EA039949B1 - Methods for the treatment of hepatitis b and hepatitis d infections - Google Patents

Abstract

It is disclosed a method for the treatment of HBV infection or HBV/HDV co-infection, the method comprising administering to a subject in need of such treatment a first pharmaceutically acceptable agent that removes the hepatitis B surface antigen from the blood and a second pharmaceutically acceptable agent which stimulates immune function.

Description

The technical field to which the invention belongs

The description relates to methods of treating a subject infected with hepatitis B virus or co-infected with hepatitis B virus/hepatitis D; the treatment comprises a first pharmaceutically acceptable agent that removes hepatitis B surface antigen from the blood and a second pharmaceutically acceptable immunotherapeutic agent that stimulates immune function.

State of the art

Hepatitis B virus (HBV) is estimated to infect 400 million people worldwide and cause 600,000 deaths each year as a result of complications arising from HBV infection. Although several antiviral treatments have been approved for use, none have been able to elicit a therapeutically effective immune response capable of providing reliable infection control except in a small proportion of treated patients. In fact, there is a clear unmet medical need for a treatment regimen that can establish reliable immunological control of HBV infection in a large proportion of patients receiving this treatment.

HBV infection results in the production of two different particles: 1) the HBV virus itself (or Dane particles) containing a viral capsid assembled from the HBV core antigen (HBcAg) protein coated with the hepatitis B surface antigen (HBsAg) and capable of re-infecting cells and 2) subviral particles (or SVPs), which are high-density lipoprotein-like particles composed of lipids, cholesterol, cholesterol esters, and small to medium forms of hepatitis B surface antigen (HBsAg), that are non-infectious. For every viral particle produced, 1000-10000 SVPs are released into the blood. In fact, SVPs (and the HBsAg protein they carry) represent the vast majority of viral protein in the blood. In addition, HBV-infected cells secrete a soluble proteolytic product of the pre-core protein called HBV e-antigen (HBeAg).

Hepatitis D virus (HDV) uses HBsAg to form its viral structure (Taylor, 2006, Virology, 344: 71-76) and, in fact, HDV infection can be observed in subjects co-infected with HBV. In countries with a low incidence of HBV infection, the incidence of HDV coinfection in asymptomatic HBV carriers and in patients with chronic liver disease associated with HBV is low, whereas in countries with a high incidence of HBV infection, HDV coinfection is a significant complication in HBV-infected individuals. subjects and may increase the rate of disease progression to fulminant hepatitis. As such, there is a clear unmet medical need for methods of treating HBV infection, which is even more urgent in the case of HBV/HDV co-infected subjects.

Current standard treatments for HBV include immunotherapy based on interferon or thymosin α1 and suppression of viral production by inhibiting HBV polymerase. HBV polymerase inhibitors are effective in reducing viral production, but do little to rapidly reduce HBsAg or may slowly decrease HBsAg over long-term treatment in a limited number of patients (as with tenofovir disoproxil fumarate). Interferon-based immunotherapy can reduce both viral production and early clearance of HBsAg from the blood, but only in a small percentage of treated subjects. The generally accepted role of HBsAg in the blood is to block anti-HBsAg antibodies and allow infectious viral particles to escape detection by the immune system, which is probably one of the reasons why HBV infection persists in a chronic condition. In addition, HBsAg, HBeAg, and HBcAg all have immuno-inhibitory properties, as discussed below, and the persistence of these viral proteins in the blood of patients after administration of any of the currently available HBV treatments, as described below, is likely to have a significant impact, preventing the achievement of immunological control of HBV infection in patients.

Although all three primary HBV proteins (HBsAg, HBeAg, and HBcAg) have immuno-inhibitory properties (see below), HBsAg makes up the vast majority of the HBV protein in the circulation of HBV-infected subjects. In addition, although removal (by seroconversion) of HBeAg or reduction in serum viremia does not correlate with development of sustained control of HBV infection without treatment, removal of serum HBsAg from the blood (and seroconversion) in HBV infection is a well-known excellent predictor of antiviral response. treatment that will control HBV infection without treatment (although this is only seen in a small proportion of patients receiving immunotherapy). Thus, while reduction of all three major HBV proteins (HBsAg, HBeAg, and HBcAg) may result in optimal reversal of inhibitory effects, removal of HBsAg alone is sufficient to reverse major viral inhibition of immune function in subjects with HBV infection.

Therefore, in the absence of currently a treatment regimen that can restore immunological control of HBV in a large proportion of patients, it is necessary to provide an effective treatment for HBV infection and HBV/HDV co-infection that can restore immunological control in the majority of patients.

- 1 039949

Disclosure of invention

As described herein, a method is provided for treating an HBV infection or HBV/HDV co-infection in a subject in need of such treatment, comprising administering a first pharmaceutically acceptable agent that eliminates HBsAg from the blood of an HBV-infected host and a second pharmaceutically acceptable immunotherapeutic agent that stimulates immune function. .

Also provided is a method for treating HBV infection or HBV/HDV co-infection in a subject in need of such treatment, comprising administering a first pharmaceutically acceptable agent that inhibits the release of HBsAg from infected cells and a second pharmaceutically acceptable immunotherapeutic agent that stimulates immune function.

Also provided is a method for treating HBV infection or HBV/HDV co-infection in a subject in need of such treatment, comprising administering an effective dosing regimen of a first pharmaceutically acceptable agent that inhibits the release of HBV subviral particles from infected cells, and an effective dosing regimen of a second pharmaceutically acceptable immunotherapeutic agent that stimulates immune function.

Also provided is a method for treating HBV infection or HBV/HDV co-infection in a subject in need of such treatment, comprising administering a first pharmaceutically acceptable agent that inhibits the formation of HBV subviral particles in infected cells and a second pharmaceutically acceptable immunotherapeutic agent that stimulates immune function.

Also provided is a method for treating HBV infection or HBV/HDV co-infection in a subject in need of such treatment, comprising administering a first pharmaceutically acceptable agent that inhibits the synthesis or lowers the intracellular concentration of HBsAg in infected cells, and a second pharmaceutically acceptable immunotherapeutic agent that stimulates immune function. .

Also provided is a method for treating HBV infection or HBV/HDV co-infection in a subject in need of such treatment, comprising administering the first and second pharmaceutically acceptable agents described above together in one pharmaceutical composition or in two different pharmaceutical compositions administered with the same way.

Also provided is a method for treating HBV infection or HBV/HDV co-infection in a subject in need of such treatment, comprising simultaneous administration to the patient of the first and second pharmaceutical agents described above, which are administered in the same way or in different ways.

In accordance with the present description, the use of a first pharmaceutically acceptable agent that removes hepatitis B surface antigen from the blood, in combination with a second pharmaceutically acceptable immunotherapeutic agent that stimulates immune function, is provided for the treatment of infection with hepatitis B virus or coinfection with hepatitis B/hepatitis D viruses.

Also provided is the use of a first pharmaceutically acceptable agent that inhibits the release of HBsAg from infected cells and a second pharmaceutically acceptable immunotherapeutic agent that stimulates immune function for the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection.

Also provided is the use of a first pharmaceutically acceptable agent that inhibits the release of HBV subviral particles from infected cells and a second pharmaceutically acceptable immunotherapeutic agent that stimulates immune function for the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection.

Also provided is the use of a first pharmaceutically acceptable agent that inhibits the formation of HBV subviral particles in infected cells and a second pharmaceutically acceptable immunotherapeutic agent that stimulates immune function for the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection.

Also provided is the use of a first pharmaceutically acceptable agent that inhibits the synthesis or lowers the intracellular concentration of HBsAg in infected cells, and a second pharmaceutically acceptable immunotherapeutic agent that stimulates immune function for the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus co-infection.

Also proposed is the use of a first pharmaceutically acceptable agent that removes hepatitis B surface antigen from the blood, in combination with a second pharmaceutically acceptable immunotherapeutic agent that stimulates immune function, in the manufacture of a medicament intended for the treatment of infection with hepatitis B virus or coinfection with hepatitis B/hepatitis D viruses.

The present disclosure also provides a composition for treating hepatitis B virus infection or hepatitis B/hepatitis D co-infection, comprising an effective dose of a first pharmaceutically acceptable agent that removes hepatitis B surface antigen from the blood, and an effective dose of a second pharmaceutically acceptable immunotherapeutic agent,

- 2 039949 stimulating immune function.

In one embodiment, an agent that eliminates hepatitis B surface antigen from the blood inhibits the formation of HBV subviral particles.

In another embodiment, an agent that eliminates the surface antigen of the hepatitis virus

In blood, inhibits intracellular transport of HBV subviral particles.

In another embodiment, an agent that eliminates hepatitis B surface antigen from the blood inhibits the release of HBV subviral particles into the blood.

In another embodiment, an agent that eliminates hepatitis B surface antigen from the blood inhibits the release of hepatitis B surface antigen from an infected cell.

In another embodiment, the agent that eliminates the hepatitis B surface antigen from the blood inhibits the synthesis of HBsAg and/or other viral protein.

In another embodiment, an agent that clears the hepatitis B surface antigen from the blood inhibits the synthesis or function of apolipoprotein H.

In another embodiment, the agent that eliminates the hepatitis B surface antigen from the blood is a low molecular weight (small molecule) agent.

In another embodiment, the agent that eliminates the hepatitis B surface antigen from the blood is a nucleic acid polymer containing a 20-120 nucleotide long phosphorothioate oligonucleotide containing repeats of the AC sequence.

In another embodiment, the agent that eliminates the hepatitis B surface antigen from the blood is a nucleic acid polymer containing a 20-120 nucleotide long phosphorothioate oligonucleotide containing repeats of the CA sequence.

In another embodiment, the agent that eliminates the hepatitis B surface antigen from the blood is a nucleic acid polymer containing a 20-120 nucleotide long phosphorothioate oligonucleotide containing repeats of the TG sequence.

In another embodiment, the agent that eliminates the hepatitis B surface antigen from the blood is a nucleic acid polymer containing a 20-120 nucleotide long phosphorothioate oligonucleotide containing repeats of the GT sequence.

In another embodiment, the nucleic acid polymer further comprises at least one 2' ribose modification.

In another embodiment, all riboses contained in the nucleic acid polymer have the 2' modification.

In another embodiment, the nucleic acid polymer further comprises at least one 2'-O-methylribose modification.

In another embodiment, all riboses contained in the nucleic acid polymer have the 2'-O-methyl modification.

In another embodiment, the nucleic acid polymer further comprises at least one 5'methylcytosine.

In another embodiment, all of the cytosines in the nucleic acid polymer are present as 5' methylcytosine.

In another embodiment, the nucleic acid polymer further comprises at least one 2' ribose modification and at least one 5' methylcytosine.

In another embodiment, all riboses contained in the nucleic acid polymer are in the 2'-O-methyl modification and all cytosines are present as 5' methylcytosine.

In another embodiment, an agent that eliminates the surface antigen of the hepatitis virus

B from blood is an oligonucleotide selected from the group consisting of SEQ ID NOs: 1-10.

In another embodiment, the agent that eliminates hepatitis B surface antigen from the blood is an oligonucleotide chelate complex containing an oligonucleotide selected from the group consisting of SEQ ID NOs: 1-10.

In another embodiment, the agent that eliminates the hepatitis B surface antigen from the blood is an oligonucleotide consisting of SEQ ID NO: 2.

In another embodiment, the agent that eliminates the hepatitis B surface antigen from the blood is an oligonucleotide chelate complex containing SEQ ID NO: 2.

In another embodiment, the agent that eliminates the hepatitis B surface antigen from the blood is an oligonucleotide consisting of SEQ ID NO: 3.

In another embodiment, the agent that eliminates the hepatitis B surface antigen from the blood is an oligonucleotide chelate complex containing an oligonucleotide selected from the group consisting of SEQ ID NO: 3.

In another embodiment, the agent that eliminates the hepatitis B surface antigen from the blood is an oligonucleotide consisting of SEQ ID NO: 10.

In another embodiment, the agent that eliminates the hepatitis B surface antigen from the blood is an oligonucleotide chelate complex containing an oligonucleotide,

- 3 039949 selected from the group consisting of SEQ ID NO: 10.

In another embodiment, an agent that eliminates the surface antigen of the hepatitis virus

B from the blood, is an antisense oligonucleotide that targets any site of any

HBV mRNA.

In another embodiment, the agent that eliminates the hepatitis B surface antigen from the blood is an antisense oligonucleotide that targets any region of the human apolipoprotein H mRNA.

In another embodiment, the agent that eliminates the hepatitis B surface antigen from the blood is a siRNA that targets any region of any HBV mRNA.

In another embodiment, the agent that eliminates the hepatitis B surface antigen from the blood is a siRNA that targets any region of the human apolipoprotein H mRNA.

In another embodiment, the agent that clears the hepatitis B surface antigen from the blood is an shRNA targeting any region of any HBV mRNA or human apolipoprotein H mRNA.

In another embodiment, the nucleic acid polymer, antisense oligonucleotide, or siRNA is designed as an oligonucleotide chelate complex.

In another embodiment, the agent that eliminates HBV surface antigen from the blood is an oligonucleotide aptamer or Spiegelmer targeted to HBV surface antigen.

In another embodiment, the agent that clears the hepatitis B surface antigen from the blood is an oligonucleotide aptamer or spiegelmer targeted to apolipoprotein H.

In another embodiment, the agent that eliminates hepatitis B surface antigen from the blood is an antibody or antibody fragment that recognizes hepatitis B surface antigen.

In another embodiment, the immunotherapeutic agent that stimulates immune function contains one or more compounds selected from the group consisting of thymosin α1;

any α-interferon or its pegylated derivatives;

any β-interferon or its pegylated derivatives;

any γ-interferon or its pegylated derivatives;

any λ-interferon or its pegylated derivatives;

interferon a-2a or a-2b or a-N3;

interferon b-1a or e-1b;

interferon γ-10;

interferon λ1 or λ2 or λ3;

pegylated interferon a-2a or a-2b or λ1 or λ2;

any antiviral cytokine or its pegylated derivatives;

thymus protein A;

any polypeptide having antiviral activity or immunostimulatory activity;

an immunostimulatory non-CpG oligonucleotide, including IMO-2055 or IMO-2125;

a small molecule Toll-like receptor (TLR) agonist, including GS-9620 or ANA-773; and any antiviral or immunostimulatory hormone, including DHEA or its metabolites.

In another embodiment, the immunotherapeutic agent that stimulates immune function contains one or more compounds selected from the group consisting of an immunostimulatory oligonucleotide, including IMO-2055 or IMO-2125;

a small molecule Toll-like receptor (TLR) agonist, including GS-9620 or ANA-773; and any antiviral or immunostimulatory hormone, including DHEA or its metabolites.

In a further embodiment, the first and second pharmaceutically acceptable agents are formulated in the same pharmaceutical composition.

In a further embodiment, the first and second pharmaceutically acceptable agents are formulated into separate pharmaceutical compositions.

In a further embodiment, the first and second agents are intended to be administered simultaneously.

In a further embodiment, the first and second agents are intended to be administered by different routes.

In a further embodiment, the first and second agents are formulated for use by one or more of the following: oral ingestion, aerosol inhalation, subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection, and intravenous infusion.

In an additional embodiment, the agent that eliminates HBsAg from the blood contains one or more molecules selected from the group consisting of an antibody or antibody fragment that binds to the surface antigen of the hepatitis B virus;

triazolopyridine derivatives listed below

5-(4-chlorophenyl)-7-(2,6-difluorophenyl)-4,7-dihydro[1,2,4]triazolo[1,5-a]pyrimidine; oligonucleotide selected from among

SEQ ID NO: 2;

SEQ ID NO: 3;

SEQ ID NO: 10;

SEQ ID NOs: 1 and 4-9;

nucleic acid polymer selected from among

thiophosphate oligonucleotide, 20-120 nucleotides long, containing repeats follow- AC power; thiophosphate oligonucleotide, 20-120 nucleotides long, containing repeats follow- the validity of SA; thiophosphate oligonucleotide, 20-120 nucleotides long, containing repeats follow- TG; and thiophosphate oligonucleotide, 20-120 nucleotides long, containing repeats follow-

the validity of the GT;

an antisense oligonucleotide targeting any portion of any HBV mRNA;

an antisense oligonucleotide targeting any portion of the human apolipoprotein H mRNA;

siRNA targeting any site on any HBV mRNA;

siRNA targeting any region of human apolipoprotein H mRNA;

shRNA targeting any site on any HBV mRNA;

shRNA targeting any region of human apolipoprotein H mRNA;

a spiegelmer or aptamer targeting the hepatitis B surface antigen; and a spiegelmer or aptamer targeting human apolipoprotein H;

and the second immunotherapeutic agent contains one or more molecules from the group consisting of thymosin α1;

any α-interferon or its pegylated derivatives;

any β-interferon or its pegylated derivatives;

any γ-interferon or its pegylated derivatives;

any λ-interferon or its pegylated derivatives;

interferon a-2a or a-2b or a-N3;

interferon b-1a or β-16;

interferon γ-16;

interferon λ1 or λ2 or λ3;

pegylated interferon a-2a or α-2b or λ1 or λ2;

any antiviral cytokine or its pegylated derivatives;

thymus protein A;

- 5 039949 any polypeptide with antiviral activity or immunostimulatory activity;

immunostimulatory oligonucleotide, including IMO-2055 or IMO-2125;

a small molecule Toll-like receptor (TLR) agonist, including GS-9620 or ANA-773; And any antiviral or immune-stimulating hormone, including DHEA, or its metabolites.

In addition, in a further embodiment, the following oligonucleotides may be formulated as an oligonucleotide chelate complex:

SEQ ID NO: 2;

SEQ ID NO: 3;

SEQ ID NO: 10;

SEQ ID NOs: 1 and 4-9;

The nucleic acid polymer is selected from the following:

a thiophosphate oligonucleotide 20-120 nucleotides long containing repeats of the AC sequence;

a thiophosphate oligonucleotide 20-120 nucleotides long containing repeats of the CA sequence;

a thiophosphate oligonucleotide 20-120 nucleotides long containing repeats of the TG sequence and a thiophosphate oligonucleotide 20-120 nucleotides long containing repeats of the GT sequence;

an antisense oligonucleotide targeting any portion of any HBV mRNA;

an antisense oligonucleotide targeting any portion of the human apolipoprotein H mRNA;

siRNA targeting any portion of any HBV mRNA; and siRNA targeting any part of human apolipoprotein H mRNA.

In another embodiment, the uses or methods of treatment described above further comprise the administration or concomitant use of a third pharmaceutically acceptable agent selected from the following:

tenofovir zizoproxil fumarate;

entecavir;

telbuvidin;

adefovir dipivoxil; and lamivudine.

Also provided is a method of treatment or use of those listed in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that comprises SEQ ID NO: 2 and a second pharmaceutically acceptable agent , which contains pegylated interferon a-2a.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D co-infection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that contains an oligonucleotide chelate complex of SEQ ID NO: 2 and a second pharmaceutically acceptable agent. an acceptable agent that contains pegylated interferon a-2a.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that comprises SEQ ID NO: 3 and a second pharmaceutically acceptable agent, which contains pegylated interferon a-2a.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that contains an oligonucleotide chelate complex of SEQ ID NO: 3 and a second pharmaceutically acceptable agent. an acceptable agent that contains pegylated interferon a-2a.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that comprises SEQ ID NO: 10 and a second pharmaceutically acceptable agent, which contains pegylated interferon a-2a.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that contains an oligonucleotide chelate complex of SEQ ID NO: 10 and a second pharmaceutically acceptable agent. an acceptable agent that contains pegylated interferon a-2a.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D co-infection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that comprises SEQ ID NO: 2 and a second pharmaceutically acceptable agent, containing thymosin α1.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D co-infection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that contains an oligonucleotide chelate complex of SEQ ID NO: 2 and a second pharmaceutically acceptable agent. an acceptable agent containing thymosin α1.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that comprises SEQ ID NO: 3 and a second pharmaceutically acceptable agent, containing thymosin α1.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that contains an oligonucleotide chelate complex of SEQ ID NO: 3 and a second pharmaceutically acceptable agent. an acceptable agent containing thymosin α1.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that comprises SEQ ID NO: 10 and a second pharmaceutically acceptable agent, containing thymosin a1a.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that contains an oligonucleotide chelate complex of SEQ ID NO: 10 and a second pharmaceutically acceptable agent. an acceptable agent containing thymosin α1.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D co-infection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that comprises SEQ ID NO: 2 and a second pharmaceutically acceptable agent, containing interferon a-2b.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D co-infection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that contains an oligonucleotide chelate complex of SEQ ID NO: 2 and a second pharmaceutically acceptable agent. an acceptable agent containing interferon a-2b.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that comprises SEQ ID NO: 3 and a second pharmaceutically acceptable agent, containing interferon a-2b.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that contains an oligonucleotide chelate complex of SEQ ID NO: 3 and a second pharmaceutically acceptable agent. an acceptable agent containing interferon a-2b.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that comprises SEQ ID NO: 10 and a second pharmaceutically acceptable agent, containing interferon a-2b.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that contains an oligonucleotide chelate complex of SEQ ID NO: 10 and a second pharmaceutically acceptable agent. an acceptable agent containing interferon a-2b.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D co-infection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that comprises SEQ ID NO: 2 and a second pharmaceutically acceptable agent, containing interferon λ1.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of

- 7 039949 in such treatment, the first pharmaceutically acceptable agent, which is the oligonucleotide chelate complex of SEQ ID NO: 2, and the second pharmaceutically acceptable agent, containing pegylated interferon λ1.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that comprises SEQ ID NO: 3 and a second pharmaceutically acceptable agent, containing pegylated interferon λ1.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that contains an oligonucleotide chelate complex of SEQ ID NO: 3 and a second pharmaceutically acceptable agent. an acceptable agent containing pegylated interferon λ1.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that comprises SEQ ID NO: 10 and a second pharmaceutically acceptable agent, containing pegylated interferon λ1.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that contains an oligonucleotide chelate complex of SEQ ID NO: 10 and a second pharmaceutically acceptable agent. an acceptable agent containing pegylated interferon λ1.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D co-infection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that comprises SEQ ID NO: 2 and a second pharmaceutically acceptable agent, containing GS-9620.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D co-infection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that contains an oligonucleotide chelate complex of SEQ ID NO: 2 and a second pharmaceutically acceptable agent. an acceptable product containing GS-9620.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that comprises SEQ ID NO: 3 and a second pharmaceutically acceptable agent, containing GS-9620.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that contains an oligonucleotide chelate complex of SEQ ID NO: 3 and a second pharmaceutically acceptable agent. an acceptable product containing GS-9620.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that comprises SEQ ID NO: 10 and a second pharmaceutically acceptable agent, containing GS-9620.

Also provided is a method of treatment or use in the treatment of hepatitis B virus infection or hepatitis B/hepatitis D virus coinfection, which comprises administering to a patient in need of such treatment a first pharmaceutically acceptable agent that contains an oligonucleotide chelate complex of SEQ ID NO: 10 and a second pharmaceutically acceptable agent. an acceptable product containing GS-9620.

Brief description of the drawings

Fig. 1 represents two triazolopyrimidine derivatives blocking HBsAg release from the HBV producing cell line HepG2.2.15 as described in Yu et al., 2011, J. Med. Chem. 54: 56605670;

fig. 1A - main potential compound 1a (PBHBV-001); and fig. 1B - the main potential compound 3c (PBHBV-2-15) established in this work.

Implementation of the invention

HBsAg plays a key role in HBV infection and HBV/HDV coinfection. In addition to its role as a major structural component for virion formation, HBsAg is also released in large quantities into the blood of infected subjects in the form of subviral particles (SVPs), which lack the viral capsid and genome, and whose function appears to be mainly delivery of HBsAg to the blood. SVPs are secreted from infected cells at 1000-10000 times the secretion of the virus, which allows SVPs to effectively block HBsAg antibodies (anti-HBs), so that the HBV or HDV virus in the blood can avoid recognition by the adaptive immune system. Although some studies have shown that the action of HBsAg may also be associated with a direct blocking of the activation of the adaptive and innate immune responses to HBV infection (Cheng et al., 2005, Journal of hepatology, 43:4 65-471; Op den Brouw et al., 2009 , Immunology, 126: 280-289, Vanlandschoot et al., 2002, The Journal of general virology, 83: 1281-1289, Wu et al. Molecular immunology, 46: 2640-2646), the presence of this functionality in HBV infection and HBV/HDV coinfection in humans and its effect on the activity of immunotherapeutic agents has not been studied or established. HBeAg and HBcAg have also been shown to have immunoinhibitory properties (Kanda et al., 2012 J. Inf. Dis. 206: 415-420; Lang et al., 2011 J. Hepatol. 55: 762-769; Gruffaz et al. 2013 , J. Hepatol 58 (suppl), p sl55, Abstract 378).

In addition to the recognized activity of excess HBsAg in the blood (as SVPs) to isolate anti-HB, the ability of HBsAg to block cytokine signals in some in vitro and in vivo systems indicates that these immunoinhibitory properties of HBsAg may also be present in HBV infection and HBV/HDV coinfection in humans. Due to the large excess of HBsAg in the blood of infected patients, there is likely to be a real deterioration in many signaling mechanisms important for the optimal functioning of the immune system (both adaptive and innate). In addition, the new findings presented herein confirm for the first time that many of these signaling mechanisms are also essential for the full realization of the effects of immunotherapeutic agents. In addition, these disclosures for the first time justify the essential role of circulating HBsAg in inhibiting the action of immunotherapeutic agents.

This document contains a demonstration of an effective treatment for HBV infection and HBV/HDV co-infection, which includes the first pharmaceutically acceptable agent capable of eliminating HBsAg from the blood, and an immunotherapeutic agent that stimulates immune function. This combined treatment allows circulating anti-HBsAg antibodies to directly attack the circulating virus and virus-producing cells, which in the absence of the immuno-inhibitory properties of HBsAg leads to a significant improvement in the effect of immunotherapy, which in turn leads to a significant increase in the proportion of patients achieving immunological control of HBV infection than with immunotherapy alone.

This document provides a new demonstration that, in addition to its previously described ability to block cytokine signals in vitro, circulating HBsAg unexpectedly also directly inhibits the function of HBV-approved immunotherapy in addition to suppressing the host's immune response against HBV infection. Thus, a therapy that combines a first agent capable of clearing HBsAg from the blood and a second agent that stimulates immune function results in a first shown synergistic effect between the two agents, which significantly improves the impact associated with restoring immunological control of HBV infection. .

This document provides results from human patients showing that removal of HBsAg (and other HBV antigens) from the blood of chronic HBV patients (using nucleic acid polymers or NAPs) can provide some measure of immunological recovery, however this level of immunological reactivation is not sufficient to establish reliable control in a large proportion of patients. These data clearly show that it is not expected that any other pharmaceutically acceptable agent or method that reduces or removes HBsAg (or additionally other HBV proteins) in the blood of infected patients can achieve a better treatment outcome than , which is achieved using NAP.

This document further provides results in human patients showing for the first time that the presence of HBsAg in the blood of patients with HBV infection inhibits the biochemical activity of immunotherapeutic agents like thymosin α1 or pegylated interferon a-2a. Using NAP REP 2139, HBsAg in the blood of patients with HBV infection was removed prior to treatment with thymosin α1 or pegylated interferon a-2a. In an HbsAg-negative environment, treatment with either of these two immunotherapies resulted in a significant and unexpected synergism in the activation of the immunological response (as measured by the production of anti-HBsAg antibodies in the blood), with the response being significantly stronger and much faster than the response with use of these immunotherapeutic agents in monotherapy. Most importantly, the removal of HBsAg from the blood of these patients allowed for these very strong responses to immunotherapy in the majority of patients. Any type of positive immunological response is rare in patients treated with immunotherapeutic agents as monotherapy.

These results also demonstrate for the first time that the removal of HBsAg from the blood of patients with HBV infection or HBV/HDV infection will have a synergistic effect on the ability of any immunotherapeutic agent to induce a stronger immunological response in pain.

- 9 039949 most or all patients receiving immunotherapy, with a shorter treatment regimen than usually used. These results clearly indicate to one of skill in the art that any method or pharmaceutically acceptable agent that removes HbsAg from the blood of HBV-infected or HBV/HDV co-infected patients can be expected to have a similar beneficial and synergistic effect in improving the biochemical activity of any pharmaceutical drug. acceptable immunotherapeutic agent. This improvement in the activity of the immunotherapeutic agent can be realized when reduction or removal of HBsAg has been achieved prior to immunotherapy or concurrently with immunotherapy, or when reduction or removal of HBsAg has been achieved after immunotherapy that has been started previously and is ongoing.

The discovery of a significant synergistic antiviral effect in patients treated with a pharmaceutically acceptable agent that clears HBsAg from the blood (by any means) in combination with a pharmaceutically acceptable agent that stimulates immune function represents a new approach to achieve a significantly improved antiviral response using available immunotherapy, which is not was predicted and not reported in the prior art.

The term oligonucleotide (ON) refers to an oligomer or polymer of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA). The term includes ONs containing modified nucleotide bases (including 5' methylcytosine and 4' thiouracil), sugars, and covalent internucleoside (backbone) bonds, as well as ONs having non-naturally occurring sites with a similar function.

Such modified or substituted ONs may be preferred over native forms due to desirable properties such as, for example, reduced immunoreactivity, increased cellular uptake, increased affinity for the nucleic acid target (in the context of antisense ONs, siRNA and shRNA), and/or increased stability to nuclease mediated degradation. ONs can also be double stranded. ONs also include single-stranded molecules such as antisense oligonucleotides, spiegelmers and aptamers, as well as double-stranded molecules such as small interfering RNA (siRNA) or small hairpin RNA (shRNA).

The ON may contain various modifications, such as stabilizing modifications, and thus may contain at least one phosphodiester bond and/or sugar and/or base modification. For example, ON may contain, without limitation, one or more modifications, or may be completely modified so that all bonds or sugars or bases contain the listed modifications. Modified bonds may include thiophosphate bonds, dithiophosphate bonds and/or methylphosphonate bonds. Because modified linkages are used, ONs may contain phosphodiester linkages. Additional suitable modifications include, without limitation, modifications at the 2' position of the sugar, including 2'-O-alkyl modifications such as 2'-O-methyl modifications, 2'O-methoxyethyl (2' MOE), 2'-amino modifications, 2'-halo modifications such as 2'-fluoro; acyclic nucleotide analogs. Other 2' modifications, such as locked nucleic acids, are also known in the art and can be used. In particular, ON has modified bonds everywhere or each of its bonds is modified, for example, thiophosphate; has a 3'- and/or 5'-cap; includes a terminal 3'-5' bond; ON is or includes a concatemer consisting of two or more ON sequences joined by linker(s). Base modifications may include 5' methylation of the cytosine base (5' methylcytosine or in the case of the nucleotide 5' methylcytidine) and/or 4' thionation of the uracil base (4' thiouracil or in the case of the nucleotide 4' thiouridine). Where the synthesis conditions are chemically compatible, various chemically compatible modified linkages can be combined, for example, a thiophosphate linked oligonucleotide, a 2' ribose modification (such as 2'O-methylation), and a modified base (such as 5' methylcytosine). The ON can be completely modified with all of these various modifications (eg every bond is thiophosphate, every 2' ribose and every base is modified).

As used herein, the term nucleic acid polymer or NAP defines any single-stranded ON that does not contain a sequence with special functionality, designed to either hybridize to a nucleic acid target or adapt a specific secondary structure of the sequence, resulting in binding to a specific protein. The biochemical activity of NAP is independent of ON recognition at a Toll-like receptor, hybridization to a target nucleic acid, or aptamer interaction requiring a specific secondary/tertiary ON structure resulting from the particular order of nucleotides present. NAPs may include a base or bond and/or sugar modifications as described above. Examples of NAP compounds include a 20-120 nucleotide long phosphorothioate oligonucleotide containing repeats of the AC sequence;

phosphorothioate oligonucleotide 20-120 nucleotides long containing repeats of the CA sequence;

- 10 039949 phosphorothioate oligonucleotide, body length TG;

phosphorothioate oligonucleotide, length GT;

phosphorothioate oligonucleotide, length UG;

phosphorothioate oligonucleotide, length

20-120 nucleotides containing sequence repeats

20-120 nucleotides containing sequence repeats

20-120 nucleotides containing sequence repeats

20-120 nucleotides containing sequence repeats; and

SEQ ID NO: 1-10.

ON chelate complexes are two or more ONs intermolecularly linked via a divalent or multivalent metal cation. ON chelate complexes neutralize the intrinsic chelating properties of ON, which may contribute to the side effects associated with the administration of these compounds. The administration of ON chelate complexes is a method of administering ON to a subject that reduces administration-related side effects due to non-chelated ONs (which are ONs administered as sodium salts, as commonly used in the art). These side effects may include tremor, fever, and chills when administered intravenously, or induration, inflammation, and pain at the injection site when injected subcutaneously. In addition, by preparing ONs as chelates, their pharmacokinetic behavior can be improved, resulting in increased therapeutic activity at the same dosage compared to non-chelated ONs, as described in International Publication No. WO 2012/021985 and US Publication No. 2012/ 0046348, which are incorporated herein by reference in their entirety. The introduction of ON chelate complexes does not interfere with the biochemical activity of ON during normal use in the form of sodium salts. Thus, any antisense ON, siRNA or NAP described in this work can optionally be obtained as an ON chelate complex without affecting their biochemical activity.

ON chelate complexes may contain various multivalent metal cations, including calcium, magnesium, cobalt, iron, manganese, barium, nickel, copper, zinc, cadmium, mercury, and lead. This further shows that chelation of these multivalent metal cations results in the formation of ON chelate complexes consisting of two or more ONs linked via metal cations and occurs in ONs longer than 6 nucleotides and in the presence of ONs or with phosphodiester or thiophosphate bonds. Optionally, each ON bond may be phosphorothioate. Chelation can occur at ONs containing 2' modifications (such as 2'O methyl) of ribose, or containing modified bases such as 5' methylcytosine or 4-thiouracil. These 2' modifications may be present on one or more or all of the riboses, and the modified bases may be present on one or more bases, or present ubiquitously on each base (ie, all cytosines are present as 5' methylcytosine). In addition, ON chelate complexes may include ONs containing several modifications, such that each bond is thiophosphate, each 2' ribose, and each base is modified. ON modifications compatible with the formation of an ON chelate complex are specified above. Moreover, chelation of metal cations does not depend on the sequence of the nucleotides present, but instead depends on the physiochemical properties that are common to all ONs.

Although ON chelation can be achieved using any divalent metal cation, ON chelates for drug use should preferably only contain calcium and/or magnesium, but may also contain trace amounts of iron, manganese, copper or zinc, but must not include cobalt, barium, nickel, cadmium, mercury, lead, or any other divalent metal not listed here.

ONs can exert their therapeutic effects through multiple mechanisms that are sequence dependent or sequence independent. Sequence dependent mechanisms are those that require a specific nucleotide sequence for their activity, and in case the activity is reduced by one or more changes in the nucleotide sequence present. This specific sequence may include the full length of ON or only a portion of it (sequence motif). Examples of sequence dependent ONs include:

1) Antisense ONs or single-stranded or double-stranded (e.g., synthetic interfering RNA (siRNA) or small hairpin RNA (shRNA)) are designed to target a specific site of interest in messenger RNA (mRNA) or micro RNA (miRNA) through specific hybridization between antisense ON and the sequence at the target site of the mRNA of interest. Introduction of antisense ONs into a cell results in the formation of a dual site on or with miRNA mRNA that determines the degradation of that specific mRNA or miRNA by RNAse H. When siRNA is introduced into a cell (or shRNA is expressed in a cell), the antisense strand ) integrates into RISC (RNA-induced silencing complex), which uses guide-chain-directed hybridization

- 11 039949 a complementary region on the target mRNA to cleave it with a RISC catalytic component called Argonaute;

2) steric blocking ONs are single-stranded antisense ONs complementary to a specific region of mRNA or immature mRNA, which are created in such a way as not to activate RNase H, or by incorporating a 2' modification of each ribose, this modification is known to prevent the action of RNase H, or by using chemically modified ONs (such as morpholino ONs) that are not recognized by RNase H. Hybridization of these ONs to their target mRNAs results in a double-stranded region that provides steric hindrance to proteins normally targeting RNA (such as splicing proteins or ribosomes). ). Such ONs can be used to block translation of a particular mRNA or to modify post-transcriptional splicing and maturation of a particular mRNA;

3) aptamers are ONs that adopt a specific three-dimensional conformation that is capable of interacting with specific proteins and interacts with host DNA or RNA with difficulty. Aptamers can also include spiegelmers, which use L nucleotides instead of D nucleotides to confer high enzyme resistance on the ON;

4) immunostimulatory ONs contain specific modifications that lead to binding to or activation of toll-like receptors 7, 8, and 9 through a mechanism mediated by a non-CpG motif and are capable of stimulating immune function (Kandimalla et al., 2011. Cell. Immunol. 270: 126-134; Struthers et al., 2010 Cell Immunol 263: 105-113).

When designing antisense ON, siRNA or shRNA, the sequence of these molecules is designed to be 100% complementary to the intended target sequence of the specific RNA within the following guidelines:

Antisense ONs are 15-25 nucleotides in length and contain sequences that are 100% complementary to the intended target sequence.

The guide strand siRNA contains one oligoribonucleotide, 19-21 nucleotides long, that is 100% complementary to the target region of the mRNA of interest, while the accompanying strand (the other strand in the duplex) contains the same length of the ribonucleotide sequence, which is 100 % complementary to the guide chain. Both the guide strand and the accompanying strand also have two additional deoxythymidine nucleotides at the 3' end of each strand.

shRNA molecules are produced by an expression vector, such as a plasmid or expression construct based on a virus (e.g., lentivirus or adenovirus), which produces a long RNA containing a guide and escort strand sequence (as described above for siRNA, but which can be 19-29 nucleotides long). ), in one contiguous oligonucleotide but separated by a short non-complementary oligonucleotide sequence designed to form a hairpin. Transcription of the RNA from this expression construct results in the formation of short hairpin RNA, which is processed by the dicer enzyme and introduced into the RISC as described above for siRNA.

As used herein, the term antiviral ON refers to any antisense ON, siRNA, shRNA, or NAP that, through its specific biochemical activity (sequence-dependent or sequence-independent), has the ability to directly or indirectly inhibit some aspect of viral replication, or directly or indirectly enhance the ability the host to eliminate the viral infection by immunological or other mechanisms.

In the present disclosure, the term ON chelate complex refers to a complex of two or more ONs in solution bound intermolecularly by a divalent metal cation as described in International Publication No. WO 2012/021985 and US Publication No. 2012/0046348, which are hereby incorporated in their entirety. by sending. ON chelate complexes can be formed with antisense ON, siRNA or NAP.

In the present disclosure, the term antiviral ON chelate complex refers to a complex of two or more antiviral ONs in solution bound intermolecularly by a multivalent metal cation.

Phosphorothioate NAPs are a new class of ON-based broad-spectrum antivirals (Bernstein et al., 2008, Antimicrobial Agents and Chemotherapy, 52: 2727-2733; Cardin et al., 2009, Virology Journal, 6: 214; Guzman et al. ., 2007, Antiviral Therapy, 12: 1147-1156; Lee et al., 2008, Virology, 372: 107-117; Matsumura et al., 2009, Gastroenterology, 137: 673-681; Vaillant et al., 2006, Antimicrobial Agents and Chemotherapy, 50: 1393-1401 and US Pat. Since SVPs make up >99.9% of HBsAg in the blood of HBV patients, blocking the formation and/or release of SVPs from infected hepatocytes with NAP is a highly effective way to remove HBsAg from the blood of HBV infected patients.

As described in example II, the removal of HBsAg from the blood of infected patients using NAP

- 12 039949 leads to a partial restoration of the immune response, which in turn eliminates the HBV antigen (HBeAg) from the blood, and a significant decrease in blood levels of the virus during treatment, however, in most patients this effect does not persist after stopping treatment. Since this partial restoration of the immune response (in the absence of HBsAg and other viral antigens) can lead to reliable immunological control of HBV infection in a small proportion of patients after treatment is stopped, it is not enough to achieve such control in the absence of treatment in the majority of treated patients. Thus, the approach of simply removing HBsAg from the blood by any method, or by any other pharmaceutically acceptable agent with a similar effect, will provide only some moderate level of immunological recovery, which will lead to the establishment of reliable control without treatment in a limited part of patients.

There is no publicly disclosed agent other than NAP that has the ability to rapidly remove HBsAg from the blood of HBV-infected human patients. However, there are several other methods other than the use of NAP and well known in the art that can be used to remove HBsAg from the blood. Such methods include (but are not limited to) the following.

A. Use of small molecules to target regions of the HBsAg protein or other viral or host factors involved in SVP formation to block SVP formation, block SVP transport by exploiting the infected cell's secretory apparatus, block the release of SVP from infected hepatocytes into the blood, or generally blocking the release of HBsAg from infected cells. The small molecules used in this approach may include triazolopyrimidine derivatives as described in Yu et al. (2011, J. Med Chem. 54: 56605670), and may include specific triazolopyrimidine derivatives as described in FIG. 1 which have been shown to block HBsAg release from HBV producing cell lines. Other small molecules that target the Apo H protein, which may be important for SVP production, can also be used (as described in Canadian Application No. 2746981).

B. Using an antisense ON approach that includes antisense oligonucleotides, siRNA or shRNA molecules to target specific mRNAs and thereby accelerate their degradation, inhibit HBsAg synthesis (i.e. catalyze the degradation of the mRNA that is used to produce HBsAg protein), or other viral or host factors involved in SVP formation (including Apo H as described in Canadian Application No. 2746981), transport of SVPs through the secretory apparatus of an infected cell, or release of SVP from infected hepatocytes into the blood. This antisense ON approach can also be used to hybridize with viral or host mRNAs required for the synthesis of proteins important for the formation, intracellular transport, or release of SVPs from infected hepatocytes, and to cause degradation of these mRNAs through the mechanisms described above. . In particular, some antisense-based approaches to HBV may be useful in the form of single antisense molecules, such as siRNA, which can be designed to interfere with all HBV mRNAs produced by the viral genome by hybridizing to a specific site on the HBV genome, causing degradation of all mRNAs produced by the HBV genome that simultaneously affect the synthesis of HBsAg, HBeAg, and HBcAg, as described in Fu et al. (2008, Acta Pharmacol. Sin. 29: 15221528). Two or more antisense molecules can also be used simultaneously or as separate molecules or as molecules produced by a single expression vector introduced into an infected host, as described by Snyder et al. (2008, Antiviral Res., 80: 36-44). Examples known in the art for antisense inhibition of HBV protein synthesis include:

a. Altritol-modified siRNA lipoplexes (Hean et al., 2010, Artificial DNA: PNA & XNA, 1: 17-26).

b. One or more siRNA sequences (Xin et al., 2008, World J. Gastroenterol., 14: 38493854; Zhe et al., 2005, J. Zhejiang Univ. Sci., 6B: 236-241 and reviewed in Chen et al. , 2008, Pharmaceutical Res., 25: 72-86).

c. One or more siRNA sequences and a polyconjugated system (ARC-520), http://www.arrowheadresearch.com/press-releases/arrowhead-research-advances-arc-520-ind-enabling-studiestreatrnent-hepatitis-b-and; http://www.arrowheadresearch.com/sites/default/files/press_releases/pdf/whitepaperhbv-3-7-12.pdf.

d. Locked nucleic acid modified antisense molecules (Sum et al., 2011, Biochem. Biophys. Res. Comm., 409: 430-435).

e. One or more shRNAs (Zhang et al., 2010, BMC Microbiol. 10:214; Starkey et al., 2009, J. Gen Virol. 90: 115-126 and reviewed in Chen et al., 2008, Pharmaceutical Res., 25:72-86).

C. Use of an ON-based aptamer (including classical aptamers or Spiegelmers) to target regions of HBsAg, HBeAg or HBcAg proteins or other viral or host factors (including Apo H) present in the circulation to accelerate their removal from the blood. Classical aptamers and spiegelmers can be further pegylated as described in

- 13 039949

Waters et al. 2011 Blood 117: 5514-5522 and Wlotzka et al. 2002 Proc. Nat. Acad. sci. U.S.A. 99: 8898-8902, in order to improve their stability and half-life from the circulation.

D. Use of an antibody-based approach targeting HbsAg directly to accelerate its removal from the blood.

As used herein, the term removal of HBsAg from the blood means any statistically significant reduction in blood HBsAg concentration relative to pre-treatment blood HbsAg concentrations, as determined by the Abbott Architect™ quantitative HbsAg assay. This serum HBsAg test is the accepted standard for measuring blood levels of HBsAg and is approved for use in human diagnosis.

Examples of ONs that may be used in the current disclosure are provided in Table 1. one.

Table 1. Examples of ONs that may be used in the current disclosure

ON class Nucleic acid type Sequence(5' - 3') Modifications NAP DNA (AC) ₂₀ (SEQ ID NO: 2) All links PS NAP DNA (CA) ₂₀ (SEQ ID NO: 1) All links PS NAP DNA (A-5'MeC) ₂₀ (SEQ ID NO: 3) All links PS NAP DNA (5'MeC-A) ₂₀ (SEQ ID NO: 4) All links PS NAP RNA (2'OMeA-2'OMeC) ₂₀ (SEQ ID NO: 5) All links PS NAP RNA (2OMeC-2'OMeA) ₂₀ (SEQ ID NO: 6) All links PS NAP DNA (TG) ₂₀ (SEQ ID NO: 7) All links PS NAP DNA (GT)20 (SEQ ID NO: 8) All links PS NAP RNA (2'OMe, 5'MeC-2'OMeA) ₂₀ (SEQ ID NO: 9) All links PS NAP RNA (2'OMeA-2'OMe, 5'MeC) ₂₀ (SEQ ID NO: 10) All links PS Antisense DNA/RNA Sequence is 100% complementary to viral or host mRNA (e.g. Apo H) or HBV mRNA All PS bonds, may contain 2' ribose or LNA modified RNA siRNA Double stranded RNA/DNA Contains a 100% complementary sequence viral or host mRNA (eg, Apo H) or HBV mRNA May contain 2' ribose modified RNA, may contain PS shRNA Double-stranded RNA produced by an expression vector Contains a 100% complementary sequence viral or host mRNA (eg, Apo H) or HBV mRNA RNA

PS - thiophosphate, 2'OMe - 2'O methyl, 5'MeC - 5'methylcytosine.

Typical effective dosing regimens for the various pharmaceutically acceptable agents described above that can be used to remove HBsAg from the blood are:

For all NAPs and phosphorothioate antisense oligonucleotides that degrade HBV or apoH mRNA, the art-established weekly parenteral administration of 100-500mg of the compound is commonly used to achieve therapeutically active levels of these compounds in the liver, as described for NAP in the examples below, and with respect to phosphorothioate antisense ON causing degradation of specific mRNAs in the liver (for B100 apolipoproteins), see Akdim et al. 2010 Journal of the American College of Cardiology 55: 1611-1618).

To achieve therapeutic levels of activity in the liver, siRNAs are typically encapsulated and dosed to degrade PCSK9 mRNA in the liver of human patients, as described in

- 14 039949 http://www. google.com/ur l?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=0CDgQFjAB&u rl=http%3A%2F%2Fwww.alnylarn.com%2Fcapella%2Fwpcontent%2Fuploads%2F2012%2F01 %2FALN Y-PCS -Phl-PrelimResutlsJan2012. pdf&ei=ii6UUZnBLZbe4AO8roHoBA&usg=AFQjCNGiN3K7 _fVEcY_2F91 nDuepwraZ A&sig2=3ZAZvGQO JXQ1 xU4WRb5w&bvm=bv.46471029,d.dmg&cad=rja

As described above, encapsulated siRNAs can provide a therapeutic effect in the liver of human patients at parenteral doses in the range of 0.015 - 0.20 mg/kg and are thought to provide a sustained effect at biweekly or monthly dosing. .

shRNA has not yet been used in humans, but works in vivo in rodents, it has been shown that expression vectors designed to express shRNA, when dosed to mice at concentrations comparable to siRNA, can be as effective as siRNA (McAnuff et al. 2007 Journal of Pharmaceutical Science 96: 2922-2930 Therefore, it is expected that dosing shRNA with either viral-based or encapsulated delivery systems as described above will provide a comparable effect in the liver when using dosing regimens comparable to those used in relation to siRNA.

Aptamers are typically pegylated to achieve better stability and can be therapeutically effective at weekly parenteral doses in the range of 100-600 µg/kg as described by Waters et al., 2011 Blood 117:5514-5522.

Spiegelmers are also pegylated in most cases to achieve a better circulatory half-life and may be therapeutically effective at parenteral doses >1.2 mg/kg every other day, as described by Riecke et al., 2012 Abstract 2432 presented at the 54th ^Annual ASH Meeting and Exposition, Atlanta, GA, USA With respect to binding to and accelerating the removal of HBsAg or other HBV proteins from the blood of HBV-infected patients, higher doses of aptamers or Spiegelmers may be required due to the high concentrations of HbsAg commonly present in HBV patients -infection.

As described above, immunotherapeutic approaches to the treatment of HBV infection have limited efficacy. One limitation of interferon-based monotherapy is the ability to remove HBsAg from the blood in a very small proportion of patients (Moucari et al., 2009, Antiviral Ther., 14: 1183-1188; Reijnders et al., 2011, J. Hepatol., 54: 449-454). This removal of HBsAg may underlie the achievement of reliable HBV DNA control with and without treatment in this small subset of treated patients (Moucari et al., 2009, Hepatology, 49: 1151-1157). Another important limitation of interferon-based therapy is that it induces only modest levels (<50 mS/mL) of anti-HBs production in a very small proportion of patients on treatment (Reijnders et al., 2011, J. Hepatol., 54: 449454 ; Harayiannis et al., 1990, J. Hepatol., 10: 350-352) after 48 weeks of exposure. These important limitations are likely to be critical factors underlying the achievement of a sustained virological response in only a limited number of patients after immunotherapy.

As described above, HBsAg can block signaling pathways important for cytokine-mediated stimulation of immune function. It is well known in the art that many different classes of immunotherapeutic agents use some common signal transduction pathways to efficiently activate the immune system. The disclosures presented here further demonstrate that many (or most) of these signal transduction pathways used by immunotherapeutic agents can also be blocked by the action of HBsAg. The novel disclosures herein indicate that the action of various immunotherapeutic agents is specifically inhibited by the presence of HbsAg and that the therapeutic effects of these various immunotherapeutic agents, when provided in a treatment regimen, are synergistically enhanced in the absence of HBsAg. Therefore, the removal of HBsAg from the blood, in turn, should lead to weaker inhibition of the signaling pathways required for the optimal activity of various immunotherapeutic agents. Thus, the use of immunotherapy in patients who have previously had HBsAg removed from their blood, or who actively remove HBsAg from their blood during immunotherapy, would likely have a similar synergistic effect on the immunostimulatory effects of any immunotherapy.

In the present disclosure, the term immunotherapeutic agent refers to a small molecule or polypeptide or cytokine or hormone that, through its specific biochemical activity, has the ability to directly or indirectly enhance the host's immune function. The polypeptide may be natural or recombinant. The polypeptide can be obtained recombinantly from a portion of the polypeptide of natural origin. The polypeptide may or may not be pegylated.

Methods for pegylation of polypeptides and compatibility of pegylation with the biochemical activity of these polypeptides are well known in the art and consist of linking chains at

- 15 039949 polyethylene glycol (PEG) with the considered polypeptide on specific amino acid residues. The main purpose of pegylation is to increase the lifetime of the polypeptide in the circulation and also to reduce its immunogenicity. These properties improve the tolerability of the polypeptide in question and reduce the frequency of dosing required to obtain an optimal therapeutic effect. Furthermore, it is known in the art that attachment of PEG residues to a polypeptide can be achieved without affecting the specific biochemical activity of the polypeptide in question. It is also known that pegylation increases the water solubility of the polypeptide in question, which facilitates the preparation of the drug. Numerous examples of pegylated polypeptides are known in the art and include: Mircera™ the pegylated form of erythropoietin; Neulasta™, a pegylated form of human granulocyte colony stimulating factor; Pegasys™ pegylated form of human interferon a-2a; Peg-intron™, a pegylated form of human interferon a-2b; and pegylated interferon λ1 (which is currently in clinical trials).

In addition, immunotherapeutic agents that have not previously shown suitable immunotherapeutic activity in the presence of HBV proteins (e.g., in infected patients, chimpanzees, or cell models) may now have suitable immunotherapeutic activity when HBsAg is removed from the blood and be used in the treatment of HBV in combination with any agent that eliminates HBsAg from the blood.

The demonstration of antiviral activity of any immunotherapeutic agent is considered to be a generally accepted proxy for its ability to stimulate immune function, whereby this stimulated immune function has an antiviral effect. Therefore, any immunotherapeutic agent with antiviral activity has the ability to stimulate immune function.

Several immunotherapies are currently approved for viral infections, including pegylated interferon a-2a (Pegasys™) for HBV and hepatitis C (HCV)), interferon a-2b (Intron-A™) for HBV and HCV, and thymosin a1 (Zadaxin™) for the treatment of HBV in most Asian countries. There are also other immunotherapeutic agents with demonstrated antiviral activity, including cytokines, interferon λ1, λ2, λ3 and γ, and TNFa (Friborg et al., 2013, Antimicrobial Agents and Chemotherapy, 57: 1312-1322; Lau et al., 1991, Hepatology 14: 975-979; McClary et al., 2000. Journal of Virology 74: 2255-2264; Robek et al., 2005. Journal of Virology 79: 38513854), pegylated interferon λ1 (Muir et al., 2010, Hepatology, 52: 822-832) and small molecule Toll-like receptor agonists like GS-9620 (currently under development by Gilead Sciences), ANA-773 (currently under development by Anadys), and the immunostimulatory oligonucleotides IMO-2055 and IMO-2125 ( currently being developed by Idera Pharmaceuticals) (Wu et al., 2007, Hepatology, 46: 1769-1778; Horscroft et al., 2012, J. Antimicrob. Chemotherapy, 67: 789-801; Lanford et al. 2013 Gastroenterology Feb 12 in press;

www.natap.org/2010/AASLD/AASLD_39.htm). In addition, the hormone dehydroepiandrosterone (5-androsten3b-17-one, DHEA) and many of its metabolites (including androstenediol (5-androsten-3b-17b-diol, PAED), androstentriol (5-androsten-3b-7b-17b- triol PAET) have a clear, well-established immunostimulatory functional ability to improve the formation of a protective vaccine response against viral infections and provide direct antiviral activity against many viral infections in vivo (Araeno et al., 1993, J. Inf. Dis., 167: 830-840 ; Danenberg et al., 1995, Vaccine, 13: 1445-1448; Khorram et al., 1997, J. Gerontol. A. Biol. Sci. Med. Sci., 52: M1-M7; Loria and Padgett, 1998, Rinsho Byori 46: 505-517 Loria 2002 Steroids 67 953-966 Knoferl et al 2003 J Appl Physiol 95 529-535 Oberbeck et al 2007 Inten. Car Med., 33: 2207-2213 Burdnick et al., 2009, Int. Immunopharmacol., 9: 13421346, Hazeldine et al., 2010, J. Steroid Biochem. Mol. Biol., 120: 127-136, Schmitz et al., 2013, Med. Chem., Feb 15, Epub ahead of p print).

Evaluation of the stimulation of immune function, as described in the current disclosure and in the case of HBV infection, is easily carried out (but not limited to) by changing the levels of free anti-HBsAg antibodies produced in a patient receiving immunotherapy. The use of the Abbott Architect™ anti-HBsAg antibody quantitation is a worldwide accepted method for assessing free anti-HBsAg antibody levels in the serum of patients with chronic HBV infection, and the appearance or increase in anti-HBsAg antibody production in patients with HBV infection is accepted replacement measure of the immune response in these patients who receive immunotherapy or therapy that inhibits HBV polymerase.

There are other accepted criteria for assessing immune function that can be used to monitor improvement in immune function in the presence of combination treatment, as described above. These criteria may include an increase in the transcriptional activity of interferon-response genes or an increase in blood levels of HBV-specific CD4+ or CD8+ T cells or elevated blood levels of various cytokines such as IL2 (Liang et al., 2011. Virology Journal 8: 69) .

Use of HBV vaccination (usually using HBsAg as antigen)

- 16 039949 on) is a well-known method for effectively preventing HBV infection and is a method accepted worldwide for preventing the spread of HBV infection. However, vaccination against HBV antigens has a moderate to negligible therapeutic effect, even when the vaccine combines two different HBV antigens, such as HBsAg and HBcAg (Mahtab et al., 2013. J. Hepatol. 58 (supp 1 ) abstract 760). According to the disclosure provided herein, this weak effect may be due to the circulating levels of HbsAg present in the blood of these patients and therefore the ability of the vaccine to stimulate the production of new antibodies to HBsAg (or other HBV proteins) can be greatly improved by removing HBsAg from blood.

Thus, there are many immunotherapies known to stimulate immune function that can be used when administered before, during, or after elimination of HBsAg from the blood, these immunotherapies include (but are not limited to):

thymosin α1;

any α-interferon or its pegylated derivatives;

any β-interferon or its pegylated derivatives;

any γ-interferon or its pegylated derivatives;

any λ-interferon or its pegylated derivatives;

interferon a-2a or a-2b or a-N3;

interferon e-1a or e-1b;

interferon γ-ib;

interferon λ1 or λ2 or λ3;

pegylated interferon a-2a or a-2b or λ1 or λ2;

any antiviral cytokine or its pegylated derivatives;

thymus protein A;

any antiviral cytokine or its pegylated derivatives;

any polypeptide having antiviral activity or immunostimulatory activity;

an immunostimulatory oligonucleotide such as IMO-2125 and IMO-2055;

vaccine directed to any HBV antigen;

a small molecular weight Toll-like receptor agonist such as GS-9620 and ANA-773; and any hormone having antiviral activity or immunostimulatory activity such as DHEA or its metabolites.

Examples of effective dosing regimens for immunotherapeutic agents used to achieve stimulation of immune function may include:

weekly doses of 90-180 mcg for Pegasys™ (according to package insert);

weekly doses of 1.6 mg for Zadaxin™ (according to package insert);

weekly doses of 1x10'U for Intron-A™ (according to package insert);

weekly doses of 1.5-3.0 µg/kg for pegylated interferon λ1 as described in Muir et al., 2010, Hepatology, 52: 822-832;

similar weekly doses as described above for any cytokine or immunotherapeutic peptide, whether pegylated or non-pegylated;

weekly doses of 0.16 - 0.48 mg/kg/week for the non-CpG immunostimulatory oligonucleotide IMO-2125 as described at http://www. google. com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=4&ved=0CEsQFj

IAD&url=http%3 A%2F%2Fwww.iderapharma.com%2Fdevelopment%2FIMO-2125-AASLD10312010.pdf&ei=WTiUUfnxHdWp4AO9vYGoBw&usg=AFQjCNENcwjLWM 1 B2r8GfOJmW

PRaeh-Xbw&sig2=kyENERyNTYmQoADWGoPluQ&bvm=bv.46471029,d.dmg&cad=rja

The commonly administered vaccine doses are in accordance with conventional practice in the art and specifically for HBV vaccines Energix-B™, Recombivax-HB™.

Thus, in accordance with the disclosure presented in the description, any of the above methods or means capable of achieving the elimination of HBsAg from the blood of patients, in combination with stimulation of the host immune function using any of the immunotherapeutic agents listed above, is expected to have a synergistic effect. to restore immune function in patients with HBV infection or HBV/HDV coinfection. In addition to restoring immune function better able to maintain infection control without treatment, it is also expected that such synergy would allow for a reduction in the dose of one or more agents and even the duration of treatment with any agent needed to provide a therapeutically effective immune response in the majority of patients. Example III illustrates the synergistic effect on restoration of immunological function when HBsAg is removed with NAP REP 2139

- 17 039949 accompanied by additional therapy using either thymosin a1 or pegylated interferon a-2a.

In the context of combining an agent that can clear HBsAg from the blood with an immunotherapeutic agent, any amount of eliminated HBsAg may provide a synergistic improvement in immunotherapy activity, and a fractional dose of a particular immunotherapeutic agent may result in comparable or even superior immunostimulatory activity of the immunotherapeutic agent, even if the HBsAg is not removed completely. Thus, the combination of any agent that removes HBsAg from the blood with any other immunotherapeutic agent will have a synergistic effect on the action of both agents, which demonstrates the potential for improving the stability of the antiviral response (host immunological control) without treatment, in addition, when this may require reduced doses of both agents to achieve a similar or even superior effect than when used in monotherapy.

Therefore, in accordance with the disclosure provided herein, it may be beneficial to treat a subject with HBV infection or HBV/HDV co-infection with a pharmaceutically acceptable agent that reduces or eliminates HBsAg from the blood, in combination with a second pharmaceutically acceptable immunotherapeutic agent.

It may also be useful to administer two pharmaceutically acceptable agents in the same pharmaceutical composition, or to administer two pharmaceutically acceptable agents in separate pharmaceutical compositions at the same time or at different times.

It may also be useful to administer pharmaceutically acceptable agents by the same or different routes of administration.

In order to provide the best possible antiviral response in a subject, it may be necessary to add to the combination treatment described above an HBV polymerase inhibitor such as (but not limited to): tenofovir disoproxil fumarate, entecavir, telbuvidine, adefovir dipivoxil, or lamivudine. Such antiviral agents can prevent the replication of the double-stranded viral genome in HBV and reduce the concentration of HBV virus in the blood.

The compositions described herein may be administered by any suitable means, for example orally, in the form of tablets, capsules, granules or powders; sublingually; buccal; parenterally, for example by subcutaneous, intravenous, intramuscular injection or infusion (eg, as sterile injectable aqueous or non-aqueous solutions or suspensions); with the help of inhalation; topically, for example in the form of a cream or ointment; or rectally in the form of suppositories or enemas; in unit dosage formulations containing non-toxic, pharmaceutically acceptable bases or diluents. For example, the present compositions may be administered in a form suitable for immediate release or sustained release. Immediate release or sustained release can be achieved by using suitable pharmaceutical compositions or, in particular, in the case of sustained release, through the use of special devices such as subcutaneous implants or osmotic pumps. Thus, the above compositions can be adapted for administration by one of the following routes: oral ingestion, inhalation, subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection or infusion, or topically.

The present disclosure will become clearer in view of the following examples.

Example I

NAPs inhibit the transport of HBsAg out of cells.

HBsAg has been shown to block many aspects of the immune response to HBV infection (Cheng et al., 2005, J. Hepatology, 43: 465-471; Moucari et al., Hepatology 49: 1151-1157; Vanlandschoot et al., 2002, J. Gen. Virol.83: 1281-1289; Woltman et al., 2011, PloS One 6: el5324; Wu et al., 2009, Hepatology 49: 1132-1140 and Xu et al., 2009, Mol. Immunology 46 : 2640-2646). Therefore, the elimination of circulating HBsAg may be a critical factor in ensuring the restoration of immunocompetence in patients with chronic hepatitis B infection. An effective way to eliminate HBsAg in the circulation is to prevent the formation and/or release of subviral particles (SVPs) from infected cells (SVPs are the main carrier of HBsAg in the blood) . The morphogenesis and intracellular transport of SVP can be modeled in vitro in BHK-21 cells expressing a small form of the HBsAg protein (sHBsAg), which is the form especially elevated in SVP. This model is considered a surrogate model for HBV SVP morphogenesis and transport (Patient et al., 2007, J. Virology 81: 38423851). Due to the critical role of serum HBsAg in maintaining the chronic state of HBV infection, the efficacy of the compounds in this model demonstrates their antiviral activity against HBV.

Various NAP compounds have been tested on sHBsAg-expressing BHK-21 cells, including fully degraded phosphorothioate NAPs REP 2006 and REP 2107, non-phosphorothioate, fully 2' O-methylated degraded NAP (REP 2086), and NAP consisting of the polyAC sequence: REP 2055 (SEQ ID NO: 2) and REP 2148 (SEQ ID NO: 3). These

- 18 039949

NAPs were introduced into BHK-21 cells simultaneously with messenger RNA for sHBsAg expression using electroporation. Activity on the BHK model system was assessed by visualizing the location of the HBsAg protein within BHK-21 cells using immunofluorescence microscopy. SVP formation in the perinuclear space was visualized using transmission electron microscopy. Compounds were considered active if HBsAg was restricted to the perinuclear space and did not pass to the cell periphery (secretion), or if SVP formation was prevented. The activity of the various NAP compounds is summarized in Table 1. 2 below.

Table 2. Effect of various NAPs on HBsAg transport in BHK-21 cells

NAP HBsAg is retained in the perinuclear space HBsAg travels to the cell periphery Education SVP control (NAP not present) - ++++ ++++ R.E.P. 2006 ++++ • - REP2107 +++ + - REP 2086 - ++++ ++++ REP 2055 (SEQ ID NO: 2) ++++ - not explored REP 2148 (SEQ ID NO: 3) ++++ - not explored

- - the effect was not observed;

+ to ++++ - the observed effect from minimal to complete.

The results of treatment of sHBsAg-expressing BHK-21 cells with REP 2006 and REP 2107 demonstrate the ability of NAP to block SVP formation and sHBsAg transport in a sequence-independent manner. The lack of activity with REP 2086 indicates that the activity of NAPs is clearly dependent on the presence of phosphorothionation. Moreover, this ability was maintained in the presence of a 2' ribose modification (in REP 2107) and a base modification (5' methylcytosine in the case of REP 2148). In addition, REP 2107, REP 2055, known to be completely devoid of any immunostimulatory activity, were active compared to REP 2006. In turn, a certain poly AC sequence (REP 2055 and REP 2148) was relatively active relative to the degenerate sequence ( REP 2006 and REP 2107).

These results show that in the context of a degenerate sequence and a sequence containing alternating purine/pyrimidine nucleotide repeats such as AC (and therefore also CA) and also such as TG and GT or UG and GU and optionally containing 2' ribose and /or modification of the base, it is assumed that NAPs will be able to block the formation, intracellular transport and secretion of SVPs from infected cells at a length of oligonucleotides of 20-120 nucleotides in accordance with the sequence-independent properties of NAPs, as described in US patents No. 8008269 B2, 8008270 B2 and 8067385 B2.

Example II.

Effect of elimination of HBsAg from the blood of HBV-infected patients on elimination of other HBV proteins and restoration of immunological potential.

Patients with chronic HBV infection were treated with NAP REP 2055 (also known as REP 9AC, SEQ ID NO: 2) to eliminate HBsAg from the blood. The effect of REP 2055 administration (typically 400 mg weekly dosing) on blood HBsAg levels was monitored using the Abbott Architect™ quantitative HbsAg assay, the results are shown in Table 1. 3.

Table 3. Effect of NAP REP 2055 on blood levels of HBsAg in patients with chronic HBV infection

Patient HBsAg Pretreatment (W/mL) At the end of the course of HBsAg treatment (N/ml) one 934 0.14 Patient HBsAg Pretreatment (W/mL) At the end of the course of HBsAg treatment (N/ml) 2 1885.4 0.38 3 384.1 0.00 4 126465.07 0.03 5 158180 0.00 6 36996 7.00 7 4672.5 43.7

Removal of HBsAg from the blood caused an immunological recovery, as evidenced by a decrease in circulating HBeAg (measured in two patients by quantitative HbeAg

- 19 039949 test Abbott Architect™ - see tab. 4), the appearance of free anti-HbsAg antibodies (measured with the Abbott Architect™ quantitative HbeAg test - see Table 5) and the decrease in HBV virus in the blood (HBV DNA measured with the Roche Cobas™ quantitative test - see Table 6 ). A decrease in circulating HBV virus was observed in all patients, however, to varying degrees. Moreover, the levels of free anti-HBsAg antibody titers measured during treatment in most of these patients were moderate at best and in most cases worse than the blood anti-HBsAg antibody titers observed during HBsAg vaccination of healthy uninfected adults.

Table 4. Effect of eliminating HBsAg on eliminating HBeAg

Patient HBeAg Pretreatment (IU/ml*) During HBeAg treatment (N/ml*) one 1181.29 7.63 2 78.25 8.211

*measured using Abbott Architect™ quantitative analysis.

Table 5 Effect of HBsAg elimination on detection of free anti-HBsAg antibodies in patients with chronic HBV infection

Patient Pre-treatment with anti-HBsAg* (mS/ml) At the end of anti-HBsAg treatment* (mS/ml) one 0 13.2 2 one 22.8 3 5.68 277 4 3 5.39 5 4.99 385.7 6 one 19.7 7 2 19.2

*measured using Abbott Architect™ quantitative analysis.

Table 6. Effect of HBsAg elimination on blood levels of HBV virus (HBV DNA)

Patient HBV DNA pretreatment* (copies/ml serum) At the end of treatment HBV DNA* (copies/ml serum) one 2x 10 ⁶ <500 2 1.4x10' 1.39 x 10 ⁴ 3 4.5 x 10' <116 4 1.9 x 10 ¹² 3.1 x 10 ^b 5 7.9 x 10" <116 6 4.8 x 10" 372 7 1.8 x 10' 3.5 x 10 ⁶

*measured using Roche Cobas™ analysis.

Discontinuation of REP 2055 treatment in these patients resulted in a subsequent prolonged resurgence of viremia in 5/7 patients who experienced clearance of serum HBV proteins (recurrence of HBsAg in the blood, decrease or disappearance of anti-HBsAg antibodies in the blood, and an increase in HBV DNA to levels up to start of treatment). Thus, treatment with NAPs, or any other agent that removes HBsAg (and other HBV antigens) from the blood, is expected to undergo a similar viral resurgence if treatment is stopped in the absence of any concomitant immunotherapy.

Example III.

Combination therapy with a NAP chelate complex and two different immunotherapies in the treatment of human patients with chronic hepatitis B.

REP 2139-Ca is a NAP REP 2139 calcium chelate complex (SEQ ID NO: 10) prepared in saline at a ratio of 30 mg CaCl ₂ for every 100 mg of oligonucleotide present. REP 2139 in the form of a calcium chelate complex is used to improve the tolerability of ON administration (see International Published Application No. WO 2012/021985 and US Published Application No. 2012/0046348), while not interfering with its specific antiviral activity. REP 2139-Ca (usually administered weekly at a dose of 500 mg) eliminates blood HBsAg (and subsequently HBeAg) and HBV virions (HBV DNA) in HBV-infected patients in a similar manner to REP 2055 through the same mechanism of action (see below). 3, 4, and 6 vs. 7, 8, and 9, respectively) and therefore also demonstrates that NAPs containing both 2' ribose modifications and modified bases (e.g., REP 2139) can reduce blood HBsAg, and that ON formulated in the form of chelate complexes (for example, REP 2139-Ca), can be used to reduce or eliminate HBsAg from the blood.

- 20 039949

Table 7. Effect of REP 2139-Ca monotherapy on serum

HBsAg in patients with chronic HBV infection

Patient Serum HBsAg (mIU/ml)* Preliminary processing REP 2139-Ca one 70050 0.19 2 13400 0 3 3654.3 0.34 4 47689.7 180.44 5 107659 32.15 6 58937.87 9.91 7 17988 29.21 eight 125000 0.01 nine 1288.56 0.02

*measured with the Abbott Architect™ HBsAg quantitation assay.

Table 8 Effect of HbsAg Eliminating REP 2139-Ca on Serum HBeAg Levels

Patient Serum HBeAg (index*) Preliminary processing REP 2139-Ca one 1.488 0.38 2 556.27 0.34 3 662.09 1.62 4 1100.43 0.31 5 1815.75 1,I 6 561.96 0.32 7 15.27 18.27 eight 1767.85 0.40 nine 101.73 19.35

*<1 - not detected, >1 - highly infectious condition.

Table 9 Effect of REP 2139-Ca monotherapy on serum HBV DNA (virions) in patients with chronic HBV infection

Patient Serum HBV (copies/ml)* Preliminary processing REP 2139-Ca one 9.89x10“” 791 2 1.66x10" 1680 3 2.01x10" 3643 4 1.28x10" 9060 5 9.89x10" 2.52x 10 ⁶ 6 8.71 x 10" 558 7 7,lxl0 ^{5 1.94x104} eight 9.89x10" 552 nine ^9.9x106 3250

*measured using Roche Cobas™ analysis;

**upper limit of quantitation.

As described in Example II, a limitation of NAP therapy (or any therapy that can eliminate HBsAg) is that although the patient's existing levels of anti-HBs production are released to clear the virus during NAP therapy, this level of antibody production ( and elimination of HBsAg-induced immuno-inhibition) in most patients is insufficient to ensure complete control of HBV infection after completion of NAP treatment. HbsAg clearance mediated by REP 2139 achieved the same total blood levels of anti-HbsAg antibodies as with REP 2055 monotherapy (see Tables 5 and 10 [monotherapy termination]), and indeed, as would be expected resulted in the same poor retention of immunological control as was the case with NAP REP 2055 when treatment was discontinued (see Example II above). The results of the use of NAPs in monotherapy indicate that an underlying (and previously unrecognized) defect in the ability of the immune system to restore a fully competent immune response to HBV infection even in the absence of these HBV proteins is likely the result of chronic exposure to HBsAg, HBeAg and HbcAg, which causes long-term immunological damage that persists in HBV-infected subjects even after elimination of these antigens from the blood.

To test whether NAP treatment (which eliminates HBsAg from the blood) can be enhanced by immunotherapy (stimulating immune function), patients who had their serum HBV proteins eliminated or reduced while on REP 2139-Ca monotherapy received either thymosin α1 (Zadaxin™ 1.6mg SC injection twice weekly) or pegylated interferon a-2a (Pegasys™ 90-180mcg SC injection once weekly) as add-on therapy to ongoing REP 2139-Ca. Pegylated interferon a-2a is sold under the trade name Pegasys™ by Roche Inc. (Basel, Switzerland) and approved for the treatment of chronic HBV infection. Thymosin α1 is sold under the trade name Zadaxin™ by SciClone Pharmaceuticals (Foster City, California, USA) and is also approved for the treatment of chronic HBV infection.

Interferon-based monotherapy in most cases results in only a moderate level (<50 mIUml) of anti-HBsAg antibodies in the blood in a very small proportion of patients (<10%) after 48 weeks of treatment (Reijnders et al., 2011, J. Hepatol. , 54: 449-454; Harayiannis et al., 1990, J. Hepatol., 10: 350352), and the antiviral effects of thymosin α1 are similarly limited (Yang et al., 2008, Antiviral Res. 77: 136-141) . However, after the addition of thymosin α1 or pegylated interferon α-2a to REP 2139-Ca treatment, HBsAg clearance from the blood was achieved, with a significant increase in anti-HBsAg antibody levels achieved in all patients, with levels significantly exceeding the anti-HbsAg levels observed with using NAP-mediated elimination of HBsAg alone, or levels reported using immunotherapy alone (see Table 10). Furthermore, this apparent synergistic effect on increasing anti-HBsAg antibody levels was achieved with as little as 13 weeks of immunotherapy in combination with REP 2139-Ca (compared to the 48 weeks routinely prescribed for such immunotherapy) and was also observed in two patients who received half the dose of Pegasys (90 mcg) usually prescribed for the treatment of HBV. In addition, this synergistic response was observed in 9/9 (100%) patients. Significant reactivation of anti-HbsAg production, observed with the use of additional immunotherapy after the removal of HBV protein from the blood, is the only direct criterion for a synergistically improved functioning of immunotherapy in the absence of HBsAg. Based on these findings, it is possible to predict synergistically improved activity in other areas of immunostimulation, such as T-cell mediated immunity and innate immunity, which may also be required to achieve complete immunological control of HBV infection.

Table 10. Synergistic effect on anti-HBsAg production after combined treatment with REP 2139-Ca and thymosin α1 or pegylated interferon a-2a

Immunotherapeutic agent given in combination Patient Serum anti-HBsAg antibody (mW/ml)* REP 2139-Ca (end of monotherapy) REP 2139-Ca + additional immunotherapy ** Thymosin al one 19.36 987.03 2 365 1302 3 44.64 1108 Pegylated interferon a-2a 4*** 5.36 381.57 2.12 288.85 6 1.64 798.22 7 19.69 223.29 eight 42.07 242.31 nine 42.61 499.05

*measured with Abbott Architect™ quantitative anti-HBsAg ELISA assay;

**13 weeks of continuous immunotherapy (additional) after HBsAg reduction achieved with REP 2139-Ca monotherapy;

***These two patients received 90 mcg Pegasys/week.

The results presented in table. 10 demonstrate that the elimination of serum HBsAg has a significant synergistic effect on the ability of thymosin α1 or pegylated interferon a-2a to stimulate immune function, which has not previously been suggested in the art. All patients achieved very high anti-HBsAg antibody titers (and thus apparently more effective overall immune stimulation) using a significantly shorter immunotherapy regimen than is normally required to achieve even much lower blood anti-HBsAg antibody titers at use in monotherapy, and even then only in a small proportion of patients. In table. 10, all patients with eliminated serum HBsAg responded strongly to immunotherapy. In many cases, a clear increase in anti-HBsAg production was found as early as 6-10 weeks of immunotherapy. These synergistic effects on stimulation of host immunity resulted in the control of HBV infection in the absence of treatment in 8/9 patients and clearly demonstrates the synergistic effect of restoring a competent immune response in the majority of patients with HBV infection when immunotherapy is administered in the absence of circulating HBsAg.

- 22 039949

These results demonstrate that any pharmaceutically acceptable agent capable of reducing or eliminating HBsAg from the blood, when administered in combination with an immunotherapeutic agent, will have a beneficial and synergistic effect on the stimulation of immune function (such as, but not limited to, the production of anti-HBsAg antibodies) in patients with chronic HBV. Example III clearly shows that removal of HBsAg from the blood synergistically improves the ability of immunotherapy to elicit a strong host secondary antiviral immune response and strongly suggests that persistently circulating HBsAg in the blood of immunotherapy-only patients has a significant inhibitory effect on immunotherapy activity and likely contributes to low the effectiveness of the adopted immunotherapy in achieving immunological control of the infection, which continues in the absence of treatment. Example III also clearly shows that a synergistic effect on the restoration of immune function was observed in all patients in whom anti-HBsAg blood levels were reached much faster and at higher levels than the levels observed with immunotherapy alone, in all cases exceeding the levels anti-HBsAg antibodies that are commonly seen in healthy, uninfected HBsAg vaccinated individuals. These effects can be achieved even with low doses of immunotherapy (Pegasys at 90 µg/week), which are known to be insufficient (suboptimal) when used alone. In the case of removal of only the HBV protein or in the case of only one immunotherapy, these strong protective levels of anti-HBsAg antibodies are rarely observed.

The removal of serum HBsAg achieved using the NAPs in the current disclosure is an example of the best effect that can be achieved using any other agent designed to reduce or eliminate HBsAg (or other HBV antigens) from the blood, regardless of its mechanism of action. In fact, the resulting synergy between HBsAg clearance from the blood and stimulation of immune function observed with NAP and the immunotherapeutic agents Pegasys™ and Zadaxin™ clearly demonstrates to the person skilled in the art that the occurrence of such synergistic effects in combination with immunotherapy can now be reliably predicted when the use of any agent capable of reducing or eliminating HBsAg from the blood, regardless of the chemical structure or mechanism of action of the agent. Therefore, this disclosure provides a clear and novel idea that, in HBV infection, a synergistic effect on immune stimulation in HBV-infected patients using NAPs in combination with Zadaxin™ or Pegasys™ can be obtained by a combination of any agent or method capable of reduce or eliminate HBsAg from the blood, and any other agent capable of stimulating immune function. It can also be expected that the synergistic effect demonstrated with REP 2139-Ca and thymosin α1 or pegylated interferon a-2a would be observed with other combinations of agents as set forth in this disclosure, in cases where the first agent is able to reduce or eliminate HBsAg from the blood, and the second agent is able to stimulate immune function. In addition, it can be assumed that combinations of two or more different agents capable of reducing or eliminating HBsAg from the blood with two or more different immunotherapeutic agents may also have a similar or superior effect.

While the removal of HBsAg alone from the blood appears to provide most of the synergistic effect on immunotherapy, the additional removal of HBeAg and HBcAg may also contribute slightly to the synergistic effect due to their inherent immunoinhibitory properties. Thus, additional removal of HBeAg and HBcAg from the blood (an effect also achieved with NAPs) may provide minimal benefit, but will have little or no effect without elimination of HBsAg.

The striking effect that the removal or elimination of HBsAg from the blood has an impact on conventional immunotherapy can also significantly improve the effect of vaccines against HBV antigens when administered therapeutically. Based on the disclosure provided, one of skill in the art can reasonably assume that circulating HBsAg inhibits a fully stimulated vaccine response in HBV-infected subjects, and furthermore that vaccine response can be significantly improved when administered in the absence or at reduced levels HBsAg.

Example IV.

REP 2139-Ca/Pegasys™ Combination Treatment at the Beginning of Therapy in Patients with Chronic Hepatitis B.

In a new cohort of HBV-infected patients, both REP 2139-Ca (500 mg once weekly) and Pegasys™ (180 mg weekly) were administered from the start of therapy to see if the synergistic effects seen in example III would be achieved. , in the early stages of the treatment regimen. These three patients experienced a rapid and dramatic decrease in serum HBsAg and a rapid rise in free anti-HBsAg antibody titers (see Table 11).

- 23 039949

Table 11. Synergism of REP 2139-Ca and Pegasys™ when used in combination at the beginning of treatment

Patient Serum HBsAg (U/ml*) Serum anti-HBsAg (mIU/ml*) Pretreatment Treatment week 9 Pretreatment Against the backdrop of treatment one 2510.66 0.17 0.48 123.75 (week 17) 2 4789.73 0.02 0.4 521.57 (week 15) 3 3338.24 0.05 0.8 646.42 (week 11)

*measured with Abbott Architect™.

The results of Example IV show that the synergy of the simultaneous combination of HBsAg removal from the blood with immunotherapy can be observed very early in the course of treatment when NAPs and Pegasys™ are both administered at the start of treatment. In addition, these results and the results presented in Example III show that a significant improvement in the effect of immunotherapy can be observed when immunotherapy is added after the removal of HBsAg from the blood is achieved, or when immunotherapy is added at the time of removal of HBsAg from the blood (i.e. . at the beginning of treatment).

Although Examples II, III, and IV refer to patients with HBV monoinfection, in view of the critical and well-established role that HBsAg plays in HDV virus generation and immunosuppression in HBV/HDV coinfection, these examples provide a clear idea that the concepts The guidelines set forth in this disclosure should be applicable to both HBV monoinfection and HBV/HDV coinfection.

The above description is for illustrative purposes only, and those skilled in the art will appreciate that changes may be made to the described embodiments within the scope of the invention as defined by the appended claims. However, other modifications that fall within the scope of the present invention, as defined in the appended claims, will be apparent to those skilled in the art in light of and in view of this disclosure.

CLAIM

Claims (30)

CLAIM 1. A method of treating hepatitis B infection or hepatitis B/hepatitis D co-infection, comprising administering to a patient in need thereof at least one first pharmaceutically acceptable agent selected from an oligonucleotide chelate complex containing the following SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and a nucleic acid polymer selected from a phosphorothioate oligonucleotide 20-120 nucleotides long containing repeats of the AC sequence; a phosphorothioate oligonucleotide 20-120 nucleotides long containing repeats of the CA sequence; a phosphorothioate oligonucleotide 20-120 nucleotides in length containing repeats of the UG sequence; a phosphorothioate oligonucleotide 20-120 nucleotides in length containing repeats of the GU sequence; a phosphorothioate oligonucleotide 20-120 nucleotides in length containing repeats of the TG sequence; or a phosphorothioate oligonucleotide 20-120 nucleotides in length containing repeats of the GT sequence; and at least one second pharmaceutically acceptable immunotherapeutic agent selected from thymosin a1; interferon a2a; interferon a2b; interferon aN3; interferon β 1a; interferon β 1b; interferon Y1b; interferon λ1; interferon λ2; interferon λ3; pegylated interferon a2a; pegylated interferon a2b; pegylated interferon λ1; pegylated interferon λ2. 2. The method of claim 1, wherein the nucleic acid polymer further comprises at least one 5' methylcytosine. 3. The method of claim 1, wherein all of the cytosines contained in the nucleic acid polymer are present as 5' methylcytosine. 4. The method of claim 1, wherein the nucleic acid polymer further comprises at least one 2'-O-methyl ribose modification and at least one 5' methylcytosine. 5. The method according to claim 1, where all the riboses contained in the nucleic acid polymer have a modification of 2'-O-methyl, and all cytosines are present in the form of 5' methylcytosine. 6. A method for treating hepatitis B infection or hepatitis B/hepatitis D co-infection, comprising administering to a patient in need thereof at least one first pharmaceutically acceptable agent selected from an oligonucleotide selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 9 or 10; and a nucleic acid polymer selected from a 20-120 nucleotide long phosphorothioate oligonucleotide consisting of repeats of the AC sequence; and a phosphorothioate oligonucleotide 20-120 nucleotides long, consisting of repeats of the CA sequence; and at least one second pharmaceutically acceptable agent selected from thymosin α1; any pegylated interferon α; any pegylated interferon β; any pegylated interferon γ; any pegylated interferon λ, where all the cytosines contained in the nucleic acid polymer are present as 5' methylcytosine. 7. The method of claim 1 or 6, wherein the nucleic acid polymer further comprises at least one 2' ribose modification. 8. The method according to claim 1 or 6, wherein all riboses contained in the nucleic acid polymer have a 2' modification. 9. The method of claim 1 or 6, wherein the nucleic acid polymer further comprises at least one 2'-O-methyl ribose modification. 10. The method according to claim 1 or 6, wherein all the riboses contained in the nucleic acid polymer have the 2'O-methyl modification. 11. A method of treating hepatitis B infection or hepatitis B/hepatitis D co-infection, comprising administering to a patient in need thereof at least one first pharmaceutically acceptable agent selected from an oligonucleotide selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 9 or 10; and a nucleic acid polymer selected from a 20-120 nucleotide long phosphorothioate oligonucleotide consisting of repeats of the AC sequence; and a phosphorothioate oligonucleotide 20-120 nucleosides long, consisting of repeats of the CA sequence; and at least one second pharmaceutically acceptable agent selected from thymosin α1; any pegylated interferon α; any pegylated interferon β; any pegylated interferon γ; any pegylated interferon λ; where in these oligonucleotides, all riboses contained in the nucleic acid polymer have a 2'-O-methyl modification, and all cytosines are present in the form of 5' methylcytosine. 12. The method according to claim 6 or 11, wherein said at least one second pharmaceutically acceptable agent is pegylated interferon a2a, pegylated interferon a2b, pegylated interferon λ1, or pegylated interferon λ2. 13. The method according to any one of claims 1, 6 or 11, wherein said at least one first pharmaceutically acceptable agent and at least one second pharmaceutically acceptable agent are part of the same pharmaceutical composition. 14. The method according to any one of claims 1, 6 or 11, wherein said at least one first pharmaceutically acceptable agent and at least one second pharmaceutically acceptable agent are part of separate pharmaceutical compositions. 15. The method according to any one of claims 1, 6 or 11, wherein said at least one first pharmaceutically acceptable agent and at least one second pharmaceutically acceptable agent are administered simultaneously. 16. The method according to any one of claims 1, 6 or 11, wherein said at least one first pharmaceutically acceptable agent and at least one second pharmaceutically acceptable agent are administered by different routes. 17. The method according to any one of claims 1, 6, or 11, wherein said first and second agents are administered by one or more of the following routes: oral ingestion, aerosol inhalation, subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection, and intravenous injections. 18. The method of claim 6 or 11 which further comprises administering a third pharmaceutically acceptable agent comprising one or more pharmaceutically acceptable agents selected from tenofovir disoproxil fumarate, entecavir, telbuvidine, adefovir dipivoxil and lamivudine. 19. The method of claim 1, comprising administering to a patient in need thereof a first pharmaceutically acceptable agent that contains the oligonucleotide chelate complex of SEQ ID NO: 2 and a second pharmaceutically acceptable agent that contains pegylated interferon a2a. 20. The method of claim 1, comprising administering to a patient in need thereof a first pharmaceutically acceptable agent that contains the oligonucleotide chelate complex of SEQ ID NO: 3 and a second pharmaceutically acceptable agent that contains pegylated interferon a2a. 21. The method of claim 1, comprising administering to a patient in need thereof a first pharmaceutically acceptable agent that contains the oligonucleotide chelate complex of SEQ ID NO: 10 and a second pharmaceutically acceptable agent that contains pegylated interferon a2a. 22. The method of claim 1, comprising administering to a patient in need thereof a first pharmaceutically acceptable agent that contains the oligonucleotide chelate complex of SEQ ID NO: 2 and a second pharmaceutically acceptable agent that contains thymosin α1. 23. The method of claim 1, comprising administering to a patient in need thereof a first pharmaceutically acceptable agent that contains the oligonucleotide chelate complex of SEQ ID NO: 3 and a second pharmaceutically acceptable agent that contains thymosin α1. - 25 039949 24. The method of claim 1, comprising administering to a patient in need thereof a first pharmaceutically acceptable agent that contains the oligonucleotide chelate complex of SEQ ID NO: 10 and a second pharmaceutically acceptable agent that contains thymosin otl. 25. The method of claim 1, comprising administering to a patient in need thereof a first pharmaceutically acceptable agent that contains the oligonucleotide chelate complex of SEQ ID NO: 2 and a second pharmaceutically acceptable agent that contains interferon a2b. 26. The method of claim 1, comprising administering to a patient in need thereof a first pharmaceutically acceptable agent that contains the oligonucleotide chelate complex of SEQ ID NO: 3 and a second pharmaceutically acceptable agent that contains interferon oc2b. 27. The method of claim 1, comprising administering to a patient in need thereof a first pharmaceutically acceptable agent that contains the oligonucleotide chelate complex of SEQ ID NO: 10 and a second pharmaceutically acceptable agent that contains interferon oc2b. 28. The method of claim 1, comprising administering to a patient in need thereof a first pharmaceutically acceptable agent that contains an oligonucleotide chelate complex of SEQ ID NO: 2 and a second pharmaceutically acceptable agent that contains pegylated interferon λΐ. 29. The method of claim 1, comprising administering to a patient in need thereof a first pharmaceutically acceptable agent that contains an oligonucleotide chelate complex of SEQ ID NO: 3 and a second pharmaceutically acceptable agent that contains pegylated interferon λΐ. 30. The method of claim 1, comprising administering to a patient in need thereof a first pharmaceutically acceptable agent that contains the oligonucleotide chelate complex of SEQ ID NO: 10 and a second pharmaceutically acceptable agent that contains pegylated interferon λΐ.