CN110179785B

CN110179785B - Application of widmanone in preparation of medicine for treating or preventing hand-foot-and-mouth disease

Info

Publication number: CN110179785B
Application number: CN201910403234.3A
Authority: CN
Inventors: 肖伟; 曹泽彧; 徐君; 许治良; 曹亮; 王振中
Original assignee: Jiangsu Kanion Pharmaceutical Co Ltd
Current assignee: Jiangsu Kanion Pharmaceutical Co Ltd
Priority date: 2019-05-15
Filing date: 2019-05-15
Publication date: 2021-06-29
Anticipated expiration: 2039-05-15
Also published as: CN110179785A

Abstract

The invention provides application of ketoferulone or solvate or pharmaceutically acceptable salt thereof in preparing a medicament for treating or preventing hand-foot-and-mouth disease. The widmanone or pharmaceutically acceptable salt thereof has small toxic and side effects, and can effectively inhibit cytopathic effect caused by hand-foot-and-mouth disease virus, inhibit virus replication, reduce virus load, reduce or eliminate death of infected mice, prolong survival experiment, suppress inflammation and the like. The compound or the pharmaceutically acceptable salt thereof can be further developed into a medicine for treating or preventing hand-foot-and-mouth diseases, has various dosage forms and administration modes, and has wide application prospects.

Description

Application of widmanone in preparation of medicine for treating or preventing hand-foot-and-mouth disease

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of ketoferulone in preparation of a medicine for treating or preventing hand-foot-and-mouth disease.

Background

The hand-foot-and-mouth disease is a child infectious disease caused by viruses and is a third infectious disease in China. The disease mainly infects infants of 0-6 years old, and is most common when children of 2-3 years old infect, herpes or ulcer is caused at the extremities and oral cavity of the sick children in the early stage of the hand-foot-and-mouth disease, and the sick children are mostly cured within 1-2 weeks. However, in a few cases, the disease progresses rapidly, meningitis, aseptic encephalitis, brainstem encephalitis, encephalomyelitis, pulmonary edema, respiratory and blood circulation disorders and the like appear in about 1 to 5 days of the disease, and in a few cases, the disease is critical and can cause death, and sequelae can be remained in survival cases.

Although the vaccine for the hand-foot-and-mouth disease is already approved to be marketed, the safety and the effectiveness of the vaccine are still required to be further clinically verified; on the other hand, no specific medicine for directly targeting the viruses causing the hand-foot-and-mouth disease exists clinically at present, and a symptomatic treatment strategy is mainly adopted clinically; the broad-spectrum antiviral drug ribavirin has the risks of teratogenesis and growth inhibition when applied to infants. Therefore, there is still a need to develop new compounds to remedy the deficiencies of the prior art in this respect.

Disclosure of Invention

The invention aims to make up the defects of the prior art and provides the application of the active ingredient extracted from the liquorice in preparing the medicine for treating or preventing the hand-foot-and-mouth disease, thereby providing a small molecular compound for clinically treating or preventing the hand-foot-and-mouth disease.

The purpose of the invention is realized by the following technical scheme:

the invention provides application of widmanone or solvate or pharmaceutically acceptable salt thereof in preparing a medicament for treating or preventing hand-foot-and-mouth disease.

Further, in the application of the invention, the hand-foot-and-mouth disease comprises hand-foot-and-mouth disease caused by enterovirus 71, coxsackievirus A group 16, coxsackievirus A group 4, coxsackievirus A group 5, coxsackievirus A group 7, coxsackievirus A group 9, coxsackievirus A group 10, coxsackievirus B group 2, 5 or echovirus.

Preferably, in the application of the present invention, the hand-foot-and-mouth disease is a hand-foot-and-mouth disease caused by enterovirus 71 or coxsackievirus group a 16.

For drugs for treating or preventing hand-foot-and-mouth disease, the compound of the present invention may be used as it is or in the form of a pharmaceutical composition, when used as a drug. The pharmaceutical composition comprises 0.1-99%, preferably 0.5-90% of the compound of the invention, the rest can be other active ingredients with similar or synergistic effect, and can also or further comprise pharmaceutically acceptable carriers and/or excipients which are nontoxic and inert to human and animals.

Furthermore, the medicament is a clinically allowable dosage form prepared by taking the ketoferyl or the solvate or the pharmaceutically acceptable salt thereof as an active ingredient and pharmaceutically acceptable auxiliary materials.

Specifically, the dosage form comprises various dosage forms such as tablets, capsules, granules, dispersing agents, injections or sprays. The administration modes of the ketofern or the salt thereof are various, and the ketofern or the salt thereof can be administered by injection or oral administration.

In the present invention, the pharmaceutically acceptable salt includes, but is not limited to, hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate of the compound.

The pharmaceutically acceptable auxiliary materials are one or more of solid, semi-solid and liquid diluents, fillers and pharmaceutical product auxiliaries. The medicament of the present invention is used in the form of a dose per unit body weight. The medicine of the invention is prepared into various dosage forms, such as liquid preparations (injection, suspension, emulsion, solution, syrup and the like), solid preparations (tablets, capsules, granules, medicinal granules and the like), sprays, aerosols and the like by adopting a method which is generally recognized in the fields of pharmacy and food. The medicine can be used for corresponding prevention or treatment through administration routes such as injection (intravenous injection, intravenous drip, intramuscular injection, intraperitoneal injection and subcutaneous injection), oral administration, sublingual administration, mucosal dialysis and the like.

The invention firstly detects the toxicity condition of the ferulone to Vero cells (African green monkey kidney cells) by an MTS method; secondly, the protective effect of the ketoferulone on Vero cells infected by enterovirus 71 and coxsackievirus A group 16 is researched, and the virus inhibition rate is in a dose-dependent effect; thirdly, the influence of the drug on the virus nucleic acid replication is detected by utilizing PCR, and the result shows that the ketoferyl can intervene in the early event of virus infection, inhibit virus replication, reduce the virus load in host cells and promote the virus to turn negative; fourthly, the study finds that the ketoferyl has broad-spectrum antiviral effect; finally, the fact that the welferone can prolong the survival time of the infected mice, improve the survival rate, inhibit the release of inflammatory factors of the infected mice and stabilize inflammation is discovered through various administration modes. And through comprehensive experiment result analysis, the fact that the widmanone can be used for preventing and treating the hand-foot-and-mouth virus infection is determined.

Compared with the prior art, the invention has the following advantages and effects:

1. widmanone is a natural small molecule compound, TC50 is about 20.2 μmol/L;

2. the widmanone can inhibit EV71 and CoxA16 virus infection in a dose-dependent manner and has a dose-dependent effect;

3. intervening in early virus infection events, inhibiting virus replication, reducing EV71 and CoxA16 virus loads in infected cells, and promoting virus to turn negative;

4. the human hand-foot-and-mouth disease virus inhibitor has broad spectrum in the aspect of resisting hand-foot-and-mouth disease viruses, and has obvious inhibiting effects on viruses of coxsackie virus A groups 4, 5,7, 9 and 10, coxsackie virus B groups 2 and 5 and echovirus which can cause hand-foot-and-mouth disease symptoms;

5. relieving symptoms caused by viruses, relieving or eliminating inflammation and death caused by virus infection, and the like; the survival time of the mice infected with the virus is prolonged, and the survival rate of the mice is improved;

6. the release of the mouse inflammatory factors caused by virus infection is inhibited;

7. the administration mode is various, and the injection administration or the oral administration can be carried out.

In conclusion, the widmanone can be developed as a new medicine for treating or preventing the hand-foot-and-mouth disease, and a new way and means are provided for treating or preventing the hand-foot-and-mouth disease.

Detailed Description

According to the 2015 pharmacopoeia, the traditional Chinese medicine licorice is the dried root and rhizome of licorice (Glycyrrhiza uralensis Fisch), Glycyrrhiza inflata Bat or Glycyrrhiza glabra L of Leguminosae. The widmanone is a natural flavonoid compound extracted from liquorice, and has the following structural formula:

according to the systematic nomenclature, the chemical name of the Widmanone is 5,7, 4' -trihydroxy-6-isopentenyl isoflavone (Wigneone, CAS number: 51225-30-0), the beneficial effects of the Widmanone are less reported at present, and the application of the Widmanone in resisting hand-foot-and-mouth disease is not reported in documents.

The model is a carrier of compound activity and action mechanism, and hand-foot-and-mouth virus infection can induce a plurality of apoptosis, including human malignant embryo rhabdomyoma cells (RD), Jurkat cells, neuroepithelial tumor cells (SK-N-MC), human neuroblastoma cells (SK-N-SH), glioblastoma cells (SF268), African green monkey kidney cells (Vero), human microvascular endothelial cells, HeLa cells and the like. In animal models, 1-7 day old mice are sensitive to viruses and are commonly used as animal models. 3-5 days after virus inoculation of the mice, symptoms such as milk refusal and weight loss appear, obvious symptoms such as catalepsy appear in limbs 6-8 days, and the mice self-heal or die after 9-14 days according to different virus inoculation amounts.

The following examples are provided to further illustrate the essence of the present invention, but are not intended to limit the invention thereto:

example 1 Vero cytotoxicity of Widmanone

1. Materials and methods

1.1 cells and culture methods

The Vero cell was used as a cell model (provided by institute of microbial epidemiology, the same applies below) and DMEM medium (from Jiangsu Kayji Biotech Co., Ltd.) containing 10% fetal bovine serum (from Gibco Co.) was placed at 37 ℃ and 5% CO₂Culturing in an incubator, and when the cells grow to 90% density and then are passaged, the cell passage ratio is 1/3-1/4.

1.2 reagents

MTS cell proliferation quantitative detection kit (purchased from Promega corporation); widmanone (available from Shanghai leaf Biotech Co., Ltd., the same applies hereinafter).

1.3 instruments

Microplate reader (available from Perkin Elmer, model: EnSpire, supra); an inverted microscope (from Olympus, model: CKX41, infra); carbon dioxide incubator (available from Forma Scientific, model: Forma Steri-Cycle 371, supra); a biological safety cabinet (purchased from Shanghai Jiejia air purification technology Co., Ltd., the same below).

1.4 design of the experiment

Vero cells cultured to 90% density were taken, digested with 0.25% trypsin, and then adjusted to 1X 10 cell density in DMEM medium containing 10% fetal bovine serum⁵each.mL^-1Each well was seeded with 100 μ L of cell suspension in 96-well plates. Adding 200 μ L of DMEM medium containing medicine with different concentrations (containing 2 times of continuous gradient dilution 5 gradients from 400 μmol/L of ketoferulon concentration, total 6 administration concentrations, and final fetal calf serum concentration of 2.5%) as sample group, and setting control group (containing no tested medicine in culture medium), wherein each treatment group contains 3 multiple wells, and CO₂After culturing for 72h in the incubator, quantitative detection of cell proliferation is carried out, and the absorbance (A) of each well is detected at 490nm by a microplate reader. With [ A (sample group)/A (control group)]The x 100% was taken as the cell viability of each group and the median Toxic Concentration (TC) of the samples to Vero cells was calculated₅₀)。

1.5 statistical treatment

All data were processed with SPSS 19.0 statistical software, experimental data are expressed as mean. + -. standard error, and data comparisons between groups were performed using one-way analysis of variance. P <0.05 is statistically significant for differences.

2. Results

Table 1 influence of weiterketone on Vero cell viability (% mean ± sem, n ═ 3)

Note: indicates that each sample group data had significant differences at a level of P <0.05 compared to the control group.

The study defines the toxicity condition of the witness ketone on Vero cells, as shown in Table 1, the toxicity effect of the witness ketone on Vero cells is in an obvious dose-effect relationship, the cell activity is gradually reduced along with the increase of the administration concentration, the toxicity of the witness ketone on the cells is gradually increased, and the toxicity of the witness ketone on the Vero cells TC is calculated according to the data in Table 1₅₀＝20.2μmol/L。

Example 2 protection of West's ketone against EV71 and CoxA16 virus infection of Vero cells and inhibition of viral replication

1. Materials and methods

1.1 cells and culture methods

Adopting Vero cell as cell model, adopting DMEM medium containing 10% fetal calf serum, placing at 37 deg.C and 5% CO₂Culturing in an incubator, and when the cells grow to 90% density and then are passaged, the cell passage ratio is 1/3-1/4.

1.2 Virus strains

EV71 virus BJ09/07 strain, GenBank accession number JQ 319054.1; CoxA16 virus TS10/08 strain, GenBank accession number JX068829, is provided by the institute of microbial epidemics, military medical academy of sciences, the same below. Half cytopathic dose (TCID) of EV71 virus was measured just before use₅₀) Is 10⁸/mL，CoxA16 TCID₅₀Is 10^8.5/mL。

1.3 reagents

MTS cell proliferation quantitative detection kit; ketoferyl; total RNA extraction kit (TRIzol method) (purchased from Invitrogen, usa); reverse transcription kit Prime Script^TM(available from precious bioengineering (Dalian) Co., Ltd.); enterovirus 71 and Coxsackie virus 16 nucleic acid combined assay kit (fluorescence PCR method) (purchased from Biotech GmbH, Inc., of Yangtze, Shanghai).

1.4 instruments

A microplate reader; inverting the microscope; a carbon dioxide incubator; a biological safety cabinet; a general PCR instrument (purchased from ABI, Inc., model: 2720); fluorescent quantitative PCR instrument (purchased from ABI, Inc.: model: StepOne Plus)^TM)。

1.5 design of the experiment

1.5.1 taking and culturing to 90% densityVero cells, 0.25% pancreatin, cell density adjusted to 1X 10 in DMEM medium containing 10% fetal bovine serum⁵each.mL^-1Each well was seeded with 100 μ L of cell suspension in 96-well plates. Add 50. mu.L CoxA16 or EV71 virus suspension (100 TCID)₅₀) Adding 50 μ L of DMEM medium containing drug at different concentrations (the concentration of ketoferulon is 2 times of the gradient from 20 μmol/L and is serially diluted for 5 concentrations, the total concentration is 6 administration concentrations, the final concentration of fetal calf serum is 2.5%), setting control group (containing neither virus nor drug) and model group (containing only virus), each treatment group contains 3 multiple wells, CO₂After culturing for 72h in the incubator, quantitative detection of cell proliferation is carried out, and the absorbance (A) of each well is detected at 490nm by a microplate reader. With [ A (sample group) -A (model group)]/[ A (control group) -A (model group)]X100% inhibition for each group, half maximal Effect Concentration (EC) of samples on CoxA16 and EV71 viruses was calculated₅₀) And selection index (SI ═ TC)₅₀/EC₅₀)。

1.5.2 study of viral load of infected cells: vero cells cultured to 90% density are taken, 0.25% pancreatin is digested, and the cell density is adjusted to 2 x 10 by using DMEM medium containing 10% fetal calf serum⁵each.mL^-11mL of the solution was inoculated into 6-well plates per well, and 1mL of the solution containing 2000TCID was added₅₀DMEM medium of CoxA16 or EV71 virus and drug Widmanetone (final concentration of 10 mu mol/L), and control group (containing neither virus nor drug) and model group (containing only virus) were set, and each group was treated with 2 replicate wells, CO₂After culturing for 8h in an incubator, RNA is extracted, cDNA is synthesized and the virus load is detected respectively according to the instruction of the kit.

1.6 statistical treatment

2. Results

2.1 inhibition of EV71 and CoxA16 viruses by Widmanone

Table 2 inhibition of EV71 and CoxA16 viruses by widmanone (%, mean ± sem, n ═ 3)

The study discusses the inhibition effect of the widescreenone on EV71 and CoxA16 viruses, as shown in Table 2, the inhibition effect of the widescreenone on EV71 and CoxA16 viruses is dose-dependent, the inhibition rate is stable after being gradually increased to the maximum value along with the increase of the administration dose, and the virus inhibition effect at the concentration of 10 mu mol/L is the best. Wescone inhibition of EV71 virus half-EC as calculated from the data in Table 2₅₀5.3 μ M, SI 3.8, CoxA16 virus EC suppression₅₀＝3.9μM，SI＝5.2。

2.2 inhibition of EV71 and CoxA16 Virus replication by Widmantone

Table 3 inhibition of EV71 and CoxA16 virus replication by widmanone (mean ± sem, n ═ 6)

Note: ND means not detected; indicates that each group had significant differences in P <0.05 levels compared to the control group

The study discusses the inhibition effect of the widescreenone on EV71 and CoxA16 viruses, as shown in Table 3, 10 mu mol/L of widescreenone can significantly inhibit the replication of EV71 and CoxA16 viruses in cells, and reduce the virus load, which indicates that the widescreenone has the effect of promoting the virus to turn negative.

Example 3 broad-spectrum study of the resistance of widmanone to hand-foot-and-mouth virus

1. Materials and methods

1.1 cells and culture methods

The Vero cell is taken as a cell model, and the cell with the passage number of 130-145 is taken for experiment. The cells were cultured in DMEM medium containing 10% fetal bovine serum at 37 deg.C under 5% CO₂Culturing in an incubator, and when the cells grow to 90% density, carrying out passage at a passage ratio of 1/3-1/4.

1.2 Virus strains

Coxsackievirus group a types 4, 5,7, 9 and 10 (CoxA4, CoxA5, CoxA7, CoxA9 and CoxA10), coxsackievirus group B types 2 and 5 (CoxB2 and CoxB5) and Echovirus (ECHO). Determination of the TCID of the respective viruses just before use₅₀Are respectively 10⁸、10^7.5、10⁷、10⁶、10^8.5、10^7.5、10⁷And 10^7.5mL, provided by the institute for Wuhan Virus, Chinese academy.

1.3 reagents

MTS cell proliferation quantitative detection kit; and (3) ketoferyl.

1.4 instruments

A microplate reader; inverting the microscope; a carbon dioxide incubator; a biological safety cabinet.

1.5 design of the experiment

Vero cells cultured to 90% density were taken, digested with 0.25% trypsin, and then adjusted to 1X 10 cell density in DMEM medium containing 10% fetal bovine serum⁵each.mL^-1Each well was inoculated with 100. mu.L of cell suspension in a 96-well plate, and 50. mu.L of virus suspension (100 TCID) was added₅₀) Adding 50 μ L of DMEM medium containing drug (final concentration of Widmanone is 250 μ g/mL, final concentration of fetal calf serum is 2.5%), setting control group (containing neither virus nor drug) and model group (containing only virus) at the same time, each treatment group contains 3 multiple wells, and CO₂After culturing for 72h in the incubator, quantitative detection of cell proliferation is carried out, and the absorbance (A) of each well is detected at 490nm by a microplate reader. With [ A (sample group) -A (model group)]/[ A (control group) -A (model group)]X 100% as inhibition for each group, EC of West Ketone for each strain was calculated₅₀And SI.

1.6 statistical treatment

All data were processed with SPSS 19.0 statistical software.

2. Results

TABLE 4 Wesetone EC against various hand-foot-and-mouth viruses₅₀And SI

As shown in Table 4, 10 mu mol/L of Widmanone has obvious inhibitory effect on various viruses which can cause hand-foot-and-mouth disease symptoms, and EC₅₀From 3.46 to 5.49. mu. mol/L. This example shows that widmanone has a broad spectrum in inhibiting hand-foot-and-mouth virus.

Example 4 West ketone administration by injection prolongs survival time of virus infected mice, inhibits death of mice and release of inflammatory factors

1. Materials and methods

1.1 animals

5 day old ICR mice (Mus musculus) purchased from Experimental animals, Inc. of Wei Tong Li Hua, Beijing.

1.2 Virus strains

EV71 virus BJ09/07 strain, GenBank accession number JQ319054.1, CoxA16 virus TS10/08 strain, GenBank accession number JX 068829. Half cytopathic dose of EV71 virus (TCID50) was determined to be 10 just before use⁸CoxA16 Virus TCID/mL₅₀Is 10^8.5/mL。

1.3 reagents and consumables

Lupin widenin (Lupiwighteone, abbreviation: LWT, CAS No. 104691-86-3, available from shanghai-source leaf biotechnology ltd.), ribavirin injection (abbreviation: RBV, available from lukangxin pharmaceutical industry ltd.), normal saline for injection, disposable sterile syringe. The cytokine interleukin-6 (IL-6) ELISA assay kit (from eBioscience), the tumor necrosis factor-alpha (TNF-alpha) ELISA assay kit (from eBioscience), and the monocyte chemoattractant factor-1 (MCP-1) ELISA assay kit (from eBioscience).

1.4 Experimental methods

(1) Preparing a virus suspension: according to the virus TCID₅₀Value, two diseases are treated by DMEM medium before useRespectively diluting the toxin to 10⁷TCID₅₀/mL。

(2) Preparing a liquid medicine: dissolving the widmantone and the lupin widmantone in 5% (w/v) sodium bicarbonate, diluting with normal saline for injection to the required dosage, and filtering with 0.22 μm microporous membrane. The ribavirin injection is shaken well before use, and is diluted to the required dosage (10mg/kg) by using normal saline.

(3) Grouping, modeling and dosing: the 5-day-old mice were grouped, molded, and dosed according to table 5. Grouping: the mice were randomly divided into 13 groups: blank control group, EV71 model group and CoxA16 model group, 10mg/kg lupin widetrone treatment EV71 group (EV71+ LWT), 10mg/kg lupin widetrone treatment CoxA16 group (CoxA16+ LWT), widetrone high, medium and low dose groups (20, 10 and 5mg/kg), ribavirin group (RBV)10mg/kg, each group containing 13 mice. Molding: mice in each group were injected intraperitoneally with either EV71 or CoxA16 virus suspension, 0.1mL each, and the blank control group was replaced with saline injections. Administration: after completion of the molding, each mouse was administered by intraperitoneal injection (the blank control group, EV71 model group, and CoxA16 model group were replaced with saline injection), 0.1mL each time, 1 time daily, and 13 consecutive days. On day 6 after model building, 3 mice were taken from each group, and after eye-picking, serum was taken to determine the contents of cytokines IL-6, TNF- α and MCP-1 (these 3 mice were sacrificed in advance, so the end-point survival was not taken into account). All operations in the animal experiment process were performed according to the guidance for animal health in 2006, published by the Ministry of technology.

(4) And (3) statistical indexes are as follows: the survival condition of the mice is recorded every day, and the death rate, the average survival time and the content of each cell factor of the mice are counted.

2. Results

Table 5 administration of weiterketone injection alleviates mouse death caused by EV71 and CoxA16 viruses (n ═ 10)

Note:

and represents P for this group compared to the EV71 model group<0.05；^$And^#representing that the group of data is compared with the CoxA16 model group, P<0.05。

As shown in Table 5, similar to the positive ribavirin injection, the dose group (10mg/kg) of the ketoferulone significantly inhibits the death of mice caused by EV71 virus, and significantly prolongs the survival time of the mice by 42.9%; the dose (10mg/kg) of the widmanone obviously inhibits the death of mice caused by CoxA16 virus, and obviously prolongs the survival time of the mice to 47.4 percent. Meanwhile, other doses of the Widmanone also have similar drug effects, so that the death of the mice caused by viruses is relieved to different degrees, and the survival time of the mice is prolonged. Lupin widenin, an isopentenyl isoflavone compound with a similar structure to widenin, was administered at 10mg/kg without improvement in mortality of mice infected with EV71 and CoxA16 viruses, and only weakly prolonged mean survival in mice, but with no significant difference compared to the respective model groups (P > 0.05).

TABLE 6 mouse serum cytokine levels (ng/L, n ═ 3)

Note: a and^#represents P of the group data compared with EV71 model group and CoxA16 model group<0.05。^&And^Δindicates that the group data is P compared with EV71+ RBV group and CoxA16+ RBV group<0.05。

As shown in Table 6, the EV71 virus infection causes the abnormal increase of IL-6, TNF-alpha and MCP-1 content in mouse serum, and the high, medium and low dose groups (20, 10 and 5mg/kg) of the Widmanone inhibit the increase of mouse cytokines (P <0.05) caused by EV71 virus or CoxA16 virus to different degrees, which is obviously superior to the inhibition effect of the ribavirin injection as a positive medicine; the inhibition effect is most obvious by using a middle-dose widmanone group, and the middle-dose widmanone group (10mg/kg) obviously inhibits the increment of mouse cytokines caused by EV71 virus (P is less than 0.05); similarly, the dose group (10mg/kg) in the widmanone significantly inhibited the rise in mouse cytokines caused by the CoxA16 virus (P < 0.05). The 10mg/kg lupin widmantone injection administration only has weak inhibition effect on the inflammatory factor secretion caused by EV71 and CoxA16 virus infection, but has no significant difference compared with the respective model group (P > 0.05).

As shown by tables 5 and 6: the injection administration of the Widmantone can obviously reduce the death rate of mice caused by EV71 and CoxA16 viruses, prolong the survival time of the mice, obviously inhibit the release of inflammatory factors caused by the viruses, and relieve the symptoms of inflammation and the like. While lupin widetone with a structure similar to that of lupin has no significant inhibitory activity against hand-foot-and-mouth virus.

Example 5 oral administration of West's ketone prolongs survival time, inhibits death and inflammatory factor release in virally infected mice

The research aims are as follows: animal experiments prove that the oral administration of the ketoferulone can prolong the survival time of virus-infected mice and inhibit the death of the mice and the release of inflammatory factors.

1. Materials and methods

1.1 animals

1.2 Virus strains

EV71 virus BJ09/07 strain, GenBank accession number JQ319054.1, CoxA16 virus TS10/08 strain, GenBank accession number JX 068829. Half cytopathic dose (TCID) of EV71 virus was measured just before use₅₀) Is 10⁸CoxA16 Virus TCID/mL₅₀Is 10^8.5/mL。

1.3 reagents and consumables

Ribavirin oral liquid (abbreviated: RBVO, from Coprinus group, Inc., physiological saline, gavage device. cytokine interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and monocyte chemotactic factor-1 (MCP-1) ELISA assay kits were purchased from eBioscience, Inc.

1.4 Experimental methods

(1) Virus suspensionLiquid preparation: according to the TCID50 value of the virus, the two viruses are diluted to 10 by adopting a DMEM medium before use⁷TCID₅₀/mL。

(2) Preparing a liquid medicine: the Widmanone is pre-dissolved by 5% (w/v) sodium bicarbonate, and diluted to the required dosage by normal saline. The ribavirin oral liquid is shaken well before use, and is diluted to the required dosage (0.32g/kg) by using normal saline.

(3) Grouping, modeling and dosing: the 5-day-old mice were grouped, molded, and dosed according to table 7. Grouping: the mice were randomly divided into 13 groups: blank control group, EV71 model group and CoxA16 model group, high, medium and low dose of ketoferulone groups (50, 20 and 10mg/kg) and ribavirin group (RBVO)0.32g/kg, each group containing 13 mice. Molding: mice in each group were intraperitoneally injected with either EV71 or CoxA16 virus suspension, 0.1mL each, and the blank control group was replaced with saline injections. Administration: after the molding was completed, each mouse was administered by gavage (the blank control group, EV71 model group, and CoxA16 model group were replaced with physiological saline) 0.1mL each time, 1 time daily, and 13 consecutive days. On day 6 after model building, 3 mice were taken from each group, and after eye-picking, serum was taken to determine the contents of cytokines IL-6, TNF- α and MCP-1 (these 3 mice were sacrificed in advance, so the end-point survival was not taken into account). All operations in the animal experiment process were performed according to the guidance for animal health in 2006, published by the Ministry of technology.

2. Results

Table 7 oral administration of weiterketone ameliorated death in mice caused by EV71 and CoxA16 viruses (n ═ 10)

Note:

and means that P compares this group data with EV71 group<0.05；^$And^#indicating that the group data is compared with CoxA16 group, P<0.05。

As shown in Table 7, similar to the positive drug ribavirin oral liquid, the dose group (20mg/kg) of the ketoferulone obviously inhibits the death of mice caused by EV71 virus, and obviously prolongs the survival time of the mice to 36.1%; the dose (20mg/kg) of the widmanone obviously inhibits the death of the mice caused by CoxA16 virus, and obviously prolongs the survival time of the mice to 46.2 percent. Meanwhile, other doses of the Widmanone have similar drug effects, so that the death of the mice caused by viruses is reduced to different degrees, and the survival time of the mice is prolonged.

TABLE 8 mouse serum cytokine levels (ng/L, n ═ 3)

Note: a and^#indicate that P compares this group data with EV71 and CoxA16 groups, respectively<0.05。^&And^Δrespectively, the data of the group show that P is compared with EV71+ RBVO combination CoxA16+ RBVO<0.05。

As shown in Table 8, the EV71 virus infection causes the abnormal increase of IL-6, TNF-alpha and MCP-1 content in mouse serum, and the high, medium and low dose groups (50, 20 and 10mg/kg) of the Widmanone inhibit the increase of mouse cytokines (P <0.05) caused by EV71 virus or CoxA16 virus to different degrees, which is obviously better than the inhibition effect of the ribavirin oral liquid as a positive medicine; the inhibition effect is most obvious by using a middle-dose widmanone group, and the middle-dose widmanone group (20mg/kg) obviously inhibits the increment of mouse cytokines caused by EV71 virus (P is less than 0.05); similarly, the dose group (20mg/kg) in the widmanone significantly inhibited the rise in mouse cytokines caused by the CoxA16 virus (P < 0.05).

The data from tables 7 and 8 show that: by oral administration of the Widmantone, the death rate of mice caused by EV71 and CoxA16 viruses can be obviously reduced, the survival time of the mice is prolonged, the release of inflammatory factors caused by the viruses can be obviously inhibited, and the symptoms such as inflammation and the like can be relieved.

The viruses used in the embodiments of the present invention are only for illustrating the protection of Vero cells infected by Enterovirus 71 and Coxsackie virus A16 and the inhibition of virus replication, and have significant inhibitory effects on Coxsackie virus A (including but not limited to 4, 5,7, 9 and 10), Coxsackie virus B (including but not limited to 2 and 5) and Eicoviruses which can cause hand-foot-and-mouth disease symptoms, and can reduce the mouse mortality caused by Enterovirus 71 and Coxsackie virus A16, inhibit the release of inflammatory factors caused by viruses, and relieve inflammation, but the viruses targeted by the compounds of the present invention are not limited to the sources of the above embodiments. It will be clear to the skilled person that any virus having the corresponding pathogenicity and identified as enterovirus type 71, coxsackievirus type a4, 5,7, 9, 10 and 16, coxsackievirus type B2 and 5, echovirus is suitable for use in the present invention.

Moreover, the compound provided by the invention has small toxic and side effects, and can effectively inhibit cytopathic effect caused by hand-foot-and-mouth disease viruses, inhibit virus replication, reduce virus load, reduce or eliminate death of infected mice, prolong survival experiments, stabilize inflammation and the like. Therefore, the compound or the pharmaceutically acceptable salt thereof can be further developed into a medicine for treating or preventing hand-foot-and-mouth disease, and has various dosage forms and administration modes and wide application prospects.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. Use of widmanone or pharmaceutically acceptable salt in preparing medicine for treating or preventing hand-foot-and-mouth disease is provided.

2. The use of claim 1, wherein the hand-foot-and-mouth disease comprises hand-foot-and-mouth disease caused by enterovirus 71, coxsackievirus a group 16, coxsackievirus a group 4, coxsackievirus a group 5, coxsackievirus a group 7, coxsackievirus a group 9, coxsackievirus a group 10, coxsackievirus B group 2, 5 or echovirus.

3. The use of claim 1, wherein the hand-foot-and-mouth disease is a hand-foot-and-mouth disease caused by enterovirus type 71 or coxsackievirus type a group 16.

4. The use according to claim 1, wherein the pharmaceutically acceptable salt comprises a hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate salt.

5. The use of any one of claims 1-4, wherein the medicament is a clinically acceptable dosage form prepared from the active ingredient of ketoferulone and pharmaceutically acceptable adjuvants.

6. The use according to claim 5, wherein the dosage form comprises a tablet, capsule, granule, injection or spray.