CN110812357A - Application of biapenem in preparation of medicine for preventing and treating bovine enterovirus infection - Google Patents
Application of biapenem in preparation of medicine for preventing and treating bovine enterovirus infection Download PDFInfo
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Abstract
The invention provides application of biapenem in preparation of a medicament for preventing and treating bovine enterovirus infection. The invention discovers for the first time that the compound biapenem can effectively inhibit the multiplication of bovine enterovirus viruses and has low toxicity to cells, and experiments prove that the biapenem has half toxicity concentration (CC) to MDBK cells50) Greater than 100. mu.M, and a half-Effective Concentration (EC) against bovine enterovirus virus50) 60 mu M; the therapeutic index of biapenem to bovine enterovirus is more than 1.67, which shows that the biapenem has the prospect of developing drugs for preventing and/or treating bovine enterovirus infection, opens up new drug application for biapenem, also lays an experimental foundation for developing high-efficiency specific anti-BEV drugs and provides a new visual field.
Description
Technical Field
The invention belongs to the technical field of medicine, and particularly relates to application of biapenem in preparation of a medicine for preventing and treating bovine enterovirus 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 Enterovirus (BEV) is a member of the picornaviridae, Enterovirus genus. The picornavirus is a very complicated virus family, can be divided into 5 genera of rhinovirus, foot and mouth disease virus, enterovirus, cardiovirus, hepatitis A virus and the like, and has more than 200 viruses in total, can cause various diseases of human and animals, such as hepatitis A, foot and mouth disease and the like, and has great significance in medical and veterinary research. Early studies separated bovine enteroviruses into two serotypes, BEV-1 and BEV-2; later, with the development of molecular biology, Roland Zell et al performed systematic gene sequence comparison analysis of bovine enteroviruses that have been isolated at present in 2006, and further classified the bovine enteroviruses into two families A and B, namely BEV-A and BEV-B, which respectively contain 2 and 3 genotypes within BEV-A and BEV-B.
Bovine enterovirus infection occurred in cattle farms in Beijing at the beginning of 2005, which severely affected animal husbandry production. The cows in some cattle farms commonly (about 80%) develop severe diarrhea symptoms: watery diarrhea, foamy, few (10-15%) severe with fresh blood, insignificant changes in body temperature, decreased appetite, and a large reduction in milk production (about 30%). After about two weeks recovery, the milk yield of most cows after recovery cannot be restored to the level before illness, particularly high-producing cows producing more than 35 kg daily, and there is also a risk of contaminating the milk product. However, the prevention and treatment of bovine intestinal diseases in China are very weak, and no vaccine for the bovine intestinal diseases exists, so that the research and development of the drug capable of inhibiting and killing bovine intestinal viruses has great significance for the dairy cow breeding industry in China.
Biapenem (Biapenem) is a carbapenem synthetic antibiotic which exerts antibacterial effects by inhibiting the synthesis of bacterial cell walls and has broad-spectrum antibacterial activity against gram-positive, gram-negative aerobic and anaerobic bacteria. However, there has been no report on whether or not it has antiviral effects.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the application of biapenem in preventing and/or treating bovine enterovirus related diseases, and the invention proves that the biapenem can effectively inhibit the proliferation of bovine enterovirus for the first time, and has low toxicity to cells, so the biapenem has a prospect of being developed into a medicament for preventing and/or treating bovine enterovirus infection. The application of the invention is disclosed for the first time and is different from the known clinical application of biapenem.
Specifically, the invention relates to the following technical scheme:
in a first aspect of the invention, the application of biapenem in preparing a medicament for preventing and/or treating bovine enterovirus infection is provided.
According to the present invention, the concept of "prevention and/or treatment" means any measure suitable for the treatment of bovine enterovirus-related diseases, or the prophylactic treatment of such manifested diseases or manifested symptoms, or the avoidance of recurrence of such diseases, such as recurrence after the end of a treatment period or treatment of symptoms of diseases that have already occurred, or the prevention or inhibition or reduction of the occurrence of such diseases or symptoms with prior intervention.
Meanwhile, in the medicine for preventing and/or treating bovine enterovirus infection, biapenem has the effective concentration (E) not lower than halfC50) The drug concentration of (a), the median Effective Concentration (EC) of biapenem against bovine enterovirus50) 60 μ M.
In a second aspect of the invention, the application of biapenem in preparing a medicament for resisting bovine enterovirus is provided.
In a third aspect of the invention, the application of biapenem in preparing a medicament for inhibiting and/or killing bovine enterovirus is provided.
According to the present invention, not only is the use of biapenem for the preparation of a medicament for the prevention and/or treatment of bovine enterovirus infection disclosed, but also the use of biapenem in combination with at least one other pharmaceutically active ingredient to enhance such an effect is disclosed. Biapenem may also be used in combination with other non-pharmaceutically active ingredients, as an alternative or supplement to other 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 intestinal viral infection, the pharmaceutical composition comprising biapenem and at least one other pharmaceutically active ingredient and/or at least one other non-pharmaceutically active ingredient.
Wherein biapenem has Effective Concentration (EC) not less than half50) The drug concentration of (a), the median Effective Concentration (EC) of biapenem against bovine enterovirus50) 60 mu M; the therapeutic index of biapenem to bovine enterovirus is greater than 1.67.
In the meaning of the invention, the pharmaceutical composition provided by the invention represents a substance, the contained biapenem has obvious inhibiting and/or killing effects on bovine enteroviruses, and mainly acts on multiple stages of the life cycle of the bovine enteroviruses, so that the direct killing effect on the bovine enteroviruses can be realized.
The invention has the beneficial technical effects that:
the invention discovers for the first time that the penem antibiotic compound biapenem can effectively inhibit the multiplication of bovine enterovirus viruses and has low toxicity to cells, and experiments prove that the biapenem has half toxicity concentration (CC) to MDBK cells50) Greater than 100. mu.M, and half of bovine enterovirus virusNumber Effective Concentration (EC)50) 60 mu M; the therapeutic index of biapenem to bovine enterovirus is more than 1.67, which shows that the biapenem has the prospect of developing drugs for preventing and/or treating bovine enterovirus infection, opens up new drug application for biapenem, lays an experimental foundation for developing high-efficiency specific anti-bovine enterovirus drugs and provides a new visual field.
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 showing the effect of biapenem of the present invention on BEV-damaged cells;
wherein: wherein FIG. 1A is a viral control group; FIG. 1B is a set of MDBK normal cells; FIG. 1C is a test group of drugs for infected cells (100. mu.M biapenem administration);
FIG. 2 is the half Cytotoxic Concentration (CC) of biapenem against MDBK cells in example 250) A drawing;
FIG. 3 is the half-maximal Effective Concentration (EC) of biapenem against BEV in example 350) A drawing;
FIG. 4 is a graph of the effect of biapenem administered at different time points on BEV inhibition in example 4;
FIG. 5 is a graph showing the effect of biapenem on BEV direct killing in example 5.
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 on biapenem for preventing and/or treating bovine enterovirus-related diseases in the prior art.
In view of the above, the invention establishes a drug screening system of bovine enterovirus on a cell level, and determines the half toxicity concentration (CC) of biapenem to MDBK cells50) Greater than 100. mu.M, half the Effective Concentration (EC) for bovine enterovirus50) At 60 μ M, the therapeutic index of biapenem against bovine enterovirus is greater than 1.67.
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 embodiment of the invention, the application of biapenem in preparing a medicament for preventing and/or treating bovine enterovirus infection is provided. And therefore biapenem is effective for preventing and/or treating bovine enterovirus-associated diseases.
The compound Biapenem (Biapenem, CAS number: 120410-24-4) of the invention is an injectable carbapenem antibiotic developed by Lederle corporation and cyanamide corporation in 1989, Japan, and it is directed to G+Bacteria, G-The bacteria, the anaerobe and the like have broad-spectrum and strong antibacterial activity, and the structural formula is as follows:
wherein, in the medicine for preventing and/or treating bovine enterovirus infection, biapenem has Effective Concentration (EC) not less than half50) The drug concentration of (a), the median Effective Concentration (EC) of biapenem against bovine enterovirus50) 60 mu M;
according to the present invention, the concept of "prevention and/or treatment" means any measure suitable for the treatment of bovine enterovirus-related diseases, or the prophylactic treatment of such manifested diseases or manifested symptoms, or the avoidance of recurrence of such diseases, such as recurrence after the end of a treatment period or treatment of symptoms of diseases that have already occurred, or the prevention or inhibition or reduction of the occurrence of such diseases or symptoms with prior intervention.
In another embodiment of the invention, the application of biapenem or a composition containing biapenem or a preparation thereof in preparing a medicament for resisting bovine enterovirus is provided.
In another embodiment of the invention, the application of biapenem or a composition containing biapenem or a preparation of the composition in preparing a medicament for inhibiting and/or killing bovine enterovirus is provided.
According to the present invention, not only is the use of biapenem for the preparation of a medicament for the prevention and/or treatment of bovine enterovirus infection disclosed, but also the use of biapenem in combination with at least one other pharmaceutically active ingredient to enhance such an effect is disclosed. Biapenem may also be used in combination with other non-pharmaceutically active ingredients, as an alternative or supplement to other pharmaceutically active ingredients.
In yet another embodiment of the present invention, there is provided a pharmaceutical composition for the prevention and/or treatment of bovine intestinal viral infection, said pharmaceutical composition consisting of biapenem and at least one other pharmaceutically active ingredient and/or at least one other non-pharmaceutically active ingredient.
Wherein biapenem has Effective Concentration (EC) not less than half50) The drug concentration of (a), the median Effective Concentration (EC) of biapenem against bovine enterovirus50) 60 mu M; of course, when biapenem has inhibition and/or killing or other effectWhen drugs or active ingredients for aiding the inhibition and/or killing of bovine enteroviruses and the like are used in combination with the same applications as described above, the drug concentration may theoretically be lower than the effective concentration described above, but special exceptions are not excluded.
According to the embodiment of the invention, the inhibiting or killing effect of biapenem is improved along with the increase of the concentration in the safe range of the drug concentration. The medicine of the invention has no harm to susceptible cells of the bovine enterovirus when the medicine inhibits or kills the bovine enterovirus within the concentration safety range.
In the meaning of the invention, the pharmaceutical composition provided by the invention represents a substance, the contained biapenem has obvious inhibiting and/or killing effects on bovine enteroviruses, and mainly acts on multiple stages of the life cycle of the bovine enteroviruses, so that the direct killing effect on the bovine enteroviruses can be realized.
In still another embodiment of the present invention, the other pharmaceutically active ingredient comprises a substance having the ability to inhibit and/or kill bovine enteroviruses or to assist in inhibiting and/or killing bovine enteroviruses.
In yet another embodiment of the present invention, the other non-pharmaceutically active ingredient comprises a pharmaceutically acceptable carrier, excipient and/or diluent. Such as pharmaceutically compatible inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and non-ionic surfactants or lipids, pharmacologically innocuous salts such as sodium chloride, flavoring agents, vitamins such as vitamin a or vitamin E, tocopherols or provitamins, antioxidants such as ascorbic acid, and stabilizers and/or preservatives for extending the use and shelf life of the pharmaceutically active ingredient or formulation, and other common non-pharmaceutically active ingredients or adjuvants and additives known in the art, and mixtures thereof.
The administration form of the pharmaceutical composition can be solid oral preparation, liquid oral preparation or injection.
The administration dosage form of the pharmaceutical composition can be further tablets, dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, capsules, sugar-coated agents, granules, dry powders, oral solutions, small water injection for injection, freeze-dried powder injection for injection, large infusion or small infusion.
The technical solution of the present invention is further described with reference to the following specific examples. It should be noted that the bovine enterovirus used in the examples of the present application was isolated from the research center for cows of the academy of agricultural sciences of Shandong province and was identified as belonging to the BEV-A family genotype 1.
The specific separation and identification method is as follows: diluting fresh cattle feces 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 BEV virus genome RNA according to virus genome DNA/RNA extraction kit specification, reverse transcribing into cDNA, performing PCR amplification with BEV specific primer, filtering and sterilizing the feces supernatant identified as positive by PCR with 0.22 μm filter membrane, collecting 1mL inoculated with 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 with 5% CO2Culturing in a cell culture box, observing cytopathic condition every 12h, and continuously observing for 4 d. And (3) repeatedly freezing and thawing cells with pathological Changes (CPE) for 3 times, collecting cell virus liquid, simultaneously identifying whether the obtained cell virus liquid is BEV single infection or not, and obtaining the BEV single infection cell virus liquid by using a plaque purification method if the cell virus liquid is not the BEV single infection. This isolate was characterized by phylogenetic tree analysis of the BEV structural proteins VP1 and VP3 as belonging to the BEV-A family genotype 1.
Example 1 Virus TCID50Measurement of (2)
MDBK cells (stored in Dairy research center of Oncology institute of agriculture, Shandong province) were digested and then digested at 3X 10 per well5Cell density of one/mL was seeded into 96-well cell culture plates and placed at 37 ℃ in 5% CO2Culturing the bovine enterovirus in the cell culture box to form monolayer cells, discarding the growth medium of the cells in the pores, and continuously diluting the bovine enterovirus by 10 times to obtain virus diluent (the dilution is 10 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 incubator of (1) was continued, and the CPE of the cells was observed day by day, and cytopathic wells were recorded in detailAnd (4) counting. 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. Cell lesion wells were wells corresponding to the above cell lesions, and virus TCID50 was calculated according to the Karber method.
TABLE 1TCID50of BEV
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 effects or death (CPE) in a well or tube of a culture plate.
As a result: morphological observation under a microscope shows that virus diluents with different concentrations all cause cytopathic effect when the virus diluents are used for 48 hours, the refractive index of cells is changed, the single-layer structure is damaged, the cells are subjected to circular 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 ratios with different concentrations are calculated, and the TCID of the bovine enteroviruses 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×(3.25-0.5)＝-3.75
TCID50＝10-3.75/0.1mL
I.e. diluting the virus 103.75Inoculation with 100. mu.L resulted in 50% of the cells being diseased.
Example 2 toxicity test of biapenem on MDBK cells:
MDBK cells are susceptible cells to bovine enterovirus. Therefore, biapenem was first tested for cytotoxicity against MDBK cells, and the specific experimental procedures were as follows:
(1) mu.L of cells (MDBK 3X 10) were seeded in 96-well plates4One/hole).
(2) After about 12h of incubation, 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: 100 μ L of 2% FBS DMEM medium was added. 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 4h 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 the median Cytotoxic Concentration (CC) of biapenem was calculated using GraphPad Prism550) The value is obtained. The results are shown in FIG. 2.
As a result: the biapenem has a dose-dependent relationship, namely, the cell pathological changes are more obvious along with the increase of the concentration of the medicine. Statistical analysis confirmed that the biapenem median poisoning concentration was greater than 100 μ M.
Example 3 inhibition experiment of biapenem against bovine enteroviruses:
(1) 3X 10 inoculations in each well of a 96-well plate4MDBK cells, 37 ℃, 5% CO2Culturing in an incubator overnight;
(2) the medium was discarded and 100. mu.L of 100TCID was added to each well50The bovine enterovirus diluent (virus diluent is added after 2% FBS DMEM cells are full, diluted and added according to 100 mu M initial concentration and double concentration gradient, and 5% CO is added2Culturing in an incubator;
(3) after 48h, the OD at 490nm was measured with a microplate reader, following the instructions of the CCK-8 kit.
(4) The data were analyzed by using Gr, the viral inhibition (%) of which (drug-treated group D450nm value-viral control group D450nm value)/(normal cell control group D450nm value-viral control group D450nm value) × 100%Half the Effective Concentration (EC) of the compound from the aphPad 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 CCK-8 kit is used for detecting the cell activity, so that the effective inhibition rate of the medicine on the bovine enterovirus can be calculated. From the results, the effective inhibition rate of biapenem is increased along with the increase of the concentration of the drug in a safe concentration range, and the biapenem has a certain dose-effect relationship. Half Effective Concentration (EC) of bovine enterovirus by analytical software50) 60 μ M. The therapeutic index of biapenem to bovine enterovirus is greater than 1.67.
EXAMPLE 4 preliminary investigation of the mechanism of action
Through different administration time, namely corresponding time points of firstly administering and then infecting viruses (before 0h), firstly infecting viruses and then administering (after 0h), and simultaneously adding the viruses and the medicaments into cells (0h), the compound to be detected is added into sensitive cells MDBK cells inoculated with the bovine enteroviruses, and then the action period of biapenem is preliminarily judged. The specific experimental steps are as follows:
(1) 3X 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 of the other wells were aspirated off, and the diluted bovine enterovirus diluent was added to each well of columns 2-11 at a sample addition volume of 50 μ 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 biapenem has obvious inhibition effect on viruses when the biapenem is added in the virus infected cells for-2 h and 0h, which shows that the biapenem can be mainly used in the early stage of the bovine intestinal tract virus life cycle.
EXAMPLE 5 Effect of Biapenem Compound addition at different times on bovine enterovirus replication
We further research the action mechanism of the screened biapenem with better effect and lower toxicity. The in vitro antiviral inhibition test is carried out by 3 different action modes of adding medicine first and then adding virus, adding virus first and then adding medicine, and pre-acting medicine and virus.
(1) Direct killing effect of medicine on virus
Equal amount of 100TCID50Mixing 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: under the administration mode of the preaction of biapenem and bovine enterovirus, the cytopathy of the experimental drug-adding group of each concentration is slight compared with that of a virus control group, the pathological phenomena of cell rounding, cell shedding, vacuolation and the like are relieved, and the effect of biapenem on bovine enterovirus is shown in figure 5 through analysis software. As can be seen from the figure, in the action formula, biapenem has a certain inhibition effect on bovine intestinal tract viruses, and the effective inhibition rate of the drug is increased along with the increase of the drug concentration within a safe concentration range, so that a certain dose-effect relationship exists. The biapenem has a certain direct inactivation effect on bovine enteroviruses.
(2) Blocking effect of medicine on bovine intestinal tract virus adsorption
Inoculating the digested cells into a pore plate according to the cell density of each pore, removing the supernatant after the cells grow into a monolayer, adding the drug diluents with different concentrations into a 96-pore cell culture plate with the monolayer, removing the supernatant after the drug diluents with different concentrations act for 4 hours in an incubator in advance, washing twice with PBS, adding 100TCID with the same amount50Placing 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: CPE observations showed that there was no significant change in the extent of cytopathic effect in the drug pre-treated group compared to the virus control group. Cell viability detection results show that the effective inhibition rate of the drug on bovine enteroviruses is below 20%, which indicates that biapenem cannot prevent the adsorption of bovine enteroviruses on cells.
(3) Blocking effect of medicine on bovine enterovirus replication
Inoculating the digested cells into a well plate according to the cell density of each well, removing the supernatant after the cells grow into a monolayer, and adding 100TCID (total dissolved solids)50Adding 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: CPE observations showed that there was no significant change in the extent of cytopathic effect in the virus pretreatment group compared to the virus control group. The detection result shows that the effective inhibition rate of biapenem on bovine intestinal tract virus is lower than 20%, which indicates that biapenem can not block the replication function in cells.
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 biapenem is found to have a certain inhibiting effect on bovine intestinal viruses, and the direct killing effect on the viruses is better than the adsorption blocking effect and the replication blocking effect.
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 (10)
1. Application of biapenem in preparation of medicine for preventing and/or treating bovine enterovirus infection.
2. The use of claim 1, wherein the biapenem is in a concentration of no less than 60 μ M.
3. The use as claimed in claim 1, wherein biapenem is used in the preparation of a medicament against bovine intestinal tract viruses.
4. Use according to claim 1, of biapenem in the preparation of a medicament for inhibiting and/or killing bovine enterovirus.
5. A pharmaceutical composition for preventing and/or treating bovine intestinal viral infection, wherein the pharmaceutical composition comprises biapenem and 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 biapenem is in a concentration of no less than 60 μ M.
7. The pharmaceutical composition of claim 5, wherein the other pharmaceutically active ingredient comprises a substance that inhibits and/or kills bovine enteroviruses or assists in inhibiting and/or killing bovine enteroviruses.
8. A pharmaceutical composition according to claim 5, wherein the other non-pharmaceutically active ingredients comprise pharmaceutically acceptable carriers, excipients and/or diluents.
9. The pharmaceutical composition of claim 5, wherein the administration form comprises a solid oral formulation, a liquid oral formulation, or an injection.
10. The pharmaceutical composition of claim 9, wherein the administration forms comprise tablets, dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, capsules, sugar-coated agents, granules, dry powders, oral solutions, small water-injection solutions for injection, freeze-dried powder injections for injection, large infusion solutions and small infusion solutions.
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