CYCLIC NTCP-TARGETING PEPTIDES AND THEIR USES AS ENTRY INHIBITORS

Abstract

The present invention relates to cyclic NTCP targeting peptides which are preS-derived peptides of hepatitis B virus (HBV). The present invention further relates to pharmaceutical compositions comprising at least one cyclic peptide. The present invention further relates to medical uses of said cyclic peptides and the pharmaceutical compositions, such as in the diagnosis, prevention and/or treatment of a liver disease or condition, and/or in the inhibition of HBV and/or HDV infection. The present invention further relates to methods of diagnosis, prevention and/or treatment of a liver disease or condition and/or the inhibition of HBV and/or HDV infection.

Claims

1. A cyclic peptide of the general formula I
(X).sub.mP(Y).sub.n (I) wherein P is the amino acid sequence NPLGFXaaP (SEQ. ID NO: 1), with Xaa being F or L, X is an amino acid sequence having a length of m amino acids, wherein m is 0 or at least 1; Y is an amino sequence having a length of n amino acids, wherein n is 0 or at least 1; and wherein m+n is 0 or at least 1; or a pharmaceutically acceptable salt thereof.

2. The cyclic peptide of claim 1, which is hydrophobically modified, wherein the hydrophobic modification(s) is/are at amino acid side chain(s) of X and/or Y, and wherein the hydrophobic modification is acylation and/or addition of hydrophobic moieties.

3. The cyclic peptide of claim 1, wherein the peptide is cyclized (a) via thiol oxidation (disulfide bridge formation) of two cysteines (C) in the peptide, (b) amide condensation of two amino acid side chains (lactam), (c) via head-to-tail cyclization, (d) via backbone cyclization, (e) via thioether formation, and/or (f) via hydrogen bond formation and/or bond-forming derivatives of amino acids.

4. The cyclic peptide of claim 1, wherein m+n is 0 to 42, m=0 to 8 and n=0 to 34.

5. The cyclic peptide of claim 1, further comprising an accessory domain, which is part of the cyclic peptide or is acyclic.

6. The cyclic peptide of claim 1, having the general formula Ia
cyclo[(X).sub.mP(Y).sub.n] (Ia) and carrying at least one hydrophobic modification at one or more amino acid side chains of X and/or P, wherein said cyclic peptide is represented by the formula:
H-cyclo[(X).sub.mP(Y).sub.n] wherein H is the hydrophobic modification.

7. The cyclic peptide of claim 1, wherein the peptide comprises an amino acid sequence selected from the group of TABLE-US-00032 HBVpreS9-15 (SEQIDNO.:2) NPLGFFP Cys-HBVpreS9-15-Cys (SEQIDNO.:4) C-NPLGFFP-C Cys-HBVpreS9-15-Cys-D-Tyr (SEQIDNO.:5) C-NPLGFFP-C-y HBVpreS9-16 (SEQIDNO.:6) NPLGFFPD Cys-HBVpreS9-16-Cys (SEQIDNO.:7) C-NPLGFFPD-C Cys-HBVpreS9-16-Cys-D-Tyr (SEQIDNO.:8) C-NPLGFFPD-C-y HBVpreS8-16 (SEQIDNO.:9) PNPLGFFPD Cys-HBVpreS8-16-Cys (SEQIDNO.:10) C-PNPLGFFPD-C Cys-HBVpreS8-16-Cys-D-Tyr (SEQIDNO.:11) C-PNPLGFFPD-C-y HBVpreS9-17 (SEQIDNO.:12) NPLGFFPDH Cys-HBVpreS9-17-Cys (SEQIDNO.:13) C-NPLGFFPDH-C Cys-HBVpreS9-17-Cys-D-Tyr (SEQIDNO.:14) C-NPLGFFPDH-C-y HBVpreS8-17 (SEQIDNO.:15) PNPLGFFPDH Cys-HBVpreS8-17-Cys (SEQIDNO.:16) C-PNPLGFFPDH-C Cys-HBVpreS8-17-Cys-D-Tyr (SEQIDNO.:17) C-PNPLGFFPDH-C-y HBVpreS2-21 (SEQIDNO.:18) GTNLSVPNPLGFFPDHQLDP Cys-HBVpreS2-21-Cys (SEQIDNO.:19) C-GTNLSVPNPLGFFPDHQLDP-C Cys-HBVpreS2-21-Cys-D-Tyr (SEQIDNO.:20) C-GTNLSVPNPLGFFPDHQLDP-C-y HBVpreS2-48 (SEQIDNO.:21) GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANQVG Cys-HBVpreS2-48-Cys (SEQIDNO.:22) C-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNHDHWPEANQ VG-C Cys-HBVpreS2-48-Cys-D-Tyr (SEQIDNO.:23) C-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANQVG- C-y MyrcludexB (SEQIDNO.:24) GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANKVG Cys-MyrcludexB-Cys (SEQIDNO.:25) C-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANKVG- Cand Cys-MyrcludexB-Cys-D-Tyr (SEQIDNO.:26) C-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANK VG-C-y.

8. The cyclic peptide of claim 1, comprising one or more further moieties, selected from drugs and their respective prodrugs; tags; labels; recombinant viruses and derivatives thereof; carrier or depots for drugs, prodrugs or labels; immunogenic epitopes; hormones; inhibitors; and toxins.

9. A pharmaceutical composition comprising: (i) at least one cyclic peptide of claim 1, and (ii) optionally, a pharmaceutically acceptable carrier and/or excipient.

10. (canceled)

11. A method for one or more of inhibition of HBV and/or HDV infection, prevention of a primary HBV and/or HDV infection, and inhibiting HBV and/or HDV entry, wherein said method comprises the use of a compound of claim 1.

12. A method for: A) diagnosis, prevention and/or treatment of a liver disease or condition, and/or B) prevention and/or treatment of a cardiovascular disease (CVD), wherein said method comprises the use of a compound of claim 1.

13. The method, according to claim 12, wherein said method is used for prevention and/or treatment of a cardiovascular disease (CVD), wherein said method comprises the control or modification of the cholesterol level or cholesterol uptake, and wherein the cholesterol level or uptake is controlled or modified by decreasing or blocking the NCTP-mediated bile salt transport, by the cyclic peptide.

14. The method according to claim 12, wherein the cyclic peptide is administered in a therapeutically effective amount, in the range of from about 0.01 mg to about 50 mg per patient, or is applied to a patient in a dose ranging from 10 pmol per kg to 20 mol per kg body weight.

15. The method according to claim 12, wherein the route of administration is selected from oral, subcutaneous, intravenous, nasal, intramuscular, transdermal, inhalative, and by suppository.

16. The method, according to claim 12, wherein, the liver disease or condition is selected from hepatitis, cirrhosis, and haemochromatosis, and/or wherein the liver disease or disorder is a disease which involves malaria, schistosomiasis, leishmaniasis, and/or Morbus Wilson, and/or wherein the liver disease or disorder is a liver tumor, and/or wherein the liver disease or disorder is a post-transplantation complication after liver transplantation related to bile salt accumulation within the biliary pathway, and/or wherein the liver disease or condition is related to sodium taurocholate cotransporter polypeptide (NTCP)-mediated transport of compounds into hepatocytes, wherein the compounds that are transported into hepatocytes via NTCP are bile acids; steroids; conjugated and non-conjugated thyroid hormones; liver toxins; or compounds that are covalently bound to taurocholate, bromosulphophthalein, or drugs.

17. The cyclic peptide, according to claim 1, wherein the hydrophobic modification is an acylation with a C8 to C22 and/or the hydrophobic moiety is selected from cholesterol, cholesterol derivatives, phospholipids, glycolipids, glycerol esters, steroids, ceramids, and isoprene derivatives.

18. The cyclic peptide, according to claim 4, wherein m+n is at least 1.

19. The cyclic peptide, according to claim 7, wherein the peptide comprises an amino acid sequence selected from TABLE-US-00033 (SEQ.IDNO:2) cyclo[NPLGFFP] (SEQ.IDNO:6) cyclo[NPLGFFPD] (SEQ.IDNO:9) cyclo[PNPLGFFPD] (SEQ.IDNO:12) cyclo[NPLGFFPDH] (SEQ.IDNO:15) cyclo[PNPLGFFPDH] (SEQ.IDNO:18) cyclo[GTNLSVPNPLGFFPDHQLDP] (SEQ.IDNO:21) cyclo[GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPE ANQVG] (SEQ.IDNO:24) cyclo[GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPE ANKVG] (SEQIDNO.3) cyclo[NPLGFLP] (SEQIDNO.27) cyclo[NPLGFLPD] (SEQIDNO.28) cyclo[PNPLGFLPD] (SEQIDNO.29) cyclo[NPLGFLPDH] (SEQIDNO.30) cyclo[PNPLGFLPDH] (SEQIDNO.31) cyclo[GTNLSVPNPLGFLPDHQLDP] (SEQIDNO.32) cyclo[GTNLSVPNPLGFLPDHQLDPAFGANSNNPDWDFNPNKDHWPE ANQVG] (SEQIDNO.33) cyclo[GTNLSVPNPLGFLPDHQLDPAFGANSNNPDWDFNPNKDHWPE ANKVG].

20. The cyclic peptide, according to claim 7, wherein the peptide comprises an amino acid sequence selected from TABLE-US-00034 Myr-cyclo[HBVpreS9-15] (SEQ.IDNO:2) myr-cyclo[NPLGFFP] Myr-cyclo[HBVpreS9-16] (SEQ.IDNO:6) myr-cyclo[NPLGFFPD] Myr-cyclo[HBVpreS8-16] (SEQ.IDNO:9) myr-cyclo[PNPLGFFPD] Myr-cyclo[HBVpreS9-17] (SEQ.IDNO:12) myr-cyclo[NPLGFFPDH] Myr-cyclo[HBVpreS8-17] (SEQ.IDNO:15) myr-cyclo[PNPLGFFPDH] Myr-cyclo-[HBVpreS2-21] (SEQ.IDNO:18) myr-cyclo[GTNLSVPNPLGFFPDHQLDP] Myr-cyclo-[HBVpreS2-48] (SEQ.IDNO:21) myr-cyclo[GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDH WPEANQVG] Myr-cyclo-[HBVpreS2-48] = cyclicMyrcludexB (SEQ.IDNO:24) myr-cyclo[GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDH WPEANKVG].

21. The cyclic peptide, according to claim 7, wherein the peptide consists of an amino acid sequence selected from TABLE-US-00035 (SEQ.IDNO:2) cyclo[NPLGFFP] (SEQ.IDNO:6) cyclo[NPLGFFPD] (SEQ.IDNO:9) cyclo[PNPLGFFPD] (SEQ.IDNO:12) cyclo[NPLGFFPDH] (SEQ.IDNO:15) cyclo[PNPLGFFPDH] (SEQ.IDNO:18) cyclo[GTNLSVPNPLGFFPDHQLDP] (SEQ.IDNO:21) cyclo[GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWP EANQVG] (SEQ.IDNO:24) cyclo[GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWP EANKVG] (SEQIDNO.3) cyclo[NPLGFLP] (SEQIDNO.27) cyclo[NPLGFLPD] (SEQIDNO.28) cyclo[PNPLGFLPD] (SEQIDNO.29) cyclo[NPLGFLPDH] (SEQIDNO.30) cyclo[PNPLGFLPDH] (SEQIDNO.31) cyclo[GTNLSVPNPLGFLPDHQLDP] (SEQIDNO.32) cyclo[GTNLSVPNPLGFLPDHQLDPAFGANSNNPDWDFNPNKDHWP EANQVG] (SEQIDNO.33) cyclo[GTNLSVPNPPGFLPDHQLDPAFGANSNNPDWDFNDNKDHWP EANKVG].

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0261] FIG. 1 Myrcludex B and derivatives

[0262] FIG. 2 A covalently bridged cyclic derivative of Myrcludex B shows inhibitory potential.

[0263] A, The sequence and structure of the covalently bridged cyclic derivative of Myrcludex B (Myr-2-48 Cyc).

[0264] B, The inhibitory activity of Myr-2-48 Cyc is comparable to other Myrcludex B derivatives, such as a linear Myrcludex B derivative with myristoyl group within the peptide sequence (2-Myr-48) and a linear preS-derives peptide 2-21 with a C-terminal myristoyl group (2-21-Myr).

[0265] FIG. 3 Further cyclic Myrcludex B derivates

[0266] FIG. 4 Further cyclic Myrcludex B derivates

[0267] A, Overview of synthesized cysteine cyclized peptides and control peptides

[0268] B, Experimental setup for testing of peptides:

[0269] HepG2 NTCP cells were seeded in 24 well plates. When cells reached 60-70% confluency, they were subjected to HBV infection or TC uptake assay. Cell supernatant was collected from day 5 to 7 for HBeAg measurement and cells were fixed with 4% PFA at day 7 for immunofluorescence with an anti-HBc antibody.

[0270] FIG. 5 Coronary PET images 40-60 minutes post injection of .sup.68Gallium labeled peptides.

[0271] A,

TABLE-US-00030 myr-CNPLGFFPDCK(DOTA[.sup.68Ga])

[0272] B, Liver blocked with cold Myrcludex B 1 g/g bodyweight 30 minutes prior to injection with myr-CNPLGFFPDCK(DOTA[.sup.68Ga])

[0273] C, Myr-K(DOTA[.sup.68Ga])HBVpres3-48y (WT peptide)

[0274] D, Myr-K(DOTA[.sup.68Ga])HBVpres3-48y Ala11-15 (Binding incompetent control peptide) See also Slijepcevic et al., 2015.

[0275] FIG. 6 .sup.3H-Taurocholate uptake in HepG3 NTCP cells comparison of different cyclic peptides with Myrcludex B

[0276] FIG. 7 .sup.3H-Taurocholate uptake in HepG3 NTCP cells: IC 50 values and curves of different cyclic peptides with Myrcludex B

[0277] FIG. 8 HBV infection inhibition assay on HepG2 NTCP cells.

[0278] with Myrcludex B (GMP grade), Myrcludex B-y (selfmade) and (+H) cyclic peptide.

[0279] A, IC 50 curves and values.

[0280] B, Absolute values of HBeAg measurement of supernatants diluted 1:2

EXAMPLES

Example 1 Materials & Methods

Abbreviations

[0281] COMU (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium-hexafluorophosphate [0282] DCM Dichloromethane [0283] DIPEA N,N-diisopropylethylamine [0284] DMF Dimethylformamide [0285] DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid [0286] Fmoc Fluorenylmethyloxycarbonyl chloride [0287] Ga Gallium [0288] HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate [0289] HBeAg Hepatitis B Virus Early Antigen [0290] HBcAg Hepatitis B Core Antigen [0291] HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate [0292] HBV Hepatitis B Virus [0293] HPLC High performance liquid chromatography [0294] LC/MS Liquid chromatography-mass spectrometry [0295] NHS N-Hydroxysuccinimide [0296] NMP N-Methyl-2-pyrrolidone [0297] NTCP Sodium taurocholate cotransporting polypeptide [0298] PBS Phosphate buffered saline [0299] PET Positron emission tomography [0300] PFA Paraformaldehyde [0301] TC Taurocholate [0302] TFA Trifluoro acetic acid [0303] TIS Triisopropylsilane

1. Peptide Synthesis

1.1 Disulfide Bridge Peptide

Solid Phase Peptide Synthesis

[0304] Peptides were synthesized on solid phase (Tentagel R RAM resin, capacity 0.22 mmol/g, Rapp Polymere, Tibingen, Germany) using Fmoc/tBu chemistry in peptide synthesizer (Applied Biosystems 443A, Foster City, Calif., USA). Before beginning peptide synthesis the resin (0.05 mmol) was preswollen in DCM. Fmoc-protected amino acids were used in a 10-fold excess (0.5 mmol) and activated with HBTU/DIPEA in NMP.

[0305] See also Schieck et al., 2010.

Myristoylation

[0306] Peptide on the solid support was swollen in DCM and washed with NMP. Myristic acid (4 eq.) and HATU or COMU (4 eq.) were dissolved in NMP and 10 eq. DIPEA were added. The mixture was added to the resin and was incubated for 30 min. Afterwards the resin was washed three times with NMP, three times with DCM and dried.

Deprotection and Cleavage from Resin

[0307] The peptide was cleaved and deprotected with TFA/TIS/H.sub.2O (95:2.5:2.5). The deprotected peptide was precipitated with diethyl ether, pelleted by centrifugation (3000 rpm, 5 min) and washed twice with fresh diethyl ether. The peptide was dried.

MyrB:

[0308]

TABLE-US-00031 [SEQIDNO.24] Myr-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANK VG-amide

Disulfide Bridge Formation

[0309] 5 mg of raw peptide were dissolved in 5 ml 80% acetic acid. 1 mg of iodine in glacial acetic acid was slowly dropped into the peptide solution. 10 l of saturated ascorbic acid solution were added. The solvent was evaporated and the peptide redissolved in 1:1 acetonitril:H.sub.2O and purified with preparative HPLC. The success of the reaction was confirmed by LC/MS.

Coupling of Fluorescent Dye/Compounds

[0310] Peptides were dissolved in DMF and reacted with NHS-ester-activated compounds (2 eq.) and DIPEA (10 eq.). The reaction was controlled with HPLC.

1.2 Aminoproline-Peptide

[0311] 200 mg (0.32 mmol) 2-Chlorotrityl chloride resin was charged with 41 mg (0.1 mmol) Fmoc-Glu(OAll)-OH and 52 mg (68 L; 0.4 mmol) DIPEA in 2 mL Dichloromethane. After capping with methanol the resin was subjected to automated peptide synthesis (ABI 433A). 109 mg (0.25 mmol) Fmoc-L-Pro(4-NH-Alloc)-OH (2S,4S) were coupled at the desired amino acid position by COMU-activation. Solid phase cyclization was achieved by 110 mg (86 mL; 0.4 mmol) DPPA and 77 mg (102 L; 0.6 mmol) in 2 mL NMP after linear assembly as well as catalytic allyl-deprotection with 5 mg tetrakis(triphenylphosphine)palladium(0) and 30 mg borane dimethylamine complex. The cyclic peptide was cleaved and deprotected by 95:2.5:2.5 TFA/water/TIS and purified by HPLC; occasionally a portion of the raw peptide was modified with DOTA or fluorescent dye active esters prior to purification

2. .SUP.68.Ga-Labeling and PET Imaging

[0312] Ca. 1 mL of [.sup.68Ga]Ga.sup.3+ eluate (ca. 600-800 MBq) was added to a mixture of 20 L of a 1 mM solution of compound xy in DMSO and 10 L of saturated solution of ascorbic acid in water. The pH of the resulting mixture was adjusted to 3.5-4.0 by careful addition of a 2.5 M sodium acetate solution in water. Complexation was achieved by heating to over 95 C. for 5-10 minutes under constant stirring. The product was isolated by solid phase extraction with ethanol followed by evaporation. The residue was taken up in 1% bovine serum albumin solution and an appropriate amount (ca. 20-50 MBq) was used for the individual experiment in a volume not exceeding 100 l. Mice were anesthetized with 1% sevoflurane (Abbott, Wiesbaden, Germany) and images were recorded using an Inveon small animal positron emission tomographic (PET) scanner (Siemens, Knoxville, Tenn.) up to 60 minutes postinjection.

3. .SUP.3.H-Taurocholate Uptake Assay

[0313] HepG2 NTCP cells seeded in a 24 well format were preincubated with the indicated peptide for 30 min at 37 C. in culture medium. 150 M taurocholate (containing 450 cpm/fmol .sup.3H taurocholate) were added to each well and the cells were incubated an additional 15 minutes at 37 C. Uptake was stopped by removal of the cell culture medium and addition of ice cold PBS. The cells were washed three times with cold PBS and lysed (0.2 M NaOH, 0.05% SDS). Cell lysates were mixed with Ultima Gold liquid scintillation solution (Perkin Elmer, Rodgau, Germany) and the radioactivity measured in a liquid scintillation counter (Packard Instruments, Frankfurt, Germany).

4. HBV Infection Inhibition Assay

[0314] HepG2 NTCP cells seeded in a 24 well format were preincubated with the indicated peptide at indicated concentrations for 30 min at 37 C. in culture medium. The cells were subsequently infected with HBV (GE 1.810.sup.8) overnight in cell culture medium containing 4% PEG for 16 h at 37 C. in the presence of the peptides followed by a washing step with PBS. The medium was changed every two days and supernatant collected from day 5 to 7 post infection for HBeAg measurement.

[0315] 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

[0316] Chan, H L. & Sung, J J. Hepatocellular carcinoma and hepatitis B virus. Semin Liver Dis 26, 153-161 (2006). [0317] Cho M H, Song J S, Kim H J, Park S G, Jung G. Structure-based design and biochemical evaluation of sulfanilamide derivatives as hepatitis B virus capsid assembly inhibitors. J Enzyme Inhib Med Chem 2013; 28:916-925. [0318] Dawson S, et al. (2012) In vitro inhibition of the bile salt export pump correlates with risk of cholestatic drug-induced liver injury in humans. Drug Metab Dispos 40: 130-138. [0319] Doring B, Lutteke T, Geyer J, Petzinger E. The SLC10 carrier family: transport functions and molecular structure. Curr Top Membr 2012; 70:105-168. [0320] Fattinger K, Funk C, Pantze M, et al. The endothelin antagonist bosentan inhibits the canalicular bile salt export pump: a potential mechanism for hepatic adverse reactions. Clin Pharmacol Ther. 2001; 69:223-31. [0321] Funk C, Pantze M, Jehle L, et al. Troglitazone-induced intrahepatic cholestasis by an interference with the hepatobiliary export of bile acids in male and female rats. Correlation with the gender difference in troglitazone sulfate formation and the inhibition of the canalicular bile salt export pump (Bsep) by troglitazone and troglitazone sulfate. Toxicology. 2001; 167:83-98. [0322] Funk C, Ponelle C, Scheuermann G, et al. Cholestatic potential of troglitazone as a possible factor contributing to troglitazone-induced hepatotoxicity: in vivo and in vitro interaction at the canalicular bile salt export pump (Bsep) in the rat. Mol Pharmacol. 2001; 59:627-35. [0323] Gausepohl, H. et al. Int. J. Prot. Pept. Res. 34, 287-294 (1989). [0324] Gripon P, Cannie I, Urban S. Efficient inhibition of hepatitis B virus infection by acylated peptides derived from the large viral surface protein. J Virol 2005; 79:1613-1622. [0325] Kotani N, Maeda K, Debori Y, Camus S, Li R, Chesne C, Sugiyama Y. Expression and Transport Function of Drug Uptake Transporters in Differentiated HepaRG Cells. Mol Pharm 2012; 9(12):3434-41. [0326] Lempp F A, Urban S. Inhibitors of hepatitis B virus attachment and entry. Intervirology 2014; 57:151-157. [0327] Mailly L, Xiao F, Lupberger J, Wilson G K, Aubert P, Duong F H, Calabrese D, Leboeuf C, Fofana I, Thumann C, Bandiera S, Litgehetmann M, Volz T, Davis C, Harris H J, Mee C J, Girardi E, Chane-Woon-Ming B, Ericsson M, Fletcher N, Bartenschlager R, Pessaux P, Vercauteren K, Meuleman P, Villa P, Kaderali L, Pfeffer S, Heim M H, Neunlist M, Zeisel M B, Dandri M, McKeating J A, Robinet E, Baumert T F. Clearance of persistent hepatitis C virus infection in humanized mice using a claudin-1-targeting monoclonal antibody. Nat Biotechnol. 2015; 33(5):549-54. doi: 10.1038/nbt.3179. Epub 2015 Mar. 23. [0328] Meier A, Mehrle S, Weiss T S, Mier W, Urban S. The myristoylated preS1-domain of the Hepatitis B Virus L-protein mediates specific binding to differentiated hepatocytes. Hepatology 2012; 58:31-42. [0329] Mitchell A R, Erickson B W, Ryabtsev M N, Hodges R S, and Merrifield R B, Tert-butoxycarbonylaminoacyl-4-(oxymethyl)-phenylacetamidomethyl-resin, a more acid-resistant support for solid-phase peptide synthesis. J Am Chem Soc. 1976; 98(23):7357-62. [0330] Morgan R E, et al. (2010) Interference with bile salt export pump function is a susceptibility factor for human liver injury in drug development. Toxicol Sci 118: 485-500. [0331] Muiller T, Mehrle S, Schieck A, Haberkorn U, Urban S, Mier W. Liver imaging with a novel hepatitis B surface protein derived SPECT-tracer. Mol Pharm. 2013; 10(6):2230-6. [0332] Ni Y, Lempp F A, Mehrle S, Nkongolo S, Kaufman C, Falth M, Stindt J, et al. Hepatitis B and D Viruses Exploit Sodium Taurocholate Co-transporting Polypeptide for Species-Specific Entry into Hepatocytes. Gastroenterology 2014; 146:1070-1083. [0333] Ogimura E, et al. (2011) Bile salt export pump inhibitors are associated with bile acid-dependent drug-induced toxicity in sandwich-cultured hepatocytes. Biochem Biophys Res Commun 416: 313-317. [0334] Schieck A, Mller T, Schulze A, Haberkorn U, Urban S and Mier W. Solid-Phase Synthesis of the Lipopeptide Myr-HBVpreS/2-78, a Hepatitis B Virus Entry Inhibitor. Molecules 2010, 15(7), 4773-4783. [0335] Schieck A, Schulze A, Gahler C, Muller T, Haberkorn U, Alexandrov A, Urban S, Mier W. Hepatitis B virus hepatotropism is mediated by specific receptor recognition in the liver and not restricted to susceptible hosts. Hepatology 2013; 58(1): 43-53. [Epub ahead of print: 2013 Jan. 4.] [0336] Schulze A, Schieck A, Ni Y, Mier W, Urban S. Fine mapping of pre-S sequence requirements for hepatitis B virus large envelope protein-mediated receptor interaction. J Virol 2010; 84:1989-2000. [0337] Shepard, C. W., Simard, E. P., Finelli, L., Fiore, A. E. & Bell, B. P. Hepatitis B virus infection: epidemiology and vaccination. Epidemiol Rev 28, 112-125 (2006). [0338] Slijepcevic, D., Kaufman, C., Wichers, C. G. K., Gilglioni, E. H., Lempp, F. A., Duijst, S., de Waart, D. R., Oude Elferink, R. P. J., Mier, W., Stieger, B., Beuers, U., Urban, S., van de Graaf, S. F. J., 2015. Impaired uptake of conjugated bile acids and hepatitis b virus pres1-binding in na+-taurocholate cotransporting polypeptide knockout mice. Hepatology 62, 207-219 [0339] Stieger B, Fattinger K, Madon J, et al. Drug- and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver. Gastroenterology. 2000; 118:422-30. [0340] Stray S J, Zlotnick A. BAY 41-4109 has multiple effects on Hepatitis B virus capsid assembly. J Mol Recognit 2006; 19:542-548. [0341] Urban S, Future Virol. 2008, 3(3), 253-264. [0342] Urban S, Bartenschlager R, Kubitz R, Zoulim F. Strategies to Inhibit Entry of HBV and HDV into Hepatocytes. Gastroenterology 2014; 147(1):48-64. [0343] Wang Y J, Lu D, Xu Y B, Xing W Q, Tong X K, Wang G F, et al. A Novel Pyridazinone Derivative Inhibits Hepatitis B Virus Replication by Inducing Genome-Free Capsid Formation. Antimicrobial agents and chemotherapy 2015; 59:7061-7072. [0344] White C J and Yudin A K. Contemporary strategies for peptide macrocyclization, Nature Chemistry 2011; 3, 509-524. [0345] Yan H, Zhong G, Xu G, He W, Jing Z, Gao Z, Huang Y, et al. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. elife. 2012; 1:e00049. [0346] Zoulim, F. Antiviral therapy of chronic hepatitis B. Antiviral Res 71, 206-215 (2006).