WO2016145840A1 - 抗乙型肝炎病毒x蛋白多肽药物 - Google Patents

抗乙型肝炎病毒x蛋白多肽药物 Download PDF

Info

Publication number
WO2016145840A1
WO2016145840A1 PCT/CN2015/092132 CN2015092132W WO2016145840A1 WO 2016145840 A1 WO2016145840 A1 WO 2016145840A1 CN 2015092132 W CN2015092132 W CN 2015092132W WO 2016145840 A1 WO2016145840 A1 WO 2016145840A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
polypeptide
hepatitis
ttk001
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2015/092132
Other languages
English (en)
French (fr)
Inventor
张晓东
叶丽虹
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tianjin Toptech Bio-Science & Technology Co Ltd
Original Assignee
Tianjin Toptech Bio-Science & Technology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tianjin Toptech Bio-Science & Technology Co Ltd filed Critical Tianjin Toptech Bio-Science & Technology Co Ltd
Priority to ES15885210T priority Critical patent/ES2819238T3/es
Priority to KR1020177029558A priority patent/KR102017973B1/ko
Priority to US15/557,209 priority patent/US10570176B2/en
Priority to EP15885210.3A priority patent/EP3269727B1/en
Priority to JP2017566181A priority patent/JP6660966B2/ja
Publication of WO2016145840A1 publication Critical patent/WO2016145840A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • 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
    • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the field of polypeptide medicine, in particular to a polypeptide containing a D-type amino acid against hepatitis B virus X protein and an application thereof.
  • Liver cancer is one of the malignant tumors that cause death of patients. Its malignant degree is high.
  • the mortality rate of liver cancer in China is second only to gastric cancer, ranking second in the mortality rate of malignant tumors in China. According to statistics, the number of new liver cancers in China is 300,000 per year, and the number of liver cancer deaths per year is 110,000.
  • Hepatitis B virus (HBV) infection can lead to hepatitis, cirrhosis and primary liver cancer. More than 80% of liver cancer patients in China are liver cancer after hepatitis B infection, which can be called liver cancer after hepatitis B.
  • HBV is a DNA virus of about 3.2 kb in length, and its open reading frame expresses hepatitis B virus surface antigen (HBsAg), hepatitis B virus core antigen (HBcAg), hepatitis B virus polymerase and hepatitis B virus X antigen (HBxAg).
  • HBsAg hepatitis B virus surface antigen
  • HBcAg hepatitis B virus core antigen
  • HBxAg hepatitis B virus polymerase
  • HBxAg hepatitis B virus X antigen
  • HBxAg hepatitis B virus X protein
  • HBx acts as a trans-acting factor to promote the growth and proliferation of liver cancer, which is called an oncoprotein.
  • Studies at the molecular, cellular and animal levels have also found that HBx has a strong role in promoting proliferation and migration of liver cancer cells.
  • Transgenic mouse experiments have also demonstrated that HBx has a significant role in promoting the development of liver cancer.
  • a large number of studies have shown that the persistence of HBV infection can lead to chronic liver disease, including cirrhosis of the liver tissue in the liver tissue caused by recurrent episodes of chronic hepatitis and chronic hepatitis, and most of them occur on the basis of cirrhosis.
  • Liver cancer In the development of chronic liver disease (including hepatitis, cirrhosis and liver cancer), HBx plays an important role. Therefore, HBx is an important target for the prevention and treatment of liver diseases.
  • HBx is an important pathogenic factor for the development of liver cancer, and its specific inhibitors have important theoretical significance and practical clinical application value.
  • HBx since the three-dimensional conformational analysis of HBx is not yet completed, it is difficult to design its chemical inhibitor by the stereoscopic three-dimensional conformation of HBx.
  • thymopeptide is a thymopentin extracted from calf thymus, which promotes lymphocyte transformation and enhances macrophage phagocytic activity and can be used to treat various immunodeficiency diseases.
  • Peptide drugs are characterized by clear pharmacological effects, high safety, and ease of production.
  • the efficacy of a polypeptide drug is generally not ideal because it is susceptible to degradation by proteases in the living body, with low stability and short half-life.
  • Amino acids may exist in the conformation of a left-handed (“L-amino acid”) or a right-handed (“D-amino acid”). Natural amino acids that make up proteins in humans or animals are all L-formed and do not have D-form amino acids. There is a lack of proteolytic enzymes acting on D-type amino acids in the body. Therefore, when a polypeptide drug is produced, if the natural amino acid on the polypeptide chain is replaced by the L form to the D form, the stability of the polypeptide drug in the blood can be improved even without changing the amino acid sequence. However, how to improve the anti-degradation of peptide drugs without destroying their binding properties and thus retaining the efficacy is still a big problem that plagues the development of peptide drugs.
  • the inventors of the present invention have found a polypeptide having the function of inhibiting hepatitis B virus X protein (HBx) and consisting of natural L-form amino acids (see Chinese National Invention Patent No. ZL201110061840 for details). 5. The disclosure of which is incorporated herein by reference in its entirety.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1.
  • the polypeptide has an inhibitory effect on HBx activity at the molecular, cellular and animal levels, and can be used for preventing and treating hepatitis caused by inhibition of hepatitis B virus infection, Cirrhosis and liver cancer.
  • the present invention relates to a polypeptide against hepatitis B virus X protein and uses thereof.
  • the polypeptide is a polypeptide comprising a D-type amino acid, which has the function of inhibiting hepatitis B virus X protein (HBx), and has the activity of inhibiting HBx at the molecular level, the cellular level and the animal level, and inhibiting the type B Hepatitis virus DNA replication and expression of related antigens (such as HBeAg), and in turn, inhibition of hepatitis B, cirrhosis caused by hepatitis B virus infection, and liver cancer on the basis of cirrhosis.
  • HBx hepatitis B virus X protein
  • the invention provides an isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or a functional fragment thereof, or a functional variant of said amino acid sequence and functional fragments thereof, and said amino acid sequence,
  • the functional fragment, or one or more L-form amino acids in the amino acid sequence of the functional variant is substituted with a D-form amino acid which inhibits the function of the hepatitis B virus X protein and inhibits the hepatitis B virus The occurrence and development of chronic liver disease after infection.
  • the amino acid sequence of the polypeptide of the invention has the same amino acid sequence as SEQ ID NO: 1 of at least 70% (preferably at least 80%, 85%, 90%, 95%, 99% or higher) Sex.
  • the amino acid sequence of the polypeptide is any one of the amino acid sequences set forth in SEQ ID NOs: 2-11.
  • the chronic liver disease after the hepatitis B virus infection includes hepatitis, cirrhosis, and liver cancer.
  • the invention provides the use of the polypeptide in the manufacture of a medicament for chronic liver disease following infection with hepatitis B virus.
  • the drug may be a therapeutic vaccine for hepatitis B.
  • the medicament may be a pharmaceutical composition comprising any of the polypeptides of the invention, which may comprise any Selected drug carrier.
  • the polypeptides of the present invention Compared with polypeptides having the same amino acid sequence but consisting entirely of L-form amino acids, the polypeptides of the present invention not only have good stability, but also have surprisingly significant pharmaceutical effects, replication of hepatitis B virus DNA and related antigens ( The inhibitory effect of expression such as HBeAg) is particularly pronounced.
  • the polypeptides are therefore widely used for the prevention and treatment of chronic liver diseases caused by hepatitis B virus infection, including hepatitis, cirrhosis and liver cancer.
  • FIG. 3 Effect of detecting the polypeptide of the present invention on the secretion of HBeAg in the integrated HBV DNA hepatoma HepAD38 cells at the cellular level.
  • the results showed that the artificially synthesized 0.1, 1, 10 and 100 ⁇ M peptides D-TTK001 and L-TTK001 (ie anti-HBxP1#) were used to inhibit the HBeAg level secreted by HBV DNA hepatoma HepAD38 cells for 48 hours. , in a dose-dependent manner.
  • the minimum effective dose of D-TTK001 is 0.1 ⁇ M; the minimum effective dose of L-TTK001 is 10 ⁇ M. Therefore, the effect of D-TTK001 on HBeAg is better than that of L-TTK001 on HBeAg.
  • *P ⁇ 0.05, **P ⁇ 0.01, and Student's t test statistical analysis was performed.
  • Figure 4 Effect of detecting the polypeptide of the present invention on the secretion of HBeAg in HepG2.2.15 cells at the cellular level.
  • the results showed that the artificially synthesized 0.1, 1, 10 and 100 ⁇ M peptides D-TTK001 and L-TTK001 (ie anti-HBxP1#) were used to inhibit the secretion of HBeAg in HepG2.2.15 cells for 48 hours.
  • the minimum effective dose of D-TTK001 is 0.1 ⁇ M; the minimum effective dose of L-TTK001 is 10 ⁇ M. Therefore, the effect of D-TTK001 on HBeAg is better than that of L-TTK001 on HBeAg.
  • *P ⁇ 0.05, **P ⁇ 0.01, and Student's t test statistical analysis was performed.
  • Figure 5 Effect of detection of the polypeptide of the present invention on proliferation of HepAD38 cells integrated with HBV DNA at the cellular level using MTT assay.
  • the results show that artificially synthesized 0.1, After treatment with 1, 10 and 100 ⁇ M polypeptides D-TTK001 and L-TTK001 (ie anti-HBxP1#) for 48 hours, the proliferation of HepAD38 cells integrated with HBV DNA was inhibited in a dose-dependent manner. *P ⁇ 0.05, **P ⁇ 0.01, and Student's t test statistical analysis was performed.
  • FIG. 7 Therapeutic effect of the synthetic peptide D-TTK001 on HBV transgenic mouse hepatitis.
  • the left picture shows the liver pathology of HBV transgenic mice.
  • Six of the 10 HBV transgenic mice in the untreated control group showed typical pathological characterization of viral hepatitis such as hepatic balloon-like degeneration;
  • the right picture shows the application of D-TTK001 (5 mg/ Kg) Liver tissue after 3 weeks of tail vein injection, no hepatic balloon-like degeneration in the experimental group, suggesting that D-TTK001 has a significant therapeutic effect on pathological hepatitis lesions in liver tissue.
  • Figure 8 Effect of synthetic peptide D-TTK001 on survival rate of liver cancer-bearing nude mice.
  • the results of animal experiments showed that the artificially synthesized D-TTK001 polypeptide 0.1 mg/kg group and 5.0 mg/kg group had a significant prolongation of the survival rate of HepG2-X cell retrophage nude mice, suggesting that D-TTK001 has Inhibition of the malignant phenotype of HepG2-X cells in nude mice.
  • Figure 9 Effect of detecting the polypeptide of the present invention on the secretion of HBeAg in HepAD38 cells integrated with HBV DNA at the cellular level.
  • the results showed that the artificially synthesized 10 ⁇ M polypeptide D-TTK001 and SEQ ID NOs: 2-11 (such as D-TTK001-1, D-TTK001-2, D-TTK001-3, D-TTK001-4, respectively) were used.
  • the symbol "D-*" indicates any D-type amino acid which replaces the original L-form amino acid at the specific position of the polypeptide.
  • amino acids may exist in a left-handed (“L-amino acid”) and a right-handed (“D-amino acid”) conformation.
  • L-form amino acids are naturally found in nature and in most biological systems.
  • naturally occurring polypeptides are composed of L-form amino acids and thus may be referred to as L-form polypeptides.
  • the D-type amino acid is a "mirror image" of its L-form amino acid counterpart, and although the D-form polypeptide (a polypeptide consisting entirely of D-form amino acids) is not naturally occurring, these polypeptides can be artificially synthesized to form a three-dimensional protein structure.
  • the term "separated” refers to the separation of a substance from its original environment (for example, if it is naturally produced, its natural environment).
  • a naturally occurring polypeptide is present in a living organism that is not isolated, and the same polypeptide is separated from some or all of the substances that coexist in the natural system.
  • Such a polypeptide may be part of a certain carrier or part of a certain composition. Since the carriers and compositions are not components of their natural environment, they are still isolated.
  • purified means that it has been improved in purity. "Purity” is a relative term in this and is unnecessarily interpreted as absolute purity. For example, the purity can be at least about 50%, or can be greater than 60%, 70%, 80%, 90%, or can be 100%.
  • the separated material is separated from its original environment.
  • the polypeptide in the native state in living cells is not isolated, but the same polypeptide, if separated from other substances present in the natural state, is isolated, and the purity is improved, and thus purified.
  • amino acid sequence or “polypeptide” is meant an amino acid in which a peptide, oligopeptide, polypeptide or protein and a partial fragment thereof are linked by a peptide bond.
  • amino acid sequence in the present invention relates to a naturally occurring protein molecule, or a synthetic polypeptide which has been described, such a naturally occurring protein molecule or a known polypeptide is not meant to The amino acid sequence is limited to the complete, native amino acid sequence identical to the protein molecule or known polypeptide.
  • the amino acid sequence of the present invention may contain an additional peptide such as a poly-histidine tag (His-tag), or an epitope-labeled peptide such as Myc or FLAG; the amino acid sequence of the present invention may also comprise a synthetic D-type amino acid, Substituting one or more L-form amino acids in the amino acid sequence of the protein molecule or known polypeptide.
  • additional peptide such as a poly-histidine tag (His-tag), or an epitope-labeled peptide such as Myc or FLAG
  • the amino acid sequence of the present invention may also comprise a synthetic D-type amino acid, Substituting one or more L-form amino acids in the amino acid sequence of the protein molecule or known polypeptide.
  • a “functional fragment” of a polypeptide refers to any portion of a polypeptide of the invention that retains substantially similar or identical biological activities and functions of the original polypeptide (i.e., "parent" polypeptide) as part of it.
  • a “functional variant” of a polypeptide refers to an amino acid sequence that substantially retains a biological function or activity that is identical or similar to the polypeptide or amino acid sequence, and which may include, for example, 1) one or more amino acids in the original amino acid sequence a deletion and/or one or more amino acids are added; or 2) one or more amino acids in the original amino acid sequence are substituted with a conservative or non-conservative amino acid; or 3) a group on one or more amino acids in the original amino acid sequence Substituted by other groups; or 4) fusion of the original amino acid sequence with another molecule or compound (such as sugar, lipid, polyethylene glycol, etc.); or 5) fusion of the original amino acid sequence with the added amino acid sequence to form a polypeptide sequence (such as a leader or secretion sequence or a sequence used to purify the polypeptide, etc.); or 6) a reverse analog of the original amino acid sequence; or 7) a mixture of the above.
  • deletion refers to the deletion of one or more amino acids in the amino acid sequence.
  • “Insertion” or “addition” refers to an alteration in an amino acid sequence that results in an increase in one or more amino acids as compared to a molecule that is naturally occurring or altered.
  • Replacement refers to the replacement of one or more amino acids by a different amino acid.
  • “Deletion, substitution or addition of one or more amino acids” means that a deletion, substitution or addition of a known mutant polypeptide such as a site-directed mutagenesis method can be deleted, replaced or added. The degree of amino acid added.
  • the above mutation is not limited to an artificially induced mutation by a known site-directed mutagenesis method, and may be obtained by isolating and purifying a mutation of a naturally occurring nucleic acid or protein.
  • the "identity” or “identity” percentage of an amino acid sequence refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acids. There are a number of methods well known to those skilled in the art to determine percent identity, such as by the MEGALIGN program (Lasergene software package, DNASTA, Inc., Madison, WI). The MEGALIGN program can compare two or more sequences according to different methods such as the Cluster method (see Higgins & Sharp, Gene 73:237-244, (1988)). The Gluster method arranges the sequences of each group into clusters by examining the distance between all pairs. And then assign the clusters in pairs or groups. The percent identity between two amino acid sequences, such as sequence A and sequence B, can be calculated by:
  • chronic liver disease after hepatitis B virus infection refers to liver disease caused by hepatitis B virus infection, including cirrhosis caused by hyperplasia of fibrous connective tissue in liver tissue caused by recurrent chronic hepatitis and chronic hepatitis, and Liver cancer that occurs on the basis of cirrhosis.
  • treatment and “prevention” and the terms derived therefrom are not meant to be 100% or complete treatment or prevention and may be considered to be a degree of treatment or prevention as recognized by those skilled in the art.
  • “Prophylaxis” in the present invention is understood to delay the onset of a disease, or a symptom or condition thereof.
  • the invention provides isolated or purified polypeptides.
  • the polypeptide is a polypeptide comprising the amino acid sequence Gly-Ser-Ala-Val-Met-Phe-Ser-Ser-Lys-Glu-Arg-Gly (as shown in SEQ ID NO: 1, referred to as L-
  • L- The amino acid sequence obtained after replacement of one or more natural L-form amino acids in TTK001) with a synthetic D-form amino acid.
  • the present invention demonstrates that such a polypeptide can significantly inhibit the activity of HBx, Thereby, it has the effects of inhibiting the replication of hepatitis B virus DNA and the expression of related antigens (such as HBeAg), and further inhibits the occurrence and development of chronic liver diseases after hepatitis B virus infection, especially the occurrence and development of hepatitis, liver cirrhosis and liver cancer.
  • related antigens such as HBeAg
  • any L-form amino acid in the L-TTK001 polypeptide may be, for example, one, two, three, four, five, six, seven, eight, nine or ten L
  • the type of amino acid is replaced by the corresponding D-type amino acid.
  • the amino acid sequence of the polypeptide of the present invention is that all of the L-form amino acids other than glycine in L-TTK001 are substituted by the corresponding D-type amino acids (ie, Gly-D-Ser-D- Ala-D-Val-D-Met-D-Phe-D-Ser-D-Ser-D-Lys-D-Glu-D-Arg-Gly, as shown in SEQ ID NO: 2, referred to as D-TTK001) .
  • D-form amino acid is an optical isomer of L-form amino acid
  • a polypeptide that is more stable in vivo can be obtained. For example, studies have shown that the presence of a D-type amino acid at the N-terminus or C-terminus may increase the in vivo stability of the polypeptide (Powell et al, Pharm. Res. 10: 1268-1273 (1993)).
  • a polypeptide containing a D-form amino acid residue is taken from a D-form amino acid residue.
  • the binding force and biological potency of the polypeptide will increase or even lead to complete loss.
  • the inventors of the present invention have found through experiments that one or several L-form amino acids in the polypeptide L-TTK001 are substituted with a D-form amino acid, and the polypeptide still has substantially the same biological functions as the L-TTK001 polypeptide, including: at the cellular level and Animal levels inhibit hepatitis virus replication and expression, as well as viral hepatitis caused by treatment of hepatitis virus at the animal level, as well as inhibition of late liver cancer cell growth.
  • the inventors of the present invention have also surprisingly found that even if all of the L-form amino acids in the L-form polypeptide L-TTK001 are replaced with synthetic D-form amino acids (the obtained polypeptide is D-TTK001), the polypeptide and the HBx protein are in vitro. There was no significant change in binding. For example, according to the experimental results of in vitro binding force, the in vitro binding ability of the L-form polypeptides L-TTK001 and HBx (8 nM) was not significantly different from the in vitro binding force of D-TTK001 and HBx (35 nM) under the same conditions.
  • the D-TTK001 polypeptide Since all of the amino acids contained therein are D-form amino acids, the D-TTK001 polypeptide has good pharmacokinetic properties at the same time. Therefore, the inventors have found that a polypeptide different from L-type peptide L-TTK001 can be administered, for example, by tail vein administration of an animal, and can be administered at a dose much lower than that of L-TTK001, such as D-. TTK001 still has a clear effect.
  • the inventors have found that due to the good pharmacokinetic properties and stability of the polypeptide of the present invention, it inhibits the DNA replication of HBV and the expression of related antigens (such as HBeAg), and thereby inhibits chronic liver disease after hepatitis B virus infection.
  • HBeAg related antigens
  • the occurrence and development of hepatitis has a more significant effect than L-form polypeptides (such as L-TTK001).
  • a D-form polypeptide may have an order of magnitude difference in its effective dose for inhibiting the secretion level of HBeAg compared to an L-form polypeptide; and because of its improved stability, the polypeptide of the present invention is also selected in terms of the mode of administration. All Polypeptides composed of L-form amino acids also have a greater advantage. .
  • the invention also provides various functional fragments of the polypeptides of the invention.
  • the functional fragment may be any fragment of a contiguous amino acid sequence of a polypeptide of the invention, provided that it retains the biological activity of the parent polypeptide to a similar extent, to the same extent, or to a greater extent than the parent polypeptide, eg, inhibits HBx active.
  • the functional fragment may comprise, for example, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% of the parent polypeptide, Activity of 105%, 110%, 120%, 150%, 200% or higher.
  • the functional fragment may further comprise an additional amino acid, for example, an amino acid different from the amino acid sequence of the parent polypeptide, at the amino or carboxy terminus of the various fragments of the contiguous amino acid sequence, or the amino and carboxy termini thereof.
  • the additional amino acid does not interfere with the biological function of the functional fragment, for example, inhibiting the activity of HBx, inhibiting the replication and expression of HBV virus, and effectively inhibiting the occurrence and development of hepatitis and the development of liver cancer.
  • the additional amino acid can result in enhanced biological activity when compared to the biological activity of the parent polypeptide.
  • the amino acid sequence of the functional fragment of the polypeptide of the present invention has at least 70% sequence identity with the amino acid sequence of the polypeptide of the present invention; more preferably, the amino acid sequence of the functional fragment of the polypeptide of the present invention has at least the amino acid sequence of the polypeptide of the present invention. 75%, or 80%, or 85%, or 90%, or 95% sequence identity.
  • the functional variants of the polypeptides of the invention and functional fragments thereof are also included within the scope of the invention.
  • the functional variants of the polypeptides of the invention and functional fragments thereof should retain substantially similar or identical biological activities as the parent polypeptide or the parental functional fragment, for example, inhibiting the activity of HBx and inhibiting the replication and expression of HBV virus, Treatment of HBV at the level of HBV transgenic mouse animals results in the role of viral hepatitis and the inhibition of the malignant phenotype of liver cancer cells.
  • the functional variant may be at least about 50%, 60%, 70%, 80%, 90%, 95% or 100 with the amino acid sequence of the parent polypeptide or the parent functional fragment.
  • the amino acid sequence of the functional fragment has at least 70% sequence identity to the polypeptide of the invention and functional fragments thereof; more preferably, a functional variant of a polypeptide of the invention and a functional fragment thereof and the parent polypeptide or
  • the parental functional fragment differs by only 1-3 amino acids; most preferably, the work of the polypeptide of the invention and its functional fragments
  • the variant is only one amino acid different from the parent polypeptide or the parental functional fragment.
  • the invention encompasses amino acid sequences obtained by subjecting a polypeptide of the invention and functional fragments thereof to at least one conservative amino acid substitution.
  • the polypeptides of the invention and functional fragments thereof may undergo 1, 2, 3, 4, 5, or more conservative amino acid substitutions, but still fall within the scope of the invention.
  • the invention also encompasses amino acid sequences of the parent polypeptide or parental functional fragment of the polypeptide of the invention and functional fragments thereof that undergo at least one non-conservative amino acid substitution.
  • an amino acid sequence of a parent polypeptide or a parental functional fragment having 2, 3, 4, 5, or more non-conservative amino acid substitutions can be included.
  • these amino acid substitutions do not prevent or inhibit the biological function or activity of the polypeptide of the present invention or a functional fragment thereof; more preferably, the amino acid substitution can also enhance the The biological function or activity of the polypeptides of the invention and functional fragments thereof.
  • amino acid substitutions are well known in the art and refer to amino acid substitutions in which one amino acid having a certain physical and/or chemical property is exchanged for another amino acid having the same chemical or physical properties. Those skilled in the art understand that conservative amino acid substitutions may not result in significant changes in the structure or function of the protein.
  • Typical conservative substitutions include, for example, replacing another acidic amino acid (eg, AsP or Glu) with an acidic amino acid, and replacing another amino acid having a non-polar side chain with an amino acid having a non-polar side chain (eg, Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp or Val, etc., replacing another basic amino acid (Lys, Arg, etc.) with a basic amino acid, replacing the other with a polar chain
  • the amino acid of the side chain is substituted with another aromatic amino acid or the like by an aromatic amino acid (Trp, Phe or Tyr).
  • polypeptides (including functional fragments) of the invention and functional variants thereof can be of any length, i.e., can include any number of amino acids, provided that the polypeptide (including functional fragments) and functional variants thereof retain the necessary biological activity.
  • inhibition of HBx activity, inhibition of HBV viral replication and expression at the cellular and animal levels, and treatment of viral hepatitis caused by HBV Use including inhibition of the growth of liver cancer cells in the later stage.
  • a polypeptide of the invention (including functional fragments and functional variants) can be from 4 to 2000 amino acids in length, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 in length.
  • the polypeptide of the invention is 6-20 amino acids in length and meets the requirements for pharmacodynamics and half-life of the polypeptide drug.
  • a functional variant of a polypeptide of the invention and a functional fragment thereof has an amino acid difference from the parent polypeptide SEQ ID NO: 1, which has similar biological activities and functions as the parent polypeptide SEQ ID NO: 1.
  • inhibition of the activity of HBx and effective inhibition of cancer cells are preferably liver cancer cells, particularly liver cancer cells expressing HBx.
  • the polypeptides (including functional fragments) and functional variants thereof of the invention described herein may also comprise a cell-penetrating peptide (CPP).
  • CPP cell-penetrating peptide
  • the CPP promotes passage of a polypeptide of the invention across a cell membrane and into a cell.
  • CPPs are known in the art. See, for example, Deshayes et al., Cell. Mol. Life Sci. 62: 1839-1849 (2005); EI-Andaloussi et al., Modern Drug Design (Curr. Pharm. Design.) 11: 3597-3611 (2005).
  • the CPP can be any of those known in the art.
  • Polypeptides (including functional fragments) and functional variants thereof of the invention may also, for example, be lipidated (eg, fatty acidated), glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, Cyclization by disulfide bonds, conversion to acid addition salts, dimerization or multimerization, and/or conjugation.
  • lipidated eg, fatty acidated
  • glycosylated glycosylated, amidated, carboxylated, phosphorylated, esterified
  • N-acylated N-acylated
  • Cyclization by disulfide bonds conversion to acid addition salts, dimerization or multimerization, and/or conjugation.
  • Polypeptides (including functional fragments) of the invention and functional variants thereof, including derivatives thereof such as fatty acid derivatives, may also be monomeric peptides, dimeric peptides or multimeric peptides.
  • Polypeptides (including functional fragments) and functional variants thereof of the invention can be obtained by methods known to those skilled in the art (see, for example, Chan et al, Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, UK, 2005; Reid, R., Peptide and Protein Drug Analysis, Marcel Dekker Company, 2000; and U.S. Patent No. 5,449,752). Furthermore, it can also be obtained by producing a polypeptide by a nucleic acid recombinant method (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Port, New York, 2001).
  • polypeptides of the invention can be isolated and/or purified from, for example, plants, bacteria, insects, mammals such as rats, humans, and the like. Methods of separation and purification are also well known in the art.
  • polypeptides described herein, including functional fragments thereof, as well as functional variants can be purchased commercially from commercial companies.
  • the invention also encompasses conjugates, for example, bioconjugates comprising the polypeptides of the invention (including functional fragments and functional variants) as well as peptidomimetics.
  • conjugates for example, bioconjugates comprising the polypeptides of the invention (including functional fragments and functional variants) as well as peptidomimetics.
  • Conjugates, as well as methods for the general synthesis of conjugates are also known in the art (see, for example, Hudecz, F., Methods Mol. Biol. 298:209-223 (2005) and Kirin et al., Inorg. Chem. 44(15): 5405-5415 (2005).
  • polypeptides including functional fragments and functional variants thereof, and conjugates (collectively referred to as "the substances of the present invention") may be isolated, purified, synthesized, and/or Or reorganized.
  • compositions such as pharmaceutical compositions.
  • the invention provides pharmaceutical compositions comprising any of the polypeptides (including functional fragments and functional variants) as well as peptidomimetics, and pharmaceutically acceptable carriers.
  • a pharmaceutical composition of the invention comprising any of the agents of the invention may comprise more than one substance of the invention, for example two or more different polypeptides.
  • the pharmaceutical composition may include a combination with another pharmaceutically active agent or drug.
  • the other or more pharmaceutically active agents or drugs may preferably include, for example, a chemotherapeutic agent, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine , hydroxyurea, methotrexate, paclitaxel, rituxima Infliximab, vinblastine, vincristine, etc.
  • a chemotherapeutic agent for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine , hydroxyurea, methotrexate, paclitaxel, rituxima Infliximab, vinblastine, vincristine, etc.
  • the pharmaceutical composition comprises a substance of the invention in combination with a lipid.
  • the lipid may be any lipid, including fatty acids, phospholipids, sterols, sphingolipids, guanidines, glycerides, glycerophosphates, prenol lipids, glycolipids, and polyketides. Such lipids are known in the art.
  • the pharmaceutically acceptable carrier may be any of those conventionally used, and they may be defined only by chemical physical factors such as solubility and lack of reactivity with the active compound, and by route of administration.
  • the pharmaceutical carriers of the present invention for example, vehicles, adjuvants, excipients, diluents and the like, are well known to those skilled in the art and are readily available to the public.
  • the pharmaceutically acceptable carrier is chemically inert to the active agent and does not have deleterious side effects or toxicity under the conditions of use.
  • the choice of carrier should be determined by the particular material of the invention, as well as by the particular method used to administer the materials of the invention.
  • suitable formulations of the pharmaceutical compositions of the invention are exemplary and are not intended to be limiting in any way.
  • the agents of the invention may be administered using more than one route, and in certain instances, a particular route may provide a more direct and effective method than the other route.
  • the pharmaceutical composition is a topical formulation, an intravenous formulation, or a subcutaneous formulation.
  • the pharmaceutical combination is a topical formulation.
  • Topical formulations are well known to those skilled in the art.
  • the formulation is particularly suitable.
  • the topical preparation of the present invention may be, for example, a paste, lotion, ointment, patch, oil, paste, spray, such as an aerosol spray, a gel, a roll-on liquid, a solid stick or the like.
  • the topical formulations of the invention are ointments, lotions, ointments, or patches.
  • Formulations suitable for oral administration can be composed of: a) a liquid solution, such as An effective amount of a substance of the invention dissolved in a diluent such as water, saline, or orange juice; b) capsules, tablets, troches, etc., each comprising a predetermined amount of a solid or granular active ingredient; c) a powder ; d) a suspension in a suitable liquid; and e) a suitable emulsion.
  • Liquid preparations may include diluents such as water and alcohols, for example, ethanol, benzyl alcohol, and polyethylene glycol, with or without the addition of a pharmaceutically acceptable surfactant.
  • the capsule form can be a conventional hard or soft shell gelatin type comprising, for example, a surfactant, a lubricant, and an inert filler such as lactose, sucrose, calcium phosphate, and corn starch.
  • Tablet forms may include one or more of the following: lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, gum arabic, gelatin, guar gum, gelatinous dioxide Silicon, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffers, disintegrants, wetting agents, preservatives, flavoring An agent that is compatible with other drugs.
  • Tablet forms may include the materials of the invention in which the flavoring agent is typically in sucrose or gum arabic, and soft lozenges comprising the materials of the invention in an inert matrix such as gelatin and glycerin, or sucrose and gum arabic, additionally comprising Emulsions, gels and the like of excipients known in the art.
  • the flavoring agent is typically in sucrose or gum arabic
  • soft lozenges comprising the materials of the invention in an inert matrix such as gelatin and glycerin, or sucrose and gum arabic, additionally comprising Emulsions, gels and the like of excipients known in the art.
  • the agents of the invention can be formulated as an aerosol formulation for administration by inhalation.
  • aerosol formulations can be placed in a pressurized, usable medium such as dichlorodifluoromethane, propane, nitrogen, and the like. They can also be formulated as non-pressurized formulations, such as in nebulizers or nebulizers.
  • the spray formulation can also be used to spray into the mucosa.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injections, which may contain an antioxidant, a buffer, a bacteriostatic agent, and a solute which renders the formulation isotonic with the blood of the intended recipient, and Aqueous and non-aqueous sterile suspensions, which may include suspensions, solubilizers, thickeners, stabilizers, and preservatives.
  • the materials of the invention may be employed in physiological diluents in pharmaceutical carriers such as sterile liquid or liquid mixtures including water, saline, aqueous dextrose and related sugar solutions, alcohols such as ethanol or cetyl alcohol, glycols such as propylene glycol Or polyethylene glycol, dimethyl sulfoxide, glycerol, ketal such as 2,2-dimethyl-1,3-dioxolan-4-methanol, ether, oil, fatty acid, fatty acid ester or glyceride, Or acetylated fatty acid glycerides, with or without the addition of pharmaceutically acceptable surfactants such as soaps or detergents, suspensions if glued, Methylcellulose, hydroxypropylmethylcellulose, or nitromethylcellulose, or an emulsifier and other pharmaceutically acceptable adjuvants.
  • pharmaceutical carriers such as sterile liquid or liquid mixtures including water, saline, aqueous dextrose and related sugar solutions, alcohol
  • Oils that can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of the oil include peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petroleum, and mineral oil. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid.
  • Suitable soaps for use in parenteral formulations include fatty alkali metal and triethanolamine salts and the like, and contain suitable soil release agents, for example a) cationic detergents: dimethyldialkylammonium halides and alkyl halide pyridines; b) anionic detergents: alkyl, aryl, olefin sulfonates, alkyls, olefins, ethers, monoglycerol sulfates and sulfosuccinates; c) nonionic detergents: fatty amine oxides , fatty acid alkanolamides and polyoxyethylene polypropylene copolymers, etc.; d) amphoteric detergents: alkyl- ⁇ -aminopropionates and 2-alkyl-imidazoline quaternary ammonium salts; e) mixtures thereof.
  • suitable soil release agents for example a) cationic detergents: dimethyldialkylammonium hal
  • the parenteral preparation may contain from about 0.5% to about 25% by weight of the substance of the invention in solution. Preservatives and buffers can be used. To minimize or eliminate irritation at the site of injection, the composition may comprise one or more hydrophilic-lipophilic balance (HLB) nonionic surfactants.
  • HLB hydrophilic-lipophilic balance
  • the surfactant is used in the formulation in an amount of from about 5 to 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters and high molecular weight adducts of ethylene oxide and hydrophobic groups formed by the condensation of propylene oxide and propylene glycol.
  • parenteral formulation can be presented in unit or multi-dose sealed containers and can be stored under lyophilized (lyophilized) conditions, requiring only the addition of sterile liquid excipients such as water for injection prior to use.
  • Temporary injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • the substance of the invention or a composition comprising the substance of the invention may also be formulated as an injectable preparation.
  • the need for an injectable composition for an effective pharmaceutical carrier is known to those of ordinary skill in the art. Known.
  • the cell when administered to a cell, such as a dendritic cell, the cell is administered by injection.
  • inventive substance of the present invention can be prepared as a suppository by mixing with various bases such as an emulsifying base or a water-soluble base.
  • bases such as an emulsifying base or a water-soluble base.
  • Formulations suitable for, for example, vaginal administration may be presented as pessaries, tampons, ointments, gels, pastes, foams, or spray formulations, in addition to the active ingredients thereof, those which are known in the art .
  • inventive materials of the present invention may be formulated to comprise a complex, such as a cyclodextrin comprising a complex, or a liposome.
  • the amount or dose of the substance of the invention administered should produce, for example, a therapeutic or prophylactic response in a subject or animal within a reasonable time frame.
  • the dose of the substance of the present invention should be sufficient to inhibit the proliferation of diseased cells during the period of about 2 hours or more, such as 12-24 hours or longer from the time of administration, to treat or prevent the disease (eg, tumor, cancer). The role of etc.). In certain embodiments, the time period may even be longer.
  • the dosage should be determined by the efficacy of the particular substance of the invention and the condition of the animal (e.g., human) to be treated, as well as the weight of the animal (e.g., human). Many assays for determining the dosage to be administered are known in the art.
  • the dosage of the substance of the invention will also be determined by the presence, nature and extent of any side effects that may accompany the administration of a particular subject of the invention.
  • a substance of the invention can be conjugated to a targeted moiety either directly or indirectly via a linker.
  • the practice of conjugating a compound, for example, a substance of the invention, to a targeting moiety is well known in the art. See, for example, Wadwa et al., J. Drug Targeting 3: 111, (1995) and U.S. Patent No. 5,087,616.
  • the substance of the invention may be modified to a depot shape
  • the manner in which the substance of the invention is released into the body to which it is administered is controlled in terms of time and body parts (see, e.g., U.S. Patent No. 4,450,150).
  • the depot form of the substance of the invention may be, for example, an implantable composition comprising a substance of the invention and a porous or non-porous material such as a polymer, wherein the substance of the invention is degraded by said substance and/or said non-porous substance And spread.
  • the depot is then implanted into the desired site in the body and the material of the invention is released from the implant at a predetermined rate.
  • the pharmaceutical composition of the present invention comprising a polypeptide (including a functional fragment) and a functional variant thereof, can be used for preventing and inhibiting chronic liver disease induced by hepatitis B virus infection, including viral hepatitis, and resulting cirrhosis and In the method of liver cancer.
  • chronic liver disease induced by the hepatitis B virus of the present invention may be present in any host.
  • the host is a mammal.
  • the host is a human.
  • the invention synthesizes the amino acid sequence Gly-D-Ser-D-Ala-D-Val-D-Met-D-Phe-D-Ser-D-Ser-D-Lys-D-Glu- by artificial synthesis.
  • D-Arg-Gly (SEQ ID NO: 1) type D polypeptide hereinafter referred to as D-TTK001.
  • the preparation of the polypeptide adopts a solid phase synthesis method, such as the application of AAPPTEC Apex 396 polypeptide synthesis instrument (purchased from Hong Kong Global Analytical Testing Instrument Co., Ltd.), and the amino acid according to the sequence shown in SEQ ID NO: 1 in a closed explosion-proof glass reactor.
  • the synthetic peptide D-TTK001 was analyzed by high pressure liquid chromatography (HPLC) (using a PLC Agela C18 column), and Figure 1 shows that the purity obtained was 97.2%.
  • Embodiment 2 is a diagrammatic representation of Embodiment 1:
  • the recombinant plasmid expressing HBx gene (pET-30a-HBx) was self-constructed and preserved (Zhang H, et al. J Biomed Biotechnol, doi:10.1155/2009/289068), and the expression and purification of HBx were completed by the method in this literature.
  • For in vitro binding force detection The in vitro binding of the polypeptide D-TTK001 to the recombinantly expressed HBx protein was measured using a Biacore 3000 Biomacromolecule Interaction Analyzer (manufactured by GE Healthcare). Different concentrations of polypeptide were injected at a rate of 30 ⁇ L/min for 180 seconds.
  • HBS-EP buffer was injected at a rate of 30 ⁇ L/min for 900 seconds, followed by injection of 2 needles of 1 mM NaOH for 20 seconds at a flow rate of 30 ⁇ L/min for chip regeneration. All signals are corrected via channel 1 as a control channel. Binding force assays were tested twice, and the first test of HBX was injected into the channels and blank channels of the coupled polypeptide at concentrations of 0 nM, 10 nM, 50 nM, 100 nM, 500 nM, and 600 nM, respectively. The combination time is 180 seconds and the dissociation time is 400 seconds.
  • the surface of the chip was regenerated by injecting 1 mM NaOH for 20 seconds at a flow rate of 30 ⁇ l/min. Results The kinetic analysis was performed via software as shown in Figure 2. The second test, HBX, was injected into the channels and blank channels of the coupled polypeptide at concentrations of 0 nM, 10 nM, 50 nM, 588 nM, 1 uM, and 2 uM, respectively. The combination time is 180 seconds and the dissociation time is 400 seconds. The surface of the chip was regenerated by injecting 1 mM NaOH for 20 seconds at a flow rate of 30 ⁇ L/min. The experimental results were subjected to kinetic analysis using BIA Evaluation Software software.
  • Enzyme-linked immunosorbent assay was used to detect the effect of D-TTK001 on the secretion of HBeAg from hepatoma cells.
  • Paving 96-well plate Under sterile conditions, pour off the old medium, add 3 mL of PBS each time, wash twice, add 1 mL of 0.05% trypsin, digest it under the microscope to observe the rounding of the cells, add 10 mL of medium, The cells were blown off, counted on a hemocytometer plate, and diluted with DMEM/F12 (1:1) medium, and 100 ⁇ L of the cell suspension was added to each well to make the number of cells reach 1-2 ⁇ 10 3 cells/well; After the culture is carried out until the cells are attached (usually 12h), the angle is extended, and the cells are in good condition, the subsequent test can be performed.
  • the 10 mM D-TTK001 (or L-TTK001) mother liquor was diluted stepwise from high concentration to low concentration, and added to the liver cancer HepAD38 cells (or HepG2.2.15 cells) culture medium to make each group D-TTK001 (or L-TTK001)
  • the concentrations were 0.1 ⁇ M, 1 ⁇ M, 10 ⁇ M, and 100 ⁇ M; negative control group: 10 ⁇ L of sterile ddH 2 O was added; and culture was carried out for 72 hours at 37 ° C in a 5% CO 2 incubator.
  • Termination 50 ⁇ L of the stop solution was added to each well, and the mixture was gently shaken and mixed. The measurement result was measured within 10 minutes: the wavelength of the microplate reader was set at 450 nm, and the OD value of each well was measured after blanking with a blank hole.
  • D-TTK001 inhibited HBeAg levels secreted by HepAD38 cells (or HepG2.2.15 cells) in a dose-dependent manner.
  • the minimum effective dose of D-TTK001 is 0.1 ⁇ M; the minimum effective dose of L-TTK001 is 10 ⁇ M. Therefore, the effect of D-TTK001 on HBeAg is better than that of L-TTK001 on HBeAg. *P ⁇ 0.05, **P ⁇ 0.01, and Student's t test statistical analysis was performed.
  • Inoculated cells HepAD38 cells in logarithmic growth phase were mixed into a single cell suspension with RPMI1640 medium containing 10% fetal bovine serum, and seeded into 96-well cell culture plates at 4000-5000 cells per well. The pore volume was 100 ⁇ L.
  • D-TTK001 and L-TTK001 had a significant inhibitory effect on the growth and proliferation of HepAD38 cells in a dose-dependent manner.
  • the minimum effective dose of D-TTK001 and L-TTK001 was 10 ⁇ M. *P ⁇ 0.05, **P ⁇ 0.01, and Student's t test statistical analysis was performed.
  • HBV transgenic mice were purchased from Guangzhou Junketai Pharmaceutical Technology Co., Ltd. The animal model was successfully established by Guangzhou Junketai Pharmaceutical Technology Co., Ltd. by using microinjection method to cultivate a high expression replicative 1.3 copy HBV whole gene transgenic mouse Tg (HBV1.3genome) Swb. Serum HBsAg and HBeAg can be detected by conventional ELISA kits; serum HBV DNA of 93.93% positive transgenic mice has reached 10 4 -10 6 copy/mL; liver tissue immunohistochemistry has HBsAg (cytoplasmic type) and HBcAg (karyotype) expression. .
  • D-TTK 001 was prepared aseptically, and the drug was injected into the tail vein at a dose of 2.5 mg/kg (total of 10) and 5.0 mg/kg (total of 42 mice), respectively, in a total volume of 50 ⁇ L; 50 ⁇ L of sterile PBS was used as a blank control group (10 in total); 6-8 weeks old mice, half male and half female.
  • Sample processing (negative control, positive control, quantitative reference and sample to be tested simultaneously); add 5 ⁇ L of nucleic acid release agent to each PCR reaction tube, then add 5 ⁇ L of each sample to be tested, and mix for 3-5 times; At intervals of 10 minutes or more, 40 ⁇ L of PCR-mix was added to each tube, and the tube lid was capped and centrifuged at 2000 rpm for 30 seconds.
  • the real time PCR detection HBV DNA kit produced by Sun Yat-sen University Daan Gene Co., Ltd. was used for the detection.
  • the specific steps were carried out according to the kit instructions as follows: The PCR reaction tube was placed in the sample chamber of the amplification instrument, and the negative control was set in the corresponding order.
  • fluorescence detection channel selection Take ABI 7300 instrument as an example: 1) select FAM channel (Reporter: FAM, Quencher: none) HBV-DNA was detected; 2) HBV-internal standard was selected by selecting HEX/VIC channel (Reporter: HEX/VIC, Quencher: none); 3) Passive Reference was set to ROX.
  • the loop parameters are set as follows:
  • Analysis condition setting According to the analyzed image, adjust the start value, stop value and Threshold value of Baseline (user can adjust according to the actual situation, the start value can be in 1 ⁇ 10, the stop value can be in 5 ⁇ 10, the Value value can be in 0.01 to 0.2 range selection), the standard curve under the Std curve window is optimized, that is, the correlation value is between -1.0 and -0.97. Select Analyze to automatically analyze the results under the Analysis menu. Go to the Tray window and record the unknown variable value (C). "C" indicates the concentration or content of the sample.
  • Negative control no Ct value display; but HBV-inner wall test is positive (Ct value ⁇ 40).
  • Positive control product The detected concentration value is between 1.26 ⁇ 10 5 and 1.26 ⁇ 10 6 IU/ml.
  • the real time PCR method was used to detect the HBV DNA copy number in the serum of HBV transgenic mice.
  • the results of the 2.5mg/kg group the effective rate from the first week (T1+ group) was 30% (3/10), showing a time-dependent manner; the effective rate of 5.0 mg/kg group was 54.8% (23/ 42)).
  • the effective rate from the first week (T1+ group) was 46.2% (18/39), with a significant time-dependent decline rate of 30.4% (7/23).
  • a time-dependently decreasing comparison of three 2.5 mg/kg groups and seven 5.0 mg/kg results in a dose-dependent manner in which D-TTK001 inhibits HBV replication at the level of HBV transgenic mice.
  • mice were sacrificed, liver tissues were taken, formalin was fixed, and embedded in a conventional method to prepare tissue sections for pathological observation.
  • the therapeutic effect of the synthetic polypeptide D-TTK001 on HBV transgenic mouse hepatitis As shown in Figure 7, the therapeutic effect of the synthetic polypeptide D-TTK001 on HBV transgenic mouse hepatitis.
  • the left picture shows the liver pathology of HBV transgenic mice, and 6 of 10 HBV transgenic mice in the untreated control group showed typical viral hepatitis such as hepatic balloon-like degeneration. Pathological characterization; the right picture shows liver tissue after 3 weeks of tail vein injection with D-TTK001 (5mg/kg). There was no hepatic balloon-like degeneration in the experimental group, suggesting that D-TTK001 is a pathological hepatitis in liver tissue. The lesion has a significant therapeutic effect.
  • the HepG2-X cells in the logarithmic growth phase were digested with trypsin to prepare a cell suspension, and the number of cells was counted, diluted to 1 ⁇ 10 7 cells/mL with sterile physiological saline cells, and stored in ice water.
  • Two female BALB/C nude mice of 4 to 6 weeks were used: 1 control group 1 , 0.2 mL of the above diluted cells were injected subcutaneously into the right forelimb of each mouse, and only 0.5 mL of sterilized distilled water (without polypeptide) was injected.
  • Drug 2 experimental group 1 (administered dose is 10 mg / kg body weight).
  • 0.2 mL of the above diluted cells were injected subcutaneously into the right forelimb of the mouse. After 20 days of injection, the tumor tissues were aseptically removed, cut into small tissue blocks, and returned to female BALB/C nude mice for 4 to 6 weeks, a total of 18 rats.
  • D-TTK001 has the effect of inhibiting the malignant phenotype of HepG2-X cells in nude mice.
  • P ⁇ 0.5 Log-Rank statistical analysis.
  • the invention also explores the role of functional variants of polypeptides and functional fragments thereof.
  • the sequence shown in the table below is a D-type amino acid substitution artificially synthesized based on L-TTK001 (ie, anti-HBxP1#).
  • the polypeptide fragment was artificially synthesized according to the sequence (method as above), and then the change in the action of the obtained polypeptide was observed by the above ELISA method.
  • D-TTK001 and D-TTK001-1, D-TTK001-2, D-TTK001-3, D-TTK001-4, D-TTK001-5, D-TTK001-6, D- TTK001-7, D-TTK001-8, D-TTK001-9 and D-TTK001-10 have different degrees of inhibitory effect on HBeAg levels secreted by HepAD38 cells, but D-TTK001 has the most obvious effect, suggesting single replacement artificial Synthetic D-amino acid makes L-TTK001 (ie anti-HBxP1#) Changes in the conformation of a polypeptide have varying degrees of effect on its biological activity.
  • D-TTK001 is similar to L-TTK001 in biological activity, suggesting that this symmetrical isomer retains its ability to bind to the target protein HBx. *P ⁇ 0.05, **P ⁇ 0.01, and Student's t test statistical analysis was performed.
  • D-TTK001 solution was administered to the tail vein at 3.6 mg/kg (equivalent to 5 mg/kg of mice).
  • Blood was taken from heparinized tubes at the time of blood collection, and the rats were determined by Triple-TOF mass spectrometry. D-TTK001 blood concentration.
  • Example 6 Acute toxicity test of polypeptide drugs
  • the acute toxicity test of the peptide drug showed that the mouse showed no abnormal performance. There was no abnormal change in body weight compared with the control group.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Virology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Biophysics (AREA)
  • Communicable Diseases (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Oncology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本申请提供了一种抗乙型肝炎病毒X蛋白的多肽及其药物组合物。所述多肽包含D型氨基酸，可抑制乙型肝炎病毒DNA的复制和相关抗原的表达，进而抑制乙型肝炎病毒感染所造成的肝炎、肝硬变以及肝癌。

Description

抗乙型肝炎病毒X蛋白多肽药物 技术领域
本发明涉及多肽药物领域，具体涉及含D型氨基酸的抗乙型肝炎病毒X蛋白的多肽及其应用。
背景技术
肝癌是导致病人死亡的恶性肿瘤之一，其恶性程度高，在我国肝癌死亡率仅次于胃癌，居我国恶性肿瘤死亡率第二位。据统计，我国每年新增肝癌人数30万人，而每年肝癌死亡人数达11万。乙型肝炎病毒(hepatitis B virus，HBV)感染可导致肝炎、肝硬变和原发性肝癌的发生，在我国80％以上的肝癌患者为乙肝感染后肝癌，可称之为乙肝后肝癌。
HBV为长约3.2Kb的DNA病毒，其开放阅读框表达乙型肝炎病毒表面抗原(HBsAg)、乙型肝炎病毒核心抗原(HBcAg)、乙型肝炎病毒多聚酶和乙型肝炎病毒X抗原(HBxAg)(或称之为乙型肝炎病毒X蛋白,即HBx)，其中HBx是HBV DNA复制所必须的因子。由于HBx是HBV DNA复制所必须的因子，因此抑制HBx的功能，意味着可抑制HBV的感染，以及由此导致的肝炎和肝硬变。
另外，HBx作为反式作用因子，促进肝癌的生长和增殖，称之为癌蛋白。在分子水平、细胞水平和动物水平的研究也发现，HBx具有很强的促肝癌细胞增殖和迁移的作用。转基因鼠实验也证明HBx具有显著的促进肝癌发生的作用。大量研究结果显示，HBV感染的持续存在可导致慢性肝病，包括慢性肝炎、慢性肝炎反复发作引起的肝组织中纤维结缔组织增生后发生的肝硬变，及大部分在肝硬变基础上发生的肝癌。在慢性肝病(包括肝炎、肝硬变和肝癌)的发生发展过程中， HBx发挥了重要的作用。因此，HBx是预防和治疗肝病的重要靶点。
目前，肝癌的治疗以手术为主，辅以介入疗法，而化疗效果不理想。在临床手术的肝癌组织中HBsAg和HBxAg的表达阳性检出率高达80％甚至90％以上。因此，HBx作为肝癌发生发展的重要致病因子，而发现其特异性抑制剂，则具有重要的理论意义和实际临床应用价值。然而，由于目前HBx的三维构象解析尚未完成，故难以通过HBx立体三维构象设计其化学抑制剂。
多肽片段可作为药物，其在临床上已广为应用。例如，胸腺肽(thymopeptide)是从小牛胸腺中提取的胸腺五肽，具有促进淋巴细胞转化、增强巨噬细胞吞噬活性的作用，可用于治疗多种免疫缺陷病。多肽药物的特点是药理作用明确，安全性高，并易于生产。但是，多肽药物的药效通常并不理想，因其在生物体内易受蛋白酶的影响而降解，稳定度低、半衰期短。
氨基酸可以左旋(“L型氨基酸”)或右旋(“D型氨基酸”)的构象存在。在人体或动物体内构成蛋白质的天然氨基酸全部都是L型的，并没有D型氨基酸。机体中缺乏作用于D型氨基酸的蛋白水解酶。因此，在生成多肽药物时，如果将多肽链上的天然氨基酸由L型替换为D型，即使在不改变氨基酸序列的情况下，也能提高多肽药物在血液中的稳定性。但是如何在提高多肽药物抗降解的同时，不破坏其结合特性，从而保留药效，仍然是困扰着多肽药物开发的大问题。
本发明的发明人曾经发现了一种具有抑制乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)功能的、且均由天然L型氨基酸组成的多肽(详见中国国家发明专利号ZL201110061840.5，其公开内容以全文引用的方式并入于此)。该多肽包含如SEQ ID NO：1所示的氨基酸序列。该多肽在分子水平、细胞水平和动物水平上具有抑制HBx活性的作用，可用于预防和治疗抑制乙型肝炎病毒感染所造成的肝炎、 肝硬变和肝癌。
发明内容
本发明涉及一种抗乙型肝炎病毒X蛋白的多肽及其应用。所述多肽为包含D型氨基酸的多肽，其具有抑制乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)功能，可在分子水平、细胞水平和动物水平上具有抑制HBx活性，抑制乙型肝炎病毒DNA的复制和相关抗原(如HBeAg)的表达，并进而抑制乙型肝炎病毒感染所造成的肝炎、肝硬变，以及在肝硬变基础上发生肝癌。
一方面，本发明提供了分离的多肽，其包含如SEQ ID NO:1所示的氨基酸序列或其功能片段、或者所述氨基酸序列及其功能片段的功能变体，并且所述氨基酸序列、所述功能片段、或所述功能变体的氨基酸序列中的一个或多个L型氨基酸被D型氨基酸所取代，所述多肽具有抑制乙型肝炎病毒X蛋白的功能，并能抑制乙型肝炎病毒感染后的慢性肝病的发生和发展。
优选地，本发明的所述多肽的氨基酸序列具有与SEQ ID NO:1所示的氨基酸序列至少70％(优选至少80％、85％、90％、95％、99％或更高)的相同性。
在本发明的一些具体实施例中，所述多肽的氨基酸序列为如SEQ ID NOs:2-11所示的氨基酸序列中的任一种。
在本发明中，所述乙肝病毒感染后的慢性肝病包括肝炎、肝硬变和肝癌。
另一方面，本发明提供了所述多肽在制造抗乙肝病毒感染后的慢性肝病的药物中的应用。具体来说，所述药物可为乙肝的治疗性疫苗。所述药物可以为包含本发明任一种多肽的药物组合物，其可以包含任 选的药物载体。
和具有相同氨基酸序列但全部由L型氨基酸组成的多肽相比，本发明的多肽不单具有良好的稳定性，更有着令人惊异的显著药学效果，对乙型肝炎病毒DNA的复制和相关抗原(如HBeAg)的表达的抑制效果尤其明显。所述多肽因此可广泛用于防治乙型肝炎病毒感染所引发的慢性肝病，包括肝炎、肝硬变和肝癌。
附图说明
图1.应用高压液相色谱(HPLC)对纯化后人工合成多肽D-TTK001的分析结果。
图2.应用Biacore 3000检测本发明的多肽与靶蛋白HBx的体外结合力的实验结果。
图3.检测本发明的多肽在细胞水平对整合HBV DNA肝癌HepAD38细胞分泌HBeAg水平的影响。结果显示，分别应用人工合成的0.1、1、10和100μM多肽D-TTK001和L-TTK001(即anti-HBxP1#)作用48小时后，对整合HBV DNA肝癌HepAD38细胞分泌的HBeAg水平均具有抑制作用，呈剂量依赖性。D-TTK001的最低有效剂量为0.1μM；L-TTK001的最低有效剂量为10μM。因此，D-TTK001对HBeAg的作用优于L-TTK001对HBeAg的作用。*P<0.05，**P<0.01，进行Student’s t test统计学分析。
图4.检测本发明的多肽在细胞水平对HepG2.2.15细胞分泌HBeAg水平的影响。结果显示，分别应用人工合成的0.1、1、10和100μM多肽D-TTK001和L-TTK001(即anti-HBxP1#)作用48小时后，对HepG2.2.15细胞分泌的HBeAg水平均具有抑制作用，呈剂量依赖性。D-TTK001的最低有效剂量为0.1μM；L-TTK001的最低有效剂量为10μM。因此，D-TTK001对HBeAg的作用优于L-TTK001对HBeAg的作用。*P<0.05，**P<0.01，进行Student’s t test统计学分析。
图5.应用MTT检测本发明的多肽在细胞水平对整合HBV DNA肝癌细胞HepAD38增殖的影响。结果显示，分别应用人工合成的0.1、 1、10和100μM多肽D-TTK001和L-TTK001(即anti-HBxP1#)作用48小时后，对整合HBV DNA肝癌细胞HepAD38的增殖均具有抑制作用，呈剂量依赖性。*P<0.05，**P<0.01，进行Student’s t test统计学分析。
图6.检测本发明多肽D-TTK001对HBV转基因鼠的影响。结果显示，分别应用人工合成的多肽D-TTK001尾静脉注射HBV转基因鼠，分为2.5mg/kg和5.0mg/kg两个剂量组，阴性对照组为尾静脉注射PBS。然后，应用real-time PCR方法检测转基因鼠血清中HBV DNA的拷贝数。结果显示，D-TTK001导致HBV转基因鼠血清中HBV DNA的拷贝数明显减少，呈时间依赖和剂量依赖性，提示D-TTK001具有抑制HBV DNA复制的作用。*P<0.05，**P<0.01，进行Student’s t test统计学分析。
图7.人工合成的多肽D-TTK001对HBV转基因鼠肝炎的治疗效果。左图为HBV转基因鼠肝脏病理组织，未处理对照组中10只HBV转基因鼠中有6只，表现为典型的肝气球样变性等病毒性肝炎病理表征；右图为应用D-TTK001(5mg/kg)尾静脉注射3周后的肝组织，实验组6只中均无肝气球样变性表现，提示D-TTK001对肝组织中的病理性肝炎病变有明显的治疗作用。
图8.人工合成的多肽D-TTK001对肝癌荷瘤裸鼠生存率的影响。动物实验检测结果表明，应用人工合成的D-TTK001多肽0.1mg/kg组和5.0mg/kg组对HepG2-X细胞回接瘤裸鼠的存活率均具有明显的延长作用，提示D-TTK001具有抑制裸鼠体内HepG2-X细胞回接瘤恶性表型的作用。*P<0.5，Log-Rank统计学分析。
图9.检测本发明的多肽在细胞水平对整合HBV DNA肝癌细胞HepAD38分泌HBeAg水平的影响。结果显示，分别应用人工合成的10μM多肽D-TTK001和如SEQ ID NOs：2-11所示(如D-TTK001-1、D-TTK001-2、D-TTK001-3、D-TTK001-4、D-TTK001-5、D-TTK001-6、D-TTK001-7、D-TTK001-8、D-TTK001-8、D-TTK001-9、D-TTK001-10)作用48小时后，对整合HBV DNA肝癌细胞HepAD38分泌的HBeAg水平均具有抑制作用。*P<0.05，**P<0.01，进行Student’s t test统计 学分析。
具体实施方式
本发明中，包括说明书和权利要求书，使用的下列术语除非特别说明，具有如下的含义：
在本发明中，将用缩写来描述氨基酸，包括L-氨基酸和D-氨基酸。表1提供了这里使用的缩写表：
表1缩写表
Figure PCTCN2015092132-appb-000001
本发明中，在多肽的氨基酸序列中，以符号“D-*”表示在多肽的该特定位处取代原L型氨基酸的任何D型氨基酸。
作为参考，氨基酸可以左旋(“L型氨基酸”)和右旋(“D型氨基酸”)构象存在。L型氨基酸为天然存在于自然界和大多数生物系统中的。此外，天然存在的多肽由L型氨基酸构成，因而可称为L型多肽。D型氨基酸为其L型氨基酸对应物的“镜像”，尽管D型多肽(完全由D型氨基酸构成的多肽)不是天然存在的，可人工合成这些多肽以形成三维的蛋白质结构。
“分离的”一词是指将物质从它原始的环境(例如，若是自然产生的就指其天然环境)分离出来。比如说，一个自然产生的多肽存在于活动物中就是没有被分离出来，而同样的多肽同一些或全部在自然系统中与之共存的物质分开就是分离的。这样的多肽可以是某一载体的一部分，也可以是某一组合物的一部分。既然载体和组合物不是它的天然环境的成分，它们仍然是分离的。
“纯化的”一词意指已经在纯度上提高的。“纯度”在这是相对术语，并不必要地解释为绝对纯度。例如，纯度可以是至少约50％，或可以是大于60％、70％、80％、90％、或可以是100％。
如本发明中所用，分离的物质是从其原始环境中分离出来。活体细胞内的天然状态下的多肽是没有分离的，但同样的多肽如从天然状态中同存在的其他物质中分开，则为分离的，同时纯度上得到了提高，也因此是纯化的。
所谓“氨基酸序列”或“多肽”是指肽、寡肽、多肽或蛋白质及其部分片段，其间以肽键相连接的氨基酸。当本发明中的“氨基酸序列”涉及一种天然存在的蛋白质分子，或一种已经被描述过的人工合成的多肽时，这种天然存在的蛋白质分子或已知的多肽并不意味着将 氨基酸序列限制为与所述蛋白质分子或已知多肽完全相同的完整的、天然的氨基酸序列。本发明的氨基酸序列可以含有附加的肽，如多组氨酸标签(His-tag)、或Myc、FLAG等表位标记的肽；本发明的氨基酸序列也可以包含人工合成的D型氨基酸，其取代所述蛋白质分子或已知多肽的氨基酸序列中的一个或多个L型氨基酸。
多肽的“功能片段”是指本发明的多肽的任何部分，所述部分保留其作为一部分的原始多肽(也即“亲本”多肽)的基本相似或相同的生物学活性和功能。
多肽的“功能变体”是指基本上保持如所述多肽或氨基酸序列相同或相似的生物学功能或活性的氨基酸序列，它们可以包括，例如，1)原氨基酸序列中一个或多个氨基酸有缺失和/或一个或多个氨基酸被添加；或者2)原氨基酸序列中一个或多个氨基酸被保守或非保守氨基酸取代；或者3)原氨基酸序列中一个或多个氨基酸上的某个基团被其它基团取代；或者4)原氨基酸序列和另外的分子或化合物(比如糖、脂类、聚乙二醇等)的融合；或者5)原有的氨基酸序列与添加的氨基酸序列融合进而形成的多肽序列(如前导序列或分泌序列或用来纯化此多肽的序列等)；或者6)原氨基酸序列的逆反类似物；或者7)以上各种情况的混合。
其中，“缺失”是指在氨基酸序列中一个或多个氨基酸的缺失。
“插入”或“添加”是指在氨基酸序列中的改变导致与天然存在或改变前的分子相比，一个或多个氨基酸的增加。
“置换”是指由不同的氨基酸替换一个或多个氨基酸。
“缺失、置换或添加一个或多个氨基酸”则是指，利用定位诱变法等公知的突变多肽的制作法缺失、置换或添加能够缺失、置换或添 加的程度的数目的氨基酸。上述突变不限于利用已知的定点突变法而人为诱导的突变，也可以是对天然存在的核酸或蛋白质的突变进行分离纯化而得到的。
氨基酸序列的“相同性”或“同一性”百分率是指在两种或多种氨基酸比较中，序列相同或相似的百分率。有很多本领域技术人员熟知的方法测定相同性百分率，如通过MEGALIGN程序(Lasergene software package,DNASTA,Inc.,Madison,WI)。MEGALIGN程序可根据不同的方法如Cluster法比较两种或多种序列(参见Higgins&Sharp，Gene 73:237-244,(1988))，Gluster法通过检查所有配对之间的距离将各组序列排列成簇，然后将簇以成对或成组分配。两个氨基酸序列如序列A和序列B之间的相同性百分率可以通过下式计算：
[(序列A与序列B之间匹配的残基个数)/(序列A的残基数-序列A中间隔残基数-序列B中间隔残基数)]×100％
本发明中，“乙肝病毒感染后的慢性肝病”是指乙型肝炎病毒感染后导致的肝病，包括慢性肝炎、慢性肝炎反复发作引起的肝组织中纤维结缔组织增生后发生的肝硬变，及在肝硬变基础上发生的肝癌。
本发明中，“治疗”和“预防”以及由它们派生的词语，但并不意味是100％的或完全的治疗或预防，可以认定为本领域技术人员所认同的治疗或预防程度。本发明中的“预防”可理解为延迟疾病、或其症状或病症的发作。
多肽
本发明提供分离或纯化的多肽。所述多肽是这样一种多肽，其包含将氨基酸序列Gly-Ser-Ala-Val-Met-Phe-Ser-Ser-Lys-Glu-Arg-Gly(如SEQ ID NO:1所示，简称L-TTK001)中的一个或多个天然L型氨基酸置换为人工合成的D型氨基酸后所获得的氨基酸序列。本发明证实这样的多肽可以显著抑制HBx的活性， 从而具有抑制乙型肝炎病毒DNA复制和相关抗原(如HBeAg)表达的作用，并进而抑制乙肝病毒感染后慢性肝病的发生和发展，尤其是肝炎，肝硬化和肝癌的发生和发展。
本发明中，L-TTK001多肽中的任意个L型氨基酸，例如，可以是1个、2个、3个、4个、5个、6个、7个、8个、9个或10个L型氨基酸被相应的D型氨基酸所取代。
根据本发明的一个具体实施例，本发明所述多肽的氨基酸序列是L-TTK001中除甘氨酸以外的10个L型氨基酸均被相应的D型氨基酸所代(即Gly-D-Ser-D-Ala-D-Val-D-Met-D-Phe-D-Ser-D-Ser-D-Lys-D-Glu-D-Arg-Gly，如SEQ ID NO：2所示,简称D-TTK001)。
由于天然的肽酶不能将D型氨基酸用作底物，利用相同类型的D型氨基酸(D型氨基酸是L型氨基酸的旋光异构体)取代多肽上的一个或多个L型氨基酸，有机会能够获得在生物体内更稳定的多肽。例如，研究表明，N末端或C末端的D型氨基酸的存在，就可能提高多肽的体内稳定性(Powell等，Pharm.Res.10:1268-1273(1993))。但由于D型氨基酸和L型氨基酸的手性不同，也即空间立体构像不同，形成“镜影”结构，包含D型氨基酸的多肽并不总是具有如原L型多肽的结合能力和生物效果，因此难以预测氨基酸序列完全相同但其上的氨基酸的构象不同的两个多肽分子在功能上是否一定相同。因此，除非对改性后的包含D型氨基酸的氨基酸序列进行具体的实验，否则没法确定包含了D型氨基酸的多肽是否仍能够具有原多肽的药效。实践中，如需将含有L型氨基酸组成的药物多肽变更为含有D型氨基酸组成的药物多肽，为达到保留和增强其药效学作用，具体如何变更、是替换局部氨基酸还是全部氨基酸、具体哪个位置，这些通常需要技术人员进行大量艰苦地实验反复优化才能完成。
一般说来，含有D型氨基酸残基的多肽，其被D型氨基酸残基取 代的L型氨基酸残基的数目越少，该多肽的结合能力的改变就越小。而大量数目的L型氨基酸被取代，对多肽的结合力和生物效能的改变就会相应增大甚至导致其完全丧失。
但本发明的发明人通过实验发现，多肽L-TTK001中的一个或数个L型氨基酸被D型氨基酸取代，该多肽仍具有和L-TTK001多肽基本相同的生物功能，包括：在细胞水平和动物水平抑制肝炎病毒复制和表达，以及在动物水平治疗肝炎病毒导致的病毒性肝炎，以及抑制后期的肝癌细胞生长。
本发明的发明人还惊讶地发现，即使将L型多肽L-TTK001中的L型氨基酸全部置换为人工合成的D型氨基酸(得到的多肽即为D-TTK001)，该多肽与HBx蛋白的体外结合力并没有明显的变化。例如，根据体外结合力的实验结果，同样条件下，L型多肽L-TTK001和HBx的体外结合力(8nM)与D-TTK001和HBx的体外结合力(35nM)相比并无显著差异。
由于其所含的氨基酸全部为D型氨基酸，D-TTK001多肽同时具有良好的药代性能。因此，发明人发现，可以通过与L型多肽L-TTK001不同的给药方法，例如可以通过动物尾静脉给药，而且可以以比L-TTK001低很多的剂量施用，本发明的多肽例如D-TTK001仍然可以获得明显的药效。
与此相关的，发明人发现，由于本发明的多肽的良好的药代性质和稳定度，其在抑制HBV的DNA复制和相关抗原(如HBeAg)的表达，并进而抑制乙肝病毒感染后慢性肝病(包括肝炎，肝硬化和肝癌)的发生和发展方面，尤其是抑制肝炎的发生和发展方面，具有比L型多肽(例如L-TTK001)更为显著的效果。例如，D型多肽与L型多肽相比，其对HBeAg分泌水平的抑制的有效剂量可以有数量级的差别；而由于其稳定性的改善，在给药方式的选择上，本发明的多肽也比全部 由L型氨基酸组成的多肽，也具有更大的优势。。
本发明还提供所述本发明多肽的各种功能片段。所述功能片段可以为本发明多肽的连续氨基酸序列的任何片段，条件是其与亲本多肽相比，能以相似程度、相同程度、或更高程度保留亲本多肽的生物活性，例如，抑制HBx的活性。参照亲本多肽，所述功能片段可以包括，例如，亲本多肽的约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、100％、105％、110％、120％、150％、200％或更高的活性。所述功能片段还可以在上述连续氨基酸序列的各种片段的氨基或羧基端、或其氨基以及羧基两端，包含额外的氨基酸，例如，与亲本多肽的氨基酸序列中不同的氨基酸。优选是所述额外的氨基酸不妨碍所述功能片段的生物学功能，例如，抑制HBx的活性，抑制HBV病毒复制和表达的作用，并有效地抑制肝炎的发生和发展以及肝癌的发展。更为理想的是，当与所述亲本多肽的生物学活性相比较时，所述额外的氨基酸能够导致增强的生物学活性。优选地，本发明多肽的功能片段的氨基酸序列与本发明多肽的氨基酸序列具有至少70％的序列同一性；更优选地，本发明多肽的功能片段的氨基酸序列与本发明多肽的氨基酸序列具有至少75％、或80％、或85％、或90％、或95％的序列同一性。
另外，本发明的多肽及其功能片段的功能变体也包括在本发明范围之内。所述本发明的多肽及其功能片段的功能变体应该保留与所述亲本多肽或亲本功能片段基本相似或相同的生物活性，例如，抑制HBx的活性，抑制HBV病毒复制和表达的作用，可在HBV转基因鼠动物水平治疗HBV导致病毒性肝炎的作用，以及抑制肝癌细胞恶性表型的作用。参照本发明的多肽及其功能片段，所述功能变体可以与所述亲本多肽或亲本功能片段的氨基酸序列至少有约50％、60％、70％、80％、90％、95％或100％的同一性。优选地，所述功能片段的氨基酸序列与本发明的多肽及其功能片段具有至少70％的序列同一性；更优选地，本发明的多肽及其功能片段的功能变体与所述亲本多肽或亲本功能片段只有1-3个氨基酸的区别；最优选地，本发明的多肽及其功能片段的功 能变体与所述亲本多肽或亲本功能片段只有1个氨基酸的区别。
例如，本发明包括本发明多肽及其功能片段经历至少一个保守氨基酸置换而获得的氨基酸序列。具体来说，所述本发明的多肽及其功能片段可以经历1个、2个、3个、4个、5个、或更多个保守氨基酸置换，但仍然属于本发明的范围。另外，本发明也包括所述本发明的多肽及其功能片段经历至少一个非保守氨基酸置换的亲本多肽或亲本功能片段的氨基酸序列。具体来说，可以包括具有2个、3个、4个、5个、或更多个非保守氨基酸置换的亲本多肽或亲本功能片段的氨基酸序列。在这些情形中，对于所述的氨基酸置换，优选地，这些氨基酸置换不妨碍或抑制所述本发明多肽或其功能片段的生物学功能或活性；更优选地，所述氨基酸置换还能提高所述本发明多肽及其功能片段的生物学功能或活性。
在本领域内，保守氨基酸置换是公知的，它指的是这样的氨基酸置换，即，其中一个具有某种物理和/或化学特性的氨基酸被换为另一个具有相同化学或物理特性的氨基酸。本领域技术人员了解，保守性的氨基酸置换可以不造成蛋白质的结构或功能的显著改变。典型的保守性置换包括，例如，用酸性氨基酸置换另一个酸性氨基酸(例如，AsP或Glu)，用具有非极性侧链的氨基酸置换另一个具有非极性侧链的氨基酸(例如，Ala、Gly、Val、Ile、Leu、Met、Phe、Pro、Trp或Val等)，用碱性氨基酸置换另一个碱性氨基酸(Lys、Arg等)，用具有极性侧链的氨基酸置换另一个具有极性侧链的氨基酸(Asn、Cys、Gln、Ser、Thr或Tyr等)，用芳香氨基酸(Trp、Phe或Tyr等)置换另一个芳香氨基酸等。
本发明的多肽(包括功能片段)及其功能变体可以是任意长度的，即，可以包括任意数目的氨基酸，条件是所述多肽(包括功能片段)及其功能变体保留必要的生物学活性，例如，抑制HBx的活性，在细胞水平和动物水平抑制HBV病毒复制和表达，以及治疗HBV导致的病毒性肝炎的作 用，包括抑制后期的肝癌细胞生长的作用。举例来说，本发明的多肽(包括功能片段和功能变体)可以是4-2000个氨基酸长，如长度为5、6、7、8、9、10、11、12、13、14、15、16、18、20、25、30、40、50、60、70、80、90、100、125、150、175、200、300、400、500、700、800、1000或更多。优选地，本发明的多肽长度为6-20个氨基酸，并满足作为多肽药物的药效学和半衰期的要求。
在一个实施例中，本发明的多肽及其功能片段的功能变体与亲本多肽SEQ ID NO：1具有一个氨基酸的差别，其具有与亲本多肽SEQ ID NO：1相似的生物学活性和功能，例如，抑制HBx的活性，以及有效地抑制癌细胞，优选是肝癌细胞，尤其是表达HBx的肝癌细胞。
本文所述的本发明的多肽(包括功能片段)和其功能变体还可以包含细胞-穿透肽(CPP)。所述CPP促进本发明多肽穿过细胞膜并且进入细胞内。CPPs在本领域中是已知的。参见，例如，Deshayes等，细胞分子生命科学(Cell.Mol.Life Sci.)62:1839-1849(2005)；EI-Andaloussi等，现代药物设计(Curr.Pharm.Design.)11:3597-3611(2005)。所述CPP可以是本领域己知的那些中的任一种。
本发明的多肽(包括功能片段)和其功能变体还可以，例如，被脂化(例如，脂肪酸化)、糖基化、酰胺化、羧酸化、磷酸化、酯化、N-酰化、通过二硫键环化、转化成酸加成盐、二聚体化或多聚体化，和/或缀合化。
本发明的多肽(包括功能片段)和其功能变体，包括其衍生物例如脂肪酸衍生物，还可以是单体肽、二聚肽或多聚体肽。
本发明的多肽(包括功能片段)和其功能变体可以通过本领域技术人员已知的方法获得(参见，例如，Chan等，Fmoc Solid Phase Peptide Synthesis，牛津大学出版社，牛津，英国，2005；Reid,R.， Peptide and Protein Drug Analysis,Marcel Dekker Company，2000；以及美国专利号5,449,752)。此外，也可由核酸重组方法生产多肽而得到(参见，例如，Sambrook等，分子克隆：实验室手册(Molecular Cloning:A Laboratory Manual)，第3版，冷泉港出版社(Cold Spring Harbor Press)，冷泉港，纽约，2001)。此外，本发明的一些多肽(包括其功能片段以及功能变体)可以从如植物、细菌、昆虫、哺乳动物如大鼠、人等分离和/或纯化。分离和纯化的方法在本领域内也是公知的。备选地，本文所述的多肽(包括其功能片段以及功能变体)可以从商业公司合成后购买获得。
缀合物
本发明还包括缀合物，例如，生物缀合物，其包括本发明的多肽(包括功能片段和功能变体)以及肽模拟物。缀合物，以及通常合成缀合物的方法在本领域内也是已知的(参见，例如，Hudecz,F.，分子生物学方法(Methods Mol.Biol.)298:209-223(2005)和Kirin等，无机化学(Inorg.Chem.)44(15):5405-5415(2005)。
药物组合物
本发明所提供的上述各种物质，包括多肽(包括功能片段)和其功能变体以及缀合物等(笼统称为“本发明物质”)可以是分离的、纯化的、合成的、和/或重组的。
本发明物质还可以配制成组合物，诸如药物组合物。在这一点上，本发明提供包括任意所述多肽(包括功能片段和功能变体)以及肽模拟物，以及药用载体的药物组合物。包含任意本发明物质的本发明药物组合物可以包括多于一种本发明物质，例如：两种或更多不同的多肽。备选地，所述药物组合物可以包括与另一种或多种药物活性试剂或药物的组合。所述另一种或多种药物活性试剂或药物优选可以包括诸如化疗剂，例如，天冬酰胺酶、白消安、卡铂、顺铂、柔红霉素、多柔比星、氟尿嘧啶、吉西他滨、羟基脲、甲氨蝶呤、紫杉醇、利妥 昔单抗、长春碱、长春新碱等等。
在本发明的一个优选实施方案中，所述药物组合物包括与脂质组合的本发明物质。所述脂质可以是任何脂质，包括脂肪酸、磷脂、固醇、鞘脂、萜、甘油脂、甘油磷酸酯、异戊烯醇脂、糖脂和聚酮化合物等。所述脂在本领域内是已知的。
关于药物组合物，药用载体可以是任意常规所用的那些，它们可以仅通过化学物理因素，诸如溶解性和缺乏与活性化合物的反应性，以及通过施用的途径，并进行限定。本发明所述的药用载体，例如，媒介物、佐剂、赋形剂和稀释剂等，是本领域技术人员公知的，并且容易为公众所获得。优选地，所述药用载体对活性试剂是化学惰性的，在使用条件下不具有有害副作用或毒性。
载体的选择应该由特定的本发明物质、以及由用于施用本发明物质的特定方法决定。因此，存在本发明药物组合物的多种适当制剂。下述用于口服、气雾剂、肠胃外、皮下、静脉内、肌内、动脉内、鞘内、腹膜内、直肠、和阴道施用的制剂是示例性的，并且不以任何方式限制。可以使用多于一种途径施用本发明物质，在特定情形中，特定的途径可以提供比另一种途径更直接和更有效的方法。
在本发明的一个优选实施方案中，所述药物组合物是局部制剂、静脉内制剂、或皮下制剂。在本发明的一个优选实施方案中，所述药物组合是局部制剂。局部制剂是本领域技术人员公知的。在本发明施用到皮肽的情形中，所述制剂特别适合。本发明的局部制剂可以是，例如，膏剂、洗液、油膏、贴片、油、糊剂、喷雾剂，例如气雾剂喷雾剂、凝胶、滚抹式的液体、固体棒等。优选地，本发明的局部制剂是膏剂、洗液、油膏、或贴片。
适用于口服施用的制剂可以由下列各项组成：a)液体溶液，诸如 溶解在稀释剂如水、盐水、或橙汁中的有效量的本发明物质；b)胶囊、片剂、锭剂等，每一种包含预先确定量的，固体或颗粒状的活性成分；c)粉剂；d)在适当液体中的混悬液；和e)适当的乳剂。液体制剂可以包括稀释剂，诸如水和醇，例如，乙醇、苯甲醇、和聚乙二醇，可以添加或不添加药用表面活性剂。胶囊形式可以是普通的硬或软壳明胶型，其包含，例如，表面活性剂、润滑剂、和惰性填充剂诸如乳糖、蔗糖、磷酸钙、和玉米淀粉。片剂形式可以包括下列各项中的一种或多种：乳糖、蔗糖、甘露醇、玉米淀粉、马铃薯淀粉、褐藻酸、微晶纤维素、阿拉伯树胶、明胶、瓜耳胶、胶状二氧化硅、滑石、硬脂酸镁、硬脂酸钙、硬脂酸锌、硬脂酸、和其它赋形剂、着色剂、稀释剂、缓冲剂、崩解剂、润湿剂、防腐剂、调味剂、和其它药物相容的赋形剂。锭剂形式可以包括处在调味剂通常是在蔗糖或阿拉伯树胶的本发明物质，以及包括处在惰性基质诸如明胶和甘油、或蔗糖和阿拉伯树胶中的本发明物质的软锭剂，另外包含本领域己知的赋形剂的乳剂、凝胶等。
本发明物质，单独的或与其它适宜的成分组合，可以制成通过吸入来施用的气雾剂制剂。这些气雾剂制剂可以放置在加压可用的介质中，诸如二氯二氟甲烷、丙烷、氮等。它们还可以配制成非加压的制剂，如在喷雾器或雾化器中。所述喷雾制剂还可以用于向粘膜喷雾。
适于肠胃外施用的制剂包括水性和非水性、等渗无菌注射液，其可以包含抗氧化剂、缓冲剂、抑菌剂、和使得所述制剂与预期接收者的血液等渗的溶质，以及水性和非水性无菌混悬液，其可以包括混悬剂、增溶剂、增稠剂、稳定剂、和防腐剂。本发明物质可在药用载体中的生理用稀释剂中使用，诸如无菌液体或液体混合物，包括水、盐水、水性葡萄糖和相关的糖溶液、醇如乙醇或十六醇、二醇如丙二醇或聚乙二醇、二甲亚砜、甘油、缩酮如2,2-二甲基-1,3-二氧戊环-4-甲醇、醚、油、脂肪酸、脂肪酸酯或甘油酯，或乙酰化的脂肪酸甘油酯，添加或不添加药用表面活性剂诸如皂或去污剂，混悬剂如果胶、 甲基纤维素、羟丙基甲基纤维素、或氮甲基纤维素，或乳化剂和其它药用佐剂。
可以用在肠胃外制剂中的油包括石油、动物油、植物油、或合成油。油的具体实例包括花生油、大豆油、芝麻油、棉籽油、玉米油、橄榄油、石油和矿物油。用在肠胃外制剂中的适当的脂肪酸包括油酸、硬脂酸、和异硬脂酸。
用在肠胃外制剂中的适当的皂包括脂肪碱金属和三乙醇胺盐等，并含有适当的去污剂，例如a)阳离子去污剂：二甲基二烷基卤化铵和烷基卤化吡啶；b)阴离子去污剂：烷基、芳基、烯属磺酸酯、烷基、烯烃、醚、单甘油硫酸酯和磺基琥珀酸酯等；c)非离子去污剂：脂肪胺氧化物、脂肪酸链烷醇酰胺和聚氧乙烯聚丙烯共聚物等；d)两性去污剂：烷基-β-氨基丙酸酯和2-烷基-咪唑啉季铵盐；e)它们的混合物。
所述肠胃外制剂可在溶液中含有大约为0.5-25％(重量体积百分浓度)的本发明物质。可以使用防腐剂和缓冲剂。为了最小化或消除在注射部位的刺激性，所述组合物可以包含一种或多种亲水性-亲脂性平衡(HLB)的非离子表面活性剂。在所述制剂中表面活性剂的用量约为5-15％(重量体积百分浓度)。适当的表面活性剂包括聚乙二醇失水山梨糖醇脂肪酸酯和环氧乙烷与疏水基的高分子量加合物，所述加合物是通过缩合环氧丙烷和丙二醇而形成的。所述肠胃外制剂可以存在于单位剂量或多剂量密封容器中，并且可以保存在冷冻干燥(冻干)的条件下，仅需要在即将使用之前加入无菌液体赋形剂如水用于注射。可以由先前所述种类的无菌粉剂、颗粒剂、和片剂制备临时的注射溶液和混悬液。
本发明物质或包括所述本发明物质的组合物还可以制成可注射制剂。可注射组合物对有效的药用载体的需要是本领域普通技术人员公 知的。优选地，当施用于细胞例如树突细胞时，所述细胞通过注射施用。
另外，本发明的发明物质，或包括所述发明物质的组合物，可以通过与各种基质如乳化基质或水溶性基质混合而制备成栓剂。适于例如阴道施用的制剂可以以阴道栓剂、卫生棉条、膏剂、凝胶、糊剂、泡沫剂、或喷雾制剂存在，除了其活性成分之外，还包含本领域中己知合适的那些载体。
本领域技术人员应该了解，除了上述药物组合物之外，本发明的发明物质还可以配制为包含复合物，诸如环糊精包含复合物，或脂质体。
为了达到本发明的目的，施用的本发明物质的量或剂量应该在合理时间范围内在受试者或动物内产生例如治疗或预防响应。例如，本发明物质的剂量应该足以在从施用时刻起约2小时以上如12-24小时或更长的时间期间内，抑制患病细胞的增殖，起到治疗或预防疾病(例如，肿瘤，癌症等)的作用。在某些实施方案中，所述时间期间甚至可以更长。剂量应该由特定的本发明物质的功效和待治疗的动物(例如，人)的状况，以及动物(例如，人)的体重所确定。确定施用剂量的许多测定方法是本领域内己知的。本发明物质的剂量还将通过施用特定的本发明物质可能伴随的任何副作用的存在、性质和程度而确定。
本领域普通技术人员应该容易理解，本发明物质可以以任何方式修饰，以提高本发明物质的治疗或预防功效。例如，本发明物质可以直接或通过连接体间接与靶向的部分缀合。使化合物，例如，本发明物质,与靶向部分缀合的实践，在本领域内是公知的。如参见，Wadwa等，药物靶向杂志(J.Drug Targeting)3:111,(1995)和美国专利号5,087,616。再另一个实施方案中，本发明物质可以被修饰成贮存库形 式，以便使本发明物质释放到其所施用的体内的方式在时间和体内部位方面受到控制(参见，例如，美国专利号4,450,150)。本发明物质的贮存库形式可以是，例如，包括本发明物质和多孔或非多孔物质如聚合物的可植入组合物，其中本发明物质通过所述物质和/或所述非多孔物质的降解而扩散。然后，将贮存库植入体内的理想部位，并且本发明物质以预先确定的速率从所述植入物释放。
本发明的药物组合物，包含多肽(包括功能片段)及其功能变体，可以用在防止和抑制乙型肝炎病毒感染诱发的慢性肝病，包括病毒性肝炎，以及由此产生的肝硬变和肝癌的方法中。本领域普通技术人员应该容易理解，本发明所述的由乙型肝炎病毒诱发的慢性肝病可以存在于任何宿主中。优选地，所述宿主是哺乳动物。特别优选地，所述宿主是人。
下面结合具体实施例，对本发明作进一步的阐述说明。应理解，这些实施例仅用于说明本发明而不在于限制本发明的范围。下述实施例中的实验方法，如无特殊说明，均为常规方法；所用的材料，如无特殊说明，均为自常规生化试剂公司购买得到的。
实施例一：多肽的设计和制备
人工合成多肽功能片段D-TTK001:
本发明用人工合成的方法合成了氨基酸序列为Gly-D-Ser-D-Ala-D-Val-D-Met-D-Phe-D-Ser-D-Ser-D-Lys-D-Glu-D-Arg-Gly(SEQ ID NO:1)的D型多肽(以下称D-TTK001)。该多肽的制备采用固相合成方法，如应用AAPPTEC Apex396型多肽合成仪器(购自香港环球分析测试仪器有限公司)，在密闭的防爆玻璃反应器中使氨基酸按SEQ ID NO:1所示的序列，从C端-羧基端向N端-氨基端，这是指第一个被加入到该氨基酸序列Gly-D-Ser-D-Ala-D-Val-D-Met-D-Phe-D-Ser-D-Ser-D-Lys-D-Glu-D-Arg-Gly的氨基酸单体是C末端的Gly，然后再是D-Arg，再是D-Glu，一 直到最后的D-Ser以及N末端的Gly，不断添加、反应、合成，操作最终得到所要具有的氨基酸序列。固相合成法，大大的减轻了每步产品提纯的难度。为了防止副反应的发生，参加反应的氨基酸的侧链都是受保护的。羧基端是游离的，并且在反应之前必须活化。
人工合成多肽D-TTK001用高压液相色谱(HPLC)(应用PLC Agela C18柱)分析，图1显示所获得的纯度为97.2％。
实施例二：
A.体外结合力检测
重组表达HBx基因的质粒(pET-30a-HBx)为自行构建保存(Zhang H,et al.J Biomed Biotechnol,doi:10.1155/2009/289068)，进而应用该文献中的方法完成HBx的表达与纯化，用于体外结合力检测。应用Biacore 3000生物大分子相互作用分析仪(GE Healthcare生产)进行多肽D-TTK001与重组表达的HBx蛋白体外结合力的测定。不同浓度的多肽分别以30μL/min的速度注入180秒。在解离阶段，HBS-EP缓冲液以30μL/min的速度注入900秒，接着在30μL/min的流速下注入2针1mM NaOH 20秒对芯片再生。所有信号都经由通道1作为对照通道来进行校正。结合力测定分别测试两次，第一次测试HBX分别以浓度0nM、10nM、50nM、100nM、500nM和600nM注入偶联多肽的通道和空白通道。结合时间180秒，解离时间400秒。芯片表面以流速30μl/min注入1mM NaOH 20秒再生。结果经由软件进行动力学分析，如图2。第二次测试HBX分别以浓度0nM、10nM、50nM、588nM、1uM和2uM注入偶联多肽的通道和空白通道。结合时间180秒，解离时间400秒。芯片表面以流速30μL/min注入1mM NaOH 20秒再生。应用BIA Evaluation Software软件对实验结果进行动力学分析。
如表1所示两次结合力测试结果：
表1亲和力分析结果
Figure PCTCN2015092132-appb-000002
该数据表明D-TTK001和HBx的体外结合力和L型多肽anti-HBxP1#相比虽然略小，但并不存在数量级的差别。
B.体外有效性实验
1.细胞系：
表2实验中应用的细胞系
Figure PCTCN2015092132-appb-000003
2.主要试剂：
Figure PCTCN2015092132-appb-000004
3.应用酶联免疫吸附实验(enzyme-linked immunosorbent assay，ELISA)方法检测D-TTK001对肝癌细胞分泌HBeAg的影响
(1)细胞培养
1)生物安全柜紫外灭菌30min，吹风30min；取出含15％胎牛血清的DMEM/F12(1:1)培养基，HepAD38细胞(或HepG2.2.15细胞)，75％乙醇消毒，放入生物安全柜内；
2)无菌条件下，倒掉旧培养基，每次加入3mL 1×PBS，清洗2次，加入1mL 0.25％的胰酶，消化至显微镜下观察细胞变圆，加入5mL培养基，吹散细胞，血球计数板计数，并用培养基稀释，使细胞数达到1～2×104个/mL；
3)铺96孔板：无菌条件下，倒掉旧培养基，每次加入3mL PBS，清洗2次，加入1mL 0.05％的胰酶，消化至显微镜下观察细胞变圆，加入10mL培养基，吹散细胞，血球计数板计数，并用DMEM/F12(1:1)培养基稀释，每孔加100μL细胞悬液，使细胞数达到1～2×103个/孔；边孔不加细胞。培养至细胞贴壁(一般为12h)，伸角，细胞状态良好后即可做后续试验。
(2)应用D-TTK001(或L-TTK001,即anti-HBxP1#)作用肝癌HepAD38细胞(或HepG2.2.15细胞)
将10mM D-TTK001(或L-TTK001)母液从高浓度到低浓度逐级稀释，加入肝癌HepAD38细胞(或HepG2.2.15细胞)培养液中，使各组D-TTK001(或L-TTK001)终浓度为0.1μM、1μM、10μM和100μM；阴性对照组：加10μL无菌ddH2O；于37℃、5％CO2培养箱中培养72小时。
(3)应用ELISA法检测HepAD38细胞(或HepG2.2.15细胞)培养液中HBeAg的含量
1)平衡：取HBeAg检测试剂盒至室温环境30分钟。
2)配液：将浓缩洗涤液用纯化水25倍稀释。
3)取板：根据实验要求，取反应板条(设阴性对照孔3孔，阳性 对照孔1孔和空白对照孔1孔)。
4)加样：分别在相对应孔中加入待测样品或阴性、阳性对照75μL，轻轻振荡混匀。
5)温育：用封板膜封后，置37℃温育60分钟。
6)加酶：每孔加入酶结合物50μL，空白孔除外，轻轻振荡混匀。
7)温育：用封板膜封后，置37℃温育30分钟。
8)洗板：小心揭掉封板膜，手工洗板5遍，拍干。
9)显色：每孔加入显色剂A、B液各50μL，轻轻振荡混匀，37℃避光显色30分钟。
10)终止：每孔加入终止液50μL，轻轻振荡混匀，十分钟内测定结果测定：设定酶标仪波长于450nm处，用空白孔凋零点后测定各孔OD值。
如图3和图4所示，D-TTK001(或L-TTK001)对HepAD38细胞(或HepG2.2.15细胞)分泌的HBeAg水平均具有抑制作用，呈剂量依赖性。D-TTK001的最低有效剂量为0.1μM；L-TTK001的最低有效剂量为10μM。因此，D-TTK001对HBeAg的作用优于L-TTK001对HBeAg的作用。*P<0.05，**P<0.01，进行Student’s t test统计学分析。
4.MTT检测
1)接种细胞：将对数生长期的HepAD38细胞，用含10％胎牛血清的RPMI1640培养液配成单个细胞悬液，以每孔4000-5000个细胞接种到96孔细胞培养板中，每孔体积100μL。
2)培养细胞：待12h后细胞贴壁，加入不同浓度的在实施例1中所获得的D-TTK001(或L-TTK001)合成多肽(0.1μM、1μM、10μM和100μM)，每个浓度重复8个孔，同一般培养条件，培养48h。
3)呈色：每孔加MTT溶液(5mg/mL用PBS<pH＝7.4>配)20μL。
4)继续孵育4小时，终止培养，小心吸弃孔内培养上清液。每孔加150μL DMSO，振荡10分钟，使结晶物充分融解。
5)比色：选择490nm波长，在酶联免疫监测仪上测定各孔光吸 收值，记录结果，进行Student’s t test统计学分析。
如图5所示，D-TTK001和L-TTK001对HepAD38细胞生长和增殖具有明显的抑制作用，呈剂量依赖性。D-TTK001和L-TTK001的最低有效剂量均为10μM。*P<0.05，**P<0.01，进行Student’s t test统计学分析。
实施例三：动物体内(in vivo)多肽有效性实验
1.D-TTK001对HBV转基因鼠中HBV DNA的影响
(1)HBV转基因小鼠广州市军科泰特医药科技有限公司的来源和特性
HBV转基因小鼠购于广州市军科泰特医药科技有限公司。该动物模型是广州市军科泰特医药科技有限公司应用显微注射法成功建立培育了高表达复制型1.3拷贝HBV全基因转基因小鼠Tg(HBV1.3genome)Swb。血清HBsAg和HBeAg可用常规ELISA试剂盒检测；93.93％阳性转基因小鼠血清HBV DNA已达104-106copy/mL；肝组织免疫组化有HBsAg(胞浆型)、HBcAg(核型)表达。性别、不同月龄、一日内不同时间对HBsAg表达无显著影响即稳定表达；且血清HBV DNA阳性率长期保持较高阳性率(检测至39周龄转基因小鼠)，性别无显著影响。这为该转基因模型应用于抗乙肝药物评价奠定了良好基础。
(2)HBV转基因小鼠尾静脉给药(D-TTK 001)方法和取血方法
无菌配制D-TTK 001，按剂量2.5mg/kg(共10只)和5.0mg/kg(共42只)小鼠体重的两个不同剂量分别于尾静脉注射药物，总体积为50μL；取50μL无菌PBS为空白对照组(共10只)；6-8周龄小鼠，雌雄各半。每天注射，连续注射3周共21天；给药前割小鼠尾部动脉取血，待检测小鼠血液中HBV DNA的含量，记作T0组；在给药后第一周(T1+组)、第二周(T2+组)、第三周(T3+组)给药前割小鼠尾部动脉取血；停药1周后取血(记为T4-组)；取血后室温静置2-4小时，制备血清；-80℃冰箱储存样本，待检测小鼠血液中HBV DNA 的含量。
(3)应用real-time PCR方法检测HBV DNA的拷贝数
1)待测样品的准备：从-80℃冰箱取出待测样品，放置室温平衡后备用；根据待测样本、阴阳性对照品以及参考品的数量，按比例(反应液38μL/人份+酶混合液2μL/人份+内标0.2μL/人份)取相应量的反应液、酶混合液及内标，充分混匀成PCR-mix。样品处理(阴性对照、阳性对照、定量参考品与待测样本同步处理)；每个PCR反应管中加入核酸释放剂5μL，然后分别加入待测样本各5μL，吸打3-5次混匀；间隔10分钟以上，每管加入PCR-mix 40μL，盖上管盖，2000rpm离心30秒。
2)PCR扩增
应用中山大学达安基因股份有限公司生产的real time PCR检测HBV DNA试剂盒进行检测，具体步骤按试剂盒说明书进行，如下：将PCR反应管放入扩增仪样品槽，按对应顺序设置阴性对照、阳性对照、定量参考品A～D以及未知样本，并设置样品名称及定量参考品浓度；荧光检测通道选择：以ABI 7300仪器为例：1)选择FAM通道(Reporter：FAM，Quencher：none)检测HBV-DNA；2)选择HEX/VIC通道(Reporter：HEX/VIC，Quencher：none)检测HBV-内标；3)参比荧光(Passive Reference)设置为ROX。
循环参数设定如下：
Figure PCTCN2015092132-appb-000005
3)结果分析：
分析条件设置：根据分析后图像调节Baseline的start值、stop值以及Threshold的Value值(用户可根据实际情况自行调整，start值可以在1～10、stop值可以在5～10、Value值可以在0.01～0.2范围选择),使Std curve窗口下的标准曲线达到最佳，即correlation数值介于-1.0～-0.97。在Analysis菜单下选择Analyze自动分析结果。到Tray窗口，记录未知变量数值(C)。“C”表示样品的浓度或含量。
4)质量控制：
阴性质控品：无Ct值显示；但HBV-内壁检测为阳性(Ct值≤40)。
阳性质控品：检测浓度值介于1.26×105～1.26×106IU/ml。
四个阳性定量参考品：全部阳性，且线性相关系数0.98<＝|r|<＝1。
以上要求需在同一次实验中同时满足，否则，本次实验无效，需重新进行。
应用real time PCR方法检测HBV转基因鼠血清中HBV DNA拷贝数。2.5mg/kg组检测结果：从第一周(T1+组)开始有效的有效率为30％(3/10只)，呈明显时间依赖方式；5.0mg/kg组有效率为54.8％(23/42只)。从第一周(T1+组)开始有效的有效率为46.2％(18/39只)，其中呈明显时间依赖方式递减有效率为30.4％(7/23只)。如图6所示，3只2.5mg/kg组和7只5.0mg/kg呈时间依赖递减有效的比较结果提示，D-TTK001在HBV转基因鼠动物水平抑制HBV复制的药效具有剂量依赖关系。
2.D-TTK001作用HBV转基因鼠后肝组织病理观察
在上述HBV转基因鼠试验结束后，处死小鼠，取肝组织，福尔马林固定，常规方法进行包埋，制备组织切片，进行病理学观察。
如图7所示，人工合成的多肽D-TTK001对HBV转基因鼠肝炎的治疗效果。左图为HBV转基因鼠肝脏病理组织，未处理对照组中10只HBV转基因鼠中有6只，表现为典型的肝气球样变性等病毒性肝炎 病理表征；右图为应用D-TTK001(5mg/kg)尾静脉注射3周后的肝组织，实验组6只中均无肝气球样变性表现，提示D-TTK001对肝组织中的病理性肝炎病变有明显的治疗作用。
3.D-TTK001对回接瘤裸鼠存活率的影响
将对数生长期的HepG2-X细胞用胰酶消化制成细胞悬液，计算细胞数，用无菌的生理盐水细胞稀释至1×107个细胞/mL，置冰水中存放。取2只4到6周雌性BALB/C裸鼠：①对照组1只，在每只小鼠右前肢腋下皮下注射上述稀释后的细胞0.2mL，仅注射0.5mL灭菌蒸馏水(不含多肽药物)；②实验组1只(给药剂量为10mg/kg体重)。在小鼠右前肢腋下皮下注射上述稀释后的细胞0.2mL，注射20天后，无菌取出瘤组织，切成小组织块，回接4到6周雌性BALB/C裸鼠，共18只。待肿瘤体积(V＝L×W2×0.5)达到100mm3时，将18只小鼠分分为3组，每组6只；第一组为对照组，尾静脉注射无菌PBS；第二组为实验组，尾静脉注射0.1mg/kg D-TTK001；第三组为实验组，尾静脉注射5.0mg/kg D-TTK001(用0.5ml灭菌蒸馏水溶解冻干的多肽药物)，每天注射一次，共注射30天，每次注射前测量小鼠体重和肿瘤体积，观察裸鼠存活时间。待小鼠死亡后，应用Kaplan-Meier氏绘制生存曲线分析，采用Log-Rank统计学方法进行统计分析。
如图8所示，实验结果表明，应用人工合成的D-TTK001多肽0.1mg/kg组和5.0mg/kg组对HepG2-X细胞回接瘤裸鼠的存活时间均具有明显的延长作用，提示D-TTK001具有抑制裸鼠体内HepG2-X细胞回接瘤恶性表型的作用。*P<0.5，Log-Rank统计学分析。
实施例四：多肽的功能片段及变体
本发明还探讨了多肽及其功能片段的功能变体的作用。下表所示的序列为以L-TTK001(即anti-HBxP1#)为基础进行人工合成的D型氨基酸置换。按照所述序列进行人工合成多肽片段(方法如上)，然后，采用上述ELISA方法，观察所得多肽作用的变化。
Figure PCTCN2015092132-appb-000006
如图9所示，10μM的D-TTK001以及D-TTK001-1、D-TTK001-2、D-TTK001-3、D-TTK001-4、D-TTK001-5、D-TTK001-6、D-TTK001-7、D-TTK001-8、D-TTK001-9和D-TTK001-10等对HepAD38细胞分泌的HBeAg水平均具有不同程度的抑制作用，但D-TTK001的作用最为明显，提示单一置换人工合成的D型氨基酸使L-TTK001(即anti-HBxP1#) 多肽在构象上发生的变化，对其生物活性有不同程度的影响。并且多肽的稳定性随着D-型氨基酸替代数量的增加而增强。而D-TTK001与L-TTK001的生物活性相似，提示这种对称的异构体保留了与靶蛋白HBx的结合能力。*P<0.05，**P<0.01，进行Student’s t test统计学分析。
实施例五：多肽药物大鼠体内药代动力学试验
取试验动物6只，以3.6mg/kg(相当小鼠5mg/kg)尾静脉给予D-TTK001溶液，按采血时间点取血置于肝素化试管中，应用Triple-TOF质谱仪测定大鼠的D-TTK001血药浓度。
如表3所示，6只大鼠静脉推注3.5mg/kg D-TTK001后主要药代参数，半衰期为55.27分钟。
表3.6只大鼠静脉推注3.5mg/kg D-TTK001后主要药代参数
Figure PCTCN2015092132-appb-000007
实施例六：多肽药物急性毒性试验
取昆明小白鼠，每组10只，每组雌雄各5只。分实验组和对照组。实验组，以500mg/kg的浓度从小鼠尾静脉单次注射多肽D-TTK001(用0.8mL灭菌蒸馏水溶解冻干的10mg多肽)；对照组注射0.8mL灭菌蒸馏水。注射后连续观察24小时。
多肽药物急性毒性试验结果显示，小鼠无异常表现。与对照组相比体重无异常变化。
本发明引用的所有参考文献，包括出版物、专利申请、和专利，通过引用结合于此，并且完全结合于此。本文提供的任何以及所有实施例，或示例性语言(例如、诸如等)仅意欲更好地阐述本发明，并不形成对本发明范围的限制，除非另外要求。本发明描述了优选实施方案，包括本发明人已知实施本发明的最佳模式。本发明包含这些优选实施方案可能的变化，本发明人意欲以这些与本文具体描述不同的方式实施本发明，同样，本领域普通技术人员也清楚了解这些变化，并预期能熟练地利用所述变化。在法律所能允许的范围，本发明包括了在所附权利要求中引用的主题的所有修改和等价物，除非本文另外指明，或明显与上下文相抵触。

Claims (7)

  1. 分离的多肽，其包含如SEQ ID NO:1所示的氨基酸序列或其功能片段、或者所述氨基酸序列及其功能片段的功能变体，并且所述氨基酸序列、所述功能片段、或所述功能变体的氨基酸序列中的一个或多个L型氨基酸被D型氨基酸所取代，所述多肽具有抑制乙型肝炎病毒X蛋白的功能，并能抑制乙型肝炎病毒感染后的慢性肝病的发生和发展。
  2. 权利要求1所述的多肽，其氨基酸序列具有与SEQ ID NO:1所示的氨基酸序列至少70％(优选至少80％、85％、90％、95％、99％或更高)的相同性。
  3. 权利要求1所述的多肽，其氨基酸序列为如SEQ ID NOs:2-11所示的氨基酸序列中的任一种。
  4. 权利要求1-3所述的任一种多肽，所述乙肝病毒感染后的慢性肝病包括肝炎、肝硬变和肝癌。
  5. 权利要求1-3所述的任一种多肽在制造抗乙肝病毒感染后的慢性肝病的药物中的应用。
  6. 权利要求5所述的应用，该应用为制造抗乙型肝炎的治疗性疫苗。
  7. 一种药物组合物，其包含权利要求1-4所述的任一种多肽以及任选的药物载体。
PCT/CN2015/092132 2015-03-13 2015-10-16 抗乙型肝炎病毒x蛋白多肽药物 Ceased WO2016145840A1 (zh)

Priority Applications (5)

Application Number Priority Date Filing Date Title
ES15885210T ES2819238T3 (es) 2015-03-13 2015-10-16 Agente farmacéutico polipeptídico de proteína X contra el virus de la hepatitis B
KR1020177029558A KR102017973B1 (ko) 2015-03-13 2015-10-16 항-b형 간염 바이러스 x 단백질 폴리펩티드 약제
US15/557,209 US10570176B2 (en) 2015-03-13 2015-10-16 Anti-hepatitis B virus X protein polypeptide pharmaceutical
EP15885210.3A EP3269727B1 (en) 2015-03-13 2015-10-16 Anti-hepatitis b virus x protein polypeptide pharmaceutical
JP2017566181A JP6660966B2 (ja) 2015-03-13 2015-10-16 Ｂ型肝炎ウイルスｘタンパク質に対するポリペプチド薬物

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201510111013.0A CN104744564B (zh) 2015-03-13 2015-03-13 抗乙型肝炎病毒x蛋白多肽药物
CN201510111013.0 2015-03-13

Publications (1)

Publication Number Publication Date
WO2016145840A1 true WO2016145840A1 (zh) 2016-09-22

Family

ID=53584887

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/092132 Ceased WO2016145840A1 (zh) 2015-03-13 2015-10-16 抗乙型肝炎病毒x蛋白多肽药物

Country Status (7)

Country Link
US (1) US10570176B2 (zh)
EP (1) EP3269727B1 (zh)
JP (1) JP6660966B2 (zh)
KR (1) KR102017973B1 (zh)
CN (1) CN104744564B (zh)
ES (1) ES2819238T3 (zh)
WO (1) WO2016145840A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104744564B (zh) * 2015-03-13 2017-07-28 天津托普泰克生物科技有限公司 抗乙型肝炎病毒x蛋白多肽药物
CN106519004B (zh) * 2016-11-03 2019-03-19 吉林大学 一种能降解乙型肝炎病毒x蛋白的特异性多肽

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1546668A (zh) * 2003-12-12 2004-11-17 南开大学 乙型肝炎病毒x蛋白转导系统表达载体及其构建
WO2007110098A1 (en) * 2006-03-29 2007-10-04 Institut National De La Sante Et De La Recherche Medicale HBx PEPTIDES, CAPABLE OF ELICITING A PROMISCUOUS IMMUNODOMINANT CD4+ RESPONSE DIRECTED AGAINST HBV
CN102675422A (zh) * 2011-03-15 2012-09-19 天津托普泰克生物科技有限公司 抗乙型肝炎病毒x蛋白多肽药物
CN102675424A (zh) * 2011-03-15 2012-09-19 天津托普泰克生物科技有限公司 抗乙型肝炎病毒x蛋白多肽药物
CN102675423A (zh) * 2011-03-15 2012-09-19 天津托普泰克生物科技有限公司 抗乙型肝炎病毒x蛋白多肽药物
CN103992388A (zh) * 2011-03-15 2014-08-20 天津托普泰克生物科技有限公司 抗乙型肝炎病毒x蛋白多肽药物
CN104744564A (zh) * 2015-03-13 2015-07-01 天津托普泰克生物科技有限公司 抗乙型肝炎病毒x蛋白多肽药物

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5141851A (en) * 1990-04-18 1992-08-25 Board Of Regents, The University Of Texas System Isolated farnesyl protein transferase enzyme
DK53291D0 (da) * 1991-03-25 1991-03-25 Carlbiotech Ltd As Smaa peptider og peptidrelaterede stoffer samt farmaceutiske praeparater indeholdende saadanne forbindelser
HUT71860A (en) 1992-08-27 1996-02-28 Deakin Res Ltd Retro-, inverso-, and retro-inverso synthetic peptide analogues
JP3401005B2 (ja) * 1992-12-11 2003-04-28 ユニバーシティ オブ フロリダ 有害生物の防除のための材料および方法
AUPM411994A0 (en) 1994-02-25 1994-03-24 Deakin Research Limited Epitopes

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1546668A (zh) * 2003-12-12 2004-11-17 南开大学 乙型肝炎病毒x蛋白转导系统表达载体及其构建
WO2007110098A1 (en) * 2006-03-29 2007-10-04 Institut National De La Sante Et De La Recherche Medicale HBx PEPTIDES, CAPABLE OF ELICITING A PROMISCUOUS IMMUNODOMINANT CD4+ RESPONSE DIRECTED AGAINST HBV
CN102675422A (zh) * 2011-03-15 2012-09-19 天津托普泰克生物科技有限公司 抗乙型肝炎病毒x蛋白多肽药物
CN102675424A (zh) * 2011-03-15 2012-09-19 天津托普泰克生物科技有限公司 抗乙型肝炎病毒x蛋白多肽药物
CN102675423A (zh) * 2011-03-15 2012-09-19 天津托普泰克生物科技有限公司 抗乙型肝炎病毒x蛋白多肽药物
CN103992388A (zh) * 2011-03-15 2014-08-20 天津托普泰克生物科技有限公司 抗乙型肝炎病毒x蛋白多肽药物
CN104744564A (zh) * 2015-03-13 2015-07-01 天津托普泰克生物科技有限公司 抗乙型肝炎病毒x蛋白多肽药物

Also Published As

Publication number Publication date
KR20180080127A (ko) 2018-07-11
CN104744564A (zh) 2015-07-01
EP3269727A1 (en) 2018-01-17
JP2018514582A (ja) 2018-06-07
KR102017973B1 (ko) 2019-09-03
CN104744564B (zh) 2017-07-28
US20180334478A1 (en) 2018-11-22
EP3269727B1 (en) 2020-07-08
US10570176B2 (en) 2020-02-25
JP6660966B2 (ja) 2020-03-11
EP3269727A4 (en) 2018-07-25
ES2819238T3 (es) 2021-04-15

Similar Documents

Publication Publication Date Title
US12122819B2 (en) Method of treating skin tissue damage by topically administering a bi-specific protein comprising a human insulin-like growth factor variant and a human annexin A5 variant
RU2668560C2 (ru) Конъюгированная вакцина на основе пептида антигена wt1
US6602510B1 (en) HLA class I A2 tumor associated antigen peptides and vaccine compositions
US20080279924A1 (en) HLA class I A2 tumor associated antigen peptides and vaccine compositions
EP2552470B1 (en) Peptides for promoting angiogenesis and an use thereof
KR20180123064A (ko) 질환의 요인이 되는 생체내 단백질을 표적으로 하는 컨쥬게이트 백신
US11020464B2 (en) Immunogenic composition targeting S100A9
EA006423B1 (ru) Олигопептид, обладающий биологической активностью модулятора рецептора тромбопоэтина, и способы его применения
WO2018232735A1 (en) USE OF A PIEZO REGULATOR IN THE PREPARATION OF A MEDICINAL PRODUCT
CN102088992B (zh) 肽基二酰基甘油酯
WO2016145840A1 (zh) 抗乙型肝炎病毒x蛋白多肽药物
JP4254972B2 (ja) 癌、関節炎および網膜症の治療用抗脈管形成薬
EP2963053A1 (en) Peptides for promoting angiogenesis and an use thereof
CN102675422A (zh) 抗乙型肝炎病毒x蛋白多肽药物
US20230203107A1 (en) Peptide for treating sepsis derived from rv3364c protein of mycobacterium tuberculosis
EP4445910A1 (en) Vaccine composition for inducing anti-il-23 antibody
WO2012122942A1 (zh) 抗乙型肝炎病毒x蛋白多肽药物
US20090312243A1 (en) Treatment of inflammatory bowel disease (ibd) with anti-angiogenic compounds
US20140147490A1 (en) Hla class i a2 tumor associated antigen peptides and vaccine compositions
TW202225185A (zh) 再摺疊的人類血清白蛋白及其用於抗腫瘤的用途
HK40058660A (zh) 用於组织修复的双特异性治疗蛋白
HK40047414B (zh) 多肽或其衍生物的应用
WO2020000063A1 (en) Rheumatoid arthritis treatment

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15885210

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2017566181

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 20177029558

Country of ref document: KR

Kind code of ref document: A

REEP Request for entry into the european phase

Ref document number: 2015885210

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 15557209

Country of ref document: US