Safety and Efficacy of Nucleic Acid Polymers in Monotherapy and Combined with Immunotherapy in Treatment-Naive Bangladeshi Patients with HBeAg+ Chronic Hepatitis B Infection

Previous in vivo studies have suggested that nucleic acid polymers (NAPs) may reduce circulating levels of HBsAg in the blood by blocking its release from infected hepatocytes and that this effect may have clinical benefit. NAP treatment, was evaluated in two clinical studies in patients with HBeAg positive chronic HBV infection. The REP 101 study examined REP 2055 monotherapy in 8 patients and the REP 102 study examined REP 2139-Ca, in monotherapy in 12 patients, 9 of which transitioned to short term combined treatment with pegylated interferon alpha 2a or thymosin alpha 1. In both studies NAP monotherapy was accompanied by 2–7 log reductions of serum HBsAg, 3–9 log reductions in serum HBV DNA and the appearance of serum anti-HBsAg antibodies (10–1712 mIU / ml). Eight of the 9 patients transitioning to combined treatment with immunotherapy (pegylated interferon or thymosin alpha 1) in the REP 102 study experienced HBsAg loss and all 9 patients experienced substantial increases in serum anti-HBsAg antibody titers before withdrawal of therapy. For 52 weeks after removal of REP 2055 therapy, rebound of serum viremia (HBV DNA > 1000 copies / ml, HBsAg > 1IU / ml) was not observed in 3 / 8 patients. Suppression of serum virema was further maintained for 290 and 231 weeks in 2 of these patients. After withdrawal of all therapy in the 9 patients that transitioned to combination therapy in the REP 102 study, 8 patients achieved HBV DNA < 116 copies / ml after treatment withdrawal. Viral rebound occurred over a period of 12 to 123 weeks in 7 patients but was still absent in two patients at 135 and 137 weeks of follow-up. Administration tolerability issues observed with REP 2055 were rare with REP 2139-Ca but REP 2139-Ca therapy was accompanied by hair loss, dysphagia and dysgeusia which were considered related to heavy metal exposure endemic at the trial site. These preliminary studies suggest that NAP can elicit important antiviral responses during treatment which may improve the effect of immunotherapy. NAPs may be a potentially useful component of future combination therapies for the treatment of chronic hepatitis B. Trial Registration: ClinicalTrials.gov NCT02646163 and NCT02646189


INTRODUCTION
Chronic hepatitis B is a long term condition caused by infection of the body with the hepatitis B virus (HBV). This infection often results in inflammation or scarring of the liver and can eventually lead to liver cirrhosis and liver failure. These infections are also one of the major causes of the development of hepatocellular carcinoma (liver cancer).
Although some drugs have been approved to treat chronic hepatitis B infections, they do not provide a complete cure except in rare cases (a cure generally means that a person loses the hepatitis B virus from the blood and the liver and develops a durable immunological control of subsequent HBV infection). However, these drugs do significantly decrease the risk of liver damage and liver cancer arising from the presence of a chronic liver infection by slowing or stopping the production of infectious virus. Thus the primary problem associated with currently available drugs is the lack of clearance of the virus from the hepatocytes which necessitates long term treatment with these drugs. There is clearly a need to identify new drugs that can benefit patients with chronic hepatitis B infections. Nucleic acid-bases polymers (NAPs) are a new class of broad-spectrum antiviral compounds which act against HBV infection by blocking the release of the surface antigen protein (HBsAg) from infected hepatocytes. In the human patients in the REP 101 protocol, the previous NAP clinical candidate, REP 9AC (REP 2055), rapidly induced pronounced reductions in or clearance of serum HBsAg in 7 out of 8 patients. HBsAg is the major immunoinhibitory mechanism by which HBV maintains its chronicity and by reducing or eliminating HBsAg from the blood, REP 9AC appears able to elicit restoration of durable immunological responses in patients capable of clearing the HBV infection. The performance of REP 9AC in providing sustained virologic responses (SVRs) in patients with chronic HBV infection appeared to be far superior than any compound currently approved for the treatment of HBV infection REP 9AC' (REP 2139) is a modified version of its predecessor, REP 9AC. Both are 40mer phosphorothioate oligonucleotides comprised of alternating adenosine and cytidine nucleotides and in the case of REP 9AC, has been shown to have low toxicity and to be highly effective in treating hepatitis B infection in human patients. The modifications in REP 9AC' significantly improve the stability and reduce the proinflammatory activity of REP 9AC' compared to that of REP 9AC while retaining all the antiviral activity found in REP 9AC. Both these modifications (5-methylation of cytosines and 2' O methylation of the ribose sugar in each nucleotide) are naturally occurring modifications in human nucleic acid and are known to be well tolerated in clinical trials. It is expected that REP 9AC' will be able to achieve a more robust antiviral activity in patients with chronic HBV with significantly lower dosing requirements and fewer side effects than REP 9AC.
Current interim data analysis from the still ongoing REP 101 assessing the activity of REP 9AC in patients with chronic HBV infection indicates the following: 1. REP 9AC exposure in eight patients has been generally well tolerated at doses up to 600mg / week and 400mg / day (for seven continuous days). Administration related side effects include mild to moderate pro-inflammatory reactions during the drug administration (itching and fever) which disappear after drug administration is complete. Chronic side effects include mild elevations in INR (~1.5 X) and serum hypocalcemia (which is easily mitigated with a mineral supplement).
2. REP 9AC has achieved serum HBsAg reduction or clearance in all patients compliant with the proscribed dosing regimen (7 out of 7 REPLICor has validated the compatibility of the modifications in REP 9AC' with the antiviral activity present in NAPs indirectly: the 2'O methyl sugar modification was found to not affect the antiviral activity of NAPs against duck hepatitis B virus in vivo and further shown to substantially improve the stability of NAPs to nuclease degradation and substantially reduce their immunoreactivity in human PBMCs. The 5'methylation of cytosine was shown not to affect target interaction in cell free interaction assays and is well known to mitigate the immunoreactivity of nucleic acids. REPLICor has verified the cGMP manufacture of REP 9AC' which will be certified for human parenteral administration. The biodistrubution of REP 9AC' is similar to other compounds from the same chemical class (phosphorothioate oligonucleotides) which achieve long lasting, therapeutic liver concentrations typically using a single 200-400mg dose delivered once every week or once every other week in human patients. However, it is expected that REP 9AC' will not suffer from the degradation occurring in REP 9AC (shortening of the polymer from the ends) which will result in fully active compound being present for a much longer time.

Primary objective
To demonstrate that REP 9AC' is well tolerated when given intravenously or subcutaneously to patients infected with chronic hepatitis B.

Secondary objective
To evaluate the effect of REP 9AC' administration on the reduction of serum HBsAg and subsequent restoration of immunological control over chronic HBV infection (the detection of anti-HBsAg antibodies and reduction or clearance of serum HBV DNA).

Patient Screening and Surveillance
Patients will be identified as prospective candidates and entered into a surveillance of their viral infection on the basis of a preliminary HBV DNA test (which must show a serum HBV DNA ≥ 10 6 cpm. Patients will be subjected to a minimum of three surveillance visits where their viremia and fibrosis state will be assessed. Patients will also be asked to take a standard multivitamin and mineral supplement during the surveillance phase.

Patient enrollment
Once a patient has fulfilled all the criteria for enrollment, the data obtained and the enrollment submission form must be submitted to, and approved by REPLICor prior to a patient being entered into the trial. Each patient entered into the trial will receive a unique patient identifier (REP 102-X)

Management of REP 9AC' dosing
Several patients in the REP 101 protocol (REP 101-02B, 04B and 08B) rapidly cleared their serum HBsAg with 400-800mg of REP 9AC given in seven daily or every other day doses. Because of the substantially improved stability of REP 9AC' over REP 9AC, it is expected that patients receiving REP 9AC' IV infusions will benefit from a rapid and very long lasting suppression of their serum HBsAg. Therefore, patients clearing their serum HBsAg during the first week of treatment will come to the clinic during the subsequent weeks only for physical examination and blood testing but no drug will be administered until the HBsAg becomes detectable. If and when the HBsAg becomes detectable, patient will be given one additional gram of REP 9AC' by intravenous infusion which will be followed by weekly administration of 50 mg of REP 9AC' given subcutaneously. If patients have not completely cleared their serum HBsAg during the first week of treatment (but tolerated the infusions well), they will receive a second and potentially a third intravenous administration on an outpatient basis during the following two weeks. If additional REP 9AC' dosing is required to clear HBsAg or maintain HBsAg seroclearance, it will be administered by subcutaneous injection.
If a patient clears their serum HBsAg during the first week and this clearance is durable for several weeks after with no additional dosing, future serum HBsAg rebounds in this patient may be treated with outpatient IV infusions of REP 9AC' with longer intervals between doses.

Discontinuation of REP 9AC' treatment
The criterion for enrollment must be followed explicitly. In addition, patients will be discontinued from the drug study in the following circumstances: • If at any time during the study, a patient develops any conditions listed in the exclusion criterion, the principal investigator and REPLICor must be contacted to determine if patient discontinuation is warranted.
• The principal investigator decides that the patient should be discontinued. If this decision is made because of a serious adverse event (SAE) or a clinically significant laboratory value, the study drug is to be stopped and appropriate measures are to be taken. A joint decision between the principal investigator and REPLICor will be made to decide if the patient can continue receiving the drug later on.
• The patient is not capable or willing to continue participation in the study.
• The patient is non-compliant with the proscribed dosing and supplementation protocol.
• The patient meets the performance criteria (see below) for early entry into followup.

Rules for early entry into followup
Based on performance data from the REP 101 protocol, the following performance criterion for patients on REP 9AC' monotherapy will be used to identify patients which have demonstrated complete control over their infection and in which durable immunological control is likely to persist after treatment is withdrawn.

At least four continuous weeks of HBV DNA < 500 cpm
Patients meeting this criteria at any time after starting REP 9AC' monotherapy may be permitted to stop REP 9AC' treatment and patients admitted early into the follow up phase must follow the monitoring process as scheduled and would be started back on treatment only if they show signs of a relapse which they cannot self-resolve.

Missed doses
If a patient misses a treatment appointment, he should come in as soon as possible to receive the treatment that same week. Dosing appointments for subsequent weeks will not be altered.
If patient misses multiple treatments and it is decided to keep him on the study, these missed treatments can be added on past the scheduled end of treatment by extending the weekly treatment appointments by the number of treatments missed.

Adverse events
All adverse events must be promptly reported to REPLICor and a joint decision will be made in collaboration with the principal investigator regarding any actions to be taken.
The principal investigator will have the last word in any situation where there may be a disagreement with the sponsor. In every situation, the well-being of the patient shall remain the first priority.
The primary contact at REPLICor that should be contacted is:

Subcutaneous preparations
The drug will be provided in 3cc polycarbonate syringes prefilled with 2.1cc of 25mg/ml REP 9AC calcium chelate. The syringes will be identified with a label containing the following information: Name: REP 2139•Ca 25mg/ml Date of fill finish Each syringe has a luer lock cap that must be intact until use to ensure sterility. Each syringe is enclosed in a sterile plastic envelop that should only be opened at the time of administration.

Intravenous solutions
The drug will be provided in pre-filled sterile normal saline bags indicating the name of the drug and the total dose contained in the bag. These intravenous solutions will be ready to administer. The recommended speed of administration will be provide to the principal investigator for each patient. Each bag will have a label with the following information: Name: REP 2139•Ca Dose in mg Date of fill finish Each bag will be enclosed in a sterile plastic envelope that should only be opened at the time of administration. DEHP tubing for intravenous administration will be provided.

Drug storage
The drug should be stored in a refrigerator at between 4 o and 8 o degrees Celsius until use. The drug can tolerate room temperature for a long period of time without degradation but it is recommended to keep it in optimal conditions to maximize its shelf life.

Mineral Supplementation
Recent reports now provide evidence of substantial vitamin D deficiency (Fisher and In order to ensure the well-being of all future patients being enrolled on to the REP 102 study, patients will receive a daily, over the counter multivitamin and mineral / vitamin D3 supplement taken orally each day while they are participating in the REP 102 study.

Prohibited concomitant therapies
Patients cannot receive any drugs known to be active against HBV or HCV during the whole duration of the study. These drugs include interferon, ribavirin, protease inhibitors, polymerase inhibitors, nucleoside analogs, etc.

Concomitant therapies with restrictions on use
Patients should refrain from taking any drug other than REP 9AC' unless specifically authorized by the principal investigator. Every week, the investigator will question the patient for any drug taken over the last 7 days.

Safety assessments to be performed
History review of any symptoms the patients may be reporting.
Abdominal examination including liver tenderness assessment.

MONITORING AND DATA KEEPING
All the information collected during the trial about a specific patient will be collected and stored electronically in the english language.
All data obtained will be kept under the supervision of the principal investigator who will be responsible for the safekeeping of the electronic data on site. In addition, as soon as practical, every new data generated will be scanned and e-mailed to the sponsor at the following addresses: mbazinet@replicor.com and availlant@replicor.com. This will be used to maintain a duplicate electronic copy of all patient data.
Principal investigator will be responsible for drug accountability on site.

INFORMED CONSENT
The principal investigator is responsible for ensuring that the patient understands the risks and benefits of participating in the study and should answer any questions the patient may have throughout the study in a timely manner. Also, the investigator must promptly inform the patient of important new developments that may impact the patient's willingness to continue participating in the study.

COSTS OF STUDY
All the costs incurred during the screening, surveillance, for inclusion and exclusion criteria during the selection process, the weekly laboratory evaluation and the fibroscan analysis will be covered by the sponsor. The drug will be provided for free for the duration of the trial.

REGULATORY CONSIDERATIONS
This study will be conducted in accordance with the ethical principles that are consistent with good clinical practice and the applicable laws and regulations of Bangladesh.

INTRODUCTION
REP 9AC' (REP 2139) is a modified version of its predecessor, REP 9AC. Both are 40mer phosphorothioate oligonucleotides comprised of alternating adenosine and cytidine nucleotides and in the case of REP 9AC, has been shown to have low toxicity and to be highly effective in treating hepatitis B infection in human patients. The modifications in REP 9AC' significantly improve the stability and reduce the proinflammatory activity of REP 9AC' compared to that of REP 9AC while retaining all the antiviral activity found in REP 9AC. Both these modifications (5-methylation of cytosines and 2' O methylation of the ribose sugar in each nucleotide) are naturally occurring modifications in human nucleic acid and are known to be well tolerated in clinical trials.
Current interim data analysis from the still ongoing REP 102 protocol assessing the activity of REP 9AC' in patients with chronic HBV infection indicates the following: 1. REP 9AC exposure in twelve patients has been generally well tolerated at doses up to 500mg / week. Chronic side effects include mild gastrointestinal discomfort (which is easily mitigated with a PPI such as omeprazole). The pro-inflammatory side effects observed with REP 9AC in the REP 101 protocol are almost absent with REP 9AC' in the REP 102 protocol.
2. REP 9AC has achieved serum HBsAg reduction or clearance in 10 of 12 patients to date.
3. In these ten patients, all have demonstrated restoration of at least a partial immunological control of their infection (appearance of serum anti-HBs and the establishment of a lower serum HBV DNA setpoint). 4. In these ten patients, five have achieved a 4 -6 log reduction in serum HBV DNA and are approaching the limit of quantification (116 copies/ml) of the assay. 5. In these five patients, the achievement of these serum HBV DNA reductions is correlated with a transient flare of serum ALT and AST, suggesting that restoration of the immune response following HBsAg reduction and or clearance is a key factor in controlling the infection. Similar ALT and AST flares were observed in patients achieving immunological control of their HBV infection in the REP 101 protocol.
It is now a widely held notion that any successful outcome in the treatment of chronic HBV must involve immunostimulation in order to catalyze restoration of the appropriate immune responses (both adaptive and innate) in order to achieve durable control over HBV infection after therapy. The results from the 19 patients who have experienced treatment with REP 9AC or REP 9AC' clearly demonstrate that elimination of serum HBsAg removes the chronic immunosuppression mediated by this protein. However there is a heterogeneous immunological response to HBsAg reduction / clearance in the patients treated to date (as measured by serum HBV DNA decline): only about 40% of patients appear to exhibit a strong immunological response after the reduction / removal of serum HBsAg by REP 9AC / REP 9AC'. This observation strongly suggests that while HBsAg suppression is essential for establishing durable immunological control, specific immunostimulation may also be required in many patients in order for them to achieve durable immunological control while on REP 9AC / REP 9AC' therapy.

Thymosin alpha as a potential add-on therapy for immunostimulation in patients currently on REP 2139 (REP 9AC) treatment.
Thymosin alpha 1 (sold as Zadaxin ™ by SciClone Pharmaceuticals) is currently approved as a monotherapy for the treatment of chronic HBV in the following countries: Zadaxin™ is synthetically prepared thymosin alpha 1 polypeptide (28 amino acids, MW 3108) which is identical to the naturally occurring thymosin alpha 1 present in humans. Thymosin alpha 1 shares homology with the interferon alpha family of peptides but unlike interferon alpha, Zadaxin administration in human subjects is not accompanied by any significant side effects. More importantly, Zadaxin administration with other proinflammatory compounds (like interferon alpha) does not alter the side effect profiles of those compounds (1).
Zadaxin shares many of the immunostimulatory properties of interferon alpha in that it is able to stimulate the production of several cytokines important in reestablishing an immune response capable of controlling the HBV infection (1,2). Zadaxin is also able to stimulate the production of NK, CD4 and CD8 cells all known to be correlated with the establishment of a durable immunological control of HBV infection (1,2).
In the treatment of chronic HBV in the clinic, Zadaxin monotherapy is able to achieve HBeAg and HBV DNA seroclearance in 30-50% of patients which is comparable or better than that achievable with interferon alpha (1,3,4,5,6). More important is the consistent observation that the proportion of patients achieving HBV DNA seroclearance off therapy continually increases (an effect not observed with interferon alpha) (1).
Based on the very low side effect profile of Zadaxin (both as a monotherapy and in combination with interferon alpha and HBV polymerase inhibirors) and its clear ability to stimulate functional and durable immune response and control of HBV in patients with chronic HBV infections, the investigators strongly suspect that Zadaxin add-on therapy in patients currently receiving REP 9AC' will be safe and may have a synergistic effect on the ability of patients to achieve durable immunological control, thus greatly increasing the proportion of patients achieving durable immunological control with REP 9AC' -Zadaxin therapy compared to either therapy alone.

Pegasys ™ as a potential add-on therapy for immunostimulation in patients currently on REP 2139 (REP 9AC) treatment with no antiviral response on Zadaxin
It may be possible that the immunostimulation provided by Zadaxin™ may not provide a strong or broad enough immunostimulatory effect to provide an additive or synergistic antiviral response from the patient's immune system while on REP 9AC' therapy. Therefore, patients who tolerate Zadaxin™ / REP 9AC' combination therapy well but do not experience an improved or complete antiviral response after 8-10 weeks will be eligible to have the Zadaxin™ in their combination therapy replaced with Pegasys™. Since Pegasys™ is a much stronger immunostimulatory drug with significant side effects, dosing with this compound will start at 1/20 th of the normal dose (9 ug once weekly) and slowly escalate each week to a full dose (180 ug once weekly) providing no grade 3 adverse events are observed. Pegasys™ exposure will not last more than 8-10 weeks in the absence of any improved antiviral response compared to REP 9AC' alone and normally not exceed 24 weeks.

Proposed amendments to the REP 102 protocol.
Zadaxin™ combination therapy is to be implemented according to the following rules: A. Early add-on rule: Patients not achieving serum HBV DNA < 2000 copies / ml after 20 weeks of REP 9AC' therapy who have not developed any persistent grade 3 or higher side effect are eligible to begin Zadaxin™ therapy. Zadaxin™ will be administered initially once weekly (1.6 mg SC injection) during the same visit in which patients receive their REP 9AC' by IV infusion. Zadaxin™ therapy can transition to twice weekly SC injections in the third week in the absence of any side effects grade 3 or higher.
B. Late add-on rule: Patients not achieving 4 consecutive weeks of HBV DNA < 200 copies / ml after 30 weeks of REP 9AC' treatment who have not developed any significant complications are eligible to begin Zadaxin™ therapy. Zadaxin™ will be administered initially once weekly (1.6 mg SC injection) which can then transition to twice weekly SC injections in the third week in the absence of any side effects grade 3 or higher.
C. Extension of combination REP 9AC' / Zadaxin™ therapy: Patients showing response to combination therapy (continued reduction in serum HBV DNA or achievement of serum HBV DNA < 116 copies / ml) may have their combination therapy extended beyond the current 40 week REP 9AC' limit (and 24 week Zadaxin™ limit) at the Principle investigator's discretion in order to achieve the goal of 4 continuous weeks of serum HBV DNA < 116 copies / ml prior to cessation of REP 9AC' therapy. This extension can only be permitted in patients who have not developed symptoms greater than grade 3 on combination therapy.
Safety and efficacy monitoring will proceed without modification.
Pegasys ™ therapy is to be implemented according to the following rules: A. Initiation of treatment: Patients who have had 8-10 weeks of Zadaxin™ / REP 9AC' combination therapy with no adverse effects grade 3 or greater and have had no significant improvement in their antiviral response compared to REP 9AC' alone and have no significant hematological or hepatic dysfunction are eligible to start REP 9AC / Pegasys ™ treatment.
B. Dose escalation: patients eligible to start REP 9AC / Pegasys™ combination treatment will receive an initial dose of 9ug of Pegasys™ (1/20 th of the normally prescribed dose) by SC injection. Pegasys™ doses will escalate each week to 18ug, 45ug, 90ug and finally 180ug provided no adverse events grade 3 of higher are observed.
C. Extension of combination REP 9AC' / Pegasys™ therapy: Patients showing response to combination therapy (continued reduction in serum HBV DNA or achievement of serum HBV DNA < 116 copies / ml) may have their combination therapy extended beyond the current 40 week REP 9AC' limit (and 24 week Pegasys™ limit) at the Principle investigator's discretion in order to achieve the goal of 4 continuous weeks of serum HBV DNA < 116 copies / ml prior to cessation of REP 9AC' therapy. This extension can only be permitted in patients who have not experiences any adverse events greater than grade 3 on combination therapy.
Hematology will be monitored on a weekly basis while patients are on REP 9AC' / Pegasys™ combination therapy.