EP4676518A1 - Synergistic effect of an fxr agonist and ifn for the treatment of chronic hdv infection - Google Patents

Abstract

The present invention relates to a synergistic combination of Vonafexor with PEG-INFα for the treatment of hepatitis D, especially the treatment of chronic hepatitis D.

Description

SYNERGISTIC EFFECT OF AN FXR AGONIST AND IFN FOR THE TREATMENT OF CHRONIC HDV INFECTION

FIELD OF THE INVENTION

The present invention relates to a method for treating chronic Hepatitis D infection.

BACKGROUND OF THE INVENTION

Hepatitis D virus (HDV) infection is the most severe form of chronic viral hepatitis due to rapid progression towards liver-related death and hepatocellular carcinoma. The World Health Organization (WHO) estimates that 15-20 million persons are infected by HDV (www.who.int/en/news-room/fact- sheets/detail/hepatitis-d). The most recent meta-analysis of HDV burden suggests an underestimation of hepatitis D prevalence; indeed, seroprevalence might be as high as 0.98% of the worldwide population and 10.58% of global chronic hepatitis B patients (Chen H-Y et al. Gut 2019;68:512-521). In the absence of specific anti-HDV therapy, current guidelines generally recommend subcutaneous injection of Pegylated-interferon-alpha-2a (PEG-IFN-a2a), which is a non-specific immune-stimulator, rather toxic and poorly supported by patients. Since interferons can be associated with significant adverse events, consideration between prolonged therapy and drug toxicity is critical. It is often used in addition to a nucleoside analogue, which controls HBV viremia. The overall rate of sustained virological response is low. Indeed, a response rate on treatment of about 25% has been reported and a high level of relapse after cessation of treatment. It has been reported that the combination with adefovir did not improve the treatment outcome. Similarly, no increase in virologic response has been observed when nucleoside analogs such as lamivudine and ribavirin were used in combination.

Accordingly, there is an unmet need of new therapeutic agents and strategies for the treatment of infection by HDV. However, there are several difficulties in the development of therapeutic agents for these patients.

HDV genome is a single-stranded RNA (= 1700 nucleotides) of negative polarity containing a single open reading frame encoding two viral proteins: the small and the large delta antigens (HDAg-S and HDAg-L). Replication of the HDV RNA genome takes place in the nucleus of infected cells, and occurs by a rolling circle mechanism, followed by cleavage by endogenous ribozymes and ligations resulting in the formation of antigenomic circular monomers. From these circular antigenomic monomers, the same mechanisms generate new genomic circular RNA monomers. It is assumed that HDV hijacks DNA-dependent host RNA- polymerase(s) during the genome replication steps. HDV uses at least the RNA polymerase II for both replication and transcription of viral mRNA but the role of RNA polymerase I and III is also suggested (Mentha N et al. J Adv Res. 2019 May; 17:3-15). During this process, viral linear mRNAs are also synthesized resulting in synthesis of HDAg-S and HDAg-L. Compared with HDAg-S (195 amino acids), HDAg-L (214 amino acids) contains an additional domain of 19-20 amino acids at its C-terminus resulting from ADAR-l-mediated RNA editing of the antigenomic HDV RNA at a location corresponding to the stop codon of HDAg-S gene (Wong SK, Lazinski DW. Proc Natl Acad Sci U S A. 2002 Nov 12;99(23):15118-23). HDAg-S is involved in HDV accumulation during the replication step. In particular, HDAg-S is thought to interact with numerous cellular proteins (more than 100 identified interactants) and is present in a nuclear complex in association with cellular proteins involved in transcriptional regulation such as Yin Yang 1 (YY1), Histone acetyltransferase (HAT) p300 Creb Binding Protein (p300/CBP) and selectivity factor 1 (SL1), which belongs to the pre-initiation complex of RNA polymerase I (Huang W-H etal. J Virol. 2008 Aug;82(15):7313-24; Li Y-Jet al. J Virol. 2006 Jul;80(13):6478-86). Interaction of HDAg-S with cellular histone H1.4 has also been described with consequences on viral replication (Lee C-Z, Sheu J-C. Virology. 2008 May 25;375(l):197-204). Whereas HDAg-S is mainly involved in the replication step, HDAg-L is essential for virion budding. The 19-20 amino acids additional domain in HDAg-L contains a CXXX-box motif, a substrate for cellular farnesyltransferase, which adds a farnesyl group to the cysteine of this CXXX- box. This farnesylation process was shown to be essential for virion assembly (Glenn J et al. Science. 1992 May 29;256(5061):1331-3). During this step, HDV hijacks HBV surface antigens for its own use and the secretion of infectious HDV virions that can spread or maintain the infection. HBV and HDV virions contain the same envelope proteins and are undistinguishable from a humoral-response perspective. Consequently, HBV and HDV share the same entry receptor, i.e. sodium taurocholate cotransporting polypeptide (NTCP), the main transporter of bile acid (BA) at the baso-lateral membrane of hepatocytes (Yan H et al. eLife [Internet]. 2012 Nov 13 [cited 2019 Sep 3];1. Available from: https://elifesciences.org/articles/00049). As a consequence of this viral symbiosis, HDV transmission generally occurs through HBV co-infection or super-infection. However, aside from the crucial role of HB antigens (HBAgs) for hepatocyte entry, the other steps of HDV life cycle described above, in particular the replication process, are not dependent on HBV and contribute per se to the severity of the disease and rapid evolution toward cirrhosis and HCC. In addition, in the typical course of HDV super-infection, markers of HBV infection are usually inhibited, with IgM anti-HBc and HBV DNA that could test negative (Romeo R, Perbellini R. World J Hepatol 2015;7:2389-95; Schaper M et al. J Hepatol. 2010;52:658-64). HBV replication is, however, usually suppressed to low levels during the acute phase of HDV infection. This suppression becomes persistent in case of a chronic hepatitis D establishment.

The co-infection with HDV and HBV is a complex situation that mixes features specific for each virus and some that are common to both. Importantly, recent studies report that addition of the standard of care for HBV treatment, which inhibits the HBV specific polymerase by nucleotide analogues, to HDV standard of care treatment with PEG-IFN-2a does not improve HDV response rate at the end of treatment (Wedemeyer H et al. Lancet Infect. Dis. 2019;19:275-286; Mentha N et al. J. Adv. Res. 2019;17:3-17). These findings clearly highlight that alternative treatment options that target HDV replication and/or propagation steps are needed for hepatitis D. Indeed, lonafarnib, an inhibitor of the enzyme farnesyl transferase, which is a mandatory step in virion assembly, repress HDV replication independently of HBV (application US20110129549A1; Mentha N, Clnts.google.com/patAlfaiate D. J. Adv. Res. 2019;17:3-17). However, its toxicity prevents its broad use in anti-HDV therapy. Several other antiviral molecules targeting the HDV interaction with the HBV HBsAg are currently under development: Myrcludex B, which blocks HDV entry into hepatocytes by inhibiting HBsAg binding to NTCP, siRNA silencing HBV mRNA, including HBsAg mRNA, and REP 2139, which is thought to inhibit HBsAg release from hepatocytes and interact with hepatitis delta antigen (Ye X et al. ACS Infect. Dis. 2019;5:738-5:7; Mentha N et al. J. Adv. Res. 2019;17:3-17).

Then, anti-HDV treatment should inhibit at least one HDV replication or propagation step. The replication step may be HDV specific, so as the prenylation of HDAg-L, or shared with HBV by inhibiting HBsAg synthesis, release, or function. Ideally, treatment of hepatitis D should not only repress HDV replication or propagation but also HBV replication by inhibiting specific replication steps of each virus. Besides compounds targeting the common HBsAg dependency of both viruses, no such molecule that could specifically repress both HDV propagation AND HBV replication has been reported. Molecules inhibiting the two viruses are the subject of active research since it is difficult to predict how the second virus will react when a treatment is targeting only one virus. There is a risk of activation or reactivation of one virus when the other is inhibited. For instance, it has been observed in the development of antiviral agents against HCV that, in patients co-infected with HBV, reactivation of HBV has occurred when HCV was targeted (Ma et al, Gastroenterology, 2018, 154, 795-798). Therefore, patients co-infected by HBV and HDV are generally excluded from clinical trials. Indeed, a review in 2016 reports that less than 1000 patients coinfected with HDV were included in the clinical trials (Guglielmi S et al, 2016, Revue medicale Suisse, 12, 1415-1418).

WO 2021/144330 discloses the capacity of several FXR agonists to prevent HDV RNA genome replication and propagation and suggests a new way for treating chronic hepatitis D infection with a FXR agonist.

Still, there is a need for improved treatments of chronic hepatitis D infection.

SUMMARY OF THE INVENTION

The inventors surprisingly identified that Vonafexor has a synergistic effect with PEG-INFa for the treatment of hepatitis D, especially the treatment of chronic hepatitis D. Indeed, Vonafexor and PEG-INFa have a synergistic effect on the reduction of HDV propagation from an infected hepatocyte to a noninfected hepatocytes. Therefore, the present invention relates to the synergistic combination of Vonafexor or a pharmaceutically acceptable salt thereof and PEG-INFa for use in the treatment of hepatitis D infection, especially chronic hepatitis D.

The present invention relates to

Vonafexor or a pharmaceutically acceptable salt thereof or a pharmaceutic composition comprising it for use in combination with PEGylated interferon alpha (PEG-INFa) for the treatment of hepatitis D virus infection, especially chronic hepatitis D, wherein Vonafexor and PEG-INFa are used so as to obtain a synergistic effect for decreasing the HDV propagation;

PEG-INFa or a pharmaceutic composition comprising it for use in combination with vonafexor or a pharmaceutically acceptable salt thereof for the treatment of hepatitis D virus infection, especially chronic hepatitis D, wherein Vonafexor and PEG-INFa are used so as to obtain a synergistic effect for decreasing the HDV propagation; or

A pharmaceutical composition comprising vonafexor or a pharmaceutically salt thereof and PEG- IFN-a for use in the treatment of hepatitis D virus (HDV) infection, wherein vonafexor and PEG- IFN-a are used so as to obtain a synergistic effect for decreasing the HDV propagation.

It also relates to the use of Vonafexor or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising it for the manufacture of a drug for the treatment of hepatitis D virus infection, especially chronic hepatitis D, in combination with PEG-INFa, wherein Vonafexor and PEG-INFa are used so as to obtain a synergistic effect for decreasing the HDV propagation. It further relates to a method for treating a hepatitis D virus infection, especially chronic hepatitis D, in a subject in need thereof, comprising administering a therapeutically effective or sub-therapeutic amount of Vonafexor or a pharmaceutically acceptable salt thereof and administering a therapeutically effective or sub-therapeutic amount of PEG- INFa, wherein Vonafexor and PEG-INFa are administered so as to obtain a synergistic effect for decreasing the HDV propagation.

In one aspect, Vonafexor is to be administered at a dose in the range from 25 to 800 mg per day. More particularly, Vonafexor is to be administered at a dose in the range from 100 to 600 mg, preferably from 200 to 400 mg per day. Optionally, Vonafexor is to be administered at a sub-therapeutic amount.

In one aspect, Vonafexor is to be administered once a day. In another aspect, Vonafexor is to be administered twice a day.

In one aspect, the PEG-INFa is to be administered by subcutaneous route once a week. Optionally, PEG- INFa can be administered at a sub-therapeutic amount. In one particular aspect, both Vonafexor and PEG-INFa are to be administered at sub-therapeutic amounts.

In one aspect, Vonafexor and PEG-INFa are to be administered during a period of time from 5 6, 7 or 8 weeks to 52 weeks.

Optionally, the PEG-INFa is selected from the group consisting of PEG-IFN-a2a (e.g., Pegasys) or PEG-IFN- a2b (e.g., ViraferonPeg or Introna).

In one aspect, Vonafexor and PEG-INFa are to be used in combination with at least one additional active ingredient. More specifically, the at least one additional active ingredient can be selected in the group consisting of a nucleoside analog, a nucleic acid polymer, a NTCP inhibitor or a farnesyl transferase inhibitor, preferably in the group consisting of ribavirin, ritonavir, lonafarnib, EBP 921, lamivudine, adefovir, telbivudine, entecavir, tenofovir, emtricitabine, ezetimibe, myrcludex B, nucleic acid polymer REP 2139 and nucleic acid polymer REP 2165.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Synergistic effect of Vonafexor and IFN-alpha on HDV propagation. Differentiated HepaRG cells were infected with HBV at a multiplicity of infection of 250 Genome Equivalent (GE) per cell, then superinfected 5 days later with HDV at a multiplicity of infection of 25 GE/cell. From day 8 to 15 postinfection, cells were treated with Vonafexor (concentrations between 1 and 30pM), or IFN-alpha at 250 lU/mL, or with a combination of Vonafexor (lOpM) + IFN-alpha at 250 lU/mL, or with vehicle. Supernatants were harvested at day 15 and used to re-infect Huh7.5.hNTCP cells. Six days later, RNA was extracted from these cells and HDV RNA quantified by RTqPCR.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Vonafexor or EYP001 is an FXR agonist disclosed in CAS number 1192171-69-9 and having the following formula: By vonafexor is intended to refer to this compound and any pharmaceutically acceptable salt thereof.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

More particularly, the term "treating", or "treatment", means alleviating HDV infection, arresting disease development, and/or removing HDV by administering the composition. By decreasing HDV propagation, it is preferably meant that the HDV propagation is decreased by at least 10 or 100 fold in comparison with the HDV propagation in absence of treatment.

In particular, the inhibition of HDV propagation corresponds to a reduction of about 10, 20, 30, 40, 50, 60, 70, 80, 90% or 100 % of the number of HDV RNA copies in newly infected hepatocytes. Techniques for measuring the number of copies, particularly those based on Polymerase Chain Reaction (PCR), are well known to the person skilled in the art.

According to the invention, the term "subject" or "patient" and "subject in need thereof" or "patient in need thereof", is intended for a human or non-human mammal infected or likely to be infected with a hepatitis D virus. In some aspects of the invention, the subject suffers from a chronic HDV infection.

The terms "coinfected patients" refers to individuals that have been simultaneously infected with HBV and HDV. The terms "super-infected patients" refers to individuals that have been firstly infected with HBV, and then infected with HDV.

As used herein, the term "therapeutically effective amount" refers to a quantity of an active ingredient or of a pharmaceutical composition which prevents, removes or reduces the deleterious effects of the disease, particularly infectious disease. It is obvious that the quantity to be administered can be adapted by the man skilled in the art according to the subject to be treated, to the nature of the disease, etc. In particular, doses and regimen of administration may be function of the nature, of the stage and of the severity of the disease to be treated, as well as of the weight, the age and the global health of the subject to be treated, as well as of the judgment of the doctor.

As used herein, the term "sub-therapeutic amount" or "sub-therapeutic dose" refers to a dosage which is less than that dosage which would produce a therapeutic result in the subject if administered in the absence of the other agent. For instance, "sub-therapeutic amount" or "sub-therapeutic dose" can refer to a dosage which is decreased by 25, 50, 70, 80 or 90 % in comparison to the therapeutically effective amount, especially the conventional therapeutic dosage for the same indication and the same administration route when used alone. The conventional therapeutic dosages are those acknowledged by the drug approvals agencies (e.g., FDA or EMEA).

As used herein, the term "excipient or pharmaceutically acceptable carrier" refers to any ingredient except active ingredients that is present in a pharmaceutical composition. Its addition may be aimed to confer a particular consistency or other physical or gustative properties to the final product. An excipient or pharmaceutically acceptable carrier must be devoid of any interaction, in particular chemical, with the active ingredients.

By "a synergistic effect" is intended to refer to an effect for decreasing the HDV propagation which is more than the sum of the effects of each molecule alone.

Synergistic combination of vonafexor and PEG-INFa

PEG-INFa can be a pegylated INFalb, a pegylated INFa2a or a pegylated INFa2b. More specifically, the PEG-INFa can be selected in the non-exhaustive group consisting of a pegylated IFN-a2a including PEGASYS®, YPEG-IFNa-2a, PEG-INTRON® and Pegaferon, and a pegylated IFN-a2b including Pegintron®, Albuferon, AOP2014/P1101, Algeron, Pai Ge Bin. In a preferred aspect, the PEG-INFa is a pegylated IFN- a2a.

In an aspect, PEG-INFa is administered by subcutaneous route, preferably once a week; for instance, at a dosage varying from 1 pg to 500 pg, preferably from 10 pg to 500 pg, still more preferably from 100 pg to 250 pg, such as 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 pg. Optionally, the dosage is less than 180 pg, for instance from 1 pg to 170 pg, 10 pg to 160 pg or 100 pg to 150 pg.

Optionally, PEG-INFa can be administered at a sub-therapeutic amount.

Vonafexor can be administered at a therapeutic amount effective once or twice a day; more specifically, at a daily dose from 25 to 800 mg per adult per day, preferably from 100 to 600 mg per adult per day; still more preferably from 150 to 400 mg per adult per day or from 200 to 400 mg per day and for instance about 300 mg per adult per day; preferably orally.

Optionally, vonafexor can be administered at a sub-therapeutic amount.

Optionally, PEG-INFa and vonafexor are administered at a sub-therapeutic amount.

Preferably, the composition, dosage unit or dosage form contains 5, 10, 15, 25, 50, 75, 100, 200, 300, 400 and 500 mg of vonafexor for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 5 mg to about 500 mg of vonafexor, preferably from 50 mg to about 500 mg of vonafexor, from 50 mg to about 450 mg of vonafexor, from 100 mg to about 400 mg of vonafexor, or from 150 mg to about 300 mg of vonafexor.

In one aspect, the dosage form can be a scored dosage form. Alternatively, the daily dosage can be provided by administering several dosage forms.

Vonafexor may be combined with pharmaceutically acceptable excipients, and optionally sustained- release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

The pharmaceutical compositions comprising vonafexor can be suitable for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, preferably for oral administration.

Vonafexor, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

In a preferred aspect, the oral dosage form is a capsule or a tablet. Optionally, the oral dosage form is a scored dosage form. Optionally, the dosage form can be scored into four pieces, three pieces or two pieces.

Optionally, the treatment lasts from 2-4 months up to 24 months, for instance between 2 and 24 months or between 2 and 12 months, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 months. In a very specific aspect, the treatment lasts from 12 to 52 weeks, preferably from 45 to 52 weeks, for instance 48 weeks.

Vonafexor and IFN PEG-INFa can be used in combination with at least one additional active ingredient. Preferably, the additional active ingredient is an antiviral, more particularly an antiviral having an activity against HDV and/or HBV. In a preferred aspect, the at least one additional active ingredient is selected in the group consisting of a nucleoside analog, a nucleic acid polymer, a NTCP inhibitor or a farnesyl transferase inhibitor. More specifically, the at least one additional active ingredient is selected in the group consisting of ribavirin, ritonavir, lonafarnib, EBP 921, lamivudine, adefovir, telbivudine, entecavir, tenofovir, emtricitabine, ezetimibe, myrcludex B, nucleic acid polymer REP 2139 and nucleic acid polymer REP 2165. Optionally, the at least one additional active ingredient is selected in the group consisting of ribavirin, ritonavir, lonafarnib and EBP 921. Further aspects and advantages of the present invention will be described in the following example, which should be regarded as illustrative and not limiting.

EXAMPLES

EXAMPLE 1

RESULTS

Differentiated HepaRG cells were infected with HBV, then superinfected 5 days later with HDV. From day 8 to 15 post-infection, cells were treated with Vonafexor (concentrations between 1 and 30pM), or IFN- alpha at 250 lU/mL, or with a combination of Vonafexor (10pM) + IFN-alpha at 250 lU/mL, or with vehicle. Supernatants were harvested at day 15 and used to re-infect Huh7.5.hNTCP cells. Six days later, RNA was extracted from these cells and HDV RNA quantified by RT-qPCR. As shown on Figure 1, Vonafexor treatment induced a dose dependent decrease of HDV propagation. The treatment with IFN-alpha alone also reduced HDV propagation, but unexpectedly, the combination of vonafexor and IFN-alpha almost totally suppressed HDV propagation, reducing HDV propagation by 98.6%. These data demonstrated the strong synergistic effect of Vonafexor and IFN-alpha on HDV propagation.

MATERIAL & METHODS

Cell lines

The HepaRG cell line derived from a human cellular hepato carcinoma can differentiate and regain many phenotypic traits of hepatocytes after 4 weeks of culture under defined conditions¹. HepaRG cells were cultured, differentiated, and infected by HBV and HDV as previously described²-³. Briefly, for differentiation, cells were maintained for 2 weeks in standard medium then for at least 2 weeks in standard medium supplemented with 1,8% DMSO. The composition of standard medium was the following: William's E medium supplemented with 10% HyCLone FetalClone II serum (Thermo Fisher Scientific), penicillin/streptomycin, L-glutamine, Insulin-Transferrin-Selenium (Gibco) and 50 pM hydrocortisone hemisuccinate.

Huh7.5 cells were previously described by Blight and colleagues⁴. These cells were stably engineered using lentiviral vector allowing the expression of the human NTCP (sodium taurocholate cotransporting peptide), which is the entry receptor for HBV and HDV. These cells are cultured in DMEM/Glutamax supplemented with 10% HyCLone FetalClone II serum (Thermo Fisher Scientific), penicillin/streptomycin (Gibco), sodium pyruvate (Gibco) and non-essential amino acids (Gibco). Viruses

An HBV inoculum (genotype D, Genbank ID U95551) was prepared from supernatant of HepAD38 cells, which were initially described in Ladner and colleagues⁵; the 0.22 micron filtrated supernatant of HepAD38 was PEG8000 (8% final) precipitated to generate the viral stock. An HDV genotype 1 inoculum was prepared from supernatant of Huh7 cells co-transfected with pSVLD3 et pHB2.7 cells as described by C. Sureau⁶; the 0.22 micron filtrated supernatant was PEG8000 (8% final) precipitated to generate the HDV stock. The titers of these stocks, in virus genome equivalent (GE)/mL, were determined by either qPCR for HBV or RT-qPCR for HDV as described in previous report by Michelet et colleagues⁷.

Infection and treatment procedures

The conditions of infection have been previously described by Michelet and colleagues⁷. In brief, a multiplicity of infection of 250 HBV GE/cell and 25 HDV GE/cell were used to infect and super-infect HepaRG cells respectively. The infection procedure consists of an exposure of cells overnight with an inoculum containing the desired amount of virus (from viral stocks) and PEG8000 at 4% final. Superinfection were mainly performed as following: infection with HBV, then 5 days later super-infection with HDV. After 3 days of infection with HDV, cells were treated with drugs. Drugs were extra-temporally diluted in complete medium from mother stocks. Treatments were repeated 3 times every 2 to 3 days. Huh7.5-hNTCP cells were used for the secondary infections. Cells were infected with supernatants, recovered from HepaRG cultures, and adjusted to 4% final with PEG8000.

Chemicals

Vonafexor was provided by ENYO Pharma. Interferon alpha-2 (ROFERON-A) was purchased from Roche.

Quantification of viral RNAs by qPCR

Total RNA was extracted from infected (and treated) cells with « NucleoSpin RNA Plus, Mini kit for RNA purification with DNA removal column" (Macherey-Nagel), according to manufacturer's instructions, then retro-transcribed with Superscript III kit (Thermo Fisher Scientific), following manufacturer's instructions. Complementary DNA was quantified by qPCR with HDV specific primers and the "Luna* Universal qPCR Master Mix" reagent (BioLabs). More details can be found in Michelet et al.⁷.

REFERENCES

1. Hantz, O. et al. Persistence of the hepatitis B virus covalently closed circular DNA in HepaRG human hepatocyte-like cells. J. Gen. Virol. 90, 127-135 (2009).

2. Gripon, P. et al. Infection of a human hepatoma cell line by hepatitis B virus. Proc. Natl. Acad. Sci. U. S. A. 99, 15655-15660 (2002). 3. Alfaiate, D. et al. HDV RNA replication is associated with HBV repression and interferon- stimulated genes induction in super-infected hepatocytes. Antiviral Res. 136, 19-31 (2016).

4. Blight KJ, McKeating JA, Marcotrigiano J, Rice CM. Efficient replication of hepatitis C virus genotype la RNAs in cell culture. J Virol. 2003 Mar;77(5):3181-90. 5. Ladner, S. K. et al. Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication. Antimicrob. Agents Chemother. 41, 1715-1720 (1997).

6. Sureau C. The use of hepatocytes to investigate HDV infection: the HDV/HepaRG model. Methods Mol Biol. 2010;640:463-73. 7. Michelet M, Alfaiate D, Chardes B, Pons C, Faure-Dupuy S, Engleitner T, Farhat R, Riedl T,

Legrand AF, Rad R, Rivoire M, Zoulim F, Heikenwalder M, Salvetti A, Durantel D, Lucifora J. Inducers of the NF-KB pathways impair hepatitis delta virus replication and strongly decrease progeny infectivity in vitro. JHEP Rep. 2021 Dec 14;4(3):100415.

Claims

1- A pharmaceutical composition comprising vonafexor or a pharmaceutically acceptable salt thereof for use in combination with a pegylated interferon alpha (PEG-IFN-a) in the treatment of hepatitis D virus (HDV) infection, wherein vonafexor and PEG-IFN-a are used so as to obtain a synergistic effect for decreasing the HDV propagation.

2- A pharmaceutical composition comprising PEG-IFN-a for use in combination with vonafexor or a pharmaceutically salt in the treatment of hepatitis D virus (HDV) infection, wherein vonafexor and PEG- IFN-a are used so as to obtain a synergistic effect for decreasing the HDV propagation.

3- A pharmaceutical composition comprising vonafexor or a pharmaceutically salt thereof and PEG-IFN-a for use in the treatment of hepatitis D virus (HDV) infection, wherein vonafexor and PEG-IFN-a are used so as to obtain a synergistic effect for decreasing the HDV propagation.

4- The pharmaceutical composition for use according to any one of claims 1-3, for use in the treatment of chronic HDV infection.

5- The pharmaceutical composition for use according to any one of claims 1-4, wherein the PEG-INFa is selected from the group consisting of PEG-IFN-a2a (e.g., Pegasys) or PEG-IFN-a2b (e.g., ViraferonPeg or Introna).

6- The pharmaceutical composition for use according to any one of claims 1-5, wherein vonafexor or a pharmaceutically acceptable salt thereof is to be administered at a dose in the range from 25 to 800 mg per day or from 100 to 600 mg per day or 200 to 400 mg per day.

7- The pharmaceutical composition for use according to any one of claims 1-5, wherein vonafexor or a pharmaceutically acceptable salt thereof is to be administered at a sub-therapeutic amount.

8- The pharmaceutical composition for use according to any one of claims 1-7, wherein PEG-IFN-a is to be administered at a sub-therapeutic amount.

9- The pharmaceutical composition for use according to any one of claims 1-8, wherein vonafexor or a pharmaceutically acceptable salt thereof and the pegylated IFN-a are to be administered during a period of time from 5, 6, 7 or 8 weeks to 52 weeks.

10- The pharmaceutical composition for use according to any one of claims 1-9, wherein the pharmaceutical composition is to be used in combination with at least one additional active ingredient, preferably selected in the group consisting of a nucleoside analog, a nucleic acid polymer, a NTCP inhibitor or a farnesyl transferase inhibitor, preferably in the group consisting of ribavirin, ritonavir, lonafarnib, EBP 921, lamivudine, adefovir, telbivudine, entecavir, tenofovir, emtricitabine, ezetimibe, myrcludex B, nucleic acid polymer REP 2139 and nucleic acid polymer REP 2165.

11- Use of a pharmaceutical composition comprising vonafexor or a pharmaceutically acceptable salt thereof for the manufacture of a medicine for the treatment of hepatitis D virus (HDV) infection in combination with a pegylated interferon alpha (PEG-IFN-a), wherein vonafexor and PEG-IFN-a are used so as to obtain a synergistic effect for decreasing the HDV propagation.

12- Use of a pharmaceutical composition comprising a pegylated interferon alpha (PEG-IFN-a) for the manufacture of a medicine for the treatment of hepatitis D virus (HDV) infection in combination with vonafexor or a pharmaceutically acceptable salt thereof, wherein vonafexor and PEG-IFN-a are used so as to obtain a synergistic effect for decreasing the HDV propagation.

13- Use of a pharmaceutical composition comprising vonafexor or a pharmaceutically acceptable salt thereof and a pegylated interferon alpha (PEG-IFN-a) for the manufacture of a medicine for the treatment of hepatitis D virus (HDV) infection, wherein vonafexor and PEG-IFN-a are used so as to obtain a synergistic effect for decreasing the HDV propagation.

14- Use according to any one of claims 11-13, wherein the HDV infection is a chronic HDV infection.

15- Use according to any one of claims 11-14, wherein the PEG-INFa is selected from the group consisting of PEG-IFN-a2a (e.g., Pegasys) or PEG-IFN-a2b (e.g., ViraferonPeg or Introna).

16- Use according to any one of claims 11-15, wherein vonafexor or a pharmaceutically acceptable salt thereof is to be administered at a dose in the range from 25 to 800 mg per day or from 100 to 600 mg per day or 200 to 400 mg per day.

17- Use according to any one of claims 11-15, wherein vonafexor or a pharmaceutically acceptable salt thereof is to be administered at a sub-therapeutic amount.

18- Use according to any one of claims 11-17, wherein PEG-IFN-a is to be administered at a sub- therapeutic amount.

19- Use according to any one of claims 11-18, wherein vonafexor or a pharmaceutically acceptable salt thereof and the pegylated IFN-a are to be administered during a period of time from 5, 6, 7 or 8 weeks to 52 weeks.

20- Use according to any one of claims 11-19, wherein the pharmaceutical composition is to be used in combination with at least one additional active ingredient, preferably selected in the group consisting of a nucleoside analog, a nucleic acid polymer, a NTCP inhibitor or a farnesyl transferase inhibitor, preferably in the group consisting of ribavirin, ritonavir, lonafarnib, EBP 921, lamivudine, adefovir, telbivudine, entecavir, tenofovir, emtricitabine, ezetimibe, myrcludex B, nucleic acid polymer REP 2139 and nucleic acid polymer REP 2165.

21- A method for treating a hepatitis D virus infection, especially chronic hepatitis D, in a subject in need thereof, comprising administering a therapeutically effective or sub-therapeutic amount of Vonafexor or a pharmaceutically acceptable salt thereof and administering a therapeutically effective or sub- therapeutic amount of PEG-INFa, wherein Vonafexor and PEG-INFa are administered so as to obtain a synergistic effect for decreasing the HDV propagation.

22- The method of claim 21, wherein the PEG-INFa is selected from the group consisting of PEG-IFN-a2a (e.g., Pegasys) or PEG-IFN-a2b (e.g., ViraferonPeg or Introna).

23- The method of claim 21 or 22, wherein vonafexor or a pharmaceutically acceptable salt thereof is to be administered at a dose in the range from 25 to 800 mg per day or from 100 to 600 mg per day or 200 to 400 mg per day.

24- The method of any one of claims 21 to 23, wherein vonafexor or a pharmaceutically acceptable salt thereof and the pegylated IFN-a are to be administered during a period of time from 5, 6, 7 or 8 weeks to 52 weeks.

25- The method of any one of claims 21 to 23, wherein the method further comprises administering a therapeutically effective of at least one additional active ingredient, preferably selected in the group consisting of a nucleoside analog, a nucleic acid polymer, a NTCP inhibitor or a farnesyl transferase inhibitor, preferably in the group consisting of ribavirin, ritonavir, lonafarnib, EBP 921, lamivudine, adefovir, telbivudine, entecavir, tenofovir, emtricitabine, ezetimibe, myrcludex B, nucleic acid polymer REP 2139 and nucleic acid polymer REP 2165.