(12) Patent Application Publication (10) Pub. No.: US 2016/0220586 A1 ANDRE Et Al

US 2016O220586A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0220586 A1 ANDRE et al. (43) Pub. Date: Aug. 4, 2016

(54) METHODS AND PHARMACEUTICAL (86). PCT No.: PCT/EP2014/069312 COMPOSITIONS FOR THE TREATMENT OF S371 (c)(1) HEPATITIS B VIRUS INFECTION (2) Date Mar 10, 2016 (71) Applicants: INSERMLA. SANTE (INSTITUT ET DE LA NATIONAL RECHERCHE DE (30) Foreign ApplicationO O Priority Data MEDICALE),UNIVERSITE ParisCLAUDE (FR): BERNARD - Sep. 11, 2013 (EP) ...... 13306245.5 LYON 1, Villeurbanne (FR); ENS - Publication Classification ECOLE NORMALE SUPERIEURE DELYON, Lyon (FR); CENTRE (51) Int. Cl. NATIONAL DE LARECHERCHE A 6LX3/575 (2006.01) SCIENTIFIOUE (CNRS), Paris (FR): A613 L/53 (2006.01) EDELRIS, Lyon (FR); POXEL, Lyon A613 L/496 (2006.01) (FR) A613 L/4525 (2006.01) A613 L/42 (2006.01) (FR): VINCENT LOTTEAU, LYON (52) U.S. Cl CEDEX 07 (FR); PAULINE CPC ...... A61 K3I/575 (2013.01); A61K3I/42 RADREAU, LYON CEDEX 07 (FR): MARINE GILARDONE, MILLERY (2013.01); A61 K3I/551 (2013.01); A61 K (FR): AMAURY PATIN, LAUSANNE 3 1/496 (2013.01); A61 K3I/4525 (2013.01): (CH); DIDIER ROCHE, ECULLY A6 IK3I/513 (2013.01) (FR), DANIEL CRAVO, MONTESSON (57) ABSTRACT (FR); SOPHIE HALLAKOU-BOZEC, ANTONY (FR) The invention relates to methods and pharmaceutical compo sitions for the treatment of hepatitis B virus infection. In (21) Appl. No.: 14/917,958 particular, the present invention relates to farnesoid X recep tor (FXR) agonists for use in a method for the treatment of (22) PCT Filed: Sep.10, 2014 hepatitis B virus infection in a subject in need thereof. Patent Application Publication Aug. 4, 2016 Sheet 1 of 10 US 2016/0220586 A1

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METHODS AND PHARMACEUTICAL need for better therapies to meet the treatment goals in HBV COMPOSITIONS FOR THE TREATMENT OF infection, in particular CHB infection. Indirectly acting anti HEPATITIS B VIRUS INFECTION virals (IAD), besides interferons, arise as a very promising alternative class of antivirals. Small molecules blocking the FIELD OF THE INVENTION interaction of a cellular protein with a viral protein have been successfully developed to prevent HIV entry and HCV repli 0001. The present invention relates to methods and phar cation. Viral entry and innate immunity are obvious cellular maceutical compositions for the treatment of hepatitis B virus functions to be screened for the identification of new thera infection. peutic targets. However and unlike HIV and HCV, our knowl edge of specific cellular functions used by HBV to replicate in BACKGROUND OF THE INVENTION hepatocytes remains very limited and systematic screening 0002 HBV is an enveloped virus containing a 3.2-kb par for the identification of these essential host factors is neces tially double stranded DNA genome with four open reading sary to increase the diversity of potential therapeutic targets frames. These open reading frames encode the reverse tran and molecules. A major goal is therefore to identify these Scriptase, precore, and core proteins; three surface antigen functions for preventing their use and-or perturbation by the proteins (pre-S1, pre-S2, and S); and the X protein. Regula virus by safer and broad-spectrum molecules with high bar tion of HBV transcription is under the control of four pro rier to resistance. moters (the core, pre-S1, pre-S2/S, and X promoters) and two 0004 Recent data strongly suggest that farnesoid X recep enhancer regions (EN1 and EN2). Eight genotypes of HBV. tor (FXR) which is a member of the nuclear receptor super designated A to H, have been determined, with Some geo family is implicated in the regulation of HBV core promoter graphical distribution. The virus is non-cytopathic, with activity and that bile acids could play an important role in the virus-specific cellular immunity being the main determinant natural history of HBV infection (Ramiére C. Scholtés C, for the outcome of exposure to HBV acute infection with Diaz, O, Icard V. Perrin-Cocon L, Trabaud MA, Lotteau V. resolution of liver diseases within 6 months, or chronic HBV André P. Transactivation of the hepatitis B virus core pro infection that is frequently associated with progressive liver moter by the nuclear receptor FXRalpha. Journal of Virology, injury. Detection of HBS Ag in the serum by conventional 2008: 82: 10832-10840). Specifically in the particular cellu diagnostic immunoassays, is the key diagnostic marker for lar model of infection in the Huh-7 cell line with various HBV infection with HBV and persistent detection of HBSAg in infection vectors, data Suggested that FXRC agonists increase serum for more than 6 months is the hallmark of chronic HBV viral replication while antagonists of FXRC. may represent a infection. The best marker for clinically significant HBV new class of compounds useful for the treatment of HBV replication is the level of HBV DNA in serum, as detected by infection by inhibiting HBV replication. sensitive polymerase chain reaction (PCR)-based assay. Worldwide more than 350 million people are chronically SUMMARY OF THE INVENTION infected with HBV and are thus at increased risk of develop 0005. The present invention provides new methods for the ing serious liver disease—such as chronic hepatitis, cirrhosis, treatment of patients with hepatitis B virus infections. In liver failure and hepatocellular carcinoma (HCC). particular, the present invention is defined by the claims. 0003. The primary goal of treatment for chronic hepatitis B (CHB) is to permanently suppress HBV replication and DETAILED DESCRIPTION OF THE INVENTION prevent or improve liver disease. Seven drugs are currently available for treatment of CHB infection—conventional 0006. The present invention relates to farnesoid X receptor interferon, pegylated interferon and direct antiviral agents. (FXR) agonists for use in a method for the treatment of The directantivirals (nucleos/tide analogues) belong to three hepatitis B virus infection in a subject in need thereof. classes: L-nucleosides (lamivudine, telbivudine and emitric 0007 As used herein a “Hepatitis B virus infected patient” itabine); deoxyguanosine analogue (entecavir) and nucleo means a patient being infected with any Hepatitis B virus side phosphonates (adefovir and tenofovir) which directly genotype, e.g., genotype A, B, C, D etc. interfere with HBV DNA replication, primarily as chain ter 0008 According to the invention, the term “subject' or minators. The key limitations for interferon treatment are “patient' and “subject in need thereof or “patient in need major side-effects, low rate of HBV DNA suppression and thereof, is intended for a human or non-human mammal low rate of ALT normalization; key limitations of the treat infected or likely to be infected with a hepatitis B virus. In ment with direct antivirals are: development of resistance: some embodiments, the subject suffers from a chronic HBV rebound of HBV replication after stopping therapy requiring infection. prolonged, life-long therapy, very low rate of HBS Ag clear 0009. As used herein, the term “treatment' or “treat refer ance, increasing the risk of adverse events with prolonged, to both prophylactic or preventive treatment as well as cura life-long therapy. Importantly, current direct antivirals tive or disease modifying treatment, including treatment of repress the reverse transcription of the pregenomic viral RNA patient at risk of contracting the disease or Suspected to have into the genomic DNA. They thus act down stream to the contracted the disease as well as patients who are ill or have formation of the cccDNA that is formed after virus entry into been diagnosed as Suffering from a disease or medical con hepatocytes.cccDNA reside in the cell nucleus as additional dition, and includes Suppression of clinical relapse. The treat minichromosomes that are transcribed into viral mRNAs and ment may be administered to a Subject having a medical transmitted to daughter cells when hepatocytes divide. Cur disorder or who ultimately may acquire the disorder, in order rent direct antivirals have no or very little effect on the HBV to prevent, cure, delay the onset of, reduce the severity of, or cccDNA reservoir and the expression of the viral genes. Thus, ameliorate one or more symptoms of a disorder or recurring the currently available treatments are suboptimal and may be disorder, or in order to prolong the survival of a subject associated with severe side effects. Accordingly there is a beyond that expected in the absence of such treatment. By US 2016/0220586 A1 Aug. 4, 2016

“therapeutic regimen’ is meant the pattern of treatment of an target genes fall into two main groups (Edwards PA. et al. illness, e.g., the pattern of dosing used during HBV therapy. A 2002, Kapadia S.B. Et al. 2005). The first group functions to therapeutic regimen may include an induction regimen and a decrease hepatic bile acids concentrations by increasing maintenance regimen. The phrase “induction regimen” or export and decreasing their synthesis. The second group of “induction period’ refers to a therapeutic regimen (or the FXR target genes Such as the phospholipid transport protein portion of a therapeutic regimen) that is used for the initial PLTP and apolipoproteins modulates lipoprotein levels in the treatment of a disease. The general goal of an induction regi serum and decreases plasma triglyceride concentration. For a men is to provide a high level of drug to a patient during the more detailed list of FXR-regulated genes, see, e.g., WO initial period of a treatment regimen. An induction regimen 03/016288, pages 22-23. U.S. Pat. No. 6,005,086 discloses may employ (in part or in whole) a "loading regimen’, which the nucleic acid sequence coding for a mammalian FXR may include administering a greater dose of the drug than a protein. The human polypeptide sequences for FXR are physician would employ during a maintenance regimen, deposited in nucleotide and protein databases under acces administering a drug more frequently than a physician would sion numbers NM_005123, Q96RI1, NP_005114 administer the drug during a maintenance regimen, or both. AAM53551, AAM53550, AAK60271. The phrase “maintenance regimen” or “maintenance period” 0012. In this specification, the term “FXRagonist' has its refers to a therapeutic regimen (or the portion of a therapeutic general meaning in the art and refers in particular to com regimen) that is used for the maintenance of a patient during pounds that function by targeting and selectively binding the treatment of an illness, e.g., to keep the patient in remission farnesoid X receptor (FXR) and which activate FXR by at for long periods of time (months or years). A maintenance least 40% above background in the assay described in Mal regimen may employ continuous therapy (e.g., administering oney et al. (J. Med. Chem. 2000, 43:2971-2974). a drug at a regular intervals, e.g., weekly, monthly, yearly, 0013. In some embodiments, the FXR agonist of the etc.) or intermittent therapy (e.g., interrupted treatment, inter invention is a selective FXRagonist. As used herein, the term mittent treatment, treatment at relapse, or treatment upon “selective FXRagonist” refers to an FXRagonist that exhib achievement of a particular predetermined criteria e.g., pain, its no significant cross-reactivity to one or more, ideally Sub disease manifestation, etc.). stantially all, of a panel of nuclear receptors consisting of 0010. The efficacy of the therapy regimen may be moni LXRC, LXRB, PPARC, PPARy, PPAR8, RXRC, RARY, tored using standard protocols. Treatment may be followed VDR, SXR, ERO, ERB, GR, AR, MR and PR. Methods of by determinations of HBV levels in serum and measurement determining significant cross-reactivity are described in J. of serum ALT levels. For example, the patients may be Med. Chem. 2009, 52,904-907. assessed for the presence of HBV DNA in their serum. HBV 0014 FXRagonists are well known to the skilled person. DNA (IU/mL) can be measured at regular intervals during the For example the skilled person may easily identified FXR treatment, e.g., at Day 1 (pre-dose and 4, 8, and 12 hours agonist from the following publications: post-dose) and pre-dose at Day 2, Day 3, Day 8, Day 15, Day (0.015 Adorini L, Pruzanski M, Shapiro D. Farnesoid X 29, and at Week 12, Week 24, Week 36, Week 48, Week 72 receptor targeting to treat nonalcoholic Steatohepatitis. (when applicable), and at follow up. Accordingly, the efficacy Drug Discov Today. 2012 September; 17(17-18):988 of therapy will be monitored using internationally accepted 97. doi:10.1016/j.drudis.2012.05.012. Epub 2012 May parameters: a) Serum HBV DNA levels are monitored using 29. Review. sensitive quantitative PCR-based assays to assess the effect 0016 Akwabi-Ameyaw A, Bass J Y. Caldwell R D, on viral replication, b) In HBeAg-positive patients—HBeAg Caravella JA, Chen L, Creech K L., Deaton D N, is monitored along with the corresponding anti-HBe to deter Madauss K. P. Marr H B. McFadyen R B, Miller A B, mine whether HBe-seroconversion has occurred, c) Serum Navas F3rd, Parks DJ, Spearing PK, Todd D, Williams levels of ALT and/or AST are monitored to assess impact on SP, Bruce Wisely G. FXRagonist activity of conforma liver inflammation and liver cell death and d) Serum HBs.Ag tionally constrained analogs of GW 4064. Bioorg Med is monitored—qualitatively and quantitatively along with the Chem Lett. 2009 Aug. 15; 19(16):4733-9. doi:10.1016/ corresponding anti-HBs to determine whether HBs-serocon j. bmcl.2009.06.062. Epub 2009 Jun. 21. version has occurred as HBS Ag clearance and seroconversion 0017 Akwabi-Ameyaw A, Bass J Y. Caldwell R D, would indicate optimal treatment outcome. Ultimately, even Caravella JA, Chen L. Creech KL, Deaton DN, Jones if not of actual clinical routine practice, cccDNA persistence SA, Kaldor I, Liu Y. Madauss K P. Marr HB, McFadyen might be assessed by specific PCR to quantify the level of RB, Miller AB, Iii FN, Parks DJ, Spearing P K, Todd viral minichromosome in liver biopsies. D, Williams S P Wisely G. B. Conformationally con 0011. The term “FXR” refers to the farnesoid X receptor, strained farnesoid X receptor (FXR) agonists: Naph which is a nuclear receptor that is activated by Supraphysi thoic acid-based analogs of GW 4064. Bioorg Med ological levels of farnesol (Forman et al., Cell, 1995, 81,687 Chem Lett. 2008 Aug. 1; 18(15):4339-43. doi:10.1016/ 693). FXR, is also known as NR1H4, retinoid X receptor j. bmcl.2008.06.073. Epub 2008 Jun. 28. interacting protein 14 (RIP 14) and bile acid receptor (BAR). 0.018 Akwabi-Ameyaw A, Caravella JA, Chen L, Containing a conserved DNA-binding domain (DBD) and a Creech KL, Deaton DN, Madauss KP, Marr HB, Miller C-terminal ligand-binding domain (LBD), FXR binds to and A B, Navas F 3rd, Parks DJ, Spearing P K, Todd D, becomes activated by a variety of naturally occurring bile Williams S P Wisely G. B. Conformationally con acids (BAS), including the primary bile acid chenodeoxy strained farnesoid X receptor (FXR) agonists: alterna cholic acid (CDCA) and its taurine and glycine conjugates tive replacements of the stilbene. Bioorg Med Chem (Makishima et al., 1999; Parks et al., 1999: Wanget al., 1999). Lett. 2011 Oct. 15: 21 (20):6154-60. doi: 10.1016/j. Upon activation, the FXR-RXR heterodimer binds the pro bmcl.2011.08.034. Epub 2011 Aug. 11. moter region of target genes and regulates the expression of 0.019 Baghdasaryan A. Claudel T. Gumhold J. Silbert several genes involved in bile acid homeostasis. Hepatic FXR D, Adorini L, Roda A, Vecchiotti S. Gonzalez, F J, US 2016/0220586 A1 Aug. 4, 2016

Schoonjans K. StraZZabosco M. Fickert P. Trauner M. 0030 Iguchi Y. Kihira K. Nishimaki-Mogami T. Une Dual farnesoid X receptor/TGR5 agonist INT-767 M. Structure-activity relationship of bile alcohols as reduces liver injury in the Mdr2-/- (Abcb4-/-) mouse human farnesoid X receptor agonist. Steroids. 2010 cholangiopathy model by promoting biliary HCO out January: 75(1): 95-100. doi:10.1016/j.steroids.2009.11. put. Hepatology. 2011 October; 54(4): 1303-12. doi: 002. Epub 2009 Nov. 12. 10.1002/hep.24537. 0.031 Lin HR. Triterpenes from Alisma Orientalis act as 0020 Bass J Y. Caldwell RD, Caravella JA, Chen L, farnesoid X receptor agonists. Bioorg Med Chem Lett. Creech K L., Deaton D N, Madauss K P. Marr H B, 2012 Jul. 15; 22(14):4787-92. doi: 10.1016/j.bmcl. McFadyen RB, Miller A B, Parks DJ, Todd D, Williams 2012.05.057. Epub 2012 May 23. SP, Wisely GB. Substituted isoxazole analogs of farne 0032) Lefebvre P. Cariou B, Lien F. Kuipers F. Staels B. soid X receptor (FXR) agonist GW4064. Bioorg Med Role of bile acids and bile acid receptors in metabolic Chem Lett. 2009 Jun. 1; 19(11):2969-73. doi:10.1016/ regulation. Physiol Rev. 2009 January: 89(1): 147-91. j. bmcl.2009.04.047. Epub 2009 Apr. 18. doi:10.1152/physrev.00010.2008. 0021 Bass J Y. Caravella JA, Chen L. Creech K L, 0033 Lundquist JT, Harnish DC, Kim CY, Mehlmann Deaton DN, Madauss K P. Marr HB, McFadyen R B, JF, Unwalla R.J. Phipps KM, Crawley ML, Commons Miller AB, Mills WY. Navas F 3rd, Parks DJ, Smalley T. Green DM, Xu W. HumWT, Eta JE, Feingold I, Patel TLJr, Spearing PK, Todd D, Williams SP, Wisely GB. V. Evans MJ, Lai K. Borges-Marcucci L, Mahaney PE, Conformationally constrained farnesoid X receptor Wrobel J.E. Improvement of physio chemical properties (FXR) agonists: heteroaryl replacements of the naphtha of the tetrahydroazepinoindole series of farnesoid X lene. Bioorg MedChem Lett. 2011 Feb. 15:21(4): 1206 receptor (FXR) agonists: beneficial modulation of lipids 13. doi:10.1016/j. bmcl.2010.12.089. Epub 2010 Dec. in primates. JMed Chem. 2010 Feb. 25:53(4): 1774-87. 23. doi:10.1021/im901650u. (0022. Buijsman et al., Curr. Med. Chem. 2005, 12, 1017 0034. MaY. Huang Y.Yan L, Gao M, Liu D. Synthetic (0023 Chiang PC, Thompson DC, Ghosh S, Heitmeier FXR agonist GW4064 prevents diet-induced hepatic MR. A formulation-enabled preclinical efficacy assess ment of a farnesoid X receptor agonist, GW4064, in steatosis and insulin resistance. Pharm Res. 2013 May; hamsters and cynomolgus monkeys. J Pharm Sci. 2011 30(5): 1447-57. doi:10.1007/s11095-013-0986-7. Epub November; 100(11):4722-33. doi:10.1002/jps.22664. 2013 Feb. 1. Epub 2011 Jun. 9. 0035 Marinozzi M, Carotti A, Sardella R. Buonerba F. (0024 Crawley, Expert Opin. Ther. Pat. 2010, 20, 1047 Ianni F, Natalini B, Passeri D, Rizzo G., Pellicciari R. 0025 Feng S. Yang M. Zhang Z. Wang Z. Hong D, Asymmetric synthesis of the four diastereoisomers of a Richter H, Benson GM, Bleicher K, Grether U, Martin novel non-steroidal farnesoid X receptor (FXR) agonist: RE, Plancher J. M. Kuhn B. Rudolph M. G., Chen L. Role of the chirality on the biological activity. Bioorg Identification of an N-oxide pyridine GW4064 analog as Med Chem. 2013 Jul. 1; 21 (13):3780-9. doi:10.1016/j. a potent FXR agonist. Bioorg Med Chem Lett. 2009 bmc.2013.04.038. Epub 2013 Apr. 23. May 1, 19(9):2595-8. doi:10.1016/j. bmcl.2009.03.008. 0.036 Misawa T. Hayashi H. Makishima M. Sugiyama Epub 2009 Mar. 9. Y. Hashimoto Y. E297G mutated bile salt export pump (0026. Flatt B, Martin R, Wang TL, Mahaney P. Murphy (BSEP) function enhancers derived from GW4064: B, Gu X H, Foster P. Li J, Pircher P, Petrowski M, structural development study and separation from farne Schulman I, Westin S, Wrobel J. Yan G, Bischoff E, soid X receptor-agonistic activity. Bioorg Med Chem Daige C. Mohan R. Discovery of XL335 (WAY Lett. 2012 Jun. 15:22(12):3962-6. doi:10.1016/j.bmcl. 362450), a highly potent, selective, and orally active 2012.04.099. Epub 2012 Apr. 30. agonist of the farnesoid X receptor (FXR). JMed Chem. 0037 Mudaliar S, Henry R. R. Sanyal AJ, Morrow L, 2009 Feb. 26; 52(4):904-7. doi:10.1021/im8014 124. Marschall HU, Kipnes M. Adorini L. Sciacca CI, Clop 0027 Ghebremariam YT, Yamada K, Lee J C, Johnson ton P. Castelloe E. Dillon P. Pruzanski M., Shapiro D. CL, Atzler D, Anderssohn M. Agrawal R. Higgins JP, Efficacy and safety of the farnesoid X receptor agonist Patterson A. J. Boger R H. Cooke J P FXR agonist obeticholic acid in patients with type 2 diabetes and INT-747 upregulates DDAH expression and enhances nonalcoholic fatty liver disease. Gastroenterology. 2013 insulin sensitivity in high-salt fed Dahl rats. PLoS One. September; 145(3):574-82. el. doi: 10.1053/igastro. 2013 Apr. 4; 8(4):e60653. doi:10.1371/journal.pone. 2013.05.042. Epub 2013 May 30. OO60653. Print 2013. 0038 Richter H.G. Benson GM, Bleicher KH, Blum D, 0028 Gioiello A. Macchiarulo A, Carotti A, Filipponi P. Chaput E. Clemann N, Feng S. Gardes C. Grether U. Costantino G, Rizzo G, Adorini L, Pellicciari R. Extend Hartman P. Kuhn B. Martin RE, Plancher J. M. Rudolph ing SAR of bile acids as FXR ligands: discovery of MG, Schuler F. Taylor S. Optimization of a novel class 23-N-(carbocinnamyloxy)-3C,7C.-dihydroxy-6C.-ethyl of benzimidazole-based farnesoid X receptor (FXR) 24-nor-5 B-cholan-23-amine. Bioorg Med Chem. 2011 agonists to improve physicochemical and ADME prop Apr. 15; 19(8):2650-8. doi:10.1016/j.bmc.2011.03.004. erties. Bioorg Med Chem Lett. 2011 Feb. 15: 21 (4): Epub 2011 Mar. 10. 1134-40. doi:10.1016/j.bmcl.2010.12.123. Epub 2010 0029. Hoekstra M. vander Sluis RJ, Li Z, Oosterveer M Dec. 31. H, Groen A K. Van Berkel T.J. FXR agonist GW4064 0039. Rizzo G. Passeri D, De Franco F, Ciaccioli G, increases plasma glucocorticoid levels in C57BL/6 Donadio L. Rizzo G., Orlandi S, Sadeghpour B. Wang X mice. Mol Cell Endocrinol. 2012 Oct. 15:362(1-2):69 X, Jiang T. Levi M. Pruzanski M., Adorini L. Functional 75. doi:10.1016/j.mce.2012.05.010. Epub 2012 May characterization of the semisynthetic bile acid derivative 27. INT-767, a dual farnesoid X receptor and TGR5 agonist. US 2016/0220586 A1 Aug. 4, 2016

Mol Pharmacol. 2010 October; 78(4):617-30. doi: 0071 WO2003015771; 10.1124/mol. 110.064501. Epub 2010 Jul. 14. 0072 WO2003016280; 0040 Schuster D, Markt P. Grienke U, Mihaly-Bison J, 0073 WO2003016288; Binder M, Noha SM, Rollinger J M. Stuppner H, Boch 0.074 WO2003030612: kov V. N. Wolber G. Pharmacophore-based discovery of 0075) WO2OO3O60.078 FXR agonists. Part I: Model development and experi 0076 WO2003080803; mental validation. Bioorg Med Chem. 2011 Dec. 1; 0.077 WO2003090745: 19(23):7168-80. doi:10.1016/j.bmc.2011.09.056. Epub 0078 WO2004.007521 2011 Oct. 4. 0079 WO2004O46.162 0080 WO2004.048349; 0041) Soisson SM, Parthasarathy G, Adams AD. Sahoo 0081 WO2005082925; S, Sitlani A, Sparrow C. Cui J. Becker J.W. Identification 0082 WO2005092328; of a potent synthetic FXR agonist with an unexpected 0083 WO2005097097; mode of binding and activation. Proc Natl Acad Sci 0084 WO2OO6O2O68O USA. 2008 Apr. 8: 105(14):5337-42. doi:10.1073/pnas. 0085 WO2007076260 07.10981105. Epub 2008 Apr. 7. 0086 WO2007076260: 0042 Watanabe M, HoraiY. Houten S M, Morimoto K, 0087 WO2007092751: Sugizaki T, Arita E, Mataki C, Sato H. Tanigawara Y. 0088 WO2OO714O174 Schoonjans K, Itoh H. Auwerx J. Lowering bile acid 0089 WO2OO714O183 pool size with a synthetic farnesoid X receptor (FXR) 0090 WO2O08OOO643 agonist induces obesity and diabetes through reduced 0091 WO2008002573; energy expenditure. J Biol Chem. 2011 Jul. 29:286(30): 0092 WO2O08025539 26913-20. doi:10.1074/bc.M111.248203. Epub 2011 0093 WO2O08025540 Jun. 1. 0094) WO2008051942: 0043 Yu D, Mattern D L. Forman B M. An improved 0.095 WO2008073825; synthesis of 6C.-ethylchenodeoxycholic acid 0.096 WO2008 157270 (6ECDCA), a potent and selective agonist for the Far O097 WO2009005998 nesoid X Receptor (FXR). Steroids. 2012 November; 0098 WO2009012125, 77(13): 1335-8. doi: 10.1016/j.steroids.2012.09.002. 0099 WO2009027264; Epub 2012 Sep. 21. 01.00 WO2009062874, 0044 Zhang S. Wang J, Liu Q. Harnish D C. Farnesoid 0101 WO2009080555; X receptoragonist WAY-362450 attenuates liver inflam 01.02 WO2009 127321; mation and fibrosis in murine model of non-alcoholic (0103) WO2009149795, steatohepatitis. J Hepatol. 2009 August; 51(2):380-8. 0104 WO2010028981; doi:10.1016/j.ijhep.2009.03.025. Epub 2009 May 18. 01.05 WO2010034649, 0045 Typically FXR agonists include the class of steroid 01.06 FXR agonists and non steroid FXR agonists. 01.07 WO2010069604, 0046. In certain embodiments of the invention the FXR 0108 WO2011020615, agonist is selected from Small molecule compounds which act 0109 WO20130.07387, as FXR modulators that have been disclosed in the following 0110 and WO2013037482. publications: 0111 Specific examples of FXR agonists include but are not limited to GW4064 (as disclosed in PCT Publication No. 0047 EP1392714; WO 00/37077 or in US2007/0015796), 6-ethylchenodeoxy 0048 EP1568706; cholic acids (6ECDCA), especially 3C, 7C.-dihydroxy 7o-di 0049 EP2128158, hydroxy-6.O-ethyl-5f-cholan-24-oic acid, also referred to as 0050 EP2289883, INT-747; 6-ethyl-ursodeoxycholic acids, INT-1103, UPF 0051 JP2005281155; 987, WAY-362450, MFA-1, GW9662, T0901317, fex 0052 US20030203939; aramine, a cholic acid, a deoxycholic acid, a glycocholic acid, 0053 US2005080064; a glycodeoxycholic acid, a taurocholic acid, a taurodihydro 0054 US2006128764; fusidate, a taurodeoxycholic acid, a cholate, a glycocholate, a 0055 US20070010562 deoxycholate, a taurocholate, a taurodeoxycholate, a cheno 0056 US20070015796; deoxycholic acid, a 7-B-methyl cholic acid, a methyl litho 0057 US20080038435: cholic acid. 0058 US20080300235 0112. In some embodiments, the FXR agonist is not 0059 US20090062526, selected from natural bile acids, preferably chenodeoxy 0060 US20090163552, cholic acid CDCA or taurine- or glycine-conjugated CDCA 0061 US20100093818, tauro-CDCA or glyco-CDCA and synthetic derivatives of 0062 US20100184809; natural bile acids, preferably 6-Ethyl-CDCA or taurine- or 0063 US20110077273, glycine-conjugated 6-Ethyl-CDCA, natural non-steroidal 0064 US20110105475; agonists, preferably Diterpenoids such as Cafestol and Kah 0065 .S. Pat. No. 6,984,560; weol, or synthetic non-steroidal FXRagonists. 0066 .S. Pat. No. 7,671,085, 0113. In some embodiments, the FXR agonist is selected 0067 WO2000037077; from the group consisting of GW4064, 6ECDCA and the 0068 WO200040965; compound identified by the CAS REGISTRY NUMBER 0069. WO200076523; 1192171-69-9 (described in WO 2009127321 also named 0070 WO2001017994 PXL007): US 2016/0220586 A1 Aug. 4, 2016 5

0117. In some embodiments, the FXR agonist is selected C from the group consisting of

O 18 \ O / So F N

C N -

0114. In some embodiments, the FXRagonist is the com pound having the formula of

CH3

HO "wo NCH,

0115. In some embodiments, the FXRagonist is the com- F pound having the formula of CH3 HC O 0118. In some embodiments, the FXR agonist is selected W \ from the group consisting of the compounds disclosed in O 2N WO2013007387, namely: HOC O C 2 O N C C O N A 0116. In some embodiments, the FXRagonist is the com- Cl, pound having the formula of

HNN/ OY. N F A O N Cl,

O O F US 2016/0220586 A1 Aug. 4, 2016

-continued -continued

Cl,

Cl, US 2016/0220586 A1 Aug. 4, 2016 7

-continued -continued

O O

O MN Z HO Cl, Cl, O W C Os? / C

O O AN O Cl, 7 C Cl,

O AN HO Cl, O s C Y. O A s C C M s HO ry

O MN Cl, O C M N O M

Cl, W C

HO Cl, s C Cl, C C

HO \ |

US 2016/0220586 A1 Aug. 4, 2016 9

-continued N N O N) C N 2N C HO

N 2 \, NH N O S. C 2N C HO

0148. In some embodiments, the FXR agonist is selected from the group consisting of the compound disclosed in HO O O WO2008025539, namely: N N 7. Cl

N O 2

O CF C -N. O S. O N1 n C O 21 C 0149. In some embodiments, the FXR agonist is selected from the group consisting of the compounds described in WO2008025540, namely:

US 2016/0220586 A1 Aug. 4, 2016 22 medical judgment. The specific therapeutically effective dose intravenous, transdermal, local or rectal administration, the level for any particular patient will depend upon a variety of active principle, alone or in combination with another active factors including the disorder being treated and the severity of principle, can be administered in a unit administration form, the disorder; activity of the specific compound employed; the as a mixture with conventional pharmaceutical Supports, to specific composition employed, the age, body weight, gen animals and human beings. Suitable unit administration eral health, sex and diet of the patient; the time of adminis forms comprise oral-route forms such as tablets, gel capsules, tration, route of administration, and rate of excretion of the powders, granules and oral Suspensions or solutions, Sublin specific compound employed; the duration of the treatment; gual and buccal administration forms, aerosols, implants, drugs used in combination with the specific agonist Subcutaneous, transdermal, topical, intraperitoneal, intra employed; and like factors well known in the medical arts. muscular, intravenous, Subdermal, transdermal, intrathecal For example, it is well within the skill of the art to start doses and intranasal administration forms and rectal administration of the compound at levels lower than those required to achieve forms. the desired therapeutic effect and to gradually increase the 0796. In particular, the pharmaceutical compositions con dosage until the desired effect is achieved. However, the daily tain vehicles which are pharmaceutically acceptable for a dosage of the products may be varied over a wide range from formulation capable of being injected. These may be in par 0.01 to 1,000 mg per adult per day. Preferably, the composi ticular isotonic, sterile, saline solutions (monosodium or tions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, disodium phosphate, Sodium, potassium, calcium or magne 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for sium chloride and the like or mixtures of Such salts), or dry, the symptomatic adjustment of the dosage to the patient to be especially freeze-dried compositions which upon addition, treated. A medicament typically contains from about 0.01 mg depending on the case, of sterilized water or physiological to about 500 mg of the active ingredient, preferably from 1 mg saline, permit the constitution of injectable solutions. to about 100 mg of the active ingredient. An effective amount 0797 The pharmaceutical forms suitable for injectable of the drug is ordinarily Supplied at a dosage level from use include Sterile aqueous solutions or dispersions; formu 0.0002 mg/kg to about 20 mg/kg of body weight per day, lations including sesame oil, peanut oil or aqueous propylene especially from about 0.001 mg/kg to 7 mg/kg of body weight glycol; and sterile powders for the extemporaneous prepara per day. tion of sterile injectable solutions or dispersions. In all cases, 0791) Any of the above treatment regimens can be admin the form must be sterile and must be fluid to the extent that istered to individuals who have been diagnosed with an HBV easy Syringability exists. It must be stable under the condi infection. Any of the above treatment regimens can be admin tions of manufacture and storage and must be preserved istered to individuals who have failed previous treatment for against the contaminating action of microorganisms. Such as HBV infection (treatment failure patients). “Treatment fail bacteria and fungi. ure patients' as used herein generally refers to HBV-infected 0798 Solutions comprising compounds of the invention patients who failed to respond to previous therapy for HBV as free base or pharmacologically acceptable salts can be (referred to as “non-responders’) or who initially responded prepared in water Suitably mixed with a Surfactant, Such as to previous therapy, but in whom the therapeutic response was hydroxypropylcellulose. Dispersions can also be prepared in not maintained (referred to as “relapsers'). The previous and glycerol, liquid polyethylene glycols, and mixtures thereof currently available therapy generally can include treatment and in oils. Under ordinary conditions of storage and use, with antiviral drugs such as lamivudine (Epivir), adefovir these preparations contain a preservative to prevent the (Hepsera), tenofovir (Viread), telbivudine (Tyzeka) and ente growth of microorganisms. cavir (Baraclude), and the two immune system modulators 0799. The FXRagonist of the invention can beformulated interferon alpha-2a, PEGylated interferon alpha-2a (Pegasys) into a composition in a neutral or salt form. Pharmaceutically and interferon alpha-2b (ViraferonPegou Introna). acceptable salts include the acid addition salts (formed with 0792. In particular the FXR agonist according to the the free amino groups of the protein) and which are formed invention may be administered to the Subject in combination with inorganic acids such as, for example, hydrochloric or with currently available therapy, including treatment with phosphoric acids, or such organic acids as acetic, oxalic, antiviral drugs such as the reverse transcriptase inhibitors, tartaric, mandelic, and the like. Salts formed with the free lamivudine (Epivir), adefovir (Hepsera), tenofovir (Viread), carboxyl groups can also be derived from inorganic bases telbivudine (Tyzeka) and entecavir (Baraclude), and such as Such as, for example, Sodium, potassium, ammonium, cal immune system modulators, interferon alpha-2a, PEGylated cium, or ferric hydroxides, and Such organic bases as isopro interferon alpha-2a (Pegasys) or interferon alpha-2b (Viraf pylamine, trimethylamine, histidine, procaine and the like. eronPegou Introna). 0800 The carrier can also be a solvent or dispersion 0793. The FXRagonist of the invention may be combined medium containing, for example, water, ethanol, polyol (for with pharmaceutically acceptable excipients, and optionally example, glycerol, propylene glycol, and liquid polyethylene Sustained-release matrices. Such as biodegradable polymers, glycol, and the like), Suitable mixtures thereof, and Veg to form pharmaceutical compositions. etables oils. The proper fluidity can be maintained, for 0794 "Pharmaceutically” or “pharmaceutically accept example, by the use of a coating, such as lecithin, by the able” refers to molecularentities and compositions that do not maintenance of the required particle size in the case of dis produce an adverse, allergic or other untoward reaction when persion and by the use of surfactants. The prevention of the administered to a mammal, especially a human, as appropri action of microorganisms can be brought about by various ate. A pharmaceutically acceptable carrier or excipient refers antibacterial and antifungal agents, for example, parabens, to a non-toxic Solid, semi-solid or liquid filler, diluent, encap chlorobutanol, phenol, Sorbic acid, thimerosal, and the like. Sulating material or formulation auxiliary of any type. In many cases, it will be preferable to include isotonic agents, 0795. In the pharmaceutical compositions of the present for example, Sugars or Sodium chloride. Prolonged absorp invention for oral, Sublingual, Subcutaneous, intramuscular, tion of the injectable compositions can be brought about by US 2016/0220586 A1 Aug. 4, 2016

the use in the compositions of agents delaying absorption, for (0809 FIG. 2 Secretion of HBV surface (HBSAg), core example, aluminium monostearate and gelatin. (HBeAg) antigens and HBV DNA in supernatant of HBV 0801 Sterile injectable solutions are prepared by incorpo infected HepaRG. rating the active polypeptides in the required amount in the 0810. Differentiated HepaRG cells were infected with appropriate solvent with various proportions of the other HBV (100 geq/cell for 24hr), then treated 3 successive times ingredients enumerated above, as required, followed by fil (days 4, 7 and 11 post infection) with FXR agonists and tered sterilization. Generally, dispersions are prepared by antagonists or FXR inactive bile acid UDCA at indicated incorporating the various sterilized active ingredients into a concentrations (LM). Cell supernatants were collected 14 sterile vehicle which contains the basic dispersion medium days post infection for quantification of HBs.Ag, HBeAg and the required other ingredients from those enumerated (Architec Abbott) or HBV DNA by quantitative PCR using above. In the case of sterile powders for the preparation of rcDNA primers (n=3+SEM). A Both FXR agonists, sterile injectable solutions, the preferred methods of prepa GW4064 and 6ECDCA, inhibit the secretion of HBs.Ag and ration are vacuum-drying and freeze-drying techniques HBeAg in the Supernatant in a dose-dependent manner which yield a powder of the active ingredient plus any addi whereas UDCA and 052EDL133 have no effect on the anti tional desired ingredient from a previously sterile-filtered gen secretion. B. Both FXR agonists, GW4064 and solution thereof. 6ECDCA, inhibit the secretion of infectious HBV DNA posi 0802. Upon formulation, solutions will be administered in tive viral particles in the Supernatant in a dose-dependent a manner compatible with the dosage formulation and in Such manner. UDCA and 052EDL133 have no or limited effect on amount as is therapeutically effective. The formulations are the virion secretion. C. The FXRagonist PXL007 represses easily administered in a variety of dosage forms, such as the HBSA.g., HBeAg and HBV DNA in a dose-dependent manner. type of injectable solutions described above, but drug release 0811 FIG. 3 Expression of HBV core protein (HBc) capsules and the like can also be employed. within HepaRG cells in presence or not of FXRagonists. 0803 For parenteral administration in an aqueous solu 0812. Differentiated HepaRG cells grown on coverslips tion, for example, the solution should be suitably buffered if were infected and treated as described in FIG. 1 legend necessary and the liquid diluent first rendered isotonic with (n=3+SEM). Cells were fixed on day 14 post infection and Sufficient Saline or glucose. These particular aqueous solu immunocytochemistry using anti-HBc antibody was carried tions are especially suitable for intravenous, intramuscular, out. Fluorescent microscopy reveals that FXR agonists, Subcutaneous and intraperitoneal administration. In this con GW4064 and 6ECDCA, drastically reduce the expression of nection, sterile aqueous media which can be employed will be HBc in the infected cells. UDCA and 052EDL133 do not known to those of skill in the art in light of the present appear to modify the expression of HBc. disclosure. For example, one dosage could be dissolved in 1 0813 FIG. 4 HBV Pregenomic/precore mRNA and ml of isotonic NaCl solution and either added to 1000 ml of cccDNA expression in HBV infected HepaRG cell line in hypodermoclysis fluid or injected at the proposed site of presence or not of FXR agonists. infusion. Some variation in dosage will necessarily occur 0814) Differentiated HepaRG cells were infected and depending on the condition of the Subject being treated. The treated as described in FIG. 1 legend. Cells were lysed and person responsible for administration will, in any event, RNA was extracted, then either reverse transcribed into determine the appropriate dose for the individual subject. cDNA for quantitative PCR (qRT-PCR) (A) or used in North 0804. The FXR agonist of the invention may be formu ern Blot experiment (B). The same experiment was repeated lated within a therapeutic mixture to comprise about 0.0001 and DNA was extracted. Following plasmid-safe DNase to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about treatment, cccDNA expression was quantified by qPCR 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple experiment using specific HBV cccDNA primers and Taq doses can also be administered. Man probe (n=3+SEM) (C).cccDNA quantification was nor 0805. In addition to the compounds of the invention for malized to the number of Bglobin gene. The expression levels mulated for parenteral administration, Such as intravenous or of HBV pregenomic gene were quantified, as well as 3 house intramuscular injection, other pharmaceutically acceptable keeping genes for normalization (n=3+SEM). Both FXRago forms include, e.g. tablets or other Solids for oral administra nists, GW4064 and 6ECDCA, inhibit the expression of HBV tion; liposomal formulations; time release capsules; and any pregenomic/precore mRNA in a dose-dependent manner. The other form currently used. reduction is confirmed in the northern blot (3.4-3.5 Kb band). (0806. The invention will be further illustrated by the fol The expression of the other HBV mRNAs (S: 2.1-2.4 Kb: X: lowing FIGS. and examples. However, these examples and 0.7 Kb) is also reduced, as seen on the densitometry graph FIGS. should not be interpreted in any way as limiting the (n=3+SEM). cccDNA levels were also reduced by more than Scope of the present invention. 50% following treatment with FXR agonists. 0815 FIG. 5 Effect of the HBV entry inhibitor FIGURES Cyclosporin A on FXRagonists modulation of HBSAg and HBeAg secretion in the supernatant of HBV infected Hep (0807 FIG. 1-Secretion of surface HBs antigen (HBSAg) aRG cells. in the supernatant of HBV infected HepaRG cell line in 0816. Differentiated HepaRG cells were infected and presence of FXR modulators. treated as described in FIG. 1 legend. In addition to the usual (0808 Differentiated HepaRG cells were infected with protocol previously described, cells were treated with HBV (100 geq/cell for 24hr), then treated 3 successive times cyclosporin A (CyA) either during HBV infection (i.e. for 24 (days 4, 7 and 11 post infection) with FXR modulators at hr) or during the 1st treatment with FXRC. agonists (i.e. for 72 indicated concentrations in LM. Cell Supernatants were col hr; from day 4 to 7 post infection). Cell supernatants were lected 14 days post infection for quantification of HBs.Ag collected 14 days post infection for quantification of HBSAg (Architec Abbott). and HBeAg (n=3+SEM). A) CyA treatment during HBV US 2016/0220586 A1 Aug. 4, 2016 24 infection inhibits viral entry in a dose-dependent manner and cccDNA, transcription of pregenomic and viral mRNAs as does not impair the decrease in HBS Ag and HBeAg secre well as later stages of the replication cycle with the synthesis tions following treatment with FXR agonists. B) Cy A treat of viral proteins, the assembly and secretion of infectious ment post infection has no effect on HBV antigen secretion virions and the secretion of the viral proteins HBs and HBe. whatever the presence or not of FXR agonists. 0824. The HepaRG system thus allows the monitoring of 0817 FIG. 6—Co-Immunoprecipitation assay of FXR the secretion of HBS Ag and HBeAg and virion incorporated and HBX proteins DNA into the cell culture supernatant after infection with 0818 HEK293T cells were co-transfected with fusion HBV infectious virion stocks prepared from the HepG2.2.15 proteins 3XF-HBX and Gluc-FXR encoding plasmids. 48 h. cell line. This system also allows the monitoring of the Syn post-transfection, cells were lysed and co-immunoprecipita thesis of pregenomic and viral mRNAS as well as cytoplasmic tion was performed with Dynabeads(RProtein G beforehand replication intermediates and cccDNA. The effects of mol coupled with anti-3XF antibody. Cells lysates and co-immu ecules on cell physiology and cell differentiated functions noprecipitation products were analyzed by western blot; FXR were explored including the quantification of hepatic markers expression was detected with anti-Glue antibody. FXR as albumin and apo lipoprotein B. The effects of the com expression in control, Gluc-FXR alone, or in the test cells, pounds on the cellular bile acid pathway were monitored by co-expression of Gluc-FXR and 3XF-HBX, was similar as analysis and quantification of the FXR mRNA as well as the shown on the left western blot. After immunoprecipitation SHP and apoA1 encoding mRNAs the expression of which is with anti-3XF antibody, FXR fusion protein was evidently under the control of FXR detected in the test condition and not in the control (right western blot). These observations strongly suggest an inter Results action between the viral HBX protein and the nuclear receptor FXR. 0825 FXRAgonists are Potent HBV Replication Inhibi 0819 FIG.7 mRNA expression of FXR and two of its tOrs regulated genes 0826. A panel of molecules, not previously described and original or reference modulators of FXR activity were first 0820) Differentiated HepaRG cells were infected and tested on the expression of a reporter gene under the control of treated as described in FIG. 1 legend. Cells were lysed and the HBV Enh2/core promoter region that contains two FXR RNA was extracted, then reverse transcribed into cDNA for response elements in the Huh-7 cell line as described in qPCR. The expression levels of 3 genes of interest were Ramiére C, et al., 2008, JVirol; 82: 10832-10840. Molecules quantified: FXRC, SHP and APOA1, as well as 3 housekeep were then classified as FXR agonists or antagonists on the ing genes for normalization (n=3+SEM). FXR agonists, basis respectively of the expression increase or decrease of GW4064 and 6ECDCA, inhibit the expression of FXR the reporter gene under the transcription control of FXR. mRNA in a dose-dependent manner. SHP and APOA1 are Some molecules had an intermediate profile, being moder two genes under the regulation of FXR: SHP is induced by ately agonist when tested separately and weak antagonist FXR while APOA1 is repressed. Here, SHP mRNA expres when tested in competition against a reference agonist (data sion increases with GW4064 and 6ECDCA treatments, while not shown). The most potent and representative compounds APOA1 mRNA expression decreases. This suggests an acti were first evaluated for their effect on the synthesis and secre vation of FXR despite its reduced expression. tion of HBs.Ag in the culture supernatant of HepaRG cell 0821 FIG. 8 Co-treatment of FXRagonist with lamivu culture system naturally infected with HBV produced in dine, a potent nucleoside analog reverse transcriptase inhibi HepG2.2.15 (FIG. 1). Unexpectedly, the most potent antago tOr nists (i.e. 100ED0038, 100ED0136, 100ED0137, and 0822. Differentiated HepaRG cells were infected and 100ED0166) as well as the reference antagonist 052EDL133 treated as described in FIG. 1 legend. Cell supernatants were described in patent WO 2007052843: Takeda Pharmaceutical collected 14 days post infection. (A) Quantification of Co. Ltd., Osaka, Japan, had little or no effect on HBSAg HBS Ag and HBeAg secretion. (B) Quantification of secreted secretion. Surprisingly, the agonist GW4064 such as dis infectious particles by DNA extraction and quantification by closed in PCT Publication No. WOOO/37077 or in US2007/ qPCR. Treatment with lamivudine at 10 uM has very limited 001 5796 had a strong and dose dependent inhibitory effect on effect on the secretion of HBV antigens whereas its effect on HBSAg secretion (around 70% inhibition at 10 uM. Partial HBV DNA secretion is nearly complete with 97% reduction agonists such as PXL0914 (4-Bromo-5-4-(2-chloro-benze of HBV DNA in the cell supernatant. nesulfonyl)-1,4-diazepan-1-yl-benzofuran-2-carboxylic acidfrom patent WO2009127321), PXL0924 (5-4-(2,6- EXAMPLE Dichloro-benzoyl)-piperazin-1-yl)-4-methyl-benzofuran-2- carboxylic acid from patent WO2009127321) and PXL0743 Methods (4-Bromo-5-4-(2,6-dichloro-benzenesulfonylamino)-pip 0823. The HepaRG line derived from a human cellular eridin-1-yl)-benzofuran-2-carboxylic acid from patent hepato carcinoma can differentiate and regain many pheno WO2009127321) had an intermediate profile of inhibition. typic traits of hepatocytes after 4 weeks of culture under Thus some molecules decreased the production of HBSAg in defined conditions (Hantz, O. et al., 2009. J Gen Virol 90: a dose-dependent manner. If the active molecules are classi 127-135). After differentiation, these cells are susceptible to fied by their antagonist, agonist or agonist partial” by the infection at high MOI of HBV virions produced by HepG2. screening test in Huh-7 cell with the reporter gene construct, 2.15 line. Under these conditions viral production can be it appears, against previous odds, that the inhibitory effect on observed in the second week post infection. This system the production of HBs.Ag grew with the tendency of the allows the study of most steps of the viral replication cycle, molecule to be a potent FXR agonist. including penetration into the cell, translocation of the viral 0827. To confirm this finding, we next tested several mol genome into the nucleus, the repair and synthesis of the ecules, the reference FXR antagonist 052EDL133 (see US 2016/0220586 A1 Aug. 4, 2016

above), two well characterized FXR agonists, which belong 2014 April: 60(4):723-31. doi:10.1016/j.ijhep.2013.11.022. to different chemical class, GW4064 (see above) and Epub 2013 Dec. 1). When increasing concentrations of CSA 6ECDCA (a biliary salt derivative and potent FXR agonist, were introduced into the culture medium during infection, we currently in clinical trial for primary biliary cirrhosis and observed a dose dependant competition with HBV entry with insulin resistance, see above) and the biliary salt analogue an inhibition of HBs.Ag and HBeAg secretion as expected. ursodesoxycholic acid, which is not a FXR ligand (Parks D Treatment with GW4064 or 6ECDCA further reduced the J1, et al. Science. 1999 May 21; 284(5418): 1365-8. Mak secretion of the viral proteins (FIG. 5A). On the opposite, ishima M1, et al. Science. 1999 May 21; 284(5418): 1362-5). addition of CSA after the infection period, had no effect on FIG. 2A shows that only GW4064 and 6ECDCA had a dose HBV replication with a conserved HBs.Ag and HBeAg secre dependent and strong inhibitory effect on the secretion of tion nor did modify the effect of FXR agonists (FIG. 5B). HBS Ag and HBeAg in HepaRG supernatant of infected cells Taking into account, as previously reported, that there is little after 10 days of treatment. The bile salt ursodeoxycholate did or no viral spread during the culture in this system, we con not inhibit the secretion of the viral protein at any doses and clude that antiviral activity of FXRagonists is not related to a the FXRantagonist 052EDL133 had little or no effect. Simi direct inhibition of NTCP but rather modulate later steps of lar findings were observed when testing the effect of these the infection cycle. molecules on the secretion of the viral DNA in the superna 0832. We next investigated if viral proteins could interfere tant (FIG. 2B). Strong inhibition, up to 80%, was observed with FXR by co-immunoprecipitation using tagged viral pro with the two agonists, while UDCA did not modify the secre teins and FXR. We found that HBx viral protein and FXR tion of the viral DNA. It should be noted however that the could be immunoprecipitated by antibody directed against antagonist 052EDL133 moderately reduced the amount of one or the other protein (FIG. 6). This data suggested an viral DNA secreted at 10 uM (close to 20% inhibition). interaction between HBX and FXR, strengthening the hypoth Finally the activity on viral replication of a chemically dif esis that the virus tightly regulates FXR activity. ferent FXRagonist, PXL007, indentified by the CAS registry 0833 Next we investigated the effect of FXR agonists on number 1192171-69-9 (described in WO 2009127321) was the expression level of mRNA encoding FXR itself as well as tested in the same assay. This FXR agonist also strongly SHP and ApoA1, two genes the expression of which is respec inhibited viral protein and DNA secretion (FIG. 2C). tively under the positive and negative control of FXR. We 0828 We further explored the effect of GW4064 and found that 10 days treatment with FXRagonists GW4064 and 6ECDCA on the cellular expression of the viral core protein 6ECDCA increased the expression of SHP mRNA and HBc by immunofluorescence (FIG.3). Again both FXRago decreased that of ApoA1 mRNA indicating that indeed FXR nists strongly inhibited HBc expression in infected cells agonists boosted FXR activity (FIG. 7). Interestingly, FXR whereas UDCA and 052EDL133 did not significantly modify mRNA expression was also strongly repressed by both ago HBc synthesis. nists likely as a result of the SHP dependent negative control 0829. Finally we quantified the amount of viral RNA by loop on FXR expression. Thus treatment with FXR agonists quantitative RT-PCR and Northern blotting in infected cells significantly and durably modifies bile acid metabolism with treated or not by GW4064 and 6ECDCA as well as the varia an increased FXR activity along with a decreased FXR tions of the cccDNA reservoir (FIG. 4). The presence of the expression. two 3.4 and 3.5 pre-core and pre-genomic RNA was reduced 0834) Effect of Combined Treatment with FXR Agonist in a dose dependent manner by FXR agonists up to 75% and Reverse Transcriptase Inhibitor on HBV Replication. (panel A) as measured by quantitative RT-PCR. The presence 0835 FXR agonists thus appear to repress HBV replica of the three classes of viral mRNA, i.e. the 3.4 and 3.5 pre tion at steps that occur after viral entry and mainly on core and pre-genomic mRNA, the 2.1-2.4 S mRNA and the cccDNA reservoir stability and expression, thus before the 0.7xmRNA, was reduced to similar extent at 10uM measured reverse transcription step. We thus tested the effect of com by Northern blotting (panel B). Interestingly, the cccDNA bination treatment of HBV infected HepaRG cells on viral reservoir was also reduced by more than 50% after treatment replication (FIG. 8). We observed that treatment with the with the two agonists at 10 uM (panel C). nucleoside analogue reverse transcriptase inhibitor lamivu 0830 Mechanism of Action dine even at high concentration (10M with an IC50 at 6 nM. 0831. The sodium taurocholate cotransporter peptide Lada O, et al. Antivir Ther. 2004 June; 9(3):353-63) only (NTCP) was recently identified as a receptor for HBSAgat the weakly repressed the secretion of HBS Aget HBeAg but very baso-lateral plasma membrane of hepatocytes. NTCP expres efficiently decreased HBV DNA positive virions secretion as sion is mandatory for virus entry into hepatocytes. Sodium expected. Addition of FXRagonist did not seem in this con taurocholate cotransporting polypeptide is a functional recep dition to further decrease the secretion of viral DNA but tor for human hepatitis B and D virus (Yan H. et al. Elife potently repressed the synthesis and secretion of viral pro (Cambridge). 2012 Nov. 13; 1:e()0049. doi:10.7554/eLife. teins. 00049). HBV and bile acids share common binding site on NTCP and compete for the receptor (Yan H. et al. J. Virol. DISCUSSION 2014 March; 8806):3273-84. doi: 10.1128/JVI.03478-13. 0836. We showed that FXR is an essential hostfactor in the Epub 2014 Jan. 3). As FXR agonists are molecules that development of HBV in hepatocytes and that, unexpectedly, directly derived from bile acids or share some molecular FXRagonists are more potent inhibitors than the antagonists determinants with bile acids, FXRagonists might just inhibit on HBV replication in HepaRG cell lines. This antiviral activ the virus entry through competition for the receptor. We thus ity was demonstrated with structurally very diverse and selec tested the effect of addition to FXRagonists of cyclosporin A tive FXRagonists, GW4064, PXL007 (molecule having the (CSA), a molecule which inhibits NTCP mediated bile acids CAS Registry number: 1192171-69–9), the bile acid deriva uptake, binds NTCP at a site identical or overlapping with the tive 6ECDCA and others. This reduces the probability of an pre-S1 peptide binding site (Nkongolo S, et al. J Hepatol. “off-target effect. FXR agonists seem to primarily act on US 2016/0220586 A1 Aug. 4, 2016 26 viral mRNAs transcription and expression from the viral min REFERENCES ichromosome and on the cccDNA stability. Overall FXR 0839 Throughout this application, various references agonists beside reducing viral DNA replication and the pro describe the state of the art to which this invention pertains. duction of infectious virions, an effect that can be efficiently The disclosures of these references are hereby incorporated and safely obtained with reverse transcriptase polymerase by reference into the present disclosure. inhibitors, have the unique capacity to decrease the synthesis 1-7. (canceled) and secretion of the viral proteins and to reduce the cccDNA 8. A method for the treatment of hepatitis B virus (HBV) reservoir size. These two late effects are not obtained by infection in a subject in need thereof, comprising administer polymerase inhibitors and can only be reached for a low ing to the Subject a therapeutically effective amount of a percentage of patients treated with interferons. Reducing the farnesoid X receptor (FXR) agonist. viral protein secretion and cccDNA reservoir are two majors 9. The method of claim 8, wherein the subject is infected goals to cure HBV infection since on the one hand, viral with any Hepatitis B virus genotype including genotype A, B, proteins have been shown to dampen the innate immune C, or D. response, mainly through interaction with TLR, and maintain 10. The method of claim 8, wherein the subject suffers an immune-tolerant status against the virus and, on the other from a chronic HBV infection. hand, viral persistence and latency depend on the continuous 11. The method of claim 8, wherein the farnesoid X recep presence of cccDNA. tor (FXR) agonist is a selective FXR agonist. 12. The method of claim 8, wherein the farnesoid X recep 0837 Persistence of HBV replication also requires the tor (FXR) agonist is selected from the group consisting of the presence of a Supportive cellular environment providing, in compounds identified by the CAS REGISTRY NUMBERS particular, an active transcriptional cellular machinery for the 1192171-69-9, 6ECDCA, GW4064, PXL0914, and expression of the viral genes. Regulation of FXR activity by PXLO743. the virus may be part of the viral strategy to control its own 13. The method of claim 8, wherein the farnesoid X recep replication. Indeed, the competition between HBV virions tor (FXR) agonist is selected from the group consisting of the and bile acids for NTCP decreases the intracellular bile acids compounds identified by the CAS REGISTRY NUMBERS pool with the Subsequent consequences of a decreased FXR 11921 71-69-9 and 6ECDCA. activity and an increased level of FXR expression (Oehler N. 14. The method of claim8, wherein the subject has failed to et al. Hepatology. 2014 Apr. 8. doi: 10.1002/hep.27159. respond to a previous treatment for HBV infection. Epub ahead of print). Treatment with FXRagonists proved 15. The method of claim 14, wherein the previous treat to reverse the bile acid metabolism modification induced by ment is selected from the group consisting of lamivudine the virus, which thus appears as a beneficial condition for (Epivir), adefovir (Hepsera), tenofovir (Viread), telbivudine Supporting viral replication. (Tyzeka), entecavir (Baraclude), interferon alpha-2a, PEGy 0838. The discovery of the antiviral effect of 6ECDCA, a lated interferon alpha-2a (Pegasys) and interferon alpha-2b molecule in clinical development in two separate indications (ViraferonPeg or Introna). (i.e. primary biliary cirrhosis and insulin resistance), with 16. The method of claim 8, wherein the FXR agonist is good tolerance during long-term treatment, offers the oppor administered in combination with a treatment selected from tunity for “repositioning the molecule in the treatment of the group consisting of lamivudine (Epivir), adefovir (Heps HBV infection. In conclusion, we have identified new mol era), tenofovir (Viread), telbivudine (Tyzeka), entecavir ecules (i.e. FXRagonists) that regulate (reduce) HBV infec (Baraclude), interferon alpha-2a, PEGylated interferon tion. This should allow the selection of candidates who could alpha-2a (Pegasys) and interferon alpha-2b (ViraferonPeg or be tested in a mouse model or directly in humans with FXR Introna). agonists already in clinical trials.