阅读说明：本技术 治疗b型肝炎和d型肝炎病毒感染的方法 (Methods of treating hepatitis B and hepatitis D virus infections ) 是由 安德鲁·瓦利恩特 于 2015-07-07 设计创作，主要内容包括：本申请涉及治疗B型肝炎和D型肝炎病毒感染的方法。具体地,本申请公开了治疗B型肝炎病毒感染或B型肝炎病毒/肝炎δ病毒共感染的方法,所述方法包含向需要该治疗的对象施用包含至少一种硫代磷酸酯化核酸聚合物的第一药学上可接受的药剂和包含至少一种核苷/核苷酸类似物HBV聚合酶抑制剂的第二药学上可接受的药剂。(The present application relates to methods of treating hepatitis B and hepatitis D virus infections. In particular, the present application discloses a method of treating a hepatitis B virus infection or a hepatitis B virus/hepatitis delta virus co-infection, said method comprising administering to a subject in need of such treatment a first pharmaceutically acceptable agent comprising at least one phosphorothioate nucleic acid polymer and a second pharmaceutically acceptable agent comprising at least one nucleoside/nucleotide analog HBV polymerase inhibitor.)

1. A composition for treating an HBV infection or HBV/HDV co-infection in a subject comprising a first pharmaceutically acceptable agent comprising a chelating complex of at least one phosphorothioated nucleic acid polymer and a second pharmaceutically acceptable agent comprising at least one nucleoside/nucleotide analog HBV polymerase inhibitor.

2. A composition for treating HBV infection or HBV/HDV co-infection comprising a first pharmaceutically acceptable agent comprising a chelating complex of one or more nucleic acid polymers selected from the group consisting of:

SEQ ID NO：2；

SEQ ID NO：10；

SEQ ID NO：13；

SEQ ID NO: 1. 3-9, 11, 12 and 14-20;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of sequence AC;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of sequence CA;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising repeats of the sequence TG; and

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of the sequence GT;

and a second pharmaceutically acceptable agent comprising one or more of:

lamivudine;

adefovir dipivoxil;

entecavir;

telbivudine;

tenofovir disoproxil fumarate;

emtricitabine;

(ii) clevudine;

besnfovir;

tenofovir alafenamide fumarate;

AGX-1009；

elvucitabine;

vallatrine, vallatate;

(ii) peradfovir mesylate;

(ii) valtocitabine; and

any nucleoside/nucleotide analogs that inhibit HBV polymerase.

3. The composition of claim 1 or 2, wherein the nucleic acid polymer further comprises at least one 2' ribose modification.

4. The composition of any one of claims 1 to 3, wherein the nucleic acid polymer further comprises at least one 5' methylcytosine.

5. Use of a first pharmaceutically acceptable agent comprising at least one phosphorothioate nucleic acid polymer chelate complex and a second pharmaceutically acceptable agent comprising at least one nucleoside/nucleotide analogue HBV polymerase inhibitor for the treatment of HBV infection or HBV/HDV co-infection in a subject.

6. Use of a first pharmaceutically acceptable agent comprising at least one phosphorothioate nucleic acid polymer chelate complex and a second pharmaceutically acceptable agent comprising at least one nucleoside/nucleotide analogue HBV polymerase inhibitor in the manufacture of a medicament for treating an HBV infection or HBV/HDV co-infection in a subject.

7. Use of a first pharmaceutically acceptable agent comprising one or more chelating complexes of nucleic acid polymers selected from the group consisting of:

SEQ ID NO：2；

SEQ ID NO：10；

SEQ ID NO：13；

SEQ ID NO: 1. 3-9, 11, 12 and 14-20;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of sequence AC;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of sequence CA;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising repeats of the sequence TG; and

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of the sequence GT; and

the second pharmaceutically acceptable agent comprises one or more of:

lamivudine;

adefovir dipivoxil;

entecavir;

telbivudine;

tenofovir disoproxil fumarate;

emtricitabine;

(ii) clevudine;

besnfovir;

tenofovir alafenamide fumarate;

AGX-1009；

elvucitabine;

vallatrine, vallatate;

(ii) peradfovir mesylate;

(ii) valtocitabine; and

any nucleoside/nucleotide analogs that inhibit HBV polymerase.

8. Use of a first pharmaceutically acceptable agent comprising one or more chelate complexes of nucleic acid polymers selected from the group consisting of:

SEQ ID NO：2；

SEQ ID NO：10；

SEQ ID NO：13；

SEQ ID NO: 1. 3-9, 11, 12 and 14-20;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of sequence AC;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of sequence CA;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising repeats of the sequence TG; and

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of the sequence GT; and

the second pharmaceutically acceptable agent comprises one or more of:

lamivudine;

adefovir dipivoxil;

entecavir;

telbivudine;

tenofovir disoproxil fumarate;

emtricitabine;

(ii) clevudine;

besnfovir;

tenofovir alafenamide fumarate;

AGX-1009；

elvucitabine;

vallatrine, vallatate;

(ii) peradfovir mesylate;

(ii) valtocitabine; and

any nucleoside/nucleotide analogs that inhibit HBV polymerase.

9. The use of any one of claims 6 to 8, wherein the nucleic acid polymer further comprises at least one 2' ribose modification.

10. The use of any one of claims 6 to 9, wherein the nucleic acid polymer further comprises at least one 5' methylcytosine.

Technical Field

The present description relates to methods of treating a subject having a Hepatitis B Virus (HBV) infection or HBV/Hepatitis Delta Virus (HDV) co-infection comprising administering a first pharmaceutically acceptable formulation of a phosphorothioate nucleic acid polymer and a second pharmaceutically acceptable formulation of a nucleoside/nucleotide analog that inhibits HBV polymerase.

Background

HBV afflicts 4 billion individuals worldwide and causes an estimated 600,000 deaths annually due to complications from HBV infection. Although some antiviral therapies have been approved for use, none of these therapies elicit a therapeutically effective immune response that can provide long-lasting control of infection, except in a small fraction of patients undergoing treatment.

HBV infection results in the production of two distinct particles: 1) infectious HBV viruses themselves (or dyne particles) comprising a viral capsid assembled from HBV core antigen protein (HBcAg) and covered by HBV surface antigen (HBsAg), and 2) subviral particles (or SVPs) which are high density lipoprotein-like particles comprising lipids, cholesterol esters and HBV surface antigen (HBsAg) in a non-infectious, small to medium form. For each viral particle produced, 1,000 to 10,000 SVP are released into the blood. Thus, SVP (and its carried HBsAg protein) represents the vast majority of viral proteins in the blood. HBV infected cells also secrete soluble protein hydrolysates of the precore protein known as HBV e-antigen (HBeAg).

HDV uses HBsAg to form its viral structure (Taylor, 2006, Virology, 344: 71-76), and thus HDV infection can only be seen with concomitant HBV infectionOccurs in stained subjects. While the incidence of HDV co-infection in asymptomatic HBV carriers and chronic HBV-associated liver disease is low in countries with low incidence of HBV infection, it is a significant complication in subjects infected with HBV in countries with high incidence of HBV infection and can increase the rate of progression of liver disease to cirrhosis. In HBV/HDV co-infected subjects, the unmet medical need for HBV infection is even more pressing; there are no specific approved agents that directly target the HDV virus, and patients respond worse than in patients with HBV simple infection even with combination therapy of approved agents for HBV treatment (Wedemeyer et al, 2014, Oral abstrate 4, 49)^thAnnual Meeting of the European Association for the Study of the Liver,April 9-14,London,UK)。

Currently approved therapies for HBV include interferon-alpha or thymosin alpha 1 based immunotherapy, and inhibition of viral production by inhibition of HBV polymerase by nucleoside/nucleotide analogs. HBV polymerase inhibitors can effectively reduce the production of infectious virions, but have little to no effect on reducing HBsAg, or reduce HBsAg very slowly with only a limited number of patients under long-term treatment (Fung et al, 2011, am.J.Gasteroenterol.,106:1766-^thAnnual Meeting of The European Association for The Study of The Liver, April 24-28, Amsterdam, The Netherlands). The primary role of HBV polymerase inhibitors is to block the conversion of pregenomic viral mRNA into partially double-stranded DNA that is present in infectious virions. Interferon-based immunotherapy can achieve a reduction in infectious virus and removal of HBsAg from the blood, but only in a small percentage of treated subjects.

HBsAg in the blood can sequester anti-HBsAg antibodies and allow infectious viral particles to escape immunodetection, which may be one of the reasons that HBV infection remains a chronic condition. In addition, HBsAg, HBeAg and HBcAg all have immunosuppressive properties as described below, and the persistence of these viral proteins in the patient's blood after administration of any of the currently available treatments for HBV described above may have a significant impact on the immune control that prevents the patient from achieving their HBV infection.

Although all three major HBV proteins (HBsAg, HBeAg and HBcAg) have immunosuppressive properties (see below), HBsAg accounts for the vast majority of HBV proteins in the circulation of HBV infected subjects and is likely to be a major mediator of the host immune response to HBV infection. Although removal of HBeAg, appearance of anti-HBe, or reduction of serum viremia is not associated with the development of continued control of HBV infection after cessation of treatment, removal of serum HBsAg from the blood (and appearance of free anti-HBsAg antibodies) in HBV infection is a well-recognized superior prognostic indicator of antiviral response at the time of treatment, which, after cessation of treatment, will result in control of HBV infection (although this will only occur in a small fraction of patients receiving immunotherapy or HBV polymerase inhibitors). Thus, while reduction of all three major HBV proteins (HBsAg, HBeAg and HBcAg) may lead to optimal removal of the inhibitory effect, removal of HBsAg is necessary and its removal alone may be sufficient to remove most of the inhibition of immune function in subjects with HBV infection.

Another key feature of chronic HBV infection is the establishment of a stable repository of HBV genetic information (reservoir), called covalently closed circular dna (cccdna), in the nucleus of infected cells. cccDNA exists in multiple copies in the nucleus as an extrachromosomal episome, which serves as a transcription template for the production of mRNA encoding all viral proteins and as an immature genome (pregenomic mRNA) for the production of new virions. After encapsidation in the cytoplasm, the immature pregenomic mRNA is converted to a mature partially double-stranded DNA genome by HBV polymerase (which is co-encapsidated with the pregenomic mRNA), thereby enabling the mature HBV genome to have the capacity to establish or replenish the cccDNA reservoir in the original or previously infected cells. The end of the infection process consists of the transport of this part of the double stranded genomic HBV template into the nucleus and its conversion into cccDNA.

cccDNA can be replenished in the nucleus of infected cells via nuclear import of HBV capsid containing mature HBV genome that supplements cccDNA copy number. This nuclear cccDNA supplementation can be achieved by two mechanisms: the assembled capsid is either transfused directly from the cytoplasm into the nucleus, or the previously infected hepatocyte is reinfected, and the internalized capsid is then shuttled into the nucleus (Rabe et al, 2003, proc. natl. acad. sci. usa,100: 9849-. Transcriptional inhibition or elimination of this genomic HBV reservoir in the nucleus is crucial to establishing long-term control of HBV infection after treatment.

Long-term treatment with nucleoside/nucleotide HBV polymerase inhibitors can reduce cccDNA copy number in the nucleus, consistent with HBV polymerase inhibitors being able to block the recruitment of cccDNA through the nucleus into the capsid containing the mature HBV genome. However, although cccDNA copy number/hepatocytes is reduced, it still retains transcriptional activity, so HBsAg levels are largely unaffected (Werle-Lapostole et al, 2004, Gastronerol., 126:1750-^thAnnual Meeting of the European Association for the Study of the Liver, April 9-14, London, UK). cccDNA transcription can be inactivated by immune-mediated processes (Belloni et al, 2012, j.clin.inv.,122: 529-.

Thus, there is a clear medical need for a treatment regimen that can elicit durable immune control of HBV infection in a large proportion of patients receiving such treatment.

Disclosure of Invention

In accordance with the present description, there is now provided a composition for treating HBV infection or HBV/HDV co-infection in a subject comprising a first pharmaceutically acceptable agent comprising at least one phosphorothioate nucleic acid polymer; and a second pharmaceutically acceptable agent comprising at least one nucleoside/nucleotide analog HBV polymerase inhibitor.

Also provided are compositions for treating HBV infection or HBV/HDV co-infection in a subject comprising a first pharmaceutically acceptable agent comprising a chelating complex of at least one phosphorothioate nucleic acid polymer; and a second pharmaceutically acceptable agent comprising at least one nucleoside/nucleotide analog HBV polymerase inhibitor.

Further provided is the use of a first pharmaceutically acceptable agent comprising at least one phosphorothioate nucleic acid polymer and a second pharmaceutically acceptable agent comprising at least one nucleoside/nucleotide analogue HBV polymerase inhibitor for the treatment of HBV infection or HBV/HDV co-infection in a subject.

There is further provided the use of a first pharmaceutically acceptable agent comprising at least one phosphorothioate nucleic acid polymer and a second pharmaceutically acceptable agent comprising at least one nucleoside/nucleotide analogue HBV polymerase inhibitor in the manufacture of a medicament for the treatment of HBV infection or HBV/HDV co-infection in a subject.

Further provided is the use of a first pharmaceutically acceptable agent comprising a chelating complex of at least one phosphorothioated nucleic acid polymer and a second pharmaceutically acceptable agent comprising at least one nucleoside/nucleotide analog HBV polymerase inhibitor for treating HBV infection or HBV/HDV co-infection in a subject.

There is further provided the use of a first pharmaceutically acceptable agent comprising at least one phosphorothioate esterified nucleic acid polymer chelate complex and a second pharmaceutically acceptable agent comprising at least one nucleoside/nucleotide analogue HBV polymerase inhibitor in the manufacture of a medicament for the treatment of HBV infection or HBV/HDV co-infection in a subject.

In another embodiment, there is provided a composition for treating HBV infection or HBV/HDV co-infection comprising a first pharmaceutically acceptable agent comprising one or more chelating complexes of nucleic acid polymers selected from the group consisting of:

SEQ ID NO：2；

SEQ ID NO：10；

SEQ ID NO：13；

SEQ ID NO: 1. 3-9, 11, 12 and 14-20;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of sequence AC;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of sequence CA;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising repeats of the sequence TG; and

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of the sequence GT;

and a second pharmaceutically acceptable agent comprising one or more of:

lamivudine (lamivudine);

adefovir dipivoxil (adefovir dipivoxil);

entecavir (entecavir);

telbivudine (telbivudine);

tenofovir disoproxil fumarate (tenofovir disoproxil fumarate);

emtricitabine (entricitabine);

clevudine (clevudine);

besnfovir (besifovir);

tenofovir alafenamide fumarate (tenofovir alafenamide fumarate);

AGX-1009；

elvucitabine (elvucitabine);

vallatate (lagociclovir valactate);

pradefovir mesylate (pradefovir mesylate);

valtocitabine (valtocitabine); and

any nucleoside/nucleotide analogs that inhibit HBV polymerase.

In an embodiment, there is provided the use of a first pharmaceutically acceptable agent comprising a chelating complex of one or more nucleic acid polymers selected from the group consisting of:

SEQ ID NO：2；

SEQ ID NO：10；

SEQ ID NO：13；

SEQ ID NO: 1. 3-9, 11, 12 and 14-20;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of sequence AC;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of sequence CA;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising repeats of the sequence TG; and

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of the sequence GT;

and the second pharmaceutically acceptable agent comprises one or more of:

lamivudine;

adefovir dipivoxil;

entecavir;

telbivudine;

tenofovir disoproxil fumarate;

emtricitabine;

(ii) clevudine;

besnfovir;

tenofovir alafenamide fumarate;

AGX-1009；

elvucitabine;

vallatrine, vallatate;

(ii) peradfovir mesylate;

(ii) valtocitabine; and

any nucleoside/nucleotide analogs that inhibit HBV polymerase.

In another embodiment, there is provided a use of a first pharmaceutically acceptable agent comprising a chelating complex of one or more nucleic acid polymers selected from the group consisting of:

SEQ ID NO：2；

SEQ ID NO：10；

SEQ ID NO：13；

SEQ ID NO: 1. 3-9, 11, 12 and 14-20;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of sequence AC;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of sequence CA;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising repeats of the sequence TG; and

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of the sequence GT;

and the second pharmaceutically acceptable agent comprises one or more of:

lamivudine;

adefovir dipivoxil;

entecavir;

telbivudine;

tenofovir disoproxil fumarate;

emtricitabine;

(ii) clevudine;

besnfovir;

tenofovir alafenamide fumarate;

AGX-1009；

elvucitabine;

vallatrine, vallatate;

(ii) peradfovir mesylate;

(ii) valtocitabine; and

any nucleoside/nucleotide analogs that inhibit HBV polymerase.

In another embodiment, the nucleic acid polymer comprises a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of sequence AC.

In another embodiment, the nucleic acid polymer comprises a phosphorothioate oligonucleotide 20-120 nucleotides in length comprising a repeat of the sequence CA.

In another embodiment, the nucleic acid polymer comprises a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of the sequence TG.

In another embodiment, the nucleic acid polymer comprises a phosphorothioate oligonucleotide 20-120 nucleotides in length comprising a repeat of the sequence GT.

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

In another embodiment, the phosphorothioate nucleic acid polymer further comprises all ribose sugars with a 2' modification.

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

In another embodiment, the phosphorothioate nucleic acid polymer further comprises all ribose sugars with a 2' O methyl modification.

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

In another embodiment, the phosphorothioate nucleic acid polymer further comprises all cytosines present as 5' methylcytosine.

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

In another embodiment, the phosphorothioate nucleic acid polymer further comprises all riboses having a 2'O methyl modification and all cytosines present as 5' methylcytosine.

In another embodiment, the nucleic acid polymer is selected from the group consisting of SEQ ID NOs: 1-20.

In another embodiment, the nucleic acid polymer is prepared to comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-20 oligonucleotide chelate complexes.

In another embodiment, the nucleic acid polymer consists of SEQ ID NO: 2.

In another embodiment, the nucleic acid polymer is prepared to comprise SEQ ID NO: 2.

In another embodiment, the nucleic acid polymer consists of SEQ ID NO: 10.

In another embodiment, the nucleic acid polymer is prepared to comprise SEQ ID NO: 10 of an oligonucleotide chelate complex.

In another embodiment, the nucleic acid polymer consists of SEQ ID NO: 13 in a pharmaceutically acceptable carrier.

In another embodiment, the nucleic acid polymer is prepared to comprise SEQ ID NO: 13 to the oligonucleotide chelate complex.

In one embodiment, the chelating complex is a calcium chelating complex.

In another embodiment, the chelate complex is a magnesium chelate complex.

In further embodiments, the chelate complex is a calcium/magnesium chelate complex.

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

In further embodiments, the first agent and the second agent are formulated in separate pharmaceutical compositions.

In further embodiments, the first agent and the second agent are formulated for administration simultaneously.

In further embodiments, the first agent and the second agent are formulated for administration by different routes.

In further embodiments, the first and second agents are formulated for administration using one or more of the following: oral ingestion, aerosol inhalation, subcutaneous injection, intravenous injection, and intravenous infusion.

In further embodiments, the nucleic acid polymer is at least one of:

SEQ ID NO：2；

SEQ ID NO：10；

SEQ ID NO：13；

SEQ ID NO: 1. 3-9, 11, 12 and 14-20;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of sequence AC;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of sequence CA;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising repeats of the sequence TG; and

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of the sequence GT.

In further embodiments, the following nucleic acid polymers may be further formulated as oligonucleotide chelate complexes:

SEQ ID NO：2；

SEQ ID NO：10；

SEQ ID NO：13；

SEQ ID NO: 1. 3-9, 11, 12 and 14-20;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of sequence AC;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of sequence CA;

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising repeats of the sequence TG; and

a phosphorothioate oligonucleotide of 20-120 nucleotides in length comprising a repeat of the sequence GT.

In another embodiment, the nucleoside/nucleotide analog HBV polymerase inhibitor comprises one or more of:

lamivudine;

adefovir dipivoxil;

entecavir;

telbivudine;

tenofovir disoproxil fumarate;

emtricitabine;

(ii) clevudine;

besnfovir;

tenofovir alafenamide fumarate;

AGX-1009；

elvucitabine;

vallatrine, vallatate;

(ii) peradfovir mesylate;

(ii) valtocitabine; and

any nucleoside/nucleotide analogs that inhibit HBV polymerase.

Drawings

FIG. 1 shows the synergistic effect of treatment with a combination of NAP REP 2055(SEQ ID NO:2) and Entecavir (ETV) on the reduction of serum levels of HBsAg.

Fig. 2A shows the antiviral activity of NAP administered as a calcium chelating complex to a beijing duck (Pekin duck) infected with DHBV, measured by monitoring serum DHBsAg by ELISA at the end of treatment.

Fig. 2B shows the antiviral activity of NAP administered as a calcium-chelating complex to a beijing duck infected with DHBV, assessed by monitoring liver DHBV DNA by quantitative PCR at the end of treatment.

Figure 3A shows DHBV DNA levels in duck serum treated with saline for 28 days at the following times: A) before treatment, B) when treatment is half complete, C) at the end of treatment, D) one month after treatment and E) two months after treatment. The lower limit of quantization (LLOQ) was 3.1X 10⁴VGE/ml. The value less than LLOQ is set at 3X 10³VGE/ml. VGE ═ viral genome equivalents.

Figure 3B shows DHBV DNA levels in duck serum treated with Tenofovir Disoproxil Fumarate (TDF) for 28 days at the following times: A) before treatment, B) when treatment is half complete, C) at the end of treatment, D) one month after treatment and E) two months after treatment. The lower limit of quantization (LLOQ) was 3.1X 10⁴VGE/ml. The value less than LLOQ is set at 3X 10³VGE/ml. VGE ═ viral genome equivalents.

FIG. 3C shows DHBV DNA levels in duck serum treated with REP 2139-Ca for 28 days at the following times: A) before treatment, B) when treatment is half complete, C) at the end of treatment, D) one month after treatment and E) two months after treatment. The lower limit of quantization (LLOQ) was 3.1X 10⁴VGE/ml. The value less than LLOQ is set at 3X 10³VGE/ml. VGE ═ viral genome equivalents.

FIG. 3D shows the following time advantageDHBV DNA levels in duck serum treated with REP 2139-Ca and TDF for 28 days: A) before treatment, B) when treatment is half complete, C) at the end of treatment, D) one month after treatment and E) two months after treatment. The lower limit of quantization (LLOQ) was 3.1X 10⁴VGE/ml. The value less than LLOQ is set at 3X 10³VGE/ml. VGE ═ viral genome equivalents.

FIG. 3E shows DHBV DNA levels in duck serum treated with REP 2139-Ca, TDF and Entecavir (ETV) for 28 days at the following times: A) before treatment, B) when treatment is half complete, C) at the end of treatment, D) one month after treatment and E) two months after treatment. The lower limit of quantization (LLOQ) was 3.1X 10⁴VGE/ml. The value less than LLOQ is set at 3X 10³VGE/ml. VGE ═ viral genome equivalents.

Detailed Description

Provided herein is a combination therapy against HBV infection consisting of administering a first pharmaceutically acceptable agent capable of removing HBsAg from the blood and a second pharmaceutically acceptable agent that inhibits HBV polymerase. Such combined treatment allows restoration of host immune function (by removal of serum HBsAg), which in turn leads to immune-mediated transcriptional inactivation of cccDNA and or reduction of cccDNA copy number in infected hepatocytes, while simultaneously either infusing capsid containing mature HBV genome via the nucleus or producing infectious virus (by inhibiting HBV polymerase) blocking cccDNA supplementation. The synergistic effect of the combination of these two drugs can accelerate the antiviral response to treatment and or eliminate cccDNA of infected cells, thereby shortening the treatment time needed to obtain sustained inhibition of infection after treatment is stopped. Importantly, these effects can be achieved in the absence of immunotherapy. Such combination therapy would be effective against both HBV-only infection and HBV/HDV co-infection.

HBsAg plays a key role in HBV infection and HBV/HDV co-infection. In addition to its role as an essential structural component of virosome formation, HBsAg is also released in large quantities into the blood of infected subjects in the form of subviral particles (SVP) which lack viral capsid and genome and which appear to be primarily used for the delivery of HBsAg into the blood. Infected cells secrete SVP in excess of 1,000-10,000 fold over virus secretion, allowing SVP to effectively sequester HBsAg antibodies (anti-HB), allowing HBV or HDV viruses in the blood to escape recognition due to adaptive immunity. Some studies have also shown that HBsAg can also be used to directly block The adaptation of HBV infection and activation of innate immune responses (Cheng et al, 2005, Journal of Hepatology,43: 465-471; Op den Brouw et al, 2009, Immunology,126: 280-289; Vanlandschoot et al, 2002, The Journal of genetic virology,83: 1281-1289; Wu et al, 2009, Hepatology,49: 1132-1140; Xu et al, 2009, Molecular Immunology,46: 2640-2646). The presence of this functionality in human HBV infection and its effect on the activity of immunotherapeutic agents and the additional applicability of these antiviral effects in HBV/HDV co-infection have been previously described in US 2014/0065102 a1, which is incorporated herein by reference in its entirety. Although HBeAg and HBcAg have also been shown to have immunosuppressive properties (Kanda et al 2012, j.inf.dis.,206: 415-.

Nucleoside/nucleotide analogue inhibitors of HBV polymerase (NRTI) are a well-known class of antiviral agents, the activity against HBV infection occurring through the same mechanism of action: such compounds act as immediate or delayed chain terminators by competing with the natural nucleotide substrates during DNA strand elongation (Menendez-Arias et al, 2015Curr. Op. Virol.8: 1-9). Such compounds may retain the basic core nucleotide/nucleoside core structure consisting of a nitrogenous base and a sugar, or may be acyclic nucleotides, or may lack a sugar or pseudo-sugar ring, or may have a phosphonate group substituted for the alpha-phosphate and may have a number of other additional modifications, as described in michaillis et al, 2012int.j.biochem.cell.biol.44: 1060-.

Duck HBV Virus (DHBV) infected ducks are a well-established HBV infection model and have been used to evaluate several HBV NRTI currently used in the treatment of human patients (Schultz et al, 2004, Adv Virus Res,63: 1-70; Foster et al, 2005, J Virol,79: 5819-. Phosphorothioate Nucleic Acid Polymers (NAP) have been shown to have antiviral activity in DHBV infected ducks (Noordeen et al, 2013Anti-Microb. Agents Chemother.57: 5291-. In addition, therapeutic intervention with NAP REP 2055(SEQ ID NO:2) in previously established DHBV infections in vivo, REP 2055 can clear serum duck HBsAg (DHBsAg), which is accompanied by transcriptional inactivation of cccDNA and a reduction in cccDNA copy number (Noordeen et al, 2009, Abstract 88 HEPPAT recording Dec 6-9, HI, USA). This priming and elimination of cccDNA is caused by the removal of DHBsAg-mediated inhibition of host immune function, and then the cccDNA of infected cells can be inactivated and cleared by a well-recognized immune-mediated mechanism (Levrero et al, 2009, j.hepatol.,51: 581-592; Belloni et al, 2012, j.clin.inv.,122: 529-537).

NAP for effective removal of HBsAg from the blood of human patients as described in US 2014/0065102. In a recognized preclinical HBV infection model (duck HBV infected beijing duck), NAP treatment can eliminate serum duck hbsag (DHBsAg), and restoration of immune function in the absence of serum DHBsAg can transcriptionally inactivate and eliminate cccDNA of infected hepatocytes (Noordeen et al, 2009, Abstract 88, HEPDART meeting Dec 6-10, HI, USA). Therefore, removal of HBsAg from the serum of HBV infected patients is expected to have the same effect on cccDNA inactivation in situ infected human hepatocytes.

Thus, described herein is an effective means for faster establishment of control of serum viremia or for establishment of persistent control of cccDNA activity and or elimination of cccDNA of HBV infected hepatocytes consisting of a novel combinatorial approach as follows: wherein HBsAg is reduced or removed from the blood by using a pharmaceutically acceptable phosphorothioate NAP preparation and cccDNA supplementation and infectious virus production is blocked by a second pharmaceutically acceptable nucleotide/nucleoside analogue preparation that inhibits HBV polymerase. This combined approach has the following novel and important advantages:

1) it combines the following capabilities: improving host immune function (caused by removal of serum HBsAg) to transcriptionally inactivate intracellular cccDNA and or reduce intracellular cccDNA copy number, and blocking cccDNA replenishment (by preventing capsid containing mature genome from entering nucleus (by inhibiting HBV polymerase activity) or infectious virion production (by preventing pregenomic RNA from converting to partially double stranded DNA within HBV capsid);

2) it has a synergistic effect on the reduction of the removal of infected hepatocytes from the liver, elimination or establishment of transcriptional inhibition of cccDNA or the duration of treatment required for control of serum viremia due to the overlapping effect of the two pharmaceutically acceptable agents; and

3) it does not require the use of immunotherapy (as taught specifically needed in u.s.2014/0065102) to achieve sustained control of HBV infection after treatment, which would be an important therapeutic improvement given that immunotherapy is poorly tolerated in many patients.

The antiviral effect improved by the above method will have the same therapeutic benefit in patients with HBV-only infection and HBV/HDV co-infection, since HDV infection cannot be present in the absence of the above HDV infection.

Thus, in the absence of any current treatment regimen that can abrogate or establish persistent control of cccDNA activity in a large proportion of patients without the use of immunotherapy, the present document provides for the first time an effective combination therapy against HBV infection and HBV/HDV co-infection that simultaneously reduces or eliminates HBsAg from the blood and which blocks cccDNA recruitment in the nucleus of HBV infected cells. These effects can be achieved by using a pharmaceutically acceptable formulation of phosphorothioate NAP in combination with a pharmaceutically acceptable nucleoside/nucleotide analog HBV polymerase inhibitor.

This novel combination approach is effective in the absence of immunotherapy, which has the important advantages of improved therapeutic tolerance and reduced incidence of known hematological and other side effects that occur with immunotherapy.

The term Oligonucleotide (ON) refers to an oligomer or polymer of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA). The term includes ONs consisting of modified nucleobases (including 5 'methylcytosine and 4' thiouracil), sugars and covalent internucleoside (backbone) linkages, as well as functionally similar ONs having non-naturally occurring moieties. Such modified or substituted ONs may be preferred over the native form due to desirable properties such as: for example, decreased immune reactivity, increased cellular uptake, increased affinity for nucleic acid targets (in the case of antisense ON, siRNA and shRNA), and/or increased stability to nuclease-mediated degradation. The ON may also be double stranded. ONs also include single-stranded molecules such as antisense oligonucleotides, Speigelmers, and aptamers, and mirnas, as well as double-stranded molecules such as small interfering rnas (sirnas) or small hairpin rnas (shrnas).

An ON may include various modifications, such as stabilizing modifications, and thus may include at least one modification in a phosphodiester linkage and/or ON a sugar and/or base. For example, an ON may include, but is not limited to, one or more modifications, or may be fully modified to contain all linkages or sugars or bases having the described modifications. Modified linkages may include phosphorothioate linkages and phosphorodithioate linkages. When a modified linkage is useful, the ON may comprise a phosphodiester linkage. Additional useful modifications include, but are not limited to, 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 are also well known in the art and may be used, such as locked nucleic acids. Specifically, ONs have modified linkages throughout or each linkage modified, e.g., phosphorothioates; having a 3 '-cap and/or a 5' -cap; including a terminal 3'-5' linkage; an ON is or comprises a concatemer of two or more ON sequences joined together by a linker. Base modifications may include 5 'methylation of cytosine bases (5' methylcytosine or 5 'methylcytidine in the case of nucleotides) and/or 4' sulfylation of uracil bases (4 'thiouracil or 4' thiouridine in the case of nucleotides). When the synthesis conditions are chemically compatible, different chemically compatible modified linkages can be combined, for example, oligonucleotides having modified bases with phosphorothioate linkages, 2' ribose modifications (e.g., 2' O-methylation), and modified bases (e.g., 5' methylcytosine). All of these different modifications (e.g., each phosphorothioate linkage, each 2' modified ribose, and each modified base) can be used to further fully modify the ONs.

As encompassed herein, the term "nucleic acid polymer" or NAP is any single-stranded ON that does not contain sequence-specific functionality to hybridize to a nucleic acid target or to employ sequence-specific secondary structures that result in binding to a particular protein. The biochemical activity of NAP is not dependent ON Toll-like receptor recognition of ONs, hybridization to target oligonucleotides, or aptamer interactions that require specific secondary/tertiary ON structures derived from the specific order of nucleotides present. NAP may include base and or bond or sugar modifications as described above. NAP requires phosphorothioate to have antiviral activity. Exemplary antiviral NAP compounds are listed in table 1:

table 1

Examples of antiviral NAPs useful in the present disclosure

dA ═ deoxyadenosine, a ═ adenosine, dC ═ deoxycytidine, C ═ cytidine, dT ═ deoxythymidine, dG ═ deoxyguanosine, PS ═ phosphorothioate, 2'OMe ═ 2' omethyl, 5'MeC ═ 5' methylcytosine modified cytidine, 5'MedC ═ 5' methylcytosine modified deoxycytidine

In the present disclosure, the term "ON chelate complex" refers to two or more ONs that are connected intermolecularly by a divalent or polyvalent metal cation and that can occur under single-or double-stranded ONs. The ON chelate complex neutralizes the inherent chelating properties of ONs, which can help take advantage of the application-related side effects of these compounds. Administration of ON chelate complexes is a method of administering ON to a subject, wherein side effects associated with administration associated with unchelated ON (which is ON administered as a sodium salt commonly used in the art) are reduced, as described in u.s.8,513,211 and 8,716,259, which are incorporated herein by reference in their entirety. These side effects may include chills, fever and cold under intravenous infusion or induration, inflammation and pain at the injection site under subcutaneous administration. The administration of the ON chelate complex does not interfere with the biochemical activity of the ON when it is used as a sodium salt in general. Thus any of the NAPs described herein may optionally be prepared as ON chelate complexes without affecting their biochemical activity.

The ON chelate complex can contain a variety of different polyvalent metal cations, including calcium, magnesium, cobalt, iron, manganese, barium, nickel, copper, zinc, cadmium, mercury, and lead. It is further shown that chelation of these polyvalent metal cations results in the formation of an ON chelate complex comprising two or more ONs linked via the metal cations, and occurs under ONs greater than 6 nucleotides in length and in the presence of ONs having phosphodiester or phosphorothioate linkages. The ONs may optionally have each phosphorothioate linkage. Chelation also occurs under ON with a 2' modification at the ribose (e.g., 2' O methyl) or with a modified base, such as 5' methylcytosine or 4-thiouracil. These 2 'modifications can be present on one or more or all of the ribose sugars, and the modified base can be present on one or more bases or ubiquitous on each base (i.e., all cytosines are present as 5' methylcytosine). In addition, the ON chelate complex may comprise an ON comprising a plurality of modifications, such as each phosphorothioate linkage, each 2' modified ribose, and each modified base. ON modifications compatible with ON chelate complex formation are further defined above. Furthermore, the chelation of metal cations is not dependent ON the sequence of nucleotides present but rather ON physicochemical features common to all ONs.

While the formation of the ON chelate complex can be achieved with any divalent metal cation, the ON chelate complex intended for use as a medicine should preferably contain only calcium and or magnesium, but may also contain minor amounts of iron, manganese, copper or zinc and should not include cobalt, barium, nickel, cadmium, mercury, lead or any other divalent metal not listed herein.

As described in u.s.2014/0065192, removal of HBsAg from the blood of infected patients by phosphorothioate-esterified NAP results in partial restoration of the immune response, thereby removing HBVe-antigen (HBeAg) from the blood and resulting in a substantial reduction in the level of virus in the blood during treatment, but these antiviral effects are not maintained in most patients after treatment is discontinued. While this partial recovery of the immune response (in the absence of HBsAg and other viral antigens) may allow the establishment of persistent immunological control of HBV infection in a small fraction of patients after cessation of treatment, it is desirable to establish persistent immunological control of infection in an even larger fraction of patients. An improvement in the proportion of patients with persistent immunological control after treatment can be achieved by: the use of phosphorothioate-esterified NAP in combination with other antiviral agents improves the speed and efficacy of the antiviral response to treatment. It may be desirable to avoid immunotherapy, such as interferon-based therapy, or other immunotherapy, as these therapies are often associated with side effects that make it more difficult for patients to tolerate the therapy.

The term "removal of HBsAg from blood" as used herein refers to any statistically significant reduction in HBsAg concentration in blood relative to the concentration of HBsAg in blood prior to treatment, such as by Abbott Architect^TMQuantitative HBsAg analysis or other clinically approved quantitative measurements of serum HBsAg.

An exemplary effective dosing regimen for phosphorothioate-NAPs follows those typically used for other phosphorothioate-ONs (such as antisense oligonucleotides) as described in u.s.2014/0065102; the conventional use of 100-500mg compounds administered parenterally weekly is well established in the art to achieve therapeutically active levels of these compounds in the liver as described for NAP in example I below and for phosphorothioate antisense ON (for apolipoprotein B100) which causes degradation of liver-specific mRNA as described by Akdim et al (2010, Journal of the American College of medicine, 55: 1611-1618).

Thus, in accordance with the disclosure presented herein, it is useful to treat subjects with HBV infection or HBV/HDV co-infection with a pharmaceutically acceptable phosphorothioate NAP formulation in combination with a pharmaceutically acceptable nucleoside/nucleotide HBV polymerase inhibitor.

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

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

In order to provide the most likely antiviral response in a subject, it may be necessary to use more than one HBV polymerase inhibitor to maximally block HBV polymerase and thus have the greatest effect on blocking cccDNA supplementation. Thus, the one or more HBV polymerase inhibitors may be selected from the following nucleoside analogues:

lamivudine;

adefovir dipivoxil;

entecavir;

telbivudine;

tenofovir disoproxil fumarate;

emtricitabine;

(ii) clevudine;

besnfovir;

tenofovir alafenamide fumarate;

AGX-1009；

elvucitabine;

vallatrine, vallatate;

(ii) peradfovir mesylate;

(ii) valtocitabine; and

any nucleoside/nucleotide analogs that inhibit HBV polymerase.

The compositions described herein may be administered by any suitable means, e.g., orally, such as in tablet, capsule, granule, or powder form; under the tongue; transbuccal; parenterally, such as by subcutaneous, intravenous, injection, or infusion techniques (e.g., as sterile injectable aqueous or nonaqueous solutions or suspensions); by inhalation; topically, e.g., in the form of a cream or ointment; or rectally, e.g., in the form of suppositories or enemas; in dosage unit formulations containing non-toxic pharmaceutically acceptable carriers or diluents. For example, the present compositions may be administered in a form suitable for immediate release or extended release. Immediate release or extended release can be achieved by the use of suitable pharmaceutical compositions or, particularly in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps. Thus, the above compositions may be suitable for administration by any of the following routes: oral ingestion, inhalation, subcutaneous injection, intravenous injection or infusion, or topical.

The disclosure will be more readily understood by reference to the following examples.

Example I

Effect of Combined NAP/ETV treatment on serum HBsAg

A pharmaceutically acceptable formulation of NAP REP 2055(SEQ ID NO:2) was administered to patients with chronic HBV infection by IV infusion of 400mg once a week. Serum HBsAg response in this patient was monitored in real time weekly using a qualified field qualitative ELISA. This ELISA method is very sensitive to low levels of HBsAg, but does not accurately quantify any significant HBsAg concentration in the blood. Although no detectable reduction in serum HBsAg was observed using this HBsAg assay during the REP 2055 monotherapy (fig. 1, square), the patient experienced a very mild (about 1log) reduction in serum viremia (serum HBV DNA), indicating that some antiviral response had occurred. Thus, after 29 weeks of REP 2055 monotherapy, the patient receives HBV polymerase inhibition therapy in addition to the current REP 2055 therapy consisting of 0.5mg entecavir orally per day.

An immediate reduction in serum HBsAg was detected by qualitative analysis within two weeks of the initial combination REP 2055/ETV treatment and serum HBsAg became undetectable in the qualitative ELISA within 4 weeks after the start of the combination treatment (figure 1, squares). This synergistic control of serum HBsAg by the combined REP 2055/ETV treatment is maintained over many weeks of treatment.

To demonstrate the synergistic activity of the combination REP 2055/ETV treatment on inhibiting HBsAg, serum samples from this patient were re-analyzed using the IMPACT platform to accurately quantify serum HBsAg levels as described in de Neit et al (2014, anti ther.,19: 259-267). This quantitative analysis showed an initial approximately 2log reduction in serum HBsAg that occurred with REP 2055 monotherapy (fig. 1 circle), which was undetectable by qualitative ELISA and which may be responsible for the approximately 1log reduction in viremia observed after the REP 2055 monotherapy described above. Importantly, serum HBsAg reduction in this patient reaches a plateau where significant serum HBsAg is stable from the start of 10 weeks of REP 2055 treatment until the start of combined REP 2055/ETV treatment at 29 weeks of treatment. Quantitative analysis of serum HBsAg at the start of combined REP 2055/ETV treatment showed almost identical and rapid reductions in serum HBsAg, as observed with field qualitative testing, and these additional reductions exceeded 1.5 logs, which could also be achieved within 4 weeks after the start of combined REP 2055/ETV treatment.

Persistence of low serum HBsAg levels in the presence of REP 2055 monotherapy indicates that transcriptionally active cccDNA is still present in the liver of the patient. The very rapid additional clearance of serum HBsAg by adding ETV to the existing REP 2055 treatment indicates a synergistic effect on cccDNA transcriptional control and or that elimination has occurred. Importantly, the occurrence of this additionally controlled cccDNA occurred much faster than observed with HBV polymerase inhibitors used in monotherapy, which only took 4 weeks to achieve. Thus, these observations demonstrate a novel synergistic antiviral effect on serum HBsAg reduction (achieved using NAP REP 2055 in this case) when combined with HBV polymerase inhibitors (entecavir in this case).

Example II

Antiviral effects of various NAPs in DHBV infected Beijing ducks

Various NAPs containing different nucleic acid modifications were tested in DHBV infected beijing ducks to establish their antiviral activity. These NAPs are REP 2055(SEQ ID NO:2), REP 2139(SEQ ID NO: 10), REP 2163(SEQ ID NO: 11) and REP 2165(SEQ ID NO: 13). Table 2 provides a chemical description of these NAPs.

TABLE 2

Description of NAP used in example II

dA ═ deoxyriboadenosine

dC ═ deoxyribonucleotide

A-riboadenosine

5 'MeC-Ribose-5' Methylcytidine

5 'MedC-5' methylcytidine

By 2X 10¹¹DHBV of individual Viral Genome Equivalents (VGE)/ml infects three days old beijing ducklings. It has been established that NAP treatment is initiated 11 days after infection. NAP was administered via intraperitoneal injection of 10mg/kg NAP (formulated as a calcium chelate complex) 3 times per week for three weeks, followed by analysis of antiviral effect at the end of treatment. The control group was treated with saline via the same route of administration and with the same dosing regimen. Antiviral activity was assessed by monitoring serum DHBsAg by ELISA (fig. 2A) and liver DHBV DNA by quantitative PCR (fig. 2B).

All NAPs resulted in a reduction of serum DHBsAg and liver DHBV DNA, suggesting that different NAPs containing multiple different oligonucleotide modifications would have comparable antiviral effects. This in turn suggests that the synergistic antiviral activity observed with particular NAPs and HBV polymerase inhibitors based on one or more nucleoside analogs (as observed with REP 2055 and entecavir in example I above) will occur under any other phosphorothioate NAP and also under any of the phosphorothioate NAPs formulated as chelating complexes (as described in u.s.8,513,211 and 8,716,259).

Example III

Antiviral effects of NAP in combination with TDF and ETV in DHBV infected Beijing ducks

The antiviral effect of calcium chelate complex of REP 2139 (REP 2139-Ca) and TDF or combination of REP 2139-Ca and TDF with ETV treatment in DHBV infected beijing ducks was examined by assessing changes in serum and liver DHBV DNA levels during and after treatment by quantitative PCR. Infection of ducks was performed as described in example II, except that treatment was initiated one month after infection. The treatment regimen was as follows:

1) physiological saline, administered by IP injection 3 times per week for 4 weeks

2) TDF administered by oral gastric tube feeding at a dose of 15 mg/day for 28 days

3) REP 2139-Ca, administered by IP injection at 10mg/kg 3 times a week for 4 weeks.

4) REP 2139-Ca and TDF (administered as above)

5) REP 2139-Ca and TDF (administered as above), and ETV, were administered by orogastric tube feeding at 1 mg/day for 28 days.

Serum DHBV DNA was evaluated before treatment (time point a), on day 14 of treatment (time point B), at the end of treatment (time point C) and one and two months after treatment cessation (follow-up, time points D and E).

In the saline treated group, no control of DHBV DNA was observed during treatment, but DHBV DNA became spontaneously controlled in 3 ducks in this group during follow-up (fig. 3A). In the TDF treatment group, serum DHBV DNA was reduced in all ducks, but control was not achieved in all ducks until treatment was complete. DHBV DNA rebounded in all ducks in the group during the follow-up period (fig. 3B). In the REP 2139-Ca treatment group, no DHBV DNA changes were observed in both ducks throughout the study, with DHBV DNA being controlled in only two ducks at the end of treatment and becoming spontaneously controlled in two additional ducks during follow-up (fig. 3C). When REP 2139-Ca is combined with TDF, control of DHBV DNA occurs in all but one duck at half the time of treatment and is generally faster than control achieved in groups treated with REP 2139-Ca or TDF alone. When REP 2139-Ca is combined with TDF and ETV, DHBV can be controlled in all ducks half the time the treatment is performed (3E). The proportion of ducks that maintained control of serum DHBV DNA during follow-up with combined REP 2139-Ca and TDF (or TDF and ETV) was greater than with TDF alone or REP 2139-Ca.

These observations teach that the antiviral response during treatment in HBV infection can be synergistically improved by combining REP 2139-Ca and TDF or TDF and ETV and can lead to an improvement in sustained virological response after cessation of treatment, compared to that achieved with REP 2139-Ca or TDF alone. The synergistic activity observed in the above examples can be reliably predicted to occur with any NAP anti-HBV activity as described herein and with any nucleotide/nucleoside analog-based HBV polymerase inhibitor as described herein. In addition, NAP in combination with more than one nucleoside/nucleotide HBV polymerase inhibitor may also be used, with similarly generated synergistic antiviral effects.

The synergistic effect of the combined NAP/TDF/ETV treatment may improve the speed of antiviral response, suggesting the potential to shorten the treatment regimen that enables a sustained virologic response after treatment discontinuation. This potential can also be achieved using any combination of NAP and nucleotide/nucleoside analog HBV polymerase inhibitor therapy as described herein.

Thus, these observations teach that any pharmaceutically acceptable phosphorothioate NAP formulations that reduce or remove HBsAg from the blood (as described in u.s.2014/0065102 and u.s.8,008,269, 8,008,270 and 8,067,385) can be combined with any nucleoside/nucleotide HBV polymerase inhibitors as listed above and are expected to achieve synergistic effects on the rate of controllable serum viremia and or transcriptional inactivation and or elimination of HBV cccDNA. The observed synergistic effect also teaches that lower doses of the pharmaceutically acceptable agents can be combined and still achieve synergistic activity as well as useful antiviral effects.

In view of the synergistic antiviral effects observed above with one phosphorothioate-esterified NAP used in combination with one nucleoside/nucleotide HBV polymerase inhibitor, excellent synergistic effects may also be achieved with one or more phosphorothioate-esterified NAPs used in combination with one or more nucleoside/nucleotide HBV polymerase inhibitors as described above.

The above description is intended to be exemplary only, and those skilled in the art will recognize that changes may be made to the described embodiments without departing from the scope of the invention, which is defined by the appended claims. Other modifications which fall within the scope of the invention as defined by the appended claims will be appreciated by persons skilled in the art from a review of this disclosure without departing from the scope of the invention as defined by the appended claims.

Sequence listing

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