatedly, collecting cell virus liquid, identifying whether the obtained cell virus liquid is single BPIV3 infection or not, and obtaining the cell virus liquid with single BPIV3 infection by using a plaque purification method if the cell virus liquid is not single virus infection. The isolate was identified to be of BPIV 3C type by phylogenetic tree analysis of BPIV 3.

Example 1Virus TCID₅₀Measurement of (2)

MDBK cells (stored in Dairy research center of Oncology institute of agriculture, Shandong province) were digested at 1X 10 per well⁵Cell density of one/mL was seeded into 96-well cell culture plates and placed at 37 ℃ in 5% CO₂After culturing the cells in the cell culture chamber of (1) to form a monolayer of cells, the cell growth medium in the wells was discarded, and BPIV3 was continuously diluted 10-fold with virus dilutions (10 dilutions, respectively)^-1～10^-10) Inoculating to a 96-well plate full of monolayer cells, each well having a volume of 100 μ L, placing at 37 deg.C and 5% CO₂The culture was continued in the incubator, and the cells were observed day by day for CPE and the number of cytopathic wells was recorded in detail. And setting a normal cell control group and a blank control group at the same time, setting 8 repeats in each group, and judging the result when the cytopathic effect is not continued. The above cytopathic hole corresponds to the cytopathic effectCell wells and virus TCID calculated by Karber method₅₀。

TABLE 1 TCID₅₀ of BPIV3

Note: TCID₅₀Tissue culture infectious dose, also known as 50% Tissue cell infectious dose; i.e., the amount of virus required to cause half of the cytopathic effect or death (CPE) in a well or tube in culture.

As a result: morphological observation under a microscope shows that virus diluents with different concentrations all cause cytopathic effect when 36 hours elapse, the refractive index of cells changes, the monolayer structure is destroyed, cells are subjected to round shrinkage necrosis and gradually take the shape of a net and form vacuoles, some cells are cracked and fall off into fragments, cytopathic effect of each hole is not continued after 72 hours, the number of CPE holes with different concentrations is counted, the CPE ratio with different concentrations is calculated, and the TCID of BPIV3 is calculated according to a Karber method₅₀The value:

LgTCID₅₀＝L-D(S-0.5)

(L: logarithm of highest dilution; D: difference between logarithm of dilutions; sum of S-positive well ratios)

LgTCID₅₀＝L-D(S-0.5)＝-1-1×(5.75-0.5)＝-6.25

TCID₅₀＝10^-6.25/0.1mL

I.e. diluting the virus 10^6.25Inoculation with 100. mu.L resulted in 50% of the cells being diseased.

Example 2Toxicity assay of metacycline on MDBK cells:

MDBK cells are susceptible cells to BPIV 3. Therefore, the cytotoxicity of metacycline on MDBK cells is firstly detected, and the specific experimental steps are as follows:

(1) mu.L of cells (MDBK 1X 10) were seeded in 96-well plates⁴One/hole).

(2) After incubation to MDBK monolayer, the next dosing analysis was performed. Media was discarded and 100 μ L of 2% FBS DMEM containing different drug concentrations were added to each well, 3 replicates for each concentration. At the same time, control wells: add 100. mu.L of 2% FBS DMEM medium. Zero setting hole: cells were not plated.

(3) At 37 ℃ 5% CO₂After culturing for 48h under the condition, the OD value at 450nm is measured by an enzyme-labeling instrument according to the instruction of a CCK-8 kit.

(4)37℃，5％CO₂After further incubation for 2h under these conditions, the absorbance was measured at 450 nm. A450nm for normal growing cells was set as a 100% cell control.

(5) Data were analyzed and half-Cytotoxic Concentration (CC) of methacycline was calculated using GraphPad Prism5₅₀) The value is obtained. The results are shown in FIG. 2.

As a result: the metacycline has a dose-dependent relationship, namely, the metacycline shows more obvious cytopathic effect along with the increase of the concentration of the drug. Statistical analysis confirmed that half toxic metacycline concentrations were greater than 100 μ M.

Example 3Inhibition of BPIV3 by metacycline:

(1) 1X 10 inoculations in each well of a 96-well plate⁴MDBK cells, 37 ℃, 5% CO₂Culturing in an incubator overnight;

(2) discard medium, add 100. mu.L of 2% DMEM per well, dilute and add drug at 25. mu.M initial concentration, two-fold concentration gradient, 5% CO₂Culturing in an incubator; after 2h drug incubation, 100. mu.L 1000 TCID per well was added₅₀BPIV3 dilution.

(3) After 48h, the OD at 450nm was measured with a microplate reader, following the instructions of the CCK-8 kit.

(4) The data were analyzed, and the rate of virus inhibition (%) (drug-treated D450nm value-virus control D450nm value)/(normal cell control D450nm value-virus control D450nm value) × 100%, half Effective Concentration (EC) of the compound was obtained using GraphPad Prism5 software₅₀) The value is obtained. The results are shown in FIG. 3. Then according to the formula TI ═ CC₅₀/EC₅₀And calculating the corresponding therapeutic index TI value.

As a result: the effective inhibition of the drug to the BPIV3 can be calculated by detecting the cell viability through a CCK-8 kitAnd (5) preparing the rate. From the results, the effective inhibition rate of the metacycline in a safe concentration range is increased along with the increase of the drug concentration, and the metacycline is in a certain dose-effect relationship. Half Effective Concentration (EC) of BPIV3 by analytical software₅₀) It was 9.08. mu.M. The therapeutic index of methacycline to BPIV3 is greater than 10.

Example 4Preliminary study of mechanism of action

The compound to be tested is added into MDBK cells inoculated with BPIV3 through different administration time, namely corresponding time points of first administration and then infection of virus (before 0 h), first infection of virus and then administration (after 0 h), and simultaneous addition of virus and medicine into the cells (0 h), so as to preliminarily judge the action period of the metacycline. The specific experimental steps are as follows:

(1) 1X 10 inoculations in each well of a 96-well plate⁴MDBK cells, 37 ℃, 5% CO₂Culturing in an incubator.

(2) According to the measured pharmacodynamic evaluation result of the related medicine, the concentration of the medicine required by the experiment is determined, and the medicine is diluted to the required concentration by using a maintenance medium.

(3) After overnight incubation, the cell supernatants from the second three duplicate wells of the 96-well plate were aspirated and the cells were washed 2 times with phosphate buffer. Then 50. mu.L of the drug to be tested was added, and the time was recorded as-2 h.

(4) After 2h, the cell supernatants from the other wells were aspirated off, and diluted BPIV3 was added to each well in columns 2-11 at a volume of 50. mu.L per well. At the same time, 50. mu.L of the corresponding analyte was added to the three duplicate wells in column 3, which was recorded as 0 h.

(5) And adding corresponding compounds to be detected into the three compound holes in the next row at regular intervals, and marking the corresponding time. MDBK cells from column 11 were used as virus control.

(6) After 48 hours of incubation, OD measurements were performed. The data were analyzed and concluded, the results of which are shown in fig. 4.

As a result: according to the analysis of the administration experiment results at different time points, the metacycline has obvious inhibition effect on viruses when added with medicine when the viruses infect cells for-2 h.

Example 5Effect of Compound addition at different times on BPIV3 replication

The metacycline is subjected to in vitro antiviral inhibition tests by 3 different action modes of firstly adding medicine and then adding virus, firstly adding virus and then adding medicine, and pre-acting the medicine and the virus.

(1) Direct killing effect of medicine on virus

Equal amount of 1000 TCID₅₀Mixing the virus solution with the medicinal diluent at different concentrations, and standing at 37 deg.C with 5% CO₂Pre-acting in an incubator for 4h, adding into a 96-well cell culture plate with a monolayer, allowing each liquid medicine to have a gradient of 100 μ L/well, acting in the incubator for 2h, discarding the supernatant, and adding cell maintenance liquid to continue culturing. The test simultaneously sets a normal cell control group, a virus control group and a blank control group, each concentration is set to be 3 times, cell viability detection is carried out for 48 hours, and GraphPad Prism5 software is used for obtaining EC of the compound₅₀。

As a result: in the pre-treatment regimen of methacycline with BPIV3, it can be seen that 25 μ M methacycline had 40% direct inactivation of BPIV3, and less than 25 μ M methacycline had no direct inactivation of BPIV 3.

(2) Blocking effect of drug on adsorption of BPIV3

At a rate of 1X 10 per hole⁴Inoculating digested cells into a pore plate at a certain cell density, removing supernatant after monolayer cells grow, adding medicinal diluent with different concentrations into a 96-pore cell culture plate with a monolayer, performing pre-treatment in an incubator for 4h, removing supernatant, washing twice with PBS, adding 1000 TCID with equal amount, and collecting supernatant₅₀Placing the virus liquid at 37 ℃ and 5% CO₂Culturing in an incubator. The test is simultaneously provided with a normal cell control group, a virus control group and a blank control group, each concentration is set for 3 times, cell activity detection is carried out after 48 hours, and the antiviral effective rate of the drugs with different concentrations under the action mode is calculated.

As a result: the effect of methacycline on BPIV3 is shown in figure 5 by analysis software, and the result shows that the concentration of 9.08 mu M and above can completely and effectively inhibit the proliferation of BPIV3 in a safe concentration range, which indicates that the methacycline can prevent the adsorption of BPIV3 on cells. (3) Blockade of BPIV3 replication by drugs

At a rate of 1X 10 per hole⁴Cell density digested cells were seeded into well plates, supernatant discarded after monolayer growth, and an equivalent amount of 1000 TCID was added₅₀Adding virus solution into 96-well cell culture plate, and standing at 37 deg.C under 5% CO₂Pre-acting in an incubator for 2h, removing supernatant, washing cells for 2 times with PBS, adding drug diluents with different concentrations, each drug solution gradient being 100 μ L/well, setting a normal cell control group, a virus control group and a blank control group at the same time in the test, setting 3 repeats for each concentration, placing at 37 ℃ and 5% CO₂Culturing in an incubator, detecting cell viability after 48h, analyzing data and obtaining a conclusion.

As a result: metacycline was found by data analysis to be unable to block replication of BPIV 3.

In the application embodiment of the invention, the bovine kidney cells (MDBK) are used as a carrier, and 3 different action modes of adding medicine first and then adding virus, adding medicine first and then adding medicine, and pre-acting virus and then adding medicine are adopted on a cytopathogenic model to carry out in-vitro antiviral inhibition research. The novel antiviral effect of the metacycline is found, and the metacycline has a certain inhibition effect on BPIV 3.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. 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. Application of metacycline in preparation of medicines for preventing and/or treating bovine parainfluenza virus type 3 infection.

2. The use of claim 1, wherein the metacycline drug is at a concentration of not less than 9.08 μ M.

3. Application of metacycline in preparing anti-bovine parainfluenza virus type 3 drugs.

4. Application of metacycline in preparation of drugs for inhibiting and/or killing bovine parainfluenza virus type 3.

5. A pharmaceutical composition for the prevention and/or treatment of bovine parainfluenza virus type 3 infection, characterized in that it consists of metacycline together with at least one other pharmaceutically active ingredient and/or at least one other non-pharmaceutically active ingredient.

6. The pharmaceutical composition of claim 5, wherein the drug concentration of metacycline is not less than 9.08 μ M.

7. The pharmaceutical composition of claim 5, wherein the additional pharmaceutically active ingredient comprises a substance that inhibits and/or kills bovine parainfluenza virus type 3 or assists in inhibiting and/or killing bovine parainfluenza virus type 3.

8. The pharmaceutical composition of claim 5, wherein the other non-pharmaceutically active ingredients comprise pharmaceutically acceptable excipients and/or carriers.

9. A method of preventing and/or treating a disease associated with infection by bovine parainfluenza virus type 3, the method comprising: administering to the subject a therapeutically effective dose of metacycline or the pharmaceutical composition of any one of claims 6-8.

10. The method of claim 9, wherein the therapeutically effective dose is not less than 9.08 μ Μ.