WO2019080919A1 - 充分剂量条件下治疗乙肝病毒相关肝病的药物和方法 - Google Patents

充分剂量条件下治疗乙肝病毒相关肝病的药物和方法

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WO2019080919A1
WO2019080919A1 PCT/CN2018/112062 CN2018112062W WO2019080919A1 WO 2019080919 A1 WO2019080919 A1 WO 2019080919A1 CN 2018112062 W CN2018112062 W CN 2018112062W WO 2019080919 A1 WO2019080919 A1 WO 2019080919A1
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polypeptide
amino acid
dose
pharmaceutical composition
hbv
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PCT/CN2018/112062
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English (en)
French (fr)
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刘宏利
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上海贺普药业股份有限公司
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Application filed by 上海贺普药业股份有限公司 filed Critical 上海贺普药业股份有限公司
Priority to EP18871510.6A priority Critical patent/EP3701966A4/en
Priority to CN201880068783.3A priority patent/CN111417402A/zh
Priority to US16/759,200 priority patent/US20200316166A1/en
Priority to JP2020543680A priority patent/JP2021500411A/ja
Publication of WO2019080919A1 publication Critical patent/WO2019080919A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses

Definitions

  • the present disclosure relates to drugs and methods for treating hepatitis B virus-associated liver disease under adequate dosage conditions.
  • HBV hepatitis B virus
  • the HBV viral envelope protein contains three surface antigen proteins: large surface antigen protein (L), medium surface antigen protein (M), and small surface antigen protein (S). These proteins are encoded by a single open reading frame on the S gene, starting at three different translation initiation sites, namely L(Pre-S1+Pre-S2+S), M(Pre-S2+S) and S(S). HBV is divided into 9 genotypes (A-I), and each genotype has a certain difference in the amino acid sequence of Pre-S1 region.
  • L large surface antigen protein
  • M medium surface antigen protein
  • S small surface antigen protein
  • Large HBsAg large surface proteins
  • NTCP sodium taurocholate cotransporting polypeptide
  • 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 animal models (Petersen J et al. Nat Biotechnol. 2008 Mar; 26(3): 335-41.).
  • NTCP is responsible for the physiological function of bile acid transport from the portal vein to the liver, and is one of the important proteins of the bile acid enterohepatic circulation (Alrefai W et al. Pharmaceutical Research, 2007; 24(10): 1803-1823).
  • HBV Pre-S1 a polypeptide derived from HBV Pre-S1
  • Myrcludex B a peptide derived from HBV Pre-S1
  • has completed a single-dose clinical phase Ia clinical study and pharmacokinetic study (lank A et al. J Hepatol. 2016 Sep; 65(3): 483-9.).
  • the study set 12 dose groups respectively, a single 2h continuous intravenous injection of 0.3ug, 3ug, 10ug, 100ug, 800ug, 3mg, 5mg, 10mg, 20mg and a single subcutaneous injection of 800ug, 5mg, 10mg dose.
  • a total of 36 healthy subjects were enrolled in the study and assigned to the above 12 dose groups, 3 in each group. All 36 subjects completed clinical trials. Due to the effect of continuous intravenous administration on pharmacokinetic parameters, it is difficult to analyze the pharmacological parameters of intravenous administration.
  • V-analysis of apparent distribution volume parameter V of subcutaneous administration the apparent volume of distribution after 800 ug and 5 mg subcutaneous administration was greater than 100L, much larger than body weight, showing that the polypeptide was distributed in the target organ; when the dose was increased to 10 mg, the blood concentration was high.
  • the apparent distribution volume is 43L. Due to the low dose setting of the subcutaneous administration of this test, the lack of reference and comparison failed to obtain the conclusion that the polypeptide was saturated in the liver target organ.
  • the accumulation coefficient R AUC was 1.79 after 6 consecutive days of administration, with an accumulation effect; however, the apparent volume distribution parameter was not reported (Blank A et al.
  • the present disclosure provides a method for treating and preventing hepatitis B virus-associated liver disease, the method comprising administering to a subject in need thereof a daily dose of a polypeptide or a pharmaceutical composition thereof, wherein the polypeptide contains a derivative derived from hepatitis B virus (HBV) The amino acid sequence of Pre-S1; said daily dose of said polypeptide which is saturated in the liver target organ after said polypeptide is administered in a sufficient dose.
  • HBV hepatitis B virus
  • the sufficient dose is a daily dose of 777.95-926.13 nmol, preferably a daily dose of 777.95 nmol or 926.13 nmol.
  • the sufficient 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.
  • the polypeptide comprises the amino acid sequence of the pre-S1 region of HBV.
  • the N-terminus of the polypeptide contains a hydrophobic group modifying group.
  • the hydrophobic group is selected from the group consisting of myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, cholesterol, and arachidonic acid.
  • the hydrophobic group is myristic acid.
  • the polypeptide has a C-terminal modification or no modification.
  • the C-terminal modification is amidation or isopentylation.
  • the polypeptide comprises the amino acid sequence of the pre-S1 region of the HBV genotype A, B, C, D, E, F, G, H or I.
  • the polypeptide comprises the sequence of amino acid sequence 13-59 of the pre-S1 region of HBV genotype C, or contains the HBV genotype A, B, D, E, F, G, H or I.
  • one or more amino acid residues of the polypeptide are deleted, substituted or inserted; preferably, 1-30, 1-20, 1-10, 1-8, 1- of the polypeptide 5 or 1-3 amino acid residues are deleted, substituted or inserted.
  • the polypeptide comprises a native flanking amino acid sequence from the pre-S1 region of HBV at its N-terminus and/or C-terminus; preferably, the natural flanking amino acid of the pre-S1 region of the HBV The sequence is 1-10, 1-8, 1-5 or 1-3 amino acids in length.
  • the polypeptide comprises 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 of the pre-S1 region of HBV genotype C or Lysine of amino acid 57.
  • polypeptide in certain embodiments, the polypeptide:
  • polypeptide comprising an amino acid sequence selected from any one of SEQ ID NO: 21-40 or 49; preferably, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 23;
  • polypeptide is as set forth in any one of SEQ ID Nos: 1-20 or 51.
  • the polypeptide comprises SEQ ID NO: 23 or 49, wherein the polypeptide further comprises an N-terminal myristate modification and a C-terminal amination modification, or wherein the polypeptide comprises SEQ ID NO: 3 or 51.
  • the hepatitis B virus-associated liver disease comprises 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 comprises HBeAg-positive chronic hepatitis B and HBeAg-negative chronic hepatitis B.
  • the hepatitis B-related liver transplantation includes protection of HBV infection of the donor liver before, during, and after transplantation.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 23, the N-terminus of which is modified by myristic acid and C-terminal amination; the sufficient dose is a daily dose of 4.2-5.0 mg, A daily dose of 4.2 mg is preferred, the method comprising at least 7 consecutive days of administration.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 49, the N-terminus of which is modified with myristic acid and C-terminal amination; the sufficient dose is a daily dose of 4.2-5.0 mg, A daily dose of 5.0 mg is preferred, the method comprising at least 7 consecutive days of administration.
  • the polypeptide is an amino acid sequence comprising SEQ ID NO: 23, the N-terminus of which is modified with myristic acid and C-terminal amination, the method comprising administering to the subject N a daily dose
  • the pharmaceutical composition achieves a daily dose of 4.2 mg, wherein N is any integer from 1 to 42.
  • the polypeptide is an amino acid sequence set forth in SEQ ID NO: 49, the N-terminus of which is modified with myristic acid and C-terminal amination, the method comprising administering the N agent to the subject daily.
  • the pharmaceutical composition achieves a daily dose of 5.0 mg, wherein N is any integer from 1 to 50.
  • the present disclosure also provides a pharmaceutical composition comprising an amino acid sequence derived from hepatitis B virus (HBV) Pre-S1; the pharmaceutical composition being a pharmaceutical composition containing an amount of the polypeptide by administering an integer number of preparations
  • HBV hepatitis B virus
  • the pharmaceutical composition of the specification achieves saturation of the polypeptide in the target organ of the liver.
  • the pharmaceutical composition contains a therapeutically effective amount of a polypeptide, wherein the therapeutically effective amount is 777.95 of the total daily dose of the polypeptide after administration of one or several doses of the pharmaceutical composition per day. -926.13nmol.
  • the therapeutically effective amount is a daily total dose of the polypeptide of from 4.2 to 5.0 mg, preferably a total daily dose of 4.2 mg or 5.0 mg, after administration of one or several doses of the pharmaceutical composition per day. .
  • 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 per dose, ie, the pharmaceutical composition specification Is 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 the pharmaceutical composition contains 5.0 mg, 2.5 mg, 1.0 mg, 0.5 mg, 0.25 mg or 0.1 per dose.
  • the polypeptide of mg, i.e., the pharmaceutical composition is 5.0 mg, 2.5 mg, 1.0 mg, 0.5 mg, 0.25 mg or 0.1 mg.
  • polypeptide is as described in any of the embodiments of the present disclosure.
  • the present disclosure also provides a kit comprising one or more doses of a medicament containing a polypeptide as an active ingredient for administration to a patient for one or more days, wherein the one or more doses of the medicament are contained
  • the polypeptide is administered in an amount such that 777.95-833.52 nmol of the polypeptide is administered daily after administration of one or several doses of the drug, wherein the polypeptide contains an amino acid sequence derived from hepatitis B virus (HBV) Pre-S1.
  • HBV hepatitis B virus
  • the one or more doses of the medicament comprise a polypeptide in an amount such that 4.2 or 5.0 mg, preferably 4.2 mg or 5.0 mg, is administered daily after administration of one or more doses of the medicament.
  • polypeptide is as described in any of the embodiments of the present disclosure.
  • the present disclosure also provides the use of a pharmaceutical composition described herein in the manufacture of a medicament for the treatment or prevention of hepatitis B virus-associated liver disease.
  • polypeptides described herein or pharmaceutical compositions thereof for use in the treatment and prevention of hepatitis B virus associated liver disease.
  • the method of treating and preventing hepatitis B virus-associated liver disease is the method of any of the embodiments herein.
  • FIG. 1 FIFC-labeled hepalatide binds to HEK293 cells expressing NTCP (NTCP-293) and does not bind to HEK293 cells (BLANK-293) as a control.
  • NTCP-293 NTCP-293
  • BLANK-293 HEK293 cells
  • a FIFC-tagged polypeptide derived from Heron HBV was used as a control polypeptide.
  • FIG. 2 Effect of Cmyr-47 on bile acid uptake in vitro.
  • Cyclosporine A (CsA) was used as a positive control.
  • A TA entry cells are NTCP-specific, positive control cyclosporine A has a significant inhibitory effect on NTCP;
  • B hepalatide has a two-way effect on NTCP bile acid transport function.
  • Figure 3 Precipitation radioactivity time curve after subcutaneous injection of 125 I-heptapeptide.
  • Figure 4 Mean plasma concentration-time curve for the Phase Ia PK study (mean + SD).
  • Figure 6 Mean plasma concentration-time curve (mean + SD) before dosing on days 2-7 of the Phase Ib PK study.
  • Figure 7 Final dose mean plasma concentration-time curve (mean + SD) for the Phase Ib PK study.
  • the article “a” refers to one or more (ie, at least one) of the grammatical terms of the article.
  • an element means one element or more than one element.
  • the term “about” refers to a quantity, level, value, value, frequency, percentage, size, size, quantity, weight, or length that is compared to the amount, level, value, value, frequency, percentage, size, size of the reference. , quantity, weight or length of approximately 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% Variety.
  • the term “about” modifies the range and extends its limits above or below the value.
  • the term “about” when used to modify a value means within 10% of the value above and below.
  • polypeptides derived from HBV Pre-S1 for use in treating or preventing HBV-associated liver disease.
  • the polypeptide blocks the infection of HBV in vitro.
  • the polypeptide is capable of binding to NTCP in vitro, for example in solution or in a cell-free system (such as a cell lysate or in a reconstituted system), or in a cell, such as an ex vivo cultured cell (eg, expression) NTCP is bound to cells in NTCP, or in hepatocytes, or in cells of the subject.
  • the subject can be a mammal. In some embodiments, the subject can be a human.
  • polypeptide polypeptide
  • peptide protein
  • proteins are used interchangeably, including 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 of the biological activities of the polypeptide.
  • Polypeptide may include natural and/or non-natural 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 (eg, N-terminal and/or amino acid residues at the C-terminus).
  • the N-terminus of the polypeptides described herein can be modified with hydrophobic groups such as myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, cholesterol, and arachidonic acid.
  • the C-terminus of the polypeptides described herein can be modified.
  • the C-terminal modification can be selected from amidation (amination), isopentylation or no modification.
  • polypeptide derived from HBV Pre-S1 or "HBV Pre-S1 derived polypeptide” as used herein means that the origin or source of the polypeptide is HBV and may include natural, recombinant, synthetic or purified polypeptides.
  • polypeptide derived from HBV or "HBV-derived polypeptide” refers to a full-length native HBV polypeptide or fragment thereof, as well as variants of a full-length native polypeptide or fragment thereof.
  • 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 all of the native sequence.
  • the polypeptides described herein can be derived from the pre-S1 region of any HBV subtype L protein. In some embodiments, a polypeptide described herein can contain the entire pre-S1 region of any HBV subtype L protein. In certain embodiments, the polypeptides described herein can be derived from the pre-S1 region of the L protein of any of the 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.
  • polypeptides described herein can 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 for the corresponding native HBV polypeptide.
  • a "variant" associated with a polypeptide described herein, a polypeptide derived from HBV Pre-S1, or a HBV Pre-S1 derived polypeptide refers to a polypeptide derived from a given polypeptide (ie, a polypeptide described herein, derived from HBV Pre-S1). Or the HBV Pre-S1 derived polypeptide) differs in amino acid sequence but retains one or more of the biological activities described herein for a given polypeptide.
  • a variant polypeptide as described herein may have one or more amino acid additions (e.g., insertions), deletions, or substitutions relative to a given polypeptide.
  • the variant polypeptides described herein can have 1-30, 1-20, 1-10, 1-8, 1-5, or 1-3 (including these ranges) relative to a given polypeptide. All integers) amino acid additions (such as insertions), deletions or substitutions.
  • a polypeptide sequence can contain conservative substitutions of amino acids.
  • a conservative substitution of an amino acid i.e., the replacement of an amino acid with a different amino acid having similar properties (e.g., hydrophilicity and degree of charge and distribution of charged regions), typically involves minor changes and therefore does not significantly alter the biological activity of the polypeptide. These small changes can be identified in part by considering the hydropathicity of the amino acid based on the hydrophobicity and charge of the amino acid.
  • Amino acids having a similar hydrophilicity index and hydrophilicity value can be substituted for each other and still retain protein function.
  • the hydropathic and hydrophilic values of an amino acid are affected by the particular side chain of the amino acid. Consistent with this observation, amino acid substitutions that match biological functions depend on the relative similarity of amino acids, especially the side chains of those amino acids, which are characterized by hydrophobicity, hydrophilicity, chargeability, size, and other properties.
  • variant also includes a certain identity with a given polypeptide, such as having at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a given polypeptide.
  • a “variant” as used herein also includes a polypeptide comprising a portion of the native sequence of a given polypeptide corresponding to the HBV protein.
  • Variant can also refer to a fusion protein or chimeric protein comprising a polypeptide derived from two or more proteins of different origin.
  • Non-limiting examples of fusion proteins described herein can include two fusion proteins, such as one derived from HBV Pre-S1, and another polypeptide derived from a non-HBV Pre-S1 protein, derived from two different HBV subtypes.
  • variant also encompasses the amino acid sequence comprising the same amino acid sequence as a given polypeptide (ie, a polypeptide described herein, derived from HBV Pre-S1 or a HBV Pre-S1 derived polypeptide), retaining one or more of the given polypeptide A polypeptide that is biologically active but chemically and/or post-translationally modified in a manner different from the given polypeptide. "Variant" can also be used to describe that it has been treated differently, such as proteolysis, phosphorylation or other post-translational modifications, but retains one or more of the biological activities described herein. As used herein, “variant” includes fragments of variants, unless otherwise indicated.
  • variant also includes homologous polypeptide sequences found in different viral species, strains or hepadnavirus subtypes. Based on the antigenic epitopes on its envelope protein, HBV is divided into four major serotypes (adr, adw, ayr, and ayw). According to the variability of all nucleotide sequences in the genome, HBV is divided into 9 genotypes ( AI). Thus, the term “variant” includes any of these homologous polypeptides found in the HBV subtype. "Variant” can also include a polypeptide having a native flanking amino acid sequence from any of these HBV subtypes added at the N and/or C terminus.
  • conservative amino acid substitution and “conservative substitution” are used interchangeably herein to refer to a specified amino acid exchange within a group of amino acids, wherein one amino acid differs from a different amino acid having a similar size, structure, charge and/or polarity. exchange.
  • a family of amino acid residues having similar side chains are well known in the art and include basic side chains (such as leucine, arginine and histidine), acidic side chains (such as aspartic acid, glutamic acid), Non-electrode side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (such as alanine, proline, bright Amino acid, isoleucine, valine, phenylalanine, methionine, tryptophan), beta side chain (such as threonine, valine, isoleucine) and aromatic side chain (such as tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains such as leucine, arginine and histidine
  • acidic side chains such as aspartic acid, glutamic acid
  • Non-electrode side chains such as glycine, asparagine,
  • an amino acid residue in a polypeptide can be replaced with another amino acid residue from the same side chain family.
  • an amino acid sequence can be replaced with an amino acid sequence that is structurally similar but differs in the order and/or composition of the members of the side chain family.
  • mutations can be introduced randomly at the entire sequence or partial sequence of the polypeptide.
  • conservative amino acid substitutions include, for example, replacing one of the four amino acids of the group with one of the aliphatic or hydrophobic amino acids Ala, Val, Leu, and Ile; substitution between the hydroxyl-containing residue Ser and Thr; Substitution between residues Asp and Glu; substitution between amide residues Asn and Gln; substitution between basic residues Lys, Arg and His; substitution between aromatic residues Phe, Tyr and Trp; Replacement between small amino acids Ala, Ser, Thr, Met and Gly.
  • Conservative substitutions can be reasonably foreseen, such as substitution of a conservative amino acid with a similar, structurally related amino acid, which will not substantially affect the biological activity of the polypeptide.
  • sequence identity refers to the extent to which the sequences are identical when compared to amino acids in a comparison window.
  • a polypeptide described herein can contain an amino acid sequence that is at least about 30%, 40%, 50%, 60%, 70%, 80% identical to the sequence of a given polypeptide. 85%, 90%, 95%, 96%, 97%, 98% or 99%, and still retain one or more biological activities of the given polypeptide.
  • the result Multiplying by 100 to obtain a percent sequence identity, "percent identity” or "% identity” can be calculated.
  • the optimal sequence arrangement for arranging in a comparison window can be performed by using a computer to perform algorithms known in the art, for example Family procedures, either by visual inspection, produce an optimal alignment using either method selected (ie, yielding the highest percentage of homology in the comparison window). For sequence comparison, one sequence is used as a reference sequence and the test sequence is compared to the reference sequence.
  • sequence comparison algorithm When using the sequence comparison algorithm, enter the test and reference sequences into the computer and, if necessary, design the subsequence coordinates and set the sequence algorithm program parameters. The sequence comparison algorithm then calculates the current sequence identity of the test sequence relative to the reference sequence based on the programmed parameters.
  • the setting of sequence algorithm program parameters is well known in the art.
  • the comparison window can be set to cover the full length of any one or two of the comparison sequences, such as covering the entire length of the reference sequence, while allowing for a difference of up to 5% of the total amino acid number of the reference sequence.
  • the polypeptides described herein may comprise the amino acid sequence of the pre-S1 region of any of the HBV subtypes. In some embodiments, the polypeptide described herein comprises amino acids 13-59 of the pre-S1 region of HBV genotype C: GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANQVG (SEQ ID NO: 23) or GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANKVG (SEQ ID NO: 49).
  • polypeptides described herein may contain a corresponding one from another HBV genotype (eg, from any of genotypes A, B, C, D, E, F, G, H, or I) Pre-S1 sequence.
  • the polypeptides described herein can contain:
  • GKNISTTNPLGFFPDHQLDPAFRANTRNPDWDHNPNKDHWTEANKVG SEQ ID NO: 37;
  • GKNLSASNPLGFLPDHQLDPAFRANTNNPDWDFNPKKDPWPEANKVG SEQ ID NO: 39
  • a polypeptide described herein can comprise a portion of a pre-S1 region of HBV, the portion comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 23, 34-40, or 49.
  • the polypeptides described herein may contain the entire pre-S1 region of HBV.
  • the polypeptides described herein can be from 10 to 100 amino acids in length.
  • the polypeptide may be 15-100, 15-80, 20-100, 20-80, 20-60, 25-60, 30-60, 35-60 or 40-60 (including these ranges) All integers between) amino acids.
  • the polypeptides described herein can be at least 20 amino acids in length, such as at least 25, 30, 35 or 40 amino acids.
  • the polypeptides described herein can be 20, 25, 30, 35, 40, 47, 55 or 60 amino acids in length.
  • the polypeptides described herein can be 47 amino acids in length. Variants of different lengths of the polypeptides described herein retain one or more biological activities associated with the corresponding polypeptide.
  • the N-terminus of a polypeptide described herein can contain a hydrophobic group modification.
  • the hydrophobic group can be selected, for example, from myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, cholesterol, and arachidonic acid.
  • the hydrophobic group can be selected from the group consisting of myristic acid, palmitic acid, stearic acid, and cholesterol.
  • the hydrophobic group can be myristic acid.
  • the polypeptides described herein may comprise an amino acid sequence selected from any one of SEQ ID NOs: 23, 34-40, and 49, wherein the N-terminus of the polypeptide may be selected from myristic acid, Hydrophobic group modification of palmitic acid, stearic acid and cholesterol.
  • the polypeptides described herein may comprise an amino acid sequence selected from any one of SEQ ID NOs: 23, 34-40, and 49, wherein the N-terminus of the polypeptide may be myristoylated.
  • a polypeptide described herein can comprise the amino acid sequence set forth in SEQ ID NO: 23, wherein the N-terminus of the polypeptide can be myristoylated.
  • the polypeptides described herein may contain a C-terminal modification, or no modification.
  • the C-terminal modification can be selected, for example, from amidation (amination), isopentylation, or no C-terminal modification.
  • the C-terminal modification can be amidation (amination).
  • a polypeptide described herein may comprise the amino acid sequence set forth in SEQ ID NO: 23, which may be acylated at the N-terminus and/or amidated (aminated) at the C-terminus.
  • a polypeptide described herein can comprise the amino acid sequence set forth in SEQ ID NO:23.
  • the polypeptides described herein may comprise an amino acid sequence selected from any one of SEQ ID NOs: 34-40, 49, wherein the N-segment of the polypeptide may be myristoylated, and/or C The ends can be modified by amidation (amination).
  • the polypeptides described herein may comprise an amino acid sequence selected from any one of SEQ ID NOs: 14-20, 51. Variants of the polypeptides described herein that are modified at the N-terminus and/or C-terminus retain one or more biological activities of the corresponding polypeptide that are not modified in the same manner.
  • Variants of the polypeptides described herein are also included in the disclosure, including one or more amino acid deletions, substitutions or insertions, while retaining one or more biological activities of the polypeptide.
  • the polypeptide described herein preferably retains 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).
  • the polypeptide described herein retains an asparagine or amino acid 57 lysine corresponding to amino acid 20 of the pre-S1 region of HBV genotype C.
  • the polypeptides described herein can have one or more naturally occurring mutations in the pre-S1 region of HBV.
  • the polypeptides described herein can have from 1 to 30, such as from 1 to 20, from 1 to 10, from 1 to 8, and from 1 to 5, relative to sequences from the pre-S1 region of HBV. 1-3 (including all integers within these ranges) amino acid deletions, substitutions or insertions. In some embodiments, the polypeptide described herein can have from 1 to 30, such as from 1 to 20, from 1 to 10, relative to the amino acid sequence selected from any one of SEQ ID NOs: 23, 34-40, and 49. 1-8, 1-5 or 1-3 (including all integers within these ranges) amino acid deletions, substitutions or insertions.
  • the polypeptide described herein has from 1 to 30, such as from 1 to 20, from 1 to 10, from 1 to 8, from 1 to 5, or from 1 to 3, relative to the amino acid sequence of SEQ ID NO:23. Amino acid deletions, substitutions or insertions, including all integers within these ranges. In some embodiments, the polypeptide described herein has 1-3 amino acid deletions, substitutions or insertions relative to the amino acid sequence of SEQ ID NO:23. In certain embodiments, the polypeptides described herein have from 1 to 30, for example, 1 to 20, 1- at the C-terminus relative to an amino acid sequence selected from any one of SEQ ID NOs: 23, 34-40, and 49.
  • a polypeptide described herein can contain an amino acid sequence selected from any one of SEQ ID NOs: 21, 22, and 24-28.
  • the polypeptides described herein may contain the amino acid sequence of any of the polypeptides listed in Table 1.
  • the polypeptides described herein can be selected from any of the post-translationally modified polypeptides listed in Table 1.
  • the polypeptides described herein can have at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% with any of the polypeptides described herein. , 96%, 97%, 98% or 99% identity.
  • the polypeptide may comprise an amino acid sequence having at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, and any one of SEQ ID NOs: 21-40, 90%, 95%, 96%, 97%, 98% or 99% identity.
  • the polypeptide may comprise an amino acid sequence having at least about 30%, 40%, 50%, 60%, 70 of any of SEQ ID NOs: 23, 34-40, and 49.
  • the polypeptide may comprise an amino acid sequence having at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% with SEQ ID NO:23. , 95%, 96%, 97%, 98% or 99% identity.
  • a variant having a certain sequence identity to a polypeptide described herein retains one or more biological activities of the corresponding polypeptide.
  • aspects of the disclosure also include variants of the polypeptides described herein having a native flanking amino acid sequence of an L protein from HBV (eg, from the pre-S1 region of the L protein), wherein the flanking amino acid sequence is in the variant N and / or C end.
  • the natural flanking amino acid sequence refers to the native sequence of the N or C terminus of a polypeptide described herein flanking the pre-S1 region of the corresponding HBV genotype or any other HBV genotype.
  • the polypeptides described herein may comprise an amino acid sequence selected from any one of SEQ ID NOs: 23, 34-40, and 49, and a pre-S1 derived from any of the HBV genotypes AH.
  • the region is flanked by natural and amino acid sequences at the N and/or C terminus.
  • the natural flanking amino acid sequence can be derived from GenBank accession number 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 ( The consensus sequence of the HBV strain of genotype G; SEQ ID NO: 47) or AB179747 (genotype H; SEQ ID NO: 48).
  • a polypeptide described herein may comprise the amino acid sequence set forth in SEQ ID NO: 23 and, at its N and/or C terminus, a native flanking amino acid sequence from the pre-S1 region of HBV genotype C.
  • the polypeptide described herein may comprise the amino acid sequence set forth in SEQ ID NO: 23 and contain at its N and/or C terminus any of the HBV genotypes A, B, D, E, F, G and H. The natural flanking amino acid sequence of the pre-S1 region of the genotype.
  • the N and/or C terminus of the polypeptides described herein may independently comprise from 1 to 10, such as from 1 to 8, from 1 to 5, or from 1 to 3 in length (including all integers within these ranges) A natural flanking amino acid sequence of an amino acid.
  • a polypeptide described herein may comprise the amino acid sequence set forth in SEQ ID NO: 23 and, at its N-terminus, a natural amino acid sequence of 10 amino acids in length from the pre-S1 region of HBV genotype C.
  • the polypeptide may contain amino acids 2-59 (SEQ ID NO: 29) of the pre-S1 region of HBV genotype C.
  • the polypeptide described herein may comprise the amino acid sequence set forth in SEQ ID NO: 23 and, at its N-terminus, a natural amino acid of 9 amino acids in length from the pre-S1 region of HBV genotype E or G. sequence.
  • the polypeptide may contain amino acids 13-59 of the pre-S1 region of HBV genotype C and amino acids 2-11 (SEQ ID NO: 30) of the pre-S1 region of HBV genotype E or G.
  • any of the polypeptides described herein may have a natural flanking amino acid sequence of any length extending from its N and/or C terminus, and the resulting polypeptide retains one or more organisms of the original polypeptide. Learning activity.
  • polypeptides described herein can be prepared using chemical synthesis or recombinant techniques.
  • a synthetic gene can be constructed from scratch, or a natural gene can be mutated using, for example, cassette mutagenesis.
  • the polypeptides described herein can be prepared using recombinant DNA techniques. Briefly, these techniques involve obtaining a native or synthetic gene encoding the polypeptide, inserting it into a suitable vector, transferring the vector into a suitable host cell, culturing the host cell to express the gene, and recovering or isolating it. The peptide produced. In some embodiments, the recovered peptide is purified to a suitable purity.
  • a DNA sequence encoding a polypeptide described herein is cloned and manipulated such that it can be expressed in a suitable host.
  • the DNA encoding the parent polypeptide can be obtained from the cDNA of the mRNA of the cell expressing the polypeptide from the HBV genomic library, or the DNA sequence can be constructed synthetically to obtain the DNA encoding the parent polypeptide.
  • the parental DNA is then inserted into a suitable plasmid or vector for transformation of the host cell.
  • a species plasmid vector derived from a host cell is compatible and contains replication and control sequences for use in such hosts.
  • Vectors typically carry a replication site and a sequence encoding a protein or peptide that provides phenotypic selection for the transformed cell.
  • Vectors can be those commonly used in the art, or constructed using standard techniques and in conjunction with functional fragments of vectors commonly used in the art.
  • the host cell can be a prokaryotic cell or a eukaryotic cell.
  • prokaryotic host cells can include Escherichia coli, Bacillus subtilis, and other Enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, as well as various Pseudomonas species.
  • eukaryotes such as yeast cultures, or cells derived from multicellular organisms, such as insect or mammalian cell cultures, can 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.
  • the polypeptides described herein can be prepared using solid phase synthesis or equivalent chemical synthesis methods known in the art.
  • solid phase synthesis of the C-terminus of the polypeptide is initiated by coupling a pre-protected alpha-amino acid to a suitable resin.
  • the starting material can be prepared by attaching an alpha amino protected amino acid to a chloromethylated resin or a hydroxymethylated resin via an ester bond, or via an amide bond to a BHA resin or MBHA resin.
  • Amino acids are linked to the peptide chain using techniques well known in the art for forming peptide bonds.
  • One method involves converting an amino acid into a derivative that will provide a carboxyl group that is more readily reactive with the free N-terminal amino group of the peptide fragment.
  • amino acids can be converted to a mixture by reaction between a protected amino acid and ethyl chloroformate, phenyl chloroformate, t-butyl chloroformate, isobutyl chloroformate, pivaloyl chloride or a similar acid chloride.
  • Anhydride Anhydride.
  • the amino acid can be converted to an active ester such as 2,4,5-trichlorophenyl ester, pentachlorophenyl ester, pentafluorophenyl ester, p-nitrophenyl ester, N-hydroxysuccinimide ester, or by An ester formed by hydroxybenzotriazole.
  • an active ester such as 2,4,5-trichlorophenyl ester, pentachlorophenyl ester, pentafluorophenyl ester, p-nitrophenyl ester, N-hydroxysuccinimide ester, or by An ester formed by hydroxybenzotriazole.
  • Another coupling method involves the use of a suitable coupling agent such as N,N'-dicyclohexylcarbodiimide or N,N'-diisopropylcarbodiimide.
  • the alpha amino groups of each amino acid used in peptide synthesis are protected in a coupling reaction to prevent side reactions involving their active alpha amino function.
  • certain amino acids containing reactive side chain functional groups e.g., sulfhydryl, amino, carboxyl, and hydroxy
  • a suitable protecting group to prevent chemical reactions at the site during the initial and subsequent coupling steps.
  • Those skilled in the art are aware of how to select a suitable side chain protecting group. After obtaining the peptide of the desired amino acid, the protecting group can be easily removed under the reaction conditions which do not change the structure of the peptide chain.
  • the remaining alpha amino and side chain protected amino acids are coupled stepwise in the desired order.
  • some of the amino acids may be coupled to each other before being added to the solid phase synthesizer.
  • Each protected amino acid or amino acid sequence is added to the solid phase reactor in excess, a suitable coupling medium in dimethylformamide (DMF) or CH 2 Cl 2 or mixtures thereof. If the coupling is incomplete, the coupling process is repeated first in the presence of the N-amino protecting group prior to coupling the next amino acid. The success of the coupling reaction at each stage of the synthesis was monitored.
  • the method may be used as known BIOSEARCH 9500 TM automatic peptide synthesizer coupling reaction.
  • the protected peptide After obtaining the desired peptide sequence, the protected peptide must be cleaved from the resin support and all protecting groups must be removed. The cutting and removal of the protecting groups can be carried out simultaneously or sequentially.
  • the resin support is a chloromethylated polystyrene resin
  • the peptide is anchored to the resin.
  • the bond is a free carboxyl group of the C-terminal residue and one of a plurality of chloromethyl groups on the resin matrix is formed. Ester bond. It should be understood that the anchoring bond can be cut using an agent known to break the ester bond and penetrate the resin matrix.
  • polypeptide may be modified before or after cleavage of the polypeptide from the support, such as with hydrophobic groups including, for example, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, cholesterol, and Arachidonic acid) to modify the N-terminus, or to modify the C-terminus with amidation (amination), isopentylation or other modifications that stabilize the C-terminus.
  • hydrophobic groups including, for example, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, cholesterol, and Arachidonic acid
  • compositions comprising a polypeptide described herein.
  • the composition may contain any one or more of the polypeptides described herein.
  • the composition may also contain a suitable pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” means an inactive ingredient, such as a solid, semi-solid, or liquid filler, diluent, coating material, formulation, excipient or carrier, in combination with a therapeutic agent,
  • a "pharmaceutical composition” is administered to a subject.
  • the pharmaceutically acceptable carrier is non-toxic to the subject at the dosages and concentrations employed and is compatible with the other ingredients in the formulation.
  • a pharmaceutically acceptable carrier is suitable for the formulation employed.
  • the carrier can be a gel capsule. If the therapeutic agent is administered subcutaneously, it is desirable that the carrier is non-irritating to the skin and does not cause an injection site reaction.
  • a pharmaceutical composition of the polypeptide can be prepared by mixing a polypeptide described herein in a desired purity with one or more optional pharmaceutically acceptable carriers.
  • Pharmaceutically acceptable carriers can include, for example, buffering agents (such as phosphates, citrates, and other organic acids); antioxidants (such as ascorbic acid and methionine); preservatives (such as octadecylbenzyl dimethyl) Ammonium chloride, hexamethylene diammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl p-hydroxybenzoate such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate, Catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol); low molecular weight (eg less than about 10 residues) of the polypeptide; protein (eg serum albumin, gelatin or immunoglob
  • Exemplary pharmaceutical carriers may also include binders such as pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.; fillers such as lactose or other sugars, microcrystalline cellulose, pectin , gelatin, calcium sulfate, ethyl cellulose, polyacrylate or calcium hydrogen phosphate; lubricants such as magnesium stearate, talc, silica, colloidal silica, stearic acid, metal stearic acid Salt, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.; disintegrants such as starch, sodium starch glycolate, etc.; and wetting agents such as sodium lauryl sulfate.
  • binders such as pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.
  • fillers such as lactose or other sugars, microcrystalline cellulose, pectin
  • Exemplary pharmaceutically acceptable carriers may also include interstitial drug dispersing agents such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as the human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Baxter International, Inc.).
  • sHASEGP soluble neutral active hyaluronidase glycoprotein
  • the sHASEGP can be mixed into a pharmaceutical composition containing one or more other glycosammoglycanases such as chondroitinase.
  • compositions may also contain more than one active ingredient which is suitable for the particular indication being treated, and such active agents may be, for example, active agents of complementary activity without adverse effects. Such active agents may suitably be present in combination in amounts effective to meet the intended purpose.
  • the active agent can be encapsulated in microcapsules (such as hydroxymethylcellulose or gel microcapsules or polymethylmethacrylate microcapsules) prepared by, for example, coacervation techniques or interfacial polymerization, or wrapped In a colloidal drug delivery system (such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in a bulk emulsion.
  • microcapsules such as hydroxymethylcellulose or gel microcapsules or polymethylmethacrylate microcapsules
  • colloidal drug delivery system such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules
  • the pharmaceutical composition can include a sustained release formulation.
  • sustained release formulations include semipermeable matrices such as solid hydrophobic polymers containing the polypeptides described herein, wherein the matrices can be in the form of shaped articles, such as films or microcapsules.
  • the pharmaceutical composition can be used for in vivo administration and can be sterile. Sterility can be readily achieved by filtration, for example by sterile filtration.
  • the pharmaceutical composition can be prepared in any of a variety of possible dosage forms such as tablets, capsules, gel capsules, powders or granules.
  • the pharmaceutical compositions may also be prepared as solutions, suspensions, emulsions or mixed media.
  • the pharmaceutical composition can be prepared as a lyophilized formulation or an aqueous solution.
  • the pharmaceutical composition can be prepared as a solution.
  • a polypeptide described herein can be administered by adding it to an unbuffered solution, such as saline or water.
  • the polypeptide can also be administered by adding it to a suitable buffer solution.
  • the buffer solution can contain acetate, citrate, prolamin, carbonate or phosphate, or any combination thereof.
  • the buffer solution can be phosphate buffered saline (PBS). The pH and osmotic pressure of the buffer solution containing the polypeptide can be adjusted to a level suitable for administration to a subject.
  • PBS phosphate buffered saline
  • the pharmaceutical composition can be prepared as an aqueous suspension, a non-aqueous suspension, or a mixed substrate suspension.
  • Aqueous suspensions may also contain materials which increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the pharmaceutical composition can be prepared as an emulsion.
  • An exemplary emulsion includes a heterogeneous system in which a liquid is dispersed in another liquid in a droplet typically having a diameter in excess of 0.1 ⁇ m.
  • the emulsion may contain other ingredients in addition to the dispersed phase and the active drug which may be present in the aqueous phase solution, the oil phase solution or itself as a separate phase.
  • Microemulsions may also be included as an embodiment of the present disclosure.
  • the pharmaceutical composition can also be prepared as a liposomal formulation.
  • the pharmaceutical composition is prepared in a dosage form suitable for administration, such as an injection suitable for subcutaneous administration.
  • the pharmaceutical compositions contain an amount of a polypeptide described herein to achieve a therapeutically effective amount of the daily dose of one or more of the pharmaceutical compositions administered daily.
  • a therapeutically effective amount refers to the amount of polypeptide that achieves liver target organ saturation after administration of the therapeutically effective amount of the polypeptide for several days (>7 days).
  • 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 to 5.0 mg.
  • the polypeptide in the pharmaceutical composition is hepalatide, and the therapeutically effective amount is from 4.2 to 5.0 mg/day, more preferably 4.2 mg/day. In certain embodiments, the polypeptide in the pharmaceutical composition is Myrcludex B, and the therapeutically effective amount is from 4.2 to 5.0 mg/day, more preferably from 5.0 mg/day.
  • the pharmaceutical composition is administered several times a day to achieve a therapeutically effective amount.
  • the pharmaceutical compositions herein are formulated into a plurality of unit dosage forms, for example, containing up to 18.52 nmol per unit dosage form, or may contain 37.05 nmol, 55.57 nmol, 111.14 nmol, 129.66 nmol, 259.32 nmol or 388.98nmol.
  • the corresponding amount of unit dosage form can be administered daily such that the total amount administered on that day is within the therapeutically effective amount.
  • the unit dosage form may contain at least 0.1 mg of hepalatide, or may contain 0.2 mg, 0.3 mg, 0.6 mg, 0.7 mg, 1.4 mg, 2.1.
  • the specification of the mg or 4.2 mg of hepalatide, i.e., the pharmaceutical composition is 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.
  • a pharmaceutical composition of 42 unit dosage forms can be administered daily.
  • 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, a drug.
  • the composition has a specification of 0.1 mg, 0.25 mg, 0.5 mg, 1.0 mg, 2.5 mg or 5.0 mg.
  • a pharmaceutical composition of 50 unit dosage forms can be administered daily. Accordingly, the amount of the unit dosage form in the pharmaceutical composition of the present invention may range from 1 to 50, such as from 1 to 42 doses.
  • each unit dosage form contains 777.95-926.13 nmol or 4.2-5.0 mg of the polypeptide described herein.
  • the therapeutically effective amount can be achieved by administering 1 unit dosage form per day.
  • compositions herein may be in lyophilized form.
  • a lyophilized dose of a drug may contain from 37.05 to 926.13 nmol or from 0.1 to 5 mg of a polypeptide described herein, such as 259.32 to 926.13 nmol or 1.4 to 5 mg, 388.98 to 926.13 nmol or 2.1 to 5 mg, or 777.95 to 926.13 nmol or 4.2 to 5 mg of the polypeptide described herein.
  • it can be administered as an injection by dissolving in an appropriate amount of a solvent.
  • One or more doses of the injection may be administered such that the total daily dose is in the range of 777.95-926.13 nmol or 4.2-5 mg.
  • the lyophilized preparation may contain 2 bottles of the drug, each containing 388.98 nmol or 2.1 mg of the lyophilized polypeptide, which may be administered to the subject twice daily to achieve a total daily dose of 777.95 nmol or 4.2 mg.
  • kits containing the pharmaceutical compositions described herein may contain one or more doses of the pharmaceutical compositions described herein for administration over one or more days to prevent or treat hepatitis B virus-associated liver disease.
  • the kit contains at least 7 doses of the pharmaceutical composition, each dose containing 777.95-926.13 nmol or 4.2-5.0 mg of the polypeptide described herein for at least 7 consecutive days of administration. In certain embodiments, the kit satisfies 7 consecutive administrations.
  • the kit contains at least 7 doses of the pharmaceutical composition, each dose of the pharmaceutical composition comprising 4.2 to 5.0 mg, preferably 4.2 mg of hepalatide or 5.0 mg of Myrcludex B, for at least 7 consecutive days. medicine.
  • the kit contains at least two doses of the pharmaceutical composition, and the sum of the polypeptides contained in all of the pharmaceutical compositions in the kit amounts to 777.95-926.13 nmol or 4.2-5.0 mg as the total dose on the 1st.
  • each dose of the pharmaceutical composition may contain, 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.
  • the pharmaceutical composition contained in the kit may be at least 42, 21, 14, 7, 7, 6, or 2; or, each of the pharmaceutical compositions may contain 18.52 Nmol, 46.31nmol, 92.61nmol, 185.23nmol or 463.06nmol, or containing 0.1mg, 0.25mg, 0.5mg, 1.0mg or 2.5mg of polypeptide, the pharmaceutical composition containing at least 50, 20, 10 doses , 5 doses, 2 doses.
  • the kit contains two or more doses of the pharmaceutical composition, and the polypeptide in the pharmaceutical composition is hepalatide, and the content of hepalatide in each dose of the pharmaceutical composition is 0.1 mg, 0.2 mg.
  • the ratio of the content of the polypeptide contained in all the pharmaceutical compositions in the kit was 0.3 mg, 0.3 mg, 0.6 mg, 0.7 mg, 1.4 mg or 2.1 mg.
  • the kit contains two or more doses of the pharmaceutical composition, the polypeptide in the pharmaceutical composition is Myrcludex B, and the amount 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, the sum of the contents of the polypeptides contained in all the pharmaceutical compositions in the kit is 5.0 mg.
  • the kit contains multiple doses of the pharmaceutical composition that are administered for at least 7 consecutive days, and the sum of the amounts of the polypeptides contained in the plurality of doses of the pharmaceutical composition meets the daily dose of 777.95. -926.13 nmol or 4.2 to 5.0 mg.
  • 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, each of which meets the daily dose.
  • the kit contains at least 21 doses of the pharmaceutical composition, each of which meets the daily dose.
  • the pharmaceutical composition comprises a polypeptide that is hepalatide or Myrcludex B.
  • the drug is administered for 7 consecutive days.
  • compositions and kits described herein are used to administer a polypeptide of the subject described herein to achieve saturation of the polypeptide in its liver target organ after at least 7 consecutive days of administration.
  • Embodiments of the present disclosure include the therapeutic and prophylactic uses of the polypeptides described herein.
  • the use of a polypeptide described herein as a drug raft is provided.
  • the use of a polypeptide described herein to treat and prevent HBV-associated liver disease is provided.
  • Hepatitis B virus-associated liver diseases described herein include 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.
  • the hepatitis B-related liver transplantation includes protection of HBV infection of the donor liver before, during, and after transplantation.
  • atient and “subject” are used interchangeably to refer to an animal (eg, a mammal or a human) to be treated or assessed for a disease, disorder, or condition, or to assess whether there is a risk of developing a disease, disorder, or condition, or An animal (such as a mammal) or human suffering from a disease, disorder, or condition.
  • these diseases, disorders, or conditions can include metabolic diseases.
  • the metabolic disease can be involved in a disorder of lipid metabolism.
  • a polypeptide of the invention described herein, or a pharmaceutical composition thereof, can be administered to a subject in need thereof for the prophylaxis or treatment of an HBV-associated liver disease.
  • "sufficient dose” refers to a daily dose of a polypeptide that is administered daily at a dose that achieves hepatic target organ saturation after several consecutive days (>7 days).
  • the "sufficient dose” is typically 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 of the day.
  • the target organ is not saturated when the amount of the polypeptide is administered for the first time, but the target organ is saturated after 7 consecutive administrations.
  • the prophylactic or therapeutic methods described herein comprise the step of administering a sufficient dose of the polypeptide or a pharmaceutical composition thereof for at least 7 days in succession.
  • Target organ saturation as described herein refers to an apparent distribution volume of less than 100 L after administration of a certain amount of the polypeptide.
  • the apparent volume of distribution is typically from 10 to 100 L, such as from 10 to 50 L or from 20 to 40 L.
  • the term "treating" includes treating a subject (eg, a mammal, such as a human) or a cell to alter the current progression of the subject or cell.
  • Treatment includes, for example, administration of a polypeptide described herein or a pharmaceutical composition comprising the polypeptide, the treatment being carried out in a prophylactic manner, or may be initiated after a pathological event has occurred or has been contacted with the infectious agent.
  • Treatment also includes “prophylactic” treatment, which refers to reducing the rate of progression of the disease or condition to be treated, delaying the onset of the disease or condition, or reducing the severity of the onset.
  • Treatment does not necessarily mean the complete eradication, cure, or prevention of the occurrence of a disease or condition or related condition.
  • the term “treating” can include alleviating, alleviating or reversing the pathological course of an HBV-associated liver disease.
  • the term “treating” can include ameliorating at least one symptom or at least one measurable parameter of a metabolic disease. It will be apparent to those skilled in the art that biological and/or physiological parameters can be used to assess the pathological course of a metabolic disease. These pathological processes or symptoms may include one or more metabolically related chemical or biological molecules, such as ALT, HBV DNA, or an excess or increase in one or more measured metabolic changes, as compared to a healthy subject. Physiological parameters such as liver function index.
  • administering includes administering a polypeptide described herein as a topical or systemic administration.
  • Administration may be topical (including mucosal administration of the eye or vagina and rectum), lung (eg by inhalation or insufflation by powder or aerosol, including by nebulizer, intratracheal, intranasal administration), epidermis , transdermal, oral or parenteral.
  • Parenteral administration includes intravenous, subcutaneous, intraperitoneal or intramuscular injection or infusion, or intracranial, such as intrathecal or intraventricular administration.
  • the present disclosure also provides methods of practicing the above-described uses of the polypeptides described herein in a subject. These methods can comprise administering to the subject a therapeutically or prophylactically effective amount of a polypeptide described herein or a pharmaceutical composition comprising the polypeptide.
  • the subject can be a mammal.
  • the subject can be a human.
  • the subject can have or be at risk of developing an HBV-associated liver disease.
  • the combination therapies described herein can include co-administration (wherein the two or more therapeutic agents can be the same formulation or separate formulations), as well as separate administrations, in which case the polypeptides described herein can be administered in other treatments.
  • the agent is administered before, at the same time or after.
  • These drugs include interferons, nucleotides, hepatitis B immunoglobulin, and hepatitis B vaccine.
  • interferon includes common interferon and PEG long-acting interferon.
  • Common interferons include interferon alpha-2a, interferon alpha-2b, and PEG long acting interferons include PEG interferon alpha-2a, PEG interferon alpha-2b.
  • Nucleotide drugs include lamivudine LAM, entecavir ETV, tenofovir disoproxil TDF, telbivudine TBV, adefovir dipivoxil ADV, and tenofovir alafenamide TAF.
  • polypeptides described herein may be administered in a suitable manner; they may also be administered topically or intrathecally.
  • the polypeptides described herein can be administered parenterally.
  • Parenteral administration can include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.
  • the polypeptides described herein can be administered subcutaneously.
  • the polypeptides described herein can be administered intravenously.
  • Administration can be by any suitable means, such as by injection or infusion, such as intravenous or subcutaneous injection or infusion, depending in part on whether the administration is short-lived or chronic.
  • Various dosage regimens are also contemplated, including single or multiple administrations, such as at various time points, high dose administration, and pulsed injection.
  • the polypeptides described herein or pharmaceutical compositions thereof for use in the methods of treating and preventing hepatitis B virus-associated liver diseases described herein are also disclosed herein. More specifically, the polypeptide or a pharmaceutical composition thereof is used in the therapeutic and prophylactic method at a daily dose (in terms of polypeptide) of 777.95 to 926.13 nmol or 4.2 to 5.0 mg for several days to achieve liver
  • the target organ is saturated, preferably for at least 7 consecutive days.
  • the use herein includes a hepalatide or a pharmaceutical composition thereof for use in a method of treating or preventing hepatitis B virus-associated liver disease, wherein the method comprises 777.95-926.13 nmol per day.
  • the target amount of heptapride preferably 926.13 nmol or 5.0 mg
  • the pharmaceutical composition can be as described in any of the embodiments herein.
  • the polypeptide can be synthesized by the method of Example 1.1 of PCT/CN2017/086558. Specifically, the polypeptides shown in Table 1 were synthesized according to the standard Fmoc protocol for polypeptide synthesis. Typically, starting from the MBHA resin, a single amino acid residue extends from the carboxy terminus to the amino terminus. The N-terminus is then subjected to myristoylation. After completion of the peptide synthesis, the polypeptide is cleaved from the resin using a cleavage solution, and the C-terminus of the polypeptide is further aminated. The resin was filtered off through a G6 sand funnel and the filtrate containing the peptide was evaporated in vacuo.
  • the explorer 100 high pressure liquid chromatograph was used for purification, and the main peak was collected step by step.
  • the samples collected from the target peaks were analyzed for purity using an Agilent Model 1100 reverse phase high pressure liquid chromatography (HPLC) equipped with a C18 column, and their molecular weights were identified by mass spectrometry.
  • HPLC reverse phase high pressure liquid chromatography
  • the collected solution was purified by medium pressure liquid chromatography for lyophilization.
  • the dried sample was dissolved in PBS and filtered through a 0.20 ⁇ M membrane.
  • the peptide stock solution dissolved in PBS was stored at -80 ° C until use.
  • the amino acid sequence of hepalatide is represented by SEQ ID NO: 23, which is modified by N-terminal myristic acid and modified by C-terminal amination, and has a molecular weight of 5398.8 Da and a molecular formula of C 247 H 352 N 65 O 73 .
  • PBS control group 50 adult male tree shrews were randomly divided into 5 groups: PBS control group, hepalatide high (2 mg/kg), medium (0.4 mg/kg), low (0.08 mg/kg) dose group and hepatitis B immunoglobulin.
  • Protein HBIG blocking group 60 IU/kg. 1 ml of HBV virus serum was intraperitoneally injected with 108 HBV DNA/ml titer to infect tree shrews, and the hepalatide blocking group was given different doses on days 0, 1, 2, 3, 5, 7, 9, 11, and 13 after infection. Palladium peptide is injected subcutaneously. The HBIG blocking group was given intramuscular injection of immunoglobulin HBIG on the day of infection and on the third day after infection.
  • Tree sputum serum was collected 4 days before infection and on days 9, 14, 21 and 42 after infection to detect HBsAg, HBeAg, HBV DNA titer and alanine aminotransferase (ALT) in serum; liver tissue was taken 21 days after infection. Pathological examination.
  • HBsAg peaked on the 9th day after infection in the PBS control group, and HBsAg was substantially cleared after the 15th day.
  • Hepalatide high, medium and low dose groups
  • HBIG could not effectively block the HBV infection of this infection.
  • HBsAg was not significantly decreased compared with the PBS control group on the 9th day after infection, and there was a tendency to prolong the secretion of HBsAg.
  • the HBsAg level was higher than that of the PBS control group.
  • the level of HBsAg in the peaks of hepalatide (high, medium and low doses) was lower than that of the PBS control group and the HBIG blocking group, and the hepatic (high, medium and low dose) groups had HBsAg on the 9th day.
  • the water level was significantly lower than that of the HBIG blocking group (P less than 0.05).
  • HBeAg peaked on the 9th day after infection in the PBS control group, and HBeAg disappeared after the 21st day.
  • Hepalatide can effectively block HBV infection, and high-dose and medium-dose of hepalatide can control HBsAg during infection.
  • HBIG could not effectively block the HBV infection of this infectious dose.
  • the HBsAg was equivalent to the PBS control group on the 9th and 15th day after infection.
  • Heparin (high, medium and low doses) had lower levels of HBeAg on day 9 than PBS control group and HBIG block group, and hepalatide (high, medium and low dose) group 9 day HBsAg
  • the water level was significantly lower than that of the PBS control group (P less than 0.05).
  • the HBV DNA peaked on the 9th day after infection in the PBS control group, and HBV DNA was cleared after the 15th day. Hepalatide can effectively block HBV infection, and high-dose and medium-dose of hepalatide can control HBV DNA during infection. However, HBIG could not effectively block the HBV infection of this infection dose, and the HBV DNA was further increased on the 15th day after infection than the PBS control group, and it was not cleared until 21 days after infection.
  • the level of HBV DNA in the peaks of hepalatide (high, medium and low doses) was lower than that of the PBS control group and the HBIG blocking group.
  • the HBV DNA level of the high dose group of hepalatide was significantly lower than that of the HBIG block group. (P is less than 0.05).
  • ALT before infection was 56.8 IU/ml, which was set to be the normal level of tree ALT.
  • ALT showed a peak on the 21st day after infection in the PBS control group, and ALT returned to normal level after the 42nd day.
  • Hepalatide can effectively block HBV infection, and high-dose and medium-dose hepalatide can control ALT during infection.
  • HBIG could not effectively block the HBV infection of this infection dose.
  • the ALT was still higher than the normal value on the 21st day after infection, and returned to normal after 42 days of infection.
  • the ALT level of hepalatide (high, medium and low doses) on the 21st day was lower than that of the PBS control group and the HBIG block group.
  • the ALT level in the high and middle dose groups of hepalatide was significantly lower than that in the HBIG block group. (P is less than 0.05).
  • HBV infection caused obvious pathological changes in the liver tissue of the tree shrew, mainly due to hepatocyte turbidity and balloon-like changes in the portal area around the hepatic lobules, accompanied by a small amount of lymphocyte infiltration, and its pathological changes and hepatitis caused by acute HBV infection in adults. Similar. However, HBIG could not alleviate the pathological changes caused by HBV infection in the liver tissue of tree shrews.
  • liver pathological changes of the low dose group of hepalatide were similar to those of the PBS group, and the degree of lesions was slightly reduced.
  • High-dose and medium-dose hepalatide completely blocked the pathological damage caused by HBV on hepatocytes, and the liver tissue was basically consistent with the liver of normal tree shrew liver.
  • hepalatide can effectively block HBV infection in serology (HBsAg, HBeAg), virology (serum HBV DNA copy number), enzymology (algae aminotransferase ALT) and pathological levels.
  • Non-hepatocyte derived 293 cells were transfected with NTCP to form an NTCP-293 cell line.
  • FITC-labeled heptapline (Cmyr-47) 37th amino acid or irrelevant control peptide (47 peptide derived from heron hepatitis B virus heron HBV Pre-S1 region, myristoylation-GLNQSTFNPLGFFPSHQLDPLFKANAGSADWDKNPNKDPWP QAHDTA-amidation, SEQ ID NO: 50).
  • FITC-hepalatide binds to NTCP-293 cells but does not bind to 293 cells (BLANK-293) that have not been transfected with NTCP, indicating that the binding of hepalatide to cells is NTCP-specific. .
  • the FITC-labeled irrelevant control peptide did not bind to NTCP-293 cells, indicating that the binding has hepalatide sequence specificity.
  • Example 5 Effect of polypeptide and NTCP bile acid transport function
  • hepalatide The effect of hepalatide on the transport function of NTCP bile acid was studied in the reference method of NTCP bile acid transport function (Kim RB et al. J Pharmacol Exp Ther. 1999 Dec; 291(3): 1204-9).
  • hepalatide was administered to rats in a single subcutaneous injection at a dose of 40 ug ⁇ kg -1 to detect the distribution of hepalatide in each tissue.
  • the results of the study (Figure 3) show that hepalatide is mainly distributed in the liver and urinary system, in which the urinary system is dominated by metabolites. The drug reached a peak in the liver for 2 hours, and the Cmax was 44.3 ⁇ 28.9 ng-Equ.mL -1 .
  • the half-life of the end of the liver was reduced to 8.7 h at the end of the liver, and there was a higher concentration in the liver at 24 h ( 4.5 ⁇ 1.2 ng-Equ.mL -1 ), the drug concentration in the liver for 48 h was estimated to be about 0.66 ng-Equ.mL -1 , which is equivalent to the serum drug concentration level 4 hours after subcutaneous injection.
  • the serum was cleared rapidly and was substantially reduced to baseline levels 6 hours after administration.
  • Preclinical pharmacokinetic results show that hepalatide has significant liver target organ pooling.
  • Example 7 Phase Ia clinical trial and clinical pharmacokinetic study of single-dose climb of polypeptide
  • the single-dose climb phase Ia clinical trial of hepalatide was a randomized, double-blind, blank, single-center, dose-climbing trial with six dose groups (0.525 mg, 2.1 mg, 4.2 mg, 6.3mg, 8.4mg and 10.5mg), the plan was to enroll 5 patients, 5 patients, 10 patients, 10 patients, 10 patients and 5 healthy subjects, the ratio of male to female 22:23. After screening, healthy volunteers were randomly selected into the experimental group and the blank control group in a 4:1 ratio, and received a single subcutaneous injection of hepalatide. After stopping the drug for 7 days. The dose of hepalatide in the test and the degree of exposure are shown in Table 2.
  • test population 45 adult healthy subjects were enrolled, and 45 subjects were actually enrolled. All of the trials were completed and included in the safety analysis population.
  • the safety analysis population of Phase Ia clinical studies is shown in Table 3. There were no significant differences in demographic indicators such as age, gender, ethnicity, occupation, height, weight, BMI, and past history.
  • Phase Ia clinical pharmacokinetic study Clinical pharmacokinetic study of subcutaneous single dose dose Ia was performed in 45 healthy subjects. The dose setting and PK analysis population are shown in Table 5. Blood samples were taken before administration and 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, 12 h and 24 h after administration. The biological sample was tested by liquid chromatography-mass spectrometry, and the lower limit of quantification was 0.2 ng/ml. The blood drug concentration curve is shown in Figure 4. The main parameters of blood pharmacokinetics are shown in Table 4.
  • the results of the study showed that after a single subcutaneous injection of hepalatide, the subject received a single subcutaneous injection of hepalatide in a dose range of 0.525 mg-4.2 mg, and the blood curve was low, while 6.3 mg-10.5 mg.
  • the blood drug curve was significantly high in the dose range.
  • the apparent distribution volume Vz/F is greater than 100 L in the dose range of 0.21-4.2 mg, suggesting that the drug is mainly distributed in the target organ.
  • hepalatide specifically binds to NTCP (Example 4), and preclinical pharmacokinetic studies show significant hepatic aggregation of hepalatide (Example 6), liver specificity of drug target NTCP Expression, therefore, it is determined that the drug is mainly collected in the liver of the target organ.
  • Vz/F is close to the whole body fluid volume of the human body, and shrinks with the increase of dose, suggesting that the drug is distributed to body fluid after the target organ is saturated. Therefore, a single dose of 0.525 mg-4.2 mg and 6.3 mg-10.5 mg was divided into a target organ liver unsaturation dose range and a saturation dose range, respectively.
  • the apparent volume of distribution of Myrcludex B after subcutaneous administration of 5 mg in a single dose was 135 L (lank A et al. J Hepatol. 2016 Sep; 65(3): 483-9). According to the results of this example, Myrcludex B was singled at this dose. Subcutaneous administration did not reach saturation of the target organ of the liver.
  • Example 8 Clinical trial and clinical pharmacokinetic study of multi-dose climb Ib of peptide
  • Hepalapide multi-dose climb Ib clinical trial was a randomized, double-blind, blank, single-center clinical study. Three dose groups (4.20 mg, 6.30 mg, 8.40 mg) were set up in the trial. The plan was to enroll 10 patients, 10 patients, and 15 healthy subjects, with a male to female ratio of 18:17. After screening, healthy volunteers were randomly selected into the experimental group and the blank control group in a 4:1 ratio, and received once a day for 7 consecutive days. Observe for 3 days after stopping the drug. The levels of hepalatide administration and exposure in the test are shown in Table 6.
  • the heparin Ib clinical study plan enrolled 35 adult healthy subjects, and actually enrolled 35 subjects, all of which were completed and included in the safety analysis population.
  • the safety analysis population of the Phase Ib clinical study, as well as the demographic indicators of each trial group, are shown in Table 7. There were no significant differences in demographic indicators such as age, gender, ethnicity, occupation, height, weight, BMI, and past history.
  • Table 7 Basic situation of demographics in Phase Ib clinical trials
  • Table 8 Distribution of adverse events in stage Ib (certainly relevant, likely relevant, possibly relevant) in each trial group
  • Clinical pharmacokinetic study Subcutaneous multiple dose-drafting Phase Ib clinical pharmacokinetic study was performed in 35 healthy subjects. Subjects received one daily 7-day dosing, dose setting and The PK analysis population is shown in Table 9. Blood samples were collected as follows: blood samples before 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, 12 h before administration; Day 2: 24 h blood samples of Day 1 (before Day 2 administration) Day3 ⁇ 6: before administration; Day7: blood samples before administration and 2min, 5min, 15min, 30min, 45min, 1h, 1.5h, 2h, 3h, 4h, 6h, 8h, 12h after administration.
  • the biological sample was tested by liquid chromatography-mass spectrometry, and the lower limit of quantification was 0.2 ng/ml.
  • the blood dose-time curve of the first dose, 2-7 days before and after the dose is shown in Figure 5-7, and the main pharmacokinetic parameters are shown in Table 10.
  • the results showed that the blood drug curve of each dose group was consistent with stage Ia after the first dose, the first dose of 4.2 mg did not reach the target organ saturation, and the first dose of 6.3 and 8.4 mg reached the target organ saturation.
  • the concentration of the drug valley reached steady state during the continuous administration on days 2-6.
  • the accumulation coefficients R AUC of the 4.2 mg, 6.3 mg, and 8.4 mg dose groups were 4.43 ⁇ 1.60, 2.76 ⁇ 0.76, and 1.91 ⁇ 0.39, respectively, indicating that there was accumulation during the continuous administration of hepalatide.
  • the apparent volume of 4.2 mg decreased from 153.6025 ⁇ 107.1807 liters at the first dose to 22.1880 ⁇ 8.0721 liters at the end dose, indicating that the 4.2 mg dose reached the target organ saturation after continuous administration.
  • the apparent volume of distribution was 15.1021 ⁇ 4.4743 liters, which was close to the space of 14 liters of extravascular fluid in the body, indicating that the drug outside the target organ was mainly distributed in the extracellular fluid.
  • Clinical pharmacodynamic study In the multi-dose climb Ib clinical trial, subjects received a daily subcutaneous injection of hepalatide for 7 consecutive days in the dose range of 4.2-8.4 mg, which effectively increased blood total bile acid during the administration period. Level, blood total bile acid returned to normal after stopping the drug. Total bile acid can be used as an ideal bioactive biomarker. The results of the study showed that hepalatide 4.2 mg daily, subcutaneous injection for 7 consecutive days, can achieve a sufficient dose to achieve pharmacodynamic response.
  • Example 9 Summary of clinical pharmacokinetics of phase I clinical trials of peptides
  • Hepalatide was mainly distributed in the target organ after a single subcutaneous injection in the dose range of 0.525-4.2 mg; the target organ was saturated after a single subcutaneous injection in the dose range of 6.3-10.5 mg, and the drug was distributed to the plasma.
  • the daily dose of 4.2 mg is the lowest target organ saturation dose for continuous administration of hepalatide. Based on the results of the present disclosure, it was judged that Myrcludex B did not reach the target organ saturation in a single subcutaneous administration at a dose of 5 mg, and was administered continuously at this dose, and the liver target organ saturation was also achieved after accumulation.
  • Subjects were well tolerated with a single subcutaneous injection of hepalatide in the 0.525-10.5 mg dose range, with no dose-limiting toxicity and no maximum tolerated dose. Subjects were well tolerated for 7 consecutive days of subcutaneous injection of hepalatide in the daily dose range of 4.2-8.4 mg without dose-limiting toxicity and not reaching the maximum tolerated dose.
  • the dose is determined to be a clinically recommended dose.

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Abstract

充分剂量条件下治疗和预防乙型肝炎病毒相关肝病的药物和方法。具体而言，提供一种治疗和预防乙型肝炎病毒相关肝病的方法，所述方法包括每日给予需治疗和预防对象以充分剂量的多肽或其药物组合物，其中，所述多肽含有衍生自乙肝病毒(HBV)Pre-S1的氨基酸序列；所述充分剂量为给予所述多肽数日后所述多肽在肝脏靶器官中达到饱和的日剂量。

Description

充分剂量条件下治疗乙肝病毒相关肝病的药物和方法 技术领域
本公开涉及充分剂量条件下治疗乙肝病毒相关肝病的药物和方法。
背景技术
全世界每年约有100万人死于乙型肝炎病毒(HBV)感染所致的肝衰竭、肝硬化和原发性肝细胞癌。目前估计我国现存慢性乙肝患者2000万人，每年新发病例100万例，国每年乙肝导致肝硬化发生500万例，每年死亡100万例。每年乙肝相关性肝癌发生30万例，死亡30万例。
HBV病毒包膜蛋白含有三个表面抗原蛋白：大表面抗原蛋白(L)、中表面抗原蛋白(M)、和小表面抗原蛋白(S)。这些蛋白由S基因上的单一的开放读框编码，起始于三个不同的翻译起始位点，即L(Pre-S1+Pre-S2+S)，M(Pre-S2+S)和S(S)。HBV分为9种基因型(A-I)，各基因型在Pre-S1区氨基酸序列存在一定差异。研究证实，HBV通过病毒表面大蛋白(large HBsAg)与肝细胞表面的钠离子-牛磺胆酸共转运蛋白(sodium taurocholate cotransporting polypeptide,NTCP)结合，介导对肝细胞的感染(Yan等，eLife,1：e00049(2012))。含有HBV Pre-S1氨基酸序列的衍生多肽可在体外(Gripon P等.J.Virol.2005；79(3):1613–1622)和动物模型体内阻断HBV对肝细胞的感染(Petersen J等.Nat Biotechnol.2008Mar；26(3):335-41.)。此外，NTCP承担胆汁酸自门静脉血向肝内转运的生理功能，是胆汁酸肠肝循环的重要蛋白之一(Alrefai W等.Pharmaceutical Research,2007；24(10):1803-1823)。
NTCP特异性表达于肝脏，其他组织鲜有表达，HBV Pre-S1来源的多肽在动物体内显示明显的肝脏汇集性(Schieck A等.Hepatology.2013Jul；58(1):43-53.)。来源于HBV Pre-S1的多肽Myrcludex B，已完成人体单剂量爬升Ia期临床研究和药代动力学研究(lank A等.J Hepatol.2016Sep；65(3):483-9.)。该研究设置12个剂量组，分别给予单次2h持续静脉注射0.3ug、3ug、10ug、100ug、800ug、3mg、5mg、10mg、20mg和单次皮下注射800ug、5mg、10mg剂量。研究共入组36例健康受试者，被分配进入上述12个剂量组，每组3人。36例受试者全部完成临床试验。由于持续静脉给 药方式对药代参数的影响，静脉给药药代参数难以分析。皮下给药表观分布容积参数V分析，800ug和5mg皮下单次给药后表观分布容积均大于100L，远大于体重，显示多肽分布于靶器官；当剂量提高至10mg时，血药浓度高耸，表观分布容积43L。由于该试验皮下给药剂量设置少，缺乏参照和对比，未能获得多肽在肝脏靶器官饱和情况的结论。在Myrcludex B以10mg剂量连续6天皮下给药临床药代动力学研究中，连续6天给药后蓄积系数R AUC为1.79，具有蓄积作用；但未报告表观分布容积参数(Blank A等.Clin Pharmacol Ther.2017 May 24.doi:10.1002/cpt.744.)。Myrcludex B以每天2mg剂量联合干扰素治疗丁肝患者的临床试验正在进行，其剂量选择依据该多肽在猩猩体内的药代研究结果，但临床研究结果在HBsAg清除的主要研究终点指标上未显示治疗效果(Bogomolov P等.J Hepatol.2016Sep；65(3):490-8.doi:10.1016/j.jhep.2016.04.016.Epub 2016Apr 27)。HBV Pre-S1衍生肽治疗HBV相关肝病的充分剂量尚未确定。
发明内容
本公开提供一种治疗和预防乙肝病毒相关肝病的方法，所述方法包括每日给予需治疗和预防对象以充分剂量的多肽或其药物组合物，其中，所述多肽含有衍生自乙肝病毒(HBV)Pre-S1的氨基酸序列；所述充分剂量给予所述多肽数日后所述多肽在肝脏靶器官中达到饱和的日剂量。
在某些实施方案中，所述充分剂量为777.95-926.13nmol的日剂量，优选为777.95nmol或926.13nmol的日剂量。
在某些实施方案中，所述充分剂量为4.2～5.0mg的日剂量，更优选为4.2mg或5.0mg的日剂量。
在某些实施方案中，所述多肽含有HBV的pre-S1区域的氨基酸序列。
在某些实施方案中，所述多肽的N末端含有疏水基团修饰基团。
在某些实施方案中，所述疏水基团选自：肉豆蔻酸、棕榈酸、硬脂酸、油酸、亚油酸、胆固醇和花生四烯酸。
在某些实施方案中，所述疏水基团是肉豆蔻酸。
在某些实施方案中，所述多肽具有C末端修饰或无修饰。
在某些实施方案中，所述C末端修饰是酰胺化或异戊二醇化。
在某些实施方案中，所述多肽含有HBV基因型A、B、C、D、E、F、G、H或I的pre-S1区域的氨基酸序列。
在某些实施方案中，所述多肽含有HBV基因型C的pre-S1区域氨基酸序列13-59的序列，或含有来自HBV基因型A、B、D、E、F、G、H或I的pre-S1区域中与HBV基因型C的pre-S1区域氨基酸序列13-59相对应的序列。
在某些实施方案中，所述多肽的一个或多个氨基酸残基缺失、被取代或插入；优选地，所述多肽的1－30、1－20、1－10、1－8、1－5或1－3个氨基酸残基缺失、被取代或插入。
在某些实施方案中，所述多肽在其N末端和/或C末端含有来自HBV的pre-S1区域的天然侧接氨基酸序列；优选地，所述HBV的pre-S1区域的天然侧接氨基酸序列长1－10、1－8、1－5或1－3个氨基酸。
在某些实施方案中，所述多肽含有对应于HBV基因型C的pre-S1区域的氨基酸13的甘氨酸，和/或对应于HBV基因型C的pre-S1区域的氨基酸20的天冬酰胺或氨基酸57的赖氨酸。
在某些实施方案中，所述多肽：
(1)含有选自SEQ ID NO：21－40或49中任一所示的氨基酸序列；优选地，所述多肽含有SEQ ID NO：23所示的氨基酸序列；或
(2)与SEQ ID NO：21－40或49任一所示的氨基酸序列具有至少约30％、40％、50％、60％、70％、80％、85％、90％、95％、96％、97％、98％或99％的相同性。
在某些实施方案中，所述多肽如SEQ ID NO:1－20或51任一所示。
在某些实施方案中，所述多肽含有SEQ ID NO：23或49，其中所述多肽还含有N末端肉豆蔻酸修饰和C末端胺化修饰，或其中所述多肽含有SEQ ID NO：3或51。
在某些实施方案中，所述乙肝病毒相关肝病包括慢性乙肝病毒感染、慢性乙型肝炎、乙肝相关肝纤维化和肝硬化、乙肝相关肝癌、乙肝相关肝移植和乙肝相关母婴传播。
在某些实施方案中，所述慢性乙型肝炎包括HBeAg阳性慢性乙型肝炎和HBeAg阴性慢性乙型肝炎。
在某些实施方案中，所述乙肝相关肝移植包括移植术前、术中和术后对供肝的HBV感染的保护。
在某些实施方案中，所述多肽含有SEQ ID NO：23所示的氨基酸序列，其N末端被肉豆蔻酸修饰和C末端胺化修饰；所述充分剂量为4.2-5.0mg的日剂量，优选4.2mg的日剂量，所述方法包括至少连续给药7天。
在某些实施方案中，所述多肽含有SEQ ID NO：49所示的氨基酸序列，其N末端被肉豆蔻酸修饰和C末端胺化修饰；所述充分剂量为4.2-5.0mg的日剂量，优选5.0mg的日剂量，所述方法包括至少连续给药7天。
在某些实施方案中，所述多肽为含有SEQ ID NO：23所示的氨基酸序列，其N末端被肉豆蔻酸修饰和C末端胺化修饰，所述方法包括每日给予所述对象N剂的所述药物组合物，实现4.2mg的日剂量，其中，N为1～42中的任一整数。
在某些实施方案中，所述多肽为含有SEQ ID NO：49所示的氨基酸序列，其N末端被肉豆蔻酸修饰和C末端胺化修饰，所述方法包括每日给予所述对象N剂的所述药物组合物，实现5.0mg的日剂量，其中，N为1～50中的任一整数。
本公开还提供一种药物组合物，所述多肽含有衍生自乙肝病毒(HBV)Pre-S1的氨基酸序列；所述药物组合物为含有一定量所述多肽的药物组合物，通过给予整数个制剂规格的该药物组合物，实现所述多肽在肝脏靶器官中达到饱和。
在某些实施方案中，所述药物组合物含有治疗有效量的多肽，其中，所述治疗有效量为每日给予1剂或几剂所述药物组合物后所述多肽的日总剂量达777.95-926.13nmol。
在某些实施方案中，所述治疗有效量为每日给予1剂或几剂所述药物组合物后所述多肽的日总剂量达4.2～5.0mg，优选日总剂量为4.2mg或5.0mg。
在某些实施方案中，每剂所述药物组合物含有4.2mg、2.1mg、1.4mg、0.7mg、0.6mg、0.3mg、0.2mg或0.1mg的所述多肽，即所述药物组合物规格为4.2mg、2.1mg、1.4mg、0.7mg、0.6mg、0.3mg、0.2mg或0.1mg；或每剂所述药物组合物含有5.0mg、2.5mg、1.0mg、0.5mg、0.25mg或0.1mg的所述多肽，即所述药物组合物规格为5.0mg、2.5mg、1.0mg、0.5mg、0.25mg或0.1mg。
在某些实施方案中，所述多肽如本公开任一实施方案所述。
本公开还提供一种药盒，所述药盒含有1剂或多剂含有多肽作为活性成分的药物，用于患者服用1日或多日，其中，所述1剂或多剂药物所含的多肽的量使得每日给予1剂或几剂所述药物后日给药量达777.95-833.52nmol的所述多肽，其中，所述多肽含有衍生自乙肝病毒(HBV)Pre-S1的氨基酸序列。
在某些实施方案中，所述1剂或多剂药物所含的多肽的量使得每日给予1剂或几剂所述药物后日给药量达4.2～5.0mg优选4.2mg或5.0mg。
在某些实施方案中，所述多肽如本公开任一实施方案所述。
本公开还提供本文所述的药物组合物在制备用于治疗或预防乙肝病毒相关肝病的药品中的应用。
还公开的是用于治疗和预防乙肝病毒相关肝病的本文所述的多肽或其药物组合物。
在某些实施方案中，所述乙肝病毒相关肝病的治疗和预防方法为本文任一实施方案所述的方法。
附图说明
图1：FIFC标记的贺普拉肽结合表达NTCP的HEK293细胞(NTCP-293)，不结合作为对照的HEK293细胞(BLANK-293)。FIFC标记的衍生自苍鹭HBV的多肽用作对照多肽。
图2：Cmyr-47对体外胆汁酸摄取的作用。环孢霉素A(CsA)用作阳性对照。A、TA进入细胞是NTCP特异性介导的，阳性对照环孢素A对NTCP发挥显著抑制作用；B、贺普拉肽对NTCP胆汁酸转运功能具有双向性影响。
图3： 125I-贺普拉肽皮下注射后沉淀放射性时间曲线。
图4：Ia期PK研究平均血药浓度-时间曲线(平均值+SD)。
图5：Ib期PK研究首剂量平均血药浓度-时间曲线(平均值+SD)。
图6：Ib期PK研究第2-7天给药前平均血药浓度-时间曲线(平均值+SD)。
图7：Ib期PK研究末剂量平均血药浓度-时间曲线(平均值+SD)。
具体实施方式
除非另有说明，否则本文所用的所有技术和科学术语与本公开所属技术领域的普通技术人员通常所理解的含义一致。出于本公开的目的，下述术语如下文所定义。
冠词“一”指一个或一个以上(即至少一个)的该冠词的语法宾语。例如，“一元件”指一个元件或一个以上的元件。
除非文中另有清楚说明，否则术语“或者”与术语“和/或”可互换使用。
就术语“含有”、“包括”、“具有”或其语法变体在本公开或权利要求书中使用的范畴而言，这些术语可以与术语“包含”于权利要求中被用作过渡词时所解释的相类似的方式使用。术语“包括”或其语法变体指“包括但不限于”，且可与“包括但不限于”互换使用。
术语“约”指一定量、水平、值、数值、频率、百分比、尺寸、大小、数量、重量或长度，其相较于参比的量、水平、值、数值、频率、百分比、尺寸、大小、数量、重量或长度具有差不多30％、25％、20％、15％、10％、9％、8％、7％、6％、5％、4％、3％、2％或1％的变化。当术语“约”与一数值范围一起用时，该术语修饰该范 围，将其界限扩大到高于或低于该数值。通常，术语“约”修饰一数值时，意指高于和低于该数值10％以内。
本申请包含一份以ASCII格式电子递交的序列表，其整体被纳入作为参考。
本文所披露的各实施方案中的各技术特征可任意组合，构成优选的技术方案。
I、多肽
本公开的某些方面提供用于治疗或预防HBV相关肝病的衍生自HBV Pre-S1的多肽。优选地，所述多肽可体外阻断HBV的感染。更优选地，所述多肽能体外结合NTCP，例如在溶液中或在无细胞体系中(如细胞裂解液或在重新构建的体系中)，或在细胞中，如离体培养的细胞(如表达NTCP的细胞，或肝细胞)中，或在对象的体内细胞中结合NTCP。所述的对象可以是哺乳动物。在一些实施方案中，所述的对象可以是人。
术语“多肽”、“肽”和“蛋白质”可交互使用，包括全长蛋白和片段，以及全长蛋白和片段的变体。本文所述多肽的这些片段和变体至少保留了所述多肽的一种或多种生物学活性。“多肽”、“肽”和“蛋白质”可包括天然的和/或非天然的氨基酸残基。这些术语还包括翻译后修饰的蛋白质，包括例如糖基化、唾液酸化、乙酰化、和/或磷酸化的蛋白质。这些术语还包括在一个或多个氨基酸残基(例如N末端和/或在C末端的氨基酸残基)进行了化学修饰的蛋白质。例如，本文所述多肽的N末端可被疏水基团(如肉豆蔻酸、棕榈酸、硬脂酸、油酸、亚油酸、胆固醇和花生四烯酸)修饰。在一些实施方案中，本文所述的多肽的C末端可被修饰。C末端修饰可选自酰胺化(胺化)、异戊二醇化或无修饰。
本文所用术语“衍生自HBV Pre-S1的多肽”或“HBV Pre-S1衍生多肽”指多肽的起源或来源是HBV，可包括天然的、重组的、合成的或纯化的多肽。术语“衍生自HBV的多肽”或“HBV衍生多肽”指全长的天然HBV多肽或其片段，以及全长天然多肽或其片段的变体。在一些实施方案中，片段可由至少3－5个氨基酸、至少5－10个氨基酸、至少10－20个氨基酸、至少20－30个氨基酸、至少30－50个氨基酸组成，或由天然序列的全部氨基酸组成，或可由本领域普通技术人员鉴别为来源于所述天然序列。在一些实施方案中，本文所述的多肽可衍生自任何HBV亚型L蛋白的pre-S1区域。在一些实施方案中，本文所述的多肽可含有任意HBV亚型L蛋白的整个pre-S1区。在某些实施方案中，本文所述的多肽可衍生自HBV基因型A、B、C、D、E、F、G、H或I中的任意一种的L蛋白的pre-S1区。这些HBV基因型的基因组序列可分别 见于GenBank登录号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)和AB179747(SEQ ID NO：48)。在某些实施方案中，本文所述的多肽可衍生自HBV基因型C的L蛋白的pre-S1区。本文所述的衍生自HBV的多肽保留了相应天然HBV多肽的本文所述的一种或多种生物学活性。
本文所用的与本文所述多肽、衍生自HBV Pre-S1的多肽或HBV Pre-S1衍生多肽相关的“变体”指与给定多肽(即本文所述多肽，衍生自HBV Pre-S1的多肽或HBV Pre-S1衍生多肽)在氨基酸序列上存在差异但保留了给定多肽的本文所述的一种或多种生物学活性。本文所述的变体多肽相对于给定多肽可具有一个或多个氨基酸增加(如插入)、删除或取代。在一些实施方案中，相对于给定多肽，本文所述的变体多肽可具有1-30、1-20、1-10、1-8、1-5或1-3个(包括这些范围内的所有整数)氨基酸增加(如插入)、删除或取代。例如，多肽序列可含有氨基酸的保守取代。氨基酸的保守取代，即用具有相似性质(如亲水性和带电程度和带电区域的分布)的不同氨基酸取代一氨基酸，通常涉及较小的变化，因此不会明显改变多肽的生物学活性。这些小变化可部分通过基于该氨基酸的疏水性和电荷来考虑氨基酸的亲水指数而得以鉴定。具有类似亲水指数和亲水性数值的氨基酸可相互取代，并仍然保留蛋白质功能。氨基酸的亲水指数和亲水性数值受该氨基酸的特定侧链的影响。与此观察一致，与生物学功能相匹配的氨基酸取代依赖于氨基酸的相对相似性，尤其是那些氨基酸的侧链，表现为疏水性、亲水性、带电情况、大小和其它特性。
术语“变体”还包括与给定多肽具有一定相同性，如具有至少约30％、40％、50％、60％、70％、80％、85％、90％、95％、96％、97％、98％或99％与给定多肽的相同性。本文所用的“变体”还包括含有给定多肽对应于HBV蛋白的天然序列的一部分的多肽。“变体”还可指融合蛋白或嵌合蛋白，含有衍生自两种或多种不同来源的蛋白的多肽。本文所述的融合蛋白的非限制性例子可包括如衍生自HBV Pre-S1的一条多肽与衍生自非HBV Pre-S1蛋白的另一条多肽的融合蛋白，衍生自不同的HBV亚型的两条多肽的融合蛋白，以及衍生自任一HBV亚型L蛋白的不同区域的两条多肽或衍生自任一HBV亚型L蛋白pre-S1区域内的不同序列的两条多肽的融合蛋白。
术语“变体”还包括含有与给定多肽(即本文所述多肽，衍生自HBV Pre-S1的多肽或HBV Pre-S1衍生多肽)相同的氨基酸序列，保留该给定多肽的一种或多种生物学活性，但以不同于该给定多肽的方式进行了化学和/或翻译后修饰的多肽。“变体”还 可用于描述已被差别化处理，如蛋白质水解、磷酸化或其它翻译后修饰，但保留了本文所述的一种或多种生物学活性。本文中，除非另有说明，“变体”包括变体的片段。术语“变体”还包括在不同病毒种类、菌株或嗜肝病毒属亚型中发现的同源性多肽序列。基于其包膜蛋白上的抗原性表位，HBV分为四种主要血清型(adr、adw、ayr和ayw)，根据基因组中全部核苷酸序列的变异性，HBV分为9种基因型(A-I)。因此，术语“变体”包括这些发现于HBV亚型中的任意一种同源性多肽。“变体”还可包括在N和/或C末端添加有来自任一这些HBV亚型的天然侧接氨基酸序列的多肽。
术语“保守性氨基酸取代”和“保守取代”在本文中可交互使用，指一组氨基酸范围内的指定氨基酸交换，其中，一氨基酸与具有类似大小、结构、电荷和/或极性的不同氨基酸交换。具有类似侧链的氨基酸残基家族为本领域所周知，包括碱性侧链(如亮氨酸、精氨酸和组氨酸)，酸性侧链(如天冬氨酸、谷氨酸)，未带电极性侧链(如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)，非极性侧链(如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)，β侧链(如苏氨酸、缬氨酸、异亮氨酸)和芳族侧链(如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此，在一些实施方案中，多肽中的氨基酸残基可用来自相同侧链家族的另一氨基酸残基替代。在其它实施方案中，一段氨基酸序列可用结构相似、但侧链家族成员的顺序和/或组成不同的一段氨基酸序列替代。在其它实施方案中，可在该多肽的全部序列或部分序列随机引入突变。保守性氨基酸取代的例子包括如用脂族或疏水氨基酸Ala、Val、Leu和Ile中的一个氨基酸替换该组4个氨基酸中的另一个氨基酸；含羟基残基Ser和Thr之间的替换；酸性残基Asp和Glu之间的替换；酰胺残基Asn和Gln之间的替换；碱性残基Lys、Arg和His之间的替换；芳族残基Phe、Tyr和Trp之间的替换；和小氨基酸Ala、Ser、Thr、Met和Gly之间的替换。可合理预见到保守性取代，如用类似的、结构相关的氨基酸取代一保守的氨基酸，将不会对多肽的生物学活性产生实质性影响。
术语“序列相同性”(如与某序列具有50％的相同性)指在一比较窗口中以氨基酸对氨基酸的方式比较时序列相同的程度。在一些实施方案中，本文所述的多肽可含有一氨基酸序列，该氨基酸序列与给定多肽的序列的相同性至少约为30％、40％、50％、60％、70％、80％、85％、90％、95％、96％、97％、98％或99％，并仍然保留了该给定多肽的一种或多种生物学活性。通过在比较窗口中最优排列两条序列，测定两条序列中出现相同氨基酸的位置的数量、以获得匹配位置的数量，并将匹配位置的数量除以比较窗口中总的位置数量，将结果乘以100以得到序列相同性百分数，可计算 得到“百分比相同性”或“％相同性”。用于排列于一比较窗口的最优序列排列可通过采用计算机执行本领域已知的算法，例如
Figure PCTCN2018112062-appb-000001
家族的程序，或通过肉眼观察来进行，采用所选的任一种方法产生最佳的排列(即，在比较窗口中产生最高同源性百分比)。对于序列比较，将一条序列用作参比序列，将测试序列与该参比序列进行比较。当使用序列比较算法时，将测试和参比序列输入电脑，如果有必要，可设计子序列坐标，并设定序列算法程序参数。然后，序列比较算法基于设定的程序参数计算该测试序列相对于该参比序列的当前的序列相同性。序列算法程序参数的设定为本领域所周知。例如，比较窗口可设定为涵盖任一或两条比较序列的全部长度，如涵盖参比序列的全部长度，同时允许存在达参比序列总氨基酸数量5％的差异。
在一些实施方案中，本文所述多肽可含有任一HBV亚型的pre-S1区域的氨基酸序列。在一些实施方案中，本文所述多肽含有HBV基因型C的pre-S1区域的氨基酸13-59：GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANQVG(SEQ ID NO：23)或GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANKVG(SEQ ID NO：49)。在其它实施方案中，本文所述的多肽可含有来自另一种HBV基因型(如来自基因型A、B、C、D、E、F、G、H或I中的任意一种)的相应的pre-S1序列。例如，在某些实施方案中，本文所述的多肽可含有：
HBV基因型A的pre-S1氨基酸13-59：
GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPVKDDWPAANQVG(SEQ ID NO：34)；
HBV基因型B的pre-S1氨基酸13-59：
GTNLSVPNPLGFFPDHQLDPAFKANSENPDWDLNPNKDNWPDANKVG(SEQ ID NO：35)；
HBV基因型D的pre-S1氨基酸2-48：
GQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVG(SEQ ID NO：36)；
HBV基因型E的pre-S1氨基酸12-58：
GKNISTTNPLGFFPDHQLDPAFRANTRNPDWDHNPNKDHWTEANKVG(SEQ ID NO：37)；
HBV基因型F的pre-S1氨基酸13-59：
GQNLSVPNPLGFFPDHQLDPLFRANSSSPDWDFNTNKDSWPMANKVG(SEQ ID NO：38)；
HBV基因型G的pre-S1氨基酸12-58：
GKNLSASNPLGFLPDHQLDPAFRANTNNPDWDFNPKKDPWPEANKVG(SEQ ID NO：39)；或
HBV基因型H的pre-S1氨基酸13-59：
GQNLSVPNPLGFFPDHQLDPLFRANSSSPDWDFNTNKDNWPMANKVG(SEQ ID NO：40)。
在一些实施方案中，本文所述多肽可含有HBV的pre-S1区的一部分，所述部分含有选自SEQ ID NO：23、34－40或49所示的至少一条氨基酸序列。在一些实施方案中，本文所述多肽可含有HBV的完整pre-S1区域。
在一些实施方案中，本文所述多肽长度可为10－100个氨基酸。例如，所述多肽的长度可为15－100、15－80、20－100、20－80、20－60、25－60、30－60、35－60或40－60个(包括这些范围之间的所有整数)氨基酸。在一些实施方案中，本文所述多肽的长度可以为至少20个氨基酸，例如至少25、30、35或40个氨基酸。在一些实施方案中，本文所述多肽的长度可以为20、25、30、35、40、47、55或60个氨基酸。在一些实施方案中，本文所述多肽的长度可以为47个氨基酸。本文所述多肽的长度不同的变体保留了与相应多肽相关的一种或多种生物学活性。
在一些实施方案中，本文所述多肽的N末端可含有疏水基团修饰。例如，疏水基团可选自例如肉豆蔻酸、棕榈酸、硬脂酸、油酸、亚油酸、胆固醇和花生四烯酸。在一些实施方案中，所述疏水基团可选自肉豆蔻酸、棕榈酸、硬脂酸和胆固醇。在一些实施方案中，所述疏水基团可以是肉豆蔻酸。在某些实施方案中，本文所述多肽可含有选自SEQ ID NO：23、34－40和49任一所示的氨基酸序列，其中，所述多肽的N末端可被选自肉豆蔻酸、棕榈酸、硬脂酸和胆固醇的疏水基团修饰。在某些实施方案中，本文所述多肽可含有选自SEQ ID NO：23、34－40和49任一所示的氨基酸序列，其中，所述多肽的N末端可以被肉豆蔻酰化。在一些实施方案中，本文所述多肽可含有SEQ ID NO：23所示的氨基酸序列，其中，所述多肽的N末端可以被肉豆蔻酰化。在一些实施方案中，本文所述多肽可含有C末端修饰，或无修饰。例如，C末端修饰可选自如酰胺化(胺化)、异戊二醇化或无C末端修饰。在一些实施方案中，所述C末端修饰可以是酰胺化(胺化)。例如，本文所述的多肽可含有SEQ ID NO：23所示的氨基酸序列，其N端可被肉豆蔻酰化，和/或C端可被酰胺化(胺化)。在一些实施 方案中，本文所述多肽可含有SEQ ID NO：23所示的氨基酸序列。在一些实施方案中，本文所述多肽可含有选自SEQ ID NO：34-40、49中任一所示的氨基酸序列，其中所述多肽的N段可被肉豆蔻酰化，和/或C末端可被酰胺化(胺化)修饰。在一些实施方案中，本文所述多肽可含有选自SEQ ID NO：14－20、51中任一所示的氨基酸序列。本文所述多肽的在N末端和/或C末端被修饰的变体保留了未以相同方式修饰的相应多肽的一种或多种生物学活性。
本文所述多肽的变体也包括在本公开中，包括具有一个或多个氨基酸删除、取代或插入，同时保留了所述多肽的一种或多种生物学活性。本文所述多肽优选保留有对应于HBV基因型C的pre-S1区域氨基酸13的甘氨酸(即SEQ ID NO：23的N末端甘氨酸)。在一些实施方案中，本文所述多肽保留了对应于HBV基因型C的pre-S1区域氨基酸20的天冬酰胺或氨基酸57的赖氨酸。在一些实施方案中，本文所述多肽可在HBV的pre-S1区域中具有一个或多个天然产生的突变。在一些实施方案中，相对于来自HBV的pre-S1区域的序列，本文所述多肽可具有1－30个，例如1－20个、1－10个、1－8个、1－5个或1－3个(包括这些范围内的所有整数)氨基酸缺失、取代或插入。在一些实施方案中，本文所述多肽相对于选自SEQ ID NO：23、34－40和49任一所示的氨基酸序列可具有1－30个，例如1－20个、1－10个、1－8个、1－5个或1－3个(包括这些范围内的所有整数)氨基酸缺失、取代或插入。在一些实施方案中，本文所述多肽相对于SEQ ID NO：23的氨基酸序列具有1－30个，例如1－20个、1－10个、1－8个、1－5个或1－3个(包括这些范围内的所有整数)氨基酸缺失、取代或插入。在一些实施方案中，本文所述多肽相对于SEQ ID NO：23的氨基酸序列具有1－3个氨基酸缺失、取代或插入。在某些实施方案中，本文所述多肽相对于选自SEQ ID NO：23、34－40和49任一所示的氨基酸序列在C末端具有1－30个，例如1－20个、1－10个、1－8个、1－5个或1－3个(包括这些范围内的所有整数)氨基酸缺失或插入。例如，本文所述多肽可含有选自SEQ ID NO：21、22和24－28任一所示的氨基酸序列。在一些实施方案中，本文所述多肽可含有表1所列任一多肽的氨基酸序列。在一些实施方案中，本文所述多肽可选自表1所列的任一翻译后修饰多肽。
表1：示例性多肽列表
Figure PCTCN2018112062-appb-000002
Figure PCTCN2018112062-appb-000003
Figure PCTCN2018112062-appb-000004
在各种实施方案中，本文所述多肽与本文所述的任一多肽可具有至少约30％、40％、50％、60％、70％、80％、85％、90％、95％、96％、97％、98％或99％的相同性。例如，所述多肽可含有一氨基酸序列，该氨基酸序列与SEQ ID NO：21－40中任一序列具有至少约30％、40％、50％、60％、70％、80％、85％、90％、95％、96％、97％、98％或99％的相同性。在一些实施方案中，所述多肽可含有一氨基酸序列，该氨基酸序列与SEQ ID NO：23、34－40和49中任一序列具有至少约30％、40％、50％、60％、70％、80％、85％、90％、95％、96％、97％、98％或99％的相同性。在一些实施方案中，所述多肽可含有一氨基酸序列，该氨基酸序列与SEQ ID NO：23具有至少约30％、40％、50％、60％、70％、80％、85％、90％、95％、96％、97％、98％或99％的相同性。与本文所述多肽具有一定序列相同性的变体保留了相应多肽的一种或多种生物学活性。
本公开的各方面还包括本文所述多肽的具有来自HBV的L蛋白(如来自L蛋白的pre-S1区)的天然侧接氨基酸序列的变体，所述侧接氨基酸序列在所述变体的N和/或C端。所述天然侧接氨基酸序列指侧接相应HBV基因型或任意其它HBV基因型的pre-S1区域中本文所述多肽的N或C末端的天然序列。在一些实施方案中，本文所述多肽可含有选自SEQ ID NO：23、34－40和49中任一所示的氨基酸序列，以及衍生自HBV基因型A-H中任一基因型的pre-S1区域侧接于N和/或C末端的天然氨基酸序列。在一些实施方案中，所述天然侧接氨基酸序列可衍生自GenBank登录号为KC875260(基因型A；SEQ ID NO：41)、AY220704(基因型B；SEQ ID NO：42)、AF461363(基因型C；SEQ ID NO：43)、AY796030(基因型D；SEQ ID NO：44)、AB205129(基因型E；SEQ ID NO：45)、DQ823095(基因型F；SEQ ID NO：46)、HE981176(基因型G；SEQ ID NO：47)或AB179747(基因型H；SEQ ID NO：48)的HBV株的共有序列。例如，本文所述多肽可含有SEQ ID NO：23所示的氨基酸序列，并在其N和/或C端含有来自HBV基因型C的pre-S1区域的天然侧接氨基酸序列。或者，本文所述多肽可含有SEQ ID NO：23所示的氨基酸序列，并在其N和/或C端含有来自HBV基因型A、B、D、E、F、G和H中的任一基因型的pre-S1区域的天然侧接氨基酸序列。在一些实施方案中，本文所述多肽的N和/或C末端可独立含有长度为1－10个，如1－8个、1－5个或1－3个(包括这些范围内的所有整数)氨基酸的天然侧接氨基酸序列。例如，本文所述多肽可含有SEQ ID NO：23所示的氨基酸序列， 并在其N端含有来自HBV基因型C的pre-S1区域的长10个氨基酸的天然侧接氨基酸序列。换言之，该多肽可含有HBV基因型C的pre-S1区域的氨基酸2－59(SEQ ID NO：29)。另一例子是，本文所述多肽可含有SEQ ID NO：23所示的氨基酸序列，并在其N端含有来自HBV基因型E或G的pre-S1区域的长9个氨基酸的天然侧接氨基酸序列。换言之，该多肽可含有HBV基因型C的pre-S1区域氨基酸13－59和HBV基因型E或G的pre-S1区域的氨基酸2－11(SEQ ID NO：30)。应理解的是，本文所述的任何一种多肽可具有从其N和/或C末端延伸的任意长度的天然侧接氨基酸序列，且所产生的多肽保留了原始多肽的一种或多种生物学活性。
本文所述多肽可采用化学合成或重组技术制备。
当选择重组方法时，可从头构建一合成基因，或可采用例如盒式诱变来突变天然基因。本文所述多肽可采用重组DNA技术制备。简要的说，这些技术包括获得编码该多肽的天然或合成的基因，将其插入合适的载体中，将载体转入合适的宿主细胞中，培养该宿主细胞以使该基因表达，以及回收或分离所产生的肽。在一些实施方案中，将所回收的肽纯化到合适的纯度。
例如，克隆并操作编码本文所述多肽的DNA序列，以使其可在合适的宿主中表达。可从HBV基因组文库中衍生自表达该多肽的细胞的mRNA的cDNA获得编码亲本多肽的DNA，或者采用合成方法构建该DNA序列而获得编码亲本多肽的DNA。然后将该亲本DNA插入用于转化宿主细胞的合适的质粒或载体中。通常，来源于与宿主细胞相容的物种质粒载体且含有复制和控制序列用于这些宿主中。载体通常携带复制位点以及编码能为转化的细胞提供表型选择的蛋白质或肽的序列。载体可以是本领域常用的那些载体，或使用标准技术并结合本领域常用的载体的功能性片段来构建。
宿主细胞可以是原核细胞或真核细胞。例如，原核宿主细胞可以包括大肠杆菌、枯草芽孢杆菌和其它肠杆菌科如鼠伤寒沙门氏杆菌(Salmonella typhimurium)或粘质沙雷氏菌(Serratia marcesans)，以及各种假单胞菌。除了原核生物，也可使用真核生物，如酵母培养物，或来源于多细胞生物的细胞，如昆虫或哺乳动物细胞培养物。这类真核宿主细胞系的例子包括VERO和Hela细胞，中国仓鼠卵巢(CHO)细胞系，W138，293，BHK，COS-7和MDCK细胞系。
在一些实施方案中，可采用固相合成或本领域已知的同等化学合成方法制备本文所述多肽。在一些实施方案中，通过将预先保护的α-氨基酸偶联到合适的树脂上而开始多肽C末端的固相合成。可通过将α氨基受保护的氨基酸经由酯键连接到氯甲基化树脂或羟基甲基化树脂，或经由酰胺键连接到BHA树脂或MBHA树脂而制备所述起 始材料。使用本领域熟知的用于形成肽键的技术将氨基酸连接到肽链上。一种方法涉及将氨基酸转化成一衍生物，该衍生物将提供更易于与肽片段的游离N末端氨基反应的羧基。例如，可通过受保护的氨基酸与氯甲酸乙酯，氯甲酸苯酯，氯甲酸叔丁基酯，氯甲酸异丁酯，新戊酰氯或类似的酰基氯之间的反应而将氨基酸转化成混合的酸酐。或者，可将氨基酸转化成活性酯，例如2,4,5-三氯苯酯，五氯苯酯，五氟苯酯，对硝基苯酯，N-羟基琥珀酰亚胺酯，或由1-羟基苯并三唑形成的酯。另外一种偶联方法涉及使用合适的偶联剂，如N,N′-二环己基碳二亚胺或N,N′-二异丙基碳二亚胺。
在一些实施方案中，肽合成中使用的各氨基酸的α氨基在偶联反应中被保护，以阻止涉及它们的活性α氨基功能的副反应。例如，可用合适的保护基保护含反应性侧链官能团(如巯基，氨基，羧基和羟基)的某些氨基酸，以防止在最初以及之后的偶联步骤中在该位点发生化学反应。本领域技术人员知道该如何选择合适的侧链保护基。在获得所需氨基酸的肽之后，在不会改变肽链结构的反应条件下即可容易地除去保护基。
除去α氨基保护基之后，以所需的顺序逐步将剩余的α氨基和侧链受保护的氨基酸偶联。除了在合成中分别加入各氨基酸外，作为替代，一些氨基酸可在被加到固相合成仪之前先相互偶联。本领域技术人员熟知该如何选择合适的偶联剂。
各受保护的氨基酸或氨基酸序列过量加到固相反应器中，偶联适宜于在介质二甲基甲酰胺(DMF)或CH 2Cl 2或其混合物中进行。如果偶联不完全，则在偶联下一个氨基酸前，在N-氨基保护基存在时先重复该偶联过程。监控合成各阶段的偶联反应是否成功。可使用周知的方法如BIOSEARCH 9500 TM肽合成仪自动进行偶联反应。
获得所需的肽序列之后，必须从树脂支持物上切割下受保护的肽，且必须除去所有保护基。保护基的切割和去除可以同时或依次进行。当树脂支持物是氯甲基化的聚苯乙烯树脂时，将肽锚定到该树脂的键是C末端残基的游离羧基基团与该树脂基质上众多氯甲基基团中的一个形成的酯键。应理解的是，可采用已知能断裂酯键并能渗入树脂基质的试剂切割该锚定用的键。也应认识到的是，可在从支持物上切割多肽之前或之后修饰该多肽，如用疏水基团(包括如肉豆蔻酸、棕榈酸、硬脂酸、油酸、亚油酸、胆固醇和花生四烯酸)来修饰N末端，或用酰胺化(胺化)、异戊二醇化或其它能稳定C末端的修饰来修饰C末端。
可采用常规的方法如制备HPLC(包括反相HPLC)或其它已知的层析技术(如凝胶渗透，离子交换，分配色谱法，亲和层析(包括单克隆抗体柱)或反流分布法)纯化本发明的多肽。
II、药物组合物和药盒
本公开还提供组合物，包括药物组合物，该组合物含有本文所述的多肽。在某些实施方案中，该组合物可含有本文所述的任意一条或多条多肽。在一些实施方案中，该组合物还可含有合适的药学上可接受的载体。
“药学上可接受的载体”指无活性成分，如固体，半固体，或液体填充剂，稀释剂，包衣材料，制剂佐料，赋形剂或载体，与治疗剂合用，一起构成用于给予对象的“药物组合物”。药学上可接受的载体在所用的剂量和浓度对于受试者而言是无毒的，且与制剂中的其他成分相容。药学上可接受的载体对于所采用的制剂而言是适当的。例如，如果治疗剂将口服给药，则载体可以是凝胶胶囊。如果治疗剂将皮下给药，则理想的是，载体对皮肤无刺激，且不引起注射位点反应。
可将具有所需纯度的本文所述多肽与一种或多种任选的药学上可接受的载体混合而制备所述多肽的药物组合物。药学上可接受的载体可包括如缓冲剂(如磷酸盐，柠檬酸盐和其它有机酸)；抗氧化剂(如抗坏血酸和甲硫氨酸)；防腐剂(如十八烷基苄基二甲基氯化铵、氯化六甲双铵、苯扎氯铵、苄索氯铵、苯酚、丁醇或苄醇、对羟基苯甲酸烷基酯如对羟基苯甲酸甲酯或对羟基苯甲酸丙酯、儿茶酚、间苯二酚、环己醇、3-戊醇和m-甲酚)；低分子量(如低于约10个残基)的多肽；蛋白质(如血清白蛋白，明胶或免疫球蛋白)；亲水性聚合物(如聚乙烯吡咯烷酮)；氨基酸(如甘氨酸，谷氨酰胺，天冬酰胺，组氨酸，精氨酸或赖氨酸)；单糖；二糖；和其它碳水化合物，包括葡萄糖，甘露糖，或糊精；螯合剂，如EDTA；糖类，如蔗糖，甘露醇，海藻糖或山梨醇；成盐反离子，如钠；金属复合物，如Zn-蛋白复合物；和/或非离子型表面活性剂，如聚乙二醇(PEG)。
示例性的药物载体还可包括粘合剂，如预糊化的玉米淀粉，聚乙烯吡咯烷酮或羟丙基甲基纤维素等；填充剂，如乳糖或其它糖类，微晶纤维素，果胶，明胶，硫酸钙，乙基纤维素，聚丙烯酸酯或磷酸氢钙等；润滑剂，如硬脂酸镁，滑石粉，二氧化硅，胶态二氧化硅，硬脂酸，金属硬脂酸盐，氢化植物油，玉米淀粉，聚乙二醇，苯甲酸钠，乙酸钠等；崩解剂，如淀粉，羟基乙酸淀粉钠等；和润湿剂，如十二烷基硫酸钠等。
示例性的药学上可接受的载体还可包括间质性药物分散剂，如可溶性的中性活性透明质酸酶糖蛋白(sHASEGP)，如人可溶性PH-20透明质酸酶糖蛋白，如rHuPH20 (
Figure PCTCN2018112062-appb-000005
Baxter International,Inc.)。在一些实施方案中，sHASEGP可以混合到含有一种或多种其它粘多糖酶(glycosammoglycanases)如软骨素酶的药物组合物中。
药物组合物还可含有一种以上适于所治疗的特定适应症的活性成分，这类活性剂可以是例如无不利影响的互补活性的活性剂。这类活性剂可适宜地以有效满足预期目的的量组合存在。
在一些实施方案中，活性剂可被包裹在采用如凝聚技术或界面聚合所制备的微胶囊(如羟甲基纤维素或凝胶微胶囊或聚甲基丙烯酸甲酯微胶囊)中，或包裹在胶态药物递送系统(如脂质体、白蛋白微球、微乳液、纳米颗粒和纳米胶囊)中，或包裹在粗乳液中。
在一些实施方案中，该药物组合物可包括缓释制剂。缓释制剂的合适例子包括如含本文所述多肽的固体疏水聚合物的半透性基质，其中，该基质可以是成形制品的形式，如膜或微胶囊。
在一些实施方案中，所述药物组合物可用于体内给药，且可以是无菌的。可采用例如通过无菌滤膜过滤的方式容易地实现无菌。
所述药物组合物可被制备成多种可能的剂型中的任意一种，如片剂，胶囊，凝胶胶囊，粉末或颗粒。所述药物组合物还可被制备成溶液、悬液、乳液或混合介质。在一些实施方案中，所述药物组合物可被制备成冻干制剂或水性溶液。
在一些实施方案中，所述药物组合物可被制备成溶液。例如，可将本文所述的多肽加入到非缓冲的溶液(如盐水或水)中施用。在一些实施方案中，也可将多肽加入合适的缓冲溶液中施用。例如，缓冲溶液可含有乙酸盐，柠檬酸盐，醇溶谷蛋白，碳酸盐或磷酸盐，或其任意组合。在一些实施方案中，缓冲溶液可以是磷酸盐缓冲液(PBS)。可将含所述多肽的缓冲溶液的pH和渗透压调到适于给予对象的水平。
在一些实施方案中，所述药物组合物可被制备成水性悬液、非水性悬液或混合基质悬液。水性悬液还可含有增加悬液粘度的物质，包括如羧甲基纤维素钠、山梨醇和/或葡聚糖。悬液还可含有稳定剂。
在一些实施方案中，所述药物组合物可被制备成乳液。示例性的乳液包括一种液体以通常超过0.1μm直径的液滴分散在另一种液体中形成的非均质系统。除分散相和可存在于水相溶液、油相溶液或自身作为独立相存在的活性药物外，乳液可含有其它成分。还可包括微乳液，作为本公开的一个实施方案。在一些实施方案中，所述药物组合物还可被制备成脂质体制剂。
在某些实施方案中，所述药物组合物被制备成适合给药的剂型，例如适合皮下给药的注射剂。在这些实施方案中，药物组合物含有一定量的本文所述多肽，用以在每日给予1剂或数剂所述药物组合物后该日的日剂量可达到治疗有效量。本文中，治疗有效量指给予所述治疗有效量的多肽数日(≥7天)之后能达到肝脏靶器官饱和的多肽的量。
在某些实施方案中，所述治疗有效量为777.95-926.13nmol的日剂量。在某些实施方案中，所述治疗有效量为4.2～5.0mg的日剂量。
在某些实施方案中，药物组合物中的多肽为贺普拉肽，治疗有效量为4.2～5.0mg/日，更优选为4.2mg/日。在某些实施方案中，药物组合物中的多肽为Myrcludex B，治疗有效量为4.2～5.0mg/日，更优选为和5.0mg/日。
在某些实施方案中，每日给予数次所述药物组合物，可达到治疗有效量。因此，在这些实施方案中，本文的药物组合物被制成多个单位剂型，例如，每单位剂型最低可含有18.52nmol，或可含有37.05nmol、55.57nmol、111.14nmol、129.66nmol、259.32nmol或388.98nmol。可每日给予相应数量的单位剂型，使得当日的总给药量达到治疗有效量的范围内。在这些实施方案中，当药物组合物含有贺普拉肽时，每单位剂型最低可含有0.1mg的贺普拉肽，或可含有0.2mg、0.3mg、0.6mg、0.7mg、1.4mg、2.1mg或4.2mg的贺普拉肽，即药物组合物的规格为0.1mg、0.2mg、0.3mg、0.6mg、0.7mg、1.4mg、2.1mg或4.2mg。例如，当每单位剂型含有0.1mg的贺普拉肽时，每日可给予42个单位剂型的药物组合物。在这些实施方案中，当药物组合物含有Myrcludex B时，每单位剂型最低可含有0.1mg的Myrcludex B，或可含有0.25mg、0.5mg、1.0mg、2.5mg或5.0mg的Myrcludex B，即药物组合物的规格为0.1mg、0.25mg、0.5mg、1.0mg、2.5mg或5.0mg。例如，当每单位剂型含有0.1mg的Myrcludex B时，每日可给予50个单位剂型的药物组合物。因此，本发明药物组合物中，单位剂型的数量可在1～50剂的范围内，如1～42剂。
在某些实施方案中，每个单位剂型含有777.95-926.13nmol或4.2～5.0mg的本文所述多肽。因此，每日给予1单位剂型即可达到所述治疗有效量。
本文的药物组合物可以是冻干剂型。例如，冻干的一剂药物中可含有37.05～926.13nmol或0.1～5mg的本文所述多肽，如259.32～926.13nmol或1.4～5mg、388.98～926.13nmol或2.1～5mg、或777.95-926.13nmol或4.2～5mg的本文所述多肽。使用时，可使用适量的溶剂溶解后作为注射剂给予对象。可给予1剂或数剂所述注射剂，使日总剂量在777.95-926.13nmol或4.2～5mg的范围内。例如，冻干的制剂可含 有2瓶药物，每瓶含有388.98nmol或2.1mg的冻干多肽，每日可分两次给予对象所述制剂，使得日总剂量达777.95nmol或4.2mg。
本文还包括含有本文所述药物组合物的药盒。药盒中可含有1剂或多剂本文所述的药物组合物，用于1日或多日给药，预防或治疗乙肝病毒相关的肝病。
在某些实施方案中，药盒含有至少7剂药物组合物，每剂药物组合物含有777.95-926.13nmol或4.2～5.0mg的本文所述多肽，用于连续至少7日给药。在某些实施方案中，所述药盒满足7日连续给药。
在某些实施方案中，药盒含有至少7剂药物组合物，每剂药物组合物含有4.2～5.0mg、优选4.2mg的贺普拉肽或5.0mg的Myrcludex B，用于至少连续7日给药。
在某些实施方案中，药盒含有至少2剂药物组合物，药盒中全部药物组合物所含多肽的含量之和达777.95-926.13nmol或4.2～5.0mg，作为1日的总剂量。在这些实施方案中，每剂药物组合物可含有如18.52nmol、37.05nmol、55.57nmol、111.14nmol、129.66nmol、259.32nmol或388.98nmol，或0.1mg、0.2mg、0.3mg、0.6mg、0.7mg、1.4mg或2.1mg的多肽，药盒所含药物组合物最少可达42剂、21剂、14剂、7剂、6剂、3剂或2剂；或者，每剂药物组合物可含有18.52nmol、46.31nmol、92.61nmol、185.23nmol或463.06nmol，或含有0.1mg、0.25mg、0.5mg、1.0mg或2.5mg的多肽，药物所含药物组合物最少可达50剂、20剂、10剂、5剂、2剂。
在某些实施方案中，药盒含有2剂或2剂以上药物组合物，药物组合物中的多肽为贺普拉肽，每剂药物组合物中贺普拉肽的含量为0.1mg、0.2mg、0.3mg、0.6mg、0.7mg、1.4mg或2.1mg，药盒中所有药物组合物所含多肽的含量之和为4.2mg。
在某些实施方案中，药盒含有2剂或2剂以上药物组合物，药物组合物中的多肽为Myrcludex B，每剂药物组合物中Myrcludex B的含量为0.1mg、0.25mg、0.5mg、1.0mg或2.5mg，药盒中所有药物组合物所含多肽的含量之和为5.0mg。
在某些实施方案中，药盒含有多剂药物组合物，满足至少连续7天给药，多剂药物组合物中的数剂所含多肽的含量之和满足一天的给药量，即达777.95-926.13nmol或4.2～5.0mg。例如，某些实施方案中，每剂药物组合物含有388.98nmol或2.1mg的多肽，则药盒至少含有14剂药物组合物，每2剂满足一天的给药量。当每剂药物组合物含有259.32nmol或1.4mg的多肽时，药盒至少含有21剂药物组合物，每3剂满足一天的给药量。以此类推。在某些实施方案中，所述药物组合物所含的多肽为贺普拉肽或Myrcludex B。在某些实施方案中，所述药物满足连续7日给药。
本文所述的药物组合物和药盒用于给予需要的对象本文所述的多肽，以在至少连续给予7天后在所述多肽在其肝脏靶器官中达到饱和。
III、使用方法
本公开的实施方案包括本文所述多肽的治疗和预防用途。一方面，提供了本文所述多肽作为药物的用途。另一方面，提供了本文所述多肽治疗和预防HBV相关肝病的用途。
本文所述乙肝病毒相关肝病包括慢性乙肝病毒感染、慢性乙型肝炎、乙肝相关肝纤维化和肝硬化、乙肝相关肝癌、乙肝相关肝移植和乙肝相关母婴传播。所述慢性乙型肝炎包括HBeAg阳性慢性乙型肝炎和HBeAg阴性慢性乙型肝炎。在某些实施方案中，所述乙肝相关肝移植包括移植术前、术中和术后对供肝的HBV感染的保护。
“患者”和“对象”可互换使用，指待治疗或评估是否患有疾病、失调或病症的动物(如哺乳动物或人)，或评估是否有患上疾病、失调或病症的风险，或患上了疾病、失调或病症的动物(如哺乳动物)或人。在一些实施方案中，这些疾病、失调或病症可包括代谢疾病。在一些实施方案中，该代谢疾病可涉及脂质代谢失调。
可给予需要的对象充分剂量的本文所述多肽或其药物组合物，用于预防或治疗HBV相关的肝病。本文所述的“充分剂量”指每日给予该剂量的多肽，连续给予数日(≥7天)之后能达到肝脏靶器官饱和的日剂量。在这些实施方案中，所述“充分剂量”通常为777.95-926.13nmol或4.2～5.0mg，如777.95-833.52nmol或4.2～4.5mg，或777.95nmol或4.2mg，或926.13nmol或5.0mg的日剂量，首次给予该量的多肽时靶器官不饱和，但连续给予7次后可达到靶器官饱和。因此，本文所述的预防或治疗方法包括连续给予至少7天的充分剂量的多肽或其药物组合物的步骤。
本文所述靶器官饱和指给予一定量所述多肽后，表观分布容积小于100L。在这些实施方案中，表观分布容积通常为10～100L，如10～50L或20～40L。
在各实施方案中，术语“治疗”包括对对象(如哺乳动物，如人)或细胞进行处理，以改变该对象或细胞的当前进程。治疗包括例如给予本文所述多肽或含该多肽的药物组合物，治疗可以预防性的方式进行，或可在发生了病理性事件或与发病物质接触后开始进行。治疗也包括“预防性”治疗，该治疗指向降低待治疗疾病或病症的进展速度，延缓该疾病或病症的发作，或降低其发作的严重性。“治疗”或“预防”并不必须指完全根除、治愈，或防止疾病或病症或相关症状的发生。在各实施方案中，术语“治疗”可包括减轻、缓和或反转患HBV相关肝病的病理学进程。在一些实施方 案中，术语“治疗”可包括改善代谢疾病的至少一种症状或至少一种可测量参数。对于本领域技术人员而言显而易见的是，可使用生物学和/或生理参数评估代谢疾病的病理进程。这些病理进程或症状可包括如与健康对象相比过量或增加的一种或多种与代谢相关的化学或生物分子，如ALT、HBV DNA，或过量或增加的一种或多种衡量代谢变化的生理参数，如肝功能指数。
术语“给予”或“给药”包括将本文所述多肽以局部或全身给药的方式给药。给药可以是局部的(包括眼部或阴道和直肠的粘膜给药)，肺部(如通过粉末或气雾剂的吸入或吹入，包括采用喷雾器，气管内，鼻内给药)，表皮，经皮，口服或肠胃外。肠胃外给药包括静脉内、皮下、腹膜内或肌肉内注射或输注，或颅内，如鞘内或心室内给药。
本公开还提供实施本文所述多肽在对象中的上述用途的方法。这些方法可包括给予所述对象治疗或预防有效量的本文所述多肽或含所述多肽的药物组合物。在一些实施方案中，所述对象可以是哺乳动物。在一些实施方案中，所述对象可以是人。在一些实施方案中，所述对象可患有HBV相关肝病或有患上所述疾病的风险。
本文所述的这些组合治疗可包括联合给药(其中，两者或多种治疗剂可以是同一制剂或分开的制剂)，以及分别给药，这种情况下，本文所述多肽可在其它治疗剂之前、同时或之后给予。这些药物包括干扰素、核苷酸类药物、乙肝免疫球蛋白、乙肝疫苗。其中干扰素包括普通干扰素、PEG长效干扰素。普通干扰素包括干扰素α-2a、干扰素α-2b，PEG长效干扰素包括PEG干扰素α-2a、PEG干扰素α-2b。核苷酸类药物包括拉美夫定LAM、恩替卡韦ETV、替诺福韦酯TDF、替比夫定TBV、阿德福韦酯ADV和替诺福韦艾拉酚胺TAF。
可以合适的方式给予本文所述多肽(和任意其它治疗剂)，包括肠胃外，肺内和鼻内；也可局部治疗或可病灶内给药。在一些实施方案中，本文所述多肽可肠胃外给药。肠胃外给药可包括肌肉内、静脉内、动脉内、腹膜内或皮下给药。在一些实施方案中，本文所述多肽可皮下给药。在一些实施方案中，本文所述多肽可静脉内给药。可以任何合适的手段给药，如通过注射或输注，如静脉内或皮下注射或输注，这部分取决于给药是短暂的还是长期的。还考虑各种剂量方案，包括如在各时间点上的单次或多次给药，大剂量给药，以及脉冲注射。
因此，本文还公开在本文所述的治疗和预防乙肝病毒相关肝病的方法中使用到的本文所述的多肽或其药物组合物。更具体而言，所述多肽或其药物组合物以777.95- 926.13nmol或4.2～5.0mg的日剂量(以多肽计)用于所述治疗和预防方法中，连续给药数日，以达到肝脏靶器官饱和，优选至少连续给药7天。
在某些实施方案中，本文包括在本文所述的治疗或预防乙肝病毒相关肝病的方法中使用到的贺普拉肽或其药物组合物，其中，所述方法包括以每日777.95-926.13nmol或4.2～5.0mg的量、优选777.95nmol或4.2mg的量给予对象贺普拉肽，优选926.13nmol或5.0mg给予对象Myrcludex B，连续给药数日，以达到肝脏靶器官饱和，优选至少连续给药7天。
优选地，所述药物组合物可如本文任一实施方案所述。
下述实施例可用于阐述性目的，不应理解为限缩本发明的范围。
实施例1：多肽的合成
可参照PCT/CN2017/086558实施例1.1的方法合成多肽。具体而言，根据用于多肽合成的标准Fmoc方案合成表1所示多肽。通常，起始于MBHA树脂，单个氨基酸残基从羧基端向氨基端延伸。然后对N末端实施肉豆蔻酰化。肽合成完成后，采用切割液将多肽从树脂上切下，并进一步对多肽的C末端进行胺化修饰。用G6砂芯漏斗滤除树脂，真空抽干含多肽的滤液。将多肽产物溶解在去离子水中，用装备了C18柱的
Figure PCTCN2018112062-appb-000006
explorer 100型高压液相色谱仪进行纯化，分步收集主峰。从目标峰收集的样品采用装配了C18柱的Agilent 1100型反相高压液相色谱(HPLC)分析纯度，采用质谱仪鉴定它们的分子量。中压液相色谱纯化所收集的溶液进行冻干保存。将干燥样品溶解在PBS中，用0.20μM膜过滤。溶解于PBS的多肽储备液保存在-80℃备用。贺普拉肽氨基酸序列为SEQ ID NO：23所示，其N末端肉豆蔻酸修饰，C末端胺化修饰，分子量为5398.8Da，分子式为C 247H 352N 65O 73。
实施例2：多肽对HBV感染的体外阻断
成年雄性树鼩麻醉后，利用显微外科技术采用肝门静脉胶原酶两步灌注法获得游离的树鼩原代肝细胞，并采用肝细胞专用培养体系培养。肝细胞培养3天后的肝细胞加入纯化的HBV病毒，以感染复数MOI＝0、6.25、12.5、25、50和100基因组DNA/细胞感染肝细胞，可见12天后上清中HBeAg分泌量与MOI具有良好的线性关系(R 2＝0.9873)，证实上清中HBsAg可定量反映HBV的感染程度。
在HBV感染肝细胞同时(MOI＝100基因组DNA/细胞)，加入贺普拉肽(0、2.5、5、10、20、40ng/ml)阻断HBV感染。可见上清中HBeAg分泌量伴随贺普拉肽浓度的增高而下降，经logit-log回归分析可见两者具有良好的线性相关剂量依赖关系(R 2＝0.9563)。贺普拉肽抑制HBV感染具有明显剂量依赖关系，此时计算贺普拉肽半数抑制浓度IC50为3.956ng/ml。
实施例3：多肽对HBV感染的模型动物体内阻断
将成年雄性树鼩50只，随机分为5组：PBS对照组、贺普拉肽高(2mg/kg)、中(0.4mg/kg)、低(0.08mg/kg)剂量组和乙肝免疫球蛋白HBIG阻断组(60IU/kg)。腹腔注射108HBV DNA/ml滴度的HBV病毒血清1ml感染树鼩，贺普拉肽阻断组于感染后第0、1、2、3、5、7、9、11、13天给予不同剂量贺普拉肽皮下注射。HBIG阻断组于感染当天和感染后第3天给予免疫球蛋白HBIG肌肉注射。于感染前4天和感染后第9、14、21、42天采集树鼩血清，检测血清中HBsAg、HBeAg、HBV DNA滴度和谷丙转氨酶(ALT)；于感染后第21天取肝组织病理检测。
PBS对照组感染后第9天HBsAg出现高峰，第15天后HBsAg基本被清除。贺普拉肽(高、中、低剂量组)均可有效阻断HBV感染，使感染过程中HBsAg均得到很好控制。而HBIG则不能有效阻断本感染剂量的HBV感染，感染后第9天HBsAg较PBS对照组未出现明显降低，而且有延长HBsAg分泌的趋势，第15天HBsAg水平反高于PBS对照组。贺普拉肽(高、中、低剂量)各组第9天HBsAg水平均低于PBS对照组和HBIG阻断组，其中贺普拉肽(高、中、低剂量)各组第9天HBsAg水平均明显低于HBIG阻断组(P均小于0.05)。
PBS对照组感染后第9天HBeAg出现高峰，第21天后HBeAg基本消失。贺普拉肽可有效阻断HBV感染，高剂量和中剂量的贺普拉肽使感染过程中HBsAg均得到很好控制。而HBIG则不能有效阻断本感染剂量的HBV感染，感染后第9天和第15天HBsAg与PBS对照组相当。贺普拉肽(高、中、低剂量)各组第9天HBeAg水平均低于PBS对照组和HBIG阻断组，其中贺普拉肽(高、中、低剂量)各组第9天HBsAg水平均明显低于PBS对照组(P均小于0.05)。
PBS对照组感染后第9天HBV DNA出现高峰，第15天后HBV DNA被清除。贺普拉肽可有效阻断HBV感染，高剂量和中剂量的贺普拉肽使感染过程中HBV DNA均得到很好控制。而HBIG则不能有效阻断本感染剂量的HBV感染，而且感染后第15 天HBV DNA反而较PBS对照组进一步升高，直至感染21天后才趋于清除。贺普拉肽(高、中、低剂量)各组第9天HBV DNA水平均低于PBS对照组和HBIG阻断组，其中贺普拉肽高剂量组HBV DNA水平明显低于HBIG阻断组(P小于0.05)。
感染前ALT平均值为56.8IU/ml，设定该值为树鼩ALT正常水平。PBS对照组感染后第21天ALT出现高峰，第42天后ALT恢复正常水平。贺普拉肽可有效阻断HBV感染，高剂量和中剂量贺普拉肽使感染过程中ALT均得到很好控制。而HBIG则不能有效阻断本感染剂量的HBV感染，感染后第21天ALT仍较正常值高，感染42天后才恢复正常。贺普拉肽(高、中、低剂量)各组第21天ALT水平均低于PBS对照组和HBIG阻断组，其中贺普拉肽高、中剂量组ALT水平明显低于HBIG阻断组(P小于0.05)。
随机选取各试验组树鼩各1只于感染后第21天行肝脏组织病理学检查，正常树鼩肝组织作为正常对照。PBS组显示HBV感染造成树鼩肝脏组织明显病理学改变，主要为肝小叶周围汇管区肝细胞浊肿和气球样变，伴有少量淋巴细胞浸润，其病理改变与HBV急性感染成人造成的肝炎极为相似。而HBIG不能减轻HBV感染对树鼩肝脏组织造成的病理改变，贺普拉肽低剂量组肝脏病理改变与PBS组相似，病变程度稍微减轻。而高剂量和中剂量贺普拉肽完全阻断HBV对肝细胞造成的气球样变等病理学损害，肝组织与正常树鼩肝组肝基本一致。
综上所述，贺普拉肽可在血清学(HBsAg、HBeAg)、病毒学(血清HBV DNA拷贝数)、酶学(谷丙转氨酶ALT)和病理学水平有效阻断HBV感染。
实施例4：多肽与NTCP特异性结合
将非肝细胞来源的293细胞转染NTCP，形成NTCP-293细胞系。以FITC荧光标记贺普拉肽(Cmyr-47)第37位K氨基酸或无关对照肽(来源于苍鹭乙肝病毒heron HBV Pre-S1区的47肽，肉豆蔻酰化-GLNQSTFNPLGFFPSHQLDPLFKANAGSADWDKNPNKDPWP QAHDTA-酰胺化，SEQ ID NO：50)。如图1所示，FITC-贺普拉肽与NTCP-293细胞结合，却不与未转染NTCP的293细胞(BLANK-293)结合，说明贺普拉肽与细胞的结合是NTCP特异性的。FITC标记的无关对照肽不与NTCP-293细胞结合，说明该结合具有贺普拉肽序列特异性。
实施例5：多肽与NTCP胆汁酸转运功能的影响
参照文献中NTCP胆酸转运功能研究方法(Kim RB等.J Pharmacol Exp Ther.1999Dec；291(3):1204-9)研究贺普拉肽对NTCP胆酸转运功能的影响。以转染人NTCP的293细胞(NTCP-293)为细胞模型，研究贺普拉肽对NTCP-293吸收 3H标记胆汁酸( 3H-TA)的影响。结果显示(见图2，A)：1)TA进入细胞是NTCP特异性介导的，因为293细胞本身对TA几乎不吸收；2)阳性对照环孢素A对NTCP发挥显著抑制作用，与文献报导一致；3)贺普拉肽对NTCP具有双向性影响(见图2，B)，在低浓度时(≤500ng/ml，相当于50×IC50)促进TA的吸收，在高浓度时(＞500ng/ml)抑制TA的吸收，IC50浓度为838.81ng/ml。
实施例6：多肽临床前药代动力学研究
125I标记的贺普拉肽以40ug·kg -1剂量给予大鼠单次皮下注射后，检测贺普拉肽在各组织的分布。研究结果(图3)显示，贺普拉肽主要分布于肝脏和泌尿系统，其中泌尿系统以代谢产物为主。药物在肝内2小时达峰，Cmax为44.3±28.9ng-Equ.mL -1，其在肝内的末端消除半衰期t1/2长达8.7h，在24h时肝内还有较高的浓度(4.5±1.2ng-Equ.mL -1)，推算肝内48h药物浓度约0.66ng-Equ.mL -1，相当于皮下注射后4小时的血清药物浓度水平。血清内药物清除迅速，给药后6小时基本降低至基线水平。临床前药代结果显示贺普拉肽具有显著的肝脏靶器官汇集性。
实施例7：多肽单剂量爬升Ia期临床试验及临床药代动力学研究
用药/暴露程度：贺普拉肽的单剂量爬升Ia期临床试验为随机双盲、空白对照、单中心、剂量爬坡的试验，设定6个剂量组(0.525mg、2.1mg、4.2mg、6.3mg、8.4mg和10.5mg)，计划依次入组5例、5例、10例、10例、10例和5例健康受试者，男女比例22:23。健康志愿者经过筛选后按照4:1比例双盲随机进入试验组和空白对照组，接受贺普拉肽单次皮下注射给药，停药后观察7天。试验中贺普拉肽用药和暴露程度见表2。
受试人群例数及特征：计划入组45例成年健康受试者，实际入组45例受试者，全部完成试验，均被纳入安全分析人群。Ia期临床研究安全分析人群见表3。各试验组年龄、性别、民族、职业、身高、体重、BMI和既往史等人口学指标均未见显著性差异。
表2：Ia期试验总体设计及剂量设定
Figure PCTCN2018112062-appb-000007
表3：单剂量爬升Ia期临床试验人口学指标
Figure PCTCN2018112062-appb-000008
安全性研究：在贺普拉肽单剂量爬升Ia期临床试验中未发生死亡和严重不良事件，试验过程中共记录63例不良事件，均为I级不良事件；与试验药物有关、很可能有关和可能有关的不良事件发生29例(见表4)。不良事件发生率在各剂量组和空白对照组未见组间显著差异，8.4mg剂量组不良事件发生率显著高于空白对照组，不良反应 发生率在0.525mg、2.10mg、4.20mg、6.30mg、10.50mg与空白对照之间均无显著性差异。
表4：Ia期不良事件(肯定有关、很可能有关、可能有关)在各试验组的分布
Figure PCTCN2018112062-appb-000009
Ia期临床药代动力学研究：在45例健康受试者中进行了皮下单次给药剂量爬升Ia期临床药代动力学研究，剂量设置及PK分析人群见表5。在给药前及给药后2min、5min、15min、30min、45min、1h、1.5h、2h、3h、4h、6h、8h、12h和24h采集血样。生物样本采用液质联用法检测药物浓度，定量下限为0.2ng/ml。血药浓度曲线见图4，血药代动力学主要参数见表4。研究结果显示，受试者在单次接受贺普拉肽皮下注射后，在0.525mg-4.2mg剂量范围内接受单次皮下注射贺普拉肽后血药曲线低平，而6.3mg-10.5mg剂量范围内血药曲线明显高耸。在0.21-4.2mg剂量范围内表观分布容积Vz/F大于100L，提示药物主要分布于靶器官。结合临床前研究显示贺普拉肽与NTCP特异性结合(实施例4)，临床前药代动力学研究显示贺普拉肽显著肝脏汇集性(实施例6)，药物靶点NTCP的肝脏特异性表达，因此判定药物主要汇集于靶器官肝脏。在6.3-10.5mg剂量范围内，Vz/F接近于人体全身体液体积，并随剂量增加呈缩小趋势，提示药物靶器官饱和后药物向体液分布。因此将单次给药剂量0.525mg-4.2mg和 6.3mg-10.5mg分别划分为靶器官肝脏不饱和剂量范围和饱和剂量范围。Myrcludex B皮下注射单次给药5mg后表观分布容积为135L(lank A等.J Hepatol.2016Sep；65(3):483-9)，根据本实施例结果，Myrcludex B在该剂量下单次皮下给药，未达到肝脏靶器官饱和。
表5：Ia期PK研究主要药代动力学参数
Figure PCTCN2018112062-appb-000010
实施例8：多肽多剂量爬升Ib临床试验及临床药代动力学研究
用药/暴露程度：贺普拉肽多剂量爬升Ib期临床试验为随机、双盲、空白对照、单中心临床研究。试验设定三个剂量组(4.20mg、6.30mg、8.40mg)，计划依次入组10例、10例和15例健康受试者，男女比例18:17。健康志愿者经过筛选后按照4:1比例双盲随机进入试验组和空白对照组，接受每日1次，连续7天皮下注射给药。停药后观察3天。试验中贺普拉肽用药和暴露程度见表6。
受试人群例数及特征：贺普拉肽Ib期临床研究计划入组35例成年健康受试者，实际入组35例受试者，全部完成试验，均被纳入安全分析人群。Ib期临床研究的安全 分析人群，以及各试验组人口学指标见表7。各试验组年龄、性别、民族、职业、身高、体重、BMI和既往史等人口学指标均未见显著性差异。
表6：Ib期试验总体设计及剂量设定
Figure PCTCN2018112062-appb-000011
表7：Ib期临床试验人口学基本情况
Figure PCTCN2018112062-appb-000012
安全性评价：贺普拉肽Ib期临床研究共记录74例不良事件，所有不良事件均为I度不良事件，均未予治疗自行缓解，未留有后遗症。试验过程中记录与试验药物有关、很可能有关和可能有关的不良事件合计36例(见表8)。主要不良事件为总胆汁酸升 高，共发生27例，占全部不良事件75.0％。由于胆汁酸升高为贺普拉肽药理反应，在扣除总胆汁酸增加不良事件后，4.2、6.3和8.4mg剂量组不良事件发生率分别为25.0％、37.5％和8.3％，与空白对照组不良事件发生率42.9％相当。Ib期临床研究过程中未发生贺普拉肽剂量限制性毒性DLT，剂量爬升至本研究设计最大剂量8.4mg时未达到最大耐受剂量MTD。
表8：Ib期不良事件(肯定有关、很可能有关、可能有关)在各试验组的分布
Figure PCTCN2018112062-appb-000013
临床药代动力学研究：在35例健康受试者中进行了皮下多次给药剂量爬升Ib期临床药代动力学研究，受试者接受每日1次连续7天给药，剂量设置及PK分析人群见表9。血样采集如下：给药前及给药后2min、5min、15min、30min、45min、1h、1.5h、2h、3h、4h、6h、8h、12h血样；Day2：Day1的24h血样(Day2给药前)；Day3～6：给药前；Day7：给药前及给药后2min、5min、15min、30min、45min、1h、1.5h、2h、3h、4h、6h、8h、12h血样。生物样本采用液质联用法检测药物浓度，定量下限为0.2ng/ml。首剂量、2-7天给药前及末剂量血样药-时曲线见图5-7，主要药代参数见表10。研究结果显示，首剂量后各剂量组血药曲线与Ia期一致，4.2mg首剂量未达到靶器官饱和，6.3和8.4mg首剂量达到靶器官饱和。第2-6天连续给药期间药谷浓度达到稳态。连续给药7天后，4.2mg、6.3mg和8.4mg剂量组蓄积系数R AUC分别为4.43±1.60、2.76±0.76和1.91±0.39，显示贺普拉肽连续给药时存在蓄积作用。经蓄积后4.2mg表观分布容积由首剂量时153.6025±107.1807升缩小至末剂量时22.1880±8.0721升，表明4.2mg剂量经过连续给药蓄积后末剂量达到靶器官饱和。最高剂量8.4mg连续给药靶器官充分蓄积后，表观分布容积为15.1021±4.4743升，接近14升体内血管外液的空间，表明靶器官外药物主要分布于细胞外液。
表9：Ib期临床药代动力学分析PK人群
Figure PCTCN2018112062-appb-000014
表10：Ib期PK研究主要药代动力学参数
Figure PCTCN2018112062-appb-000015
临床药效动力学研究：在多剂量爬升Ib临床试验中，在4.2-8.4mg剂量范围内受试者接受贺普拉肽每日一次连续7天皮下注射，给药期间有效提高血总胆汁酸水平，停药后血总胆汁酸恢复正常。总胆汁酸可以作为理想的药效学生物标志物。研究结果 显示，贺普拉肽每日4.2mg，连续7天皮下注射，可以达到实现药效学反应的充分剂量。
实施例9：多肽I期临床试验临床药代动力学总结
贺普拉肽在0.525-4.2mg剂量范围内单次皮下注射后主要分布于靶器官；在6.3-10.5mg剂量范围内单次皮下注射后靶器官饱和，药物向血浆分布。出人意料地，贺普拉肽以4.20mg剂量单次皮下注射给药时，未达到肝内靶器官饱和，但该剂量下皮下注射连续给药7天，经蓄积后达到肝脏靶器官达到饱和，这是从未报道过和难以预料的结果。本申请公开的结果，确定了每日4.2mg剂量为贺普拉肽连续给药最低靶器官饱和剂量。基于本公开的结果判断，Myrcludex B以5mg剂量单次皮下给药未达到肝脏靶器官饱和，以该剂量连续给药，经蓄积后也将达到肝脏靶器官饱和。
实施例10：肽I期临床试验安全性总结
受试者在0.525-10.5mg剂量范围内对贺普拉肽单次皮下注射给药耐受性良好，未出现剂量限制性毒性，未达到最大耐受剂量。受试者在每日4.2-8.4mg剂量范围内对贺普拉肽连续7天皮下注射给药耐受性良好，未出现剂量限制性毒性，未达到最大耐受剂量。
实施例11：临床推荐剂量
由于贺普拉肽以每日4.2mg剂量，经连续给药蓄积后达到肝脏靶器官饱和；同时，该剂量下临床安全性良好，并实现了总胆汁酸药效学生物标志物反应。因此确定该剂量为临床推荐剂量。

Claims (14)

  1. 一种治疗和预防乙型肝炎病毒相关肝病的方法，其特征在于，所述方法包括每日给予需治疗和预防对象以充分剂量的多肽或其药物组合物，其中，所述多肽含有衍生自乙肝病毒(HBV)Pre-S1的氨基酸序列；所述充分剂量为给予所述多肽数日后所述多肽在肝脏靶器官中达到饱和的日剂量。
  2. 如权利要求1的方法，其特征在于，所述多肽含有HBV的pre-S1区域的氨基酸序列；优选地，所述多肽的N末端含有疏水基团修饰基团；优选地，所述疏水基团选自：肉豆蔻酸、棕榈酸、硬脂酸、油酸、亚油酸、胆固醇和花生四烯酸；更优选地，所述疏水基团是肉豆蔻酸；和/或所述多肽具有C末端修饰；优选地，所述C末端修饰是酰胺化、异戊二醇化或无修饰；
    优选地，所述多肽含有HBV基因型A、B、C、D、E、F、G、H或I的pre-S1区域的氨基酸序列；更优选地，所述多肽含有HBV基因型C的pre-S1区域氨基酸序列13-59的序列，或含有来自HBV基因型A、B、D、E、F、G、H或I的pre-S1区域中与HBV基因型C的pre-S1区域氨基酸序列13-59相对应的序列。
  3. 如权利要求1－2中任一项所述的方法，其特征在于，所述多肽的一个或多个氨基酸残基缺失、被取代或插入；优选地，所述多肽的1－30、1－20、1－10、1－8、1－5或1－3个氨基酸残基缺失、被取代或插入；和/或
    所述多肽在其N末端和/或C末端含有来自HBV的pre-S1区域的天然侧接氨基酸序列；优选地，所述HBV的pre-S1区域的天然侧接氨基酸序列长1－10、1－8、1－5或1－3个氨基酸。
  4. 如权利要求3所述的方法，其特征在于，所述多肽：
    (1)含有选自SEQ ID NO：21－40和49中任一所示的氨基酸序列；或
    (2)与SEQ ID NO：21－40和49任一所示的氨基酸序列具有至少约30％、40％、50％、60％、70％、80％、85％、90％、95％、96％、97％、98％或99％的相同性；
    优选地，所述多肽的N末端含有选自肉豆蔻酸、棕榈酸、硬脂酸和胆固醇的疏水基团，C末端具有酰胺化修饰；优选地，所述多肽如SEQ ID NO:1－20或51任一所示，更优选地，所示多肽如SEQ ID NO:3或51所示。
  5. 如权利要求1－4中任一项所述的方法，其特征在于，所述充分剂量为连续给药至少7天，使得所述多肽在肝脏靶器官中达到饱和的日剂量；优选地，所述充分剂量为777.95-926.13nmol的日剂量，优选777.95nmol或926.13nmol的日剂量。
  6. 如权利要求5所述的方法，其特征在于，
    所述多肽为含有SEQ ID NO：23所示的氨基酸序列，其N末端被肉豆蔻酸修饰和C末端胺化修饰；所述充分剂量为4.2-5.0mg的日剂量，优选4.2mg的日剂量，所述方法包括至少连续给药7天；或
    所述多肽为含有SEQ ID NO：49所示的氨基酸序列，其N末端被肉豆蔻酸修饰和C末端胺化修饰；所述充分剂量为4.2-5.0mg的日剂量，优选5.0mg的日剂量，所述方法包括至少连续给药7天。
  7. 如权利要求1所述的方法，其特征在于，
    所述多肽含有SEQ ID NO：23所示的氨基酸序列，其N末端被肉豆蔻酸修饰和C末端胺化修饰，所述方法包括每日给予所述对象N剂的所述药物组合物，实现4.2mg的日剂量，其中，N为1～42中的任一整数；或
    所述多肽含有SEQ ID NO：49所示的氨基酸序列，其N末端被肉豆蔻酸修饰和C末端胺化修饰，所述方法包括每日给予所述对象N剂的所述药物组合物，实现5.0mg的日剂量，其中，N为1～50中的任一整数。
  8. 如权利要求1－7中任一项所述的方法，其特征在于，所述乙型肝炎病毒相关肝病选自：慢性乙肝病毒感染、慢性乙型肝炎、乙肝相关肝纤维化和肝硬化、乙肝相关肝癌、乙肝相关肝移植和乙肝相关母婴传播；优选地，所述慢性乙型肝炎包括HBeAg阳性慢性乙型肝炎和HBeAg阴性慢性乙型肝炎，所述乙肝相关肝移植包括移植术前、术中和术后对供肝免于HBV感染的保护。
  9. 一种用于治疗和预防乙型肝炎病毒相关肝病的含有多肽的药物组合物，所述多肽含有衍生自乙肝病毒(HBV)Pre-S1的氨基酸序列，其中，所述药物组合物含有一定量的所述多肽，通过给予整数个制剂规格的该药物组合物，能使所述多肽在肝脏靶器官中达到饱和。
  10. 如权利要求9所述的药物组合物，其特征在于，所述药物组合物所含的所述多肽的量使得每日给予1剂或数剂所述药物组合物后所述多肽的日剂量达到777.95-926.13nmol，优选777.95nmol或926.13nmol。
  11. 如权利要求9或10所述的药物组合物，其特征在于，所述多肽为含有SEQ ID NO：23或49所示的氨基酸序列，其N末端被肉豆蔻酸修饰和C末端胺化修饰；该多肽在药物组合物中的量使得每日给予1剂或数剂所述药物组合物后所述多肽的日剂量达到4.2-5.0mg，优选日剂量为4.2mg或5.0mg。
  12. 如权利要求9－11中任一项所述的药物组合物，其特征在于，每剂所述药物组合物含有4.2mg、2.1mg、1.4mg、0.7mg、0.6mg、0.3mg、0.2mg或0.1mg的所述多肽，即所述药物组合物规格为4.2mg、2.1mg、1.4mg、0.7mg、0.6mg、0.3mg、0.2mg或0.1mg；或每剂所述药物组合物含有5.0mg、2.5mg、1.0mg、0.5mg、0.25mg或0.1mg的所述多肽，即所述药物组合物规格为5.0mg、2.5mg、1.0mg、0.5mg、0.25mg或0.1mg。
  13. 如权利要求9－12中任一项所述的药物组合物，其特征在于，所述多肽如权利要求2－4中任一项所述。
  14. 一种药盒，所述药盒含有1剂或多剂如权利要求9－13中任一项所述的药物组合物，用于乙肝病毒相关肝病患者1日或多日的预防或治疗。
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