AU2021107213A4 - Use of sphondin as an effective component in preparing medicine for treating hepatitis b - Google Patents

Abstract

The present invention belongs to the technical field of biomedicine, and specifically discloses a use of sphondin as an effective component in preparation of drugs for treatment of hepatitis B. It is found for the first time in the present invention that sphondin (Sph for short) can inhibit secretion of HBsAg from HBV, the transcription level of HBV RNAs, the replication level of HBV core DNA, the transcription activity of cccDNA and levels of HBV RNAs, HBV DNA, an HBV S protein and an HBV X protein in liver tissues, has an obvious function in treatment of hepatitis B, can be used to prepare new anti-HBV drugs which can reduce the HBsAg level and inhibit transcription of cccDNA, and has a broad application prospect in treatment of hepatitis B. SEQUENCE LISTING <110> Chongqing Medical University <120> Use of sphondin as an effective component in preparation of drugs for treatment of hepatitis B <130> 210429 <160>6 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> total HBV RNAs forward <400> 1 accgaccttg aggcatactt 20 1

Description

SEQUENCE LISTING

<110> Chongqing Medical University

<120> Use of sphondin as an effective component in preparation of drugs for treatment of hepatitis B

<130> 210429

<160>6

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> total HBV RNAs forward

<400> 1

accgaccttg aggcatactt 20

USE OF SPHONDIN AS AN EFFECTIVE COMPONENT IN PREPARING MEDICINE FOR TREATING HEPATITIS B

Background of the Present Invention

Field of Invention

The present invention relates to the technical field of biomedicine, and in particular, to a use

of sphondin as an effective component in preparation of drugs for treatment of hepatitis B.

Description of Related Arts

Hepatitis B virus (HBV), a prototype member of the hepadnaviridae family, has chronically

infected more than 250 million people worldwide. HBV infection is closely related to the occurrence

and development of acute and chronic hepatitis, cirrhosis and liver cancer.

According to statistics, nearly two billion people worldwide have been infected with HBV,

of which about 350 million people are chronically infected. Chronic HBV infection is a global public

health burden. Annually, about 1 million deaths from severe disease are caused by HBV infection,

such as hepatic failure, liver cirrhosis and primary hepatocellular carcinoma.

Mother-to-child transmission can be difficult to prevent, and 10% of adults and 5% of minors

cannot get vaccine immunity and thus become the population with the highest potential infection risk.

Hepatitis B virus infection and resulting diseases not only seriously endanger people's health, but also

become a serious social public health problem. Therefore, treatment of chronic HBV infection and

prevention of development of hepatitis B-related diseases are important tasks related to global public

health.

At present, there are only two types of drugs approved by US Food and Drug Administration

(FDA) for treatment of chronic hepatitis B (CHB): nucleoside analogs (NAs) targeting a pol protein

and interferons (IFNs) targeting viral transcription, which exert an antiviral effect through immune

regulation and interference with HBV replication respectively. However, IFN-a has many application

restrictions due to its low response rate, many adverse reactions, low tolerance, high price, limited treatment targets and the like. In recent years, NAs drugs have been developed rapidly and become the first choice of antiviral therapeutic drugs and have been applied widely in the clinic practice.

Although NAs drugs have a strong ability to inhibit viral DNA, HBsAg and an HBV transcription

template cccDNA (covalently closed circular DNA) in patients cannot be completely removed by NAs.

Futhermore, NAs drugs need to be taken for a long time, drug resistance may be caused due to

mutation of an HBV polymerase, the viral load rebounds after discontinuance of most NAs drugs, and

applications of such drugs are greatly limited. Therefore, it is necessary to develop anti-HBV drugs

with new action mechanisms and action targets.

HBV has a complicated life cycle. When being in contact with liver cell membranes, virus

particles enter cells under mediation of a receptor (NTCP), and relaxed circular DNA (rc DNA)

wrapped by a viral core protein is released after internalization. After rcDNA undergoes a series of

excision repair reactions under the action of a host DNA polymerase and a topoisomerase, cccDNA

with a superhelical structure is finally formed. As a key molecule to establish persistent infection,

cccDNA is subjected to transcription under the action of a host RNA polymerase II to produce 4 kinds

of RNA transcripts. Precore RNA is used to encode an HBV core protein and form HBeAg; 2.4/2.1

kb RNA is used to form an envelope protein (L, M, S); 0.7-kb RNA is used to finally form HBx. A

largest pregenomic RNA (pgRNA) forms rcDNA under the action of an HBV polymerase and

continues to replenish a cccDNA pool. HBV is difficult to cure and is closely related to long-term

stable existence of cccDNA. In addition, the envelope protein can be integrated into host genomic

DNA. Therefore, a large amount of HBsAg in the serum of a patient is difficult to turn negative. Based

on this, the present invention intends to find new anti-HBV drugs which can effectively reduce HBsAg

and stop transcription of cccDNA.

Summary of the Present Invention

In view of the shortcomings of the prior art, the present invention provideS a use of sphondin

as an effective component in preparation of drugs for treatment of hepatitis B, so as to provide a new

anti-HBV drug and a treatment method for effectively reducing HBsAg and stopping transcription of

cccDNA.

Accordingly, in a first aspect, the present invention provides a use of sphondin as an effective component in preparation of drugs for treatment of hepatitis B.

Further, the drugs for treatment of hepatitis B have at least one of the following functions:

inhibiting secretion of HBsAg from HBV, inhibiting the transcription level of HBV RNAs,

inhibiting the replication level of HBV core DNA, inhibiting the transcription activity of cccDNA,

and inhibiting levels of HBV RNAs, HBV DNA, an HBV S protein and an HBV X protein in liver

tissues.

Further, the drugs for treatment of hepatitis B necessarily include sphondin, and sphondin is

used as an effective component to achieve the functions above.

Further, in the drugs for treatment of hepatitis B, the effective component to achieve the

functions above may only be sphondin and may also contain other molecules which may achieve

similar functions.

Further, sphondin is used as one of the effective components or the only effective component

of the drugs for treatment of hepatitis B.

Further, the drugs for treatment of hepatitis B may be single-component substances or multi

component substances.

Further, the drugs for treatment of hepatitis B are not particularly limited in form and may be

in the form of solid, liquid, gel, semi-liquid, aerosol and other substance forms.

Further, the drugs for treatment of hepatitis B are mainly used to treat mammals, such as

rodents and primates.

In a second aspect, the present invention provides a medicinal preparation for treatment of

hepatitis B, and the medicinal preparation includes a safe and effective dose of sphondin.

Further, the medicinal preparation for treatment of hepatitis B also includes a

pharmaceutically acceptable carrier and/or an adjuvant.

Further, the medicinal preparation for treatment of hepatitis B necessarily includes sphondin,

and sphondin is used as an effective component to achieve the functions above.

Further, in the medicinal preparation for treatment of hepatitis B, the effective component to

achieve the functions above may only be sphondin and may also contain other molecules which may achieve similar functions.

Further, sphondin is used as one of the effective components or the only effective component

of the medicinal preparation for treatment of hepatitis B.

Further, the medicinal preparation for treatment of hepatitis B may be a single-component

substance or a multi-component substance.

Further, the medicinal preparation for treatment of hepatitis B is not particularly limited in

form and may be in the form of solid, liquid, gel, semi-liquid, aerosol and other substance forms.

Further, the medicinal preparation for treatment of hepatitis B is mainly used to treat

mammals, such as rodents and primates.

In a third aspect, the present invention provides a method for treatment of hepatitis B, and

the method includes administering sphondin to a subject.

Further, the subject may be mammals or hepatitis B cells of mammals. The mammals are

preferably rodents, artiodactyls, perissodactyls, lagomorphs, primates and the like. The primates are

preferably monkeys, apes or humans. The hepatitis B cells may be in-vitro hepatitis B cells.

Further, the subject may be a patient suffering from hepatitis B or a hepatitis B individual

expecting treatment. Or, the subject is hepatitis B cells of a patient suffering from hepatitis B or a

hepatitis B individual expecting treatment.

Further, sphondin may be administered to the subject before, during and after treatment of

hepatitis B.

In a fourth aspect, the present invention provides a drug combination for combined treatment

of hepatitis B, and the drug combination includes a safe and effective dose of sphondin, at least one

other drug for treatment of hepatitis B and the balance of a pharmaceutically acceptable carrier and/or

an adjuvant.

Further, the drug combination for combined treatment of hepatitis B may be any one of the

following forms:

(1) Sphondin and other drugs for treatment of hepatitis B are separately prepared into

independent preparations, where dosage forms of the preparations may be the same or different, and administration routes may also be the same or different.

When other drugs for treatment of hepatitis B are antibodies, a parenteral administration route is generally used. When other drugs for treatment of hepatitis B are chemical drugs, there are many administration forms including gastrointestinal administration or non-gastrointestinal administration. It is generally recommended to administer the chemical drugs through already known administration routes.

(2) Sphondin and other drugs for treatment of hepatitis B are prepared into a compound preparation, where sphondin and other drugs for treatment of hepatitis B are administered at the same time in the same administration route, and the combination of both may be prepared into a compound preparation.

In a fifth aspect, the present invention provides a method for treatment of hepatitis B, and the method includes administering an effective dose of sphondin and an effective dose of other drugs for treatment of hepatitis B to a subject, and/or implementing other means for treatment of hepatitis B to the subject.

Further, an effective dose of sphondin and an effective dose of at least one of other drugs for treatment of hepatitis B may be administered simultaneously or sequentially.

Since sphondin is a drug for treatment of hepatitis B discovered for the first time by the present invention, at least an improved treatment effect can be achieved when sphondin is combined with other drugs for treatment of hepatitis B, and a treatment effect on hepatitis B is further improved.

Further, other drugs for treatment of hepatitis B include but are not limited to: antibody drugs, chemical drugs or targeted drugs and the like.

Further, sphondin may be administered in a gastrointestinal administration route or a parenteral administration route, and other drugs for treatment of hepatitis B may be administered in a gastrointestinal administration route or a parenteral administration route. The antibody drugs are generally administered in a parenteral administration route.

In a sixth aspect, the present invention provides uses of sphondin in preparation of substances having any one or more of the following effects: a use for preparing a substance for inhibiting secretion of HBsAg from HBV, a use for preparing a substance for inhibiting the transcription level of HBV RNAs, a use for preparing a substance for inhibiting the replication level of HBV core DNA, a use for preparing a substance for inhibiting the transcription activity of cccDNA, and a use for preparing a substance for inhibiting levels of HBV RNAs, HBV DNA, an HBV S protein and an HBV X protein in liver tissues.

As described above, in the present invention, the use of sphondin as an effective component

in preparation of drugs for treatment of hepatitis B has the following beneficial effects:

it is found for the first time by the present invention that sphondin (Sph for short) can inhibit

secretion of HBsAg from HBV, the transcription level of HBV RNAs, the replication level of HBV

core DNA, the transcription activity of cccDNA and levels of HBV RNAs, HBV DNA, an HBV S

protein and an HBV X protein in liver tissues, has an obvious function in treatment of hepatitis B, can

be used to prepare new anti-HBV drugs which can reduce the HBsAg level and inhibit transcription

of cccDNA, and has a broad application prospect in treatment of hepatitis B.

Brief Description of the Drawings

FIGs. 1A-IF show MTT assay showing the cell viability of different cell lines after a series

of Sph concentration treatment.

FIG. IG is a RNA-seq analysis of the expressions of host factors after 90 M Sph treatment

compared to DMSO treatment.

FIGs. 2A-2B show ELISA assay showing HBsAg and HBeAg secretion levels in cell

supernatant in HBV-infected PHHs (A) and HepG2-NTCP cells (B) after Sph treatment.

FIG. 2C is dot blot assay to detect HBsAg levels in cell supernatant of HBV-infected PHHs

and HepG2-NTCP cells after Sph treatment.

FIGs. 2D-2E show western blotting assay to detect the expression of intracellular HBsAg in

HBV-infected PHHs (D) and HepG2-NTCP cells (E) after Sph treatment.

FIGs. 3A-3D show detection of HBV RNAs species by RT-PCR and northern blotting in

HBV-infected PHHs (A and C) and HepG2-NTCP cells (B and D).

FIGs. 4A-4D show detection of HBV DNA by quantitative PCR and southern blotting assay

in HBV-infected PHHs (A and C) and HepG2-NTCP cells (B and D). HBV DNA was significantly

inhibited both in HBV-infected PHHs and HepG2-NTCP cells.

FIGs. 5A-5D show detection of the inhibitory effects of Sph in different treating period.

Supernatant of HBV-infected PHHs (A) and HepG2-NTCP cells (C) were collected at different points

of time to measure HBsAg levels. Western blotting detected intracellular HBsAg in HBV-infected

PHHs (B) and HepG2-NTCP cells (D) 3, 6, 9 and 12 days after Sph treatment.

FIGs. 6A-6D show detection of the expressions of intracellular HBV RNAs in HBV-infected

PHHs (A and B) and HepG2-NTCP cells (C and D) by RT-PCR and northern blotting 3, 6, 9 and 12

days after Sph treatment.

FIGs. 7A-7C show Actinomycin D assay to detect the effects of Sph on HBV RNAs

degradation in HBV-infected HepG2-NTCP cells. Results showed there was no effect on HBV RNA

stability after Sph treatment.

FIGs. 8A-8C show the nascent RNA synthesis assay to detect the effect of Sph on the

synthesis of HBV RNAs in HBV-infected HepG2-NTCP cells. Results showed Sph significantly

reduced the level of nascent HBV RNAs.

FIGs. 9A-9C show detection of HBV cccDNA in HepAD38 cells and HBV-infected HepG2

NTCP cells by quantitative PCR (A) and southern blotting assay (B-C), and results showed no

significant difference of the HBV cccDNA levels after Sph treatment compared to DMSO in both cell

lines.

FIG. 9D shows the ratio of total RNAs/cccDNA and 3.5 kb RNA/cccDNA, and results

suggested the ratios were reduced in a dose-dependent manner in HBV-infected HepG2-NTCP cells

after Sph treatment.

FIG. 9E is RNA-Seq to detect the effect of Sph on HBV transcripts, and results suggested

that Sph treatment significantly decreased the HBV transcription level.

FIGs. 10A-OB show detection of the combined effect of Sph and ETV on HBV DNA in

HBV-infected HepG2-NTCP cells. (A) RT-PCR showed the inhibitory effect of Sph combined ETV

on HBV DNA. (B) Dose response matrix showed strong inhibitory effect of Sph combined ETV on

HBV DNA.

FIGs. 11A-IIF show detection of the effect of Sph on exogenous and endogenous HBV

proteins. (A-E) Huh-7 cells were transfected with plasmids expressing core (Flag-HBc), small surface

(Flag-HBs), polymerase (Flag-Pol) or HBx (Flag-HBx and HA-HBx), respectively. Cells were treated

with Sph for 72 hours, western blotting analysis was performed using anti-Flag antibody. (F)

Detection of expression of endogenous HBV X protein in HBV-infected HepG2-NTCP cells after Sph

treatment.

FIGs. 12A-12E show detection of antiviral activity of Sph in a humanised liver mouse model

of HBV infection. HBV infected Human liver-chimeric uPA/SCID mice were randomly divided into

three groups, vehicle group, Sph monotherapy group and Sph combined ETV group. (A) Serum

albumin levels of the three groups. (B) Body weight of three groups of mice. (C) Serum HBsAg levels

of the three groups. (D) Serum HBV DNA levels of the three groups.

FIGs. 13A-13D show detection of total HBV RNAs (A), 3.5 kb HBV RNA (B), HBV DNA

(C) and HBV cccDNA (D) levels in liver tissue of the three groups.

FIG.14 shows analysis of the ratio of total RNAs/cccDNA and 3.5 kb RNA/cccDNA in liver

tissues, and results suggested the ratios were significantly reduced after Sph treatment which

compared to vehicles.

FIG.15 is an immunohistochemistry staining to detect the expressions of intrahepatic HBsAg

and HBx levels. Results suggested that Sph could significantly inhibit the expressions of intrahepatic

HBsAg and HBx levels compared to vehicle group while had no influence on albumin levels.

Detailed Description of the Preferred Embodiments

Sphondin, Sph for short, is also named as 6-methoxyangelicin. In the present invention, it is

found through a high-throughput ELISA experiment that Sph can effectively inhibit secretion of

HBsAg from HBV and has no obvious toxicity to multiple liver cancer cell lines and primary human

hepatocytes (PHH). It is confirmed again through Western blot and Dot blot that Sph can effectively

reduce intracellular and extracellular HBsAg. It is proved through an RT-PCR experiment and a

Northern blot experiment that Sph can inhibit the HBV RNA level. At the same time, it is also found

in the present invention that Sph also has a concentration-dependent inhibiting effect on HBV core

DNA. However, it is not found in the present invention that Sph has an obvious inhibiting effect on a

transcription template cccDNA of HBV, but it is interesting that it is found in the present invention

that Sph can effectively reduce the transcription activity of cccDNA. In addition, a human liver

chimeric mouse model is used to perform an in-vivo experiment in the present invention. After mice

are infected with HBV and continuously cultured for 8 weeks, HBV is stably replicated and then

treated with Sph. It is found by testing virus indexes in serum and liver tissues that Sph can

significantly reduce levels of HBsAg and HBV DNA in the serum of the mice and levels of HBV

RNAs, HBV DNA, an HBV S protein and an HBV X protein in the liver tissues.

It is shown through the results above that Sph affects generation of HBV RNAs by inhibiting

the transcription activity of cccDNA, virology indexes such as a viral protein and DNA are further

reduced, and Sph is a potential and new anti-HBV drug which can reduce the HBsAg level and inhibit

transcription of cccDNA at the same time.

Based on this, the present invention provides sphondin as an effective component in

preparation of drugs for treatment of hepatitis B. Generally, the drugs for treatment of hepatitis B

include not only a safe and effective dose of sphondin, but also one or more of pharmaceutically

acceptable carriers or adjuvants according to needs of different dosage forms.

"Pharmaceutically acceptable" indicates that when a molecular ontology and a composition

are properly administered to animals or humans, adverse, allergic or other adverse reactions are not

caused.

"Pharmaceutically acceptable carriers or adjuvants" should be compatible with sphondin, and

that is to say, these substances can be mixed with sphondin without greatly reducing the effect of a

pharmaceutical composition under normal circumstances. Specific examples of some substances

which can be used as pharmaceutically acceptable carriers or adjuvants include sugars, such as lactose,

glucose and sucrose; starches, such as corn starch and potato starch; cellulose and derivatives thereof,

such as sodium methylcellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin;

talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils,

such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols, such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions and phosphate buffers. These substances are used as needed to improve the stability, activity or biological effectiveness of a formula or to produce an acceptable mouthfeel or a smell under oral administration.

In the present invention, unless otherwise specified, drugs are not particularly limited in

dosage forms and can be prepared in the dosage forms such as injection, oral liquid, tablet, capsule,

dripping pill and spray through conventional methods. The dosage forms of the drugs need to be

matched with the administration routes.

In addition, the present invention also provides a drug combination for combined treatment

of hepatitis B and an administration method. The drug combination for combined treatment of

hepatitis B may be any one of the following forms:

(1) Sphondin and other drugs for treatment of hepatitis B are separately prepared into

independent preparations, where dosage forms of the preparations may be the same or different, and

administration routes may also be the same or different. During use, several drugs may be used

simultaneously or sequentially. When several drugs are used sequentially, other drugs are

administered to the body during the period when the former drug is still effective on the body.

When other drugs for treatment of hepatitis B are antibodies, a parenteral administration route

is generally used, such as intravenous injection, intravenous drip or arterial infusion. The prior art may

be taken as a reference for usage and dosage.

When other drugs for treatment of hepatitis B are chemical drugs, there are many

administration forms including gastrointestinal administration or non-gastrointestinal administration.

It is generally recommended to administer the chemical drugs through already known administration

routes.

(2) Sphondin and other drugs for treatment of hepatitis B are prepared into a compound

preparation, where sphondin and other drugs for treatment of hepatitis B are administered at the same

time in the same administration route, and the combination of both may be prepared into a compound

preparation.

It should be noted that combined administration in the present invention refers to reasonable combined administration based on a basic principle of improving the treatment effect and/or reducing adverse reactions. During combined administration, interactions of drugs should include an interaction affecting pharmacokinetics and an interaction affecting pharmacodynamics. During combined administration, drug types should be minimized, so as to reduce adverse drug reactions caused by the drug interactions and avoid reduction of the treatment effect of the drugs, increase of toxicity and opposite results.

Before further describing the specific embodiments of the present disclosure, it is understood that the scope of the present disclosure is not limited to the specific embodiments described below. It is also to be understood that the terminology of the disclosure is used to describe the specific embodiments, and not to limit the scope of the disclosure. The test methods without specific conditions noted in the following embodiments are generally based on conventional conditions or the conditions recommended by the manufacturers.

When the numerical values are given by the embodiments, it is to be understood that the two endpoints of each numerical range and any one between the two may be selected unless otherwise stated. Unless otherwise defined, all technical and scientific terms used in the present disclosure have the same meaning as commonly understood by one skill in the art. In addition to the specific method, equipment and material used in the embodiments, any method, equipment and material in the existing technology similar or equivalent to the method, equipment and material mentioned in the embodiments of the present disclosure may be used to realize the invention according to the grasp of the existing technology and the record of the invention by those skilled in the art.

Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present disclosure all employ conventional techniques of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology in the technical field and related fields.

Embodiment 1

One. Experimental methods

1. Cell culture

HepG2 cells were purchased from American Type Culture Collection (ATCC) and Huh-7

cells were purchased from Health Science Research Resource Bank. HepG2-NTCP cells were kindly

provided by Prof. Ningshao Xia (The Xiamen University, Fujian, China). HepAD38 cells were

purchased from Shanghai Second Military University. Primary human hepatocytes (PHHs) were

purchased from ScienCell (San Diego, USA) and maintained in hepatocyte medium (catalogue no.

5201; Sciencell). Huh-7 and HepG2-NTCP cells were maintained in DMEM supplemented with 10%

fetal bovine serum and 100U/ml penicillin and 100ug/ml streptomycin (Thermo, USA). HepAD38

cells were maintained in DMEM supplemented with 10% fetal bovine serum and1OOU/ml penicillin

and 100ug/ml streptomycin in the presence of 400ug/ml G418 (Merck, German). HepG2-NTCP cells

were maintained in DMEM supplemented with 10% fetal bovine serum and 100U/ml penicillin and

100ug/ml streptomycin in the presence of 2ug/ml Doxycycline (Solarbio, China).All cells were

cultured in a humidified incubator at 37°C with 5% C02.

2. Drugs and plasmids

The natural compounds library including 1124 Chinese medicine monomeric compounds

constructed by ourselves. Sphondin (CAS Number: 483-66-9) was dissolved into 50mM for storage,

and diluted into 1 M to 90 M in cell culture. Entecavir (selleck, CAS Number: 142217-69-4) was

diluted to 25nM. pCH9/3091 was obtained from Prof. Lin Lan (The Army Medical University,

Chongqing, China). HBx-deleted HBV plasmid (HBV-AHBx) was constructed by introducing a stop

codon at the beginning of the HBx gene based on pCH9/3091 (wild-type HBV, HBV WT). The

expression plasmid for 3 X Flag-HBx was amplified by PCR using pCH9/3091 plasmid as template

and subcloned into the p3 X Flag-CMV-7.1 vector using Hind III and XbaI. Transfection of plasmids

was carried out using Lipofectamine 3000TM (Invitrogen, UAS) according to the manufacturer's

instructions.

3. Virus preparation and cell infection

HBV particles were collected from the supernatant of HepAD38 cells. HBV wild type virus

and HBV HBx-deficient virus was collected from the supernatant of Huh-7 cells transfected with HBV

wild type plasmids (HBV WT) or cotransfected with HBV-AHBx and Flag-HBx plasmids.

Concentration of the virus particles was carried out as previously described. Cells were induced 24

hours with PMM before infection, then 1000 MOI of HBV particles or 500 MOI of HBV particles were added to HepG2-NTCP cells or PHHs, respectively in the presence of 5% (v/v) PEG 8000.

4. MTT experiment

Sph was dissolved in DMSO and prepared into a 50 mM stock solution. 1.5*104 Huh-7,

PLC/PRF/5, PHH cells or 2*104 HepG2-NTCP cells and the like were inoculated into a 96-well plate,

and after 24 hours, the Sph stock solution was subjected to doubling dilution with a growth culture

medium to sequentially obtain 9 concentrations including 500 [M, 250 [M, 125 [M, 62.5 [M, 31.25

ptM, 15.6 [M, 7.8 [M, 3.9 M and 1.95 [M; a drug-free treatment group was used as a control, a pure

culture medium was used as a blank, 3 replicate wells were set for each concentration, and 100 [ of

a drug solution was added into each well. After 72 hours, 10 [ of an MTT reagent was added for

incubation at 37C for 4 hours, a supernatant was completely absorbed, 100 [ of a DMSO solution

was added for shaking oscillation for 10 minutes, the OD value was tested at 490 nm, and the CC50

value was calculated.

5. Detection of HBsAg and HBeAg

The levels of HBsAg and HBeAg from cell supernatant were detected using commercial

enzyme-linked immunosorbent assay kits (KHB) according to the manufacturer's instructions. The

serum HBsAg level was quantified by using the Abbott Architect System with HBsAg Reagent Kit

(08P0877, Abbott, Chicago, USA) according to the manufacturer's instructions.

6. Western blotting and Dot blot assay

Cells were lysed by radio immunoprecipitation assay (RIPA) lysis buffer at 4 °C for 20 min.

Then, the cell lysates were subjected to 15% SDS-PAGE and transferred to poly-vinylidene difluoride

(PVDF) membranes. After blocked in 5% nonfat milk, the membrane was incubated with primary

antibody at 4 °C overnight. Next day the membrane was incubated with secondary antibody and the

Immunoreactive signals were detected by ECL western blot reagents (WBKLS0500, Millipore,

Massachusetts, USA). GAPDH was used as a loading control.

For dot blot assay, 4 microliters of the supernatant were spotted onto a nitrocellulose

membrane (GE Healthcare, Buckinghamshire, UK), air dried, soaked in 2.5% formaldehyde-PBS for

min, rinsed with water at room temperature for 5 min, and then soaked in 50% methanol for 30

min. The following procedures were the same as western blotting.

7. Extraction and detection of RNA

After cells were washed with PBS, total RNAs of the cells were extracted by using a TRizol

method, 1 g of RNA was used for reverse transcription by using a Fast KingRT Kit of TIANGEN

company to synthesize cDNA, and then qRT-PCR was performed to detect a target gene.

Total HBV RNAs forward: 5'- ACCGACCTTGAGGCATACTT-3' (SEQ ID NO. 1), and

total HBV RNAs reverse: 5'- GCCTACAGCCTCCTAGTACA-3' (SEQ ID NO. 2); HBV 3.5-kb

mRNA forward: 5'- GCCTTAGAGTCTCCTGAGCA-3'(SEQ ID NO.3), and HBV 3.5-kb mRNA

reverse: 5'-GAGGGAGTTCTTCTTCTAGG-3'(SEQ ID NO.4).

3 replicate wells were set for each sample, and each experiment was repeated 3 times. P- actin

served as an internal reference.

8. Extraction and detection of HBV core DNA

After cells were washed with PBS, 0.5 ml of a cell lysis buffer (containing 10 mM Tris-HCl

with a pH of 8.0, 1 mM EDTA, 1% of NP-40 and 2% of sucrose) was added and uniformly mixed,

the mixture was incubated at 37°C for 15 minutes, and 15000 g of a cell lysis buffer was collected and

centrifuged for 5 minutes. A supernatant was transferred, 40 U/ml of DNaseI and 10 mM MgCl2 were

added, the mixture was incubated at 37°C for 4 hours, 200 1 of 35% PEG8000 (containing 1.5 mol/L

of NaCl) was added, treatment with an ice bath was performed for 1 hour, 11,000 g of the mixture

was centrifuged at 4°C for 10 minutes, a supernatant was removed, and 0.5 ml of a proteinase K

digestion solution (containing 0.5% of SDS, 150 mM NaCl, 25 mM Tris-HCl with a pH of 8.0 and 10

mM EDTA) and 0.5 mg/ml of a proteinase K (promega) were added for a water bath at 45°C overnight.

On the next day, phenol and chloroform equal in volume were used for extraction, 70% ethanol was

used for precipitation, and sterilized ddH20 was used to dissolve HBV DNA. A product was

quantified by using q-PCR. An HBV DNA primer, F: CCTAGTAGTCAGTTATGTCAAC (SEQ ID

NO. 5), R: TCTATAAGCTGGAGGAGTGCGA (SEQ ID NO. 6). 3 replicate wells were set for each

sample, and each experiment was repeated 3 times.

Southern blotting was performed following the DIG-High Prime DNA Labeling and

Detection Starter Kit (11585614910, Roche, Germany). Briefly, DNA samples were separated on a

0.9% agarose gel and then transferred onto a nylon membrane overnight (Roche, Germany). After UV crosslinking and pre-hybridization, the membrane was hybridized with DIG-labeled full-length HBV genome probe at 42 °C overnight. Next day, the membrane was washed in different concentration of

SSC/SDS, washing buffer, detection buffer. After blocking and incubating with anti-DIG antibody,

then placed membrane with DNA side facing up on a development folder and applied 1 ml CSPD

ready-to-use. After incubating for 5 min at +15 to +25°C, the signal was detected using X-ray film.

9. Hirt cccDNA extraction and detection

Cells were lysed in 500 L cell lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 150

mM NaCl, 1% SDS) at 37C for 20 min. Then, 125 1 of 2.5 M KCl was added to the cell lysate, and

the solution was stationary overnight at 4°C. The next day, after centrifugation at 12,000 g for 20 min,

twice, the supernatant was collected and extracted by using phenol/chloroform/isopropanol reagent

for twice and DNA was precipitated by adding the same volume of isopropanol. Then, the DNA

precipitation was washed with 70% ethanol and dissolved in double distilled water.

HBV cccDNA levels were detected by Taq-man probe qRT-PCR. Before used as the

templates for Taq-man probe qRT-PCR, the sample was treated as follows: 1) Denaturate rcDNA:

heat at 800 C for 5 minutes, immediately place on ice to cool; 2) Digest rcDNA: incubating with

exonuclease V (M0345S, New England Biolabs, MA, USA) at 37 C for 30 min; 3) Denature

cccDNA: Heating at 1000 C for 20 min to denature cccDNA. After the above processing, samples

were subjected to Taq-man probe qRT-PCR for cccDNA quantification. The PCR reaction contains

0.8 M of primers and 0.2 M of probe and the thermal cycling conditions are as follow: 10 min at

°C, 45 cycles of 15 s at 95 °C and 30 s at 64 °C.

10. Northern blotting

Total RNA was detected by DIG Northern Starter Kit (12039672910, Roche, Mannheim,

Germany) following manufacturer's instructions. The extracted RNA was separated by 1.4%

formaldehyde-agarose gel. The RNA was transferred onto nylon membrane by capillary siphon

method overnight. After UV-crosslinking, the membrane was hybridized with DIG-labeled HBV

RNA probe, and washed in different concentration of SSC/SDS, washing buffer and detection buffer.

Then blocked and incubated with secondary antibody, next placed membrane with RNA side facing

up on a development folder and applied 1 ml CDP ready-to-use. After incubating for 5 min at +15 to

+25°C, the signal was detected using X-ray film. The 28S/18S RNA was set as internal control.

11. Nascent RNA synthesis assay and Actinomycin D assay

HepG2-NTCP cells in a 12-well plate were labelled by addition of 5-ethynyl uridine (EU, an

alkyne-modified nucleoside) at a final concentration of 0.5 mM to the cell culture medium. Cells were

incubated with -/+ EU at 37C for 1 h and washed once with 1x PBS. Then, the cells were lysed in

TRNzol Reagent (TIANGEN, China), and total RNA was extracted according to the manufacturer's

instructions. To enrich, EU-labelled nascent RNA was biotinylated through a click reaction. Then, the

biotinylated EU-labelled nascent RNA was precipitated by incubation overnight at -70°C. Streptavidin

TI was bound to magnetic beads, and 50 L of Dynabeads@ MyOneTM Streptavidin TI magnetic

beads was mixed with 500 ng of biotinylated total RNA. The beads were washed 3 times with 500 p

L of Click-iT@ reaction wash buffer 2 (Thermo). After the last wash, RNA was eluted into 40 pl

DEPC-treated water by heating at 95°C for 10 minutes. First-strand cDNA was synthesized from 1 g

of RNA using an iScriptTM cDNA Synthesis kit (Bio-Rad). Relative RNA expression levels were

quantified with FastStart Universal SYBR Green Master Mix (Roche), and p-actin mRNA was used

as an endogenous control. Values were analysed using the 2^-AACt method. (QuantStudio 6 Flex,

Applied Biosystems).

12. RNA-seq

After the RNA concentration and quality were determined with a [email protected] Fluorometer

(Life Technologies, U.S.A.) and a Nanodrop One spectrophotometer (Thermo Fisher Scientific Inc.,

U.S.A.), total RNA was isolated using the RNeasy mini kit (Qiagen, Germany). The integrity of total

RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., U.S.A.), and

samples with RNA integrity number (RIN) values above 7.0 were used for sequencing. One

microgram of RNA was used as input material for RNA sample preparations. RNA-seq strand-specific

libraries were constructed using a VAHTS Total RNA-seq(H/M/R) Library Prep Kit (Vazyme, China)

according to the manufacturer's instructions. Briefly, RNA was purified with magnetic beads after

removal of rRNA. Then, the RNA was fragmented into small pieces using divalent cations for 8 min

at 94oC. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase

and random primers. Second strand cDNA synthesis was subsequently performed using DNA

Polymerase I and RNase H. These cDNA fragments were then subjected to the end repair process, addition of a single 'A' base, and ligation of the adapters. The products were purified and enriched via PCR to create the final cDNA library. Purified libraries were quantified and validated using the [email protected] Fluorometer and Agilent 2100 Bioanalyzer to confirm the insert size and calculate the molar concentration. Clusters were generated with cBot after the library was diluted to 10 pM, and then were sequenced on the Illumina NovaSeq 6000platform (Illumina, U.S.A.). The library construction and sequencing were performed by Sinotech Genomics Co., Ltd (Shanghai, China).

13. Mouse model construction and drug treatment

15 human liver-chimeric Alb-uPA/SCID mice were purchased from Beijing Vitalstar Biotechnology Co., Ltd. (Beijing, China). Fresh serum of a patient was injected into the mice through a caudal vein, the replication level of HBV in the mice was tested after the mice were subjected to normal feeding for 8 weeks, and the mice were randomly divided into 3 groups: a control group (injected with an equal volume of a solute without drugs), an Sph group (2.5 mg/kg/2 days, abdominal administration) and an ETV+Sph group (ETV: 0.02 mg/kg/2 days, intragastric administration). Blood collection was performed from the eye orbits every 4 days, the serum was separated and cryopreserved at -80°C, the mice were killed by cervical dislocation at the 7th week, blunt dissection was performed on livers of the mice, a large and complete tissue piece was cut out for paraffin-embedded sectioning, the remaining tissues were to be ground, and DNA and RNA were extracted.

14. Extraction of mouse serum virus DNA

A Biospin virus DNA extraction kit (BioFlux) was used, 10 pl of serum was added into 190 ul of normal saline, 10 1 of a proteinase K was added into a new 1.5 ml centrifuge tube, and then 200 1 of a lysis buffer was added for uniform mixing and shaking for 5-10 seconds. The mixture was incubated at 56°C for 15 minutes. 200 1 of absolute ethanol was added into the centrifuge tube above for thorough and uniform mixing and shaking for 5-10 seconds. Viral DNA was extracted with a purification column in the kit according to manufacturer's instructions of the kit, and a DNA sample was preserved at -20°C.

15. Extraction of tissue DNA and RNA

Mouse livers were separated, tissue pieces were ground into dry powder in liquid nitrogen, about 20 mg of the dry powder was weighed and added into a 1.5 ml centrifuge tube, a DNA lysis buffer and Trizol were separately added, and polishing was performed. After polishing and homogenizing were performed, DNA was extracted with a Biospin tissue genomic DNA extraction kit (BioFlux), 600 1 of an FL buffer and 10 1 of a PK solution were added and mixed thoroughly and uniformly, a small disperser (T10 basic ULTRA-TURRAX@) was used for auxiliary lysis, the mixture was incubated at 56°C for 15 minutes to fully lyse tissues, viral DNA was extracted with a purification column in the kit according to manufacturer's instructions of the kit, and a DNA sample was preserved at -20°C. RNA was extracted by using a cell RNA extraction method.

16. Immunohistochemistry

Paraffin-embedded sections were put into an oven for standing overnight at 55°C; on the next

day, the paraffin-embedded sections were continuously baked at 95°C for 10 minutes; then the

paraffin-embedded sections were sequentially put into xylene and concentration gradient of ethanol

for dewaxing treatment. Antigen retrieval was performed twice in a microwave, and then 0.5% Triton

100/PBS was added to permeate a membrane for 20 minutes; an endogenous peroxidase blocker was

added to remove an endogenous peroxidase; goat working serum was added dropwise to block non

specific sites; an HBs antibody was added for incubation overnight; on the next day, after the sections

were washed, a biotin-labeled secondary antibody and a streptavidin-peroxidase reagent were added

dropwise in sequence; finally, a newly prepared DAB color development reagent was added dropwise

for color development for 3-5 minutes; hematoxylin was added for redyeing at room temperature for

1 minute, and the sections were turned blue when rinsed with tap water; the sections were dehydrated,

sealed and observed under a microscope.

17. Statistical processing

The results are expressed as the meanSD. Statistical analyses were performed using either

Student's t-test or the Mann-Whitney U test and by one-way ANOVA. Differences were considered

significant when P<0.05. All statistical analysis was performed by using SPSS 19.0 software.

Two. Experimental results

1. The cytotoxicity of sphondin detected through an MTT experiment

As shown in FIG. 1, it is shown through results of MTT that CC50 of sphondin (Sph) on

multiple liver cancer cell lines and PHH cells is higher than 500 [M, and it is shown through RNA- seq that Sph has no significant effect on human liver specific factors after action, indicating that Sph has little toxicity to cells.

2. Inhibition of the expression of HBsAg and HBeAg by sphondin

HBV-infected PHH and HepG2-NTCP cells are treated with Sph to detect the secretion level

and intracellular expression level of HBsAg and HBeAg after drug treatment. Results in FIGs. 2A-2B

indicated that sphondin could inhibit the expression of HBsAg and HBeAg. Results in FIGs. 2C-2E

suggested sphondin could significantly inhibit the expression of HBsAg both in supernatant and

intracellular in a dose-dependent manner compared to DMSO. Results in FIG. 5 suggested sphondin

decreased the HBsAg both in supernatant and intracellular in a time-dependent manner.

3. Inhibition of the transcription level of HBV RNAs by sphondin

HBsAg is translated from HBV 2.4/2.1-kb RNA and used to detect whether HBV RNAs are

inhibited by Sph, HBV-infected PHH and HepG2-NTCP cells are treated with different concentrations

of Sph, and the cells at different time points are collected to extract total RNAs. It is shown through

results of qRT-PCR and Northern blot that HBV RNAs (including 3.5-kb RNA and 2.4/2.1-kb RNA)

are reduced by Sph in a dose-dependent manner (FIG. 3) and time-dependent manner (FIG. 6), and

EC50 is 23.48 M and 19.24 M (PHHs) and 9.26 M and 6.14 M (HepG2-NTCP cells) respectively

(FIG. 3).

4. Inhibition of the replication level of HBV core DNA by sphondin

HBV 3.5-kb RNA can be further subjected to reverse transcription to generate HBV core

DNA, so as to detect whether Sph can also inhibit the HBV core DNA level; similarly, HBV-infected

PHH and HepG2-NTCP cells are treated with concentration gradient of Sph, and the HBV core DNA

level in the cells is detected by using q-PCR and Southern blot. It is shown through results that the

HBV core DNA level is reduced by Sph in a dose-dependent manner (FIG. 4).

5. Inhibition of the transcription activity of cccDNA by sphondin

After the inhibiting effect of Sph on various virology indexes of HBV is clear, the effect of

Sph on a transcription template cccDNA of HBV is further tested. It is shown through Taq-Man probe

PCR that Sph has no obvious inhibiting effect on the replication level of cccDNA, but it is interesting

that Sph can effectively inhibit the transcription activity of cccDNA; at the same time, it is also found through RNA-Seq that Sph can effectively reduce levels of HBV transcripts (FIG. 9).

6. Inhibition of the synthesis ofnascent HBV RNA by sphondin

Results of nascent RNA synthesis assay indicated that sphondin significantly reduced the

level of nascent HBV RNAs (FIG. 8).

7. Test on in-vivo toxicity of sphondin

In order to explore an in-vivo antiviral effect of Sph, a toxic effect of Sph in mice is tested

first. 15 BALB/c mice at the age of 6-8 weeks are randomly divided into 3 groups, treated with

different doses of Sph (including a 0 mg/kg group, a 2.5 mg/kg group and a 5 mg/kg group) and

administered once every two days through abdominal administration, and the body weight of the mice

is monitored at the same time. After the mice are continuously treated for 28 days, blood collection is

performed from eyeballs, and blood routine indexes and blood biochemical indexes are tested. As

shown in Table 1, treatment with Sph has no significant effect on the body weight, white blood cells,

red blood cells, hemoglobin, platelets, total proteins, albumin, ALT, AST, GGT, total bilirubin, serum

creatinine and blood urea of the mice. It is shown that sph has little toxicity in the body and has no

obvious toxicity to liver and kidney. Therefore, sph with a concentration of 2.5 mg/kg is used in

following experiments (Table 1).

Table 1 Effects of sphondin on the body weight, blood routine indexes and blood biochemical

indexes of mice

Sph Project/Unit 0 mg/kg/2 day 2.5 mg/kg/2 day 5 mg/kg/2 day

NC group Low concentration High concentration

WBC *10 3/ L 7.08+0.17 5.95±1.91ns 6.8±2.1s

RBC *106/ L 11.72+0.26 11.56±0.33"n 11.68±0.38"n

HGB g/L 165.25+1.92 162±4.06"n 162.75±6.3"n

HCT % 52.7±0.48 51.93±1.28"n 51.71±1.39"n

PLT *10 3 4/L 874.6+347.48 584.2±107.06"n 753.55+239.41"s

MCV fL 45±1.01 44.95+0.69"s 44.29±1.44"n

MCH pg 14.1+0.14 14.03±0.18"n 13.96±0.21s

MCHC g/L 313.75±4.6 312±2.24"n 314.85±9.82"s

LYMPH *10 9/FL 4.99+0.19 4.07±1.4Os 4.685±1.53"n

BUN mg/dL 13+2.92 14.75±1.3"n 14±1.87"n

TP g/dL 5.75+0.36 5.45±0.23"n 5.5±0.47"n

ALB g/dL <0.1I <0.1"ns <0.1"ns

ALT U/L 67.75+14.08 69.25±3.77"n 67.25+7.29"s

AST U/L 118.25+5.67 116.75±10.11s 124.25±19.14"n

ALKP U/L 134.75+14.18 115.5+15.79"s 139.75±21s

GGT U/L 0.75+1.3 0.25±0.43"n 1.25±0.43"n

TBIL mg/dL 0.475+0.33 0.2250.11"s 0.23±0.04"n

All values in Table 1 are expressed as mean standard deviation.

Abbreviations in Table 1: WBC, white blood cell; RBC, red blood cell; HGB, hemoglobin;

HCT, hematocrit; PLT, platelets; MCV, mean corpusular volume; MCH, mean corpusular hemoglobin;

BUN, blood urea nitrogen; TP, total protein; ALB, albumin; ALT, alanine transaminase; AST,

aspartate transaminase; ALKP, alkaline phosphatase; GGT, y-glutamyl transpeptidase; TBIL, total

bilirubin.

Compared with the sphondin group with a concentration of 0 mg/kg/2 days, the sphondin

group with a concentration of 2.5 mg/kg/2 days has no significant effect (p>0.05).

8. Test of an in-vivo antiviral effect of sphondin by using a human liver chimeric mouse

model

Human liver chimeric mice infected with HBV for 8 weeks are randomly divided into 3

groups (including a control group, a 2.5 mg/kg group and an ETV+sph group) with 4 mice in each

group. The mice are administered once every 2 days, and blood collection is performed from the eye

orbits every 4 days. The mice are killed by cervical dislocation after treatment for 7 weeks, and then

livers are taken. Levels of HBsAg and HBV DNA in serum of the mice and levels of HBV RNAs,

HBV DNA, HBs and an HBx protein in liver tissues are tested. It is shown through results that Sph

can effectively reduce the levels of HBsAg and HBV DNA in the serum and the levels of HBV RNAs,

HBV DNA and viral proteins in the liver tissues, and especially has an obvious inhibiting effect on

HBV RNAs, HBsAg and HBx; in addition, an antiviral effect can be improved after Sph is combined with ETV (entecavir) (FIGs. 12-15).

The above-mentioned embodiments are merely illustrative of the principle and effects of the

present disclosure instead of limiting the present disclosure.

Modifications or variations of the above-described embodiments may be made by those

skilled in the art without departing from the spirit and scope of the disclosure.

Therefore, all equivalent modifications or changes made by those who have common

knowledge in the art without departing from the spirit and technical concept disclosed by the present

disclosure shall be still covered by the claims of the present disclosure.

Where any or all of the terms "comprise", "comprises", "comprised" or "comprising" are used

in this specification (including the claims) they are to be interpreted as specifying the presence of the

stated features, integers, steps or components, but not precluding the presence of one or more other

features, integers, steps or components.

A reference herein to a patent document or any other matter identified as prior art, is not to

be taken as an admission that the document or other matter was known or that the information it

contains was part of the common general knowledge as at the priority date of any of the claims.

Claims (5)

What is claimed is:

1. The use of sphondin as an effective component in preparation of drugs for treatment of hepatitis

B.

2. The use according to claim 1, wherein the drugs for treatment of hepatitis B have at least one of

the following functions:

inhibiting secretion of HBsAg from HBV;

inhibiting a transcription level of HBV RNAs;

inhibiting a replication level of HBV core DNA in a concentration-dependent manner;

inhibiting a transcription activity of cccDNA;

inhibiting levels of HBV RNAs, HBV DNA, an HBV S protein and an HBV X protein in liver

tissues.

3. A medicinal preparation for treatment of hepatitis B, comprising a safe and effective dose of

sphondin.

4. A drug combination for combined treatment of hepatitis B, comprising a safe and effective dose

of sphondin and at least one other drug for treatment of hepatitis B.

5. Uses of sphondin in preparation of substances having any one or more of the following effects: a

use for preparing a substance for inhibiting secretion of HBsAg from HBV, a use for preparing a

substance for inhibiting a transcription level of HBV RNAs, a use for preparing a substance for

inhibiting a replication level of HBV core DNA, a use for preparing a substance for inhibiting a

transcription activity of cccDNA, and a use for preparing a substance for inhibiting levels of HBV

RNAs, HBV DNA, an HBV S protein and an HBV X protein in liver tissues.