Drugs and methods for limiting the use of nucleoside (nucleotide)-based drugs in the treatment of chronic hepatitis B

Abstract

The present invention relates to a drug and method for terminating nucleoside (nucleotide) drug treatment for chronic hepatitis B, specifically to the use of an HBV virus entry inhibitor and an optional immunomodulator in the preparation of a drug or kit for terminating or terminating nucleoside (nucleotide) drug treatment for chronic hepatitis B. According to the method of the present invention, after administering a nucleoside (nucleotide) drug, an HBV virus entry inhibitor and an optional immunomodulator for a certain course of combined treatment, the drugs can be completely discontinued, and the patient does not need to continue NAs treatment.

Description

[Technical field]

[0001] The present invention relates to drugs and methods for limiting the use of nucleoside (nucleotide)-based drugs in the treatment of chronic hepatitis B. [Background technology]

[0002] Currently, only nucleoside (nucleotide) drugs and interferon are approved for the treatment of chronic hepatitis B. Nucleoside (nucleotide) drugs inhibit HBV replication by blocking HBV DNA polymerase, and the nucleoside (nucleotide) drugs currently approved by the FDA for the treatment of hepatitis B include Lamivudine, Adefovir Dipivoxil, Entecavir, Tenofovir Disoproxil (TDF), Telbivudine, and Tenofovir Alafenamide Fumarate (TAF). After treatment with nucleoside (nucleotide) drugs, the patient's viral DNA level is better suppressed, for example, the recently developed Tenofovir not only has excellent safety but also has a viral DNA inhibition rate of about 70%. Nucleoside drugs can effectively inhibit viral replication, but they require long-term administration and often cause recurrence or rebound after drug discontinuation, making NAs treatment for hepatitis B an infinite therapy.

[0003] TAF is an antiviral drug developed by Gilead and was approved by the FDA in 2016 for the treatment of chronic hepatitis B. TAF is a new nucleotide reverse transcriptase inhibitor. Once it enters liver cells, it is hydrolyzed to tenofovir, which is then phosphorylated to block viral replication. As an upgraded version of tenofovir disoproxil (TDF), TAF retains the advantages of TDF's "high antiviral effect, low drug resistance", while also partially overcoming the disadvantages of TDF's nephrotoxicity and bone density damage. Compared with TDF, with the same therapeutic effect, the dosage of TAF is smaller, and the risk of osteoporosis and nephrotoxicity is lower. Although TAF has many advantages, it still has the problem of long-term administration. Currently, there is no unified standard for the withdrawal of nucleotide analogues. According to the recommendation opinion of the "Guidelines for the Prevention and Treatment of Chronic Hepatitis B (2019 Edition)", for patients with chronic hepatitis B who are HBeAg(+), if after one year of treatment, HBV DNA falls below the lower limit of detection, ALT returns to normal, and HBeAg serological conversion occurs, treatment should be continued for at least another three years (retested every six months) to consolidate the effect, and if there is no change, drug suspension may be considered; for patients with HBeAg(-), it is recommended to discontinue taking the drug and follow up after HBeAg disappears and HBV DNA becomes undetectable. It is almost impossible to eliminate HBsAg antigen simply by using nucleoside (nucleotide) drugs. A large amount of clinical data shows that the recurrence rate after drug suspension of nucleoside drugs is high. For HBeAg-positive and HBeAg-negative patients, the patient's age, HBV DNA level, consolidation period, and other factors affect the recurrence rate after drug suspension. For the majority of patients receiving NAs therapy, safe withdrawal of NAs drugs is urgently needed, and methods for finitelization of infinite NAs therapy are urgently required.

[0004] Interferon is a cytokine with a broad spectrum of antiviral effects, originally used to treat HIV and other viruses, and approved by the FDA in 1992 for the treatment of hepatitis B. Its mechanism of action is to exert its antiviral effect mainly through immune modulation and induction of antiviral proteins in hepatocytes. A meta-analysis showed that after treating HBeAg-positive patients with conventional interferon α (conventional IFNα) for 4 to 6 months, the HBV DNA negative conversion rates (hybridization method) in the treated and untreated groups were 37% and 17%, respectively, the HBeAg negative conversion rates were 33% and 12%, respectively, and the HBsAg negative conversion rates were 7.8% and 1.8%, respectively, and the therapeutic efficacy was positively correlated with baseline serum ALT levels and the extent of liver histological lesions. Four randomized controlled trials in HBeAg-negative patients showed that response rates at the end of treatment ranged from 38% to 90%, but sustained response rates were only 10% to 47% (mean 24%). A typical course of IFNα treatment takes at least 1 year to achieve better results. In an international multicenter randomized controlled clinical trial, HBeAg-positive chronic hepatitis B patients (87% Asian) were treated with pegylated interferon α-2a (PegIFNα-2a) (40KD) for 48 weeks and followed up for 24 weeks after treatment, with an HBeAg seroconversion rate of 32%; in HBeAg-negative patients (60% Asian) treated for 48 weeks and followed up for 24 weeks, 43% of patients had HBV DNA levels of 2 × 10 4 copies / ml and was shown to be 42% at 48 weeks follow-up.

[0005] In recent years, long-term follow-up of patients undergoing interferon therapy has shown that, although there are many adverse events, the long-term benefits of interferon therapy are becoming increasingly evident. After just one course of treatment with interferon, the negative conversion rate of viral surface antigens continues to increase with the extension of the follow-up period (Hannah SJ Choi et al. Clinical Gastroenterology and Hepatology, 2020). Interferon treatment has serious side effects at current doses, and patients have poor compliance with treatment. These adverse events include: influenza-like syndromes such as fever, chills, headache, myalgia, and fatigue; transient peripheral cytopenias manifesting mainly as peripheral blood leukocyte (neutrophil) and thrombocytopenia; psychiatric disorders manifesting as depression, delusions, severe anxiety, and other psychiatric symptoms; autoimmune diseases in which some patients may develop autoantibodies and only a minority of patients develop thyroid disease (hypo- or hyperthyroidism), diabetes, thrombocytopenia, psoriasis, vitiligo, rheumatoid arthritis, and systemic lupus erythematosus-like syndromes; and other rare adverse events such as renal impairment (interstitial nephritis, nephrotic syndrome, acute renal failure, etc.), cardiovascular complications (arrhythmias, ischemic heart disease, cardiomyopathy, etc.), retinopathy, hearing loss, and interstitial pneumonia. The current standard treatment course for interferon therapy is 48 weeks.

[0006] In order to achieve a finite treatment course and remove the limitations of long-term use of nucleoside drugs, industry and academia have tried combination or sequential treatment of interferon and nucleoside drugs, but no increase in therapeutic efficacy was found. Some academic studies have shown that combination therapy of interferon and tenofovir is superior to monotherapy, but the long-term treatment of this combination is unclear and is not recommended in clinical practice or included in guidelines (M Vigano et al., Alimentary Pharmacology and Therapeutics, 44(7),653-661).

[0007] The virus entry inhibitor includes a polypeptide derived from the HBV Pre-S1 region and an anti-Pre-S1 antibody or an anti-surface antigen antibody. The polypeptide derived from the HBV Pre-S1 region includes, but is not limited to, heparatide and bulevirtide. The heparatide amino acid sequence is derived from the amino acid sequence at positions 13-59 of the Pre-S1 region of genotype C HBV and has a myristic acid modification at its N-terminus. Research shows that sodium taurocholate cotransporting polypeptide (NTCP) is an HBV infection receptor, and HBV specifically binds to NTCP via the Pre-S1 region of a large protein on the surface of the viral envelope, mediating HBV infection of hepatocytes. Heparatide and bulevirtide specifically bind to NTCP and block HBV infection of hepatocytes in vitro and in vivo. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Hannah SJ Choi et al. Clinical Gastroenterology and Hepatology, 2020 [Non-Patent Document 2] M Vigano et al., Alimentary Pharmacology and Therapeutics, 44(7),653-661 Summary of the Invention [Means for solving the problem]

[0009] A first aspect of the present invention provides the use of an HBV viral entry inhibitor and an optional immunomodulator in the preparation of a medicament or kit for terminating or terminating nucleoside (nucleotide) drug treatment of chronic hepatitis B.

[0010] In one or more embodiments, the HBV viral entry inhibitor is selected from an HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region or an HBV Pre-S1 derivative peptide, an anti-Pre-S1 antibody, an anti-surface antigen antibody or other drug that inhibits HBV entry or infection of liver cells.

[0011] In one or more embodiments, the HBV entry-inhibitory polypeptide derived from the HBV pre-S1 region is derived from the pre-S1 region of a surface antigen of any one of HBV genotypes A, B, C, D, E, F, G, H and I.

[0012] In one or more embodiments, the length of the HBV entry inhibitor polypeptide derived from the HBV Pre-S1 region is 10-118 amino acid residues.

[0013] In one or more embodiments, the immunomodulatory agent is selected from an interferon, a toll-like receptor agonist, a CPG, a CPG ODN, a PD-1 inhibitor, a PD-L1 inhibitor, an interleukin, and a cytokine.

[0014] In one or more embodiments, the interferon is selected from IFN-α, IFN-β, and IFN-γ, preferably IFNα-2a and / or IFNα-2b, more preferably PEG IFNα-2a and / or PEG IFNα-2b.

[0015] In one or more embodiments, the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region is derived from the pre-S1 region of HBV genotype C surface antigen; preferably, the HBV entry-inhibiting polypeptide is a fragment or a variant thereof of the N-terminus of the pre-S1 region of HBV genotype C surface antigen, the fragment containing at least the amino acid residues at positions 13-44 of the N-terminus; preferably, the HBV entry-inhibiting polypeptide contains the amino acid residues at positions 2-119, 2-69, 2-59, 13-119, 13-88, 13-72, 13-67, 13-59, 13-52 or 13-47 of the pre-S1 region of HBV genotype C surface antigen, or a variant thereof.

[0016] In one or more embodiments, the HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region is a fragment or a variant of the N-terminus of the pre-S1 region of HBV surface antigen of genotype A, B, F, H or I, which fragment contains at least amino acid residues 13-44 of the N-terminus, and preferably contains at least amino acid residues 13-69 of the N-terminus.

[0017] In one or more embodiments, the HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region is a fragment or a variant of the N-terminus of the pre-S1 region of the D genotype HBV surface antigen, the fragment containing at least amino acid residues 2-33 of the N-terminus, and preferably containing at least amino acid residues 2-48 of the N-terminus.

[0018] In one or more embodiments, the HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region is a fragment or a variant of the N-terminus of the pre-S1 region of the E, G genotype HBV surface antigen, the fragment containing at least the amino acid residues 12-43 of the N-terminus, and preferably containing at least the amino acid residues 12-68 of the N-terminus.

[0019] In one or more embodiments, the variant has a deletion, substitution or insertion of 1-30 amino acids compared to the fragment, and the variant retains biological activity of inhibiting HBV entry or binding to NTCP.

[0020] In one or more embodiments, the variant comprises an insertion at the N- and / or C-terminus of the fragment of naturally occurring flanking amino acid sequences derived from any one of the HBV subtypes.

[0021] In one or more embodiments, the native flanking amino acid sequence is selected from the amino acid sequence of positions 2-12 at the N-terminus of the pre-S1 region of the HBV surface antigen of genotypes A, B, F, H, and I, and the amino acid sequence of positions 2-11 at the N-terminus of the pre-S1 region of the HBV surface antigen of genotypes E and G.

[0022] In one or more embodiments, the mutant is a derivative peptide derived from the amino acid sequence at positions 13-59 of the Pre-S1 region of HBV genotype C, into which one or a combination of any two or more of the following amino acid substitutions have been introduced: N15D, F25L, G35K, N39E, F45L, N46K, N48H or N48Y or N48K, D50A, H51Q or H51N, E54K or E54D, A55S, N56K or N56D, and Q57K; optionally, native flanking amino acid sequences derived from any one of the HBV subtypes are inserted at the N- and / or C-terminus of the mutant.

[0023] In one or more embodiments, the amino acid sequence of the fragment is set forth in any one of SEQ ID NOs:2-32 or has at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs:2-32.

[0024] In one or more embodiments, the HBV entry inhibitor polypeptide derived from the HBV Pre-S1 region has an N-terminal modification and / or a C-terminal modification.

[0025] In one or more embodiments, the N-terminal modification is a hydrophobic group modification.

[0026] In one or more embodiments, the hydrophobic modification is selected from myristic acid, palmitic acid, stearic acid, cholesterol, oleic acid, linoleic acid, polyethylene glycol, and arachidonic acid.

[0027] In one or more embodiments, the C modification is an amidation or isoprene glycolation modification.

[0028] In one or more embodiments, the HBV entry inhibitor polypeptide derived from the HBV Pre-S1 region is set forth in SEQ ID NO:33-44.

[0029] In one or more embodiments, the use is the use of an HBV viral entry inhibitor, a nucleoside (nucleotide) drug used in the treatment of chronic hepatitis B, and an optional immunomodulator in the preparation of a drug or kit for terminating or terminating nucleoside (nucleotide) drug treatment of chronic hepatitis B.

[0030] In one or more embodiments, the nucleoside (nucleotide) drug is selected from Lamivudine, Adefovir Dipivoxil, Entecavir, Tenofovir Disoproxil, Telbivudine, and Tenofovir Alafenamide Fumarate, preferably Tenofovir Alafenamide Fumarate.

[0031] In one or more embodiments, the kit contains one or more doses of the HBV viral entry inhibitor to administer the drug at a daily dose of 2.1-10.5 mg, preferably 4.2-8.4 mg, more preferably 4.2 mg.

[0032] In one or more embodiments, the kit optionally contains one or more doses of the interferon, and the amount satisfies a weekly dose of 1-360 micrograms, preferably 30-180 micrograms, more preferably 60-135 micrograms, more preferably 90 micrograms.

[0033] In one or more embodiments, the kit is used for co-administration with the nucleoside (nucleotide) class drug for 1-96 weeks, preferably 12-60 weeks, more preferably 24-48 weeks.

[0034] In a second aspect of the present invention, there is provided a method for limiting or terminating nucleoside (nucleotide) drug treatment for a chronic hepatitis B patient, the method including administering an HBV viral entry inhibitor and an optional immunomodulator to the chronic hepatitis B patient in addition to the nucleoside (nucleotide) drug to perform combination treatment, and further terminating the nucleoside (nucleotide) drug treatment, the HBV viral entry inhibitor treatment and the immunomodulator treatment after a certain course of combination treatment.

[0035] In one or more embodiments, the nucleoside (nucleotide) drug is as described in any one of the embodiments herein; the HBV viral entry inhibitor is as described in any one of the embodiments herein, and the immunomodulator is as described in any one of the embodiments herein.

[0036] In one or more embodiments, the duration of the combination treatment is from 1-96 weeks, preferably from 12-60 weeks, and more preferably from 24-48 weeks.

[0037] In one or more embodiments, the patient has received continuous NAs treatment prior to administering the combination treatment, preferably for more than 0 months of NAs treatment, more than 3 months of NAs treatment, more than 12 months of NAs treatment, or more than 36 months of NAs treatment.

[0038] In one or more embodiments, the daily dose of the HBV viral entry inhibitor is 2.1-10.5 mg, preferably 4.2-8.4 mg, more preferably 4.2 mg.

[0039] In one or more embodiments, the immunomodulatory agent is an interferon, the weekly dosage of which is 1-360 micrograms, preferably 30-180 micrograms, more preferably 60-135 micrograms, more preferably 90 micrograms.

[0040] In one or more embodiments, the nucleoside (nucleotide) drug is tenofovir alafenamide fumarate and the HBV viral entry inhibitor is heparatide or bulevirtide.

[0041] In one or more embodiments, the heparatide or bulevirtide treatment cycle is from 1 week to 96 weeks.

[0042] In one or more embodiments, the therapeutic dose of heparatide or bulevirtide is 2.1 mg to 8.4 mg daily.

[0043] In one or more embodiments, the immunomodulatory agent is pegylated interferon, preferably at a dose of 10-170 ug weekly. In one or more embodiments, in the method, the nucleoside (nucleotide) drug is tenofovir alafenamide fumarate; it is treated in combination with heparatide or bulevirtide, the treatment cycle is 48 weeks, and the dose is 4.2 mg daily; and simultaneously, pegylated interferon is administered at a dose of 90 ug weekly, and the treatment cycle is 48 weeks. [Brief description of the drawings]

[0044] [Figure 1] Study flow chart of the randomized, double-blind, placebo-controlled, multicenter, phase II clinical trial (finite-new) of TAF in combination with pegylated interferon and heparatide for the treatment of chronic hepatitis B virus with a finite treatment course. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as examples) can be combined with each other to form preferred technologies.

[0046] Currently, NAs is the main drug for the treatment of chronic HBV infection, but it is used for an indefinite course of treatment, and after withdrawal, rebound or recurrence of HBV occurs. The present invention provides a method for finite NAs treatment, so that chronic hepatitis B patients who are receiving NAs treatment can safely discontinue the treatment, avoid HBV recurrence, and make NAs a drug used for a finite course of treatment.

[0047] The method of finite NAs treatment provided by the present invention means that CHB patients continue to receive NAs treatment, and then load HBV entry inhibitors for combination treatment, and after a certain period of combination treatment, end both NAs and HBV entry inhibitor treatment. The present invention has found that according to the method of the present invention, HBV in CHB patients does not rebound after treatment is terminated. "Finite" as described herein should be understood to mean that using the regimen of the present invention, the course of NAs treatment is finite, the criteria for discontinuing NAs treatment are met, and HBV after discontinuance does not rebound or recur.

[0048] In the present invention, the continuous NAs treatment drug that the CHB patient receives includes, but is not limited to, Lamivudine, Adefovir Dipivoxil, Entecavir, TDF, Telbivudine, TAF and other NAs drugs. In one preferred embodiment, the NAs treatment that the CHB patient receives is Entecavir, TDF, Telbivudine or TAF. In some embodiments, the continuous NAs treatment that the CHB patient receives is a treatment course of more than 0 months of NAs treatment, preferably more than 3 months of NAs treatment, more preferably more than 12 months of NAs treatment, and even more preferably more than 36 months of NAs treatment.

[0049] In this specification, the HBV entry inhibitor includes, but is not limited to, HBV entry-inhibitory polypeptides derived from the HBV Pre-S1 region or HBV Pre-S1 derivative peptides, anti-Pre-S1 antibodies, anti-surface antigen antibodies, or other drugs that inhibit HBV entry or infection of liver cells.

[0050] As used herein, the term "HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region" or "HBV Pre-S1 derivative peptide" refers to a polypeptide originating from or derived from the HBV surface antigen Pre-S1 region and inhibiting HBV viral entry into hepatocytes, including, but not limited to, natural polypeptides, recombinant polypeptides, synthetic polypeptides, or purified polypeptides, including full-length natural HBV surface antigen Pre-S1 region or fragments and variants thereof, full-length non-natural HBV surface antigen Pre-S1 region or fragments and variants thereof, non-full-length natural HBV surface antigen Pre-S1 region or fragments and variants thereof, non-full-length non-natural HBV surface antigen Pre-S1 region or fragments and variants thereof, and other types of variants. In some embodiments, the HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region may contain the entire surface antigen pre-S1 region from the amino acid G (glycine) at position 2 of the HBV surface antigen pre-S1 region of any genotype. In some embodiments, the HBV entry inhibitory polypeptide derived from the HBV Pre-S1 region described herein is derived from the pre-S1 region of the surface antigen of any one of HBV genotypes A, B, C, D, E, F, G, H and I. Exemplary genomic sequences of these HBV genotypes are shown, for example, in GenBank Accession Nos. KC875260, AY220704, AF461363, AY796030, AB205129, DQ823095, HE981176 and AB179747. In some embodiments, the HBV Pre-S1 derivative peptide employed includes, but is not limited to, an HBV viral entry inhibitory polypeptide derived from the amino acid sequence of the Pre-S1 region of the HBV surface antigen of genotype C, including, but not limited to, the exemplary amino acid sequence of the Pre-S1 region of the HBV surface antigen SEQ ID NO:1 and variants thereof. The HBV entry inhibitory polypeptide derived from the HBV Pre-S1 region retains at least the biological activity of inhibiting HBV entry or binding to NTCP.

[0051] In some embodiments, the length of the HBV entry inhibitor polypeptides derived from the HBV Pre-S1 region described herein may be 10-118 amino acids. For example, the length of the polypeptides may be, but is not limited to, 15-100, 15-80, 20-100, 20-80, 20-60, 25-60, 30-60, 35-60, or 40-60 amino acids (including all integers within these ranges). In some embodiments, the length of the HBV entry inhibitor polypeptides derived from the HBV Pre-S1 region described herein may be at least 20 amino acids, such as at least 25, 30, 35, or 40 amino acids. In some embodiments, the length of the HBV entry inhibitor polypeptides derived from the HBV Pre-S1 region described herein may be, but is not limited to, 20, 25, 30, 35, 40, 47, 55, or 60 amino acids. In some embodiments, the length of the HBV entry inhibitor polypeptides derived from the HBV Pre-S1 region described herein can be 47 amino acids. Variants of different lengths of the HBV entry inhibitor polypeptides derived from the HBV Pre-S1 region described herein retain one or more biological activities associated with the corresponding polypeptide, including at least the biological activity of inhibiting HBV viral entry or binding to NTCP.

[0052] In one embodiment, the HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region described herein may be derived from the pre-S1 region of HBV genotype C surface antigen, and includes, but is not limited to, amino acids 2-119, 2-69, 2-59, 13-119, 13-88, 13-72, 13-67, 13-59, 13-52, 13-47, 13-42, 13-37, and 13-32 of the pre-S1 region and variants thereof, exemplary sequences of which include, but are not limited to, SEQ ID NO:2-14 and variants thereof.

[0053] In some embodiments, the HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region comprises an amino acid sequence of at least amino acid residues 13-44 of the N-terminus of the pre-S1 region of the HBV genotype C surface antigen as described herein, and variants thereof. In some embodiments, the length of the HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region may be 32-47 amino acids. In particularly preferred embodiments, an exemplary amino acid sequence of the HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region is set forth in SEQ ID NO:9; the HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region also includes variants of the amino acid sequence set forth in SEQ ID NO:9. In some embodiments, the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region contains at least the amino acid residues at positions 1 to 32 in the exemplary sequence SEQ ID NO:16 or variants thereof, and preferably, the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region contains at least the amino acid residues at positions 1 to 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46 in the exemplary sequence SEQ ID NO:16 or variants thereof.

[0054] In several embodiments, the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region comprises a fragment of at least amino acid residues 13-44 at the N-terminus of the pre-S1 region of the HBV surface antigen of A, B, F, H, or I genotypes as described herein, or a mutant thereof, a fragment of at least amino acid residues 2-33 at the N-terminus of the pre-S1 region of the HBV surface antigen of D genotypes as described herein, or a mutant thereof, or a fragment of at least amino acid residues 12-43 at the N-terminus of the pre-S1 region of the HBV surface antigen of E or G genotypes as described herein. In some embodiments, the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region comprises a fragment of at least amino acid residues 13-69 at the N-terminus of the pre-S1 region of HBV surface antigen of A, B, F, H, or I genotype or a mutant thereof, a fragment of at least amino acid residues 2-48 at the N-terminus of the pre-S1 region of HBV surface antigen of D genotype or a mutant thereof, or a fragment of at least amino acid residues 12-68 at the N-terminus of the pre-S1 region of HBV surface antigen of E or G genotype or a mutant thereof.

[0055] As used herein, a "variant" in reference to an HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region is one that differs in amino acid sequence from the designated polypeptide but retains biological activity of inhibiting HBV entry or binding to NTCP. For example, in some embodiments, a derivative peptide derived from the amino acid sequence at positions 13-59 of the HBV Pre-S1 region of genotype C may have one or a combination of any two or more of the following amino acid substitutions: N15D, F25L, G35K, N39E, F45L, N46K, N48H or N48Y or N48K, D50A, H51Q or H51N, E54K or E54D, A55S, N56K or N56D, and Q57K; exemplary sequences include, but are not limited to, those shown in SEQ ID NOs: 15-32.

[0056] As used herein, the term "variant" further includes homologous polypeptide sequences found in different virus species, strains, or subtypes of Hrpstovirus. Based on antigenic epitopes in its envelope protein, HBV is divided into four major serotypes (adr, adw, ayr, and ayw), and based on total nucleotide sequence variability in the genome, HBV is divided into nine genotypes (A, B, C, D, E, F, G, H, and I). Thus, the term "variant" includes any one of the homologous polypeptides found in these HBV subtypes.

[0057] As used herein, "variant" includes a polypeptide or variant thereof further comprising a naturally occurring adjacent amino acid sequence derived from any one of these HBV subtypes at the N-terminus and / or C-terminus. For example, in some embodiments, the naturally occurring adjacent amino acid sequence may be a fragment derived from at least amino acid residues 2-12 at the N-terminus of the pre-S1 region of the HBV surface antigen of the A, B, F, H, or I genotypes, or a fragment derived from at least amino acid residues 2-11 at the N-terminus of the pre-S1 region of the HBV surface antigen of the E or G genotypes, including but not limited to these, and may form a fusion polypeptide with a polypeptide having an amino acid sequence of amino acid sequence 13-59 at the Pre-S1 region of the C genotype surface antigen. For example, in some embodiments, the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region described herein may contain, but is not limited to, an amino acid sequence shown in any one of SEQ ID NOs:2-32, and a naturally occurring adjacent amino acid sequence at the N-terminus and / or C-terminus of the surface antigen of any one of the HBV genotypes AI. In some embodiments, the native flanking amino acid sequences may be derived from, including but not limited to, the amino acid sequence of an HBV surface antigen of GenBank Accession Nos. KC875260 (genotype A), AY220704 (genotype B), AF461363 (genotype C), AY796030 (genotype D), AB205129 (genotype E), DQ823095 (genotype F), HE981176 (genotype G), or AB179747 (genotype H). For example, an HBV entry inhibitor polypeptide derived from the HBV Pre-S1 region described herein may contain the amino acid sequence shown in SEQ ID NO:9 and may contain at its N-terminus and / or C-terminus native flanking amino acid sequences derived from the pre-S1 region of HBV genotype C. Alternatively, the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region described in the present specification may contain the amino acid sequence shown in SEQ ID NO:9 and may contain at its N-terminus and / or C-terminus naturally occurring adjacent amino acid sequences derived from the pre-S1 region of any one of HBV genotypes A, B, C, D, E, F, G and H.In some embodiments, the N- and / or C-termini of the HBV entry inhibitory polypeptides derived from the HBV Pre-S1 region described herein may independently contain a naturally occurring flanking amino acid sequence that is 1-10 amino acids in length, e.g., 1-8, 1-5, or 1-3 amino acids in length (including all integers within these ranges). For example, an HBV entry inhibitory polypeptide derived from the HBV Pre-S1 region described herein may contain the amino acid sequence shown in SEQ ID NO:9 and at its N-terminus may contain a naturally occurring flanking amino acid sequence that is 10 amino acids in length derived from the pre-S1 region of HBV genotype C. In other words, the HBV entry inhibitory polypeptide derived from the HBV Pre-S1 region may contain amino acids 2-59 (SEQ ID NO:4) of the pre-S1 region of HBV genotype C. Another example is an HBV entry inhibitory polypeptide derived from the HBV Pre-S1 region described herein, which may contain the amino acid sequence shown in SEQ ID NO:9, and may contain at its N-terminus a naturally occurring flanking amino acid sequence of 9 amino acids in length derived from the HBV pre-S1 region of E genotype. In other words, the HBV entry inhibitory polypeptide derived from the HBV Pre-S1 region may contain amino acids 13-59 of the pre-S1 region of HBV genotype C and amino acids 2-11 of the pre-S1 region of HBV genotype E. It should be understood that the HBV entry inhibitory polypeptide derived from any one of the HBV Pre-S1 regions described above may have any length of naturally occurring flanking amino acid sequence extending from its N- and / or C-terminus, and the resulting HBV entry inhibitory polypeptide derived from the HBV Pre-S1 region retains the biological activity of inhibiting HBV entry or binding to NTCP.

[0058] As used herein, the term "variant" related to the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region further includes those having one or more amino acid deletions, substitutions or insertions, while retaining the biological activity of inhibiting HBV entry or binding to NTCP. The HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region preferably retains the amino acid corresponding to glycine at position 13 of the HBV pre-S1 region of the C genotype (i.e., the N-terminal glycine of SEQ ID NO:9). In some embodiments, the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region described herein may have one or more naturally occurring mutations in the pre-S1 region of HBV. For example, in some embodiments, the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region described herein may have 1-30, e.g., 1-20, 1-10, 1-8, 1-5 or 1-3 amino acid deletions, substitutions or insertions (including all integers within these ranges) relative to the sequence derived from the HBV pre-S1 region. In some embodiments, the HBV entry inhibitor polypeptides derived from the HBV Pre-S1 region described herein may have 1-30, e.g., 1-20, 1-10, 1-8, 1-5, or 1-3 amino acid deletions, substitutions, or insertions, including all integers within these ranges, relative to any one of the exemplary amino acid sequences selected from SEQ ID NO:2-32. In some embodiments, the HBV entry inhibitor polypeptides derived from the HBV Pre-S1 region described herein may have 1-30, e.g., 1-20, 1-10, 1-8, 1-5, or 1-3 amino acid deletions, substitutions, or insertions, including all integers within these ranges, relative to the exemplary amino acid sequence of SEQ ID NO:9. In some embodiments, the HBV entry inhibitor polypeptides derived from the HBV Pre-S1 region described herein may have 1-3 amino acid deletions, substitutions, or insertions, including all integers within these ranges, relative to the exemplary amino acid sequence of SEQ ID NO:9.In one embodiment, the HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region described herein has a deletion or insertion of 1-30, e.g., 1-20, 1-10, 1-8, 1-5 or 1-3 amino acids (including all integers within these ranges) at the C-terminus relative to any one of the exemplary amino acid sequences selected from SEQ ID NO:2-32.

[0059] In various embodiments, the present disclosure includes, but is not limited to, the above polypeptides and polypeptides having at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to any one of the above polypeptides. For example, the HBV entry inhibitor polypeptide derived from the HBV Pre-S1 region may comprise an amino acid sequence having at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to any one of the exemplary sequences of SEQ ID NOs: 2-32; preferably, the identical amino acid sequence is derived from any one of the HBVs described herein (including, but not limited to, genotypes A, B, C, D, E, F, G, H and I HBV genotypes), more preferably, from genotype C. In some embodiments, the HBV entry inhibitor polypeptide derived from the HBV Pre-S1 region may comprise an amino acid sequence having at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the exemplary sequence of SEQ ID NO:9; preferably, the amino acid sequence having the identity is derived from any one of the HBV genotypes described herein, including but not limited to HBV genotypes A, B, C, D, E, F, G, H and I, and more preferably from genotype C. Variants having a certain sequence identity to the HBV entry inhibitor polypeptide derived from the HBV Pre-S1 region described herein retain one or more biological activities of the corresponding polypeptide, including biological activity that inhibits HBV viral entry or binding to NTCP.

[0060] As used herein, the N-terminus of the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region and its variants may have a hydrophobic group modification, including, but not limited to, myristic acid (myr), palmitic acid (plam), stearic acid (stearoyl), cholesterol (chol), oleic acid, linoleic acid, polyethylene glycol (PEG), arachidonic acid, or other hydrophobic groups. In some embodiments, the hydrophobic group may be selected from myristic acid, palmitic acid, stearic acid, and cholesterol. In some embodiments, the hydrophobic group is myristic acid. In some embodiments, the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region described herein may comprise any one of the amino acid sequences shown in SEQ ID NO:2-32 or variants thereof, wherein the N-terminus of the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region may be modified with a hydrophobic group selected from myristic acid, palmitic acid, stearic acid, and cholesterol. In some embodiments, the polypeptide may comprise an amino acid sequence set forth in any one of SEQ ID NO:2-32 or a variant thereof, wherein the N-terminus of the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region may be myristoylated. In some embodiments, the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region described herein may comprise an amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:32, wherein the N-terminus of the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region may be myristoylated.

[0061] As used herein, the C-terminus of the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region may be modified. The C-terminus modification may be selected from, but is not limited to, amidation (amination), isoprene glycolation, and other C-terminus modifications; or may be deleted. In some embodiments, the C-terminus modification may be amidation (NH2). For example, the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region may contain the amino acid sequence shown in SEQ ID NO:9 or SEQ ID NO:32, and the C-terminus may be amidated.

[0062] In some embodiments, the N-terminus of the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region may be modified with a hydrophobic group and / or the C-terminus may be modified in another manner. In some embodiments, the N-terminus of the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region may be modified with a hydrophobic group and / or the C-terminus may be modified by amidation. In one embodiment, the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region may contain an amino acid sequence shown in any one of SEQ ID NO:2-32 and variants thereof, and the N-terminus may be modified with a hydrophobic group and / or the C-terminus may be modified by amidation. For example, the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region may contain the amino acid sequence shown in SEQ ID NO:9 or SEQ ID NO:32, the N-terminus of which may be modified with a hydrophobic group of myristic acid (myr), palmitic acid (plam), stearic acid (stearoyl), or cholesterol (chol), and the C-terminus of which may be modified by amidation, such as the polypeptide shown in SEQ ID NO:33-37. In some embodiments, the N-terminus of the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region may be modified with a hydrophobic group of myristic acid (myr) and / or the C-terminus may be modified by amidation. In some embodiments, the polypeptide may contain any one of the amino acid sequences shown in SEQ ID NO:2-32 and variants thereof, the N-terminus of which may be modified with myristoylation and / or the C-terminus of which may be modified by amidation. For example, the HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region may contain a polypeptide as shown in SEQ ID NO:9 or 32, the N-terminus of which may be modified to a myristic acid (myr) hydrophobic group and the C-terminus of which may be modified by amidation, for example, as shown in any one of SEQ ID NOs:33, 34 and 38-44.In some embodiments of the present invention, the loaded HBV entry inhibitors include, but are not limited to, heparatide (SEQ ID NO: 33), bulevirtide (SEQ ID NO: 43) and other HBV Pre-S1 derivative peptides, of which heparatide is named N-myristoyl-glycyl-L-threonyl-L-asparaginyl-L-leucyl-L-seryl-L-valyl-L-prolyl-L-asparaginyl-L-prolyl-L-leucyl-glycyl-L-phenylalanyl-L-phenylalanyl-L-prolyl-L-aspartyl-L-histidyl-L-glutaminyl-L-leucyl-L-aspartyl-L-prolyl-L-alanyl-L-phenylalanyl-glycyl-L-alaninyl. nyl-L-asparaginyl-L-seryl-L-asparaginyl-L-asparaginyl-L-prolyl-L-aspartyl-L-tryptophanyl-L-aspartyl-L-phenylalanyl-L-asparaginyl-L-prolyl-L-asparaginyl-L-lysyl-L-aspartyl-L-histidyl-L-tryptophanyl-L-prolyl-L-glutamyl-L-alanyl-L-asparaginyl-L-glutaminyl-L-valyl-glycinamide. The name of Bulevirtide is N-myristoyl-glycyl-L-threonyl-L-asparaginyl-L-leucyl-L-seryl-L-valyl-L-prolyl-L-asparaginyl-L-prolyl-L-leucyl-glycyl-L-phenylalanyl-L-phenylalanyl-L-prolyl-L-aspartyl-L-histidyl-L-glutaminyl-L-leucyl-L-aspartyl-L-prolyl-L-alanyl-L-phenylalanyl-glycyl-L- Alanyl-L-asparaginyl-L-seryl-L-asparaginyl-L-asparaginyl-L-prolyl-L-aspartyl-L-tryptophanyl-L-aspartyl-L-phenylalanyl-L-asparaginyl-L-prolyl-L-asparaginyl-L-lysyl-L-aspartyl-L-histidyl-L-tryptophanyl-L-prolyl-L-glutamyl-L-alanyl-L-asparaginyl-L-lysyl-L-valyl-glycinamide. The sequences relevant to this application are shown below.

[0063] [Table 1] JPEG2025503152000002.jpg90170 [Table 2]

[0064] In one embodiment of the present invention, heparatide or bulevirtide treatment is loaded on top of NAs treatment. In a more preferred embodiment, heparatide treatment is loaded on top of NAs treatment.

[0065] In one embodiment, the therapeutic dose of heparatide is 2.1-10.5 mg daily. In one preferred embodiment, the therapeutic dose of heparatide is 4.2-8.4 mg daily. In a more preferred embodiment, the therapeutic dose of heparatide is 4.2 mg daily.

[0066] In one embodiment, the heparatide treatment cycle is 1-96 weeks. In one preferred embodiment, the heparatide treatment cycle is 12-48 weeks. In a more preferred embodiment, the heparatide treatment cycle is 24-48 weeks. In a more preferred embodiment, the heparatide treatment cycle is 48 weeks.

[0067] As used herein, the anti-Pre-S1 antibody includes, but is not limited to, anti-HBV Pre-S1 monoclonal antibody, anti-HBV Pre-S1 bifunctional antibody (bibody), anti-HBV Pre-S1 polyclonal antibody (polybody), anti-HBV Pre-S1 serum, and other types of antibodies and compositions thereof, which can bind to HBV Pre-S1 and inhibit HBV infection.

[0068] As used herein, the anti-surface antigen antibody includes, but is not limited to, anti-surface antigen monoclonal antibodies, anti-surface antigen bifunctional antibodies (bifunctional antibodies), anti-surface antigen polyclonal antibodies (polyantibodies), anti-surface antigen serum, HBIG and other types of antibodies and compositions thereof, which can bind to surface antigens and inhibit HBV infection.

[0069] As used herein, the agents that inhibit HBV invasion or infection of other liver cells include, but are not limited to, small molecules, proteins, and other molecules that can act on NTCP, the HBV virus, or other targets to achieve the technical effect of inhibiting HBV infection.

[0070] The NAs are loaded with an HBV entry inhibitor and administered a course of combination therapy, in one embodiment for 1-96 weeks of combination therapy, in one preferred embodiment for 12-60 weeks of combination therapy, in one more preferred embodiment for 24-48 weeks of combination therapy, and in an even more preferred embodiment for 48 weeks of combination therapy.

[0071] As used herein, the term "HBV does not rebound in the CHB patient after the end of treatment" refers to HBV not rebounding within at least 24 weeks after the patient discontinues the combination treatment. In one preferred embodiment, the term "HBV does not rebound in the CHB patient after the end of treatment" refers to HBV not rebounding within at least 48 weeks after the patient discontinues the combination treatment. In a more preferred embodiment, the term "HBV does not rebound in the CHB patient after the end of treatment" refers to HBV not rebounding within at least 96 weeks after the patient discontinues the combination treatment.

[0072] In some embodiments, the HBV in the CHB patient does not rebound after the end of treatment refers to the patient being successfully withdrawn from the combination treatment after the cessation of the combination treatment, and the patient does not need to undergo NAs treatment again (retreatment). In one preferred embodiment, the HBV in the CHB patient does not rebound after the end of treatment refers to the HBV DNA remaining below 2000 IU / ml after the patient has discontinued the combination treatment. In one preferred embodiment, the HBV in the CHB patient does not rebound after the end of treatment refers to the HBV DNA remaining below the lower limit of quantitative detection (below 20 IU / ml) after the patient has discontinued the combination treatment. In one preferred embodiment, the HBV in the CHB patient does not rebound after the end of treatment refers to the HBV DNA remaining undetectable (below 10 IU / ml) after the patient has discontinued the combination treatment.

[0073] In one embodiment of the present invention, for CHB patients undergoing NAs sequential therapy, the combination therapy is based on TDF or TAF instead of NAs and further loaded with an HBV entry inhibitor.In a more preferred embodiment, the combination therapy is based on TAF instead of NAs and further loaded with an HBV entry inhibitor.

[0074] In one embodiment of the present invention, after continuous NAs treatment, CHB patients are loaded with HBV entry inhibitors and immunomodulators for combination therapy, including but not limited to interferon (IFN), toll-like receptors (TLR) agonists, CPG (Cytidine-phosphatte-guanonine), CPG ODN (CpG oligonucleotide), PD-1 (Programmed Death Receptor-1) inhibitors, PD-L1 (Programmed Death Receptor ligan 1) inhibitors, interleukins (IL), cytokines, and other immunomodulators, among which TLR agonists include but are not limited to agonists of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, and other TLR receptors. Wherein, PD-1 inhibitors include, but are not limited to, anti-PD-1 monoclonal antibodies, anti-PD-1 bispecific antibodies, anti-PD-1 polyclonal antibodies, small molecule drugs that inhibit PD-1 or PD-1 signaling, and other PD-1 inhibitors, Wherein, PD-L1 inhibitors include, but are not limited to, anti-PD-L1 monoclonal antibodies, anti-PD-L1 bispecific antibodies, anti-PD-L1 polyclonal antibodies, PD-L1 inhibitors, recombinant PD-L1 proteins, recombinant PD-L1 fusion proteins, small molecule drugs that inhibit PD-L1 or PD-L1 signaling, and other PD-L1 inhibitors.

[0075] In one embodiment of the present invention, the loaded HBV entry inhibitor includes, but is not limited to, HBV Pre-S1 derivative peptide, anti-Pre-S1 antibody, anti-surface antigen antibody or other drugs that suppress HBV entry or infection of liver cells. The HBV Pre-S1 derivative peptide includes, but is not limited to, heparatide (SEQ ID NO: 33), bulevirtide (SEQ ID NO: 34) and other HBV Pre-S1 derivative peptides. The other HBV Pre-S1 derivative peptides include, but are not limited to, the sequences listed in SEQ ID NO: 2-44 and other polypeptides derived from the AI ​​genotype HBV Pre-S1 region, which are characterized by binding to NTCP and suppressing HBV infection.

[0076] In one embodiment, heparatide or bulevirtide therapy is loaded on top of the NAs therapy. In a more preferred embodiment, heparatide therapy is loaded on top of the NAs therapy. In one embodiment, the therapeutic dose of heparatide is 2.1-10.5 mg daily. In one preferred embodiment, the therapeutic dose of heparatide is 4.2-8.4 mg daily. In a more preferred embodiment, the therapeutic dose of heparatide is 4.2 mg daily. In one embodiment, the therapeutic cycle of heparatide is 1-96 weeks. In one preferred embodiment, the therapeutic cycle of heparatide is 12-48 weeks. In a more preferred embodiment, the therapeutic cycle of heparatide is 24-48 weeks. In a more preferred embodiment, the therapeutic cycle of heparatide is 48 weeks. In some embodiments of the invention, the immunomodulatory agent loaded is an interferon (IFN). In some embodiments of the invention, the interferon loaded is IFN-α, IFN-β, or IFN-γ. In one preferred embodiment, the interferon loaded is IFN α-2a or IFN α-2b. In one embodiment of the present invention, the interferon loaded is a general interferon or a long-acting interferon. In one preferred embodiment, the interferon loaded is a polyethylene glycol (PEG) interferon. In a further optimized embodiment, the interferon loaded is PEG IFN α-2a or PEG IFN α-2b, including but not limited to Pegasys, Pagbin, PegIntron and other interferons. Preferably, the dose of interferon used is lower than the standard dose for clinical treatment. In one embodiment of the invention, the interferon loaded is Pegasys and the dosage is 1-360 ug per week; in one embodiment, the dosage is 30-180 ug per week; in one embodiment, the dosage is 60-135 ug per week; in one embodiment, the dosage is 90 ug per week.The NAs are loaded with an HBV entry inhibitor and combined with interferon for a fixed course of treatment, in one embodiment, the combination treatment is for 1-96 weeks, in one preferred embodiment, the combination treatment is for 12-60 weeks, in one more preferred embodiment, the combination treatment is for 24-48 weeks, and in an even more preferred embodiment, the combination treatment is for 48 weeks.

[0077] The NAs treatment based on the method for finite NAs treatment provided by the present invention includes, but is not limited to, Lamivudine, Telbivudine, Adefovir Dipivoxil, Tenofovir, Entecavir, Tenofovir alafenamide fumarate and other NAs drugs; HBV entry inhibitors include, but are not limited to, Heparatide, Bulevirtide and other HBV entry inhibitors; Immunomodulators include, but are not limited to, Interferon and other immunomodulators. The method for finite NAs treatment provided by the present invention includes at least loading HBV entry inhibitor treatment on NAs treatment, and may further include loading immunomodulator treatment. The method for finite NAs treatment provided by the present invention includes, but is not limited to, the methods listed in the table below. In some embodiments of the present invention, the methods 6, 13, 27, and 34 may be adopted, but are not limited thereto.

[0078] [Table 3] JPEG2025503152000005.jpg251170JPEG2025503152000006.jpg70170

[0079] In some embodiments, the present invention provides the use of an HBV virus entry inhibitor and an optional immunomodulator in the preparation of a drug or kit for terminating or terminating chronic hepatitis B treatment with a nucleoside (nucleotide) drug for chronic hepatitis B. The HBV virus entry inhibitor, the immunomodulator, the nucleoside (nucleotide) drug, the drug, and the kit are described in any one of the embodiments herein. In some embodiments, the present invention provides a drug or kit containing an HBV virus entry inhibitor and an optional immunomodulator as described in any one of the embodiments herein, or an HBV virus entry inhibitor and an optional immunomodulator as described in any one of the embodiments herein, for terminating or terminating chronic hepatitis B treatment with a nucleoside (nucleotide) drug. In some embodiments, the drug or kit of the present invention is used in a method for terminating or terminating chronic hepatitis B treatment with a NAs drug as described in any one of the embodiments herein. In some embodiments, the kit of the present invention further contains one or more doses of a nucleoside (nucleotide) drug as described in any one of the embodiments herein. Preferably, the drug formulations contained in the kit satisfy the dosage requirements for one or more courses as described herein.

[0080] The present invention will be further described below with reference to specific examples. It should be understood that these examples are merely illustrative of the present invention, and do not limit the scope of the present invention. Experimental methods that do not show specific conditions in the following examples generally follow normal conditions or manufacturer's recommended conditions. Unless otherwise specified, percentages and parts refer to weight. EXAMPLES

[0081] Example 1: Study design for a randomized, double-blind, placebo-controlled, multicenter, phase II clinical trial of pegylated interferon and heparatide in combination with TAF for the treatment of chronic hepatitis B virus with a finite course of treatment (L47-HB-FIN-1)

[0082] Number of subjects We expect to register 30 people.

[0083] Purpose of the Test Main Purpose: CHB patients treated with NA are to be treated with a limited treatment course combining PEG interferon, heparin, and TAF, and to achieve a certain degree of cure or clinical cure after drug withdrawal. Following Purposes: 1) To study the success rate of terminating NAs treatment after being treated with a limited treatment course combining PEG interferon, heparin, and TAF; 2) To study the safety of a limited treatment course combining PEG interferon, heparin, and TAF.

[0084] Test Design This study is a multi-center, randomized, double-blind, placebo-controlled clinical trial with a two-arm parallel group comparison. The overall design is shown in Figure 1. CHB patients who had been treated with NA for more than 2 years and met the inclusion criteria were randomly assigned to the heparin group and the placebo group at a ratio of 1:1, with 15 subjects in each group participating (see Table 1). Subjects in both groups were administered TAF (25 mg / day) and PEG interferon (90 μg / week), and heparin (4.2 mg / day) and placebo were administered to each group respectively, and treated continuously for 48 weeks. After the treatment ended, the treatment of all drugs was stopped, the primary endpoint was evaluated at the 24th week after drug withdrawal, and subsequent follow-up was conducted.

[0085]

Table 4

[0086] Entry Criteria 1. Men and women aged 18 years ≤ age ≤ 60 years; 2. HBsAg(+) or HBV DNA(+) ≥ 6 months (diagnosed as "chronic hepatitis B" by the researcher); 3. HBeAg(-); 4. Having received continuous NAs treatment for ≥ 2 years; 5. HBV DNA < LLQD (lower limit of quantitative detection) in the screening test; 6. ULN < ALT < 10 × ULN; 7. Total serum bilirubin <2×ULN; 8. Not planning to give birth within 2 years, agreeing to use effective contraception during the entire treatment period and for 3 months after the final dose, and not being pregnant or breastfeeding; 9. Not be a subject of any other clinical trial within the past 3 months; 10. Good compliance with the research plan; 11. Subject agrees to understand and sign the informed consent form.

[0087] Exclusion criteria 1. Contraindications to peginterferon treatment, such as major depression, epilepsy, autoimmune disease, or uncontrolled thyroid dysfunction; 2. Clinical evidence of cirrhosis: for example, obvious cirrhosis on imaging such as abdominal color ultrasound or CT, Metavir fibrosis score of 4 on liver biopsy, or clinically diagnosed cirrhosis by the investigator; 3. Decompensated liver disease: prothrombin time >1.2 × ULN, serum albumin <35 g / L; 4. Liver function Child-Pugh grade BC or score >6; 5. Any one of the following circumstances: 1) History of decompensated liver disease (e.g., ascites, hepatic encephalopathy, variceal bleeding, hepatorenal syndrome); 2) history of serious cardiac disease (including unstable or uncontrolled cardiac disease within 6 months); 3) have uncontrolled epilepsy, severe psychiatric illness, or a history of severe psychiatric illness; 4) history of organ transplant; 5) ineffectively controlled diabetes and hypertension; 6) Subjects with autoimmune disease, immune-related extrahepatic symptoms (vasculitis, purpura, arteritis nodosa, peripheral neuropathy, glomerulonephritis), thyroid disease, malignant tumor, or immunosuppressive therapy; (7) Those with underlying diseases such as malignant tumors, serious infections, heart failure, or chronic obstructive pulmonary disease; 8) History of alcohol or drug use. 6. Creatinine clearance <60 mL / min; 7. Patients with combined hepatitis A, C, D, E virus infection, or HIV infection; 8.Subjects who must be treated with anti-HBV nucleoside (nucleotide) drugs other than TAF during the treatment period; 9.Subjects who have used interferon within 6 months prior to the screening period; 10. Anti-HBV Pre-S1 antibody positive; 11. Blood test abnormality: White blood cell count 3 x 10 9 / L, neutrophil count 1.5 × 10 9 / L, platelet count 60×10 9 / L or less; 12. A positive pregnancy test in females; 13.Those taking medication prohibited in this study and unable to discontinue taking the medication; 14. Patients with known allergies to the study drug or underlying treatment; 15. Subjects who are obviously abnormal based on other clinical or ancillary tests and are therefore unsuitable to participate in this study.

[0088] Therapeutic drugs 1. Tenofovir alafenamide (Vemlidy) Dosage form: Tablets Standard: 25mg; Transport and storage: Store below 30°C; Validity period: 24 months Approval number: Imported Drug Registration Certificate No. H20180060; Manufacturer:Patheon Inc. 2. Polyethylene glycol interferon (Pegasys) Dosage form: Injection; Standard: 180μg / 0.5ml; Transport and storage: Store in original packaging, sealed and protected from light, at 2-8°C and avoid freezing; Validity period: 24 months Approval number: National Drug Approval No. J20120075; Manufacturer: Shanghai Roche Pharmaceutical Co., Ltd. 3. Heparatide for injection Dosage form: powder injection; Standard: 2.1mg; Transportation and storage: transportation temperature ≦8℃, 2-8℃ sealed, stored in a dark place; Validity period: 36 months; Manufacturer: Hainan Shuangcheng Pharmaceutical Co., Ltd. 4. Heparatide placebo Dosage form: powder injection; Standard: 0 mg; Transportation and storage: transportation temperature ≦8℃, 2-8℃ sealed, stored in a dark place; Validity period: 36 months; Manufacturer: Hainan Shuangcheng Pharmaceutical Co., Ltd.

[0089] Basic treatment 1. Vemlidy (25 mg / day, po QD), 48 weeks of continuous treatment. 2. Pegasys (90 μg / 0.25 ml, sc QW), 48 weeks of continuous treatment.

[0090] Study treatment On top of the basal treatment, heparatide treatment (4.2 mg, sc QD) was loaded and continued for 48 weeks. There is no set time for heparatide administration, but it is recommended that it be administered subcutaneously after dinner and 30 minutes before going to bed.

[0091] Control treatment Patients were administered a single daily subcutaneous injection of heparatide placebo on top of baseline treatment for 48 weeks.

[0092] Planned treatment Treatment cycle: 48 weeks; Follow-up period: 24 weeks.

[0093] Study Endpoints Primary Endpoint 1) Sustained virologic response: serum HBV DNA <20 IU / mL 24 weeks after discontinuation of treatment (72 weeks); 2) HBsAg became negative (72W). Secondary Endpoint 1) Retreatment rate of NA (72W); 2) virological relapse (72W); 3) clinical recurrence (72W); 4) HBsAg decreased from baseline (72W); 5) HBsAg serological conversion (72W); 6) Liver elasticity changed from baseline (72W).

[0094] Efficacy Indicators Efficacy indicators: HBV DNA, HBsAg, HBsAb, HBeAb, ALT, LSM, HBcrAg, HBV pgRNA Validity definition: Virological response: serum HBV DNA <20 IU / mL during treatment (lower limit of detection); Sustained virologic response: Response at end of treatment and at 24- or 48-week follow-up after off-treatment; Virological breakthrough: In patients with good adherence to nucleoside (nucleotide) analogue therapy and no change in therapy, HBV DNA concentration increases by 1 log10 IU / mL or more from the lowest value during treatment, or becomes positive again after becoming negative, and is confirmed by retesting with the same reagent one month later to see whether ALT is elevated or not; Virological relapse: response at end of treatment but HBV DNA concentration >2000 IU / ml in two institutional laboratory tests 1 month after drug withdrawal; Clinical relapse: Patients with sustained virologic response have ALT>2×ULN and HBV DNA>2000 IU / ml after treatment discontinuation; HBsAg negative conversion: become HBsAg negative; HBsAg serological conversion: HBsAg seroconversion accompanied by HBsAb positivity; HBeAg serological conversion: HBeAg seroconversion accompanied by HBeAb seroconversion;

[0095] Safety indicators Vital signs, physical examination; Laboratory tests: blood and urine, blood biochemistry, coagulation, thyroid function, alpha-fetoprotein; Auxiliary examinations: electrocardiogram, ultrasound examination (liver, gallbladder, spleen, pancreas, kidney, ascites), liver plain CT; AE target examination; Main indicators: hematology, liver function, blood glucose, blood lipids, blood cholesterol, total bile acid, blood phosphorus, blood fibrinogen.

[0096] Safety evaluation Symptomatic adverse events were evaluated according to a three-level classification scale and classified into mild (transient symptoms that do not affect the subject's daily life), moderate (obvious symptoms that have some impact on the subject's daily life), and severe (severe impacts that are unacceptable to the subject's daily life). Objective adverse events were evaluated using a five-level judgment scale according to NCI CTCAE v5.0.

[0097] Indications for antiviral retreatment (meeting any one of the following) 1. Confirm that the results of two consecutive facility laboratory tests at one-month intervals show that HBV DNA is ≥ 2000 IU / mL and ALT is ≥ 2 × ULN; 2. Confirm that the results of two consecutive clinical facility tests show an increase in direct bilirubin from baseline > 1.5 mg / dL (25 μmol / L) and ALT > 1 × ULN; 3. Confirm that the results of two consecutive clinical facility tests show a prolonged prothrombin time ≥ 2 seconds or INR ≥ 0.5 and ALT > 1 × ULN; 4. Confirm that the results of two consecutive facility laboratory tests show ALT > 10 × ULN and other causes of liver impairment have been excluded regardless of the presence or absence of clinical symptoms; 5. Maintain 5 × ULN < ALT ≤ 10 × ULN for ≥ 4 weeks and other causes of liver impairment have been excluded; 6. Maintain 2 × ULN < ALT ≤ 5 × ULN for ≥ 12 weeks, HBV DNA > LLQD, and other causes of liver impairment have been excluded; 7. If a patient has an elevated ALT or HBV DNA and does not meet the above criteria, it is up to the investigator to decide whether the patient should resume antiviral therapy and whether to continue with scheduled visits, add additional visits, or discontinue the patient from the study.

[0098] Anti-Pre-S1 antibody Blood samples will be collected at baseline and at weeks 4, 12, 24, 48, and 72 during treatment to detect anti-Pre-S1 antibodies.

[0099] Study Endpoints All enrolled subjects will undergo 48 weeks of treatment and 24 weeks of follow-up, and will not be recruited if they drop out or are lost to treatment.

[0100] Follow-up after After the 72-week trial ended, follow-up visits continued every 6 months for 3 years.

[0101] statistical methods Primary endpoint analysis: The primary treatment effect endpoints were tested using mITT (main analysis) and PPS, respectively, and multiple comparison factors were not considered for the statistics of different primary endpoints. For the primary endpoints of the L47 4.2 mg group and the placebo group, a two-sided test between groups was performed, with the test value being 0.025 (one-sided). The confidence interval method was used to determine superiority, and if the lower limit of the one-sided 97.5% confidence interval of the difference between groups is greater than 0, the test drug in the L47 4.2 mg group is considered to be superior to the placebo. Secondary endpoint analysis: Secondary treatment efficacy endpoints were tested using mITT (primary analysis) and PPS, respectively. Between-group comparisons of secondary endpoints between the L47 4.2 mg group and the placebo group were performed with a one-sided test value of 0.025. Analysis of other endpoint measures: Depending on the characteristics of the data, parametric or non-parametric tests will be used to compare differences between groups. Safety analysis: A safety set (SS) including all subjects exposed to at least one dose of the study drug (L47, L47 placebo) will be used. Descriptive statistics will be mainly performed on safety indicators. If any subject experiences HBV virologic breakthrough or ALT SAE during the course of the study, necessitating urgent unblinding, the treatment efficacy endpoint will be treatment failure.

Claims

1. Use of an HBV viral entry inhibitor and an optional immunomodulator in the preparation of a drug or kit for terminating or terminating nucleoside (nucleotide) drug treatment for chronic hepatitis B.

2. The HBV entry inhibitor is selected from an HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region or an HBV Pre-S1 derivative peptide, an anti-Pre-S1 antibody, an anti-surface antigen antibody, or other drugs that inhibit HBV entry or infection of hepatocytes; preferably, the HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region is derived from the pre-S1 region of the surface antigen of any one of HBV genotypes A, B, C, D, E, F, G, H, and I; preferably, the length of the HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region is 10-118 amino acid residues; The immunomodulator includes an agent selected from interferon, toll-like receptor agonist, CPG, CPG ODN, PD-1 inhibitor, PD-L1 inhibitor, interleukin, and cytokine; preferably, the interferon is selected from IFN-α, IFN-β, and IFN-γ, preferably IFN-α-2a and / or IFN-α-2b, more preferably PEG-IFN-α-2a and / or PEG-IFN-α-2b.

2. The use according to claim 1 .

3. The HBV entry-inhibitory polypeptide derived from the HBV pre-S1 region is derived from the pre-S1 region of the HBV genotype C surface antigen; preferably, the HBV entry-inhibitory polypeptide is an N-terminal fragment of the pre-S1 region of the HBV genotype C surface antigen or a variant thereof, and the fragment contains at least the amino acid residues at positions 13-44 of the N-terminus; preferably, the HBV entry-inhibitory polypeptide contains the amino acid residues at positions 2-119, 2-69, 2-59, 13-119, 13-88, 13-72, 13-67, 13-59, 13-52, or 13-47 of the pre-S1 region of the HBV genotype C surface antigen, or a variant thereof; or The HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region is an N-terminal fragment of the pre-S1 region of HBV surface antigen of genotype A, B, F, H, or I, or a variant thereof, the fragment containing at least the amino acid residues 13-44 of the N-terminus, preferably at least the amino acid residues 13-69 of the N-terminus; or The HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region is an N-terminal fragment of the pre-S1 region of the HBV surface antigen of D genotype, or a variant thereof, which fragment contains at least the amino acid residues at positions 2-33 of the N-terminus, and preferably contains at least the amino acid residues at positions 2-48 of the N-terminus; or The HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region is an N-terminal fragment of the pre-S1 region of an E or G genotype HBV surface antigen or a variant thereof, the fragment containing at least the amino acid residues at positions 12-43 of the N-terminus, preferably at least the amino acid residues at positions 12-68 of the N-terminus; Preferably, the variant has a deletion, substitution or insertion of 1-30 amino acids compared to the fragment, and the variant retains the biological activity of inhibiting HBV entry or binding to NTCP; preferably, the variant includes a naturally occurring flanking amino acid sequence derived from any one of the HBV subtypes inserted at the N- and / or C-terminus of the fragment; preferably, the naturally occurring flanking amino acid sequence is selected from the amino acid sequence of positions 2-12 at the N-terminus of the pre-S1 region of the surface antigen of HBV genotypes A, B, F, H, I, and E, and the amino acid sequence of positions 2-11 at the N-terminus of the surface antigen pre-S1 region of HBV genotypes E, G; preferably, the variant is a naturally occurring flanking amino acid sequence derived from HBV genotype C. The mutant is a derivative peptide derived from the amino acid sequence at positions 13-59 of the Pre-S1 region, into which one or a combination of any two or more of the following amino acid substitutions have been introduced: N15D, F25L, G35K, N39E, F45L, N46K, N48H or N48Y or N48K, D50A, H51Q or H51N, E54K or E54D, A55S, N56K or N56D, and Q57K; and optionally, a naturally occurring adjacent amino acid sequence derived from any one of the above HBV subtypes is inserted at the N- and / or C-terminus of the mutant.

3. The use according to claim 2.

4. 4. The use according to claim 3, wherein the amino acid sequence of the fragment is set forth in any one of SEQ ID NOs: 2-32 or has at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 2-32.

5. The HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region has an N-terminal modification and / or a C-terminal modification; Preferably, the N-terminal modification is a hydrophobic group modification; preferably, said hydrophobic group modification is selected from myristic acid, palmitic acid, stearic acid, cholesterol, oleic acid, linoleic acid, polyethylene glycol and arachidonic acid; Preferably, the C modification is an amidation or isoprene glycolation modification; Preferably, the HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region is represented by SEQ ID NO: 33-44. Use according to any one of claims 2 to 4.

6. The use according to any one of claims 1 to 4, characterized in that the nucleoside (nucleotide) drug is selected from lamivudine, adefovir dipivoxil, entecavir, tenofovir disoprox, telbivudine and tenofovir alafenamide fumarate, preferably tenofovir alafenamide fumarate.

7. The kit contains one or more doses of the HBV viral entry inhibitor to administer the drug at a daily dose of 2.1-10.5 mg, preferably 4.2-8.4 mg, more preferably 4.2 mg; The kit optionally contains one or more doses of the interferon, and the amount satisfies a weekly dose of 1-360 micrograms, preferably 30-180 micrograms, more preferably 60-135 micrograms, more preferably 90 micrograms; The kit is used for combined administration with the nucleoside (nucleotide) drug for 1 to 96 weeks, preferably 12 to 60 weeks, more preferably 24 to 48 weeks. Use according to any one of claims 1 to 4.

8. A method for limiting or terminating treatment of a patient with chronic hepatitis B with a nucleoside (nucleotide) drug, comprising administering to the patient with chronic hepatitis B a combination treatment of an HBV virus entry inhibitor and an optional immunomodulator in addition to the nucleoside (nucleotide) drug, and further terminating the nucleoside (nucleotide) drug treatment, the HBV virus entry inhibitor treatment, and the immunomodulator treatment after a certain course of combination treatment.

9. The nucleoside (nucleotide) drug is selected from lamivudine, adefovir dipivoxil, entecavir, tenofovir disoproxy, telbivudine and tenofovir alafenamide fumarate, preferably tenofovir alafenamide fumarate; The HBV entry inhibitor is selected from an HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region or an HBV Pre-S1 derivative peptide, an anti-Pre-S1 antibody, an anti-surface antigen antibody, or other drugs that inhibit HBV entry or infection of hepatocytes; preferably, the HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region is derived from the pre-S1 region of the surface antigen of any one of HBV genotypes A, B, C, D, E, F, G, H, and I; preferably, the length of the HBV entry-inhibitory polypeptide derived from the HBV Pre-S1 region is 10-118 amino acid residues; The immunomodulator includes an agent selected from interferon, toll-like receptor agonist, CPG, CPG ODN, PD-1 inhibitor, PD-L1 inhibitor, interleukin, and cytokine; preferably, the interferon is selected from IFN-α, IFN-β, and IFN-γ, preferably IFN-α-2a and / or IFN-α-2b, more preferably PEG-IFN-α-2a and / or PEG-IFN-α-2b; Preferably, the duration of said combined treatment is 1-96 weeks, preferably 12-60 weeks, more preferably 24-48 weeks; Preferably, said patient has received continuous NAs treatment prior to administering said combination treatment, preferably more than 0 months of NAs treatment, more than 3 months of NAs treatment, more than 12 months of NAs treatment, more than 36 months of NAs treatment; Preferably, the daily dose of said HBV viral entry inhibitor is 2.1-10.5 mg, preferably 4.2-8.4 mg, more preferably 4.2 mg; Preferably, the immunomodulator is an interferon, the weekly dose of which is 1-360 micrograms, preferably 30-180 micrograms, more preferably 60-135 micrograms, more preferably 90 micrograms.

9. The method of claim 8.

10. The nucleoside (nucleotide) drug is tenofovir alafenamide fumarate, and the HBV viral entry inhibitor is heparatide or bulevirtide; Preferably, the heparatide or bulevirtide treatment cycle is from 1 week to 96 weeks; Preferably, the therapeutic dose of heparatide or bulevirtide is 2.1 mg to 8.4 mg daily; Preferably, the immunomodulator is PEG-interferon, the dose of which is preferably 10-170 ug weekly; Preferably, in the method, the nucleoside (nucleotide) drug is tenofovir alafenamide fumarate; it is treated in combination with heparatide or bulevirtide, with a daily dose of 4.2 mg for a 48-week treatment cycle; and simultaneously, PEG-interferon is administered at a weekly dose of 90 μg for a 48-week treatment cycle.

10. The method according to claim 8 or 9, characterized in that

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