CN113750083A

CN113750083A - Application of metformin in preparation of medicine for treating hand-foot-and-mouth disease

Info

Publication number: CN113750083A
Application number: CN202111037699.5A
Authority: CN
Inventors: 李玉环; 王辉强; 蒋建东
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2021-09-06
Filing date: 2021-09-06
Publication date: 2021-12-07
Anticipated expiration: 2041-09-06
Also published as: CN113750083B

Abstract

The metformin provided by the invention has obvious effects of resisting replication of enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) causing hand-foot-and-mouth diseases, and has different antiviral effects from the existing clinical application of the metformin, so that the metformin has wide application prospects.

Description

Application of metformin in preparation of medicine for treating hand-foot-and-mouth disease

Technical Field

The invention belongs to the field of medical application, and particularly relates to application of metformin in preparation of a medicine for treating hand-foot-and-mouth disease.

Background

Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are both open-loop single-plus-strand RNA viruses, belong to the genus Enterovirus of the family picornaviridae, are the main pathogens causing hand-foot-and-mouth disease (HFMD) in children, and can cause serious social harm.

In 2015 for 12 months, the national food and drug administration approved the registration application for the production of a new class 1 biological product-enterovirus 71 inactivated vaccine developed by Beijing Kexing biological product Co., Ltd and the institute of medical and biological research of the Chinese medical science institute, and the vaccine was put into the market in 2016 in half a year. According to the report of the legal infectious diseases in China, the number of the disease patients of the hand-foot-and-mouth disease is reduced since the vaccine is marketed, which shows that the vaccine really plays a certain role in prevention and protection. However, due to the fact that EV71 subtypes are numerous, whether the vaccine can prevent EV71 infection of all subtypes is not clear, and the vaccine has no cross protection effect on enteroviruses such as Coxsackie virus A16 and the like which can cause hand-foot-and-mouth disease. In addition, because the EV71 inactivated vaccine belongs to a non-immune programming vaccine at present, the inoculation rate of infants is not high all over the country, and therefore, the treatment of the hand-foot-and-mouth disease is still a crucial problem. At present, no specific medicine aiming at EV71 and CVA16 infection exists clinically, and Interferon (IFN), Ribavirin (RBV), traditional Chinese medicine and the like are adopted for symptomatic treatment clinically. Therefore, the research and development of the medicine for treating the hand-foot-and-mouth disease are urgently needed.

Metformin is clinically suitable for non-insulin dependent diabetics with unsatisfactory simple diet control, and has the functions of resisting tumors, regulating immunity, reducing blood sugar and resisting viruses in literature reports. Among them, the literature reports that metformin has the effect of inhibiting various viruses, including Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Human Papilloma Virus (HPV), influenza virus, coxsackie virus B3, etc., but there is no application of metformin in resisting EV71 and CVA16 causing hand-foot-and-mouth disease in the literature at present.

Disclosure of Invention

The invention provides application of metformin in preparation of a medicine for treating hand-foot-and-mouth diseases.

The specific technical scheme of the invention is as follows:

the invention provides application of metformin shown in formula I in preparing a medicine for treating hand-foot-and-mouth disease,

the metformin provided by the invention can reduce the virus titer of EV71 and CVA16 which can reflect the content of live virus in cells in a dose-dependent manner.

The metformin provided by the invention can lower the level of the receptor proteins of EV71 and CVA16, namely human scavenger receptor B2, thereby reducing the adsorption binding of viruses and playing an antiviral role.

The metformin disclosed by the invention has a remarkable effect of resisting EV71 and CVA16 replication.

Application of metformin shown in formula I, solvate or pharmaceutically acceptable salt thereof in preparing medicine for treating hand-foot-and-mouth disease.

The metformin represented by the formula one of the present invention may form solvates such as hydrates, alcoholates and the like. In general, the solvate forms with pharmaceutically acceptable solvents such as water, ethanol, and the like are comparable to the non-solvate forms.

The pharmaceutically acceptable salt can be hydrochloride, phosphate, sulfate and acetate.

The metformin of formula one of the present invention may also be in the form of a prodrug or a form that releases the active ingredient after metabolic changes in vivo. The selection and preparation of suitable prodrug derivatives is well known to those skilled in the art and is not intended to be limiting.

The compound of the formula I or the pharmaceutically acceptable salt thereof can also exist in a crystal form, and the invention comprises any crystal form of the metformin shown in the formula I or the pharmaceutically acceptable salt thereof.

The metformin represented by the formula one of the present invention, a solvate thereof or a pharmaceutically acceptable salt thereof can be administered by the following routes: parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intradermal, transdermal, rectal, intracranial, intraperitoneal, intranasal, intramuscular routes, or as inhalants.

The metformin, the solvate thereof or the pharmaceutically acceptable salt thereof shown in the formula I can be administrated in the form of a pharmaceutical preparation, and the pharmaceutical composition comprises the metformin, the solvate thereof or the pharmaceutically acceptable salt thereof shown in the formula I and a pharmaceutically acceptable carrier or auxiliary material.

The metformin represented by the formula I, the solvate thereof or the pharmaceutically acceptable salt thereof can be prepared into various suitable dosage forms according to the administration route.

When administered orally, the compounds of the present invention may be formulated in any orally acceptable dosage form, including but not limited to tablets, capsules, aqueous solutions or suspensions. Among these, carriers for tablets generally include lactose and corn starch, and additionally, lubricating agents such as magnesium stearate may be added. Diluents used in capsule formulations generally include lactose and dried corn starch. Aqueous suspension formulations are generally prepared by mixing the active ingredient with suitable emulsifying and suspending agents. Optionally, some sweetener, aromatic or colorant may be added into the above oral preparation.

When applied topically to the skin, the compounds of the present invention may be formulated in a suitable ointment, lotion, or cream formulation wherein the active ingredient is suspended or dissolved in one or more carriers. Carriers that may be used in ointment formulations include, but are not limited to: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide, emulsifying wax and water; carriers that can be used in lotions or creams include, but are not limited to: mineral oil, sorbitan monostearate, tween 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

The metformin, the solvate thereof or the pharmaceutically acceptable salt thereof shown in the formula I can also be used in the form of sterile injection preparations, including sterile injection water or oil suspensions or sterile injection solutions, and also can be in a freeze-dried form. Among the carriers and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, the sterilized fixed oil may also be employed as a solvent or suspending medium, such as a monoglyceride or diglyceride.

The pharmaceutical preparation of the present invention includes any preparation which can be pharmaceutically practiced, for example, oral preparations, parenteral preparations and the like.

1. The toxicity of the metformin on human colon cancer cells HCT-8 is detected by using a CCK cell activity detection reagent; the effect of each drug concentration on Cytopathic effect (CPE) was also observed to determine the working concentration for evaluation of anti-EV 71 and CVA16 efficacy of metformin in cells. The results show that the cell viability is not obviously different from the control group at the concentration of 50mM of metformin and below, and the cell morphology is not changed under a microscope. Thus, in subsequent experiments we selected 20mM, 5mM and 1.25mM metformin for in vitro efficacy evaluation.

2. From the fluorescent quantitative PCR results, the metformin provided by the present invention can dose-dependently reduce the RNA levels of EV71 and CVA16 in cells. From the Western blot results, the metformin provided by the invention can reduce the viral protein levels of EV71 and CVA16 in cells in a dose-dependent manner. From the viral titer results, it is seen that metformin provided by the present invention dose-dependently reduces the viral titer of EV71 and CVA16 that are able to reflect viable viral content in cells.

3. The metformin provided by the invention has no direct inactivation effect on EV71 and CVA16, but the metformin can lower the level of receptor proteins of EV71 and CVA16, namely human scavenger receptor B2(SCARB2), so that the absorption and binding of viruses are reduced to play an antiviral effect.

Therefore, the metformin provided by the invention has remarkable effects of resisting EV71 and CVA16 replication.

Drawings

FIG. 1 is a graph showing the results of the effect of metformin on cell viability in HCT-8 cells.

FIG. 2 is a graph showing the results of the effect of metformin on the inhibition of viral RNA levels in HCT-8 cells. In the figure, A shows the result of detecting the effect of metformin on EV71 RNA level by a real-time fluorescent quantitative PCR method; in the figure, B is a graph showing the effect of real-time fluorescent quantitative PCR on CVA16 RNA level.

FIG. 3 is a graph showing the effect of metformin on the inhibition of viral protein levels in HCT-8 cells; a in the figure shows a graph of the effect of the Western blot method on the level of EV71 protein detected by metformin; in the figure, B is a graph showing the effect of the Western blot method on the level of CVA16 protein in the test of metformin.

FIG. 4 is a graph showing the results of the effect of metformin on the inhibition of live virus titer in HCT-8 cells; the graph A shows the effect of the CPE method on the EV71 virus titer; panel B shows the results of CPE assay to determine the effect of metformin on CVA16 viral titer.

FIG. 5 is a graph showing the effect of metformin on direct viral inactivation in HCT-8 cells; in the figure, A shows a graph of the effect of the CPE method on the inactivation of the EV71 virus by detecting metformin; panel B shows the results of CPE assay to determine the effect of metformin on CVA16 virus inactivation.

FIG. 6 is a graph showing the effect of metformin on the expression of EV71 and CVA16 receptor protein SCARB2 in HCT-8 cells.

FIG. 7 is a graph showing the results of the effect of metformin on EV71 adsorption binding in HCT-8 cells.

Detailed Description

In order to better understand the contents of the present invention, the following description will be made in conjunction with specific embodiments, but the present invention is not limited to the following examples. Meanwhile, the following examples do not limit the present invention further.

Example 1 inhibition of EV71 and CVA16 replication by metformin

1. Cell culture

Culture solution for HCT-8 cell passage: DMEM (Gibco) medium containing 10% fetal bovine serum (Gbico), penicillin and streptomycin diabody 100U/ml (Gibco).

Culture solution for HCT-8 cell dilution virus: DMEM medium containing 100U/ml (Gibco) of penicillin and streptomycin diabesin.

HCT-8 cell dilution drug used culture solution: DMEM medium containing 2% fetal bovine serum (Gbico), penicillin and streptomycin diabody 100U/ml (Gibco).

When the confluence of HCT-8 cells reaches 90%, 0.25% pancreatin-EDTA (Gibco) is added into a culture bottle, the cells are digested for 2 minutes at 37 ℃, the pancreatin is removed, complete culture solution is added for blowing, the cells are passaged at the ratio of 1:3, and the cells are passaged once in 1-2 days.

2. Cytotoxicity assays

HCT-8 cells were seeded in 96-well plates at 3X 10⁴One/well, 37 ℃ and 5% CO₂Medium culture; after 24h, the drug metformin (purchased from Sigma) was diluted to different concentrations with the medium used for diluting the drug, added to the cells, while a blank control (medium only without cells) and a cell control (no cells added) were set, with 3 parallel wells per group. Observing the growth state of cells in each well in the inverted microscope at 3d after adding medicine, comparing the cells with different administration concentrations with normal cells, and respectively observing the cell CP of each administration wellAnd E, respectively marking the cell state change or death rate as 4+ (cell death rate is 75% -100%), 3+ (cell death rate is 50% -75%), 2+ (cell death rate is 25% -50%), 1+ (cell death rate is 0-25%) and 0+ (the cell state is not different from the control group). After the observation, CCK (Beijing Quanjin Co., Ltd.) reagent was added to each well, the plate was incubated in an incubator for 1 hour, and OD450 value was measured on a microplate reader. The results were expressed by the formula [ (cell control OD 450-blank well OD450 value) - (administration group cell OD 450-blank well OD450 value)]V (cell control OD450 value-blank well OD450 value), cell viability was calculated for each drug concentration and the results are shown in FIG. 1.

The figure shows that the cell viability at the concentration of 50mM and below is not significantly different from the control group, and the cell morphology is not changed under the microscope. Thus, in subsequent experiments we selected 20mM, 5mM and 1.25mM metformin for in vitro efficacy evaluation.

3. Inhibition of EV71 and CVA16 RNA by metformin

HCT-8 cells were seeded in 6-well plates at 9X 10⁵One/well, 37 ℃ and 5% CO₂Medium culture; after 24h, the medium in the well plate was discarded and cells were infected with either EV71(MOI 0.1) or CVA16 (MOI 0.1) for 1 h. After 1h the virus fluid was discarded and metformin was added at various concentrations. Virus control groups (culture broth for dilution of drug) and metformin drug groups (20mM, 5mM and 1.25mM) were set. Adding medicine for 24h, extracting total RNA of cells by adopting QIAGEN RNA extraction kit, and storing at-80 ℃ for detection.

EV71 and CVA16 RNA contents were detected using a kit TransScript II Green One-Step qRT-PCR Super Mix (Beijing Quanji Co., Ltd.) in an ABI7500Fast type high throughput real-time fluorescent quantitative PCR (qPCR) instrument, and the assay was repeated 2 times for each RNA sample.

EV71 VP1 RNA primer: 5'-GATATCCCACATTCGGTGA-3' (upstream); 5'-TAGGACACGCTCCATACTCAAG-3' (downstream). CVA16 VP1 RNA primer: 5'-GTTATCCCACCTTCGGAGA-3' (upstream); 5'-TCGGGCATTGACCATAATCTAG-3' (downstream). GAPDH primer: 5'-GAAGGTGAAGGTCGGAGTC-3' (upstream); 5'-GAAGATGGTGATGGGATTTC-3' (downstream).

The reaction system is as follows:

real-time fluorescent quantitative reaction system

Reaction conditions

Using SYBR Green^ERThe fluorescence detection system performs relative quantitative analysis on a target Gene (GOI), normalizes Ct values by taking a housekeeping gene GAPDH as an internal reference (IC), and adopts 2^-ΔΔCtCalculating the expression difference of the target gene. sample represents experimental group; control represents control group; ct-_GOI ^SCt values representing the target genes of the experimental group samples; ct-_GOI ^CCt value representing target gene of control group sample; ct-_IC ^SCt values representing a reference within the experimental group of samples; ct-_IC ^CCt values representing a reference in control samples; the Fold difference represents the Fold difference of target gene expression of the experimental group and the control group. The calculation method is as follows:

ΔCt_sample＝Ct-_GOI ^S-Ct-_IC ^S

ΔCt_control＝Ct-_GOI ^C-Ct-_IC ^C

ΔΔCt＝ΔCt_sample-ΔCt_control

Fold difference＝2^-ΔΔCt

from figure 2A, it can be seen that metformin significantly reduced the level of EV71 RNA in the cells and was dose-dependent: the inhibition rate of 20mM metformin to EV71 RNA is about 77%; the inhibition rate of 5mM of metformin to EV71 RNA is about 60%; while 1.25mM metformin had no inhibitory effect on EV71 RNA.

From figure 2B, it can be seen that metformin significantly reduced the level of intracellular CVA16 RNA and was dose-dependent: the inhibition rate of 20mM metformin on CVA16 RNA is about 73%; the inhibition rate of 5mM metformin on CVA16 RNA is about 44%; while 1.25mM metformin inhibited CVA16 RNA by about 25%.

4. Inhibition of EV71 and CVA16 VP1 proteins by metformin

HCT-8 cells were seeded in 6-well plates at 9X 10⁵One/well, 37 ℃ and 5% CO₂Medium culture; after 24h, the medium in the well plate was discarded and cells were infected with either EV71(MOI 0.1) or CVA16 (MOI 0.1) for 1 h. After 1h the virus fluid was discarded and metformin was added at various concentrations. Normal control groups (medium for dilution of drug), virus control groups (medium for dilution of drug) and metformin drug groups (20mM, 5mM and 1.25mM) were set. Adding medicine for 24 hr, extracting total cell protein, and storing at-20 deg.C for detection.

Western blot detection

Electrophoresis: preparing laminated gel (with the concentration of 5%) and separation gel (with the concentration of 10%), carrying out SDS-PAGE electrophoresis on the samples, adopting the voltage of 60V for the laminated gel, increasing the voltage to 130V for continuous electrophoresis after bromophenol blue enters the separation gel, and transferring the membrane after the completion of gel running is judged according to the position of a protein Marker.

Film transfer: a transfer buffer (25mM Tris, 192mM glycine, 20% (vol/vol) methanol) was prepared during electrophoresis and pre-chilled at 4 ℃. The PVDF membrane was activated with methanol for 1 min in advance, and then immersed in a membrane transfer buffer. After the electrophoresis was completed, the gel was carefully removed, transferred to a membrane transfer buffer for equilibration for 5min, and then subjected to wet transfer. 3 pieces of filter paper, a PVDF film, gel and 3 pieces of filter paper are sequentially paved on an anode of a film rotating instrument from bottom to top, and are stacked orderly, and the removal of bubbles is noticed. After the sandwich is made, the cathode of the film transfer instrument is covered, and the film transfer instrument is rotated for about 60min under the constant current of 250 mA.

Antibody incubation: after the membrane transfer was complete, the membrane was removed and washed on a shaker with TBST for 10 min. The membrane was placed in blocking solution (5% skimmed milk powder in TBST) and blocked for 1h at room temperature with shaking. anti-VP 1 (Millipore) and β -actin (cell Signaling technology) primary antibodies were diluted with primary antibody diluent 1: 1000 dilution and incubation at 4 ℃ overnight. The next day, the primary antibody solution was removed and washed 3 times with TBST for 10min each. Horseradish peroxidase-labeled anti-rabbit secondary antibody (Cell Signaling Technology) solution 1: diluting with 5000, incubating at room temperature for 1h, washing with TBST for 3 times, each time for 10 min.

Color development: finally incubation with ECL immunoblot chemiluminescent reagent (Millipore) and image capture by ChemiDoc XRS + chemiluminescence imaging analysis system (BIO-RAD) resulted in fig. 6.

From figure 3 it can be seen that metformin dose-dependently reduced intracellular levels of viral proteins of EV71 and CVA 16.

5. Inhibition of EV71 and CVA16 viral titers by metformin

HCT-8 cells were seeded in 6-well plates at 9X 10⁵One/well, 37 ℃ and 5% CO₂Medium culture; after 24h, the medium in the well plate was discarded and cells were infected with either EV71(MOI 0.1) or CVA16 (MOI 0.1) for 1 h. After 1h the virus fluid was discarded and metformin was added at various concentrations. Normal control groups (medium for dilution of drug), virus control groups (medium for dilution of drug) and metformin drug groups (20mM, 5mM and 1.25mM) were set. Adding drug for 24h, freezing and storing the culture plate at-80 deg.C, performing triple freezing and triple thawing, centrifuging at 4 deg.C and 5000rpm, taking upper layer virus solution, diluting according to 10 times ratio, adding 100 μ L/well into Vero cell in 96-well plate, adding at 37 deg.C and 5% CO₂After medium culture for 72h, CPE observation and recording are carried out, and half of tissue cell infection amount (50% tissue induced dises, TCID) of the virus is calculated according to CPE results₅₀)。

From figure 4, it can be seen that metformin dose-dependently reduced the levels of intracellular viral titers of EV71 and CVA 16.

Example 2 inactivation of EV71 and CVA16 by metformin

Vero cells were seeded in 6-well plates at 9X 10⁵One/well, 37 ℃ and 5% CO₂Medium culture; after 24h, the medium in the well plate was discarded and 5% CO at 37 ℃ was added₂EV71 or CVA16 infected cells after 1h of incubation with metformin or DMSO for 1 h. Removing virus solution after 1h, adding culture solution for diluting medicine, and treating with 5% CO at 37 deg.C₂After medium culture for 72h, CPE observation and recording are carried out, and the infection amount of half tissue cells of the virus (50% tissue culture infection) is calculated according to the CPE resulte doses，TCID₅₀)。

From figure 5 it can be seen that metformin has no direct inactivation effect on EV71 and CVA 16.

Example 3 Effect of metformin on the content of EV71 and CVA16 receptor SCARB2 proteins

HCT-8 cells were seeded in 6-well plates at 9X 10⁵One/well, 37 ℃ and 5% CO₂Medium culture; after 24h, the medium in the well plate was discarded and different concentrations of metformin were added. The normal control group (culture solution for dilution of drug) and metformin drug groups (20mM, 5mM and 1.25mM) were set. After adding the medicine for 24h, extracting the total cell protein and carrying out western blot detection, wherein the SCARB2 antibody is a product of Abcam company in the same way as in the example 1.

As can be seen in figure 6, metformin dose-dependently down-regulated the level of EV71 and CVA16 receptor SCARB2 proteins.

Example 4 Effect of metformin on EV71 and CVA16 adsorption binding

HCT-8 cells were seeded in 6-well plates at 9X 10⁵One/well, 37 ℃ and 5% CO₂Medium culture; after 24h, the medium in the well plate was discarded, and the cells were treated with 20mM metformin for 24h before the plate was cooled at 4 ℃ for 2 h. After the medium was aspirated and discarded, the cells were washed 2 times with PBS at 4 ℃ and then EV71 or CVA16 was added to the plates and left on ice for 30min to set the virus control group and metformin drug group. After 30min, the virus solution was aspirated, washed 2 times with 4 ℃ PBS, total cellular RNA was extracted and subjected to real-time fluorescent quantitative PCR detection, as in example 1.

As can be seen in fig. 7, the adsorption binding of EV71 and CVA16 to cells was reduced after metformin treatment.

Claims

1. Application of metformin, solvate or pharmaceutically acceptable salt thereof in preparing a medicament for treating hand-foot-and-mouth disease.

2. The use of claim 1, wherein metformin dose-dependently reduces the viral titer of EV71 and CVA16 in cells that reflect viable viral content.

3. The use as claimed in claim 1, wherein metformin exerts an antiviral effect by down-regulating the levels of the receptor proteins EV71 and CVA16, human scavenger receptor B2, thereby reducing the adsorptive binding of the virus.

4. Use according to claim 1, characterized in that metformin has a significant action against the replication of EV71 and CVA 16.

5. Use according to claim 1, characterized in that the solvate of metformin is: hydrate, or alcoholate with metformin.

6. The use according to claim 1, wherein metformin is also in the form of a prodrug or a form which releases the active ingredient after metabolic changes in the body.

7. Use according to claim 1, wherein metformin or a pharmaceutically acceptable salt thereof is present in the form of crystals in any form.

8. Use according to claim 1, characterized in that metformin, its solvates or pharmaceutically acceptable salts thereof is administered by the following route: parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intradermal, transdermal, rectal, intracranial, intraperitoneal, intranasal, intramuscular routes, or as inhalants.

9. The use according to claim 1, comprising a pharmaceutical composition of metformin, its solvate or pharmaceutically acceptable salt thereof, further comprising a pharmaceutically acceptable carrier or adjuvant.

10. The use according to claim 9, wherein the pharmaceutical composition is prepared in any pharmaceutical dosage form.