CONJUGATE COMPOUNDS FOR PREVENTING AND/OR TREATING HBV AND/OR HDV INFECTIONS, LIVER DISEASES ANDFOR TARGETING NTCP

Abstract

The present invention relates to conjugate compounds which comprise a peptide moiety (a) which is preferably a hydrophobic modified preS-derived peptide of hepatitis B virus or a respective cyclic peptide, and a NTCP substrate moiety (b), which is preferably a bile acid. The present invention further relates to pharmaceutical compositions comprising at least one conjugate compound. The present invention further relates to medical uses of said conjugate compounds 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 said diseases and/or infections.

Claims

1. A conjugate compound, comprising: (a) a peptide moiety, and (b) a NTCP substrate moiety, which addresses the bile acid binding site of sodium taurocholate co-transporting polypeptide (NTCP), which are covalently attached to each other.

2. The conjugate compound of claim 1, wherein the peptide moiety (a) is selected from: a hydrophobic modified preS-derived peptide of hepatitis B virus (HBV) of the general formula I
H—[(X).sub.m—P—(Y).sub.n]—R  (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 5 to 25; H is a hydrophobic modification, which is located N-terminal of P or within X or within Y, and selected from acylation and addition of hydrophobic moieties, R is a C-terminal modification, which is a moiety that protects from degradation, or a cyclic peptide of the general formula Ia
cyclo[(X).sub.m—P—(Y).sub.n]  (Ia) wherein P, X, Y, m and n are as defined above, and carrying at least one hydrophobic modification at amino acid side chain(s) of X and/or Y, wherein said cyclic peptide is not cyclized within the amino acid sequence of P of SEQ ID NO. 1 and not via amino acid side chains of P, wherein the hydrophobic modification is an acylation or addition of hydrophobic moieties, or a pharmaceutically acceptable salt thereof.

3. The conjugate compound of claim 1, wherein the hydrophobic modification of the peptide moiety (a) is an acylation with a C8 to C22 fatty acid or the hydrophobic moiety or moieties is/are selected from cholesterol, cholesterol derivatives, phospholipids, glycolipids, glycerol esters, steroids, ceramids, and isoprene derivatives.

4. The conjugate compound of claim 1, wherein the peptide moiety (a) is peptide of the general formula I, wherein m=0 to 18 and/or n=0 to 7, or wherein the peptide moiety (a) is a cyclic peptide of the general formula Ia, wherein m=0 to 18 and/or n=0 to 7, provided that m+n is at least 1.

5. The conjugate compound of according to claim 1, wherein the peptide moiety (a) comprises an amino acid sequence selected from the group of SEQ ID NOs: 2 to 25.

6. The conjugate compound according to claim 1, wherein the peptide moiety (a) comprises further amino acid(s) for covalently attaching the bile acid moiety, wherein said further amino acid(s) are L- or D amino acid(s) and can be natural or non-natural amino acids, and/or wherein the peptide moiety (a) is a cyclic peptide which comprises further amino acid(s) for cyclization, wherein said further amino acid(s) for cyclization can be natural or non-natural amino acids.

7. The conjugate compound of according to claim 1, wherein the NTCP substrate moiety (b) is selected from natural substrate(s) of sodium taurocholate co-transporting polypeptide (NTCP).

8. The conjugate compound of according to claim 1, wherein the NTCP substrate moiety (b) comprises bile acid(s) which is/are selected from monomeric and polymeric bile acids, sulfated bile acids and salts thereof, dimers of ursodeoxycholate, dimers comprising tauroursodeoxycholate (TUDCA), mixed dimers of the bile acids.

9. The conjugate compound of according to claim 1, comprising a further moiety or moieties selected from: drug(s) or their respective prodrug(s); tag(s); label(s); recombinant virus(s); carrier or depot(s) for drug(s), prodrug(s) or label(s); immunogenic epitope(s); hormones; inhibitor(s); and toxins.

10. A pharmaceutical composition comprising: (i) at least one conjugate compound of claim 1, and (ii) a pharmaceutically acceptable carrier and/or excipient.

11. (canceled)

12. A method for the diagnosis, prevention and/or treatment of a liver disease or condition wherein said method comprises the use of the conjugate compound of claim 1.

13. 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 condition is a disease which involves a liver stadium of a virus or a non-viral pathogen, and/or wherein the liver disease or condition is a liver tumor.

14. The method according to claim 12, wherein the liver disease or condition 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, or necessitates a delivery of a compound to the site or location of the disease or condition.

15. The method according to claim 12, comprising a combination therapy wherein said conjugate compound is administered with another therapeutic agent, and/or wherein the conjugate compound 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 μmol per kg body weight, and/or wherein conjugate compound is administered via a route of administration selected from oral, subcutaneous, intravenous, nasal, intramuscular, transdermal, inhalative, and by suppository.

16. The conjugate compound according to claim 1, wherein the moiety that protects from degradation is selected from amides, D-amino acids, modified amino acids, cyclic amino acids, PEG, and glycane.

17. The conjugate compound according to claim 5, wherein the peptide moiety (a) comprises an amino acid sequence selected from SEQ ID NOs: 2 to 17 and SEQ ID NOs: 18 to 25.

18. The conjugate compound according to claim 6, wherein the further amino acids are selected from lysine (K), D-lysine (k), D-tyrosine (y), cysteine (C), propargylglycine, azidophenylalanine, azidolysine, azidophenylalanine, homoallylglycine, homopropargylglycine, azidohomoalanine, azidonorleucine, azidophenylalanine, propargyloxyphenylalanine and acetylphenylalanine.

19. The conjugate compound according to claim 7, wherein the NTCP substrate moiety (b) is selected from: bile acid(s), bile acid dimer(s) and multimer(s), or non-natural substrate(s) of NTCP selected from ezetimibe, irbesartan, rosiglitazone, zafirlukast, TRIAC, and sulfasalazine.

20. The conjugate compound according to claim 8, wherein the monomeric and polymeric bile acids are selected from: cholate (CA), ursodeoxycholate (UDCA), lithocholate (LCA), taurocholate (TCA), glycocholate, taurodeoxycholate (TDCA), taurochenodeoxycholate (TCDC), and tauroursodeoxycholate (TUDCA); and/or the dimers of ursodeoxycholate are UDCA-UDCA; and/or the dimer comprising TUDCA is ##STR00005## and/or wherein the bile acid(s) are not attached to the peptide moiety (a) via the 3-hydroxyl group of ring A of the steroid skeleton.

21. The method according to claim 13, wherein the liver disease or condition is hepatitis caused by hepatitis A, B, C, D, E, F, G and H virus.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0255] FIG. 1 Examples of bile acids (for the bile acid moiety (b) of the conjugate compound A and B, monomers, indicating the preferred coupling site to the peptide moiety,

C, dimers of bile acids

[0256] FIG. 2 Preferred conjugate compounds.

A, peptide moiety is linear,
B, peptide moiety is a cyclic peptide. Also shown are possible positions for substitution with lysine (K) for coupling of the NTCP substrate moiety.

[0257] FIG. 3 HBV/HDV infection assay.

[0258] Tested were conjugate compounds comprising a truncated variant of Myrcludex B (amino acids 2-21) conjugated with bile acids cholic acid (CA) and ursodeoxy cholic acid (UDCA). They were compared with a control peptide not containing a bile acid moiety, namely HBVpreS/(-11)-21.sup.myr (Genotype B) and the two bile acids CA and UDCA alone. The conjugate compounds show a significantly higher activity than the peptide alone (1-2 nM versus 20-100 nM).

[0259] FIG. 4 HBV/HDV infection assay.

[0260] Tested were conjugate compounds comprising a truncated variant of Myrcludex B (amino acids 2-21) conjugated with cholic acid (CA) and a cyclic peptide (also comprising amino acids 2-21) conjugated with ursodeoxy cholic acid (UDCA). They were compared with a control peptide not containing a bile acid moiety, namely HBVpreS/2-21.sup.myr (Genotype B).

[0261] FIG. 5 HBV/HDV infection assay of compounds conjugated with bile acid monomers and dimers.

[0262] Tested were three compounds: two conjugated with ursodeoxy cholic acid (UDCA) and one with a dimer of ursodeoxy cholic acid (UDCA)2, wherein two comprise amino acids 2-21 of Myrcludex B in a linear form and one as cyclic peptide.

[0263] FIG. 6 In vivo planar scintigraphy imaging of iodine-125-labeled compounds of the invention.

[0264] Time course of scintigraphic imaging (planar images) of .sup.125I-labeled compounds at the indicated points in time post injection in a naïve mouse.

[0265] FIG. 7 Infection inhibition of bile acid conjugates with linear peptide sequences Reduction of HDV infection by various bile acid conjugates, control peptides, ursodeoxycholic acid and cholic acid in Huh7-hNTCP cells. The cells were pretreated with the substances for 30 minutes and then co-incubated with the substances and HDV overnight. An in-cell ELISA was carried out five days after infection (n=3).

[0266] FIG. 8 Infection inhibition of bile acid conjugates with cyclic peptide sequences

[0267] Reduction of HDV infection by various bile acid conjugates and control peptides in Huh7-hNTCP cells. The cells were pretreated with the substances for 30 minutes and then co-incubated with the substances and HDV overnight. An in-cell ELISA was carried out five days after infection (n=3).

[0268] FIG. 9 Infection inhibition of a bile acid dimer peptide conjugates

[0269] Reduction of HDV infection by a bile acid dimer peptide conjugate bile acid conjugates as compared to the monomeric bile acid conjugates and the control peptides in Huh7-hNTCP cells. The cells were pretreated with the substances for 30 minutes and then coincubated with the substances and HDV overnight. An in-cell ELISA was carried out five days after infection (n=3).

EXAMPLES

Example 1 Solid Phase Synthesis of the Peptide Moieties

[0270] All peptides were synthesized by automated Fmoc/tBu solid phase peptide synthesis on an Applied Biosystems 433A synthesizer as described previously (Schieck et al., 2010). The cyclisation was achieved under oxidative conditions to faun the respective disulfide bridge. Following the peptide synthesis myristic acid was coupled to the N-terminal end of the peptides. For this purpose the resin was shaken with a solution of myrictic acid (10 eq), HBTU (9.5 eq) and DIPEA (20 eq) in NMP for 2.5 h. The resin was then washed with NMP and DCM.

[0271] For the removal of the lysine side chain protective group (Aloc) the resin was incubated with a solution of 3 mg Pd(PPh.sub.3) and 30 mg BH.sub.3NHMe.sub.2 in DCM for 20 min. The resin was washed with DCM and MeOH and then incubated in DCM/MeOH (10:1) two times for 30 minutes. Then it was washed with DCM and Et.sub.2O and dried in vacuo.

Example 2 Conjugation of the Bile Acid Peptide Conjugates

[0272] For the coupling of the different bile acids or bile acid dimers the resin was swollen in NMP. Then a solution of bile acid (10 eq), HBTU (9.5 eg) and DIPEA (20 eq) in NMP was added and shaken for 2.5 h. After this the resin was washed with NMP, DCM and Et.sub.2O and dried under vacuo.

[0273] The conjugates were cleaved with 95% TFA/2.5% TIS/2.5% H.sub.2O for 1 h and then precipitated in cold Et.sub.2O. Then they were purified by preparative reversed-phase HPLC and the purity was confirmed by analytical HPLC and subsequently analyzed by LC-MS.

Example 3 HDAg In-Cell ELISA (Huh7-hNTCP

[0274] A. Material:

[0275] Washing Buffer: PBS+0.05% Tween 20

[0276] Permeabilization buffer: PBS+0.25% Triton X-100

[0277] Blocking Buffer: PBS+0.05% Tween 20+1% Casein

[0278] White 96-well cell culture plates: Greiner 655098

[0279] Advansta ELISABright Chemiluminscence substrate (order via Biozym #541025)

[0280] Hydrogen peroxide solution (35%, Sigma 349887)

[0281] 1.sub.st Antibody Mouse or rabbit anti HDAg (FD3A7) 1:3000

[0282] 2nd Antibody: Goat-anti-mouse or goat-anti-rabbit peroxidase conjugated 1:5000

[0283] B. Procedure:

[0284] Grow and infect cells in white 96-well plates.

[0285] 1) Fix cells with 50 μl 4% PFA at RT for 30 min

[0286] 2) Wash 2× with PBS (not washing buffer)

[0287] 3) Permeabilize with 100 μl permeabilization buffer at RT for 30 min

[0288] 4) Block with 100 μl blocking buffer at RT for 30 min

[0289] 5) Incubate with 100 μl primary antibody (1:3000) diluted in blocking buffer at RT with shaking for 1 h

[0290] 6) 3×1 min 200 μl wash (washing buffer)

[0291] 7) Incubate with 100 μl 3% (HuH7-derived cells) hydrogen peroxide solution (1:12 from stock in PBS for 3%) for 10 min at RT

[0292] 8) 4×1 min 200 μl wash (washing buffer)

[0293] 9) Incubate with 100 μl 2nd Antibody 1:5000 in blocking buffer at RT with shaking for 1 h.

[0294] 10) 3×1 min 200 μl wash (washing buffer)+2×10 min 200 μl wash (washing buffer)+1×10 min 2000 wash (permeabilization buffer) [VERY IMPORTANT]

[0295] 11) Add 50 μl luminescence substrate (mix according to manufacturer's protocol) and measure at plate reader directly after pipetting.

[0296] (Enhanced luciferase protocol, 0.1 s measurement)

[0297] B. Results:

[0298] The results are shown in FIGS. 3, 4 and 5 (genotype B sequence). The following IC50 values were measured:

[0299] UDCA: 79 μM

[0300] CA: 115 μM

[0301] HBVpreS/2-21myr: 62.8 nM

[0302] HBVpreS/2-21myr-CA: 7.27 nM

[0303] HBVpreS/2-21myr-UDCA: 8.45 nM

[0304] Cyclo2-21-UDCA: 10.1 nM

[0305] HBVpreS/2-21myr-(UDCA)2: 3.55 nM.

[0306] Further results are shown in FIGS. 7 to 9.

[0307] In the experiments shown in FIGS. 7 and 9, the following IC 50 values were measured:

[0308] UDCA: 79 μM

[0309] CA: 115 μM

[0310] MyrB-UDCA: 1.5 nM

[0311] HBVpreS/2-21myr (consensus): 70.3 nM

[0312] HBVpreS/2-21myr-CA: 12.5 nM

[0313] HBVpreS/2-21myr-UDCA: 9.2 nM

[0314] HBVpreS/2-21myr-LCA: 7.5 nM

[0315] HBVpreS/2-21myr-UDCA: 9.2 nM

[0316] HBVpreS/2-21myr-(UDCA-Dimer): 12.7 nM

[0317] The IC 50 values for the cyclic compounds, as shown in FIG. 8, were all in the range of 11 nM to 100 nM.

Example 4 Planar Imaging

[0318] Radiolabeling with iodine-125, was conducted at the tyrosine moiety of the peptide using the chloramine-T method.

[0319] 5 μL of a 1 mM peptide/peptide conjugate solution were added to 25 μl phosphate buffer (0.25 M, pH 7.5). 1-20 MBq iodine-125 in 0.05 M NaOH were added and the labeling reaction was started by addition of 5 μL of freshly prepared aqueous chloramine-T (2.8 mg/mL). After vortexing for 30 sec, the labeling reaction was quenched by adding 10 μL of a saturated aqueous methionine. The radiolabeled peptide/peptide conjugate was purified by semi-preparative radio-HPLC using a Chromolith Performance RP-18e column (100×4.6 mm; Merck) eluted with a linear gradient of 0.1% TFA in water and in acetonitrile. The solvent of the collected fraction was removed in vacuo and the labeled product was reconstituted in PBS.

[0320] For in vivo planar scintigraphy imaging of the iodine-125-labeled compounds naïve NMRI-mice (20-25 g) were anesthetized with 2% sevoflurane. For each compound an activity of 1-5 MBq was injected into the tail vein. The scintigraphic imaging was performed using a Gamma Imager SCT (Biospace Lab, Paris, France) with a parallel collimator (35 mm/1.8 mm/0.2 mm). The series of scintigraphy images were scanned at the indicated points in time post-injection. See FIG. 6.

[0321] 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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