Drug and method for eliminating cccdna from liver of patient infected with hepatitis b virus

Abstract

The present invention relates to a drug and method for eliminating cccDNA from the liver of a patient infected with the hepatitis B virus. Specifically, the present invention provides use of an HBV entry inhibitor for inhibiting the HBV from entering hepatocytes and an optional immunomodulator for modulating anti-HBV immunity in the preparation of a pharmaceutical combination or a kit for use in a method for eliminating the hepatitis B virus cccDNA from a patient with hepatitis B. The present invention further provides a corresponding pharmaceutical combination and a kit for the described use.

Description

Drugs and methods for clearing cccDNA from the liver of hepatitis B virus infected individuals Technical Field

[0001] This invention relates to drugs and methods for eliminating cccDNA from the liver of hepatitis B virus-infected individuals. Background Technology

[0002] In recent years, the cure pathway and clinical endpoints for hepatitis B have been increasingly discussed and a consensus has been reached. The main goal of chronic hepatitis B treatment is to improve survival by preventing liver fibrosis, liver failure, hepatocellular carcinoma (HCC), and liver-related death. However, these clinical endpoints are generally not observed for decades and are therefore unsuitable for evaluating new drugs. Thus, clinical research needs to rely on surrogate endpoints, using short-term visible intermediates as endpoint indicators to evaluate long-term clinical benefits. In previous pre- and post-marketing studies, transaminase (ALT levels), viral response (HBV DNA < quantitation limit LLOQ and HBsAg / HBeAg seroconversion), and liver tissue response were primarily used as response indicators. Among these surrogate endpoints, HBsAg seroconversion is considered the most relevant endpoint for clinical benefit, and HBsAg seroconversion is expected to result in lower rates of HCC and liver decompensation compared to HBV DNA suppression. However, because serum HBsAg has two sources—cccDNA and integrated HBV DNA—and the detection process cannot determine whether the detected HBsAg originates from cccDNA or integrated HBV DNA, this hinders the determination of the functional cure target. In 2022, the AASLD / EASL conference on hepatitis B treatment endpoints (Journal of Hepatology 2023, vol. 79) clarified the definition of different degrees of hepatitis B cure endpoints, establishing cccDNA clearance as the ultimate goal for achieving a complete cure of hepatitis B.

[0003] cccDNA is the template for hepatitis B virus (HBV) replication. HBV is a virus containing a 3.2kb partially double-stranded loose circular DNA (rcDNA) genome that specifically infects human hepatocytes. After infection, rcDNA is repaired by cellular enzymes to generate a double-stranded free DNA genome—covalently closed circular DNA (cccDNA). Through a series of interactions with histones and non-histone proteins, cccDNA forms a stable microchromosome within the cell nucleus, serving as a template for viral transcription. Therefore, the main goal of hepatitis B treatment is to reduce or inactivate cccDNA in hepatocytes. The long-term presence of cccDNA in the liver is a major obstacle to achieving a functional cure for hepatitis B.

[0004] Hepatitis B patients threaten the health of approximately 300 million people worldwide, posing a significant public health problem. Current hepatitis B treatments include nucleoside analogs (NAs) and interferon. While these drugs can inhibit viral replication to some extent, the functional cure rate within a limited treatment course is very low, and they cannot eliminate cccDNA (Chronic Hepatitis B Virus Infection: Developing Drugs for Treatment. FDA CDE. APRIL 2022). Clinical trial results for investigational drugs have not revealed any drugs or technologies capable of achieving cccDNA clearance. Therefore, both academia and industry are exploring ways to improve the cure rate of hepatitis B, and even how to ultimately achieve cccDNA clearance.

[0005] Interferon is a cytokine with broad-spectrum antiviral activity, initially used to treat various viruses including HIV, and approved by the FDA for the treatment of hepatitis B in 1992. Its mechanism of action primarily involves immunomodulation and induction of antiviral proteins within hepatocytes. Studies have shown that interferon can promote responses from various immune cells, including macrophages, natural killer cells, dendritic cells, and T cells. All these activated immune cells can secrete a series of cytokines, such as IL-1β, IL-6, TNF-α, and TNF-γ. Among these cytokines, IL-6, IL-12, and IL-15 are partially induced by interferon α / β, which then further regulate the differentiation and activation of B cells and T cells. Interferon can also enhance APC antigen presentation by increasing the expression of class II MHC, CD86, and CD40; moreover, IFN-α / β contributes to neutrophil survival and enhances the phagocytic activity of macrophages and neutrophils. In terms of direct antiviral action, interferon is believed to act on all parts of the HBV life cycle and enhance cell-mediated immunity. IFN-α inhibits HBV replication by reducing RNA transcription of covalently closed circular DNA (cccDNA). Interferon leads to hypoacetylation of histones bound to cccDNA and active recruitment of cccDNA transcriptional co-repressors. However, in clinical practice, interferon has failed to clear cccDNA (Chronic Hepatitis B Virus Infection: Developing Drugs for Treatment. FDA CDE. APRIL 2022). A clinical study showed that after one year of treatment with PEG-IFN-α-2a or PEG-IFN-α-2b, the HBeAg seroconversion rate at the end of treatment was 22-27%, and 29-32% at 6 months post-treatment. 7-14% of patients achieved viral suppression after discontinuation of treatment (defined as HBV DNA <400 copies / mL), and 3-5% of patients experienced HBsAg seroconversion.

[0006] Viral entry inhibitors include peptides derived from the HBV Pre-S1 region, anti-Pre-S1 antibodies, anti-surface antigen antibodies, and small molecule compounds that can bind to NTCP. Peptides derived from the HBV Pre-S1 region include, but are not limited to, hapratide and bulevirtide. The amino acid sequence of hapratide is derived from amino acids 13-59 of the Pre-S1 region of genotype C HBV, with an N-terminal myristic acid modification. Studies have shown that sodium taurocholate cotransporting polypeptide (NTCP) is the HBV infection receptor. HBV mediates HBV infection of hepatocytes by specifically binding to NTCP through the Pre-S1 region of a large protein on the viral envelope. Hapratide and bulevirtide can specifically bind to NTCP, blocking HBV infection of hepatocytes in vitro and in vivo. Anti-Pre-S1 antibodies include, but are not limited to, antibodies that can bind to large HBsAg Pre-S1, including, but not limited to, monoclonal antibodies, polyclonal antibodies, or antibody sera. Monoclonal antibodies include, but are not limited to, monospecific antibodies, bispecific antibodies, multispecific antibodies, and antibody fragments capable of binding to the Pre-S1 region. Anti-Pre-S1 antibodies include, but are not limited to, Huahui Anjian HH-001, HH-003, and HH-006. Anti-surface antigen antibodies include, but are not limited to, antibodies capable of binding to surface antigens, including, but not limited to, monoclonal antibodies, multispecific antibodies, or antibody sera. Monoclonal antibodies include, but are not limited to, monospecific antibodies, bispecific antibodies, multispecific antibodies, and antibody fragments capable of binding to surface antigens. The anti-surface antigen antibodies include, but are not limited to, Vir Biotechnology's BRII-877 (VIR-3434, tobevibart), Bluejay's BJT-778, YST's 162, KW-027, HT-102, HepB mAb19 developed by Rockefeller University and NYU Langone Institute of Health, Hepatitis B immunoglobulin HBIG, and Green Cross's GC1102. The small molecule compounds that can bind to NTCP include, but are not limited to, cyclosporine and its derivatives, bile acids and their derivatives, and other NTCP inhibitors with HBV infection-inhibiting functions. Other NTCP inhibitors include, but are not limited to, Huahui Anjian HH-1270, Ezetimibe, immunoMed's hzVSF, and Albireo's A2342. Summary of the Invention

[0007] The first aspect of the present invention provides the use of HBV entry inhibitors that inhibit HBV entry into hepatocytes and optional immunomodulators that regulate anti-HBV immunity in the preparation of medicaments, drug combinations or kits used in methods for clearing hepatitis B virus cccDNA from hepatitis B patients.

[0008] In one or more embodiments, the HBV entry inhibitor is selected from HBV entry-inhibiting peptides or HBV Pre-S1-derived peptides derived from the HBV Pre-S1 region, anti-Pre-S1 antibodies, anti-surface antigen antibodies, or other agents that can inhibit HBV entry into hepatocytes or infect hepatocytes.

[0009] 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 the surface antigen of any one of HBV genotypes A, B, C, D, E, F, G, H, and I.

[0010] In one or more embodiments, the HBV entry inhibitory peptide derived from the HBV Pre-S1 region is 10-118 amino acid residues in length.

[0011] In one or more embodiments, the immunomodulatory agent comprises a selection from interferon, Toll-like receptor agonists, CPG, CPG ODN, PD-1 inhibitors, PD-L1 inhibitors, interleukins, and cytokines.

[0012] 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.

[0013] 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 the C genotype HBV surface antigen; preferably, the HBV entry-inhibiting polypeptide is a fragment or a variant thereof at the N-terminus of the C genotype HBV surface antigen pre-S1 region, the fragment containing at least amino acid residues at positions 13-44 of the N-terminus; preferably, the HBV entry-inhibiting polypeptide contains 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 C genotype HBV surface antigen pre-S1 region, or a variant thereof.

[0014] In one or more embodiments, the HBV entry inhibitory polypeptide derived from the HBV Pre-S1 region is a fragment or a variant thereof at the N-terminus of the pre-S1 region of the HBV surface antigen of genotype A, B, F, H, or I, the fragment containing at least amino acid residues 13-44 of the N-terminus, preferably at least amino acid residues 13-69 of the N-terminus.

[0015] In one or more embodiments, the HBV entry inhibitory polypeptide derived from the HBV Pre-S1 region is a fragment or a variant thereof at 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, preferably at least amino acid residues 2-48 of the N-terminus.

[0016] In one or more embodiments, the HBV entry inhibitory polypeptide derived from the HBV Pre-S1 region is a fragment or a variant thereof at the N-terminus of the pre-S1 region of the HBV surface antigen of genotype E or G, the fragment containing at least amino acid residues 12-43 of the N-terminus, preferably at least amino acid residues 12-68 of the N-terminus.

[0017] In one or more embodiments, the variant has 1-30 amino acid deletions, substitutions, or insertions compared to the fragment, and the variant retains the biological activity of inhibiting HBV entry into or binding to NTCP.

[0018] In one or more embodiments, the variant includes inserting a natural side-joint amino acid sequence from any of the HBV subtypes at the N and / or C ends of the fragment.

[0019] In one or more embodiments, the natural side-connected amino acid sequence is selected from the amino acid sequence of the N-terminus 2-12 of the pre-S1 region of HBV surface antigen in genotypes A, B, F, H, and I, and the amino acid sequence of the N-terminus 2-11 of the pre-S1 region of HBV surface antigen in genotypes E and G.

[0020] In one or more embodiments, the variant is a variant obtained by introducing one or any combination of two or more of the following amino acid substitutions into a derived peptide derived from the amino acid sequence at positions 13-59 of the Pre-S1 region of genotype C HBV: 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, the N and / or C ends of the variant are inserted with a naturally occurring side-joined amino acid sequence from any of the HBV subtypes.

[0021] In one or more embodiments, the amino acid sequence of the fragment is as shown in any one of SEQ ID NO: 2-32, or has at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 2-32.

[0022] In one or more embodiments, the HBV entry inhibitory peptide derived from the HBV Pre-S1 region has N-terminal and / or C-terminal modifications.

[0023] In one or more embodiments, the N-terminus is modified with a hydrophobic group.

[0024] In one or more embodiments, the hydrophobic group modification is selected from myristic acid, palmitic acid, stearic acid, cholesterol, oleic acid, linoleic acid, polyethylene glycol, and arachidonic acid.

[0025] In one or more embodiments, the C modification is an amidation or isopentyl glycolation modification.

[0026] In one or more embodiments, the HBV entry inhibition polypeptide derived from the HBV Pre-S1 region is shown in SEQ ID NO: 33-44.

[0027] In one or more embodiments, the use is for the use of HBV entry inhibitors and optional immunomodulators in the preparation of medicaments or kits for clearing hepatitis B virus cccDNA.

[0028] In one or more embodiments, the kit contains one or more doses of the drug to deliver the HBV entry inhibitor at a daily dose of 2.1-10.5 mg, preferably 4.2-8.4 mg, more preferably 4.2 mg.

[0029] In one or more embodiments, the kit may optionally contain one or more doses of the interferon, and the content thereof is 1-360 micrograms per week, preferably 30-240 micrograms per week, more preferably 60-240 micrograms per week, and even more preferably 180 micrograms per week.

[0030] In one or more embodiments, the kit is used in combination with an entry inhibitor for 1-96 weeks, preferably 12-60 weeks, more preferably 24-48 weeks.

[0031] A second aspect of the present invention provides a method for clearing cccDNA from hepatitis B virus-infected hepatocytes, the method comprising administering a combination therapy of an HBV entry inhibitor and, optionally, an immunomodulatory agent to a chronic hepatitis B patient.

[0032] In one or more embodiments, the HBV entry inhibitor is as described in any embodiment herein, and the immunomodulator is as described in any embodiment herein.

[0033] In one or more embodiments, the duration of the combined treatment is 1-96 weeks, preferably 12-60 weeks, and more preferably 24-48 weeks.

[0034] In one or more embodiments, the daily dose of the HBV entry inhibitor is 2.1-10.5 mg, preferably 4.2-8.4 mg, more preferably 4.2 mg.

[0035] In one or more embodiments, the immunomodulator is interferon, with a weekly dose of 1-360 micrograms, preferably 30-240 micrograms, more preferably 60-240 micrograms, and even more preferably 180 micrograms.

[0036] In one or more embodiments, the treatment period for the hepratide or bulevirtide is from 1 week to 96 weeks.

[0037] In one or more embodiments, the therapeutic dose of the hepratide or bulevirtide is 2.1 mg to 8.4 mg daily.

[0038] In one or more embodiments, the immunomodulator is PE-modified interferon, preferably at a dose of 160-240 μg per week.

[0039] A third aspect of the present invention provides a pharmaceutical combination or kit containing an HBV entry inhibitor and an immunomodulator as described in any embodiment herein. Attached Figure Description

[0040] Figure 1: Flowchart of a randomized, double-blind, placebo-controlled, multicenter phase II clinical trial of Hepratide combined with PEG interferon for the treatment of chronic hepatitis B.

[0041] Figure 2: cccDNA clearance rate in each group at the end of 24 weeks of treatment.

[0042] Figure 3: Effectiveness indicators of typical case No. 156. Detailed Implementation

[0043] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as embodiments) can be combined with each other to form preferred technical solutions.

[0044] cccDNA (covalently closed circular DNA) is the template for hepatitis B virus (HBV) replication. HBV is a virus containing a 3.2kb partially double-stranded loose circular DNA (rcDNA) genome. After infecting human hepatocytes, the HCV rcDNA is repaired by cellular enzymes to generate a double-stranded free DNA genome—covalently closed circular DNA (cccDNA). Through a series of interactions with histones and non-histone proteins, cccDNA forms a stable miniature chromosome within the cell nucleus, serving as a template for viral transcription. Therefore, the primary goal of hepatitis B treatment is to reduce or inactivate cccDNA in hepatocytes. This invention aims to eliminate HBV cccDNA.

[0045] Therefore, this application uses HBV entry inhibitors and optional immunomodulators to clear hepatitis B cccDNA. In this document, cccDNA seroconversion refers to the inability to detect cccDNA (<LOD) after a certain period of medication.

[0046] HBV Entry Inhibitor

[0047] In this article, the HBV entry inhibitors include, but are not limited to, HBV entry inhibitory peptides or HBVVre-S1-derived peptides derived from the HBVPre-S1 region, anti-Pre-S1 antibodies, anti-surface antigen antibodies, or other agents that can inhibit HBV entry into hepatocytes or infect hepatocytes.

[0048] As used herein, the terms "HBV entry inhibitory peptide derived from the HBV Pre-S1 region" or "HBV Pre-S1-derived peptide" refer to peptides originating from or derived from the HBV surface antigen Pre-S1 region that can inhibit HBV virus entry into hepatocytes. These include, but are not limited to, natural, recombinant, synthetic, or purified peptides, including but not limited to the full-length natural HBV surface antigen Pre-S1 region or fragments thereof and their variants, the full-length non-natural HBV surface antigen Pre-S1 region or fragments thereof and their variants, the non-full-length natural HBV surface antigen Pre-S1 region or fragments thereof and their variants, and other variant forms. In some embodiments, the HBV entry inhibitory peptide derived from the HBV Pre-S1 region may contain the entire surface antigen pre-S1 region of any genotype of HBV surface antigen, starting from amino acid G (glycine) at position 2. In some embodiments, the HBV entry-inhibiting peptides derived from the HBV Pre-S1 region described herein may be derived from the pre-S1 region of the surface antigen of any of the HBV genotypes A, B, C, D, E, F, G, H, and I. Example genomic sequences of these HBV genotypes are available in, for example, GenBank accessions KC875260, AY220704, AF461363, AY796030, AB205129, DQ823095, HE981176, and AB179747. In some embodiments, the HBV Pre-S1 derived peptides used include, but are not limited to, HBV virus entry-inhibiting peptides 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 HBV surface antigen Pre-S1 region SEQ ID NO: 1 and its variants. The HBV entry-inhibiting peptides derived from the HBV Pre-S1 region retain at least the biological activity of inhibiting HBV virus entry or binding to NTCP.

[0049] In some embodiments, the HBV entry-inhibiting peptide derived from the HBV Pre-S1 region described herein may consist of 10-118 amino acids. For example, the peptide length 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 (inclusive of all integers between these ranges). In some embodiments, the HBV entry-inhibiting peptide derived from the HBV Pre-S1 region described herein may be at least 20 amino acids, for example, at least 25, 30, 35, or 40 amino acids. In some embodiments, the HBV entry-inhibiting peptide 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 HBV entry-inhibiting peptide derived from the HBV Pre-S1 region described herein may be 47 amino acids. The variants of the HBV entry-inhibiting peptides derived from the HBV Pre-S1 region described herein, with varying lengths, retain one or more biological activities associated with the corresponding peptides, including at least the biological activities that inhibit HBV entry or binding to NTCP.

[0050] In some embodiments, the HBV entry inhibitory peptide derived from the HBV Pre-S1 region described herein may be derived from the pre-S1 region of the C genotype HBV surface antigen, including but 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 their variants, with example sequences including but not limited to SEQ ID NO:2-14 and its variants.

[0051] In some embodiments, the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region comprises the amino acid sequence of at least amino acid residues 13-44 of the N-terminus of the pre-S1 region of the C genotype HBV surface antigen, and variants thereof. In some embodiments, the length of the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region may be 32-47 amino acid residues. In a particularly preferred embodiment, an exemplary amino acid sequence of the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region is shown in SEQ ID NO:9; the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region also includes variants of the amino acid sequence shown in SEQ ID NO:9. In some embodiments, the HBV entry inhibitory peptide derived from the HBV Pre-S1 region contains at least amino acid residues 1-32 of the example sequence SEQ ID NO:16 or a variant thereof, preferably the HBV entry inhibitory peptide derived from the HBV Pre-S1 region contains at least amino acid residues 1 to 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46 of the example sequence SEQ ID NO:16 or a variant thereof.

[0052] In some embodiments, the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region refers to a fragment containing at least amino acid residues 13-44 at the N-terminus of the pre-S1 region of HBV surface antigens of genotypes A, B, F, H, and I, or a variant thereof; a fragment containing at least amino acid residues 2-33 at the N-terminus of the pre-S1 region of HBV surface antigens of genotype D, or a fragment containing at least amino acid residues 12-43 at the N-terminus of the pre-S1 region of HBV surface antigens of genotypes E and G, or a variant thereof. In some embodiments, the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region contains a fragment containing at least amino acid residues 13-69 at the N-terminus of the pre-S1 region of HBV surface antigens of genotypes A, B, F, H, and I, or a fragment containing at least amino acid residues 2-48 at the N-terminus of the pre-S1 region of HBV surface antigens of genotype D, or a fragment containing at least amino acid residues 12-68 at the N-terminus of the pre-S1 region of HBV surface antigens of genotypes E or G, or a variant thereof.

[0053] The term "variant" as used herein refers to an HBV entry-inhibiting peptide derived from the HBV Pre-S1 region that differs from a given peptide in its amino acid sequence but retains the biological activity of inhibiting HBV entry or binding to NTCP. For example, in some embodiments, one or any combination of two or more of the following amino acid substitutions may be introduced into a derived peptide derived from the amino acid sequence of positions 13-59 of the HBV Pre-S1 region of genotype C: N15D, F25L, G35K, N39E, F45L, N46K, N48H or N48Y or N48K, D50A, H51Q or H51N, E54K or E54D, A55S, N56K or N56D, and Q57K. Example sequences include, but are not limited to, those shown in SEQ ID NO: 15-32.

[0054] In this article, the term "variant" also includes homologous polypeptide sequences found in different viral species, strains, or subtypes of the hepatoviral genus. Based on antigenic epitopes on its envelope proteins, HBV is classified into four major serotypes (adr, adw, ayr, and ayw), and according to the variability of the total nucleotide sequence in its genome, HBV is classified into nine genotypes (A, B, C, D, E, F, G, H, and I). Therefore, the term "variant" includes any of these homologous polypeptides found in HBV subtypes.

[0055] In this document, "variants" may also include polypeptides or variants thereof with a naturally occurring side-joined amino acid sequence from any of these HBV subtypes added to the N and / or C ends. For example, in some embodiments, a fragment including, but not limited to, from at least amino acid residues 2-12 at the N-terminus of the pre-S1 region of HBV surface antigen from genotypes A, B, F, H, and I, or from at least amino acid residues 2-11 at the N-terminus of the pre-S1 region of HBV surface antigen from genotypes E and G, may be used as the naturally occurring side-joined amino acid sequence to form a fusion polypeptide with a polypeptide containing the amino acid sequence from the 13-59th amino acid position of the pre-S1 region of the surface antigen from genotype C. For example, in some embodiments, the HBV entry inhibition polypeptide derived from the HBV pre-S1 region described herein may contain an amino acid sequence selected from, but not limited to, any of the amino acid sequences shown in SEQ ID NO: 2-32, and a naturally occurring amino acid sequence derived from the surface antigen of any HBV genotype AI, side-joined to the N and / or C ends. In some embodiments, the natural side-joint amino acid sequence may be derived from the amino acid sequence of the HBV surface antigen, including but not limited to GenBank accession numbers 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, the 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 contain a natural side-joint amino acid sequence from the pre-S1 region of HBV genotype C at its N and / or C terminus. Alternatively, the HBV entry inhibitory peptide 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 and / or C terminus, a naturally occurring side amino acid sequence from the pre-S1 region of any of the HBV genotypes A, B, C, D, E, F, G, and H. In some embodiments, the N and / or C terminus of the HBV entry inhibitory peptide derived from the HBV Pre-S1 region described herein may independently contain a naturally occurring side amino acid sequence of 1-10 amino acids, such as 1-8, 1-5, or 1-3 (inclusive of all integers within these ranges). For example, the HBV entry inhibitory peptide 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, a naturally occurring side amino acid sequence of 10 amino acids from the pre-S1 region of HBV genotype C. In other words, the HBV entry inhibitory peptide derived from the HBV Pre-S1 region may contain amino acids 2-59 (SEQ ID NO: 4) from the pre-S1 region of HBV genotype C.Another example is that the HBV entry inhibitory peptide derived from the HBV Pre-S1 region described herein may contain the amino acid sequence shown in SEQ ID NO: 9, and have a natural 9-amino acid side sequence from the HBV pre-S1 region of genotype E at its N-terminus. In other words, this HBV entry inhibitory peptide 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 any of the HBV entry inhibitory peptides derived from the HBV Pre-S1 region may have a natural side sequence of any length extending from its N and / or C-terminus, and the resulting HBV entry inhibitory peptide derived from the HBV Pre-S1 region retains the biological activity of inhibiting HBV entry or binding to NTCP.

[0056] In this document, the “variants” associated with the HBV entry-inhibiting peptide derived from the HBV Pre-S1 region may also include those with 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 peptide derived from the HBV Pre-S1 region preferably retains glycine (i.e., the N-terminal glycine of SEQ ID NO: 9), corresponding to the 13th amino acid in the pre-S1 region of genotype C HBV. In some embodiments, the HBV entry-inhibiting peptide 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 peptide derived from the HBV Pre-S1 region described herein may have 1-30, such as 1-20, 1-10, 1-8, 1-5, or 1-3 (inclusive of all integers within these ranges) amino acid deletions, substitutions, or insertions relative to the sequence from the pre-S1 region of HBV. In some embodiments, the HBV entry inhibitory peptide derived from the HBV Pre-S1 region described herein may have 1-30, for example 1-20, 1-10, 1-8, 1-5, or 1-3 (inclusive of all integers within these ranges) amino acid deletions, substitutions, or insertions relative to the amino acid sequence selected from any of SEQ ID NO: 2-32. In some embodiments, the HBV entry inhibitory peptide derived from the HBV Pre-S1 region described herein may have 1-30, for example 1-20, 1-10, 1-8, 1-5, or 1-3 (inclusive of all integers within these ranges) amino acid deletions, substitutions, or insertions relative to the exemplary amino acid sequence of SEQ ID NO: 9. In some embodiments, the HBV entry inhibitory peptide derived from the HBV Pre-S1 region described herein may have 1-3 amino acid deletions, substitutions, or insertions relative to the exemplary amino acid sequence of SEQ ID NO: 9. In some embodiments, the HBV entry inhibitory peptide derived from the HBV Pre-S1 region described herein has 1 to 30, such as 1 to 20, 1 to 10, 1 to 8, 1 to 5, or 1 to 3 (inclusive of all integers in these ranges) amino acid deletions or insertions at the C-terminus relative to an amino acid sequence selected from any of the examples of SEQ ID NO: 2-32.

[0057] In various embodiments, this document includes, but is not limited to, the stated polypeptide, and polypeptides having at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any of the stated polypeptides. For example, the HBV entry inhibitory polypeptide derived from the HBV Pre-S1 region may contain an amino acid sequence having at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any of the example sequences in SEQ ID NO: 2-32; preferably, the amino acid sequence having said identity comes from any of the HBVs described herein, including but not limited to genotypes A, B, C, D, E, F, G, H, and I HBVs, more preferably from genotype C. In some embodiments, the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region may contain an amino acid sequence having at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the example sequence in SEQ ID NO: 9; preferably, the amino acid sequence having said identity comes from any of the HBV types described herein, including but not limited to genotypes A, B, C, D, E, F, G, H, and I, and more preferably from genotype C. Variants of the HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region described herein with a certain sequence identity retain one or more biological activities of the corresponding polypeptide, including biological activities that inhibit HBV viral entry or bind to NTCP.

[0058] In this document, the N-terminus of the HBV entry-inhibiting peptide derived from the HBV Pre-S1 region and its variants may be modified with a hydrophobic group, such as, 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 peptide derived from the HBV Pre-S1 region described herein may contain an amino acid sequence selected from any of the amino acid sequences shown in SEQ ID NO: 2-32 or its variants, wherein the N-terminus of the HBV entry-inhibiting peptide 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 contain an amino acid sequence selected from any of SEQ ID NO: 2-32 or a variant thereof, wherein the N-terminus of the HBV entry inhibitory polypeptide derived from the HBV Pre-S1 region may be myristylated. In some embodiments, the HBV entry inhibitory polypeptide derived from the HBV Pre-S1 region described herein may contain an amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 32, wherein the N-terminus of the HBV entry inhibitory polypeptide derived from the HBV Pre-S1 region may be myristylated.

[0059] In this document, the C-terminus of the HBV entry inhibitory peptide derived from the HBV Pre-S1 region may be modified. C-terminal modification may be selected from, but is not limited to, amidation (amine), isopentyl glycolation, and other C-terminal modifications, or may be omitted. In some embodiments, the C-terminal modification may be amidation (NH2). For example, the HBV entry inhibitory peptide 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 C-terminus of which may be amidated.

[0060] In some embodiments, the N-terminus of the HBV entry inhibitory peptide derived from the HBV Pre-S1 region may be modified with a hydrophobic group, and / or the C-terminus may be otherwise modified. In some embodiments, the N-terminus of the HBV entry inhibitory peptide derived from the HBV Pre-S1 region may be modified with a hydrophobic group, and / or the C-terminus may be amidated. In some embodiments, the HBV entry inhibitory peptide derived from the HBV Pre-S1 region may contain an amino acid sequence selected from any of the amino acid sequences shown in SEQ ID NO: 2-32 and its variants, with its N-terminus modified with a hydrophobic group, and / or its C-terminus may be amidated. For example, the HBV entry inhibitory peptide derived from the HBV Pre-S1 region may contain an amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 32, with its N-terminus modified with a hydrophobic group of myristic acid (myr), palmitic acid (plam), stearic acid (stearoyl), or cholesterol (chol), and its C-terminus modified with an amidation, as shown in the peptides of SEQ ID NO: 33-37. In some embodiments, the N-terminus of the HBV entry inhibitory peptide derived from the HBV Pre-S1 region may be modified with a myristic acid (myr) hydrophobic group, and / or the C-terminus may be modified with amidation. In some embodiments, the peptide may contain an amino acid sequence selected from any of SEQ ID NO: 2-32 and its variants, the N-terminus of which may be modified with myristic acid (myr) hydrophobic group, and / or the C-terminus of which may be modified with amidation. For example, the HBV entry inhibitory peptide derived from the HBV Pre-S1 region may contain the peptide shown in SEQ ID NO: 9 or 32, the N-terminus of which is modified with a myristic acid (myr) hydrophobic group, and the C-terminus of which is modified with amidation, as shown in any of SEQ ID NO: 33, 34, and 38-44. In some embodiments of the present invention, the loaded HBV entry inhibitor is, but is not limited to, hopratide (SEQ ID NO: 33), bulevirtide (SEQ ID NO: 43), and other HBV Pre-S1 derived peptides.The name of Hepratide 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-asparaginyl-L-histyl-L-glutamine-L-leucyl-L-asparaginyl-L-prolyl-L-alanyl-L-phenylalanyl-glycyl-L- Alanyl-L-asparagine-L-seryl-L-asparagine-L-asparagine-L-prolyl-L-asparagine-L-tryptophanyl-L-asparagine-L-phenylalanyl-L-asparagine-L-prolyl-L-asparagine-L-lysyl-L-asparagine-L-histyl-L-tryptophanyl-L-prolyl-L-glutamine-L-alanyl-L-asparagine-L-glutamine-L-valine-glycine. Bulevirtide is named as follows: N-Myristoyl-glycyl-L-threonyl-L-asparagyl-L-leucyl-L-seryl-L-valyl-L-prolyl-L-asparagyl-L-prolyl-glycyl-L-phenylalanyl-L-phenylalanyl-L-prolyl-L-asparagyl-L-histyl-L-glutamine-L-leucyl-L-asparagyl-L-prolyl-L-alanyl-L-phenylalanyl-glycyl -L-alanyl-L-asparagyl-L-seryl-L-asparagyl-L-asparagyl-L-prolyl-L-asparagyl-L-tryptophanyl-L-asparagyl-L-phenylalanyl-L-asparagyl-L-prolyl-L-asparagyl-L-lysyl-L-asparagyl-L-histyl-L-tryptophanyl-L-prolyl-glutamine-L-alanyl-L-asparagyl-L-lysyl-L-valine-glycamide.

[0061] The sequences involved in this application are shown below.

[0062] In this article, the anti-Pre-S1 antibody includes, but is not limited to, anti-HBV Pre-S1 monoclonal antibody, anti-HBV Pre-S1 bifunctional antibody (biclonal antibody), anti-HBV Pre-S1 polyclonal antibody (polyclonal antibody), anti-HBV Pre-S1 serum, and other forms of antibodies and their combinations, which can bind to HBV Pre-S1 and inhibit HBV infection.

[0063] In this article, the anti-surface antigen antibodies include, but are not limited to, anti-surface antigen monoclonal antibodies, anti-surface antigen bifunctional antibodies (biclonal antibodies), anti-surface antigen polyclonal antibodies (polyclonal antibodies), anti-surface antigen serum, HBIG, and other forms of antibodies and their combinations, which can bind to surface antigens and inhibit HBV infection.

[0064] In this article, other agents that can inhibit HBV from entering or infecting hepatocytes include, but are not limited to, small molecules, proteins, and other molecules, which can act on NTCP, HBV virus, or other targets to achieve the technical effect of inhibiting HBV infection.

[0065] Immunomodulators

[0066] In this article, immunomodulators refer to immunomodulators that regulate anti-HBV immunity, including but not limited to various drugs commonly used in the treatment of hepatitis B, including but not limited to interferon (IFN), PEGylated interferon, Toll-like receptor (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), other cytokines, and other immunomodulators.

[0067] In this document, interferon includes one or more of IFN-α, IFN-β, and IFN-γ. In a preferred embodiment, the interferon is IFNα-2a or IFNα-2b. In some embodiments, the interferon is a conventional interferon or a long-acting interferon. In a preferred embodiment, the interferon is a polyethylene glycol (PEG)-modified interferon. In a further preferred embodiment, the interferon is PEGylated IFNα-2a or PEGylated IFNα-2b, including but not limited to Pegasys, Pegasys, Pegasys, and other interferons.

[0068] In this article, 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.

[0069] In this article, 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.

[0070] In this article, 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 protein, recombinant PD-L1 fusion protein, and small molecule drugs that inhibit PD-L1 or PD-L1 signaling, as well as other PD-L1 inhibitors.

[0071] Methods to remove cccDNA

[0072] The method for clearing cccDNA from a patient's body disclosed in this article involves administering an effective dose of an HBV entry inhibitor and, optionally, an immunomodulatory agent to the patient in need.

[0073] In this document, the dosage of HBV entry inhibitors (such as hapraviride) can be 2.1-10.5 mg daily. In a preferred embodiment, the dosage of HBV entry inhibitors (such as hapraviride) is 4.2-8.4 mg daily. In a further preferred embodiment, the dosage of HBV entry inhibitors (such as hapraviride) is 4.2 mg daily. In some embodiments, the dosage of buleviride is not less than 4 mg daily, such as 4-10 mg / day.

[0074] In this document, the dosage of the immunomodulatory agent may be the usual dosage of the immunomodulatory agent, or a lower or higher dosage than the usual dosage. For example, interferon is Pegasys, and the dosage is 1-360 μg per week; in one embodiment, the dosage is 60-240 μg per week; in another embodiment, the dosage is 150-200 μg per week; and in yet another embodiment, the dosage is 180 μg per week.

[0075] In some embodiments described herein, the treatment duration of the HBV entry inhibitor (such as hapratide) is 1-96 weeks. In a preferred embodiment, the treatment duration of the HBV entry inhibitor (such as hapratide) is 12-48 weeks. In a further preferred embodiment, the treatment duration of the HBV entry inhibitor (such as hapratide) is 24-48 weeks. In an even more preferred embodiment, the treatment duration of the HBV entry inhibitor (such as hapratide) is 48 weeks.

[0076] In some embodiments described herein, when an immunomodulatory agent (such as interferon) is administered, the treatment period is 1-96 weeks. In a preferred embodiment, the treatment period for the immunomodulatory agent (such as interferon) is 12-48 weeks. In a further preferred embodiment, the treatment period for the immunomodulatory agent (such as interferon) is 24-48 weeks. In an even more preferred embodiment, the treatment period for the immunomodulatory agent (such as interferon) is 48 weeks.

[0077] In some implementations, the dosing cycle of HBV entry inhibitors (such as hapratide) may be the same as or different from that of immunomodulators (such as interferon).

[0078] In some implementations, the treatment cycle of HBV entry inhibitors (such as hapratide) is shorter than that of immunomodulators (such as interferon).

[0079] In some implementations, an immunomodulator is administered as a basal drug, followed by an HBV entry inhibitor. In some implementations, the immunomodulator is administered for 24–96 weeks, and the HBV entry inhibitor is administered for 24–96 weeks. In other implementations, the immunomodulator is administered for a period before the HBV entry inhibitor is started; the HBV entry inhibitor may be discontinued before, simultaneously with, or later than the immunomodulator.

[0080] In some implementation schemes, the target population is given an immunomodulatory agent once a week for 48 weeks, concurrently with an HBV entry inhibitor once daily for 24 weeks. In some implementation schemes, the target population is given interferon 180 μg once a week for 48 weeks as a basal dose, concurrently with hapratide for 24 weeks, followed by discontinuation of the treatment with a daily dose of 4.2–6.3 mg, preferably 4.2 mg.

[0081] This article does not specify any particular method of administration for each drug; the appropriate method of administration can be selected based on the actual drug dosage form used. For example, interferon and hoprene are typically administered via subcutaneous injection. Generally, subjects may receive a subcutaneous injection of hoprene 30 minutes before bedtime.

[0082] Drug combinations and pillboxes

[0083] The present invention also provides a pharmaceutical combination comprising an HBV entry inhibitor and an immunomodulator as described in any embodiment herein.

[0084] The amounts of HBV entry inhibitors and immunomodulators in the drug combination are typically sufficient to sustain dosing for at least one dosing cycle, preferably for two, three, or four or more dosing cycles. A typical dosing cycle comprises 1 to 96 weeks, preferably 12 to 48 weeks, and more preferably 24 to 48 weeks. In some embodiments, a dosing cycle is 48 weeks.

[0085] As mentioned earlier, immunomodulators such as interferon are typically administered once a week, while HBV entry inhibitors are administered once daily. Therefore, for a given number of dosing weeks, this drug combination contains an immunomodulator for that corresponding number of weeks and an HBV entry inhibitor for the corresponding number of days.

[0086] In some embodiments, the drug combination is used to implement any of the methods described above for eliminating cccDNA.

[0087] In this drug combination, the HBV entry inhibitor and the immunomodulator are formulated separately, each containing the corresponding active ingredient and a pharmaceutically acceptable carrier well known in the art. For pharmaceutically acceptable carriers of the HBV entry inhibitor, see, for example, the carriers disclosed in CN 201310273122.3. In some embodiments, the HBV entry inhibitor formulation used herein is the hapratide formulation disclosed in CN 201310273122.3. The immunomodulator may be a conventional pharmaceutical formulation in the art.

[0088] The kit described herein typically contains an HBV entry inhibitor and an immunomodulator as described in any embodiment herein, for implementing the method for eliminating cccDNA as described in any embodiment herein. In some embodiments, the kit contains a combination of drugs as described in any embodiment herein.

[0089] use

[0090] In some embodiments, this document provides the use of the HBV entry inhibitor and optional immunomodulator described in any embodiment herein in the preparation of a medicament, combination of medicines, or kit for eliminating hepatitis B virus cccDNA from a patient. Preferably, the medicament, combination of medicines, or kit is suitable for the method of eliminating cccDNA described in any embodiment herein.

[0091] In some embodiments, this document provides HBV entry inhibitors and optional immunomodulators as described in any embodiment herein for use in methods for eliminating hepatitis B virus cccDNA from patients. Preferably, the method is as described in any embodiment herein.

[0092] In some implementations, the patient described herein has one or more of the following characteristics: (1) HBsAg < 10,000 IU / ml; (2) HBcrAg < 10,000 IU / ml; (3) HBeAg negative; (4) HBV DNA < 100,000 IU / ml; preferably, the patient meets all four of the above characteristics.

[0093] Drug or medicine box preparation method

[0094] This invention also provides a method for preparing a medicament or kit for clearing hepatitis B virus cccDNA from hepatitis B patients. The method includes: preparing a formulation containing an HBV entry inhibitor; packaging the formulation containing the HBV entry inhibitor and optionally a formulation containing an immunomodulatory agent into a medicament or kit suitable for administration; and providing instructions for use of the formulation containing the HBV entry inhibitor and optionally the formulation containing an immunomodulatory agent to clear hepatitis B virus cccDNA from hepatitis B patients. In some embodiments, the HBV entry inhibitor and immunomodulatory agent are as described in any embodiment herein. In some embodiments, the kit is as described in any embodiment herein. In some embodiments, the instructions for use contain the method for clearing hepatitis B virus cccDNA from hepatitis B patients as described in any embodiment herein.

[0095] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0096] Example 1: Phase II Clinical Trial Protocol for the Treatment of Hepatitis B

[0097] Clinical Trial Title: Randomized, Double-Blind, Placebo-Controlled, Multicenter, Dose-Relationship Phase II Clinical Trial of Hepratide (L47) for Injection in Combination with Pegylated Interferon for the Treatment of Chronic Hepatitis B

[0098] Research protocol number: L47-HB-II-02.

[0099] The primary objectives of this study were: 1) to explore the dose-response relationship of hapratide combined with pegylated interferon in the treatment of chronic hepatitis B; and 2) to further understand the safety of hapratide combined with pegylated interferon in the treatment of chronic hepatitis B. Secondary objectives were: 1) to further understand the pharmacokinetics of hapratide combined with pegylated interferon in the treatment of chronic hepatitis B; 2) to further understand the pharmacokinetics and drug interactions of hapratide and pegylated interferon in combination therapy; and 3) to further understand the anti-antibody response of hapratide combined with pegylated interferon.

[0100] Trial Design: This study was a 4-arm parallel design, randomized, placebo-controlled, double-blind, multicenter, dose-response phase II clinical trial. The overall design is shown in Figure 1. Chronic hepatitis B patients who met the inclusion and exclusion criteria were randomly assigned to stratified by sex into 2.1, 4.2, and 6.3 mg dose groups of hoplasty, with 32 subjects in each group. In addition to PEG-IFNα-2a treatment, subjects in each hoplasty dose group were randomly assigned in a 3:1 ratio to receive the corresponding dose of hoplasty or placebo in a double-blind manner. Subjects receiving placebo in each hoplasty dose group were combined into the placebo group. Both the hoplasty dose groups and the placebo group received continuous treatment for 24 weeks, with a 4-week follow-up. PEG-IFNα-2a treatment was then continued for another 24 weeks. Efficacy evaluation was performed at the end of the 24-week hoplasty treatment period. At week 24, if a patient's HBsAg > 20,000 IU / ml and HBV DNA decreases by < 2 log10 IU / ml from baseline (HBeAg positive) or by 1 log IU / mL (HBeAg negative), then subsequent interferon therapy will be discontinued, and NA therapy will be initiated as appropriate. Trial groupings are shown in Table 1.

[0101] Table 1: Experimental Grouping Table

[0102] Inclusion criteria: 1. Age ≤ 60 years, both male and female; 2. Persistently positive for HBsAg and / or HBV DNA for more than 6 months (investigator's diagnosis of "chronic hepatitis B"), and HBsAg < 10,000 IU / ml; 3. HBcrAg < 10,000 IU / ml; 4. HBeAg negative; 5. HBV DNA < 100,000 IU / ml; 6.2 × ULN ≤ ALT ≤ 10 × ULN; 7. Serum total bilirubin < 2 × ULN; 8. No history of decompensated liver disease (ascites, jaundice, hepatic encephalopathy, varicose hemorrhage), no history of severe heart disease (including unstable or uncontrolled heart disease within 6 months), no history of severe mental illness or severe mental illness (especially depression), no history of organ transplantation, etc.; no uncontrolled epilepsy, mental illness, and uncontrolled diabetes and hypertension; no autoimmune diseases, immune-related extrahepatic manifestations (vasculitis, purpura, nodular arteritis, peripheral neuropathy, and glomerulonephritis), autoimmune thyroid disease, malignant tumors, or immunosuppressive therapy; no underlying diseases such as severe infection, retinal disease, heart failure, and chronic obstructive pulmonary disease, or other serious diseases; no alcoholism or drug abuse. 9. No plans to have children within the next two years; if female, not pregnant or breastfeeding; 10. No blood donation or clinical trial participation within the past three months; 11. Good adherence to the research protocol; 12. Subjects understand and agree to sign the informed consent form.

[0103] Exclusion criteria: 1. Past or present decompensated liver disease: complications such as ascites, hepatic encephalopathy, variceal bleeding, and hepatorenal syndrome; 2. Prothrombin time > 1.2 × ULN, serum albumin < 35 g / L; 3. Clinical evidence of cirrhosis: such as abdominal ultrasound, CT scan, or other imaging examinations confirming cirrhosis, or a clinical diagnosis of cirrhosis by the investigator; or a Metavir fibrosis score of 4 on liver biopsy; or a Child-Pugh score > 7 on liver function tests; 4. Decreased blood cell count: neutrophils < 1 × 10⁻⁶. 9 / L, platelets <50×10 9 5. Individuals with hepatitis A, C, D, or E virus infection, or HIV infection; 6. Contraindications to pegylated interferon therapy, such as severe depression, epilepsy, autoimmune diseases, or uncontrolled thyroid dysfunction; 7. Interferon allergy; 8. History of severe retinopathy or ophthalmological disease (e.g., eye disease due to hypertension or diabetes, CMV retinitis, macular degeneration, or high myopia); 9. Positive anti-HBV Pre-S1 antibody; 10. Hamilton Depression Scale (HAMD, 17 items) score >17; 11. Positive pregnancy test in women; 12. Other significant abnormalities in laboratory or auxiliary examinations, deemed unsuitable for participation in this trial by the researchers.

[0104] Experimental drug:

[0105] 1. Hepratide for injection; Dosage form: powder for injection; Specification: 2.1mg; Transportation and storage: Store at 2-8℃ in a sealed container protected from light; Shelf life: 36 months; Manufacturer: Hainan Shuangcheng Pharmaceutical Co., Ltd.

[0106] 2. Hepratide placebo; Dosage form: powder for injection; Specification: 0mg; Transportation and storage: Store at 2-8℃ in a sealed container away from light; Shelf life: 36 months; Manufacturer: Hainan Shuangcheng Pharmaceutical Co., Ltd.

[0107] 3. Pegylated interferon (Pegasys); Dosage form: Injection; Specification: 180μg / 0.5ml; Transportation and storage: Sealed, protected from light, store in the original packaging at 2-8℃. Do not freeze. Shelf life: 24 months; Approval number: National Drug Approval Number J20120075; Manufacturer: Shanghai Roche Pharmaceuticals Co., Ltd.

[0108] Basic treatment: 180μg dose of pegylated interferon as basic treatment (once a week, subcutaneous injection) for 48 consecutive weeks.

[0109] Dosage / Intervention: Add hapratide treatment (daily dose 2.1, 4.2 or 6.3 mg; generally recommended for subjects to be injected subcutaneously 30 minutes before bedtime, once daily; dosing time may be adjusted for hospitalized subjects) to basic treatment for 24 weeks.

[0110] Controlled treatment: Double-blind placebo-controlled hopeptide therapy, with patients receiving a daily subcutaneous injection of placebo hopeptide on top of basic treatment for 24 weeks.

[0111] Trial duration: Trial duration = treatment duration (24 weeks) + safety follow-up (4 weeks)

[0112] Basic treatment = 24+4 weeks of pegylated interferon treatment during the trial + 20 weeks of pegylated interferon treatment after the trial ends.

[0113] Efficacy indicators: HBV DNA, HBsAg, HBeAg, and liver histological response. Experimental indicators: HBV pgRNA and cccDNA.

[0114] Safety indicators: vital signs, physical examination; laboratory tests: routine blood and urine tests, blood biochemistry, sex hormone levels (female only), coagulation, thyroid function, autoantibodies, drug-resistant antibodies; auxiliary examinations: electrocardiogram, B-ultrasound examination (liver, gallbladder, spleen, pancreas, kidneys, ascites, breasts, uterus and adnexa), liver CT, fundus examination; mental status assessment, targeted AE examination; key indicators: hematology, liver function, sex hormone levels (female only), blood glucose, blood lipids, blood cholesterol, total bile acids, blood phosphorus, blood fibrinogen.

[0115] Safety assessment: Adverse events are classified according to NCI CTCAE v5.0, using a 5-level judgment standard.

[0116] Example 2: Enrollment in a Phase II Clinical Trial for the Treatment of Hepatitis B

[0117] Study period: Start date May 24, 2021 (date of the first participant signing informed consent); Completion date November 29, 2023 (date of the last participant completing the trial).

[0118] Number of participants: This study enrolled 96 participants, with 24 in each of the three investigational drug dose groups (2.1 mg, 4.2 mg, and 6.3 mg) and the placebo control group. One participant in the 2.1 mg group discontinued the trial due to interferon-related adverse events after receiving one PEG-IFN treatment, and one HBeAg-positive participant in the 6.3 mg dose group withdrew informed consent and discontinued the trial after 20 weeks of treatment. The remaining 94 participants completed the trial.

[0119] Baseline characteristics: This study analyzed demographic data (age, sex, ethnicity, height, weight, BMI) based on ITT (Investigative Therapy Test). Demographic information was generally balanced between the experimental and control groups at each dosage level. The baseline HBV DNA levels in the experimental drug dosage groups (2.1, 4.2, and 6.3 mg) and the placebo group were 7.968±1.17, 7.040±1.57, 7.198±1.61, and 7.603±1.35 log10 IU / mL, respectively, with baselines balanced across groups. The average baseline HBsAg levels in the experimental drug dosage groups (2.1, 4.2, and 6.3 mg) and the placebo group were 4.157±0.97, 3.708±0.75, 3.859±0.98, and 4.090±0.74 log10 IU / mL, respectively, with baselines balanced across groups.

[0120] Example 3: Results of a Phase II Clinical Trial for the Treatment of Hepatitis B

[0121] Virological response: At the end of 24 weeks of treatment, 18 / 23, 21 / 24, 17 / 24, and 14 / 24 subjects in each group achieved a virological response (HBV DNA reduction ≥2 log10 or HBV DNA <20 IU / ml), respectively. The virological response rates at the end of 24 weeks of treatment were 78.3%, 87.5%, 70.8%, and 58.3%, respectively. The virological response rate in the 4.2 mg dose group was significantly higher than that in the placebo group (P = 0.0248). Specific results are shown in Table 2.

[0122] Table 2: Virological endpoints of subjects at the end of 24 weeks of treatment

[0123] #One subject terminated the trial due to interferon adverse events (AEs) after receiving one dose of PEG interferon and this product.

[0124] HBsAg level reduction: HBsAg levels continued to decrease in all groups during the treatment process. At the end of 24 weeks of treatment, the HBsAg levels in each group decreased by -0.82, -0.70, -0.64, and -0.38 log10 IU / mL from baseline, respectively, showing that the reduction in HBsAg from baseline in each dose group was greater than that in the placebo group.

[0125] Liver histological response: A total of 14 subjects had liver biopsies obtained at baseline and at the end of 24 weeks of treatment, with 3, 3, 5, and 3 subjects in each group, respectively. At the end of 24 weeks of treatment, the number of subjects with liver histological response in each group was 0 / 3, 1 / 3, 2 / 5, and 0 / 3, respectively, and the incidence rates of liver histological response in each group were 0.0%, 33.3%, 40.0%, and 0.0%, respectively. The incidence rates of liver histological response were higher in the 4.2 mg and 6.3 mg dose groups than in the control group.

[0126] cccDNA Detection: In this study, 21 liver biopsy samples were obtained at baseline. Among them, a total of 14 subjects had liver biopsy samples obtained at both baseline and the end of 24-week treatment, with 3, 3, 5, and 3 cases in each group respectively. The PCR method verified by the central laboratory was used to quantitatively detect cccDNA in liver biopsy samples, and the lowest limit of detection (LOD) was 5 copies / μL. The baseline levels of HBV cccDNA in each group were 3.463±1.03, 2.383±0.97, 3.850±1.08, and 3.018±0.76 log10 copies / μL respectively. The baselines of each group were basically balanced, and all baseline liver biopsy samples were positive for cccDNA (≥LOD). When the 24-week treatment was completed, the number of subjects with undetectable HBV cccDNA (<LOD) in each group was 0 / 3, 1 / 3, 2 / 5, and 0 / 3 cases respectively, and the incidence rates of undetectable HBV cccDNA were 0.0%, 33.3%, 40.0%, and 0.0% respectively (Figure 2). The HBV cccDNA clearance rates in the 4.2 mg and 6.3 mg dose groups were higher than those in the control group.

[0127] Example 4: Safety Results of Phase II Clinical Trial for the Treatment of Hepatitis B

[0128] All 96 enrolled subjects were included in the safety set SS, with a total of 95 subjects, and 1281 cases of TEAE occurred, with an incidence rate of 99%. The number of cases (incidence rate) of TEAE in each group was 23 (95.8%), 24 (100%), 24 (100%), and 24 (100%) respectively. The incidence rates of TEAE in each group were balanced, with no significant difference.

[0129] A total of 28 subjects had TEAE with CTCAE ≥ grade 3, with a total of 62 cases, and the incidence rate was 29.2%. The number of cases (incidence rate) of TEAE with CTCAE ≥ grade 3 in each group was 7 (29.2%), 8 (33.3%), 5 (20.8%), and 8 (33.3%) respectively, and there was no obvious difference in the incidence rates of each group.

[0130] AE related to Hepratide (or placebo): A total of 26 subjects had at least one TEAE related to Hepratide (or placebo), and the number of cases (incidence rate) in each group was 6 (25.0), 4 (16.7), 8 (33.3), and 8 (33.3) respectively. The severity was all grade 1 and grade 2, and there was no TEAE with CTCAE ≥ grade 3 or above.

[0131] Example 5: Typical Cases of Phase II Clinical Trial for the Treatment of Hepatitis B

[0132] Subject S7-011 (No. 156), 4.2 mg dose group. Baseline levels of HBV DNA, HBsAg, pg RNA, and cccDNA were 3.67 log10 IU / mL, 3.55 log10 IU / mL, 2.69 log10 copies / mL, and 1.89 log10 copies / μL, respectively. By the end of 24 weeks of treatment, HBsAg had decreased to 1.61 log10 IU / mL, and HBV DNA, pg RNA, and cccDNA were all below the detection limit (Figure 3).

Claims

1. Use of HBV entry inhibitors and optional immunomodulators that inhibit HBV entry into hepatocytes in the preparation of medicines, combinations of medicines or kits used in methods for clearing hepatitis B virus cccDNA from patients with hepatitis B.

2. The application as described in claim 1, characterized in that: The HBV entry inhibitor is selected from HBV entry-inhibiting peptides derived from the HBV Pre-S1 region, or HBV Pre-S1-derived peptides, anti-Pre-S1 antibodies, anti-surface antigen antibodies, or other agents that can inhibit HBV entry into hepatocytes or infection of hepatocytes; preferably, the HBV entry-inhibiting peptide 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-inhibiting peptide derived from the HBV Pre-S1 region is 10-118 amino acid residues; and / or The immunomodulatory agent includes those selected from interferon, PEGylated interferon, Toll-like receptor agonists, CPG, CPG ODN, PD-1 inhibitors, PD-L1 inhibitors, interleukins, and cytokines; preferably, the interferon is selected from IFN-α, IFN-β, and IFN-γ, more preferably IFNα-2a and / or IFNα-2b, and more preferably PEGylated IFNα-2a and / or PEGylated IFNα-2b.

3. The application as described in claim 2, characterized in that, The HBV entry-inhibiting polypeptide derived from the HBV Pre-S1 region is derived from the pre-S1 region of the C genotype HBV surface antigen; preferably, the HBV entry-inhibiting polypeptide is a fragment or a variant thereof at the N-terminus of the C genotype HBV surface antigen pre-S1 region, the fragment containing at least amino acid residues 13-44 at the N-terminus; preferably, the HBV entry-inhibiting polypeptide contains amino acid residues 2-119, 2-69, 2-59, 13-119, 13-88, 13-72, 13-67, 13-59, 13-52, or 13-47 of the C genotype HBV surface antigen pre-S1 region, or a variant thereof; or The HBV entry inhibitory polypeptide derived from the HBV Pre-S1 region is a fragment or variant thereof from the N-terminus of the pre-S1 region of HBV surface antigen of genotypes A, B, F, H, or I, and the fragment contains at least amino acid residues 13-44 of the N-terminus, preferably at least amino acid residues 13-69 of the N-terminus; or The HBV entry inhibitory polypeptide derived from the HBV Pre-S1 region is a fragment or a variant thereof at the N-terminus of the pre-S1 region of the D genotype HBV surface antigen, wherein the fragment contains at least amino acid residues 2-33 of the N-terminus, preferably at least amino acid residues 2-48 of the N-terminus; or The HBV entry inhibitory polypeptide derived from the HBV Pre-S1 region is a fragment or a variant thereof at the N-terminus of the pre-S1 region of the HBV surface antigen of genotype E or G, and the fragment contains at least amino acid residues 12-43 of the N-terminus, preferably at least amino acid residues 12-68 of the N-terminus. Preferably, the variant has 1-30 amino acid deletions, substitutions, or insertions compared to the fragment, and the variant retains the biological activity of inhibiting HBV entry into or binding to NTCP; preferably, the variant comprises inserting a natural side-joint amino acid sequence from any of the HBV subtypes at the N and / or C ends of the fragment; preferably, the natural side-joint amino acid sequence is selected from the N-terminal amino acid sequence of the pre-S1 region of HBV surface antigen in genotypes A, B, F, H, and I, and the N-terminal amino acid sequence of the pre-S1 region of HBV surface antigen in genotypes E and G; preferably, the variant is derived from HBV subtype C. Variants obtained by introducing one or any combination of two or more of the following amino acid substitutions into a derived peptide of the amino acid sequence at positions 13-59 of the Pre-S1 region: 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, the N and / or C ends of the variant are inserted with a natural side-joined amino acid sequence from any of the said HBV subtypes.

4. Use according to claim 3, wherein the compound is ###0002### The amino acid sequence of the fragment is as shown in any one of SEQ ID NO:2-32, or has at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO:2-32.

5. The application as described in any one of claims 2-4, characterized in that, The HBV entry inhibition peptide derived from the HBV Pre-S1 region has N-terminal and / or C-terminal modifications. Preferably, the N-terminal modification is a hydrophobic group modification; preferably, the 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 isopentyl glycolation modification; Preferably, the HBV entry inhibition polypeptide derived from the HBV Pre-S1 region is as shown in SEQ ID NO:33-44.

6. The application as described in any one of claims 1-5, characterized in that: The kit contains one or more doses of an HBV entry inhibitor to meet the requirement of administering the HBV entry inhibitor at a daily dose of 2.1-10.5 mg, preferably 4.2-8.4 mg, more preferably 4.2 mg; and / or The medicine box may also optionally contain one or two or more doses of an immunomodulatory agent.

7. The application as described in any one of claims 1-6, characterized in that, The method for clearing hepatitis B virus cccDNA from hepatitis B patients includes the steps of administering an effective dose of an HBV entry inhibitor and, optionally, an immunomodulatory agent to the patient in need; wherein: The dosage of the HBV entry inhibitor is 2.1-10.5 mg daily, preferably 4.0-10.0 mg daily, and more preferably 4.2-8.4 mg daily; The immunomodulatory agent is interferon or PEGylated interferon, and the dosage is 1-360ug per week, preferably 60-240ug per week. The treatment period for the HBV entry inhibitor is 1-96 weeks, preferably 12-48 weeks, and more preferably 24-48 weeks; The treatment period for the immunomodulator is 1-96 weeks, preferably 12-48 weeks, and more preferably 24-48 weeks; The treatment cycle of the HBV entry inhibitor may be the same as or different from that of the immunomodulator.

8. Use according to claim 7, wherein the compound is ###0002### The method includes: administering an immunomodulator as a basal drug, and administering an HBV entry inhibitor on the basis of the basal drug; preferably, the administration period of the immunomodulator is 24 to 96 weeks, and the administration period of the HBV entry inhibitor is 24 to 96 weeks; preferably, the immunomodulator is administered for a period of time first, and then the HBV entry inhibitor is administered, and the administration of the HBV entry inhibitor is terminated before, simultaneously with, or later than the immunomodulator. Preferably, the method includes: administering the target immunomodulator to the desired subject once a week for 48 weeks, and concurrently administering an HBV entry inhibitor once daily for 24 weeks; preferably, administering the target interferon to the desired subject once a week for 48 weeks, 180 μg each time as a basal dose, and concurrently administering hopreptide once daily for 24 weeks, ending the administration period at a dose of 4.2–6.3 mg each time.

9. The application as described in any one of claims 1-8, characterized in that, The patient has one or more of the following characteristics: (1) HBsAg <10,000 IU / ml; (2) HBcrAg <10,000 IU / ml; (3) HBeAg negative; and (4) HBV DNA <100,000 IU / ml.

10. A drug combination or kit containing an HBV entry inhibitor and an immunomodulator, wherein, The HBV entry inhibitor is as described in any one of claims 2-6, and the immunomodulator is as described in claim 2 or 7; preferably, the HBV entry inhibitor and immunomodulator contained in the drug combination or kit are sufficient to implement the method of claim 7 or 8.

11. A method for preparing a drug or kit for eliminating hepatitis B virus cccDNA from hepatitis B patients, characterized in that, The method includes: preparing a formulation containing an HBV entry inhibitor; packaging the formulation containing the HBV entry inhibitor and optionally a formulation containing an immunomodulator into a drug or kit form suitable for administration; and providing instructions for use of the formulation containing the HBV entry inhibitor and optionally the formulation containing an immunomodulator to clear hepatitis B virus cccDNA from hepatitis B patients.

12. The method as described in claim 11, characterized in that: The HBV entry inhibitor is as described in any one of claims 2-5; The immunomodulator is as described in claim 2.

13. The method of claim 11, wherein, The medicine box is as described in claim 6.

14. The method of claim 11, wherein, The instruction manual contains the method for eliminating hepatitis B virus cccDNA from hepatitis B patients as described in claim 7 or 8.

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