阅读说明：本技术 格拉非宁和/或其可药用衍生物在制备抗肠道病毒药物中的应用 (Application of glatiramer and/or pharmaceutically acceptable derivatives thereof in preparation of anti-enterovirus medicines ) 是由 张定宇 黄朝林 杨庆雨 于 2021-09-06 设计创作，主要内容包括：本发明提供了格拉非宁和/或其可药用衍生物在制备抗肠道病毒药物中的应用,本发明证实格拉非宁对于肠道病毒例如EV71、柯萨奇病毒等具有较强的抗病毒活性,可以显著的抑制肠道病毒在宿主细胞RD上产生的细胞病变效应(CPE),增强细胞存活率；能抑制病毒的复制在细胞水平表现出较强的抗病毒效果,具有明显的技术效果。本发明结果表明化合物格拉非宁有潜力制备抗肠道病毒感染的特异治疗药物,具有较好的临床应用前景。(The invention provides application of glatiramer and/or medicinal derivatives thereof in preparation of anti-enterovirus medicaments, and the application proves that the glatiramer has stronger antiviral activity on enteroviruses such as EV71, Coxsackie virus and the like, can obviously inhibit cytopathic effect (CPE) generated by the enteroviruses on host cells RD, and enhances the cell survival rate; can inhibit the replication of viruses and show stronger antiviral effect at the cellular level, and has obvious technical effect. The result of the invention shows that the compound glafenin has potential for preparing specific treatment medicines for resisting enterovirus infection, and has better clinical application prospect.)

1. Use of glatiramer and/or a pharmaceutically acceptable derivative thereof for the preparation of an anti-enterovirus medicament.

2. The use of claim 1, wherein the enterovirus comprises at least one of enterovirus type 71, coxsackievirus type B3, and coxsackievirus type B4.

3. The use according to claim 1, wherein the pharmaceutically acceptable derivative of glatiramer comprises: a pharmaceutically acceptable salt of glatiramer or a pharmaceutically acceptable acid addition salt of glatiramer.

4. The use according to claim 3, wherein the pharmaceutically acceptable salt of glatiramer comprises glatiramer hydrochloride.

5. The use of claim 1, wherein the anti-enterovirus medicament further comprises pharmaceutically acceptable excipients and carriers.

6. The use of claim 5, wherein the excipient comprises at least one of a filler, a disintegrant, a binder, an excipient, a diluent, a lubricant, a sweetener, or a coloring agent.

7. The use of claim 1, wherein the dosage form of the anti-enterovirus drug comprises at least one of granules, tablets, pills, capsules, injections or dispersions.

8. The use according to claim 7, wherein the anti-viral means of the anti-enteroviral drug comprises: inhibit enterovirus intracellular nucleic acid replication, viral protein expression and infection.

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of glatiramer and/or medicinal derivatives thereof in preparation of anti-enterovirus medicines.

Background

Enterovirus 71 (Enterovirus, EV71) is a member of the genus Enterovirus of the family Picornaviridae (Picornaviridae) and is a non-enveloped virus, the virion is in the shape of a regular icosahedral sphere with a diameter of about 30nm, the genome of the virion is a single positive RNA strand, the length of the virion is about 7.5kb, and the virion is one of the most main pathogens causing hand-foot-and-mouth disease of infants. The children with hand-foot-mouth disease are sometimes accompanied with serious central nervous system complications, including aseptic meningitis, encephalitis, poliomyelitis-like paralysis, neurogenic cardiopulmonary failure and the like, and even death. Humans are the only natural hosts of enteroviruses, which spread by close contact between humans (through fingers, cutlery and food). The infected person has viruses in the pharynx and the intestine, and the time for expelling the viruses from the feces is long and can last for several weeks. Fecal-oral is the primary route of transmission. Occasionally, it may be transmitted by droplets. The disease is most frequently found in children under 5 years old, and the incidence rate is highest in children under 1-2 years old.

Enterovirus 71 ((Enterovirus, EV 71)) belongs to a member of Enterovirus (Enterovirus) of Picornaviridae (Picornaviridae), is one of the most main pathogens causing infantile hand-foot-and-mouth diseases, sometimes is accompanied by serious central nervous system complications including aseptic meningitis, brainstem encephalitis, autonomic nervous disorders, pulmonary edema and the like, and even causes death. since the first report in 1969, EV71 infectious diseases have been frequently outbreaks and epidemics worldwide, and are severe in Asia-Pacific regions, particularly China at present, viral disease prevention and treatment mainly depend on vaccines and medicaments, related vaccines are marketed in 2015, no specific data can support marketed vaccines to protect enteroviruses of other serotypes at present, treatment methods for EV71 virus infection are quite limited, and main treatment methods are symptomatic support treatment and broad-spectrum antiviral therapy, it has limited curative effect, large individual difference and difficult popularization. Therefore, the development of related antiviral drugs is a key direction for overcoming the virus, and the development of specific and effective anti-EV 71 drugs is imperative.

Therefore, there is a need to develop a new enterovirus infectious disease drug.

Disclosure of Invention

The application finds that the glatiramer has a strong inhibiting effect on enteroviruses for the first time, and provides a direction for treating and preventing enterovirus infectious diseases.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides application of glatiramer and/or pharmaceutically acceptable derivatives thereof in preparing an anti-enterovirus medicament.

Further, the enterovirus includes at least one of enterovirus type 71, coxsackievirus type B3 and coxsackievirus type B4.

Further, the pharmaceutically acceptable derivatives of glatiramer include: a pharmaceutically acceptable salt of glatiramer, a pharmaceutically acceptable acid addition salt of glatiramer.

Further, in the pharmaceutically acceptable acid addition salt of glatiramer, the pharmaceutically acceptable acid may be selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, trifluoroacetic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, tartaric acid, maleic acid, citric acid, ascorbic acid, oxalic acid, methanesulfonic acid, benzenesulfonic acid, and camphoric acid.

Further, the pharmaceutically acceptable salt of glatiramer comprises at least one of an inorganic salt and an organic salt.

Further, the inorganic salt comprises one of hydrochloride, hydrobromide, sulfate, nitrate and phosphate; the organic salt comprises one of mesylate, maleate, tartrate, succinate, acetate, trifluoroacetate, fumarate, citrate, benzene sulfonate, benzoate, benzene sulfonate, lactate and malate. The embodiment of the invention specifically selects hydrochloride of glatiramer.

Furthermore, the anti-enterovirus medicine also comprises pharmaceutically acceptable auxiliary materials and carriers.

Further, the adjuvant includes at least one of a filler, a disintegrant, a binder, an excipient, a diluent, a lubricant, a sweetener, or a coloring agent.

Further, the dosage form of the medicament comprises at least one of granules, tablets, pills, capsules, injections or dispersing agents.

Further, the antiviral means of the drug comprises: inhibit enterovirus intracellular nucleic acid replication, viral protein expression and infection.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

the application of the glatiramer and/or the medicinal derivatives thereof in preparing the anti-enterovirus medicaments provided by the invention is that the glatiramer is always used as a compound for targeted treatment of breast cancer, and the new application of the glatiramer in preparing the anti-EV 71 virus medicaments is found for the first time. Test results show that the glatiramer has stronger antiviral activity on enteroviruses such as EV71 and CVB3, can remarkably inhibit cytopathic effect (CPE) generated by the enteroviruses such as EV71 and CVB3 on host cells RD, and enhances the cell survival rate; can inhibit the replication of viruses and show stronger antiviral effect at the cellular level, and has obvious technical effect. The result of the invention shows that the compound glafenin has potential for preparing specific treatment medicines for resisting enterovirus infection, and has better clinical application prospect.

Meanwhile, the synthesis process of the glafenin small molecular compound is simple and is easy for large-scale production and popularization; the antiviral activity of glafenin is not reported, and the glafenin has a certain guiding effect on the development of the activity of resisting enteroviruses such as EV71 and CVB 3; the anti-enterovirus medicine is searched from the compounds with similar structures, the action target of the anti-enterovirus medicine is easy to be found through the structure-activity relationship research, and certain reference significance is provided for further medicine development.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

FIG. 1 is a graph showing the analysis of the results of RD cytotoxicity test of the effect of glatiramer on EV71 virus in example 1 of the present invention;

FIG. 2 is a graph showing the effect of glatiramer in example 2 of the present invention on the cytopathic effect (CPE) produced by EV71 virus in host RD cells;

FIG. 3 is a graph showing the analysis of the results of the measurement of antiviral activity of glatiramer at different concentrations in example 3 of the present invention;

FIG. 4 is a graph showing the analysis of the results of detecting the effect of glatiramer on the propagation of EV71 virus in example 4 of the present invention;

FIG. 5 is a graph showing the analysis of the test results of the effect of glatiramer on EV71 replication in example 5 of the present invention;

FIG. 6 is a graph showing the effect of Gradepin on the replication of CVB4 in example 6 of the present invention.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, 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. If there is a conflict, the present specification will control.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be obtained by an existing method.

In order to solve the technical problems, the general idea of the embodiment of the application is as follows:

the Glafenine (Glafenane) is 2, 3-dihydroxypropyl 2- ((7-chloroquinolin-4-yl) amino) benzoic acid, is a non-narcotic analgesic and a non-steroidal anti-inflammatory drug, is used for treating pain caused by inflammation for a long time and has a good effect.

The structural formula of Glafenine (glafeneine) is as follows:

the inventor of the application combines the virus RNA level analysis, the titer determination and the MTT determination cell survival rate detection method to perform an anti-enterovirus activity research experiment on the glatiramer small molecular compound, and the data analysis in the embodiment adopts Graphpad software to perform statistical analysis, so that the glatiramer has a strong inhibition effect on enteroviruses. The enterovirus includes at least one of enterovirus 71, coxsackievirus B3 and coxsackievirus B4.

Specifically, the method comprises the following steps: glatiramer was evaluated for its inhibitory activity against enteroviruses and tested for activity by standard antiviral activity test methods. Through a large number of biological experiments, the glatiramer is found to have good activity of inhibiting replication of enteroviruses, and particularly can inhibit the replication of nucleic acid and the expression of virus protein in cells of the enteroviruses, so that the proliferation of the viruses is inhibited.

Experimental results show that the glatiramer has stronger antiviral activity on the enterovirus 71, can obviously inhibit cytopathic effect (CPE) generated by the enterovirus 71 on a host cell RD, and enhances the cell survival rate; can inhibit replication of enterovirus 71, has strong antiviral effect at cell level, and has obvious technical effect. The glatiramer has stronger antiviral activity on enterovirus 71, shows stronger antiviral effect on the cellular level, and has 100 percent of inhibition rate on the enterovirus when the glatiramer is 20 mu M.

In addition, under different concentration conditions, the glafenin has obvious inhibition effect on replication of coxsackie virus type 4 (CVB 4).

In conclusion, the results of the invention show that the compound glafenin has potential for preparing specific treatment medicines for resisting enterovirus infection, and has better clinical application prospect.

Therefore, the invention provides an application of glatiramer in preparing an anti-enterovirus medicament, wherein the application refers to that the glatiramer is added with pharmaceutically acceptable auxiliary materials and carriers and is used for preparing an anti-enterovirus preparation, the auxiliary materials comprise at least one of a filling agent, a disintegrating agent, a binding agent, an excipient, a diluent, a lubricant, a sweetening agent or a coloring agent, and different auxiliary materials are selected according to the requirements of medicament dosage forms. The preparation is granule, tablet, pill, capsule, injection or dispersant.

It can be understood that the glafenin is used as a lead compound for further structural optimization, and the glafenin is used for preparing a medicament for treating enterovirus infectious diseases and also belongs to the protection scope of the invention. Wherein the pharmaceutically acceptable derivatives of glatiramer include pharmaceutically acceptable salts of glatiramer and pharmaceutically acceptable acid addition salts of glatiramer.

The use of glatiramer in the present application for the preparation of anti-enterovirus medicaments will be described in detail below with reference to examples and experimental data.

Example 1 cytotoxicity assay for glatiramer

In RD cells, glatiramer cytotoxicity was examined. RD cells were seeded in 96 wells at 37 ℃ with 5% CO₂Culturing in an incubator for 12-16h, discarding cell culture solution, adding cell maintenance solution containing glatiramer with different concentrations, culturing, repeating 3 wells for each group, and adding PBS with same amount for control group. After 48h of action, staining with MTT and detecting OD₄₉₂nM, cell viability was analyzed.

And (4) analyzing results: as shown in FIG. 1, the Prism7 software calculates the half-toxic concentration (CC) of the drug to the cell₅₀) CC of Glafeine₅₀It was 329310 nM. In the subsequent examples, glatiramer was used at a maximum concentration of 20000nM, within a safe and non-toxic range.

Example 2 detection of antiviral Activity of glatiramer against EV71

RD cells were plated in 96-well cell culture plates at 37 ℃ with 5% CO₂After the culture in an incubator for 12-16h, 100TCID₅₀The EV71 virus liquid infects cells for 2h, cell maintenance liquid containing different concentrations of test glatiramer is added respectively to continue culturing for about 48h, and PBS with the same volume is added as negative control. When the virus control wells showed CPE lesions in about 90%, cytopathic effect (CPE) was observed under a microscope.

And (4) analyzing results: inhibition of RD cell CPE effect by EV71 by glafenin As shown in FIG. 2, RD cells infected with EV71 become round and are separated from cell plate walls, and treatment with different concentrations of test compounds has a significant inhibitory effect on the pathological effect.

Example 3 assay for inhibition of EV71 proliferation Activity by glatiramer

RD cells were seeded in 24-well cell culture plates at 37 ℃ with 5% CO₂Culturing in incubator for 12-16h with 100TCID₅₀2h after infection with EV71 virus solution, treatment was performed with cell maintenance solutions containing varying concentrations of experimental glafenin, and the same volume of PBS was added as the negative control. After 24h of treatment, pass TCID₅₀Titer assays measure the effect of inhibiting viral proliferation.

And (4) analyzing results: the detection result is shown in fig. 3, under different concentration conditions, the compound glafenin has a significant inhibition effect on EV71, and the TCID of the virus can be obviously reduced in a drug treatment group compared with a control group₅₀The value of (c). The glatiramer has stronger antiviral activity on enteroviruses, shows stronger antiviral effect on a cellular level, and has 100 percent of inhibition rate on EV71 virus at 20 mu M of the glatiramer.

Example 4 Effect of Grafenan on EV71 replication

The experiment detects that glatiramer has better inhibitory activity to EV 71:

RD cells were seeded in 24-well cell culture plates at 37 ℃ with 5% CO₂Culturing in incubator for 12-16h with 100TCID₅₀2h after infection with EV71 virus, treatment was performed with test compound (glafenin) containing different concentrations, and the same volume of PBS was added as the negative control. After 16h of treatment, collecting the cultured cells to extract total RNA, and detecting the effect of the glatiramer on inhibiting virus replication by a real-time fluorescent quantitative PCR (qPCR) method.

And (4) analyzing results: the detection result is shown in fig. 4, which shows the influence of glatiramer on EV71 replication, and glatiramer has a significant inhibitory effect on the expression of VP1 gene of EV71 virus under different concentration conditions.

Example 5 inhibition of EV71 replication by Grafenin

The experiment detects that glatiramer has better inhibitory activity to EV 71:

RD cells were seeded in 24-well cell culture plates at 37 ℃ with 5% CO₂Culturing in incubator for 12-16h with 100TCID₅₀2h after infection with EV71 virus solution, treatment was performed with test compound (glafenin) containing different concentrations, and negative controls were added to the same volume of maintenance solution without drug. After 16h of treatment, total RNA is extracted from the cultured cells, and the expression of the EV71 virus VP1 is detected by a real-time fluorescent quantitative PCR (qPCR) method.

And (4) analyzing results: as shown in FIG. 5, the Prism7 software calculated the half inhibitory Concentration (Concentration for 50% of maximum inhibition, IC) of the drug against viral replication₅₀) IC of glafenin₅₀It was 191.1 nM.

Example 6 Effect of Grafenan on replication of CVB4

Hela cells were seeded in 12-well plate cell culture plates at 37 ℃ in 5% CO₂Culturing in incubator for 12-16h with 100TCID₅₀After 2h infection with CVB4 virus solutions, the samples were treated with test compounds (glafenin) at different concentrations, and the negative control was supplemented with the same volume of glafenin-free cell maintenance solution. After 17h of treatment, the effect of the Greenin on inhibiting the CVB4 virus replication is detected by a real-time fluorescent quantitative PCR (qPCR) method.

And (4) analyzing results: the detection result is shown in fig. 6, and under different concentration conditions, glatiramer has a remarkable inhibition effect on the replication of CVB 4.

In conclusion, the glatiramer has remarkable activity of inhibiting EV71 and CVB4 replication, can strongly inhibit RD cytopathic effect caused by EV71 viruses, and enhances the cell survival rate; inhibiting the replication of the viral RNA level and having the potential to further develop and prepare a medicament which is clinically effective against the enterovirus infection.

Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.