CN113730403A

CN113730403A - Application of compound KYA1797K in preparation of anti-HBV (hepatitis B virus) medicines

Info

Publication number: CN113730403A
Application number: CN202111129158.5A
Authority: CN
Inventors: 曾木圣; 戴丹玲; 文石军; 钟茜; 冯国开; 张小勇; 谢楚
Original assignee: Sun Yat Sen University Cancer Center
Current assignee: Sun Yat Sen University Cancer Center
Priority date: 2021-09-26
Filing date: 2021-09-26
Publication date: 2021-12-03
Also published as: WO2023044967A1

Abstract

The invention discloses application of a compound KYA1797K in preparation of anti-HBV drugs, discovers the anti-viral effect, particularly anti-HBV effect, of a compound KYA1797K, and finds that a small molecule KYA1797K can inhibit HBV replication at a cell level and in a mouse. The small molecule can be used as a potential clinical test medicine for treating HBV infection and related diseases, is expected to become a new treatment means, prolongs the life cycle of patients and improves the life quality of the patients.

Description

Application of compound KYA1797K in preparation of anti-HBV (hepatitis B virus) medicines

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of a compound KYA1797K in preparation of anti-HBV medicines.

Background

Liver cancer is the fourth leading cause of cancer-related deaths worldwide, and Hepatitis B Virus (HBV) infection is a major causative agent of liver cancer. After HBV infects a host, the liver can generate hepatitis, cirrhosis, low-grade dysplasia nodules, high-grade dysplasia nodules, early liver cancer, liver cancer progression, late liver cancer and other processes, and finally liver cancer is generated. Therefore, the search for drugs that effectively inhibit HBV replication is of great significance in treating HBV-related diseases.

Disclosure of Invention

The invention aims to provide application of compound KYA1797K or a derivative thereof in effectively inhibiting HBV replication.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided a use of compound KYA1797K or a derivative thereof in any one of the following (I) to (III):

(I) preparing an antiviral or virustatic product;

(II) preparing a product that inhibits viral replication;

(III) products for treating or preventing diseases caused by infectious viruses.

In some embodiments of the invention, the derivative includes a compound, a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer, stereoisomer, isotopic derivative, or prodrug thereof.

In some embodiments of the invention, the virus is a DNA virus.

In some preferred embodiments of the invention, the virus is preferably Hepatitis B Virus (HBV).

In a second aspect of the present invention, there is provided a product comprising compound KYA1797K, which has the functions as described in any one of the following (I) to (III):

(I) preparing an antiviral or antiviral inhibitor;

(II) preparing to inhibit viral replication;

(III) treating or preventing diseases caused by infectious viruses.

In some embodiments of the invention, the product further comprises a carrier, diluent or excipient acceptable therefor.

In some embodiments of the invention, the virus is a DNA virus.

In some preferred embodiments of the invention, the virus is an HBV virus.

In some embodiments of the invention, the product is a kit or a medicament.

In some embodiments of the present invention, the medicament is formulated as a powder, pill, tablet, capsule, oral liquid, paste, granule, mixture, suppository, aerosol, or injection.

In a third aspect of the present invention, there is provided a method comprising contacting KYA1797K or a derivative thereof with a virus, the method being used for any one of the following (I) to (III):

(I) preparing an antiviral or antiviral inhibitor;

(II) preparing to inhibit viral replication;

(III) treating or preventing diseases caused by infectious viruses.

In some embodiments of the invention, the methods are not used for the diagnosis or treatment of disease.

In some embodiments of the invention, the virus is a DNA virus, preferably a hepatitis b virus.

The invention has the beneficial effects that:

the invention provides the antiviral effect of a compound KYA1797K, and mainly the small molecule KYA1797K can inhibit HBV replication at a cellular level and in a mouse. The small molecule can be used as a potential clinical test medicine for treating HBV infection and related diseases, is expected to become a new treatment means, prolongs the life cycle of patients and improves the life quality of the patients.

Drawings

FIG. 1 shows that small molecule KYA1797K inhibits HBV replication at cellular level.

FIG. 2 shows that small molecule KYA1797K inhibits HBV replication in mice. Wherein FIG. 2A is the copy number of DNA; FIG. 2B shows the expression level of HBsAg; FIG. 2C shows the body weight of the mice tested.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

Example 1

HepG2 cells were pretreated by adding different concentrations (0.375. mu.M, 1.5. mu.M, 6. mu.M) of small molecules KYA1797K and transiently transformed with HBV plasmid for 48 hours, and the cell supernatants were collected for immunoenzymatic ligation to detect the expression of HBeAg antigen in serum using the hepatitis B virus e antigen detection kit (Elisa) from Shanghai Rongsheng biological drug industries. The key experimental procedures were as follows:

(1) enzyme linked immunosorbent assay for detecting hepatitis B virus e antigen

a) Preparation of the experiment: the kit was removed from the refrigerated environment and allowed to equilibrate at room temperature for 30 minutes while the wash solution was diluted 1: 20.

b) Adding a sample to be detected: adding 50 mul of sample to be tested into each well, setting 2 positive control wells, 2 negative control wells, 50 mul of sample and 1 blank control well.

c) Adding an enzyme conjugate: 50 μ l of each well, no blank control well, mixed well, glued on paper, incubated at 37 ℃ for 30 minutes.

d) Washing the plate: discarding the liquid in the reaction plate holes, patting dry on absorbent paper, filling each hole with washing liquid, standing for 5 seconds, patting dry the washing liquid in the discarded holes, and repeating for 5 times.

e) Adding a color developing agent: adding 50 mul of color-developing agent A into each hole, then adding 50 mul of color-developing agent B into each hole, fully mixing, and placing at 37 ℃ for incubation for 15 minutes in the dark.

f) Terminating the reaction solution: add stop solution 50. mu.l per well and mix well.

g) And (3) determination: reading by a microplate reader, reading the OD value of each hole by selecting the wavelength of 450nm, and completing the reading within 10 minutes.

As a result, referring to FIG. 1, it can be seen that the small molecule KYA1797K can inhibit HBV replication in a concentration-dependent manner, wherein 50% of HBV replication can be inhibited by adding 1.5. mu.M of the small molecule KYA1797K, and 75% of HBV replication can be inhibited by adding 6. mu.M of the small molecule KYA 1797K.

Example 2

5 weeks old C57BL/6 male mice were injected with pAAV-HBV1.2(μ g) plasmid via high-pressure tail vein, injected with small molecules 25mg/kg intraperitoneally starting on the tenth day, and orbital bleeds were tested for HBV DNA copy number in serum using Daan Gene hepatitis B Virus nucleic acid quantitative determination kit (PCR-fluorescent probe method) (cat # DA0032, university of Zhongshan). The key experimental operation is as follows:

(1) detection of DNA copy number of serum HBV

a) After blood is taken from mouse eyeballs, serum is placed for 2 hours at normal temperature and centrifuged at 5000rpm for 10 minutes. The supernatant was aspirated and transferred to a 1.5ml sterile centrifuge tube.

b) A200. mu.l sample was taken, 450. mu.l of DNA extract I and 4. mu.l of the internal standard solution were added thereto, and the mixture was vigorously shaken and mixed by a shaker for 15 seconds and then subjected to instantaneous centrifugation for several seconds. The constant temperature treatment is carried out for 10 minutes at 100 ℃.

c) Preparing a PCR reaction mixed solution of each tube: 27 μ l of PCR reaction solution +3 μ l of Taq enzyme.

d) Respectively adding 20 mul of the extracted nucleic acid of the sample to be detected, the HBV negative quality control product, the HBV strong positive quality control product, the HBV critical positive quality control product and the supernatant of the positive quantitative reference product into the reaction solution. The tube cap was closed and centrifuged at 8000rpm for a few seconds.

e) PCR was performed using a lightcycler480 instrument:

after the software is opened, selecting 'new experiment', setting a detection mode as 'multicolor hydrolysics probe', and setting a detection channel as FAM and VIC. Set cycling conditions (table 1):

TABLE 1 PCR cycling conditions

Selecting "sample editor", setting the name and type of the sample, inputting the concentration of a positive quantitative reference substance in a "standard concentration" frame, saving a file, and running a program.

(2) Mouse high-pressure tail vein injection HBV plasmid

a) Mice of 5 weeks of age were fixed in a holder.

b) 2ml of plasmid of PBS + 6. mu.g of pAAV HBV1.2 were mixed well and sucked into a syringe.

c) The mouse tail was wiped with an alcohol cotton ball and the plasmid was injected into the mouse tail vein through a high pressure injection device.

The copy number detection of HBV DNA in serum is shown in FIG. 2A; detecting the expression amount of HBsAg by an immune enzyme-linked reaction, and particularly referring to FIG. 2B; the body weight of the mice was measured, see in particular fig. 2C; it can be seen that after the small molecule injection is started on the tenth day, the DNA copy number of the HBV in the mice treated by the small molecule KYA1797K group is lower and the expression level of HBsAg in serum is lower compared with the control group, which indicates that the small molecule KYA1797K can significantly inhibit the replication and expression of HBV in the mice. Meanwhile, the small molecule KYA1797K has no influence on the body weight of the mice, which indicates that the small molecule KYA1797K has no toxicity on the growth of the mice at the treatment concentration.

The present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

Claims

1. Use of compound KYA1797K or a derivative thereof in any one of the following (I) to (III):

(I) preparing an antiviral or virustatic product;

(II) preparing a product that inhibits viral replication;

2. The use according to claim 1, wherein the derivative comprises a compound, a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer, stereoisomer, isotopic derivative or prodrug thereof.

3. Use according to claim 1, wherein the virus is a DNA virus, preferably an HBV virus.

4. A product comprising compound KYA1797K, which has the functions of any one of the following (I) to (III):

(I) preparing an antiviral or antiviral inhibitor;

(II) preparing to inhibit viral replication;

(III) treating or preventing diseases caused by infectious viruses.

5. The product according to claim 4, wherein the virus is a DNA virus, preferably an HBV virus.

6. Use according to any one of claims 1 to 3 or a product according to any one of claims 4 to 5, wherein the product is a kit or a medicament.

7. Use or product according to claim 6, wherein the product further comprises a carrier, diluent or excipient acceptable therefor.

8. The use or product according to claim 6, wherein the medicament is in the form of a powder, pill, tablet, capsule, oral liquid, paste, granule, mixture, suppository, aerosol or injection.

9. A method comprising contacting KYA1797K or a derivative thereof with a virus, the method being for use in any one of (I) to (III) below:

(I) preparing an antiviral or antiviral inhibitor;

(II) preparing to inhibit viral replication;

(III) treating or preventing diseases caused by infectious viruses.

10. The method according to claim 9, wherein the virus is a DNA virus, preferably selected from HBV viruses.