Hydrophobic modified pres-derived peptides of hepatitis B virus (HBV) and their use as vehicles for the specific delivery of compounds to the liver

Abstract

The present invention relates to hydrophobic modified preS-derived peptides of hepatitis B virus (HBV) which are versatile vehicles for the specific delivery of compounds to the liver, preferably to hepatocytes, in vitro as well as in vivo. Any kind of compound, but in particular drugs, such as interferons, viral reverse transcriptase inhibitors or core assembly inhibitors, and/or labels can be specifically targeted to the liver and so be enriched in the liver. This liver targeting can further be used for the targeted diagnosis, prevention and/or treatment of liver diseases or disorders, such as hepatitis, malaria, hepatocellular carcinoma (HCC), as well as for the prevention of HAV, HBV, HCV and/or HDV infection. The present invention relates to pharmaceutical compositions comprising said hydrophobic modified preS-derived peptide(s) and the compound(s) to be specifically delivered to the liver. The present invention furthermore relates to a method for the combined treatment of a liver disease and the prevention of HAV, HBV, HCV and/or HDV infection. The present invention relates also to the use of the preS-derived peptides in gene therapy and the delivery of immunogenic epitopes for hepatocyte-mediated antigen presentation to activate liver-directed immunological responses.

Claims

1. A hydrophobic modified preS-derived peptide of hepatitis B virus (HBV) of the formula
[H.sub.m-P-R.sub.n]A.sub.0 wherein P is a preS-derived peptide consisting of: a) an N-terminally and/or C-terminally truncated variant of HBV preS consensus sequence as shown in SEQ ID NO: 1, wherein the truncated variant consists of a minimum of 10 and a maximum of 46 amino acids; or b) a truncated variant of HBV preS consensus sequence as shown in SEQ ID NO: 1, wherein the truncated variant consists of a minimum of 7 and a maximum of 46 amino acids, and wherein the truncated variant comprises the sequence of SEQ ID NO: 21; wherein, H is an acylation of the preS-derived peptide P that is N-terminal of P, m is at least 1; R is a C-terminal modification of said preS-derived peptide P, which is a moiety that protects from degradation, selected from amides, albumin, natural polymers, and synthetic polymers; n is 0 or at least 1; A is an anchor group, selected from ester, ether, disulfide, amide, thiol, and thioester; and .sub.0 is 0 or at least 1; wherein said hydrophobic modified peptide further comprises at least one compound selected from drugs; prodrugs; labels; recombinant viruses or derivatives thereof; carriers or depots for a drug, prodrugs or labels; and immunogenic epitopes; wherein the label is a contrast agent; and wherein the compound and the hydrophobic peptide form a conjugate.

2. The hydrophobic modified preS-derived peptide according to claim 1, wherein the acylation is selected from acylation with myristoyl (C 14), palmitoyl (C 16) or stearoyl (C 18).

3. The hydrophobic modified preS-derived peptide according to claim 1, wherein H and/or R is linked to P via a linker or spacer.

4. The hydrophobic modified preS-derived peptide according to claim 1, wherein P consists of N-terminally and/or C-terminally truncated variants of a minimum of 20 and a maximum of 46 consecutive amino acids of SEQ ID NO: 1.

5. The hydrophobic modified preS-derived peptide according to claim 1, wherein P consists of the N-terminally and/or C-terminally truncated variants of a maximum of 46 consecutive amino acids of SEQ ID NO: 1 and comprises an amino acid sequence selected from amino acid residues 2 to 21, residues 2 to 20, or residues 9 to 15 of the HBV preS consensus sequence as shown in SEQ ID NO: 1.

6. The hydrophobic modified preS-derived peptide according to claim 1, wherein P does not comprise amino acid substitutions and/or deletions at residues 9 to 15 of SEQ ID NO: 1.

7. The hydrophobic modified preS-derived peptide according to claim 1, wherein P consists of the N-terminally and/or C-terminally truncated variants of a maximum of 46 consecutive amino acids of SEQ ID NO: 1 and comprises an amino acid sequence of SEQ ID NO: 38.

8. The hydrophobic modified preS-derived peptide according to claim 1, wherein P comprises an amino acid sequence selected from SEQ ID NOs: 31 to 37 and variants thereof.

9. The hydrophobic modified preS-derived peptide according to claim 1, wherein P comprises an amino acid sequence selected from SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 31, and SEQ ID NO: 38.

10. The hydrophobic modified preS-derived peptide according to claim 1, wherein the acylation is selected from acylation with stearoyl (C 18) or acylation with myristoyl (C 14).

11. A pharmaceutical composition comprising; at least one hydrophobic modified preS-derived peptide of HBV according to claim 1, and at least one compound to be specifically delivered to the liver; wherein the compound may be conjugated to the peptide; and, optionally, a pharmaceutically acceptable carrier and/or excipient.

12. A method for the identification of a HBV receptor, comprising the use of at least one hydrophobic modified preS-derived peptide of HBV according to claim 1, and at least one compound to be specifically delivered to the liver, wherein the compound is conjugated to the peptide; and, optionally, a pharmaceutically acceptable carrier and/or excipient.

13. The hydrophobic modified preS-derived peptide according to claim 1, wherein the compound is selected from interferons; viral reverse transcriptase inhibitors; viral RNA polymerase inhibitors; viral core assembly inhibitors or viral nucleocapsid inhibitors; kinase inhibitors; nucleoside analogues; protease inhibitors; proteasom inhibitors; antibodies or fragments thereof; siRNA or precursors thereof; farnesylation inhibitors; alcohol dehydrogenase (ADH) or an activator thereof; cholesterol biosynthesis inhibitors; inhibitors of the liver stage of a virus or a non-viral pathogen; and antibiotics.

14. The hydrophobic modified preS-derived peptide according to claim 13, wherein the drug is in the form of a prodrug.

15. The hydrophobic modified preS-derived peptide according to claim 13, wherein the compound is interferon and is attached to a peptide consisting of the N-terminally and/or C-terminally truncated variants of a maximum of 46 consecutive amino acids of SEQ ID NO: 1 and that comprises an amino acid sequence of SEQ ID NO: 38.

16. The hydrophobic modified preS-derived peptide according to claim 15, wherein H is a hydrophobic modification by acylation.

17. The hydrophobic modified preS-derived peptide according to claim 1, wherein the conjugate is formed by covalent attachment or by complex formation.

18. The hydrophobic modified preS-derived peptide according to claim 17, wherein the conjugate is formed by covalently attaching a compound to an anchor group A.

19. The hydrophobic modified preS-derived peptide according to claim 18, wherein the anchor group A further comprises a spacer or linker.

20. The hydrophobic modified preS-derived peptide according to claim 19, wherein the spacer or linker comprises a recognition site, which is recognized by a liver protein.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Schematic representation of the HBV particle and the HBV L-, M- and S-proteins.

(2) (A) The partially double stranded DNA is covalently associated with the viral polymerase complex, consisting of the terminal protein, (TP), the reverse transcriptase (RT) and the RNaseH. The genome is encapsulated by an icosahedral shell, built of 120 core-protein dimers. The 3 HBV surface proteins L-, M- and S- are embedded into an ER-derived lipid bilayer. The L- and M-proteins contain the complete S-domain serving as a membrane anchor.
(B) Domain structure of the 3 HBV surface proteins L, M and S.
The L-protein contains the N-terminally myristoylated 107 amino acid preS1-domain, the 55 amino acid preS2-domain and the S-domain containing the 4 transmembrane segments (I-IV).

(3) FIG. 2. HBV preS1 consensus sequence.

(4) At the top: the HBV L-protein with its preS1, preS2 and S-domain is depicted. The N-terminus is myristoylated.

(5) The alignment below shows: the consensus sequence (Consensus) and the eight HBV genotypes (A-H) as well as the woolly monkey HBV (WMHBV) preS sequence encompassing amino acids 2-48. Note that the genotypes A, B, C, E, G and H have eleven additional amino acids at their N-termini, genotype F has 10 additional amino acid residues. At the bottom, the known functional subdomains are shown.
Please note that HBV genotype C refers to HBV genotype C Q46K.

(6) FIG. 3. Biodistribution and liver stability of HBVpreS-derived lipopeptides after subcutaneous application.

(7) (A) Liver-specific accumulation of myristoylated (C.sub.14) versus stearoylated (C.sub.18) HBV preS/2-48 (D) peptides after subcutaneous injection in male NMRI mice in comparison to the control Fuzeon (T-20). Per animal approximately 2.25 g of the .sup.131I-labelled peptides were subcutaneously injected. At the indicated time points animals were sacrificed and the liver specific peptide accumulation (% ID/g) was determined.
(B) Reversed phase HPLC of pure .sup.131I-labelled HBVpreS/2-48.sup.myr(D) (brown curve) in comparison to liver extracts (orange curve) obtained 24 h post subcutaneous injection into uPA+//RAG-2/Pfp/ mice of the same peptide. Note that 50% of detectable activity is eluting at fraction 13. Since the hydrophobic myristoyl residue is located at the N-terminus and the iodinated Tyr-residue at the C-terminus fraction 13 represents the unaltered full-length peptide.

(8) FIG. 4. Biodistribution of HBVpreS/2-48.sup.myr(D) after subcutaneous application in NMRI mice.

(9) Per time point 2.25 g of .sup.131I-labeled HBVpreS/2-48.sup.myr(D) were subcutaneously injected into NMRI mice (N=3). 10 min, 1 h, 4 h and 24 h post injection animals were sacrificed and the organ-specific (blood, lung, heart, spleen, liver, kidney and muscle) peptide accumulations (% ID/g) were determined. Note that most of the peptide accumulates in the liver and that the Y-axis is jolted for better resolution of the distribution in the other organs.

(10) FIG. 5. Biodistribution of HBVpreS/2-48.sup.myr(D) after subcutaneous application in uPA+//RAG-2/Pfp/ mice.

(11) Per time point 2.25 g of .sup.131I-labeled HBVpreS/2-48.sup.myr(D) were subcutaneously injected (N=3). 10 min, 1 h, 4 h and 24 h post injection animals were sacrificed and the organ-specific (liver, spleen, kidney, blood, lung, intestine, heart, muscle and brain) peptide accumulations (% ID/g) were determined. Note that like in NMRI mice most of the peptide accumulates in the liver.

(12) FIG. 6. Scintigram of .sup.125I-labelled HBVpreS/2-48.sup.stearoyl(D) after subcutaneous injection in a mouse.

(13) Specific accumulation of .sup.125I-labelled HBVpreS/2-48.sup.stearoyl(D) in the liver of mice after subcutaneous injection near the right leg; liver appears on the left side in the middle. Note that the upcoming signal near the head is the thyroid and might represent free iodine after de-iodination of the peptide.

(14) FIG. 7. The liver tropism is sequence specific and requires N-terminal hydrophobic modification.

(15) A and B. Biodistribution of a preS peptide, wherein the N-terminus is not hydrophobic modified, compared to a randomized (scrambled) sequence. Note that both HBVpreS/1-48 Tyr as well as its randomized form are located to the kidney.

(16) A: Biodistribution of HBVpreS/1-48 Tyr(D) in mice.

(17) B: Biodistribution of HBVpreS/scrambled 1-48 Tyr(D) in mice.

(18) C and D. Biodistribution of a hydrophobic modified preS peptide compared to a randomized (scrambled) sequence. Only HBVpreS/2-48 Tyr.sup.stearoyl(D) is transported to the liver, wherein its randomized form is evenly distributed.

(19) C: Biodistribution of HBVpreS/2-48 Tyr.sup.stearoyl(D) in mice.

(20) D: Biodistribution of HBVpreS/2-48 Tyr (scrambled).sup.stearoyl(D) in mice.

(21) FIG. 8. Biodistribution of variants of hydrophobic modified preS peptides, wherein the variants are N- and/or C-terminal truncated as well as point mutated.

(22) The truncated variants, HBVpreS/5-48 D-Tyr.sup.stearoyl(D), HBVpreS/2-33-D Tyr.sup.stearoyl(D) as well as HBVpreS/2-21 D-Tyr.sup.stearoyl(D), show liver tropism, whereas the variant with a point mutation at position 12 (G12E) does not.

(23) A: Biodistribution of HBVpreS/5-48 D-Tyr.sup.stearoyl(D) in mice.

(24) B: Biodistribution of HBVpreS/2-33-D Tyr.sup.stearoyl(D) in mice.

(25) C: Biodistribution of HBVpreS/2-48(G12E) D-Tyr.sup.stearoyl(D) in mice.

(26) D: Biodistribution of HBVpreS/2-21 D-Tyr.sup.stearoyl(D) in mice.

(27) FIG. 9. HBV infection inhibition by hydrophobic modified HBVpreS-derived peptides of the invention.

(28) A: HBV infection inhibition by HBVpreS/2-48.sup.myr(D), HBVpreS/2-48.sup.myr(C), HBVpreS/(11)-48.sup.myr(C), HBVpreS/2-48.sup.stearoyl(C) and HBVpreS/(11)-48.sup.stearoyl(C).

(29) B: HBV infection inhibition by HBVpreS/2-48.sup.stearoyl(D), HBVpreS/2-15.sup.stearoyl(D), HBVpreS/2-21.sup.stearoyl(D), HBVpreS/2-26.sup.stearoyl(D) and HBVpreS/2-33.sup.stearoyl(D).

(30) HepaRG cells were infected either in absence (0 nM) or in the presence of 1, 5, 25, 100 and 1000 nM of the respective hydrophobic modified HBVpreS-derived peptides of the invention. The infectious inoculum (HBV of genotype D) and the peptides were incubated overnight. After washing, cells were maintained for another 12 days to allow viral gene expression. Cell culture supernatants from day 8-12 were collected and analyzed for secreted HBSAg using a quantitative commercially available ELISA. HBsAg values from the respective uncompleted infection were set to 100% and the degree of infection inhibition is given in % of the uncompleted infection.
Wherein (C) or genotype C refers to HBV genotype C Q46K.

(31) FIG. 10. Binding of the hydrophobic modified HBVpreS-derived peptides of the invention to primary human hepatocytes (PHH) is myristoyl-dependent and sequence specific

(32) Immunofluorescence, showing confocal images Blue: dapi; Green: FITC.

(33) Primary human hepatocytes (PHH) were incubated with or without 400 nM peptide for 4 hours:

(34) A without peptide

(35) B with wt myr-FITC

(36) C with mut myr-FITC

(37) D with wt myr()-FITC

(38) Bright (green) dots show autofluorescence of the cells (A, C and D), whereas only in B binding of the peptide to the cells can be seen.

(39) wt myr-FITC refers to HBVpreS/2-48.sup.myr(C)-FITC with lysine (K) at the C-terminus [SEQ ID NO. 39], labelled with a fluorophor (FITC)

(40) mut myr-FITC HBVpreS/2-48.sup.myr(C)-FITC with two amino acid substitutions in positions L11 and F13, wherein the amino acids were substituted with the respective D amino acid residues (D-Leu and D-Phe) [SEQ ID NO. 40], labelled with a fluorophor (FITC)

(41) wt myr()-FITC refers to HBVpreS/2-48(C)-FITC without myristoylation [SEQ ID NO. 39], labelled with a fluorophor (FITC)

(42) The fluorophor FITC was attached to the sidechain (NFL; group) of the additional Lys.

(43) FIG. 11. Binding of the hydrophobic modified HBVpreS-derived peptides of the invention is not restricted to HBV-susceptible hepatocytes.

(44) Immunofluorescence, showing confocal images Blue: dapi; Green: FITC.

(45) Cells were incubated with or without 400 nM peptide for 4 hours at 37 C.:

(46) A (upper panel) Differentiated HepaRG cells

(47) B (lower panel) Primary mouse hepatocytes (PMH)

(48) Only wt myr-FITC binds to the cells. Bright (green) dots show autofluorescence of the cells

(49) wt myr-FITC refers to HBVpreS/2-48.sup.myr(C)-FITC with lysine (K) at the C-terminus [SEQ ID NO. 39], labelled with a fluorophor (FITC) attached to the sidechain (NH.sub.2 group) of the additional Lys

(50) mut myr-FITC HBVpreS/2-48.sup.myr(C)-FITC with two amino acid substitutions in positions L11 and F13, wherein the amino acids were substituted with the respective D amino acid residues (D-Leu and D-Phe) [SEQ ID NO. 40], labelled with a fluorophor (FITC)

(51) wt myr()-FITC refers to HBVpreS/2-48(C)-FITC without myristoylation [SEQ ID NO. 39], labelled with a fluorophor (FITC)

(52) The fluorophor FITC was attached to the sidechain (NH.sub.2 group) of the additional Lys.

(53) FIG. 12. Binding of the hydrophobic modified HBVpreS-derived peptides of the present invention depends on the differentiation state of the cells. The peptides only bind to differentiated hepatocytes.

(54) Immunofluorescence, showing confocal images Blue: dapi; Green: FITC.

(55) Cells were incubated with or without 400 nM peptide for 4 hours at 37 C.:

(56) A (upper panel) Comparing differentiated and undifferentiated HepaRG cells

(57) B (lower panel) Comparing differentiated and de-differentiated Primary mouse hepatocytes (PMH)

(58) wt myr-FITC binds only to differentiated cells. Bright (green) dots show autofluorescence of the cells

(59) wt myr-FITC refers to HBVpreS/2-48.sup.myr(C)-FITC with lysine (K) at the C-terminus [SEQ ID NO. 39], labelled with a fluorophor (FITC)

(60) The fluorophor FITC was attached to the sidechain (NH.sub.2 group) of the additional Lys.

(61) FIG. 13. FACS analysis of the binding of a hydrophobic modified HBVpreS-derived peptide of the present invention to primary mouse hepatocytes (PMH).

(62) A showing the protocol of the FACS analysis

(63) B showing the results of the FACS analysis (in comparison to the immunofluorescence results). Only wt myr-FITC binds to the cells.

(64) wt myr-FITC refers to HBVpreS/2-48.sup.myr(C)-FITC with lysine (K) at the C-terminus [SEQ ID NO. 41], labelled with a fluorophor (FITC)

(65) mut myr-FITC HBVpreS/2-48.sup.myr(C)-FITC with two amino acid substitutions in positions L11 and F13, wherein the amino acids were substituted with the respective D amino acid residues (D-Leu and D-Phe) [SEQ ID NO. 42], labelled with a fluorophor (FITC)

(66) The fluorophor FITC was attached to the sidechain (NH.sub.2 group) of the additional Lys.

(67) FIG. 14. Fusion constructs of HBVpreS-derived peptides with mouse interferon alpha.

(68) FIG. 15. Purification and activity test of HBVpreS/2-20.sup.myr(D)-IFNalpha

(69) Purification of the fusion protein HBVpreS/2-20.sup.myr(D)-mouse Interferon alpha 2 from the supernatant of insect cells, which were infected with a recombinant baculovirus.

(70) A First chromatography step: chromatographic purification via His-Tag, using Ni-agarose, because the HBVpreS/2-20.sup.myr(D)-IFNalpha construct carries a C-terminal His-tag.

(71) B Second chromatography step: gel filtration chromatography (S75)

(72) C The elution fractions of the first chromatography step were tested for IFN.

(73) See also Examples.

(74) For the amino acid sequence of HBVpreS/2-20(D) see SEQ ID NO. 38.

EXAMPLES

(75) Synthesis of the Hydrophobic Modified preS-Derived Peptides

(76) The synthesis was carried out as described e.g. in (10).

(77) Biodistribution of the Hydrophobic Modified preS-Derived Peptides

(78) The biodistribution of the hydrophobic modified preS-derived peptides was studied in male NMRI mice. All experiments were performed in compliance with German laws. The peptides, containing an additional Tyr-residue at the C-terminal end were labelled with .sup.131I (Amersham Biosciences, Freiburg, Germany) by the chloramine-T method and purified by HPLC. The labelled peptides were subcutaneously administered by injection of a solution in 50% DMSO. At selected times mice were sacrificed and the radioactivity contained in the blood, heart, lung, spleen, liver, kidney, muscle and brain was measured in a -counter (Canberra Packard, Rsselsheim, Germany) and expressed as a percentage of injected dose per gram of tissue (% ID/g).

(79) Stability Assessment of the Hydrophobic Modified preS-Derived Peptides after Extraction from the Liver

(80) To determine the peptide stability in the liver 131I labelled HBVpreS/2-48.sup.myr (D) was extracted from one liver lobe 24 h post subcutaneous injection. To that aim, 1 ml water per gram frozen liver tissue was added to the sample. After homogenization an equal volume of acetonitrile was added and the homogenization was repeated. After centrifugation (210 min at 4000g) this solution was separated on a reverse phase HPLC column and the radioactivity of each fraction was quantified in a gamma counter.

(81) Cell Lines and Primary Cell Cultures.

(82) HepaRG cells were grown in William's E medium supplemented with 10% fetal calf serum (FCS), 100 units/ml penicillin, 100 g/ml streptomycin, 5 g/ml insulin and 510.sup.5 M hydrocortisone hemisuccinate (10). Cells were passaged 1/5 every two weeks by trypsination. Two to three weeks before infection cell differentiation was induced by adding 2% DMSO into the maintenance medium. The medium was exchanged every 2-3 days.

(83) Infection Competition Assays.

(84) As an infectious inoculum, a 50-fold concentrated culture supernatant of HepG2 clone 2.2.15 (11) cells was used, because of an unlimited supply and a constant quality. It was prepared from freshly collected supernatants by precipitating viral particles in the presence of 6% polyethylene glycol (PEG) 8000. The pellet was resuspended in phosphate buffered saline (PBS) containing 25% FCS. Aliquots were stored at 80 C. Differentiated HepaRG cells or PHH were incubated with the concentrated infectious source, 10-fold diluted in culture medium supplemented with 4% PEG 8000 (Sigma), for 20 h at 37 C. At the end of the incubation, cells were washed three times with the culture medium and maintained in the presence of 2% DMSO and 510.sup.5 M hydrocortisone hemisuccinate and harvested at indicated times.

(85) Competition experiments were performed in 12-well plates. Approximately 110.sup.6 cells were first pre-incubated for 30 min with chemically synthesized HBV derived peptides followed by a co-incubation of cells with peptide and virus for 20 h. All competition series were performed at least twice and the results of one representative experiment are shown in each case (see FIGS. 3 to 7).

(86) HepaRG cells were infected either in absence (0 nM) or in the presence of 1, 5, 25, 100 and 2000 nM of the respective peptides of the invention. The infectious inoculum (HBV of genotype D) and the peptides were incubated overnight. After washing, cells were maintained for another 12 days to allow viral gene expression. Cell culture supernatants from day 8-12 were collected and analyzed for secreted HBSAg using a quantitative commercially available ELISA. HBsAg values from the respective uncompleted infection were set to 100% and the degree of infection inhibition is given in % of the uncompleted infection.

(87) Immunofluorescence Experiments/Microscopy

(88) Primary hepatocytes grown on cover slips were incubated with the respective peptide at 200 nM in serum-free cell culture medium. After 1 hour incubation at 37 C. cells were fixed and nuclei were stained with DAPI. Fluorescence microscopy was performed on a Spinning Disk Confocal Microscope at 600 magnification.

(89) FACS Analysis

(90) Cryo-preserved primary hepatocytes were thawed and washed with serum-free medium. In each reaction 410.sup.5 cells/ml were incubated with the respective peptide at a concentration of 200 nM in serum-free cell culture medium. The staining was performed for 30 minutes at room temperature with frequent mixing. Subsequently, cells were washed extensively and resuspended in PBS to proceed with the FACS analysis.

(91) Results are shown in the Figures.

(92) Fusion Constructs of HBVpreS1-Peptides with Mouse Interferon Alpha-Production, Purification and Activity Test

(93) HBVpreS1-peptide-mouse interferon alpha fusion proteins (see FIG. 14) were expressed in Hi5-insect cells using the baculovirus expression system. The fusion proteins secreted in the cell supernatants were harvested on day 5 post infection and purified in a first step by an affinity chromatography for the His-tag fused C-terminally to the preS1-interferon. The activity of the preS1-interferon proteins in the elution fractions was measured by their ability to inhibit Newcastle disease virus mediated cell death. The elution fractions containing functional preS1-interferon proteins were further purified by gel filtration chromatography on a S75-sepharose column with a 1M urea-PBS buffer (see FIG. 15).

(94) The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

REFERENCES

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