Pharmaceutical products and methods for treating hepatitis B virus-associated liver disease under all dose conditions

Abstract

A pharmaceutical product and method for treating and preventing hepatitis B virus-associated liver disease under full-dose conditions. Specifically, the present invention provides a method for treating or preventing hepatitis B virus-associated liver disease, the method comprising administering a full dose of a polypeptide or a pharmaceutical composition thereof to a subject in need of treatment or prevention every day, the polypeptide comprising an amino acid sequence derived from hepatitis B virus (HBV) Pre-S1; the full dose is a daily dose at which the polypeptide reaches saturation in the liver, the target organ, several days after administration.

Description

[Technical field]

[0001] The present invention relates to pharmaceutical agents and methods for treating Hepatitis B virus associated liver disease under all dosage conditions. [Background technology]

[0002] Approximately one million people die each year worldwide from liver failure, cirrhosis, and primary hepatocellular carcinoma caused by hepatitis B virus (HBV) infection. Currently, it is estimated that there are 20 million chronic hepatitis B patients in China, with one million new cases each year, five million cases of hepatitis B-related cirrhosis each year, and one million deaths each year. There are 300,000 cases of hepatitis B-related liver cancer and 300,000 deaths each year.

[0003] HBV viral envelope proteins include three surface antigen proteins: large surface antigen protein (L), middle surface antigen protein (M) and small surface antigen protein (S). These proteins are encoded by a single open reading frame in the S gene and start from three different translation initiation sites, i.e., L (Pre-S1 + Pre-S2 + S), M (Pre-S2 + S) and S (S). HBV is divided into nine genotypes (AI), and each genotype has some degree of difference in the amino acid sequence of the Pre-S1 region. Studies have confirmed that HBV mediates infection of hepatocytes by binding to sodium taurocholate cotransporting polypeptide (NTCP) on the hepatocyte surface via the large surface protein (large HBsAg) (Yan et al., eLife, 1:e00049 (2012)). Derived polypeptides containing the HBV Pre-S1 amino acid sequence can block HBV infection of hepatocytes in vitro (Gripon P et al., J. Virol. 2005;79(3):1613-1622) and in animal models (Petersen J et al., Nat Biotechnol. 2008 Mar;26(3):335-41.). NTCP also assumes the physiological function of transporting bile acids from the portal blood to the liver and is one of the key proteins in the bile acid enterohepatic circulation (Alrefai W et al., Pharmaceutical Research, 2007;24(10):1803-1823).

[0004] NTCP is specifically expressed in the liver and is rarely expressed in other tissues; HBV Pre-S1 derived polypeptides show significant liver concentration in animals (Schieck A et al., Hepatology. 2013 July;58(1):43-53). A Phase Ia clinical trial and pharmacokinetic study of a single ascending dose of HBV Pre-S1 derived polypeptide Myrcludex B in humans has been completed (Lank A et al., J Hepatol. 2016 Sep;65(3):483-9.). The study included 12 dose groups, each of which received 0.3ug, 3ug, 10ug, 100ug, 800ug, 3mg, 5mg, 10mg, and 20mg as a single 2-hour continuous intravenous infusion, and 800ug, 5mg, and 10mg as a single subcutaneous injection. A total of 36 healthy subjects were enrolled in the study and distributed to each of the 12 dose groups, 3 per group. All 36 subjects completed the clinical trial. Due to the effect of continuous intravenous administration on the pharmacokinetic parameters, it was difficult to analyze the pharmacokinetic parameters of intravenous administration. Analysis of the apparent volume of distribution parameter V for subcutaneous administration showed that the apparent volume of distribution after a single subcutaneous dose of 800ug and 5mg was both over 100L, much larger than the body weight, indicating that the polypeptide was distributed to the target organs; when the dose was increased to 10mg, the blood concentration was high and the apparent volume of distribution was 43L. In this study, the subcutaneous dose set was small, so there was a lack of reference and comparison, and it was not possible to conclude that the polypeptide in the target organ liver reached a saturation situation. In a clinical pharmacokinetic study in which Myrcludex B was administered subcutaneously for 6 consecutive days at a dose of 10mg, the accumulation coefficient R after 6 consecutive days of administration was 0.01%. AUCThe β-amyloid ratio (MA) was 1.79, indicating a cumulative effect, but no apparent volume of distribution parameters were reported (Blank A et al., Clin Pharmacol Ther. 2017 May 24. doi:10.1002 / cpt.744.). Clinical trials are ongoing for the treatment of patients with hepatitis D with Myrcludex B in combination with interferon at a dose of 2 mg per day, chosen based on the results of a pharmacokinetic study of the polypeptide in orangutans; however, the results of the clinical trial showed no therapeutic effect on the primary study endpoint, clearance of HBsAg (Bogomolov et al., J Hepatol. 2016 Sep;65(3):490-8. doi:10.1016 / j.jhep.2016.04.016. Epub 2016 Apr 27). The full dose at which HBV Pre-S1-derived peptides will treat HBV-related liver disease remains to be determined. Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure provides methods of treating or preventing Hepatitis B virus-associated liver disease. [Means for solving the problem]

[0006] The method includes administering a full dose of a polypeptide or a pharmaceutical composition thereof to a subject in need of treatment or prevention daily, wherein the polypeptide contains an amino acid sequence derived from Hepatitis B virus (HBV) Pre-S1; the full dose is a daily dose that reaches saturation in the liver, the target organ of the polypeptide, several days after administration of the polypeptide.

[0007] In one embodiment, the total dose is a daily dose of 777.95 to 926.13 nmol, more preferably a daily dose of 777.95 nmol or 926.13 nmol.

[0008] In one embodiment, the total dose is a daily dose of 4.2 to 5.0 mg, more preferably a daily dose of 4.2 mg or 5.0 mg. In one embodiment, the polypeptide comprises the amino acid sequence of the pre-S1 region of HBV.

[0009] In one embodiment, the N-terminus of the polypeptide contains a hydrophobic modifying group.

[0010] In one embodiment, the hydrophobic group is selected from myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, cholesterol, and arachidonic acid.

[0011] In certain embodiments, the hydrophobic group is myristic acid.

[0012] In certain embodiments, the polypeptide has a C-terminal modification or is unmodified.

[0013] In certain embodiments, the C-terminal modification is amidation or isopentanediolation.

[0014] In one embodiment, the polypeptide comprises the amino acid sequence of the pre-S1 region of HBV genotype A, B, C, D, E, F, G, H or I.

[0015] In one embodiment, the polypeptide comprises the amino acid sequence 13-59 of the pre-S1 region of HBV genotype C, or a sequence corresponding to the amino acid sequence 13-59 of the pre-S1 region of HBV genotype C in the pre-S1 region of HBV genotypes A, B, D, E, F, G, H or I.

[0016] In some embodiments, one or more amino acid residues in the polypeptide are deleted, substituted or inserted; preferably, 1-30, 1-20, 1-10, 1-8, 1-5 or 1-3 amino acid residues in the polypeptide are deleted, substituted or inserted. In one embodiment, the polypeptide contains a native flanking amino acid sequence from the pre-S1 region of HBV at its N-terminus and / or C-terminus; preferably, the native flanking amino acid sequence of the pre-S1 region of HBV is 1-10, 1-8, 1-5 or 1-3 amino acids in length.

[0017] In one embodiment, the polypeptide contains a glycine corresponding to amino acid 13 of the pre-S1 region of HBV genotype C and / or an asparagine corresponding to amino acid 20 or a lysine corresponding to amino acid 57 of the pre-S1 region of HBV genotype C. In certain embodiments, the polypeptide comprises: (1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs:21-40 or 49; preferably, the polypeptide comprises an amino acid sequence as set forth in SEQ ID NO:23; or (2) having at least about 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:21-40 or 49.

[0018] In some embodiments, the polypeptide is set forth in any one of SEQ ID NOs:1-20 or 51.

[0019] In one embodiment, the polypeptide comprises SEQ ID NO:23 or 49, provided that the polypeptide further comprises an N-terminal myristic acid modification and a C-terminal amination modification, or the polypeptide comprises SEQ ID NO:3 or 51.

[0020] In one embodiment, the hepatitis B virus associated liver disease includes chronic hepatitis B virus infection, chronic hepatitis B, hepatitis B associated liver fibrosis and cirrhosis, hepatitis B associated liver cancer, hepatitis B associated liver transplantation, and hepatitis B associated mother-to-child transmission.

[0021] In certain embodiments, the chronic hepatitis B includes HBeAg-positive chronic hepatitis B and HBeAg-negative chronic hepatitis B.

[0022] In one embodiment, the Hepatitis B associated liver transplant includes protection of the donor liver from HBV infection before, during and after transplantation.

[0023] In one embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:23, the N-terminus of which is modified to myristic acid and the C-terminus of which is aminated; the total dosage is a daily dose of 4.2-5.0 mg, preferably a daily dose of 4.2 mg, and the method comprises administration for at least 7 consecutive days.

[0024] In one embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:49, and is N-terminally modified to myristic acid and C-terminally aminated; the total dosage is 4.2-5.0 mg daily dose, preferably 5.0 mg daily dose, and the method comprises administration for at least 7 consecutive days.

[0025] In one embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:23, and is N-terminally modified to myristic acid and C-terminally modified by aminatization; the method comprises administering to the subject N doses of the pharmaceutical composition each day to achieve a daily dose of 4.2 mg, where N is any integer from 1 to 42.

[0026] In one embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:49, and is N-terminally modified to myristic acid and C-terminally modified by aminatation; the method comprises administering to the subject N doses of the pharmaceutical composition each day to achieve a daily dose of 5.0 mg, where N is any integer from 1 to 50.

[0027] The present disclosure further provides a pharmaceutical composition, wherein the polypeptide comprises an amino acid sequence derived from Hepatitis B virus (HBV) Pre-S1; the pharmaceutical composition comprises a certain amount of the polypeptide, and administration of an integer number of formulations of the pharmaceutical composition results in saturation of the polypeptide in the liver, which is the target organ.

[0028] In one embodiment, the pharmaceutical composition contains a therapeutically effective amount of the polypeptide, wherein the therapeutically effective amount is such that a total daily dose of the polypeptide is 777.95-926.13 nmol after administration of one or more doses of the pharmaceutical composition daily.

[0029] In one embodiment, the therapeutically effective amount is one that results in a total daily dose of 4.2 to 5.0 mg of the polypeptide after administration of one or several doses of the pharmaceutical composition per day, preferably a total daily dose of 4.2 mg or 5.0 mg.

[0030] In certain embodiments, each dose of the pharmaceutical composition contains 4.2 mg, 2.1 mg, 1.4 mg, 0.7 mg, 0.6 mg, 0.3 mg, 0.2 mg, or 0.1 mg of the polypeptide, i.e., the pharmaceutical composition has a dosage of 4.2 mg, 2.1 mg, 1.4 mg, 0.7 mg, 0.6 mg, 0.3 mg, 0.2 mg, or 0.1 mg; or each dose of the pharmaceutical composition contains 5.0 mg, 2.5 mg, 1.0 mg, 0.5 mg, 0.25 mg, or 0.1 mg of the polypeptide, i.e., the pharmaceutical composition has a dosage of 5.0 mg, 2.5 mg, 1.0 mg, 0.5 mg, 0.25 mg, or 0.1 mg.

[0031] In certain embodiments, the polypeptide is as described in any one of the embodiments of the present disclosure.

[0032] The present disclosure further provides a kit, comprising one or more doses of a pharmaceutical product containing a polypeptide as an active ingredient, for administration to a patient for one or several days, wherein the amount of polypeptide contained in the one or more doses of the pharmaceutical product is an amount that results in a daily dosage of 777.95-833.52 nmol, calculated based on the polypeptide, after administration of the pharmaceutical product in one or more doses per day, and wherein the polypeptide contains an amino acid sequence derived from Hepatitis B virus (HBV) Pre-S1.

[0033] In one embodiment, the amount of polypeptide contained in the one or more doses of the pharmaceutical is such that after administration of the pharmaceutical in one or more doses per day, the daily dosage is 4.2 to 5.0 mg, preferably 4.2 mg or 5.0 mg.

[0034] In certain embodiments, the polypeptide is as described in any one of the embodiments of the present disclosure.

[0035] The present disclosure further provides the application of the pharmaceutical compositions described herein in the preparation of a medicament for treating or preventing Hepatitis B virus-associated liver disease.

[0036] Also disclosed are the polypeptides described herein or pharmaceutical compositions thereof for treating and preventing Hepatitis B virus associated liver disease.

[0037] In certain embodiments, the method of treating and preventing Hepatitis B virus associated liver disease is a method described in any one of the embodiments herein. [Brief description of the drawings]

[0038] [Figure 1]Hepalatide labeled with FIFC binds to HEK293 cells expressing NTCP (NTCP-293) but does not bind to control HEK293 cells (BLANK-293). A polypeptide derived from HBV labeled with FIFC is used as the control polypeptide. [Diagram 2] Effect of Cmyr-47 on bile acid uptake in vitro. Cyclosporine A (CsA) was used as a positive control. A, TA entry into cells was specifically mediated by NTCP, and cyclosporine A, used as a positive control, exerted a significant inhibitory effect on NTCP; B, Heparatide bidirectionally affected the bile acid transport function of NTCP. [Diagram 3] Precipitation radioactivity time curves after subcutaneous injection of 125I-heparatide. [Figure 4] Mean plasma concentration-time curves (mean + SD) from a Phase Ia PK study. [Diagram 5] Mean plasma concentration-time curves (mean + SD) of the first dose from a Phase Ib PK study. [Figure 6] Pre-dose mean plasma concentration-time curves (mean + SD) on days 2-7 of a Phase Ib PK study. [Figure 7] Mean plasma concentration-time curves (mean + SD) of the final dose of the Phase Ib PK study. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For purposes of this disclosure, the following terms are defined below.

[0040] The article "a" or "an" refers to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "a part" refers to one part or to more than one part. Unless otherwise stated, the term "or" is used interchangeably with the term "and / or."

[0041] When the terms "comprise," "have," "contain," or grammatical variations thereof are used in this disclosure or claims, these terms may be used in the same manner as the term "comprise" when used as a transitional term in the claims. The term "comprise" or grammatical variations thereof means "including, but not limited to," and is used interchangeably with "including, but not limited to."

[0042] The term "about" refers to a specific amount, level, value, number, frequency, percentage, size, magnitude, quantity, weight, or length, which has a difference of approximately 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% when compared to a reference amount, level, value, number, frequency, percentage, size, magnitude, quantity, weight, or length. When the term "about" is used in conjunction with a numerical range, the term modifies the range and extends its limits above or below the numerical value. In general, when the term "about" modifies a value, it means a range from 10% lower than the value to 10% higher than the value.

[0043] This application includes an electronically submitted Sequence Listing in ASCII format, which is incorporated by reference in its entirety.

[0044] Each technical feature in the embodiments disclosed in this specification can be arbitrarily combined to form a preferred technical solution.

[0045] I, Polypeptides An aspect of the present disclosure provides a polypeptide derived from HBV Pre-S1 for use in treating or preventing HBV-associated liver disease. Preferably, the polypeptide is capable of blocking HBV infection in vitro. More preferably, the polypeptide is capable of binding NTCP in vitro, for example in solution or in a cell-free system (e.g., in a cell lysate or reconstituted system), or in cells, such as cells in in vitro culture (e.g., cells expressing NTCP or hepatocytes), or in vivo in cells of a subject. The subject may be a mammal. In some embodiments, the subject may be a human.

[0046] The terms "polypeptide", "peptide" and "protein" are used interchangeably and include full-length proteins and fragments, as well as variants of full-length proteins and fragments. These fragments and variants of the polypeptides described herein retain at least one or more biological activities of the polypeptides. "Polypeptide", "peptide" and "protein" may include naturally occurring and / or non-naturally occurring amino acid residues. These terms also include post-translationally modified proteins, including, for example, glycosylated, sialylated, acetylated, and / or phosphorylated proteins. These terms also include proteins that have been chemically modified at one or more amino acid residues, such as amino acid residues at the N-terminus and / or C-terminus. For example, the N-terminus of the polypeptides described herein may be modified with hydrophobic groups, such as myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, cholesterol and arachidonic acid. In some embodiments, the C-terminus of the polypeptides described herein may be modified. The C-terminus modification may be selected from amidation (amination), isopentanediolation, or no modification.

[0047] As used herein, the term "polypeptide derived from HBV Pre-S1" or "polypeptide derived from HBV Pre-S1" refers to a polypeptide whose origin or source is HBV, and may include natural, recombinant, synthetic, or purified. The term "polypeptide derived from HBV" or "polypeptide derived from HBV" refers to a full-length naturally occurring HBV polypeptide or a fragment thereof, and a variant of a full-length naturally occurring polypeptide or a fragment thereof. In some embodiments, the fragment may consist of at least 3-5 amino acids, at least 5-10 amino acids, at least 10-20 amino acids, at least 20-30 amino acids, at least 30-50 amino acids, or may consist of the entire amino acid sequence of the naturally occurring sequence, or may be recognized by one of skill in the art as being derived from the naturally occurring sequence. In some embodiments, the polypeptides described herein may be derived from the pre-S1 region of any HBV subtype L protein. In some embodiments, the polypeptides described herein may include the entire pre-S1 region of the L protein of any one of the HBV subtypes. In certain embodiments, the polypeptides described herein may be derived from the pre-S1 region of the L protein of any one of HBV genotypes A, B, C, D, E, F, G, H, or I. The genomic sequences of these HBV genotypes can be found in GenBank Accession Nos. KC875260 (SEQ ID NO:41), AY220704 (SEQ ID NO:42), AF461363 (SEQ ID NO:43), AY796030 (SEQ ID NO:44), AB205129 (SEQ ID NO:45), DQ823095 (SEQ ID NO:46), HE981176 (SEQ ID NO:47), and AB179747 (SEQ ID NO:48), respectively. In some embodiments, the polypeptides described herein may be derived from the pre-S1 region of the L protein of HBV genotype C. The HBV-derived polypeptides described herein retain one or more of the biological activities described herein of the corresponding native HBV polypeptide.

[0048] As used herein, a "variant" in relation to a polypeptide described herein, a polypeptide derived from HBV Pre-S1, or a polypeptide derived from HBV Pre-S1 refers to a polypeptide that differs in amino acid sequence from a given polypeptide (i.e., a polypeptide described herein, a polypeptide derived from HBV Pre-S1, or a polypeptide derived from HBV Pre-S1) but retains one or more biological activities of the given polypeptide as described herein. A variant polypeptide described herein may have one or more amino acid gains (e.g., insertions), deletions, or substitutions relative to a given polypeptide. In some embodiments, a variant polypeptide described herein may have 1-30, 1-20, 1-10, 1-8, 1-5, or 1-3 amino acid gains (e.g., insertions), deletions, or substitutions (including all integers within these ranges) relative to a given polypeptide. For example, a polypeptide sequence may have conservative substitutions of amino acids. Conservative substitutions of amino acids, i.e., replacement of one amino acid with another having similar properties (e.g., hydrophilicity, degree of charge, distribution of charged regions, etc.), generally result in minor changes and do not significantly alter the biological activity of a polypeptide. These minor changes may be identified by considering the hydrophilicity index of an amino acid, which is based in part on the hydrophobicity and charge of that amino acid. Amino acids with similar hydrophilicity indexes and hydrophilicity values ​​can be substituted for one another and still retain protein function. The hydrophilicity index and hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with this observation, amino acid substitutions that match biological function depend on the relative similarity of the amino acids, particularly the side chains of those amino acids, as manifested in their hydrophobicity, hydrophilicity, charge status, size, and other properties.

[0049] The term "variant" further includes those that have a degree of identity with a given polypeptide (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with a given polypeptide). As used herein, "variant" further includes polypeptides that contain a portion of a given polypeptide that corresponds to the native sequence of an HBV protein. "Variant" may also refer to a fusion protein or chimeric protein that contains polypeptides derived from two or more different sources of proteins. Non-limiting examples of fusion proteins described herein include fusion proteins of one polypeptide derived from HBV Pre-S1 and another polypeptide derived from a non-HBV Pre-S1 protein, fusion proteins of two polypeptides derived from different HBV subtypes, and fusion proteins of two polypeptides derived from different regions of any one HBV subtype L protein or two polypeptides derived from different sequences within the pre-S1 region of any one HBV subtype L protein.

[0050] The term "variant" further includes polypeptides that contain the same amino acid sequence as a given polypeptide (i.e., a polypeptide described herein, a polypeptide derived from HBV Pre-S1, or a polypeptide derived from HBV Pre-S1) and retain one or more biological activities of the given polypeptide, but are chemically and / or post-translationally modified in a different manner than the given polypeptide. "Variant" can also be used to describe polypeptides that have been differentially processed (e.g., by proteolysis, phosphorylation, or other post-translational modification), but retain one or more biological activities described herein. As used herein, unless otherwise stated, "variant" includes fragments of variants. The term "variant" further includes homologous polypeptide sequences found in different virus species, strains, or hepatotropic virus subtypes. Based on antigenic epitopes in its envelope protein, HBV is divided into four main serotypes (adr, adw, ayr, and ayw), and based on the variability of the total nucleotide sequence in the genome, HBV is divided into nine genotypes (AI). Thus, the term "variant" includes any one of the homologous polypeptides found in these HBV subtypes. "Variant" further includes polypeptides having the addition of naturally occurring flanking amino acid sequences from any one of these HBV subtypes at the N- and / or C-terminus.

[0051] The terms "conservative amino acid substitution" and "conservative substitution" are used interchangeably herein and refer to the replacement of a particular amino acid within a group of amino acids, where the amino acid is replaced with a different amino acid that is similar in size, structure, charge and / or polarity. Families of amino acid residues having similar side chains are well known in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). And, in some embodiments, an amino acid residue in a polypeptide may be replaced with another amino acid residue from the same side chain family. In other embodiments, a stretch of an amino acid sequence may be replaced with an amino acid sequence that is structurally similar but differs in the order and / or composition of side chain family members. In other embodiments, mutations may be randomly introduced into the entire or partial sequence of the polypeptide. Examples of conservative amino acid substitutions include, for example, replacing one amino acid from the aromatic or hydrophobic amino acids Ala, Val, Leu, and Ile with another one of the four amino acids in the group; replacement between the hydroxyl-containing residues Ser and Thr; replacement between the acidic residues Asp and Glu; replacement between the amide residues Asn and Gln; replacement between the basic residues Lys, Arg, and His; replacement between the aromatic residues Phe, Tyr, and Trp; and replacement between the small amino acids Ala, Ser, Thr, Met, and Gly. It is reasonably expected that conservative substitutions (e.g., replacement of a conservative amino acid with a similar, structurally related amino acid) will not have a substantial effect on the biological activity of the polypeptide. The term "sequence identity" (e.g., having 50% identity with a given sequence) refers to the degree of sequence identity when compared in an amino acid-to-amino acid manner within a comparison window. In some embodiments, the polypeptides described herein may contain an amino acid sequence that is at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a sequence of a given polypeptide and retains one or more biological activities of the given polypeptide. The number of matching positions is obtained by optimizing the alignment of the two sequences within the comparison window and counting the number of positions where the same amino acid occurs in the two sequences, and then dividing the number of matching positions by the total number of positions in the comparison window and multiplying the result by 100 to obtain the percentage of sequence identity, which can be calculated as "percentage identity" or "% identity". The optimal sequence alignment in the comparison window can be selected from the optimal alignment (i.e., the alignment that leads to the highest identity percentage in the comparison window) obtained by any method selected by running an algorithm known in the art (e.g., a program of the BLAST@ family) on a computer or by visual inspection. For sequence comparison, one sequence is used as a reference sequence, and a test sequence is compared with the reference sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are designed as necessary, and parameters of the sequence algorithm program are set. The sequence comparison algorithm then calculates the current sequence identity of the test sequence to the reference sequence based on the set program parameters. The setting of sequence algorithm program parameters is known in the art. For example, the comparison window can be set to cover the entire length of one or two comparison sequences (e.g., cover the entire length of the reference sequence), while allowing a difference of 5% of the total amino acid number of the reference sequence.

[0052] In some embodiments, the polypeptides described herein may contain the amino acid sequence of the pre-S1 region of any one of the HBV subtypes. In some embodiments, the polypeptides described herein contain amino acids 13-59 of the pre-S1 region of HBV genotype C: GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANQVG (SEQ ID NO:23) or GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANKVG (SEQ ID NO:49). In other embodiments, the polypeptides described herein may contain the corresponding pre-S1 sequence from another HBV genotype (e.g., from any one of genotypes A, B, C, D, E, F, G, H, or I).

[0053] For example, in some embodiments, the polypeptides described herein may contain: HBV genotype A pre-S1 amino acids 13-59: GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPVKDDWPAANQVG(SEQ ID NO:34); HBV genotype B pre-S1 amino acids 13-59: GTNLSVPNPLGFFPDHQLDPAFKANSENPDWDLNPNKDNWPDANKVG(SEQ ID NO:35); HBV genotype D pre-S1 amino acids 2-48: GQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVG(SEQ ID NO:36); HBV genotype E pre-S1 amino acids 12-58: GKNISTTNPLGFFPDHQLDPAFRANTRNPDWDHNPNKDHWTEANKVG(SEQ ID NO:37); HBV genotype F pre-S1 amino acids 13-59: GQNLSVPNPLGFFPDHQLDPLFRANSSSPDWDFNTNKDSWPMANKVG(SEQ ID NO:38); HBV genotype G pre-S1 amino acids 12-58: GKNLSASNPLGFLPDHQLDPAFRANTNNPDWDFNPKKDPWPEANKVG (SEQ ID NO:39); or HBV genotype H pre-S1 amino acids 13-59: GQNLSVPNPLGFFPDHQLDPLFRANSSPDWDFNTNKDNWPMANKVG (SEQ ID NO:40).

[0054] In some embodiments, the polypeptides described herein may contain a portion of the pre-S1 region of HBV, said portion comprising at least one amino acid sequence selected from SEQ ID NOs: 23, 34-40, or 49. In some embodiments, the polypeptides described herein may contain the entire pre-S1 region of HBV.

[0055] In some embodiments, the polypeptides described herein may be 10-100 amino acids in length. For example, the polypeptides may be 15-100, 15-80, 20-100, 20-80, 20-60, 25-60, 30-60, 35-60, or 40-60 amino acids in length (including all integers within these ranges). In some embodiments, the polypeptides described herein may be at least 20 amino acids in length, such as at least 25, 30, 35, or 40 amino acids in length. In some embodiments, the polypeptides described herein may be 20, 25, 30, 35, 40, 47, 55, or 60 amino acids in length. In some embodiments, the polypeptides described herein may be 47 amino acids in length. Different variants of the polypeptides described herein retain one or more biological activities associated with the corresponding polypeptide.

[0056] In some embodiments, the N-terminus of the polypeptides described herein may contain a hydrophobic group modification. For example, the hydrophobic group may be selected from, for example, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, cholesterol, and arachidonic acid. In some embodiments, the hydrophobic group may be selected from, for example, myristic acid, palmitic acid, stearic acid, and cholesterol. In some embodiments, the hydrophobic group may be myristic acid. In some embodiments, the polypeptides described herein may contain an amino acid sequence set forth in any one of SEQ ID NOs:23, 34-40, and 49, except that the N-terminus of the polypeptide may be modified with a hydrophobic group selected from, for example, myristic acid, palmitic acid, stearic acid, and cholesterol. In some embodiments, the polypeptides described herein may contain an amino acid sequence as set forth in any one of SEQ ID NOs:23, 34-40, and 49, provided that the N-terminus of the polypeptide may be myristoylated. In some embodiments, the polypeptides described herein may contain an amino acid sequence as set forth in SEQ ID NO:23, provided that the N-terminus of the polypeptide may be myristoylated. In some embodiments, the polypeptides described herein may contain a C-terminal modification or may be unmodified. For example, the C-terminal modification may be selected from amidation (amination), isopentanediolation, or no C-terminal modification. In some embodiments, the C-terminal modification may be amidation (amination). For example, the polypeptides described herein may contain an amino acid sequence as set forth in SEQ ID NO:23, provided that the N-terminus of the polypeptide may be myristoylated and / or the C-terminus of the polypeptide may be amidated (amination). In some embodiments, the polypeptides described herein may comprise the amino acid sequence shown in SEQ ID NO:23.In some embodiments, the polypeptides described herein may contain an amino acid sequence set forth in any one of SEQ ID NOs:34-40, 49, except that the N-terminus may be myristoylated and / or the C-terminus may be amidated (aminated). In some embodiments, the polypeptides described herein may contain an amino acid sequence set forth in any one of SEQ ID NOs:14-20, 51. N- and / or C-terminally modified variants of the polypeptides described herein retain one or more biological activities of the corresponding polypeptide that is not modified in the same manner.

[0057] Variants of the polypeptides described herein are also included in the present disclosure, including those that have one or more amino acid deletions, substitutions, or insertions, but retain one or more biological activities of the polypeptides. The polypeptides described herein preferably retain a glycine corresponding to amino acid 13 of the pre-S1 region of HBV genotype C (i.e., the N-terminal glycine of SEQ ID NO:23). In some embodiments, the polypeptides described herein retain an asparagine corresponding to amino acid 20, or a lysine corresponding to amino acid 57, of the pre-S1 region of HBV genotype C. In some embodiments, the polypeptides described herein may have one or more naturally occurring mutations in the pre-S1 region of HBV. In some embodiments, the polypeptides 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 from the pre-S1 region of HBV. In some embodiments, the polypeptides 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 amino acid sequence set forth in any one of SEQ ID NOs:23, 34-40 and 49. In some embodiments, the polypeptides described herein 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 amino acid sequence set forth in SEQ ID NO:23. In some embodiments, the polypeptides described herein have 1-3 amino acid deletions, substitutions or insertions relative to the amino acid sequence set forth in SEQ ID NO:23. In some embodiments, the polypeptides described herein have 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 the amino acid sequence set forth in any one of SEQ ID NOs:23, 34-40 and 49.For example, a polypeptide described herein may comprise an amino acid sequence set forth in any one of SEQ ID NOs:21, 22, and 24-28. In some embodiments, a polypeptide described herein may comprise an amino acid sequence of any one of the polypeptides set forth in Table 1. In some embodiments, a polypeptide described herein may be selected from any one of the post-translationally modified polypeptides set forth in Table 1.

[0058] [Table 1-1] [Table 1-2] [Table 1-3]

[0059] In various embodiments, the polypeptides described herein may have at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to any one of the polypeptides described herein. For example, the polypeptides may comprise an amino acid sequence that has at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NOs:21-40. In some embodiments, the polypeptide may comprise an amino acid sequence that is at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 23, 34-40 and 49. In some embodiments, the polypeptide may comprise an amino acid sequence that is at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 23. Variants that share a degree of sequence identity with the polypeptides described herein retain one or more biological activities of the corresponding polypeptide.

[0060] Aspects of the present disclosure further include variants of the polypeptides described herein having naturally occurring flanking amino acid sequences from the L protein of HBV (e.g., the pre-S1 region of the L protein), which are located at the N-terminus and / or C-terminus of the variant. The naturally occurring flanking amino acid sequences refer to the naturally occurring sequences that are adjacent to the N- or C-terminus of the polypeptides described herein in the pre-S1 region of the corresponding HBV genotype or any other HBV genotype. In some embodiments, the polypeptides described herein may contain an amino acid sequence as set forth in any one of SEQ ID NOs: 23, 34-40, and 49, and naturally occurring amino acid sequences adjacent to the N-terminus and / or C-terminus from the pre-S1 region of any one of HBV genotype AH. In some embodiments, the naturally occurring flanking amino acid sequences may be derived from the consensus sequences of HBV strains having GenBank accession numbers KC875260 (genotype A; SEQ ID NO:41), AY220704 (genotype B; SEQ ID NO:42), AF461363 (genotype C; SEQ ID NO:43), AY796030 (genotype D; SEQ ID NO:44), AB205129 (genotype E; SEQ ID NO:45), DQ823095 (genotype F; SEQ ID NO:46), HE981176 (genotype G; SEQ ID NO:47) or AB179747 (genotype H; SEQ ID NO:48). For example, a polypeptide described herein may contain the amino acid sequence set forth in SEQ ID NO:23 and may contain at its N-terminus and / or C-terminus naturally occurring flanking amino acid sequences from the pre-S1 region of HBV genotype C. Alternatively, a polypeptide described herein may contain the amino acid sequence set forth in SEQ ID NO:23 and may contain at its N-terminus and / or C-terminus naturally occurring flanking amino acid sequences from the pre-S1 region of any one of HBV genotypes A, B, D, E, F, G and H.In some embodiments, the N-terminus and / or C-terminus of the polypeptides described herein may independently contain a native 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, a polypeptide described herein may contain the amino acid sequence set forth in SEQ ID NO:23 and at its N-terminus, a native flanking amino acid sequence that is 10 amino acids in length from the pre-S1 region of HBV genotype C. In other words, the polypeptide may contain amino acids 2-59 (SEQ ID NO:29) of the pre-S1 region of HBV genotype C. As another example, a polypeptide described herein may contain the amino acid sequence set forth in SEQ ID NO:23 and at its N-terminus, a native flanking amino acid sequence that is 9 amino acids in length from the pre-S1 region of HBV genotype E or G. In other words, the polypeptide 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 genotypes E or G (SEQ ID NO:30).It is understood that any one of the polypeptides described herein may have any length of naturally occurring flanking amino acid sequence extending from its N-terminus and / or C-terminus, and the resulting polypeptide retains one or more biological activities of the native polypeptide.

[0061] The polypeptides described herein can be prepared by chemical synthesis or by recombinant techniques.

[0062] When recombinant methods are selected, a de novo synthesized gene may be constructed or a natural gene may be mutated, for example, by cassette mutagenesis. The polypeptides described herein may be prepared by recombinant DNA techniques. Briefly, these techniques involve obtaining a natural or synthetic gene encoding the polypeptide, inserting it into a suitable vector, transferring the vector into a suitable host cell, expressing the gene by culturing the host cell, and recovering or isolating the peptide produced. In some embodiments, the recovered peptide is purified to a suitable degree of purity.

[0063] For example, a DNA sequence encoding a polypeptide described herein may be cloned and manipulated for expression in a suitable host. The DNA encoding the parent polypeptide may be obtained from a cDNA derived from the mRNA of a cell expressing the polypeptide in an HBV genomic library, or may be obtained by constructing the DNA sequence by synthetic methods. The parent DNA is then inserted into a suitable plasmid or vector for transforming a host cell. Generally, replication and control sequences from a plasmid vector of a type compatible with the host cell are used in these hosts. The vector generally contains a replication site and a sequence encoding a protein or peptide that provides phenotypic options to the transformed cell. The vector may be any vector commonly used in the art, or may be constructed using standard techniques in combination with functional fragments of vectors commonly used in the art.

[0064] Host cells may be prokaryotic or eukaryotic. For example, prokaryotic host cells may include Escherichia coli, Bacillus subtilis, and other Enterobacteriaceae (e.g., Salmonella typhimurium or Serratia marcesans), as well as various pseudomonas. In addition to prokaryotes, eukaryotes such as yeast cultures, or cells derived from multicellular organisms such as insect or mammalian cell cultures may also be used. Examples of such eukaryotic host cell lines include VERO and Hela cells, Chinese Hamster Ovary (CHO) cell lines, W138, 293, BHK, COS-7, and MDCK cell lines.

[0065] In some embodiments, the polypeptides described herein can be prepared by solid phase synthesis or equivalent chemical synthesis methods known in the art. In some embodiments, solid phase synthesis of the C-terminus of the polypeptide begins by coupling a pre-protected α-amino acid to a suitable resin. The starting material can be prepared by attaching an α-amino protected amino acid to a chloromethylated or hydroxymethylated resin via an ester bond, or to a BHA or MBHA resin via an amide bond. The amino acid is attached to the peptide chain by techniques known in the art for forming peptide bonds. One method is to convert the amino acid to a derivative that provides a carboxyl group that is amenable to reaction with the free N-terminal amino group of the peptide fragment. For example, the amino acid can be converted to a mixed acid anhydride by reaction of the protected amino acid with ethyl chloroformate, phenyl chloroformate, tert-butyl chloroformate, isobutyl chloroformate, pivaloyl chloride, and similar acid chlorides. Alternatively, the amino acid may be converted to an active ester (e.g., 2,4,5-trichlorophenyl ester, pentachlorophenyl ester, pentafluorophenyl ester, p-nitrophenyl ester, N-hydroxysuccinimide ester, or esters based on 1-hydroxybenzotriazole). Another coupling method involves the use of a suitable coupling agent (e.g., N,N'-dicyclohexylcarbodiimide or N,N'-diisopropylcarbodiimide).

[0066] In some embodiments, the alpha amino group of each amino acid used in the peptide synthesis is protected from the coupling reaction to prevent side reactions involving its active alpha amino group function. For example, certain amino acids that contain reactive side chain functional groups (e.g., sulfhydryl, amino, carboxyl, and hydroxyl) may be protected with appropriate protecting groups to prevent chemical reactions at that site during the first and subsequent coupling steps. Those skilled in the art know how to select appropriate side chain protecting groups. After obtaining a peptide of the desired amino acids, the protecting groups can be easily removed under reaction conditions that do not alter the peptide chain structure.

[0067] After removing the α-amino protecting group, the remaining α-amino and side-chain protected amino acids are coupled stepwise in the required order. In addition to adding each amino acid individually to the synthesis, alternatively, some amino acids may be pre-coupled to each other before being added to the solid phase synthesis apparatus. Those skilled in the art know how to select the appropriate coupling agent.

[0068] Each protected amino acid or amino acid sequence is added in excess to a solid phase reactor; coupling is suitably carried out in a medium such as dimethylformamide (DMF) or CH2Cl2 or a mixture thereof. If the coupling is not complete, the coupling process is repeated in the presence of an N-amino protecting group before coupling the next amino acid. The coupling reaction at each stage of the synthesis is monitored for success. The coupling reaction can be carried out automatically by known methods (e.g., on a BIOSEARCH 9500™ peptide synthesizer).

[0069] After obtaining the desired peptide sequence, the protected peptide must be cleaved from the resin support and all protecting groups must be removed. Cleavage and removal of the protecting groups can be performed simultaneously or sequentially. When the resin support is a chloromethylated polystyrene resin, the bond that anchors the peptide to the resin is an ester bond consisting of the free carboxyl group of the C-terminal residue and one of the abundant chloromethyl groups in the resin matrix. It should be understood that the anchoring bond may be cleaved with an agent capable of cleaving the ester bond and permeating the resin matrix. It should be recognized that the polypeptide may be modified before or after cleavage of the polypeptide from the support; for example, modified at the N-terminus with a hydrophobic group (including, for example, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, cholesterol and arachidonic acid), or modified at the C-terminus with amidation (amination), isopentanediolation or other modifications capable of stabilizing the C-terminus.

[0070] The polypeptides of the invention can be purified by conventional methods such as preparative HPLC (including reverse-phase HPLC) or other known chromatographic techniques, such as gel permeation, ion exchange, partition chromatography, affinity chromatography (including monoclonal antibody columns), or countercurrent partitioning.

[0071] II. Pharmaceutical Compositions and Kits The present disclosure further provides compositions, including pharmaceutical compositions, that contain a polypeptide as described herein. In some embodiments, the composition may contain any one or more of the polypeptides described herein. In some embodiments, the composition may further contain a suitable pharma- ceutically acceptable carrier.

[0072] A "pharmaceutical acceptable carrier" refers to an inactive ingredient, such as a solid, semi-solid, or liquid filler, diluent, coating agent, formulation auxiliary, excipient, or carrier, which is used in combination with a therapeutic agent to form a "pharmaceutical composition" administered to a subject. The dosage and concentration at which the pharmaceutical acceptable carrier is used is non-toxic to the subject and compatible with the other ingredients in the formulation. The pharmaceutical acceptable carrier is appropriate for the formulation employed. For example, if the therapeutic agent is administered orally, the carrier may be a gel capsule. If the therapeutic agent is administered subcutaneously, it is desirable that the carrier not irritate the skin or cause a reaction at the injection site.

[0073] Pharmaceutical compositions of the polypeptides described herein can be prepared by mixing the polypeptides described herein at the desired purity with any one or more pharma- ceutically acceptable carriers. Pharmaceutically acceptable carriers include buffers (e.g., phosphates, citrates and other organic acids), antioxidants (e.g., ascorbic acid and methionine); preservatives (e.g., octadecylbenzyldimethylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl p-hydroxybenzoates (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate), catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol); polypeptides of low molecular weight (e.g., less than about 10 residues); proteins, and the like. The preferred molecular weight molecules include proteins (e.g., serum albumin, gelatin, immunoglobulins, etc.); hydrophilic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides; disaccharides; other carbohydrates (e.g., glucose, mannose, or dextrin); chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); salification counterions (e.g., sodium); metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants (e.g., polyethylene glycol (PEG)).

[0074] Exemplary pharmaceutical carriers may include binders such as pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose; fillers such as lactose or other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates or calcium hydrogen phosphate; lubricants such as magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate; disintegrants such as starch, sodium starch glycolate, and wetting agents such as sodium lauryl sulfate.

[0075] Exemplary pharma- ceutically acceptable carriers may further include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), human soluble PH-20 hyaluronidase glycoprotein, rHuPH20 (HYLENEX R, Baxter International, Inc.). In some embodiments, sHASEGP may be mixed into a pharmaceutical composition that includes one or more other glycosaminoglycanases (e.g., chondroitinases).

[0076] The pharmaceutical composition may further comprise multiple active ingredients appropriate for the particular indication being treated, such active agents may be, for example, complementary active agents that have no deleterious effects, suitably such active agents may be present in combination therewith in amounts effective to achieve the intended purpose.

[0077] In some embodiments, the active agent may be encapsulated in microcapsules (such as hydroxymethylcellulose, gel microcapsules, polymethylmethacrylate microcapsules, etc.) prepared using techniques such as aggregation or interfacial polymerization, or in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules, etc.), or in coarse emulsions.

[0078] In some embodiments, the pharmaceutical compositions may comprise a sustained release formulation. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing a polypeptide as described herein, which matrices may be in the form of shaped articles, such as films or microcapsules.

[0079] In some embodiments, the pharmaceutical compositions may be for internal administration and may be sterile, which may be readily accomplished, for example, by filtration through a sterile filter.

[0080] The pharmaceutical composition may be formulated in any one of a variety of possible dosage forms, such as tablets, capsules, gel capsules, powders, or granules. The pharmaceutical composition may be formulated in a solution, suspension, emulsion, or mixed medium. In some embodiments, the pharmaceutical composition may be formulated in a lyophilized formulation or an aqueous solution.

[0081] In some embodiments, the pharmaceutical composition may be prepared in a solution. For example, the polypeptides described herein may be used in a non-buffered solution (such as saline or water). In some embodiments, the polypeptide may be used in a suitable buffer solution. For example, the buffer solution may include acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In some embodiments, the buffer solution may be phosphate buffered saline (PBS). The pH and osmolality of the buffer solution containing the polypeptide may be adjusted to a suitable level for administration to a subject. In some embodiments, the pharmaceutical compositions may be prepared as aqueous suspensions, non-aqueous suspensions, or mixed matrix suspensions. Aqueous suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or glucan. Suspensions may also contain stabilizers.

[0082] In some embodiments, the pharmaceutical composition may be formulated as an emulsion. Exemplary emulsions include heterogeneous systems of one liquid dispersed in another, usually in droplets with diameters greater than 0.1 μm. Emulsions may contain other components in addition to the dispersed phase and the active drug, which may be in aqueous phase solution, oil phase solution, or as a separate phase itself. In one embodiment of the present disclosure, emulsions may also include microemulsions. In some embodiments, the pharmaceutical composition may be formulated as a liposomal formulation.

[0083] In some embodiments, the pharmaceutical composition is prepared in a suitable dosage form for administration, for example, an injectable solution suitable for subcutaneous administration. In these embodiments, the pharmaceutical composition comprises an amount of the polypeptide described herein such that one or more doses of the pharmaceutical composition are administered daily to achieve a therapeutically effective daily dose. As used herein, a therapeutically effective amount refers to an amount of polypeptide that achieves saturation in the target organ, the liver, after administration of the therapeutically effective amount of the polypeptide over several days (≧7 days).

[0084] In certain embodiments, the therapeutically effective amount is a daily dose of 777.95-926.13 nmol. In certain embodiments, the therapeutically effective amount is a daily dose of 4.2-5.0 mg.

[0085] In one embodiment, the polypeptide in the pharmaceutical composition is heparatide and the therapeutically effective amount is 4.2-5.0 mg / day, more preferably 4.2 mg / day. In one embodiment, the polypeptide in the pharmaceutical composition is Myrcludex B and the therapeutically effective amount is 4.2-5.0 mg / day, more preferably 5.0 mg / day. In some embodiments, the pharmaceutical composition can be administered several times daily to reach a therapeutically effective amount. Thus, in these embodiments, the pharmaceutical composition herein is prepared in multiple unit dosage forms, for example, each unit dosage form may contain at least 18.52 nmol, or may contain 37.05 nmol, 55.57 nmol, 111.14 nmol, 129.66 nmol, 259.32 nmol, or 388.98 nmol. A corresponding number of unit dosage forms can be administered daily to reach a therapeutically effective amount in total administered on that day. In these embodiments, when the pharmaceutical composition contains heparatide, each unit dosage form may contain at least 0.1 mg of heparatide, or may contain 0.2 mg, 0.3 mg, 0.6 mg, 0.7 mg, 1.4 mg, 2.1 mg, or 4.2 mg of heparatide, i.e., the dosage of the pharmaceutical composition may be 0.1 mg, 0.2 mg, 0.3 mg, 0.6 mg, 0.7 mg, 1.4 mg, 2.1 mg, or 4.2 mg. For example, if each unit dosage form contains 0.1 mg of heparatide, 42 unit dosage forms of the pharmaceutical composition may be administered daily. In these embodiments, when the pharmaceutical composition contains Myrcludex B, each unit dosage form may contain at least 0.1 mg of Myrcludex B, or may contain 0.25 mg, 0.5 mg, 1.0 mg, 2.5 mg, or 5.0 mg of Myrcludex B, i.e., the dosage of the pharmaceutical composition may be 0.1 mg, 0.25 mg, 0.5 mg, 1.0 mg, 2.5 mg, or 5.0 mg. For example, if each unit dosage form contains 0.1 mg of Myrcludex B, 50 unit dosage forms of the pharmaceutical composition may be administered daily. Thus, the number of unit dosage forms in the pharmaceutical composition of the present invention may range from 1 to 50 doses, for example, from 1 to 42 doses.

[0086] In one embodiment, each unit dosage form contains 777.95-926.13 nmol or 4.2-5.0 mg of a polypeptide described herein, such that the therapeutically effective amount is achieved by administering one unit dosage form daily.

[0087] The pharmaceutical composition of the present specification may be in a lyophilized dosage form. For example, a dose of the lyophilized pharmaceutical product may contain 37.05-926.13 nmol or 0.1-5 mg of the polypeptide described herein, for example, 259.32-926.13 nmol or 1.4-5 mg, 388.98-926.13 nmol or 2.1-5 mg, or 777.95-926.13 nmol or 4.2-5 mg of the polypeptide described herein. When used, it can be dissolved in an appropriate amount of solvent and administered to a subject as an injection. One or several doses of the above injection can be administered so that the total daily dose is in the range of 777.95-926.13 nmol or 4.2-5 mg. For example, a lyophilized formulation may contain two vials of pharmaceutical agent, each containing 388.98 nmol or 2.1 mg of lyophilized polypeptide, which may be administered to a subject twice daily to achieve a total daily dose of 777.95 nmol or 4.2 mg.

[0088] The present disclosure may also include kits containing the pharmaceutical compositions described herein, the kits containing one or more doses of the pharmaceutical compositions described herein for use in single or multiple day administration to prevent or treat Hepatitis B virus associated liver disease.

[0089] In some embodiments, the kit contains at least 7 doses of the pharmaceutical composition, each dose of the pharmaceutical composition containing 777.95-926.13 nmol or 4.2-5.0 mg of a polypeptide described herein for use in at least 7 consecutive days of administration, in some embodiments, the kit satisfies 7 consecutive days of administration.

[0090] In one embodiment, the kit contains at least 7 doses of the pharmaceutical composition, each dose of the pharmaceutical composition containing 4.2 to 5.0 mg, preferably 4.2 mg of heparatide or 5.0 mg of Myrcludex B, for use in at least 7 consecutive days of administration.

[0091] In some embodiments, the kit includes at least two doses of the pharmaceutical composition, and the total amount of the polypeptide contained in all the pharmaceutical compositions in the kit is 777.95-926.13 nmol or 4.2-5.0 mg per day. In these embodiments, each dose of the pharmaceutical composition includes, for example, 18.52 nmol, 37.05 nmol, 55.57 nmol, 111.14 nmol, 129.66 nmol, 259.32 nmol, or 388.98 nmol, or 0.1 mg, 0.2 mg, 0.3 mg, 0.6 mg, 0.7 mg, 1.4 mg, or 2.1 mg of the polypeptide, and the pharmaceutical compositions in the kit include 42 doses, 21 doses, 14 doses, 7 doses, 8 doses, 10 doses, 12 doses, 14 doses, 16 doses, 18 doses, 21 ... or each dose of the pharmaceutical composition may contain 18.52 nmol, 46.31 nmol, 92.61 nmol, 185.23 nmol or 463.06 nmol, or may contain 0.1 mg, 0.25 mg, 0.5 mg, 1.0 mg or 2.5 mg of the polypeptide; the pharmaceutical composition contained in the medicament may be at least 50 doses, 20 doses, 10 doses, 5 doses or 2 doses.

[0092] In one embodiment, the kit contains two or more doses of the pharmaceutical composition, the polypeptide in the pharmaceutical composition is heparatide, the content of heparatide in each dose of the pharmaceutical composition is 0.1 mg, 0.2 mg, 0.3 mg, 0.6 mg, 0.7 mg, 1.4 mg or 2.1 mg, and the total content of the polypeptide contained in all of the pharmaceutical compositions in the kit is 4.2 mg.

[0093] In one embodiment, the kit contains two or more doses of the pharmaceutical composition, the polypeptide in the pharmaceutical composition is Myrcludex B, the content of Myrcludex B in each dose of the pharmaceutical composition is 0.1 mg, 0.25 mg, 0.5 mg, 1.0 mg or 2.5 mg, and the total content of the polypeptide in all pharmaceutical compositions in the kit is 5.0 mg.

[0094] In one embodiment, the kit contains multiple doses of the pharmaceutical composition, and is sufficient for at least 7 days of continuous administration, and the total content of the polypeptide contained in several doses of the multiple doses of the pharmaceutical composition reaches the daily dosage, i.e., 777.95-926.13 nmol or 4.2-5.0 mg. For example, in one embodiment, if each dose of the pharmaceutical composition contains 388.98 nmol or 2.1 mg of the polypeptide, the kit contains at least 14 doses of the pharmaceutical composition, and every two doses meet the daily dosage. If each dose of the pharmaceutical composition contains 259.32 nmol or 1.4 mg of the polypeptide, the kit contains at least 21 doses of the pharmaceutical composition, and every three doses meet the daily dosage. This can be inferred from the above. In one embodiment, the polypeptide contained in the pharmaceutical composition is heparatide or Myrcludex B. In one embodiment, the pharmaceutical product meets the 7-day continuous administration.

[0095] The pharmaceutical compositions and kits described herein are used to administer the polypeptides described herein to a subject in need thereof such that the polypeptides reach saturation in the target organ, the liver, after at least seven consecutive days of administration.

[0096] III.How to use Embodiments of the present disclosure include therapeutic and prophylactic uses of the polypeptides described herein. In one aspect, the use of the polypeptides described herein as pharmaceutical agents is provided. In another aspect, the use of the polypeptides described herein for treating and preventing HBV-associated liver disease is provided.

[0097] As used herein, the hepatitis B virus-related liver disease includes chronic hepatitis B virus infection, chronic hepatitis B, hepatitis B-related liver fibrosis and cirrhosis, hepatitis B-related liver cancer, hepatitis B-related liver transplantation, and hepatitis B-related mother-to-child transmission. The chronic hepatitis B includes HBeAg-positive chronic hepatitis B and HBeAg-negative chronic hepatitis B. In one embodiment, the hepatitis B-related liver transplantation includes protection of the donor liver from HBV infection before, during, and after transplantation.

[0098] "Patient" and "subject" are used interchangeably and refer to an animal (e.g., a mammal or a human) being treated or evaluated for a disease, disorder, or condition, or an animal (e.g., a mammal) or human being being evaluated for risk of or having a disease, disorder, or condition. In some embodiments, these diseases, disorders, or conditions may include metabolic disorders. In some embodiments, the metabolic disorder may involve lipid metabolism disorders.

[0099] A full dose of the polypeptide or pharmaceutical composition thereof described herein may be administered to a subject in need thereof to prevent or treat HBV-associated liver disease. As used herein, a "full dose" refers to a daily dose of the polypeptide administered daily, where the target organ is the liver, which is saturated after several consecutive days (≧7 days). In these embodiments, the "full dose" is generally a daily dose of 777.95-926.13 nmol or 4.2-5.0 mg, such as 777.95-833.52 nmol or 4.2-4.5 mg, or 777.95 nmol or 4.2 mg, or 926.13 nmol or 5.0 mg, where the target organ is not saturated the first time the polypeptide is administered, but after seven consecutive doses the target organ is saturated. Thus, the methods of prevention or treatment described herein include the step of administering a full dose of the polypeptide or pharmaceutical composition thereof continuously for at least 7 days.

[0100] As used herein, target organ saturation refers to an apparent volume of distribution that, after administration of a given amount of the polypeptide, is less than 100 L. In these embodiments, the apparent volume of distribution is typically 10-100 L, e.g., 10-50 L or 20-40 L.

[0101] In various embodiments, the term "treatment" includes treating a subject (e.g., a mammal, such as a human) or a cell to alter an existing process in the subject or cell. Treatment includes, for example, administering a polypeptide as described herein or a pharmaceutical composition comprising the polypeptide, and treatment may be performed in a prophylactic manner or may be initiated after contact with a pathological event or agent. Treatment also includes "prophylactic" treatment, which aims to slow the progression of the disease or condition being treated, delay the onset of the disease or condition, or reduce the severity of its onset. "Treatment" or "prevention" does not necessarily mean to completely eradicate, cure, or prevent the occurrence of the disease or condition or associated symptoms. In various embodiments, the term "treatment" may include reducing, alleviating, or reversing the pathological course of HBV-associated liver disease. In some embodiments, the term "treatment" may include improving at least one symptom or at least one measurable parameter of a metabolic disease. It will be apparent to one of skill in the art that biological and / or physiological parameters can be used to assess the pathological process of a metabolic disease. These pathological processes or symptoms may include, for example, an excess or increase in one or more metabolically related chemical or biological molecules (e.g., ALT, HBV DNA) compared to healthy subjects, or an excess or increase in one or more physiological parameters that measure metabolic changes (such as liver function indices).

[0102] The terms "dosing" or "administration" include administering a polypeptide as described herein in a local or systemic manner. Administration may be topical (including ocular or vaginal and rectal mucosal administration), pulmonary (such as by inhalation or insufflation with a powder or aerosol, including nebulizer, intratracheal, or intranasal administration), epidermal, transdermal, oral, or parenteral administration. Parenteral administration includes intravenous, subcutaneous, intraperitoneal, or intramuscular injection or infusion, or intracranial administration, such as intrathecal or intraventricular administration.

[0103] The disclosure further provides methods for carrying out the above-mentioned uses of the polypeptides described herein in a subject. These methods comprise administering to the subject an effective therapeutic or prophylactic amount of a polypeptide described herein or a pharmaceutical composition comprising the polypeptide. In some embodiments, the subject may be a mammal. In some embodiments, the subject may be a human. In some embodiments, the subject suffers from or is at risk of suffering from HBV-related liver disease. These combination therapies described herein may include combined administration (where the two or more therapeutic agents may be in the same formulation or in separate formulations) and separate administration (where the polypeptides described herein may be administered before, simultaneously with, or after the other therapeutic agent). These pharmaceutical agents include interferons, nucleotide drugs, hepatitis B immune globulin, and hepatitis B vaccines, where interferons include conventional interferons and PEG-long acting interferons. Conventional interferons include interferon alpha-2a and interferon alpha-2b, and PEG-long acting interferons include PEG-interferon alpha-2a and PEG-interferon alpha-2b. Nucleotide drugs include Lamivudine (LAM), Entecavir (ETV), Tenofovir (TDF), Telbivudine (TBV), Adefovir Dipivoxil (ADV), and Tenofovir alafenamide (TAF).

[0104] The polypeptides described herein (and any other therapeutic agents) can be administered in any suitable manner, including parenteral, pulmonary, and intranasal, and can be administered topically or intralesionally. In some embodiments, the polypeptides described herein can be administered parenteral. Parenteral administration can include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, the polypeptides described herein can be administered subcutaneously. In some embodiments, the polypeptides described herein can be administered intravenously. Administration can be by any suitable means, such as injection or infusion, such as intravenous or subcutaneous injection or infusion, depending on whether administration is brief or chronic. Various dosing schedules are also contemplated, including single or multiple doses at various time points, boluses, and pulse injections.

[0105] Thus, the present specification also discloses the polypeptide or pharmaceutical composition thereof described herein for use in the method for treating and preventing Hepatitis B virus-associated liver disease described herein. More specifically, the polypeptide or pharmaceutical composition thereof is used in the method for treating and preventing Hepatitis B virus-associated liver disease described herein at a daily dose (of the polypeptide) of 777.95-926.13 nmol or 4.2-5.0 mg, provided that the target organ, the liver, is saturated after several consecutive days of administration (preferably at least 7 consecutive days of administration).

[0106] In certain embodiments, the disclosure includes heparatide or a pharmaceutical composition thereof for use in a method of treating or preventing Hepatitis B virus associated liver disease as described herein, the method comprising administering heparatide to a subject in an amount of 777.95-926.13 nmol or 4.2-5.0 mg, preferably 777.95 nmol or 4.2 mg, daily to achieve saturation of the target organ liver over several consecutive days (preferably at least 7 consecutive days), and administering Myrcludex B to a subject, preferably at 926.13 nmol or 5.0 mg.

[0107] Preferably, the pharmaceutical composition is as described in any one of the embodiments herein. EXAMPLES

[0108] The following examples can be used for illustrative purposes and should not be construed as limiting the scope of the present invention.

[0109] Example 1: Synthesis of Polypeptides The polypeptide was synthesized with reference to the method of Example 1.1 of PCT / CN2017 / 086558. Specifically, the polypeptides shown in Table 1 were synthesized according to the standard Fmoc scheme for polypeptide synthesis. In general, a single amino acid residue is extended from the carboxyl terminus to the amino terminus from the MBHA resin. The N-terminus was then myristoylated. After the synthesis of the peptide was completed, a cleavage solution was used to cut the peptide from the resin and aminated the C-terminus of the polypeptide. The resin was filtered through a G6 sand core funnel, and the filtrate containing the polypeptide was suctioned under vacuum. The peptide product was dissolved in deionized water and purified using an AKTAexplorer 100 high pressure liquid chromatograph equipped with a C18 column, and the main peak was collected stepwise. The samples collected from the target peak were analyzed for purity with an Agilent 1100 reversed-phase high pressure liquid chromatography (HPLC) equipped with a C18 column, and their molecular weights were measured by a mass spectrometer. The collected solution was purified by medium pressure liquid chromatography and lyophilized. The dried sample was dissolved in PBS and filtered through a 0.20 μM film. The peptide stock solution dissolved in PBS was stored at −80° C. until use. The heparatide amino acid sequence is shown in SEQ ID NO:23, its N-terminus is modified to myristic acid, its C-terminus is modified by amino acid, its molecular weight is 5398.8 Da, and its molecular formula is C 247 H 352 N 65 O 73 It is.

[0110] Example 2: Polypeptides block HBV infection in vitro After anesthetizing adult male treeshrews, primary free tree shrew hepatocytes were obtained by microsurgical techniques using a two-step perfusion method with hepatic portal vein collagenase and cultured in a dedicated hepatocyte culture system. After 3 days of hepatocyte culture, purified HBV virus was added to the hepatocytes, infecting them at multiple MOIs of 0, 6.25, 12.5, 25, 50, and 100 genomic DNA / cell; HBeAg secretion in the supernatant after 12 days was a function of the MOI (R 2 = 0.9873), confirming that the HBsAg in the supernatant can quantitatively reflect the degree of HBV infection.

[0111] Heparatide (0, 2.5, 5, 10, 20, 40 ng / ml) was added simultaneously with HBV infection of hepatocytes (MOI = 100 genomic DNA / cell) to block HBV infection. With increasing concentrations of heparatide, the amount of HBeAg secreted in the supernatant decreased, and logit-log regression analysis showed that the two have a good linear dose-dependence (R 2 = 0.9563). Heparatide inhibited HBV infection in a dose-dependent manner, at which point the calculated half inhibitory concentration (IC50) of heparatide was 3.956 ng / ml.

[0112] Example 3: Blockade of HBV infection in vivo in animal models by polypeptides Fifty adult male tree shrews were randomly divided into five groups: a PBS control group, high (2 mg / kg), medium (0.4 mg / kg), and low (0.08 mg / kg) doses of heparatide groups, and a hepatitis B immunoglobulin HBIG blockade group (60 IU / kg). Tree shrews were infected intraperitoneally with 1 ml of HBV virus serum with a titer of 108 HBV DNA / ml, and the heparatide blockade group was subcutaneously injected with different doses of heparatide on days 0, 1, 2, 3, 5, 7, 9, 11, and 13 after infection. The HBIG blockade group was intramuscularly injected with immunoglobulin HBIG on the day of infection and on day 3 after infection. Sera from treeshrews were collected 4 days before infection and 9, 14, 21, and 42 days after infection to detect HBsAg, HBeAg, HBV DNA titers, and alanine aminotransferase (ALT) in serum; pathological examination of liver tissue was performed on day 21 postinfection.

[0113] In the PBS control group, HBsAg peaked on day 9 after infection and was essentially cleared on day 15. Heparatide (high, medium, and low dose groups) could effectively block HBV infection, and HBsAg was always well controlled during the infection process. However, HBIG could not effectively block HBV infection at the infection dose; compared with the PBS control group, HBsAg did not significantly decrease on day 9 after infection, and on the contrary, HBsAg secretion tended to be prolonged, and on day 15, the HBsAg level was higher than that of the PBS control group. The HBsAg levels on day 9 in each heparatide group (high, medium, and low dose) were all lower than those of the PBS control group and the HBIG blocking group; however, the HBsAg levels on day 9 in each heparatide group (high, medium, and low dose) were all significantly lower than those of the HBIG blocking group (all P less than 0.05).

[0114] In the PBS control group, HBeAg peaked on day 9 after infection and was essentially cleared on day 21. Heparatide could effectively block HBV infection, and high and medium doses of heparatide could effectively control HBsAg during the infection process. However, HBIG could not effectively block HBV infection at the infection dose, and HBsAg was equivalent to that of the PBS control group on days 9 and 15 after infection. The HBeAg levels on day 9 in each heparatide group (high, medium, and low doses) were all lower than those of the PBS control group and the HBIG blocking group; however, the HBsAg levels on day 9 in each heparatide group (high, medium, and low doses) were all significantly lower than those of the PBS control group (all P less than 0.05).

[0115] In the PBS control group, HBV DNA peaked on day 9 after infection and was essentially cleared on day 15. Heparatide could effectively block HBV infection, and high and medium doses of heparatide could effectively control HBV DNA during the infection process. However, HBIG could not effectively block HBV infection at the infectious dose, and HBV DNA was even higher than that of the PBS control group on day 15 after infection and was cleared until day 21 after infection. The HBV DNA levels on day 9 in each heparatide group (high, medium, and low doses) were all lower than those of the PBS control group and the HBIG blockade group; however, the HBV DNA level in the high dose heparatide group was significantly lower than that of the HBIG blockade group (P less than 0.05).

[0116] The mean ALT value before infection was 56.8 IU / ml, which was set as the normal tree shrew ALT level. In the PBS control group, ALT peaked on day 21 after infection and returned to normal levels on day 42. Heparatide could effectively block HBV infection, and high and medium doses of heparatide could effectively control ALT during the infection process. However, HBIG could not effectively block HBV infection at the infection dose, and ALT was higher than normal values ​​even on day 21 after infection and returned to normal levels 42 days after infection. The ALT levels on day 21 in each heparatide group (high, medium, and low doses) were lower than those in the PBS control group and the HBIG blockade group; however, the ALT levels in the high and medium dose heparatide groups were significantly lower than those in the HBIG blockade group (P less than 0.05).

[0117] One tree shrew from each test group was randomly selected for histopathological examination of the liver on the 21st day after infection, and the liver tissue of a normal tree shrew was used as a normal control. In the PBS group, it was shown that HBV infection caused obvious pathological changes in the liver tissue of tree shrews, mainly causing cloudy swelling and balloon-like changes of hepatocytes in the portal area around the hepatic lobule, accompanied by a small amount of lymphocytic infiltration; the pathological changes were very similar to hepatitis caused by acute HBV infection in adults. However, HBIG could not alleviate the pathological changes in the liver tissue of tree shrews caused by HBV infection, and the pathological changes in the liver in the low-dose heparatide group were similar to those in the PBS group, with only a slight reduction in the severity of the lesions. High and medium doses of heparatide completely blocked the pathological damage, such as balloon-like changes of hepatocytes, caused by HBV, and the liver tissue was virtually the same as that of normal tree shrews.

[0118] Thus, heparatide can effectively block HBV infection at the serological (HBsAg, HBeAg), virological (serum HBV DNA copy number), enzymological (ALT) and pathological levels.

[0119] Example 4: Polypeptides specifically bind to NTCP Non-hepatocyte-derived 293 cells were transfected with NTCP to form the NTCP-293 cell line. The 37th K amino acid of heparatide (Cmyr-47) or an irrelevant control peptide (47 peptide derived from the HBV Pre-S1 region of the heron, myristoylated-GLNQSTFNPLGFFPSHQLDPLFKANAGSADWDKNPNKDPWP QAHDTA-amidated, SEQ ID NO:50) was labeled with FITC fluorescence. As shown in Figure 1, FITC-heparatide bound to NTCP-293 cells but not to 293 cells not transfected with NTCP (BLANK-293), indicating that the binding of heparatide to cells has specificity for NTCP. The FITC-labeled irrelevant control peptide did not bind to NTCP-293 cells, indicating that the binding has specificity for the heparatide sequence.

[0120] Example 5: Effect of polypeptides on NTCP bile acid transport function The effect of heparatide on NTCP bile acid transport function was examined by referring to the literature study method (Kim RB et al. J Pharmacol Exp Ther. 1999 Dec;291(3):1204-9). Using 293 cells transfected with human NTCP (NTCP-293) as a cell model, the effect of heparatide on NTCP bile acid transport function was examined. 3 H-labeled bile acids ( 3 The effect of heparatide on H-TA uptake was investigated. The results were as follows (see Figure 2A): 1) 293 cells themselves do not absorb TA significantly, so TA entry into cells is specifically mediated by NTCP; 2) the positive control, cyclosporine A, exerted a significant inhibitory effect on NTCP, consistent with literature reports; 3) heparatide had a bidirectional effect on NTCP (see Figure 2B), promoting TA uptake at low concentrations (≦500 ng / ml, equivalent to 50 × IC50) and inhibiting TA uptake at high concentrations (>500 ng / ml), with an IC50 concentration of 838.81 ng / ml.

[0121] Example 6: Preclinical Pharmacokinetic Studies of Polypeptides 125 I-labeled heparatide at 40 ug kg -1 After administration to rats by a single subcutaneous injection at a dose of 100 mg / kg / day, the distribution of heparatide in each tissue was detected. The results of the study (Figure 3) show that heparatide is distributed mainly to the liver and urinary system, but its metabolites are distributed mainly to the urinary system. The drug peaked in the liver at 2 hours, with a Cmax of 44.3 ± 28.9 ng-Equ.mL. -1 The terminal elimination half-life in the liver was long at 8.7 hours, and the liver had a relatively high concentration even at 24 hours (4.5±1.2 ng-Equ.mL -1 );The estimated hepatic drug concentration at 48 hours was approximately 0.66 ng-Equ.mL -1 This corresponds to the serum drug concentration level 4 hours after subcutaneous injection. The drug was rapidly eliminated from serum, essentially declining to baseline 6 hours after administration. Preclinical pharmacokinetic results showed that heparatide is significantly concentrated in the target organ, the liver.

[0122] Example 7: Phase Ia Clinical Trial and Clinical Pharmacokinetic Study of Single Ascending Doses of Polypeptide Drug treatment / degree of exposure: The single-ascending dose phase Ia clinical trial of heparatide was a randomized, double-blind, blank-controlled, single-center, dose-ascending study, with six dose groups (0.525 mg, 2.1 mg, 4.2 mg, 6.3 mg, 8.4 mg, and 10.5 mg) and enrolled 5, 5, 10, 10, 10, and 5 healthy subjects with a male to female ratio of 22:23. After screening, healthy volunteers were randomly placed into the experimental and blank-control groups according to a double-blind 4:1 ratio, received a single subcutaneous injection of heparatide, and were observed for 7 days after drug withdrawal. Heparatide drug treatment and degree of exposure during the study are shown in Table 2.

[0123] Number and characteristics of the subject groups: 45 healthy adult subjects were planned to participate, and 45 subjects actually participated; all subjects completed the study and were included in the safety analysis population. The safety analysis population of the Phase Ia clinical study is shown in Table 3. There were no significant differences in demographic indicators, such as age, sex, ethnicity, occupation, height, weight, BMI, and past medical history, between the study groups.

[0124] [Table 2]

[0125] [Table 3]

[0126] Safety Studies: No deaths or serious adverse events occurred in the single ascending dose Phase Ia clinical trial of heparatide; a total of 63 adverse events were recorded during the study, all of which were grade I adverse events; 29 adverse events were related, likely related, or possibly related to the study drug (see Table 4). The incidence of adverse events was not significantly different between each dose group and the blank control group; the incidence of adverse events in the 8.4 mg dose group was significantly higher than in the blank control group; the incidence of adverse events was not significantly different between the 0.525 mg, 2.10 mg, 4.20 mg, 6.30 mg, 10.50 mg doses and the blank control.

[0127] [Table 4]

[0128] Phase Ia clinical pharmacokinetic study: A dose-escalating phase Ia clinical pharmacokinetic study was conducted with a single subcutaneous dose in 45 healthy subjects, and the dose setting and PK analysis population are shown in Table 5. Blood samples were taken before and after administration at 2 minutes, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours. The biological samples were detected for drug concentration by liquid mass spectrometry, and the lower limit of quantification was 0.2 ng / ml. The blood drug concentration curves are shown in Figure 4, and the main parameters of blood pharmacokinetics are shown in Table 4. The results of the study showed that when subjects received a single subcutaneous injection of heparatide, the blood drug curves were low after receiving a single subcutaneous injection of heparatide in the dose range of 0.525 mg to 4.2 mg, but in the dose range of 6.3 mg to 10.5 mg, the blood drug curves were significantly higher. In the dose range of 0.21-4.2mg, the apparent distribution volume Vz / F was greater than 100L, indicating that the drug was mainly distributed in the target organ. Combined with preclinical studies, it was shown that heparatide specifically binds to NTCP (Example 4), and preclinical pharmacokinetic studies showed that heparatide was significantly concentrated in the liver (Example 6). Since the drug target NTCP is specifically expressed in the liver, it is determined that the drug is mainly concentrated in the target organ liver. In the dose range of 6.3-10.5mg, Vz / F was close to the total body fluid volume of the human body and decreased with increasing dose, indicating that the drug was distributed to the body fluid after reaching saturation in the target organ. Therefore, the single doses of 0.525mg-4.2mg and 6.3mg-10.5mg were respectively set as the unsaturated dose range and saturation dose range of the target organ liver. After a single subcutaneous injection of 5 mg of Myrcludex B, the apparent volume of distribution was 135 L (Lank A et al. J Hepatol. 2016 Sep;65(3):483-9), and the results of this example show that Myrcludex B did not reach saturation in the target organ, the liver, when administered subcutaneously at that dose.

[0129] [Table 5]

[0130] Example 8: Multiple ascending dose phase Ib clinical trial and clinical pharmacokinetic study of a polypeptide Drug treatment / exposure: The heparatide multiple ascending dose phase Ib clinical trial was a randomized double-blind, blank-controlled, single-center clinical trial. The study enrolled 10, 10, and 15 healthy subjects in three dose groups (4.20 mg, 6.30 mg, and 8.40 mg), respectively, with a male-to-female ratio of 18:17. After screening, healthy volunteers were randomly placed into the experimental and blank-control groups according to a double-blind 4:1 ratio and received subcutaneous injections once daily for 7 consecutive days. They were observed for 3 days after drug withdrawal. The heparatide drug treatment and exposure during the study are shown in Table 6. Number and characteristics of the subject group: In the heparatide phase Ib clinical study, 35 healthy adult subjects were planned to participate, and 35 subjects actually participated; all subjects completed the study and were included in the safety analysis population. The safety analysis population and demographic indicators of each study group in the phase Ib clinical trial are shown in Table 7. There were no significant differences in demographic indicators such as age, sex, ethnicity, occupation, height, weight, BMI, and past history between the study groups.

[0131] [Table 6]

[0132] [Table 7]

[0133] Safety evaluation: In the heparatide phase Ib clinical study, a total of 74 adverse events were recorded, all of which were grade I adverse events; all were spontaneously alleviated without treatment and left no sequelae. During the study course, a total of 36 adverse events (see Table 8) related to, likely related to, or possibly related to the study drug were recorded. The main adverse event was an increase in total bile acids, which occurred in a total of 27 cases, accounting for 75.0% of all adverse events. Because an increase in bile acids is a pharmacological response of heparatide, after subtracting the adverse event of an increase in total bile acids, the incidence of adverse events in the 4.2, 6.3, and 8.4 mg dose groups compared with the blank control was 25.0%, 37.5%, and 8.3%, respectively, corresponding to an incidence of adverse events in the blank control group of 42.9%. In the Phase Ib clinical trial, no dose-limiting toxicity DLTs of heparatide occurred, and the maximum tolerated dose MTD was not reached when the dose was escalated to 8.4 mg, the maximum dose designed in the study.

[0134] [Table 8]

[0135] Clinical Pharmacokinetic Study: A subcutaneous multiple dose escalation dose phase Ib clinical pharmacokinetic study was conducted in 35 healthy subjects, subjects received one dose daily for 7 consecutive days, and the dose setting and PK analysis populations are shown in Table 9. Blood samples were collected as follows: pre-dose and post-dose blood samples at 2 min, 5 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, and 12 h; Day 2: 24-h blood sample on Day 1 (pre-dose on Day 2); Days 3-6: pre-dose; Day 7: pre-dose and post-dose blood samples at 2 min, 5 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, and 12 h. Biosamples were subjected to liquid mass spectrometry to detect drug concentration, with a lower limit of quantification of 0.2 ng / ml. The blood concentration-time curves of blood samples from the first dose, pre-dosing on days 2-7, and the final dose are shown in Figure 5-7, and the main pharmacokinetic parameters are shown in Table 10. The study results showed that after the first dose, the blood concentration curves of each dose group were consistent with phase Ia, the first dose of 4.2 mg did not reach target organ saturation, and the first doses of 6.3 and 8.4 mg reached target organ saturation. During the continuous dosing period from days 2 to 6, the blood trough concentrations reached a steady state. After 7 days of continuous dosing, the accumulation coefficients R of the 4.2 mg, 6.3 mg, and 8.4 mg dose groups were AUC were 4.43±1.60, 2.76±0.76 and 1.91±0.39, respectively, indicating that heparatide in the case of successive administration had an accumulative effect. After accumulation, the apparent volume of distribution of 4.2 mg decreased from 153.6025±107.1807 liters at the first dose to 22.1880±8.0721 liters at the last dose, indicating that the final dose reached saturation of the target organs after accumulation with successive administration in the case of the 4.2 mg dose. With the highest dose of 8.4 mg, after sufficient accumulation in the target organs, the apparent volume of distribution was 15.1021±4.4743 liters, close to the extravascular fluid space of 14 liters in the body, which indicates that outside the target organs, the drug was mainly distributed in the extracellular fluid.

[0136] [Table 9]

[0137] [Table 10]

[0138] Clinical Pharmacokinetic Study: In a multiple-dose ascending phase Ib clinical trial, subjects received subcutaneous injections of heparatide once daily for 7 consecutive days within the dose range of 4.2-8.4 mg; during the administration period, the levels of total blood bile acids increased significantly, and after drug withdrawal, total blood bile acids returned to normal. Total bile acids can be used as an ideal pharmacodynamic biomarker. The results of the study showed that a sufficient dose to achieve a pharmacodynamic response could be achieved by subcutaneously injecting heparatide 4.2 mg daily for 7 consecutive days.

[0139] Example 9: Summary of clinical pharmacokinetics of Phase I clinical trials of polypeptides After a single subcutaneous injection of heparatide in the dose range of 0.525-4.2 mg, heparatide was distributed mainly to the target organs; after a single subcutaneous injection in the dose range of 6.3-10.5 mg, target organ saturation was reached and the drug was distributed to plasma. Surprisingly, when heparatide was administered in a single subcutaneous injection at a dose of 4.20 mg, it did not reach target organ liver saturation, but when this dose was administered subcutaneously for 7 consecutive days, it accumulated and reached target organ liver saturation, which is an unreported and unexpected result. According to the results disclosed in this application, it was confirmed that a daily dose of 4.2 mg is the lowest target organ saturation dose for continuous administration of heparatide. Based on the results disclosed in this disclosure, when Myrcludex B was administered in a single subcutaneous injection at a dose of 5 mg, it did not reach target organ liver saturation, but when this dose was administered consecutively, it accumulated and reached target organ liver saturation.

[0140] Example 10: Summary of safety from Phase I clinical trials of peptides Subjects tolerated heparatide in the dose range of 0.525-10.5 mg administered as a single subcutaneous injection well, did not experience dose-limiting toxicity, and did not reach the maximum tolerated dose.Subjects tolerated heparatide in the dose range of 4.2-8.4 mg administered as consecutive subcutaneous injections for 7 days well, did not experience dose-limiting toxicity, and did not reach the maximum tolerated dose.

[0141] Example 11: Clinically Recommended Doses At a daily dose of 4.2 mg, heparatide accumulated after continuous administration and reached target organ hepatic saturation; at the same time, the clinical safety at this dose was good and pharmacodynamic biomarker responses of total bile acids were achieved. Therefore, this dose is determined to be the clinically recommended dose.

Claims

1. 20. Use of a polypeptide or a pharmaceutical composition comprising said polypeptide in the manufacture of a medicament for use in a method for treating or preventing Hepatitis B virus associated liver disease, comprising: The method comprises administering to a subject in need of treatment or prevention a daily dose of the polypeptide daily; The polypeptide is shown in SEQ ID NO:3, said daily dose being between 6.3 and 8.4 mg daily dose; Use:

2. The use described in claim 1, characterized in that the method comprises administering 1 to 42 doses of the polypeptide or pharmaceutical composition to the subject daily to achieve a daily dose of 6.3 to 8.4 mg.

3. The use according to claim 1, characterized in that the hepatitis B virus-related liver disease is selected from the group consisting of chronic hepatitis B virus infection, chronic hepatitis B, hepatitis B-related liver fibrosis, hepatitis B-related liver cirrhosis, hepatitis B-related liver cancer, and hepatitis B-related maternal-fetal transmission.

4. The use according to claim 3, characterized in that the chronic hepatitis B is HBeAg-positive chronic hepatitis B or HBeAg-negative chronic hepatitis B.

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